Capsaicin diet drives gut inflammation and exosomal miR-17-3p elevation in idiopathic short stature

Domain: neuroscience
Published: 2025-12-30
Editorial outcome: major_revision

Abstract
Idiopathic short stature (ISS) remains a major pediatric challenge with unclear causes and inconsistent responses to growth hormone therapy. Here we show that plasma exosomes from children with ISS contain elevated hsa-miR-17-3p that disrupts growth signaling and impairs cartilage cell proliferation. Elevated miR-17-3p suppresses ZNF148/SOS1 signaling, linking molecular dysfunction to dietary exposure in ISS. To investigate environmental triggers, we developed a capsaicin-rich diet rat model that recapitulates ISS, showing normal Gh/Igf-1 levels but elevated plasma miR-17-3p. The diet induced mild gut inflammation, increasing miR-17-3p in intestinal and plasma exosomes. Fecal samples from ISS children exhibited similar elevations in miR-17-3p and inflammatory markers, linking spicy diets to ISS pathogenesis. Finally, engineered exosomes designed to silence miR-17-3p, combined with localized growth hormone therapy, restored growth plate function. These findings uncover a diet-driven exosome axis underlying ISS and suggest new therapeutic strategies for children in high-capsaicin regions.

Introduction
Short stature is defined as a child’s height being less than two standard deviations or below the 3rd percentile of the average human height of children of the same sex, age, and race1–3. This condition may have significant implications for the physical and psychological well-being of affected children. Previous research studies have shown that anxiety scores and depression scores are significantly higher in children with short stature than in the control group4. As a result, these challenges often persist into adulthood. Recombinant human growth hormone (rhGH) offers hope for treating children with short stature, especially in cases of growth hormone deficiency5. However, the majority of cases are ultimately classified as idiopathic short stature (ISS) after ruling out systemic diseases, nutritional deficiencies, psychiatric disorders, chromosomal abnormalities, and growth hormone deficiency, yet their cause remains unknown1,6,7. ISS represents 30–80% of short-stature cases1,6, but its underlying mechanisms are poorly understood. Unlike children with growth hormone deficiency-related short stature, those with ISS have normal growth hormone levels7. During clinical treatment, it is not uncommon for ISS children to show insensitivity to rhGH therapy. As a result, rhGH treatment for ISS requires higher dosages and shows varying levels of effectiveness compared to treatments for the short stature of growth hormone deficiency. The efficacy and safety of rhGH therapy continue to be topics of debate.

Hou et al.8 reported an average height gain of 9.55 ± 0.13 cm after one year of rhGH treatment in 200 Chinese children with ISS (average age 9.03 ± 0.21 years; 68.5% boys, 31.5% girls), whereas Yoo et al. noted an increase of 10.68 ± 1.95 cm9. In contrast, research by Van et al.10 and Kamp et al.11 reported that while rhGH therapy may enhance height during treatment, it could accelerate growth plate closure, resulting in adult heights that do not significantly differ from those of untreated controls. These findings highlight the variable efficacy of rhGH therapy in achieving long-term height improvement.

In terms of safety, while some studies have raised concerns about potential risks associated with rhGH therapy, such as bone and cartilage tumors, cerebrovascular diseases, hypertension, insulin resistance, and brain hemorrhages12–14, other large-scale studies, including those by Savendahl et al. and Cianfarani et al., indicate that rhGH treatment is generally safe when used appropriately, particularly in low-risk patients15,16. Nevertheless, the long-term safety profile of rhGH therapy, especially regarding specific risks, remains an area of ongoing research and surveillance.

The 2016 American Academy of Pediatric Endocrinology guidelines emphasize that rhGH therapy is not universally recommended for all ISS patients, particularly those with less severe short stature (height standard deviation score [HtSDS] above −2.25) or those without significant physical or psychosocial burdens related to their condition17. This underscores the limitation of rhGH therapy in ISS, including its variable efficacy and potential risks. Additionally, the high cost of rhGH treatment, estimated at $10 billion annually in the United States18, further limits accessibility and highlights the need for alternative therapies. Given these challenges, a deeper understanding of the molecular mechanisms underlying ISS is crucial for developing more effective and targeted treatments.

In recent years, researchers have pinpointed specific ISS susceptibility genes using whole-genome sequencing. These genes include GHR, SHOX, FGFR3, ACAN, and NPR219–23. However, the mutation rate of these genes is only 1–5%, which significantly limits their potential application in clinical treatment despite being beneficial for further ISS typing. Additionally, while the hypothalamic-GH-IGF axis is a crucial pathway regulating childhood linear growth, most ISS patients do not exhibit significant abnormalities in this area.

As the terminal regulatory organ of the hypothalamic-GH-IGF axis, the growth plate is responsible for longitudinal bone growth through endochondral ossification. It determines final adult height, serving as the “engine” of height development. Analyzing the molecular pathological changes in the growth plates of ISS children could elucidate their pathogenesis. However, the ethical challenges of obtaining growth plate samples from ISS children presents significant barriers to etiological research and the development of new treatments.

It is well known that various hormones and growth factors secreted by tissues and organs are delivered to the growth plate via the bloodstream, interacting with it to regulate growth and development through intercellular signaling processes. Previous studies have indicated no abnormalities in the secretion of growth hormone, glucocorticoids, and thyroid hormones in the circulation of ISS patients. However, blood circulation contains numerous exosomes besides hormones and growth factors.

Exosomes, small lipid-bilayer vesicles, play a critical role in cell communication and pathology by transporting proteins, miRNAs, and other molecules between cells24–26. Natural nanoparticles have significant potential for drug delivery since they can cross biological barriers without causing immunogenic reactions27. Although exosomes show potential for advancing our understanding and treatment of ISS, their specific role in the pathogenesis of ISS remains largely unclear and warrants further investigation.

Our study demonstrates that plasma-derived exosomes from ISS children markedly inhibit chondrocyte proliferation and bone formation, an effect linked to hsa-miR-17-3p overexpression. However, the origin of elevated plasma exosomal miR-17-3p in ISS remains unclear. To investigate this, we reviewed epidemiological data and found that ISS prevalence is higher in Chinese regions with high capsaicin consumption—such as Jiangxi, Chongqing, Hunan, and Guizhou—collectively home to over 200 million people28. Zou et al.29 reported that Hunan Province has the highest total annual pepper consumption (7.57 million tons), followed by Jiangxi, Guizhou, Chongqing, Yunnan, and Hubei. The combined population of these six provinces is approximately 286.8 million. Based on per capita pepper consumption, the upper and middle reaches of the Yangtze River were classified as “extreme-spicy” areas, including Hunan (111.57 kg/person/year), Guizhou (98.5 kg), Jiangxi (97.6 kg), and Chongqing (95.6 kg).

These large-scale observations suggest a potential link between habitual high-capsaicin intake and ISS pathogenesis, with significant public health implications. The capsaicin dietary model used in our animal experiments was developed to reflect the epidemiological characteristics of these high-incidence regions, where capsaicin-rich diets are a common part of daily life.

In subsequent experiments, a capsaicin-enriched diet—designed as a model to simulate the chili consumption levels observed in these high-prevalence areas—induced mild intestinal inflammation in Sprague-Dawley rats. This inflammation activated the NF-κB signaling pathway, identified as the main driver of elevated plasma exosomal miR-17-3p levels. To our knowledge, this is the first study to directly connect dietary capsaicin, gut inflammation, and ISS pathogenesis, providing new insight into ISS etiology.

Furthermore, we engineered exosomes with growth plate-targeting capabilities to deliver siRNA, effectively counteracting the inhibitory effects of miR-17-3p overexpression while allowing precise local growth hormone administration. This approach significantly enhanced therapeutic outcomes for short stature, representing a promising and innovative treatment strategy. The findings of this study not only deepen our understanding of the molecular mechanisms underlying ISS but also propose a groundbreaking avenue for targeted therapeutic interventions with the potential to revolutionize current treatment approaches. Finally, our results emphasize the necessity of increasing attention to the impact of capsaicin-rich diets on children’s longitudinal growth, particularly among high-risk ISS populations and short-statured children receiving growth hormone therapy.

Results
Characterization of plasma exosomes
Plasma exosomes from ISS patients and healthy controls were isolated via ultracentrifugation and characterized using transmission electron microscopy (TEM) and Western blotting. TEM revealed cup-shaped or spherical exosomes, with Nano-Sight analysis indicating a predominant diameter of 100 nm (Fig. 1a, b). Western blotting confirmed the presence of exosomal markers CD81, CD9, and CD63 in both ISS and control samples (Fig. 1c). Successful uptake of FAM-labeled exosomes (indicated by green fluorescence) by human chondrocytes C28/I2 (blue fluorescence) was verified (Fig. 1e).

Impaired chondrocyte proliferation and bone formation induced by plasma exosomes from ISS children
Human chondrocytes co-cultured with plasma exosomes from ISS children exhibited a marked reduction in proliferation, as shown by EdU and CCK-8 assays (Fig. 1d, f, g). Flow cytometric analysis indicated an increased proportion of cells in the G0/G1 phase and a corresponding decrease in the S phases in the ISS-Exos group compared to controls (Fig. 1h, i). RT-qPCR and Western blot analyses revealed significant downregulation of chondrocyte hypertrophic differentiation markers (COL10A1, RUNX2) and osteogenic marker genes (OPN, OCN). Moreover, ALP activity was markedly reduced, and positive staining for Von Kossa and Alizarin Red decreased in the ISS-Exos group (Fig. 1j, k, l, m). These results suggest that plasma exosomes from ISS children impair chondrocyte proliferation and bone formation.

Identification of miR-17-3p as a diagnostic biomarker in ISS via high-throughput sequencing of plasma exosomal miRNAs
High-throughput sequencing of plasma exosomal miRNAs identified 42 differentially expressed miRNAs, including 31 upregulated and 11 downregulated (Supplementary Fig. 1a, b). RT-qPCR analysis demonstrated a significant upregulation of miR-17-3p in the plasma exosomes of ISS children (Supplementary Fig. 1c). An area under the curve (AUC) of 0.976 (95% CI, 0.959–0.993) was observed for miR-17-3p, with an optimal cutoff point of >1.2955, yielding a specificity of 89.62% and sensitivity of 98.13% for distinguishing children with ISS from healthy controls. (Supplementary Fig. 1d). In situ hybridization analysis demonstrated that miR-17-3p is expressed in human chondrocytes (C28 cell line) and is also localized in the growth plate cartilage of SD rats. In situ hybridization revealed localization of miR-17-3p expression in human chondrocytes versus SD rat growth plate region (Supplementary Fig. 1e). GO and KEGG pathway analyses indicated that genes targeted by the overexpression of miR-17-3p are enriched in the PI3K-Akt, RAS, and MEK signaling pathways (Supplementary Fig. 1f, g).

miR-17-3p as the key mediator of ISS Exosome-Induced inhibition of chondrocyte proliferation and bone formation
Exosomes from ISS children inhibited chondrocyte proliferation and bone formation, which was reversed by downregulating miR-17-3p expression in ISS exosomes (Fig. 2a–j). Furthermore, the overexpression of miR-17-3p in human chondrocytes significantly suppresses proliferation, as demonstrated by CCK-8, cell cycle, and EdU assays (Supplementary Fig. 2a–f). Hypertrophic differentiation markers (COL10A1, RUNX2) and osteogenic markers genes (OPN, OCN) were also downregulated, and osteoblastic activity, indicated by ALP staining, and bone formation capacity, evidenced by Von Kossa and Alizarin Red staining, were significantly reduced (Supplementary Fig. 2g-j). This indicates that ISS exosomes inhibit chondrocyte proliferation and bone formation by overexpressing miR-17-3p.

Given that two-dimensional chondrocyte cultures do not fully replicate the biological effects of miR-17-3p overexpression in growth plate cartilage, we performed whole-knee joint organ cultures of neonatal rats. The results mirrored those observed in chondrocyte cultures. Overall bone growth and development were impaired, with reduced expression of hypertrophic differentiation marker genes (COL10A1, RUNX2) and osteogenic marker genes (OCN, OPN) (Supplementary Fig. 3a-i).

miR-17-3p Targets the ZNF148/SOS1 axis to regulate chondrocyte proliferation and bone formation
High-throughput mRNA sequencing following miR-17-3p overexpression identified 77 upregulated and 166 downregulated genes with differential expression (Supplementary Fig. 4a, b). Western blotting and RT-qPCR validated decreased ZNF148 expression in human chondrocytes after overexpressing miR-17-3p (Supplementary Fig. 4c and Fig. 5b–d). Dual luciferase assays confirmed direct interaction, with reduced luciferase activity following co-transfection with wild-type miR-17-3p/ZNF148 mimics (Supplementary Fig. 4f, g). Rescue experiments with ZNF148 overexpression countered the inhibitory effects of miR-17-3p on proliferation and bone formation (Supplementary Fig. 5a–m).

Bioinformatics analysis identified SOS1 (Son of Sevenless homolog 1) as a downstream target regulated by ZNF148 (Supplementary Fig. 7a). RT-qPCR and Western blot analyses demonstrated the downregulation of ZNF148 and SOS1 following miR-17-3p overexpression (Supplementary Fig. 6a–d). Rescue experiments with SOS1 overexpression reversed the inhibitory effects of miR-17-3p on chondrocyte proliferation and ossification, as evidenced by EdU, CCK-8 assays, cell cycle analysis, and molecular marker evaluation (Supplementary Fig. 6). ChIP-seq and agarose gel electrophoresis validated ZNF148’s binding to the SOS1 (Supplementary Fig. 7b–d). A significant reduction in SOS1 expression at both mRNA and protein levels was also detected by knockdown of ZNF148 (Supplementary Fig. 7e–g). These findings suggest that miR-17-3p regulates chondrocyte proliferation and bone formation through the ZNF148/SOS1 axis.

The Role of ZNF148 and SOS1 in regulating chondrocyte proliferation and growth plate development
Using CRISPR-Cas9 to knock out ZNF148 or SOS1 in human chondrocytes and neonatal rat growth plate cartilage, we investigated their roles in chondrocyte proliferation and bone formation. The knockout of ZNF148 or SOS1 significantly impaired chondrocyte proliferation and bone formation (Supplementary Figs. 8, 9). Rescue experiments in which SOS1 was overexpressed in ZNF148-deficient chondrocytes restored proliferation and endochondral ossification/bone formation (Supplementary Fig. 10). Ex vivo cultures of neonatal rat knee joints confirmed these findings, showing reduced chondrocyte proliferation and downregulated differentiation markers (COL10A1, RUNX2) and osteogenic markers genes (OCN, OPN) in ZNF148 or SOS1 knockout groups (Supplementary Figs. 11, 12).

miR-17-3p targets the ZNF148/SOS1 axis and modulates MEK and PI3K pathways to regulate chondrocyte proliferation and bone formation
Enrichment analysis of differentially expressed mRNAs following miR-17-3p overexpression identified significant enrichment in the MEK and PI3K pathways, highlighting their role in miR-17-3p-mediated suppression of chondrocyte proliferation and endochondral ossification/bone formation (Supplementary Fig. 4d, e). RT-qPCR and Western blot analyses revealed that SOS1 knockout did not alter the mRNA or protein expression levels of key MEK and PI3K pathway genes, including ERK1/2, PI3K, and AKT. However, SOS1 knockout significantly decreased RAS expression and the phosphorylation levels of ERK1/2, PI3K, and AKT (Supplementary Fig. 9a–e and Fig. 12g, j, k). This in vitro experiment indicates that SOS1 exerts its biological effects by regulating the phosphorylation of these proteins, thereby inhibiting the RAS/ERK and PI3K pathways. This finding was further validated by in vivo experiments in rats (Figs. 3, 4 and Supplementary Fig. 18).

Exosomal miR-17-3p inhibits growth plate chondrocyte proliferation and bone formation in vivo, leading to a short-stature phenotype
In vivo studies demonstrated that overexpression of miR-17-3p, using exosomes derived from primary rat chondrocytes isolated from the proximal tibia and distal femur growth plates, induced a short-stature phenotype in immature rats (Figs. 3–4). RT-qPCR confirmed overexpression of miR-17-3p in plasma and growth plate cartilage (Supplementary Fig. 24b). Safranin O and Fast Green staining, Calcein labeling, RT-qPCR, and Western blot analyses showed decreased overall growth plate thickness with a reduced rate of new bone formation, decreased expression of hypertrophic differentiation marker genes (Col10a1, Runx2) and osteogenic marker genes (Ocn, Opn) (Fig. 3a–p).

Capsaicin-induced short-stature phenotype and elevated plasma miR-17-3p expression in immature rats
Although we are the first to report elevated miR-17-3p levels in plasma-derived exosomes from children with ISS, the underlying causes and sources of this overexpression of miR-17-3p remain unclear. Epidemiological data reveal that the incidence of ISS is significantly higher in Chongqing, Jiangxi, Sichuan, Guizhou, Hunan provinces, and Chongqing municipalities in China, regions where high chili pepper consumption is a prevalent dietary habit28,30,31. In response, we developed an ISS animal model by administering a capsaicin-enriched diet to immature rats. Analysis of the capsaicin (0.01% supplemented) and control (capsaicin-free) diet groups revealed that during the first four days, the average daily food intake in the capsaicin group was slightly lower than that in the control group (Supplementary Fig. 25a). This reduced intake in the capsaicin group during the initial four days is likely attributable to a short-term adaptation to the transition from a capsaicin-free to a capsaicin-supplemented diet. From day six onward, the daily food intake of the capsaicin group showed no statistically significant difference compared to the control group, indicating adequate palatability of the capsaicin diet (Supplementary Fig. 25a). However, by the end of the 28-day experiment, the capsaicin group exhibited significantly lower body weight and height compared to the control group (Supplementary Fig. 25b, c). The weight-to-length ratio (WLR), calculated as WLR = weight (g) ÷ length (cm), was 6.29 g/cm for the capsaicin group and 6.03 g/cm for the control group, with no statistically significant difference between the groups. These findings suggest that the reduced stature in the capsaicin group resulted from the 0.01% capsaicin diet inhibiting growth and development, rather than reduced food intake due to poor diet palatability. Remarkably, capsaicin-diet not only suppressed growth plate chondrocyte proliferation and bone formation, thereby successfully inducing a short-stature phenotype (Fig. 5), but also elevated miR-17-3p levels in the plasma of these rats (Supplementary Fig. 24a). This finding aligns with the elevated plasma miR-17-3p observed in ISS children, providing further insight into the potential link between the capsaicin diet and miR-17-3p expression in ISS.

We further performed a comprehensive histological examination of the intestinal tissues to elucidate the mechanism behind the elevated miR-17-3p expression observed in capsaicin-fed rats. This analysis uncovered significant structural disruptions in the villi, along with marked infiltration of inflammatory cells in the small intestines and colon of the capsaicin-treated group (Supplementary Fig. 14a, b and Supplementary Fig. 16). Elevated activities of myeloperoxidase (MPO) and malondialdehyde (MDA) provided evidence of oxidative stress and a likely inflammatory response (Supplementary Fig. 14c, d). ELISA assays revealed a notable increase in pro-inflammatory cytokines Il-1β, Il-10 and Tnf-α in the capsaicin-exposed rats (Supplementary Fig. 14e–g).

Previous research identified 84 differentially expressed plasma proteins in children with ISS, with 42 of these proteins associated with inflammatory pathways. Bioinformatic analysis indicated that 43% of these inflammatory proteins are regulated by the transcription factor NF-κB, implicating its involvement in the inflammatory response observed in ISS (Supplementary Fig. 13). The P65 subunit of the NF-κB protein complex regulates up to 50% of inflammation-related proteins in children with ISS (Supplementary Fig. 13). Subsequent Western blot and RT-qPCR analyses corroborated these findings, revealing elevated expression of phosphorylated p65 and Iκb in the intestinal tissues of capsaicin-fed rats, indicative of intensified NF-κB signaling (Supplementary Fig. 14l, o, and p). This was accompanied by a concurrent increase in miR-17-3p expression in the small intestine (Supplementary Fig. 14h).

To further explore the role of capsaicin in triggering intestinal inflammation and to elucidate the regulatory interplay between NF-κB and miR-17-3p, we conducted in vitro studies using the small intestinal epithelial cell line HIEC-6 and the healthy colon mucosal epithelial cell line NCM460 subjected to capsaicin stimulation. At lower concentrations, capsaicin promoted cellular proliferation, while higher concentrations, particularly at 200 µM, significantly inhibited cell growth and induced substantial apoptosis (Supplementary Fig. 15a–d and Supplementary Fig. 17a–f). This optimal concentration induced significant apoptosis (Supplementary Fig. 15e, f and Supplementary Fig. 17g–i) and elevated expression levels of both miR-17-3p and NF-κB (Supplementary Fig. 15h–k and Supplementary Fig. 17j-n). Notably, applying the NF-κB inhibitor bortezomib reduced miR-17-3p expression, underscoring that capsaicin activates NF-κB, subsequently driving the upregulation of miR-17-3p (Supplementary Fig. 15g–k and Supplementary Fig. 17j–n).

Elevated miR-17-3p in Stool Samples of ISS Children
To investigate the presence of gut inflammation and the expression of miR-17-3p in children’s ISS, we performed RT-qPCR or ELISA analyses on stool samples collected from both ISS patients and healthy controls. The results revealed a pronounced elevation in miR-17-3p and TNF-α levels in ISS children compared to their healthy counterparts, suggesting that the heightened miR-17-3p expression in ISS may be a consequence of underlying intestinal inflammation (Supplementary Fig. 14m, n).

ET-Exo-si-miR-17-3p-oeGH Antagonizes miR-17-3p-Induced Inhibition of Chondrocyte Proliferation and Bone Formation in Human Chondrocyte and Restores Height in Rats
To effectively target and eliminate aberrant miR-17-3p expression in growth plate cartilage and enable precise delivery of growth hormone (GH) for ISS treatment, we utilized membrane fusion protein technology to engineer exosomes displaying type II collagen peptides on their surface, enhancing their specificity for growth plate cartilage. These exosomes were subsequently loaded with miR-17-3p siRNA and GH. The resulting engineered exosomes, specifically designed for this purpose, are referred to as ET-Exo-si-miR-17-3p-oeGH.

Transmission electron microscopy confirmed the morphology of the exosomes, revealing a characteristic spherical or cup-shaped structure (Fig. 6a). Nano-Sight analysis indicated that the exosomes have an average diameter of approximately 150 nm (Fig. 6b). Zeta potential measurements using Zetaview (Particle Metrix) showed a negative surface charge of about -50 mV (Fig. 6c). Western blotting confirmed the presence of the exosomal markers CD81, CD9 and CD63 (Fig. 6d). The efficiency of GH loading into ET-Exo-si-miR-17-3p-oeGH was demonstrated by a dose-dependent increase in GH content, with 200 μg of ET-Exo-si-miR-17-3p-oeGH encapsulating nearly 100 ng of GH (Fig. 6e, g). Additionally, RT-qPCR confirmed the presence of siRNA-miR-17-3p within ET-Exo-si-miR-17-3p-oeGH (Fig. 6f, h). Co-culturing human chondrocytes with ET-Exo-si-miR-17-3p-oeGH demonstrated robust exosome uptake, GH internalization, and siRNA overexpression (Fig. 6i, j).

Frozen tissue sections corroborated the effective delivery of siRNA-miR-17-3p and GH protein by ET-Exo-si-miR-17-3p-oeGH to the growth plate cartilage (Fig. 6l). In vivo bioluminescence imaging assessed cartilage targeting, revealing a significantly higher accumulation of ET-Exo-si-miR-17-3p-oeGH in the growth plates of extremities compared to NC-Exos over time. Conversely, markedly fewer exosomes were detected in the heart, liver, spleen, lungs, kidneys, and pancreas (Fig. 6k and Supplementary Fig. 22), underscoring the specificity of ET-Exo-si-miR-17-3p-oeGH for targeting growth plate cartilage. Preliminary data from histological and biochemical analyses suggest that Exo-si-miR-17-3p-oeGH exhibits no significant toxicity in major organs, including the heart, liver, spleen, lungs, and kidneys (Supplementary Fig. 23). However, further studies are needed to confirm its comprehensive biosafety profile.

Subsequent in vitro experiments showed that ET-Exo-si-miR-17-3p-oeGH attenuated miR-17-3p overexpression inhibitory effects on chondrocyte proliferation and bone formation, as illustrated in Fig. 7. Following overexpression of miR-17-3p in neonatal rats, Safranin O and Fast Green staining revealed a decrease in overall growth plate thickness (Fig. 8i), and calcein labeling further highlighted a significant reduction in new bone formation (Fig. 8g, h), as well as a reduction in the expression of markers of hypertrophic differentiation (Col10a1 and Runx2) and osteogenic genes (Ocn and Opn) (Fig. 8j, k). Consequently, the rats displayed a short stature phenotype (Fig. 8a–f). Despite administering growth hormone therapy, height did not normalize compared to the control group, reflecting insensitivity to growth hormone therapy similar to that observed in clinical cases of ISS. In contrast, treatment with ET-Exo-si-miR-17-3p-oeGH demonstrated exceptional therapeutic efficacy in addressing the short stature induced by miR-17-3p overexpression, as shown in both in vitro and in vivo experiments (Figs. 7, 8). RT-qPCR demonstrated the levels of miR-17-3p in each group (Supplementary Fig. 24c, d).

Engineered Growth Plate Cartilage-Targeted Exosomes derived from Bone Marrow Mesenchymal Stem Cells similarly counteract miR-17-3p-induced inhibition of chondrocyte proliferation and bone formation in human chondrocytes and restore height in rats
Compared to chondrocyte-derived exosomes, mesenchymal stem cell-derived exosomes are more readily obtainable and hold incredible promise for clinical applications. Therefore, we evaluated the efficacy of engineered growth plate-targeted exosomes derived from bone marrow mesenchymal stem cells in treating short stature. In the present study, we established a stable line of bone marrow mesenchymal stem cells capable of secreting exosomes with consistent properties. Morphological characterization confirmed the exosomes’ spherical or cup-shaped structure (Supplementary Fig. 19a), with an average particle size of approximately 150 nm (Supplementary Fig. 19b) and a negative zeta potential (Supplementary Fig. 19c), Western blotting confirmed the presence of the exosomal markers CD81, CD9 and CD63 (Supplementary Fig. 19d). In vitro and in vivo experiments demonstrated the targeted delivery capabilities of the engineered exosomes and load capacity and confirmed their effectiveness in treating short stature (Supplementary Figs. 19–21). RT-qPCR demonstrated the levels of miR-17-3p in each group (Supplementary Fig. 24e, f).

Methods
Clinical Sample Characteristics and Plasma Exosome Collection
These 106 ISS patients and 106 age- and gender-matched controls were recruited from the Second Affiliated Hospital of Nanchang University, China. The ISS group comprised 52 males and 54 females (median age: 9.3 years, IQR: 7.475–10.50; median height: 125.6 cm, IQR: 116.3–133). The control group included 57 males and 49 females (median age: 9.55 years, IQR: 7.7–10.8; median height: 137.5 cm, IQR: 129.7–144.7) (Supplementary Table 1). Exclusion criteria encompassed: (i) skeletal anomalies or dysmorphic features at birth, (ii) hormonal abnormalities such as growth hormone (GH) deficiency (peak level <6 ng/mL) or thyroid and pubertal disorders, (iii) environmental or chronic conditions impacting growth, (iv) chromosomal aberrations detected cytogenetically, and (v) whole-exome sequencing findings.

Whole blood samples were collected between October 2016 and March 2023, and plasma was used to isolate exosomes according to the manufacturer’s protocol (Exosome Isolation and Purification Kit, Umibio, Cat No. UR52151). Exosome samples were stored at -80°C. Five pairs of samples underwent miRNA High-Throughput Sequencing for miRNA expression analysis, while all 212 samples were used for quantitative RT-PCR validation. The study was approved by the Human Research Ethics Committee of the Second Affiliated Hospital of Nanchang University (Approval No. [2024](50)). Written informed consent was obtained from participants’ guardians if under 14 years of age.

We collected 5 ml of plasma from ISS patients, yielding approximately 200 µg of exosomes after extraction. For cell culture experiments, we co-cultured 100 µg of these ISS plasma-derived exosomes with 3×10⁵ human C28/I2 chondrocytes in a T25 flask containing 5 ml of culture medium. This setup was used to investigate whether the exosomes inhibit chondrocyte proliferation and markers of endochondral ossification.

Fecal Sample Collection and Storage for miRNA and ELISA Analyses
Fresh fecal samples (2.5 ± 0.5 g) were collected within 30 minutes of defecation using sterile, single-use plastic spoons and transferred into sterile polypropylene containers to prevent contamination. Samples were immediately divided into two aliquots for downstream analyses. For miRNA analysis, one aliquot (~1.5 g) was mixed with RNAlater stabilization solution (1:1 w/v, QIAGEN, Cat No. 76106) and stored at 4°C for 24 hours to ensure RNA stabilization, then transferred to -80°C for long-term storage. For ELISA analysis of inflammatory markers (e.g., IL-1β, TNF-α), a second aliquot (~1 g) was snap-frozen at -80°C without buffer to avoid interference with protein assays. All sample handling was performed using gloves under sterile conditions to minimize contamination.

miRNA and mRNA High-Throughput Sequencing
The miRNA sequencing and data analysis were performed by Shanghai Biotechnology Corporation (Shanghai, China). Briefly, total RNAs were isolated from each sample using the QIAamp RNeasy Serum/Plasma Midi Kit (Cat No. 77044, Qiagen). Subsequently, the sequencing library was constructed with the QIAseq miRNA Library Kit (Cat No. 1103679, Qiagen) and sequenced on the Illumina HiSeq platform (California, USA). Differentially expressed miRNAs were identified with P<0.05 and |log2(FC)| ≥ 1.5.

For mRNA analysis, total RNA was extracted using the Eastep® Super RNA easy Kit (Promega, Beijing). Libraries were constructed with the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina). Transcriptome sequencing and differential expression analysis were performed using OE Biotech (Shanghai, China) on an Illumina HiSeq X Ten sequencer. Clean reads were aligned to the human genome using HISAT2, and gene expression levels were quantified via Cufflink and HTSeq-count. Differentially expressed genes (DEGs) were identified through fold-change filtering (log2(FC) ≥ 2.0, P<0.05). Gene Ontology (GO) enrichment and KEGG pathway analyses were conducted for DEGs and target genes of DE-miRNAs. The miRNA-mRNA regulatory network was visualized based on established methods.

In-Situ Hybridization
Human chondrocytes were incubated with FAM-labeled probes for miR-17-3p for 48 hours at 65°C, according to the manufacturer’s instructions (miRCURY LNA miRNA ISH Kit, Thermo Fisher). The localization of miR-17-3p was visualized using a CY3-labeled sequence (5’-CY3-CTACCTGCACTGTAAGCACTTTG-3’ and 5’-CCACAAGTGCCTTCACTGCAGT-3’, miRCURY LNA miRNA ISH kit, Thermo Fisher, Shanghai, China). Similarly, femur samples from six-week-old rats were decalcified, sectioned, and subjected to in situ hybridization.

Cell Culture, Exosome Isolation, and Extraction of Exosomes from ISS Patient Plasma
The human healthy small intestinal epithelial cell line HIEC-6 (Otwo Biotech, Cat No. HTX1978) and human healthy colon mucosal epithelial cell line NCM460 (Otwo Biotech, Cat. No. HTX1841) were cultured in DMEM enriched with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, maintained at 37°C in a humidified incubator with 5% CO2. Human chondrocytes C28/I2 (Otwo Biotech, Cat No. HTX2308) were cultured in 89% H-DMEM with 10% FBS in a 37°C incubator with 5% CO2. The hBMSCs were purchased from Otwo Biotech (Shenzhen) Inc. (Cat No. HTX1946) and cultured in an MSC-specific medium, 37°C humidified incubator, 5% CO2 atmosphere. Exosomes were isolated from cell culture media using the Exosome Isolation Kit (Umibio, Cat No. UR52121), and protein quantification was conducted using the BCA Protein Assay Kit (Beyotime, Cat No. P0010). Details of Cell culture and exosome isolating methods can be found in our previous publication28.

Cell viability assay
The cell suspension was mixed evenly with 0.4% trypan blue solution (from Beijing Solabao Co., Ltd.) in a 9:1 ratio (final concentration 0.04%). The cells were stained for 3 min, and the cell viability was measured using a cell viability counting instrument (Shanghai Bio-Red Co., Ltd.). Only samples with a cell viability greater than 95% were used for subsequent experiments.

Capsaicin co-culture model with intestinal cells
Natural capsaicin (404-86-4) was purchased from Weilkeqi Co., Ltd. It was dissolved in PBS to prepare a capsaicin solution with an initial concentration of 1 mg/ml. The solution was diluted with complete medium to obtain concentrations of 0 μM, 25 μM, 50 μM, 75 μM, 100 μM, 150 μM, and 200 μM for the intestinal cell (HIEC-6 and NCM460) capsaicin stimulation culture experiments. After a culture period of 0 h, 24 h, 48 h, and 72 h, these cells were used in further experiments.

Cell proliferation assay
The proliferations of chondrocytes, small intestinal epithelial cell line HIEC-6, and human healthy colon mucosal epithelial cell line NCM460 were assessed using the CCK-8 assay (TransGen Biotech). Cells were seeded into 96-well plates and incubated for 24, 48, 72, and 96 hours, followed by absorbance measurements at 450 nm. Additionally, EdU assays (Guangzhou Ribo Bio Co., Ltd. China) were performed to further evaluate cell proliferation of chondrocytes and bmsc, with stained cells imaged via fluorescence microscopy.

Overexpression of miR-17-3p, ZNF148 and SOS1
Overexpression plasmids for miR-17-3p, ZNF148 and SOS1 were constructed by GENEchem Co., Ltd. Cell transfections were carried out using Lipofectamine 3000 (Invitrogen), following the manufacturer’s protocol. Infection efficiency was monitored via microscopy and cells were incubated for 72 h before further experiments. The information for all vectors used in this experiment is presented in Supplementary Fig. 31, Supplementary Fig. 32a, b.

CRISPR-Cas9 knockout system
CRISPR-Cas9 knockdown plasmids targeting ZNF148 and SOS1 for human and rat cells were obtained from Guangzhou VectorBuilder Co., Ltd. (China). Details of the plasmid sequences are provided in Supplementary Fig. 32c–f.

Lentivirus Generation
HEK293T cells were transfected with lentiviral packaging plasmids. Lentiviral supernatants were collected, filtered and concentrated using the Virus Concentration Kit (Beyotime, Cat No. C2901S). The supernatants containing lentiviral particles were extracted and concentrated by a Virus Concentration Kit (Beyotime, C2901S) to a lentiviral stock greater than 1×108 TU/ml (as determined by p24 ELISA), and then frozen at -80°C in PBS solution containing 1% BSA.

C28 Chondrocytes Stable Transfection Strain Construction
All the virus supernatants were prepared using the virus construction methods mentioned in this article. Among them, the titers of overexpression plasmids for miR-17-3p, ZNF148 and SOS1 were greater than 1×109 FU/mL, and the infectious substance concentration (MOI) was 25; the titers of CRISPR-Cas9 knockdown plasmids targeting ZNF148 and SOS1 were greater than 5×108 FU/mL, and the infectious substance concentration (MOI) was 50. Concentrated viral supernatant, mixed with polybrene (8 µg/ml), was used to transfect human chondrocytes in DMEM supplemented with 10% FBS. Cells were infected for 48 h and then screened with puromycin for 14 days.

Preparation of miRNA-Loaded Exosomes and Cell Culture Experiments
Exosomes (100 µg) derived from the human chondrocyte cell line C28 were loaded with miRNA (75 µM, ~1 µL) using the ExoFectin® sRNA-into-Exosome Kit (Guangzhou Ribo Bio Co., Ltd., China). The miRNA-17-3p and negative control (NC) mimic sequences were synthesized by Guangzhou Ribo Bio Co., Ltd. (China). The transfected exosomes were harvested using the PureExo® Kit (Cat. #: UR52172, Umbio Co., Ltd., China). Similarly, exosomes (100 µg) isolated from the plasma of ISS patients were loaded with an miRNA-17-3p inhibitor (20 µM, ~1 µL) using the same ExoFectin® sRNA-into-Exosome Kit (Guangzhou Ribo Bio Co., Ltd., China). Details of the preparation of miRNA-loaded exosomes methods can be found in our previous publication57.

For cell culture experiments, 100 µg of ISS plasma-derived exosomes loaded with the miR-17-3p inhibitor were co-cultured with 3×10⁵ human C28/I2 chondrocytes in a T25 flask containing 5 mL of culture medium. This experimental setup was designed to investigate whether suppression of miR-17-3p in ISS plasma exosomes could reverse the inhibition of chondrocyte proliferation and markers of endochondral ossification induced by exosomes of ISS patients.

To evaluate the in vivo effects of miR-17-3p overexpression on height development in immature SD rat, we utilized exosomes derived from primary rat chondrocytes, isolated from the proximal tibia and distal femur growth plates, overexpressing miR-17-3p. The exosome dosage ranged from 200 to 300 µg per injection, determined based on a ratio of 1 µg of exosomes per 0.6 g of rat body weight. Rats were divided into groups at 4 weeks postpartum, and exosomes overexpressing miR-17-3p were injected at weeks 4 and 5, respectively. The experiment was terminated at week 8, and the results were observed.

RT-qPCR and Western blot analysis
RT-qPCR and Western blot methods followed our established protocols. RT-qPCR quantified miRNA and mRNA expression in both cell and tissue samples, using U6 and GAPDH as internal controls. For miRNA RT-qPCR of plasma-derived exosomes and fecal samples, U6 small nuclear RNA was used as the endogenous reference for biological normalization, and its stability across sample types and between ISS and control groups was confirmed using geNorm and NormFinder. An exogenous spike-in (cel-miR-39) was added during RNA extraction to monitor extraction and reverse transcription efficiency, thereby controlling for technical variability. Primer sequences are listed in Supplementary Table 2. Reactions were performed in triplicate with three independent cell preparations. Relative expression levels were calculated using the 2−ΔΔCt method, with internal controls adjusting for RNA quantity and transcription efficiency.

Protein samples were extracted from harvested chondrocytes and cartilage tissues using a total protein extraction kit (Applygen Technologies Inc.). The BCA assay (Thermo Fisher Scientific, Inc.) measured total protein concentrations. Protein lysates were separated via SDS-PAGE (Beijing Biosynthesis Biotechnology, Beijing, China) and transferred to PVDF membranes. Membranes were blocked with skim milk (Solarbio Inc.) for 1 h at room temperature, followed by overnight incubation at 4°C with primary antibodies (Supplementary Table 3). After washing with 1× TBST (Solarbio Inc.), membranes were incubated for 1 h at room temperature with HRP-labeled rabbit anti-mouse secondary antibody (1:3000, Abcam, USA). For multiple protein detections, membranes were reprobed with new primary antibodies using WB Stripping buffer (NO: 21059; Thermo Fisher Scientific, USA). Grey values were quantified with Image Lab (v5.2.1).

Von Kossa staining, Alkaline phosphatase staining, and Alizarin Red staining
The Von Kossa staining kit (Shanghai Gefan Biotechnology Co., Ltd), Alkaline Phosphatase Color Development Kit (Solarbio Inc.), and Alizarin Red S solution (Solarbio, G8550) were employed to assess mineralized nodules from chondrocytes in vitro, adhering to the manufacturer’s instructions. Details of the Von Kossa, Alkaline Phosphatase, and Alizarin Red S solution staining methods can be found in our previous publication41,58. Positive calcium nodules appeared as black-brown, dark black, or violet.

Cell cycle profile
A cell cycle analysis kit (KeyGEN BioTECH, China) was used to detect the cell cycle. Chondrocyte (C28) and hBMSC cells were harvested, fixed with 70% ethanol for 2 h at 4°C, and stained with a solution containing propidium iodide (0.05 mg/ml), RNase A (1 mg/ml), and 0.3% Triton X-100 for 30 min in the dark. The percentage of cells in different cell cycle phases was analyzed using a flow cytometer (Beckman Coulter, Brea, CA) and ModFIT software, with each experiment repeated three times. The gating strategies for cell cycle analyses are available on Figshare, as indicated in the Data Availability section.

Enzyme-linked immunosorbent assay (ELISA)
ELISA kits (Meimian Technology, Jiangsu, China) measured the expression of TNF-α, IL-1β, IL-10, Capsaicin, GH, IGF-1, and IGFBP3. Standards and tissue samples (50 μl each) were added to 96-well plates, followed by incubation with HRP-labeled detection antibodies. After incubation, TMB substrate and termination solution were added, and absorbance was measured at 450 nm using a microplate reader.

Myeloperoxidase (MPO) and Malondialdehyde (MDA) Activity Assay
MPO activity in intestinal tissues was assessed by homogenizing in saline and measuring absorbance at 460 nm. MDA levels were determined using the MDA Content Assay Kit (Solarbio, China), with absorbance measured at 532 nm.

Luciferase reporter assay
The ZNF148 plasmid or its mutant portions and the miR-17-3p mimic were co-transfected into 293T cells using Lipofiter (Hanbio Biotechnology, Shanghai, China). The Dual-Luciferase Assay System (Promega Corporation) assessed firefly and Renilla luciferase activity 48 hours post-transfection.

ChIP Assay
Chromatin Immunoprecipitation (ChIP) was performed by fixing cells with formaldehyde, lysing them, and fragmenting chromatin via sonication. Specific antibodies of ZNF148 (Santa Cruz, sc-398148X, 200 µg/0.1 ml) were used to immunoprecipitate protein-DNA complexes, followed by washing, reverse crosslinking and DNA purification for subsequent RT-qPCR or ChIP-seq analysis.

Construction of Engineering-target exosomes (ET-exo)
We utilized a membrane fusion protein technique to functionalize the exosome surface with the collagen type II-targeting peptide WYRGRL. This modification enhances specificity for tissues abundant in collagen type II, such as growth plate cartilage and articular cartilage, facilitating targeted exosome delivery. The ET-Exo plasmid, encoding WYRGRL/LAMP2b/EGFP for green fluorescence and GH/mCherry for red fluorescence, was designed and constructed by Vector Builder (Guangzhou, China). The generation of stably transfected cell lines expressing the viral construct and the subsequent exosome isolation were conducted as detailed in the preceding section.

In Vitro and In Vivo bone targeting
To evaluate growth plate targeting in vivo, 4-week-old SD rats (body weight: 150 g) were administered tail vein injections of ET-Exo-si-miR-17-3p-oeGH, normal exosomes, or PBS. Each injection contained exosomes at a concentration of 1 mg in 300 µL, derived from 50 mL of cell supernatant. At predetermined time points, the rats were humanely euthanized, and their bones and organs were harvested for imaging. Fluorescence imaging was performed using an excitation wavelength of 551 nm and an emission wavelength of 567 nm.

Animal feeding and OE-miR-17-3p-rats
The Animal Ethics Committee of Nanchang University approved the experiments (Approval NO: NCULAE-20221031059). The details of all experimental animal groupings are provided in Supplementary Fig. 28. All SD rats were housed individually in cages under standard conditions. Three pregnant rats at 14 days of gestation were purchased and housed individually in three cages with a standard diet for one week until delivery. The details of all experimental animal groupings are provided in Supplementary Fig. 28. All SD rats were housed individually in cages under standard conditions (22±2°C, 50–60% relative humidity, 12 h light/dark cycle) and fed a mixed diet (AIN-93, Beijing Keao Xieli Feed Company). Eighteen pregnant rats at 14 days of gestation were purchased and housed individually in 18 cages with a standard AIN-93 diet, maintained for one week until delivery. Each pregnant rat typically produced 10-14 pups, and after three weeks post-birth—when sex could be visually identified—pups underwent the first random allocation. To clarify whether miR-17-3p exerts biological effects through ZNF148/SOS1 in vivo experiments, 4 male SD rats were randomly selected from each of the 6 litters and divided into 4 groups at random. They were housed in 4 cages, with 6 male SD rats per cage, under standard conditions and fed the AIN-93 diet. The miR-17-3p, ZNF148, and SOS1 plasmids (GV367-Ubi-MCS-SV40-EGFP-IRES-puromycin) were constructed by GENEchem Co., Ltd. In group A (n=6), the 300 µL PBS was injected into the tail vein. The miR-17-3p was overexpressed in 3 week rats via tail-vein injection in groups B to D. After the rats reached 4 weeks of age, these rats received injections via the tail vein every 3 days, with Group B (n=6) receiving 300 µl of PBS, with Group C (n=6) receiving 300 µl of ZNF148 plasmid, and Group D (n=6) receiving 300 µl of SOS1 plasmid (Supplementary Fig. 28a). To clarify the biological role of ET-Exos from chondrocytes in in vivo experiments, 6 male SD rats were randomly selected from 6 litters and divided into 6 groups at random. They were housed in 6 cages, with 6 male SD rats per cage, under standard conditions and fed the AIN-93 diet. In group A (n=6), the 300 µL PBS was injected into the tail vein. The miR-17-3p was overexpressed in 3-week-old rats via tail-vein injection in groups B to E. After the rats reached 4 weeks of age, these rats received injections via the tail vein every 3 days, with Group B (n=6) receiving 300 µl of ET-Exos (si-miR-17-3p+GH), Group C (n=6) receiving 300 µl of Exos (si-miR-17-3p+GH), Group D (n=6) receiving 300 µl of PBS, Group E (n=6) receiving 300 µl of Exos (si-miR-17-3p), and Group F (n=6) receiving 300 µl of Exos (GH) (Supplementary Fig. 28b). Furthermore, we employed the same methods and protocols to evaluate the in vivo biological effects of ET exosomes derived from bone marrow mesenchymal stem cells (Supplementary Fig. 29a). The body weight and height of rats were recorded weekly, with measurements continuing for four weeks. At the end of the 8-week experimental period, the rats were euthanized using a standard method involving CO2 asphyxiation, in accordance with institutional animal care guidelines. Knee joint tissue samples were then collected for analysis of cartilage growth and development. Height and weight analyses were conducted as single-blind assessments to minimize bias.

Safranin O and Fast Green staining and Calcein staining
For all samples in this research, one-third of the distal femur was resected and kept in 4% formaldehyde solution. 4 µm thickness, serial sections were obtained for Safranin O/Fast Green staining and calcein staining. The flow chart (Figure S10, Supporting Information) demonstrates the detailed grouping information. Details of Safranin O/Fast Green staining and calcein staining methods can be found in our previous publication41,59,60.

Immunofluorescence Staining
4 μm paraffin sections were deparaffinized, rehydrated, and subjected to antigen retrieval in sodium citrate buffer. Following PBS washes, sections were blocked with 3% BSA for 30 min at room temperature, then incubated with 3% H2O2 and 5% goat serum to block nonspecific binding. Primary antibodies (Col10a1, Runx2, Opn, Ocn, Sos1, Znf148, Ras, Pi3k, p-Pi3k, Akt, p-Akt, Erk1/2, p-Erk1/2) were applied overnight, followed by secondary antibodies (1:200 dilution). Nuclei were stained with DAPI, and images were captured using fluorescence microscopy. Image-Pro Plus 6.0 software was used for analysis.

Explant Culture of Neonatal SD Rat Femurs with miR-17-3p Overexpression and Znf148/Sos1 Knockdown
Approval for the use of rat limbs was obtained from the Animal Ethics Committee of Nanchang University. Femurs from neonatal SD rats were harvested one day postpartum. The harvested tissues were cultured statically in BGJb medium (Gibco, Australia) supplemented with 6 mg/ml bovine serum albumin (BSA; Gibco, Australia), 150 µg/ml ascorbic acid (Solarbio, Cat: A8100), 100 units/ml penicillin, and 100 µg/ml streptomycin (Gibco, Australia). Cultures were maintained at 37°C in a humidified atmosphere with 5% CO2, and the medium, replenished with fresh reagents, was replaced daily.

For gene editing and overexpression studies, CRISPR-Cas9 knockdown plasmids targeting Znf148 and Sos1 and an miR-17-3p overexpression plasmid were acquired from Guangzhou VectorBuilder Co., Ltd. and GENEchem Co., Ltd (China). Detailed plasmid sequence information is provided in Supplementary Fig. 31c–f. To generate lentiviral particles, HEK293T cells were transfected with lentiviral packaging plasmids corresponding to the knockdown and overexpression constructs. The resulting lentiviral supernatants were collected, filtered, and concentrated using a Virus Concentration Kit (Beyotime, Cat: C2901S). The concentrated supernatants, containing lentiviral particles, achieved a titer greater than 1×10^8 TU/ml (as quantified by p24 ELISA) and were stored at –80°C in PBS supplemented with 1% BSA.

For transfection of the neonatal rat growth plate tissue, a 2 ml culture system in a 24-well plate was used. The concentrated lentiviral supernatant (titer >5×10^8 FU/ml, multiplicity of infection [MOI]=200) was combined with 8 µg/ml polybrene and HiTransGA (Genechem Co., Ltd., Shanghai), a specialized viral transfection reagent, to facilitate efficient transduction. The tissue was cultured in fresh medium during transfection with lentiviral particles carrying either the Znf148/Sos1 knockdown constructs or the miR-17-3p overexpression construct. The medium was replaced 72 hours post-transfection and refreshed daily thereafter. After 14 days of culture, samples were analyzed using RT-qPCR, Western blot, and immunofluorescence. Details of the in vitro experimental groupings are provided in Supplementary Fig. 30.

Isolation of primary chondrocytes from the growth plate of neonatal SD rats
Primary chondrocytes were isolated from the growth plate of one-week-old neonatal SD rats. The rats were euthanized with isoflurane, disinfected with 75% ethanol for 30 min, wiped with iodophor three times, and rinsed with pre-cooled PBS. The upper segments of the bilateral tibia and lower segments of the femur were dissected, and the cartilage was cut into ~1 mm³ fragments. These fragments were washed three times with PBS to remove blood cells.

The cartilage fragments were first digested with 0.25% trypsin at 37°C for 20 min to remove fibers and impurities, then centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the pellet was washed with PBS. The pellet was further digested with 0.2% type II collagenase (filtered and sterilized) at 37°C for 4 h with gentle shaking every 30 minutes until fully dissolved. Digestion was stopped with DMEM/F12 medium, and the cell suspension was filtered through a 40 µm cell sieve. The filtrate was centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the pellet was washed with PBS. The isolated chondrocytes were cultured in DMEM/F12 medium with 50 µg/mL ascorbic acid. The medium was changed every 2–3 days to remove unattached cells, retaining adherent chondrocytes for experiments.

Capsaicin animal model
All SD rats were housed individually under standard conditions. Three pregnant rats at 14 days of gestation were purchased and housed individually in three cages with a standard diet for one week until delivery. The details of all experimental animal groupings are provided in Supplementary Fig. 28. All SD rats were housed individually under standard conditions. Three pregnant rats at 14 days of gestation were purchased and housed individually in three cages with a standard diet for one week until delivery. Each pregnant rat produced 4 male and 4 female pups, yielding a total of 24 offspring. At three weeks post-birth, 4 male and 4 female pups from each litter were selected and housed individually, followed by one week on a capsaicin-free diet. For group allocation, from each litter, 2 males and 2 females were randomly assigned to the capsaicin-supplemented diet group and the remaining 2 males and 2 females were assigned to the control group. Beginning in the fourth week, the 24 rats were distributed into eight cages for further rearing. Four cages, each containing three rats (12 rats in total: 6 males and 6 females), were maintained on a capsaicin-free diet, while the other four cages (also 12 rats in total: 6 males and 6 females) were switched to a capsaicin-supplemented diet. Thus, both the control and capsaicin groups consisted of 6 males and 6 females. Food intake data were measured every two days, and body weight and height were recorded weekly, by calculating the difference between the amount of food provided and the remaining food, with measurements continuing for four weeks. At the end of the 8-week experimental period, the rats were euthanized using a standard method involving CO2 asphyxiation, in accordance with institutional animal care guidelines. Knee joint and intestinal tissue samples were then collected for analysis of cartilage growth and development as well as gut inflammation-related parameters. Height, weight, food intake, and intestinal tissue inflammation analyses were conducted as single-blind assessments to minimize bias.

The information on the diet composition and form shown in Supplemental Table 4. The standard AIN-93 diet is the unmodified AIN-93G chow, while the capsaicin-supplemented diet is the AIN-93G formulation with 0.01% (w/w) capsaicin incorporated. The capsaicin-supplemented diet was purchased from Ke’ao Synergy (Tianjin) Feed Co., Ltd., Tianjin, China. To ensure accuracy, we verified the capsaicin concentration using high-performance liquid chromatography (HPLC), confirming a consistent 0.01% (w/w) level with a variation of less than 5%. For more details, please refer to the information in the Supplementary Fig. 29b.

The small intestinal samples were collected at 10 cm and 40 cm from the pylorus, while colonic samples were taken at 5 cm and 10 cm from the anus. Each segment was 4 cm in length, with 1 cm was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned at 5 μm thickness, and stained with hematoxylin and eosin (H&E) for histological analysis to assess mucosal stimulation and inflammation, and the remaining 3 cm utilized for Western blot and RT-qPCR analyses to evaluate molecular changes. Villus height and wall thickness of intestinal and colon were measured in 5-6 random fields per section under a light microscope (Olympus BX53, Japan) at 20× magnification using Image-Pro Plus 6.0 software. Villus height was determined from the villus tip to the crypt-villus junction, and intestinal wall thickness was measured from the mucosa to the serosa. Data were averaged per segment per rat, and mean values for the capsaicin and control groups were compared for each intestinal region (small intestine and colon) using Student’s t-test (p<0.05 considered significant).

Capsaicin dose selection and physiological relevance
To model physiologically relevant dietary capsaicin exposure observed in high-consumption populations—particularly those in Jiangxi and Sichuan provinces, China—a 0.01% (w/w) capsaicin-supplemented diet was administered to 4-week-old SD rats, representing the juvenile growth stage.

Capsaicin concentrations in six typical Jiangxi dishes, sourced from local restaurants, were determined by HPLC, ranging from 0.0025 to 0.6277 mg/g (mean±SD=0.2181±0.2400 mg/g). These values are consistent with those reported by Jia et al.61, who identified mean capsaicinoid concentrations of 0.1756 mg/g (grade 4) and 0.2076 mg/g (grade 5) in Sichuan dishes—levels that are common in Jiangxi diets. Assuming a 70 kg adult consumes 700 g/day of grade 4–5 dishes, the estimated capsaicin intake is 122.9–145.3 mg/day (1.76–2.08 mg/kg/day); for a 40 kg adolescent consuming 400 g/day, the intake is 70.2–83.0 mg/day (1.76–2.08 mg/kg/day).

In this study, 4-week-old SD rats (average weight ≈ 70 g) consumed 7.5 g/day of a 0.01% capsaicin-supplemented diet, with capsaicin concentration verified by HPLC, resulting in an intake of 0.75 mg/day (10.71 mg/kg/day). Based on body-surface-area normalization (FDA, 2005) and a rat-to-human conversion factor of 6.2, the human equivalent dose (HED) was calculated to be approximately 1.73 mg/kg/day—closely aligning with the estimated intake among Jiangxi residents. Furthermore, Martínez-Aceviz et al.62 reported a mean capsaicin intake of 152.4 mg/day (2.18 mg/kg/day for a 70 kg adult) in Tijuana, Mexico, further supporting the global relevance of our dosing strategy for modeling high-consumption populations.

Histopathological analysis
Colon tissues were fixed in 10% paraformaldehyde, paraffin-embedded, sectioned (10 μm), and stained with H&E. Two pathologists independently scored the samples under a light microscope.

Apoptosis assays
For the apoptosis assays, cells (5×10⁵) were seeded and exposed to a range of capsaicin concentrations (0–200 μM) for 72 hours. After trypsinization, the cells were stained with Annexin V FITC/PI and analyzed via flow cytometry using FlowJo software. The gating strategies for apoptosis analyses are available on Figshare, as indicated in the Data Availability section. These experiments were conducted independently three times, with each concentration tested in triplicate per experiment to ensure robust statistical reliability.

RNA extraction from Stool and Blood
Total RNA was isolated from stool and blood samples using commercial kits (Solarbio, R1290; Tiangen, DP443). Total RNA from fecal and plasma samples was quantified and purity-assessed using an ultra-micro ultraviolet-visible spectrophotometer (ND-100C, MIU-LAB, China).

High Performance Liquid Chromatography (HPLC)
The high-performance liquid chromatography system (WATER-E2695) was used. The chromatographic column was [column model, such as Agilent ZORBAX Eclipse Plus C18, 4.6×150 mm, 3.5 μm], and the column temperature was set at 30°C. The mobile phase consisted of phase A (ultrapure water) and phase B (methanol), with gradient elution. The flow rate was 1.0 mL/min and the injection volume was 20 μL. The detection wavelength was set at 280 nm. The standard curve was set with concentrations of 0.05, 2, 10, 20, and 200 ug/ml (Supplementary Fig. 26).

Statistical analysis
The data distribution was assessed prior to analysis using the Shapiro-Wilk test for normality. Parametric tests (Student’s t-test for two-group comparisons and one-way ANOVA followed by Tukey’s post hoc test for multiple groups) were employed when data followed a normal distribution, as confirmed in all cases. If non-normality had been detected, non-parametric alternatives (e.g., Mann-Whitney U test for two groups and Kruskal-Wallis test with Dunn’s post hoc for multiple groups) would have been used; however, this was not necessary. The diagnostic efficacy of miR-17-3p was evaluated through ROC curve analysis utilizing the Xiantao platform. All statistical tests were conducted using GraphPad software (10.1.2 version). P-values of less than 0.001 (***), 0.01 (**), and 0.05 (*) were deemed statistically significant. Not significant was ns.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The relationship of miR-17-3p/ZNF148/SOS1 pathway with capsaicin diet and idiopathic short stature is interesting and may\nhave translational potential. The exosome development is also of potential interest.\nBelow are some concerns and suggestions.\nMajor\n• The origin of the chondrocytes used in the different experiments is not clear. How were the human chondrocytes isolated?\n• Hypertrophic chondrocytes typically don’t proliferate. Were different culture timepoints used to examine proliferation and\nhypertrophic differentiation? More experimental details would be useful and showing the chondrocyte phenotype through\nphase contrast microscopy may be helpful.\n• The data of Figure 1 of this manuscript are very similar with Figures 1 and 2 from a previous study by the same research\ngroup (PMID: 36927779) so this part of the manuscript could be potentially omitted and refer to the previous published\nmanuscript. I will leave this up to the editor.\n• In that previous study only 13 differentially expressed miRNAs in the ISS exosomes were found (vs 42 in the present\nstudy). Where do the authors attribute this difference? What was the initial rationale for focusing on the miR-17-3p in the\npresent study? Were any other of the differentially identified miRNAs expressed in human chondrocytes?\n• miR-17-3p appears to have similar effects on chondrocytes as the previously published, exosome-derived, miR26b-3p.\nHowever, the authors don’t discuss this.\n• Please include in the methods details on the ex vivo growth plate cartilage experiments. How was the miRNA\noverexpressed and how were the different genes knocked out?\n• The authors hypothesize that capsaicin diet is a crucial causative factor (through the demonstrated miR-17-3p pathway) for\nISS. Is a diet history available for the individuals with ISS vs the control group?\n• The engineered cartilage targeted exosomes are mentioned as ET-exo in the text while in figure 5 they are mentioned as\nCT-Exos. This is confusing to the reader. In Figure 7a, also the exosome nomenclature is not consistent (“Target exos”\ninstead of what is mentioned on the figure label).\n• GH was co-administrated with miR-17-3p via exosomes in the vivo studies. Important controls are missing and these are\ngroups with cartilage-targeted exosomes containing GH only and miR-17-30 only. These single treatments are included only\nwith non-cartilage targeted exosomes in figures 6, 7.\n• The mice in Figure 8 must have been labeled wrong. The capsaicin labeled mouse looks bigger.\nMinor\n• In 1c a negative control could be included in this western blot (such as part of the input sample without ultracentrifugation\n• Please describe in methods how miR-17-3p was inhibited in the human exosomes\n• Ideally an additional sample with miR-17-3p inhibition for the control group should have been included in the experiments\nof Figure 2.\n• Because staining can vary significantly between areas, the ALP, Von Kossa and ARS staining are best demonstrated\nshowing full cell culture wells rather than magnification of small parts of the well. Background staining may need to be less\nintense if smaller magnification is shown.\n• The demonstration of effects on growth plate cartilage is best shown on paraffin sections that include H/E in parallel\nstaining. Immunofluorescence on dark background does not clearly show the architecture. Here the hypertrophic\nchondrocyte markers appear diffusely expressed rather than contained in the hypertrophic layer. In situ hybridization may be\na better method for these gene expressions if the IHC does not produce clear results on the paraffin sections.\n• Line 190 may need to be bolded as a title line\n• In figure 3d, the shortest bone is the 3rd from top. I suspect that is the miR-17 OE. Ideally the bone sequence should match\nthe experiment sequence of figures 3a and 3c.\n• In figures 3j, 4, 7, 8 there is a variable growth plate orientation among the different pictures. Ideally pictures should have\nconsistent orientation. The hypertrophic chondrocyte layer should be clearly obvious in its entity.\n• What data are included in the Suppl Figure 3a for the inflammatory circle? What are the percentages eg 2% in the\nparentheses?\n• The statement that the biosafety of the exosomes is guaranteed in line 290 should be toned down as there is no adequate\nevidence for this.", "reviewer_2": "Jagruti Bhagwani\nEditorial Assistant\nTitle: “Capsaicin Diet-Induced Gut Inflammation Elevates Plasma Exosomal miR-17-3p: Unveiling the ZNF148/SOS1 Axis\nas a Novel Mechanism in ISS Pathogenesis and Potential Therapeutic Target”\nAuthors: Professor Jia and colleagues\nThis manuscript presents an impressive, comprehensive, and highly innovative study, spanning from clinical observations in\nchildren with idiopathic short stature (ISS) to sophisticated experimental work using animal models, tissue cultures, and cell\ncultures. Employing a wide range of state-of-the-art methodologies, the authors provide compelling results that are well-\npresented and of significant clinical relevance.\nTo enhance the clarity and accessibility of the findings, I recommend the inclusion of a graphical summary to provide a\nvisual overview of the study's key components and outcomes.\nThe study makes a novel contribution to our understanding of the mechanisms underlying growth faltering in children, an\narea where clinicians often lack effective therapeutic options. The introduction effectively sets the stage by addressing the\nchallenges associated with ISS, including the unknown etiology and the limited access to recombinant human growth\nhormone (rhGH) therapy in many regions due to high costs and limited insurance coverage. While the authors highlight the\nrisks associated with rhGH therapy, it is important to acknowledge that these side effects are rare. For example, studies such\nas Savendhal et al. (J Clin Endocrinol Metab, 2021) and Cianfarani (Front Endocrinol, 2021) support the safety profile of\nrhGH. Providing quantitative data on the incidence of side effects would offer a more balanced perspective. Additionally, the\nstatement that ISS \"seriously affects the physical and mental health of children\" should be rephrased to avoid\novergeneralization.\nThe study’s insights into the role of subclinical inflammation in ISS, particularly in specific\nregions of China, have broader implications. Previous work, such as Menahem et al. (Nutrients, 2023), highlights the\npotential for dietary interventions to mitigate growth attenuation due to LPS induced inflammation, further supporting the authors' conclusions. Moreover, the development of\nexosome-based therapeutic strategies targeting the growth plate is highly promising and could\nrevolutionize ISS treatment.\nSpecific comments and suggestions for improvement:\n1. Introduction and Background:\nClarify the connection between environmental factors (e.g., consumption of spicy foods) and ISS. Generally spicy foods are\nhypothesized to reduce microbial growth and thus reduce gut inflammation and diarrhea - therefore it is worth further\nexplanation why children consuming spicy foods may be more susceptible to subclinical inflammation.\nRephrase unclear sentences, such as those in lines 65, 70, 78, and 85, for improved readability.\nResults and Methods sections:\nProvide additional details regarding the origin of the chondrocytes in Figure 1 (line 132) and clarify the source of exosomes\n(line 213). Were they derived from gut villi or other tissues?\nProvide details on the volume of the serum that was taken from the children to extract exosomes and how much was used\nper injection to the animals and per tissue culture and cell culture treatment.\nProvide a clearer explanation on which specific regions of the growth plate are affected by miR-17-3p. It seems that in\nseveral cases the growth pate is less organized- can you comment on this finding?\nElaborate on the procedures used for CRISPR-Cas9-mediated gene knockout in human chondrocytes and rat growth plate\ncartilage (line 191; as well as in supplementary figures 11, 12, and 25).\nExplain the targeting mechanism of collagen type II-coated exosomes to the growth plate. Did these exosomes also localize\nto other tissues, such as articular cartilage, the brain, or blood vessels? Why did they localize to the skull?\nIn line 254, provide an estimation of the amount of chili required to achieve a blood concentration of 200 μM capsaicin.\nFigures and Supplementary Material:\nPlease check figure 7A and 8, as it seems the rats fed with Caspaicin are larger.\nRevise Supplementary Figure 13 to indicate whether the differentially expressed plasma proteins were identified in this\nstudy or a prior publication, and provide references if applicable.\nAddress discrepancies in the legend of Supplementary Figure 17 (e.g., CT-Exo vs. ET-Exo, what is panel K?).\nThe title of supplementary figure 19 should be corrected. Panel b showing\nthe animals is not clear, please improve the\nvisibility of panel - it is impossible to see the differences in the length of the animals.\nDiscussion:\nHighlight the potential implications of the findings beyond the studied cohort.\nProvide a clearer explanation of the mechanism by which miR-17-3p affects specific regions of the growth plate.\nMinor Comments:\nClarify terms such as \"cell communication\" (line 99) and rephrase ambiguous phrases (e.g., lines 105, 153, 204–207).\nAdd catalog numbers for antibodies listed in Supplementary Table 3.\nIn line 213 the authors indicate that the exosomes were produced by chondrocytes, but I had the impression that they were\nproduced by the gut villi- please explain what is the origin of the exosomes? what do you think was the origin of the\nexosomes in the children?\nLine 263- Were the controls from the same geographic region? or just normal height children?\nEnsure consistent scale bars in Figures 3 and 7 and clarify the staining patterns in all other figures.\nSupp figure 3- how was overexpression in growth plate of the rats achieved? Please add the details of the infection or\ntransfection that was done.\nMinor comments;\nLine 65- if there is growth hormone deficiency it is not idiopathic short stature, per definition, therefore this should be omitted.\nLine 70- please rephrase, the connection is not clear\nLine 78- treatment with GH is quite safe (as mentioned in the two studies by Savendahl and Cianfarani S), therefore the\nsentence should be rephrased.\nLine 81- it is not clear who are the children that do not get the treatment.\nLine 85- the sentence is not clear, please rephrase.\nLine 105- what do you mean by “promising”?\nLine 160- please explain how down-regulation and how overexpression was done in chondrocytes\nLine 190 should be in bold\nSupplementary Figures 5, 6, 8 panel M- what are the arrow head showing?\nAddressing these points, especially in the introduction and discussion, would improve the clarity and depth of the study,\noffering a more comprehensive understanding of the mechanisms underlying ISS and the suggested treatment."}, "author_response": ""}, {"round": 2, "reviewer_comments": {"reviewer_1": "Most of the reviewer's concerns have been addressed and this manuscript is now suitable for publication", "reviewer_2": "The authors have addressed most of my previous concerns, and I appreciate their efforts.\nHowever, a few important issues remain and should be resolved prior to publication:\nPage 26 (rebuttal letter): The alignment between the timeline and the graphs needs correction, as the current presentation\nlacks clarity.\nCapsaicin dosage: The manuscript does not provide an estimate of how much chili pepper a child would need to ingest to\nreach a capsaicin blood concentration of 200 μM.\nMechanistic explanation: The mechanism by which miR-17-3p affects growth of specific regions of the growth plate remains\ninsufficiently detailed. A clearer and more comprehensive explanation regarding the mechanism of action would significantly\nenhance the manuscript.", "reviewer_3": "This is an interesting manuscript demonstrating a mechanism underlying inhibition of chondrocyte proliferation and bone\nformation in idiopathic short stature (ISS) via miR-17-3p and plasma exosomes. The authors also show that a capsaicin-\nsupplemented diet reproduces features of the ISS phenotype in rats and provide a comprehensive set of experiments to\nsupport their conclusions. Below, are some general and specific comments for consideration.\nGeneral comments\nThe authors report having developed a capsaicin dietary model. Though, the development and validation of the model are\nnot described. Line 749 refers to 0.01% of capsaicin-supplemented diet. It is unclear how this dose was selected and how it\nrepresents physiologically relevant intakes (including in regions of the world with high consumption). Was it adjusted by rat\nbody weight to reflect intakes in humans? The source of capsaicin, and diets composition should be provided to allow\nreproducibility. The response to previous reviewers speaks of various doses of capsaicin having been tested, though the\nprocess should be described in the methods section of this paper. Also, the methodology should indicate that body weight\nand food intake were assessed, and data shown. This is a nutritional study, and information on the diet composition and\nform, source, mode of administration, housing (how many animals per cage? Sex?), sample size must be provided. Was the\ncapsaicin diet palatable for the mice? Was the capsaicin content of the diet confirmed? How does the amount of capsaicin\nused in this study relates to studies in the literature, where this compound has beneficial effects including in conditions\nassociated with altered intestinal barrier function? Finally, because the age of the rats is not provided, it is difficult to relate\nthe dietary study and intestinal outcomes to the experiments on miR-17-3p inhibition of growth plate ossification.\nCritical details are missing or are unclear in the methods section; examples are provided below.\nThis is a dense and impressive manuscript, and the authors should be commended for undertaking such a comprehensive\nseries of experiments. The data presented are valuable, and the overall effort is clear. That said, some sections are\nchallenging to follow. The manuscript could be improved by using more specific and consistent terminology, which would\ngreatly enhance clarity. At times, it was necessary to refer repeatedly to the Methods section to determine key details, for\nexample the specific cell line or intestinal tissue used, and occasionally, this information was not clearly stated even there.\nProviding these details more explicitly in the main text and ensuring they are clearly outlined in the Methods would\nstrengthen the manuscript and improve its accessibility to readers.\nIntestinal cell line experiments: these are hard to assess without knowing which intestinal cell line was used and the\nculturing conditions. Also, there seems to be a confusion between viability and proliferation (for ex, see Supplementary Fig\n15); these are not the same. Did the authors assess viability (ie using Trypan Blue exclusion or MTT) and used viable cells\nfor their experiments? Panel A shows viability % but the legend speaks of proliferation, please clarify. The fact that cell\nviability was at most about 2.5% for 25 μM is concerning. This is important for this part of the manuscript because the authors\ndiscuss that capsaicin effects (beneficial vs detrimental) depend on low vs high dosages. It is thus important to determine if\nthe 200 μM dosage is a usable high dosage or if it is ”toxic” to the cells. Experiments are hard to assess without details on\nwhich intestinal cell line was used, culture conditions, and measures of viability and proliferation (Suppl Fig 15). Similarly to\nthe in vivo experiment, is 200 μM a physiologically relevant high concentration of capsaicin? Can it be reached in the diet? \nFor example, a calculation of the amount of chili peppers that one should eat (on average) to reach this amount should be\nprovided.\nSpecific comments.\nAbstract and Introduction\n1. Abstract line 40: it is unclear why it necessary to specify: “different from inflammatory bowel disease”. IBD does not always\ncome with diarrhea. The authors did not compare the inflammatory profile induced by capsaicin with the IBD inflammatory\nphenotype so this sentence should be revised.\n2. Line 41: intestinal secretions: are the authors referring to intestinal content? In which intestinal region? It would be best to\njust specify it.\n3. Introduction: the authors could consider making this section more concise without describing all the details of their\nfindings already in this section.\n4. Line 75: at which age and sex was this height gain assessed?\n5. Line 127-128: dietary patterns are more than one ingredient, please rephrase: “frequent consumption of a capsaicin-rich\ndiet, mirroring dietary patterns….”; also, the authors could clarify what is meant by “frequent” and “capsaicin-rich”; It would be\nhelpful to know the amount of capsaicin in this diet to better understand the dosage used in the animal model.\nMethods:\n1. The term “pairs” used to describe ISS patients and age- and gender-matched controls (line 519, line 530) is misleading,\nas those children are unrelated. Furthermore, the gender distribution between the two groups is different (NC: 57 males and\n49 females, ISS: 52 males, 54 females), which indicate that not all “pairs” can be gender matched. Please revise\naccordingly.\n2. Fecal sample collection: The methods used to collect and preserve fecal samples from ISS and control children are not\ndescribed, please include this information in the clinical sample characteristics section.\n3. Inconsistent microRNA methodology: The methods regarding microRNA profiling lack clarity. Line 535 mentions miRNA\nHigh-Throughput Sequencing, while line 531 mentions miRNA microarray. Please clarify which method was used and\ninclude appropriate method details (sequencing platform used, library preparation protocol, sequencing depth; or microarray\nplatform, miRNA labelling and amplification kit, number of miRNA analyzed).\n4. 615-620: this paragraph is unclear regarding whether the experiment was conducted in immature mice, or in rats.\n5. microRNA RT-qPCR: Line 623 does not specify which internal controls were used for 2-DeltaCt method of miRNA RT-\nqPCR for plasma and stool samples. Please include this information.\n6. The description of the in vivo models is incomplete, please ensure that the ARRIVE guidelines are followed; for example:\n- Rats strains and age are missing in a few sections of the in vivo methods description (line 682, line 692, line 748).\n- Grouping Figure: the supplementary Figure 27 and 28 (Grouping of experimental animals) seem to have been mislabeled or\nswitched with Supplementary Figure 24 or 25 (overexpression vector mapping) as the legend does not match the figure\ncontent.\n- Please provide justifications for the sample sizes used in the animal studies. Is n=3 sufficient?\n- Please clarify which tissues were studied in the various experiments; terms like “intestinal tissues” are too vague, please\nspecify which regions.\n7. More details are needed regarding the diets used in the in vivo studies. For example, what constitutes a ‘mixed diet’ (line\n683) and a ‘regular diet’ (line 748). Is this the AIN-93 diet? How was the capsaicin-supplemented diet formulated, how does it differ from the group A ‘regular diet’, and how does the capsaicin dosage relate to human consumption?\n8. The explant culture of rat femurs appears to have used samples collected at one day postpartum (line 708), indicating\nneonatal rather than fetal samples. Please revise the terminology accordingly in the manuscript.\n9. Histopathological analysis of the intestine: please specify which part of the small intestine or large intestine were used to\nassess intestinal inflammation in rats. The method section mentions colon (line 754) while the figures refer to the small\nintestine (line 1280). Please add details on the number of measurements conducted and samples size used to assess villus\nheight and intestinal wall thickness.\n10. Line 757: “Cells were treated…”: Please clarify which cells and explain how they were cultured. How many times were\nthe cell culture experiments repeated? And were they done in triplicates or else?\n11. Line 761-762: was the quality of the RNA assessed?\n12. Line 763-767. Please specify use of parametric and non-parametric tests for data analysis based on data distribution.\nResults\n1. Please ensure that sample sizes are clarified in all figures and legends.\n2. Body weight data missing: a decrease in body height was observed in immature rats fed a capsaicin diet compared to\ncontrols, but the manuscript does not report their body weight. Since intestinal inflammation can lead to weight loss, please\ninclude body weight data to support the interpretation of the growth findings.\n3. Supplementary Figure 1a: the dendrogram on top (sample-based, rather than miRNA-based) should be shown to support\nthe statement in the text.\n4. Supplementary Figure 1c: unclear what does the Y axis show, is this a Delta Ct or a fold change (and if so, please explain\nversus what it was calculated). Please explain in legend.\n5. Line 172: GO and KEGG pathway analyses indicated that overexpression of miR-17-3p upregulates the PI3K-akt etc”: a\nKEGG or GO analysis can indicate that genes targeted by miR-17-3p are enriched in these pathways, not that the pathways\nthemselves are upregulated: please edit.\n6. Line 245: immature rats: please clarify what is it meant by this\n7. Supplementary Fig 14: the panels H and I seem to be switched, please also fix the corresponding text to match the\nrevised figure (line 265). Please clarify which intestinal tissues were studied (rather than saying: intestinal tissues) and also\nclarify if you used whole thickness or the epithelial lining. What was the expression of the miR in Supplem Fig 14 relative to?\n8. Lines 267-274. Please specify the cell line in the text (instead of “mucosal epithelial cells”). The authors seem to have\nconducted a regular “dose-response” experiment of capsaicin at different dosages, which is routine good practice in cell\nculture. It would be expected that at higher concentrations, the substance studied (here capsaicin) would inhibit cell viability,\nwhich is why these experiments are usually performed to find the concentrations range to be studied. Is this what the authors\ndid? Was cell viability assessed? If so, how? How was the optimal concentration (line 270) defined, and which one is it?\n9. Line 269: Supplem Fig 15 presents data on viability but not on proliferation as mentioned in the text.\nDiscussion\nThe discussion is well thought and clear.\nLine 366: the SOS1 abbreviation should have been explained earlier in the manuscript.\nLine 385: please provide a reference for “high chili pepper consumption”\nLines 396-400: this part of the discussion is important. It could be improved by relating it to the published dietary amounts of\ndietary capsaicin showing beneficial effects. How do the concentrations used with the intestinal cell lines relate to dietary\nintake in high and normal capsaicin-diets and to the amount of capsaicin known to elicit beneficial effects in published\nanimal studies? And then versus human intake.\nLine 398: please clarify where the data on proliferation are presented. Please make sure that the discussion reflects the\nresults and that these are all presented.\nLines 431-432: we cannot find the data on mice with ulcerative colitis.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_5": "This is a well-designed study of potential clinical relevance applying a multitude of methodologies.\nMinor comments:\n1. Abstract, lines 30-31: Please correct the wrong statement that ISS is insensitive to GH treatment. As a matter of fact, many\nISS patients respond as well as many of those categorized as GHD based on conventional pituitary stimulation testing.\n2. Abstract, lines 46-47: The statement that short-statured children undergoing growth hormone therapy should be evaluated\nregarding the impact of capsaicin-rich diets should be rephrased removing 'growth hormone theraphy' as no supportive data\nare presented in the manuscript.\n3. Introuction, lines 67-68: Please rephrase 'ISS with peak GH levels exceeding 10 μg/L'. In a large number of countries, the\ncutoff for differentiating GHD from ISS is lower than 10 ug/L."}, "author_response": "Dear Reviewers,\nWe are deeply grateful for your thorough and insightful reviews of our manuscript, titled\n\"Capsaicin Diet-Induced Gut Inflammation Elevates Plasma Exosomal miR-17-3p: Unveiling the\nZNF148/SOS1 Axis as a Novel Mechanism in ISS Pathogenesis and Potential Therapeutic Target\"\n(Manuscript Number: NCOMMS-24-73070). Your valuable feedback has greatly enhanced the\nclarity, rigor, and scientific impact of our work. We have carefully addressed each comment,\nincorporating revisions to improve the manuscript’s quality and accessibility. For your\nconvenience, all revised sections in the manuscript are marked in blue. Below, we provide detailed\nresponses to each reviewer’s comments, including the changes made, their rationale, and relevant\nline numbers in the revised manuscript.\nReviewer #1 (Remarks to the Author):\nThe relationship of miR-17-3p/ZNF148/SOS1 pathway with capsaicin diet and idiopathic short\nstature is interesting and may have translational potential. The exosome development is also of\npotential interest.\nBelow are some concerns and suggestions.Major\nReviewer’s Comment: The origin of the chondrocytes used in the different experiments is not\nclear. How were the human chondrocytes isolated?\nResponse： In our study, we identified significant overexpression of miR-17-3p in plasma\nexosomes from patients with idiopathic short stature (ISS) and hypothesized that this\noverexpression is a primary contributor to the short stature phenotype. To investigate the effects of\nmiR-17-3p on human growth plate chondrocytes, we utilized the C-28/I2 human chondrocyte cell\nline (obtained from Otwo Biotech, Cat No. HTX2308), rather than primary chondrocytes. This\nclarification has been included in lines 150 and 561 of the revised manuscript. This choice was\nmade due to ethical and practical constraints: obtaining growth plate chondrocytes from children\nis not feasible due to ethical standards, and while chondrocytes could be isolated from articular\ncartilage of adult patients undergoing joint replacement for severe arthritis, such cells are in a\npathological state and not representative of healthy growth plate chondrocytes. The C-28/I2 cell\nline, derived from human juvenile costal cartilage, is widely recognized and extensively utilized in\ncartilage biology, osteoarthritis research, and studies on cartilage repair. It is well-suited for\nstudying the effects of interfering factors, such as miR-17-3p, on chondrocyte proliferation,\ndifferentiation, and ossification. We believe this cell line provides a reliable and reproducible\nmodel for our in vitro experiments, and we have clarified its origin and rationale in the revised\nmanuscript.\nReviewer’s Comment: Hypertrophic chondrocytes typically don’t proliferate. Were different\nculture timepoints used to examine proliferation and hypertrophic differentiation? More\nexperimental details would be useful and showing the chondrocyte phenotype through phase\ncontrast microscopy may be helpful.\nResponse： We fully agree with the reviewer’s point that hypertrophic chondrocytes typically do\nnot proliferate, and we have designed our experiments to distinguish between proliferation and\nhypertrophic differentiation accordingly. To assess the impact of miR-17-3p on chondrocyte\nproliferation, we overexpressed miR-17-3p in C-28/I2 chondrocytes using exosomes or adenovirus\nfor 72 hours, followed by analysis with the Cell Counting Kit-8 (CCK-8) assay. CCK-8 is a\ncolorimetric assay that measures cell viability and proliferation based on the reduction of a\ntetrazolium salt into a water-soluble formazan dye by dehydrogenase enzymes in metabolically\nactive cells, providing a quantitative readout of cell proliferation. In parallel, to evaluate\nhypertrophic differentiation, we analyzed separate samples (also overexpressing miR-17-3p for 72\nhours) using qPCR and Western blot (WB) to measure the expression of hypertrophic markers\nRUNX2 and Collagen X, as commonly reported in the literature. These markers allowed us to\nassess the effect of miR-17-3p on chondrocyte hypertrophic differentiation.\nRegarding the chondrocyte phenotype, we acknowledge that in vitro cultured C-28/I2\nchondrocytes adhere to the culture surface and exhibit a polygonal morphology, lacking the\npronounced hypertrophic phenotype observed in vivo in growth plate chondrocytes, as illustrated\nin the figure below. Therefore, consistent with\napproaches in the field, we relied on molecular\nmarkers (e.g., RUNX2, Collagen X) rather\nthan phase contrast microscopy to evaluate\nhypertrophic differentiation in vitro.\nAdditionally, to assess ossification, we\nexamined osteogenic markers (OCN and OPN)\nusing quantitative PCR (qPCR), Western blot\n(WB), and staining techniques, including\nAlizarin Red (for mineralization), Alkaline\nPhosphatase (ALP) activity, and Von kossa\nstaining (for calcium deposition).\nRecognizing the limitations of 2D in vitro culture in mimicking the 3D structure of the growth\nplate, we complemented these experiments with ex vivo whole knee joint cultures. In these models,\nmiR-17-3p was overexpressed, and proliferation was assessed using BrdU incorporation.\nMeanwhile, ossification and new bone growth rates were evaluated using Calcein staining.\nImmunofluorescence further confirmed the expression of hypertrophic markers (RUNX2, Collagen\nX) and osteogenic markers (OCN, OPN). Finally, we overexpressed miR-17-3p in mice and\nanalyzed its effects on growth plate chondrocyte proliferation, differentiation, and bone formation.\nReviewer’s Comment: In that previous study, only 13 differentially expressed miRNAs in the\nISS exosomes were found (vs 42 in the present study). Where do the authors attribute this\ndifference? What was the initial rationale for focusing on the miR-17-3p in the present study?\nWere any other of the differentially identified miRNAs expressed in human chondrocytes?\nResponse: We sincerely appreciate the reviewer’s insightful questions regarding the differences in\nthe number of differentially expressed miRNAs between our previous study and the current work,\nas well as the rationale for focusing on miR-17-3p. Below, we provide a detailed response to\naddress these points:\n1. Difference in the Number of Differentially Expressed miRNAs:\nIn our previous study (PMID: 36927779), we identified 13 differentially expressed miRNAs in ISS\nplasma exosomes using a 2-fold change threshold for significance. However, in the current study,\nwe applied a 1.5-fold change threshold, which resulted in the identification of 43 differentially\nexpressed miRNAs. This difference in the number of miRNAs is primarily attributed to the more\ninclusive threshold used in the current analysis. Shanghai Biohao Biological Co., Ltd. provided\nboth the 1.5-fold and 2-fold threshold-based miRNA lists. We have included the complete raw data\n(Excel file) containing the basic information of all differentially expressed miRNAs in the raw data\nfor transparency.\n2. Rationale for Focusing on miR-17-3p:\nThe selection of miR-17-3p as the focus of the current study was based on a combination of\nbioinformatics analysis, experimental validation, and its functional relevance to chondrocyte\nbiology. Below, we outline the key steps and rationale:\n Initial Findings and Hypothesis Development:\nIn our 2023 study, we reported that miR-26-3p was highly expressed in ISS plasma\nexosomes and could inhibit chondrocyte proliferation and ossification, contributing to the\nshort-stature phenotype. However, the origin of this abnormal exosomal miRNA\nexpression remained unclear. To address this, we conducted a comprehensive literature\nreview and meta-analysis, combined with epidemiological data from China, which\nrevealed a higher incidence of ISS in regions with high capsaicin consumption. This led\nus to hypothesize that capsaicin-induced intestinal inflammation might be a key factor\ndriving the abnormal expression of exosomal miRNAs in ISS patients.\n Experimental Validation in Animal Models:\nTo test this hypothesis, we conducted capsaicin diet experiments in rats. Through three\nindependent experiments, we consistently observed that capsaicin consumption induced a\nshort stature phenotype in rats. Subsequent analysis of plasma exosomes from these rats\nrevealed that miR-17-3p, rather than miR-26-3p, was significantly upregulated, closely\nmimicking the elevated miR-17-3p levels observed in ISS patients. Additionally, we found\nthat LCN2 (lipocalin-2) was also highly expressed in the plasma of capsaicin-fed rats,\nconsistent with our 2022 report.\n Bioinformatics and Functional Screening:\nAfter establishing the capsaicin-induced short stature model, we performed a\nbioinformatics comparison of the 43 differentially expressed miRNAs identified in ISS\npatients (with a 1.5-fold threshold) with their\nrat homologs. Only 12 miRNAs showed 100%\nsequence identity between rats and humans,\nand among these, two miRNAs (miR-17-3p\nand miR-1441-3p) were confirmed by qPCR\nto be significantly differentially expressed in\nchondrocytes.\nImportantly, miR-17-3p exhibited the most\npronounced differential expression and, when\noverexpressed in human chondrocytes,\ndemonstrated the most substantial inhibitory effects on proliferation and ossification,\noutperforming miR-1441-3p. These findings led us to select miR-17-3p as the primary\ncandidate for further investigation.\nReviewer’s Comment: miR-17-3p appears to have similar effects on chondrocytes as the\npreviously published, exosome-derived, miR26b-3p. However, the authors don’t discuss this.\nResponse: Thank you for your insightful comment. In our previous study, we found that high\nexpression of plasma exosome-derived miR-26b-3p in ISS children inhibits chondrocyte\nproliferation and cartilage ossification, resulting in a short stature phenotype in experimental\nanimals. However, the reason for the elevated miR-26b-3p levels in plasma exosomes and its\norgan of origin remains unclear. Building on the observation of a higher incidence of ISS in\nregions with high capsaicin diets in China, we established a capsaicin diet animal model. In this\nmodel, rats exhibited a short stature phenotype, with normal expression of growth hormone and\ninsulin-like growth factor, mimicking the pathological features of ISS (i.e., intact hypothalamic\ngrowth hormone and IGF axis function).\nMore importantly, in the capsaicin diet rats, plasma exosome miR-17-3p was significantly\nupregulated compared to the control group (p < 0.001), recapitulating the plasma exosome\nmiR-17-3p expression profile observed in ISS patients. While we also observed higher plasma\nexosome miR-26b-3p expression in the capsaicin diet group compared to the control group, this\ndifference did not reach statistical significance. Consequently, we did not discuss the role of\nmiR-26b-3p in the context of the capsaicin diet in the current manuscript.\nAlthough the capsaicin diet rat model did not replicate the statistically significant\nupregulation of miR-26b-3p, we identified elevated expression of the LCN2 protein in the plasma\nof these rats, consistent with our previous findings reported in 2022 (Liu X, Zhang J, Yuan J, Ding\nR, Liu T, Jia J. LCN2 is a new diagnostic biomarker and potential therapeutic target in idiopathic\nshort stature. J Cell Mol Med. 2022;26(12):3568-3581). Additionally, we observed increased\nTNXB protein expression in the plasma of capsaicin-fed rats, a finding also mirrored in the\nplasma of ISS patients. However, the specific mechanisms underlying TNXB upregulation remain\nunclear and are currently under investigation.\nThe lack of statistical significance in miR-26b-3p expression in the capsaicin diet rats could\nbe attributed to the limited sample size or potential differences in genetic backgrounds between\nhumans and rats, which may prevent identical pathological manifestations. Nevertheless, it is\nnoteworthy that our capsaicin diet model not only successfully induced a short-stature phenotype\nbut also demonstrated consistent changes in multiple molecular markers (e.g., miR-17-3p, LCN2,\nand TNXB) that align with observations in ISS patients. Furthermore, similar trends were detected\nin fecal samples from ISS patients, reinforcing the relevance of this model. These findings suggest\nthat the capsaicin diet animal model holds significant value for studying the pathogenesis of ISS in\nregions with high capsaicin consumption.\nReviewer’s Comment: Please include in the methods details on the ex vivo growth plate cartilage\nexperiments. How was the miRNA overexpressed and how were the different genes knocked out?\nResponse: Thank you for your valuable suggestion. We have addressed your request by\nincorporating detailed information about the ex vivo growth plate cartilage experiments into the\nMaterials and Methods section (Lines 706–731) of the revised manuscript. Specifically, we have\nclarified the methodology for miR-17-3p overexpression and the CRISPR-Cas9-mediated\nknockdown of ZNF148 and SOS1 in fetal SD rat femur explant cultures. These additions outline\nthe lentiviral transfection process, culture conditions, and analytical techniques employed. We\nbelieve these revisions enhance the transparency of our experimental approach and fully address\nyour comment. Please let us know if further clarification is needed.\nRevised Added Content:\nExplant Culture of Fetal SD Rat Femurs with miR-17-3p Overexpression and ZNF148/SOS1\nKnockdown\nApproval for the use of rat limbs was granted by the Animal Ethics Committee of Nanchang\nUniversity. Femurs from fetal Sprague-Dawley (SD) rats were harvested one day postpartum. The\nexplanted tissues were statically cultured in BGJb medium (Gibco, Australia) supplemented with 6\nmg/ml bovine serum albumin (BSA; Gibco, Australia), 150 µg/ml ascorbic acid (Solarbio, Cat:\nA8100), 100 units/ml penicillin, and 100 µg/ml streptomycin (Gibco, Australia). Cultures were\nmaintained at 37°C in a humidified atmosphere with 5% CO2, with the medium refreshed daily\nusing freshly prepared reagents.\nFor gene editing and overexpression, CRISPR-Cas9 knockdown plasmids targeting ZNF148\nand SOS1, along with an miR-17-3p overexpression plasmid, were obtained from Guangzhou\nVectorBuilder Co., Ltd. and Genechem Co., Ltd. (China). Detailed plasmid sequences are\nprovided in Supplementary Fig. 24c–f. Lentiviral particles were generated by transfecting\nHEK293T cells with the corresponding lentiviral packaging plasmids. The resulting supernatants\nwere collected, filtered, and concentrated using a Virus Concentration Kit (Beyotime, Cat:\nC2901S) to achieve a titer exceeding 1×10^8 TU/ml (quantified by p24 ELISA). Concentrated\nlentiviral stocks were stored at -80°C in PBS with 1% BSA.\nTransfection of fetal rat growth plate cartilage was performed in a 2 ml culture system using\na 24-well plate. Concentrated lentiviral supernatant (titer > 5×10^8 FU/ml, multiplicity of\ninfection [MOI] = 200) was mixed with 8 µg/ml polybrene and HiTransG A (Genechem Co., Ltd.,\nShanghai), a specialized transfection reagent, to enhance transduction efficiency. The explants\nwere transduced with lentiviral particles carrying either the ZNF148/SOS1 knockdown constructs\nor the miR-17-3p overexpression construct in fresh medium. The medium was replaced 72 hours\npost-transfection and refreshed daily thereafter. After 14 days of culture, samples were analyzed by\nRT-qPCR, Western blot, and immunofluorescence. Experimental groupings are detailed in\nSupplementary Fig. 27.\nReviewer’s Comment: The authors hypothesize that the capsaicin diet is a crucial causative\nfactor (through the demonstrated miR-17-3p pathway) for ISS. Is a diet history available for the\nindividuals with ISS vs the control group?\nResponse: We thank the reviewer for raising this insightful question about the availability of\ndietary history data for individuals with ISS and the control group. Below, we provide a detailed\nresponse:\nIn this retrospective study, we did not collect detailed dietary histories, including capsaicin\nconsumption, from the ISS patients or controls. This limitation stems from the challenges of\nobtaining accurate and reliable dietary records in a retrospective design. Nevertheless, our\nhypothesis that capsaicin consumption contributes to ISS via the miR-17-3p pathway is supported\nby multiple lines of indirect evidence, as outlined below.\nEpidemiological Evidence:\nWe observed a higher incidence of ISS in regions of China where capsaicin-rich diets are\nprevalent, suggesting a potential association between dietary capsaicin and ISS.\nAnimal Model Evidence:\nIn our capsaicin diet-induced rat model, we successfully recapitulated key ISS features, including\nelevated plasma exosomal miR-17-3p levels and a short stature phenotype. These findings\nreinforce the role of capsaicin in the pathogenesis of ISS.\nHuman Intervention Study:\nTo explore the causal link between capsaicin consumption and miR-17-3p upregulation, we\nconducted a preliminary intervention study with eight adult volunteers (children were excluded\ndue to ethical concerns regarding repeated blood sampling and the pilot nature of the experiment).\nParticipants followed a 4-week protocol with alternating periods of no capsaicin diet, capsaicin\ndiet, and no capsaicin diet, during which blood and fecal samples were collected. Key findings\ninclude:\n Capsaicin Diet Phase: Plasma exosomal miR-17-3p levels significantly increased and\ncorrelated positively with intestinal inflammation markers.\n Post-Capsaicin Phase: miR-17-3p levels declined after cessation of the capsaicin diet.\n Reintroduction Phase: miR-17-3p levels increased again upon resumption of the\ncapsaicin diet.\nThese results provide preliminary direct evidence of a causal relationship between\ncapsaicin intake and the upregulation of miR-17-3p.\nTo directly investigate the link between capsaicin consumption and ISS, we are planning a\nprospective, multicenter study. This will include comprehensive dietary assessments (e.g., food\nfrequency questionnaires or dietary logs) for both ISS patients and controls. Such data will enable\na more robust evaluation of our hypothesis that high capsaicin intake contributes to ISS via the\nmiR-17-3p pathway.\nIn summary, while dietary history data were not available in the current study, the\ncombination of epidemiological observations, animal model results, and human intervention\nfindings supports our hypothesis. The planned prospective study with detailed dietary data will\nfurther strengthen our understanding of capsaicin’s role in ISS pathogenesis.\nReviewer’s Comment: The engineered cartilage targeted exosomes are mentioned as ET-exo in\nthe text while in figure 5 they are mentioned as CT-Exos. This is confusing to the reader. In Figure\n7a, also the exosome nomenclature is not consistent (“Target exos” instead of what is mentioned\non the figure label).\nResponse: We thank the reviewer for pointing out the inconsistencies in exosome nomenclature\nacross the text and figures. We acknowledge that the use of \"ET-exo\" in the text, \"CT-Exos\" in\nFigure 5, and \"Target exos\" in Figure 7a may cause confusion for readers. In the revised\nmanuscript, we have standardized the terminology to \"ET-Exo\" (Engineered Cartilage Targeted\nExosomes) throughout the text, figure labels, and legends to ensure consistency and clarity. These\ncorrections have been implemented in the updated draft, and we apologize for any confusion\ncaused by the oversight.\nReviewer’s Comment: GH was co-administrated with miR-17-3p via exosomes in the vivo\nstudies. Important controls are missing and these are groups with cartilage-targeted exosomes\ncontaining GH only and miR-17-30 only. These single treatments are included only with\nnon-cartilage targeted exosomes in figures 6, 7.\nResponse: Thank you for your valuable feedback and for suggesting the inclusion of additional\ncontrol groups in our in vivo studies. We truly appreciate your recommendation to incorporate\ngroups with cartilage-targeted exosomes carrying either GH alone or si-miR-17-3p alone, as\nthese controls were initially only included with non-cartilage-targeted exosomes in Figures 6 and\n7.\nOur in vivo experiments demonstrated that overexpressing miR-17-3p inhibits growth plate\nchondrocyte proliferation and bone formation in rats, resulting in a short stature phenotype, as\nshown in Group D. When miR-17-3p was overexpressed, administering growth hormone (GH)\nalone failed to restore the rats’ height to normal levels, indicating that miR-17-3p not only induces\nshort stature but also causes insensitivity to GH therapy. However, when we knocked down\nmiR-17-3p expression using siRNA while simultaneously administering GH via exosomes, we\nobserved a significant therapeutic effect. As shown in our results, Group C (non-targeted\nexosomes carrying both si-miR-17-3p and GH) outperformed Group E (non-targeted exosomes\ncarrying only si-miR-17-3p) and Group F (non-targeted exosomes carrying only GH). This further\nconfirms that addressing the abnormally high expression of miR-17-3p is essential for GH to\nachieve an effective therapeutic outcome. In vivo imaging revealed that, due to the lack of\ncartilage targeting, non-targeted exosomes distributed not only to the growth plate but also to\nother organs, such as the intestine, heart, liver, spleen, kidneys, and lungs. This lack of specificity\nraises two concerns: first, the distribution of siRNA and GH to non-target organs could potentially\nlead to off-target effects or organ damage; second, it reduces the local concentration of exosomes\nat the growth plate, limiting their therapeutic efficacy.\nTo overcome this limitation, we designed cartilage-targeted exosomes by incorporating a type\nII collagen-targeting peptide to enhance delivery specificity to the growth plate. Our in vivo\nimaging results confirmed that cartilage-targeted exosomes exhibited improved targeting to the\ngrowth plate with reduced non-specific distribution to other organs. More importantly, this\napproach significantly enhanced height recovery, as demonstrated in Group B (cartilage-targeted\nexosomes carrying both si-miR-17-3p and GH), which outperformed Group C (non-targeted\nexosomes carrying si-miR-17-3p and GH). These findings indicate that while exosomes carrying\nboth siRNA and GH are effective for ISS treatment, cartilage-targeted exosomes provide superior\ntherapeutic outcomes compared to non-targeted exosomes, demonstrating enhanced efficacy in\npromoting height recovery and highlighting their potential for clinical applications.\nWe believe our current experimental design, through the comparison of Groups A, B, C, and\nD, robustly demonstrates the therapeutic\nadvantages of targeted exosome therapy,\nparticularly the synergistic effect of\nsi-miR-17-3p and GH. As such, we initially felt\nthat adding groups with cartilage-targeted\nexosomes carrying either GH alone or si-miR-17-3p alone might not be necessary to support our\nprimary conclusions. Nevertheless, we deeply value your suggestion, as it offers an opportunity to\nfurther enhance the comprehensiveness of our study. To address this, we initiated supplementary\nexperiments during the manuscript revision period to include these additional control groups, and\nwe expect to obtain the new experimental data by mid-May. We respectfully propose that our\nexisting data already provide a clear and sufficient demonstration of the benefits of targeted\nexosomes for ISS treatment. However, if you believe these additional controls are essential to\nfurther validate our findings, we would be happy to include the new data in May. We sincerely\nappreciate your guidance and look forward to your further insights on this matter.\nReviewer’s Comment: The mice in Figure 8 must have been labeled wrong. The capsaicin\nlabeled mouse looks bigger.\nResponse: We appreciate the reviewer’s keen observation regarding the labeling in Figure 8.\nUpon review, we confirmed that the mice were indeed mislabeled, with the capsaicin-treated\nmouse appearing larger than expected. We have corrected this error in the revised manuscript by\nupdating the labels in Figure 8 to reflect the experimental groups accurately. We regret this\noversight and thank the reviewer for bringing it to our attention.\nMinor\nReviewer’s Comment: In 1c a negative control could be included in this western blot (such as\npart of the input sample without ultracentrifugation\nResponse: We sincerely thank you for your valuable suggestion to include a negative control in\nthe Western blot analysis in Figure 1c. We agree that incorporating such a control strengthens the\nrigor of our study.\nIn our study, exosomes were extracted from the plasma of ISS patients using a commercial\nexosome isolation kit (Plasma and Serum Exosome Extraction Kit, Cat. #: UR52151, Umibio,\nChina) instead of ultracentrifugation. Following your advice, we used unprocessed ISS patient\nplasma (not treated with the exosome isolation kit) as the negative control. As a positive control,\nwe included exosomes derived from the human C28 chondrocyte cell line, which were previously\nextracted using the same exosome isolation kit and\nconfirmed to be exosomes by Western blot and\ntransmission electron microscopy. In the revised\nmanuscript, the updated Western blot in Figure 1c\nnow includes the positive control group, the ISS\npatient plasma exosome group, and the negative\ncontrol group.\nWe greatly appreciate your insightful feedback, which has significantly improved the quality\nof our study.\nReviewer’s Comment: Please describe in methods how miR-17-3p was inhibited in the human\nexosomes—inhibitor.\nResponse: We appreciate your feedback, as it helps enhance the transparency and reproducibility\nof our study. In the revised manuscript, we have added a detailed explanation in the \"Methods\"\nsection (Line 602-615).\nRevised Content:\nPreparation of miRNA-Loaded Exosomes and Cell Culture Experiments (Line 602-615)\nExosomes (100 µg) derived from the human chondrocyte cell line C28 were loaded with miRNA (75 µM,\n~1 µL) using the ExoFectin® sRNA-into-Exosome Kit (Guangzhou RiboBio Co., Ltd., China). The\nmiRNA-17-3p and negative control (NC) mimic sequences were synthesized by Guangzhou RiboBio Co.,\nLtd. (China). The transfected exosomes were harvested using the PureExo® Kit (Cat. #: UR52172,\nUmbio Co., Ltd., China). Similarly, exosomes (100 µg) isolated from the plasma of ISS patients were\nloaded with an miRNA-17-3p inhibitor (20 µM, ~1 µL) using the same ExoFectin®\nsRNA-into-Exosome Kit (Guangzhou RiboBio Co., Ltd., China). Details of the preparation of\nmiRNA-loaded exosomes methods can be found in our previous publication28.\nFor cell culture experiments, 100 µg of ISS plasma-derived exosomes loaded with the\nmiRNA-17-3p inhibitor were cocultured with 3 × 10⁵ human C28/I2 chondrocytes in a T25 flask\ncontaining 5 mL of culture medium. This experimental setup was designed to investigate whether\nsuppression of miRNA-17-3p in ISS plasma exosomes could reverse the inhibition of chondrocyte\nproliferation and markers of endochondral ossification induced by exosomes of ISS patients.\nReviewer’s Comment: Ideally an additional sample with miR-17-3p inhibition for the control\ngroup should have been included in the experiments of Figure 2.\nResponse: We appreciate the reviewer's valuable suggestion. In our study, we observed that\nco-culturing exosomes from ISS patients with the human chondrocyte cell line C28/I2 suppressed\nchondrocyte proliferation and impaired cartilage ossification. To investigate whether miR-17-3p\nin ISS patient plasma exosomes was responsible, we designed three experimental groups: a\nnormal children’s exosome group, an ISS patient exosome group, and an ISS patient exosome +\nmiR-17-3p inhibitor group. Compared to the normal children’s exosome group, ISS patient\nexosomes significantly inhibited chondrocyte proliferation and cartilage ossification. However, the\ninhibition of miR-17-3p in ISS patient exosomes markedly alleviated these effects, suggesting that\nmiR-17-3p overexpression in ISS exosomes is a key driver of the observed suppression. This was\nfurther confirmed by adenovirus-mediated overexpression of miR-17-3p in chondrocytes, which\nreplicated the suppression of proliferation and ossification.\nOur experimental design, comprising healthy exosomes, ISS exosomes, and ISS exosomes\nwith miR-17-3p inhibition, effectively delineates the role of miR-17-3p in ISS pathogenesis, with\nthe overexpression experiments providing additional validation. We fully acknowledge the\nreviewer’s insightful suggestion to include miR-17-3p inhibition in the control group, as it could\nfurther strengthen the study. However, based on the current experimental setup and the robust\nevidence supporting our conclusions, we believe that the inclusion of this additional control group\nmay not significantly alter the interpretation of our findings. Nevertheless, we sincerely appreciate\nthe reviewer’s constructive feedback and will certainly consider incorporating this suggestion in\nfuture studies to refine further our understanding of miR-17-3p’s role in ISS pathogenesis.\nReviewer’s Comment: Because staining can vary significantly between areas, the ALP, Von\nKossa and ARS staining are best demonstrated showing full cell culture wells rather than\nmagnification of small parts of the well. Background staining may need to be less intense if\nsmaller magnification is shown.\nResponse: We sincerely appreciate the reviewer’s valuable suggestion regarding the presentation\nof ALP, Von Kossa, and ARS staining results. Below, we provide a detailed explanation of our\nexperimental and imaging approach, along with our response to the reviewer’s concerns.\nWe performed ALP, Von Kossa, and ARS staining using 12-well plates (with a well diameter of\napproximately 22 mm). Each experimental group consisted of three replicates, and the staining\nresults were quantified by counting positive particles in four fields of view (upper left, lower left,\nupper right, and lower right) under a 4x magnification microscope. The average number of\npositive particles per group was calculated to quantify the staining results, as demonstrated in the\nfigure below. We have uploaded the corresponding Excel spreadsheet containing all staining data\nas supplementary material in the revised manuscript.\nAfter quantification, we retained one representative image (either upper left or lower left,\ndepending on image clarity) at 4x magnification for documentation. However, due to the\nlimitations of the 12-well plate format, we were unable to capture images of the whole cell culture\nwells under the microscope, as the reviewer suggested. During manuscript preparation, we\ninitially considered presenting the 4x magnification images of ALP, Von Kossa, and ARS staining.\nHowever, as shown in the example images provided below, the positive particles were not clearly\nvisible at this magnification, making it difficult for readers to distinguish differences between\ngroups. To address this issue, we opted to present higher-magnification images in the manuscript,\nwhich allowed for clearer visualization of the staining results.\nVon Kossa ALP ARS\nWe fully acknowledge the reviewer’s point that staining variability between areas can be\nbetter demonstrated by showing full cell culture wells. However, given the technical limitations of\nour experimental setup (12-well plates and 4x magnification imaging), we were unable to capture\nsuch images. To address this concern, we have included the following supplementary materials in\nthe revised manuscript:\n Excel Spreadsheet: Containing the raw data of positive particle counts for each group.\n Statistical Graphs: Generated from the raw data to illustrate the quantitative differences\nbetween groups.\nWe believe that these additional materials will enhance the transparency and reproducibility\nof our results and provide a more comprehensive understanding of the ALP, Von Kossa, and ARS\nstaining data.\nReviewer’s Comment: The demonstration of effects on growth plate cartilage is best shown on\nparaffin sections that include H/E in parallel staining. Immunofluorescence on dark background\ndoes not clearly show the architecture. Here the hypertrophic chondrocyte markers appear\ndiffusely expressed rather than contained in the hypertrophic layer. In situ hybridization may be\na better method for these gene expressions if the IHC does not produce clear results on the paraffin\nsections.\nResponse: Thank you for your insightful suggestion. The unique structure of the growth plate is\nindeed better highlighted by Safranin O/Fast Green staining due to its specific advantages. This\nmethod stains cartilage tissue and calcified bone in distinct colors—cartilage appears bright red,\nwhile calcified areas turn green—making it especially effective for observing the growth plate’s\nstructure. The growth plate consists of distinct zones: the resting zone, proliferative zone,\nhypertrophic zone, and calcified zone. Since the calcified zone stains green, this clear color\ncontrast enables a more precise visualization of the growth plate’s organization compared to H&E.\nDue to these benefits, Safranin O/Fast Green is widely used in growth plate research. Compared\nto H&E staining, Safranin O/Fast Green is particularly well-suited for visualizing the specialized\nzonal structure of the growth plate.\nFigure legend： Since the calcified zone stains green, this clear color contrast enables a more precise visualization\nof the growth plate’s organization compared to H&E.\nThank you for your valuable feedback. Due to their small size and suboptimal contrast adjustment,\nwe acknowledge that the immunofluorescence images we initially presented did not clearly display\nthe expression of hypertrophic chondrocyte markers. To address this, we have replaced the\nprevious images with new images that have higher clarity and resolution, as well as better\ncontrast. With these adjustments, we can now observe that the hypertrophic chondrocyte markers\nare predominantly expressed in the hypertrophic chondrocyte region, providing a clearer\nrepresentation of their localization.\nFigure Legend： This enlarged image of the growth plate cartilage, derived from Figure 7j, shows the area\nbetween the two gray dashed lines as the growth plate cartilage. The yellow box indicates the proliferative zone,\nand the yellow arrow points to RUNX2-positive hypertrophic chondrocytes. The red box marks the hypertrophic\nzone, while the white box denotes the calcified zone.\nReviewer’s Comment: Line 190 may need to be bolded as a title line.\nResponse: We appreciate your careful review and suggestions for improvement. Regarding your\nfeedback, we have bolded the title on Line 190 as suggested to improve its visibility and clarity.\nHowever, please note that this title now appears on Line 208 in the revised manuscript. This\nchange has been incorporated into the updated version of the document.\nReviewer’s Comment: In figure 3d, the shortest bone is the 3rd from top. I suspect that is the\nmiR-17 OE. Ideally the bone sequence should match the experiment sequence of figures 3a and\n3c.\nResponse: We appreciate your attention to detail in pointing out the issue with Figure 3d. We\noverlooked this detail and failed to ensure that the bone sequence matched the experimental order.\nWe have now corrected this oversight by revising Figure 3d to align the bone sequence with the\nexperimental sequence shown in Figures 3a and 3c. This updated figure is included in the revised\nmanuscript.\nReviewer’s Comment: In figures 3j, 4, 7, 8 there is a variable growth plate orientation among the\ndifferent pictures. Ideally pictures should have consistent orientation. The hypertrophic\nchondrocyte layer should be clearly obvious in its entity.\nResponse: Thank you for your insightful and detailed feedback on our manuscript. We greatly\nappreciate your suggestion regarding the consistency of growth plate orientation in Figures 3j, 4,\n7, and 8, as well as the need to display the hypertrophic chondrocyte layer clearly.\nFollowing your valuable advice, we have carefully revised the images in the aforementioned\nfigures. To ensure consistency, we have standardized the orientation of the growth plates across all\nimages. Specifically, the growth plate consists of the resting zone, proliferative zone,\nhypertrophic zone, and calcified zone. In our revised images, we have adopted a uniform orientation:\nwhen the growth plate is displayed vertically, the resting zone is positioned at the top, followed by the\nproliferative zone, hypertrophic zone, and calcified zone at the bottom. For images where the\ngrowth plate is displayed horizontally (due to the nature of the tissue section), the orientation is\nadjusted from left to right, with the proliferative zone on the left, followed by the hypertrophic zone\nand calcified zone on the right. Additionally, we have ensured that the hypertrophic chondrocyte\nlayer is prominently and clearly visible in each figure to facilitate better interpretation.\nWe believe these adjustments enhance the clarity and consistency of the figures, making them\nmore accessible and understandable for readers. The updated figures have been included in the\nrevised manuscript. We sincerely thank you for your constructive suggestion, which has\nsignificantly improved the presentation of our data.\nReviewer’s Comment: What data are included in the Suppl Figure 3a for the inflammatory circle?\nWhat are the percentages eg 2% in the parentheses?\nResponse: Thank you for your insightful comments and for drawing our attention to the data\npresented in Supplementary Figure 13a regarding the inflammatory circle. We appreciate the\nopportunity to clarify the details behind the data and the meaning of the percentages, such as the\n2% noted in parentheses.\nIn our prior report, plasma proteomic analysis of ISS patients and healthy controls identified\n84 differentially expressed proteins. For this study, we re-analyzed the proteomic data from ISS\npatients with a focus on inflammation-related proteins. To do so, we first compiled a\ncomprehensive set of 2,739 inflammation-related proteins by intersecting annotations from three\ndatabases: ImmPort, InnateDB, and MSigDB. We then cross-referenced these with the 84\ndifferentially expressed proteins from ISS patients. This analysis revealed that 42 of the 84\ndifferentially expressed proteins were inflammation-related, representing 2% of the total 2,739\ninflammation-related proteins cataloged in the databases. Thus, the 2% in parentheses indicates\nthe proportion of inflammation-related proteins among the differentially expressed proteins in ISS\npatients relative to the broader pool of inflammation-related proteins.\nWe sincerely apologize for the lack of clarity in the original figure and figure legend, as this\ndetail was not adequately presented. We are grateful to the reviewer for pointing it out. To address\nthis, we have revised the manuscript to include a clearer explanation of the data and the\nsignificance of the 2% value, ensuring that this information is now properly reflected in both the\nfigure and its accompanying description. Thank you again for your thoughtful feedback, which has\ngreatly helped us improve the clarity of our work.\nReviewer’s Comment: The statement that the biosafety of the exosomes is guaranteed in line 290\nshould be toned down as there is no adequate evidence for this.\nResponse: Thank you for your valuable feedback and for raising a concern regarding the\nstatement about the biosafety of Exo-si-miR-17-3p-oeGH on lines 306-309. We agree that the\noriginal phrasing, which stated that \"the biosafety is guaranteed,\" was overly definitive. To\naddress this, we have revised the manuscript to temper this claim. The revised text now reads:\n\"Preliminary data from histological and biochemical analyses suggest that\nExo-si-miR-17-3p-oeGH exhibits no significant toxicity in major organs, including the heart, liver,\nspleen, lungs, and kidneys (Supplementary Fig. 21). However, further studies are needed to\nconfirm its comprehensive biosafety profile (Lines 306-309).\" We sincerely appreciate your\nsuggestion, which has improved the clarity and precision of our work.\nReviewer #2 (Remarks to the Author):\nTitle: “Capsaicin Diet-Induced Gut Inflammation Elevates Plasma Exosomal miR-17-3p:\nUnveiling the ZNF148/SOS1 Axis as a Novel Mechanism in ISS Pathogenesis and Potential\nTherapeutic Target”\nAuthors: Professor Jia and colleagues\nThis manuscript presents an impressive, comprehensive, and highly innovative study, spanning\nfrom clinical observations in children with idiopathic short stature (ISS) to sophisticated\nexperimental work using animal models, tissue cultures, and cell cultures. Employing a wide\nrange of state-of-the-art methodologies, the authors provide compelling results that are\nwell-presented and of significant clinical relevance.\nTo enhance the clarity and accessibility of the findings, I recommend the inclusion of a graphical\nsummary to provide a visual overview of the study's key components and outcomes. The study\nmakes a novel contribution to our understanding of the mechanisms underlying growth faltering in\nchildren, an area where clinicians often lack effective therapeutic options. The introduction\neffectively sets the stage by addressing the challenges associated with ISS, including the unknown\netiology and the limited access to recombinant human growth hormone (rhGH) therapy in many\nregions due to high costs and limited insurance coverage. While the authors highlight the risks\nassociated with rhGH therapy, it is important to acknowledge that these side effects are rare. For\nexample, studies such as Savendhal et al. (J Clin Endocrinol Metab, 2021) and Cianfarani (Front\nEndocrinol, 2021) support the safety profile of rhGH. Providing quantitative data on the incidence\nof side effects would offer a more balanced perspective. Additionally, the statement that ISS\n\"seriously affects the physical and mental health of children\" should be rephrased to avoid\novergeneralization.\nThe study’s insights into the role of subclinical inflammation in ISS, particularly in specific\nregions of China, have broader implications. Previous work, such as Menahem et al. (Nutrients, 2023), highlights the\npotential for dietary interventions to mitigate growth attenuation due to LPS induced inflammation,\nfurther supporting the authors' conclusions. Moreover, the development of\nexosome-based therapeutic strategies targeting the growth plate is highly promising and could\nrevolutionize ISS treatment.\nReviewer’s Comment: To enhance the clarity and accessibility of the findings, I recommend the\ninclusion of a graphical summary to provide a visual overview of the study's key components and\noutcomes. The study makes a novel contribution to our understanding of the mechanisms underlying growth faltering in\nchildren, an area where clinicians often lack effective therapeutic options. The introduction\neffectively sets the stage by addressing the challenges associated with ISS, including the unknown\netiology and the limited access to recombinant human growth hormone (rhGH) therapy in many\nregions due to high costs and limited insurance coverage. While the authors highlight the risks\nassociated with rhGH therapy, it is important to acknowledge that these side effects are rare. For\nexample, studies such as Savendhal et al. (J Clin Endocrinol Metab, 2021) and Cianfarani (Front\nEndocrinol, 2021) support the safety profile of rhGH. Providing quantitative data on the incidence\nof side effects would offer a more balanced perspective. Additionally, the statement that ISS\n\"seriously affects the physical and mental health of children\" should be rephrased to avoid\novergeneralization.\nResponse: Thank you for your suggestion. We have incorporated a graphical summary, as\nrecommended, to provide a visual overview of the study’s key components and outcomes. This\naddition can be found in the revised manuscript as Supplemental Figure 28. We believe this\nenhances the clarity and accessibility of our findings.\nReviewer’s Comment: For example, studies such as Savendhal et al. (J Clin Endocrinol Metab,\n2021) and Cianfarani (Front Endocrinol, 2021) support the safety profile of rhGH. Providing\nquantitative data on the incidence of side effects would offer a more balanced perspective.\nAdditionally, the statement that ISS \"seriously affects the physical and mental health of children\"\nshould be rephrased to avoid overgeneralization.\nResponse: Thank you for your insightful feedback. We have revised the manuscript to address\nyour concerns and enhance the balance and clarity of our statements. Specifically, we have\nrephrased the description of idiopathic short stature (ISS) to avoid overgeneralization, now stating:\n\"This condition may have significant implications for the physical and psychological well-being of\naffected children\" (see Lines 60-61). This revision reflects a more nuanced perspective on its\npotential impact.\nAdditionally, we have revised the manuscript to provide a more balanced view of\nrecombinant human growth hormone (rhGH) safety. The revised text now reads: \"In terms of safety,\nwhile some studies have raised concerns about potential risks associated with rhGH therapy, such as\nbone and cartilage tumors, cerebrovascular diseases, hypertension, insulin resistance, and brain\nhemorrhages,14-16 other large-scale studies, including those by Savendahl et al. and Cianfarani et al.,\nindicate that rhGH treatment is generally safe when used appropriately, particularly in low-risk\npatients. Nevertheless, the long-term safety profile of rhGH therapy, especially regarding specific risks,\nremains an area of ongoing research and surveillance. \" (see Lines 81-86).\nSpecific comments and suggestions for improvement:\nReviewer’s Comment: Introduction and Background:\nClarify the connection between environmental factors (e.g., consumption of spicy foods) and ISS.\nGenerally spicy foods are hypothesized to reduce microbial growth and thus reduce gut\ninflammation and diarrhea - therefore it is worth further explanation why children consuming\nspicy foods may be more susceptible to subclinical inflammation.\nResponse: Thank you for your insightful comment regarding the need to clarify the connection\nbetween environmental factors, such as spicy food consumption, and ISS. We greatly value your\nfeedback, which has been instrumental in guiding us to strengthen the manuscript by addressing\nthis critical point more explicitly.\nIn the revised manuscript, we have updated the Introduction to elucidate the link between\nspicy food consumption and ISS pathogenesis. The specific revisions can be found on lines\n122-133, as follows:\n\"Although miR-17-3p overexpression significantly contributes to short stature, the origin of\nelevated plasma exosomal miR-17-3p in ISS patients remains unknown. To investigate this, we\nreviewed the published literature. We identified a higher prevalence of ISS in Chinese regions with\nhigh capsaicin consumption, such as Jiangxi, Chongqing, Hunan, and Guizhou provinces, which\ncollectively house over 200 million people [38]. This finding, drawn from a large population,\nsuggests a potential association between capsaicin-rich diets and ISS pathogenesis, with\nsignificant public health implications. Our subsequent experiments confirmed that the frequent\nconsumption of a capsaicin-rich diet, which mirrors dietary patterns in these high-prevalence\nregions, induces mild intestinal inflammation in Sprague-Dawley (SD) rats. This inflammation\nactivates the NF-κB signaling pathway, identified as the primary driver of elevated plasma\nexosomal miR-17-3p levels. Notably, this is the first study to establish a direct connection between\ncapsaicin consumption, gut inflammation, and ISS pathogenesis, offering a novel insight into the\netiology of ISS.\"\nAdditionally, we have addressed your observation that spicy foods are generally hypothesized\nto reduce microbial growth and alleviate gut inflammation and diarrhea, and the question of why\nchildren consuming such foods might instead be more susceptible to subclinical inflammation. To\nensure a concise and streamlined Introduction, we have included a detailed explanation of this\napparent paradox in the Discussion section, as shown below:\n\"While some studies report that capsaicin reduces microbial growth, alleviating gut\ninflammation and diarrhea, its gastrointestinal effects are dose-dependent. Our in vitro\nexperiments showed that at low concentrations (e.g., 25μM), capsaicin enhances intestinal\nepithelial cell proliferation\nand reduces apoptosis,\nsupporting a protective\nrole. However, at higher\nconcentrations (200μM), it\nsuppresses proliferation\nand increases apoptosis. In\nhealthy individuals, we\nfound that a capsaicin-rich\ndiet significantly increased\nplasma exosomal\nmiR-17-3p levels, which correlated with elevated intestinal inflammation markers, such as IL-1β\nand TNF-α. The cessation of the diet resulted in a decrease in miR-17-3p expression, with levels\nreturning upon reintroduction. These findings suggest that, in specific populations, the chronic\nconsumption of spicy foods may shift from a protective role to a risk factor for subtle inflammatory\nresponses that influence growth plate function.\nReviewer’s Comment: Rephrase unclear sentences, such as those in lines 65, 70, 78, and 85, for\nimproved readability.\nResponse: Thank you for your valuable feedback and for pointing out the need to rephrase\nunclear sentences for improved readability. We appreciate your careful review, which has helped\nus enhance the clarity of the manuscript. We have revised the sentences on lines 65, 70, 78, and 85\nof the original manuscript to improve readability.\nRevised Content:\nOriginal on lines 65: However, most cases, classified as idiopathic short stature (ISS) after\nexcluding systemic diseases, nutritional deficiencies, psychiatric disorders, chromosomal\nabnormalities, and overt hormonal imbalances, and demonstrating peak GH levels >10 μg/L\npost-stimulation, remain idiopathic.\nRevised: However, most cases are classified as idiopathic short stature (ISS) after ruling out systemic\ndiseases, nutritional deficiencies, psychiatric disorders, chromosomal abnormalities, and overt\nhormonal imbalances, with peak GH levels exceeding 10 μg/L post-stimulation, yet their cause remains\nunknown. (Lines 65-68)\nOriginal on lines 70: Unlike the short stature of growth hormone deficiency, ISS children do not\nlack growth hormone.\nRevised: Unlike children with growth hormone deficiency-related short stature, those with ISS have\nnormal growth hormone levels. (Lines 70-71)\nOriginal on lines 76-78: In contrast, research by Van et al. 12 and Kamp et al. 13 noted that\nalthough rhGH therapy can improve height during treatment, it may lead to premature growth\nplate closure and no significant difference in adult height compared to controls.\nRevised: In contrast, research by Van et al. 12 and Kamp et al. 13 reported that while rhGH therapy may\nenhance height during treatment, it could accelerate growth plate closure, resulting in adult heights\nthat do not significantly differ from those of untreated controls. (Lines 76-78)\nOriginal on lines 85: In recent years, scholars have identified specific susceptibility genes for ISS\nthrough whole genome scanning technology.\nRevised: In recent years, researchers have pinpointed specific ISS susceptibility genes using\nwhole-genome sequencing. (Lines 95-96)\nReviewer’s Comment: Results and Methods sections:\nProvide additional details regarding the origin of the chondrocytes in Figure 1 (line 132) and\nclarify the source of exosomes (line 213). Were they derived from gut villi or other tissues?\nResponse: Thank you for your suggestions. To address your request for additional details, we\nhave revised the manuscript to clarify the origin of the chondrocytes and the source of the\nexosomes. Specifically, the chondrocytes depicted in Figure 1 (Line 132) are from the C28/I2\nhuman chondrocyte cell line, and this information has been added to the text (see revised Line\n150). Additionally, the exosomes referenced on Line 213 are derived from primary chondrocytes\nisolated from the proximal tibia and distal femur growth plates of rats, not from gut villi or other\ntissues. This clarification has been incorporated into the manuscript (see revised Line 232-233).\nWe hope these updates provide the necessary details and improve the transparency of our\nexperimental design.\nReviewer’s Comment: Provide details on the volume of the serum that was taken from the\nchildren to extract exosomes and how much was used per injection to the animals and per tissue\nculture and cell culture treatment.\nResponse: Thank you for your question regarding serum volumes and exosome usage in our\nexperiments. We collected 5 ml of plasma from the ISS children to extract exosomes, yielding\napproximately 200 µg exosomes. For cell culture experiments, we co-cultured human chondrocyte\ncell line C28 (3 × 10⁵ cells) with 100 μg of exosomes derived from ISS patients in a T25 flask\ncontaining 5 mL of culture medium (Lines 568-572). Our results showed that, compared to the\ncontrol group, ISS-derived exosomes inhibited chondrocyte proliferation, downregulated the\nexpression of hypertrophic differentiation and osteogenic marker genes, and reduced bone\nformation, as evidenced by Von Kossa and Alizarin Red staining, which are more accurate terms\nfor indicating decreased mineralization and bone formation.\nAdditionally, to induce a short stature phenotype in rats, we utilized exosomes overexpressing\nmiR-17-3p. These exosomes were not derived from ISS patients but were instead obtained from\nprimary rat chondrocytes isolated from the proximal tibia and distal femur growth plates. To\nachieve high miR-17-3p expression, mimicking the elevated levels observed in circulating\nexosomes, we enriched these exosomes with miR-17-3p using the ExoFectin® sRNA-into-Exosome\nKit. The exosome dosage ranged from 200 to 300 µg per injection, calculated based on a ratio of\n1 µg of exosomes per 0.6 g of rat body weight. Rats were divided into groups at 4 weeks\npostpartum and received injections of miR-17-3p-overexpressing exosomes at weeks 4 and 5,\nrespectively (Lines 616-621). The experiment was terminated at week 8, at which point the results\nwere assessed. We observed suppressed proliferation of growth plate chondrocytes and reduced\nbone formation, leading to a short stature phenotype. These findings confirm that elevated\nmiR-17-3p levels in circulating exosomes can contribute to the development of short stature.\nReviewer’s Comment: Provide a clearer explanation on which specific regions of the growth\nplate are affected by miR-17-3p. It seems that in several cases the growth pate is less organized-\ncan you comment on this finding?\nResponse: Thank you for your request for clarification on the specific regions of the growth plate\naffected by miR-17-3p and the observation of a less organized structure. Our in situ hybridization\nexperiments in rat growth plates revealed that miR-17-3p is expressed across the proliferative\nzone, hypertrophic zone, and calcified zone, with a predominant expression in the hypertrophic\nzone. This distribution aligns with our experimental findings, which show that miR-17-3p inhibits\nchondrocyte proliferation, hypertrophic differentiation, and bone formation. In the control group,\nwhere miR-17-3p expression is at baseline levels, the growth plate exhibits a well-defined,\norganized structure with distinct proliferative, hypertrophic, and calcified zones. However, in the\nmiR-17-3p overexpression group, the growth plate structure appears less organized, suggesting a\ndisruption in its typical layered architecture. This observation may indicate that the suppression of\nchondrocyte hypertrophic differentiation by miR-17-3p is a key factor contributing to the less\norganized appearance of the growth plate. Specifically, the impaired transition from proliferation\nto hypertrophy and subsequent mineralization could disrupt the orderly progression of zones,\nleading to this altered morphology.\nFigure legend：The area between the two yellow dashed lines represents the growth plate chondrocyte region,\nwhich includes the resting zone chondrocytes, proliferative zone, and hypertrophic zone chondrocytes. The yellow\narrow points to the hypertrophic chondrocytes. Below the lower of the two yellow dashed lines is the calcified zone.\nOur in situ hybridization experiments in rat growth plates revealed that miR-17-3p is expressed across the\nproliferative zone, hypertrophic zone, and calcified zone, with predominant expression in the hypertrophic zone.\nReviewer’s Comment: Elaborate on the procedures used for CRISPR-Cas9-mediated gene\nknockout in human chondrocytes and rat growth plate cartilage (line 191; as well as in\nsupplementary figures 11, 12, and 25).\nResponse: Thank you for your insightful comment requesting elaboration on the procedures used\nfor CRISPR-Cas9-mediated gene knockout in human chondrocytes and rat growth plate cartilage\n(line 191), as well as in Supplementary Figures 11, 12, and 25. We value your suggestion, which\nhas guided us to provide a more detailed description of our experimental approach.\nIn the revised manuscript, we have expanded the Materials and Methods section to include a\ncomprehensive outline of the CRISPR-Cas9-mediated gene knockout procedures. Specifically, the\ndetails for human chondrocytes are described in lines 595-601, while those for rat growth plate\ncartilage are provided in lines 706-731. These revisions encompass the key steps involved,\nensuring clarity and reproducibility.\nRevised Added Content:\nC28 chondrocytes Stable Transfection Strain Construction (Lines 594-601)\nAll the virus supernatants were prepared using the virus construction methods mentioned in this\narticle. Among them, the titers of overexpression plasmids for miR-17-3p, ZNF148 and SOS1 were\ngreater than 1×109 FU/mL, and the infectious substance concentration (MOI) was 25; the titers\nof CRISPR-Cas9 knockdown plasmids targeting ZNF148 and SOS1 were greater than 5×108\nFU/mL, and the infectious substance concentration (MOI) was 50. Concentrated viral supernatant,\nmixed with polybrene (8 µg/ml), was used to transfect human chondrocytes in DMEM\nsupplemented with 10% FBS. Cells were infected for 48 hours and then screened with puromycin\nfor 14 days.\nExplant Culture of Fetal SD Rat Femurs with miR-17-3p Overexpression and ZNF148/SOS1\nKnockdown (Lines 706-731)\nApproval for the use of rat limbs was granted by the Animal Ethics Committee of Nanchang\nUniversity. Femurs from fetal Sprague-Dawley (SD) rats were harvested one day postpartum. The\nexplanted tissues were statically cultured in BGJb medium (Gibco, Australia) supplemented with 6\nmg/ml bovine serum albumin (BSA; Gibco, Australia), 150 µg/ml ascorbic acid (Solarbio, Cat:\nA8100), 100 units/ml penicillin, and 100 µg/ml streptomycin (Gibco, Australia). Cultures were\nmaintained at 37°C in a humidified atmosphere with 5% CO2, with the medium refreshed daily\nusing freshly prepared reagents.\nFor gene editing and overexpression, CRISPR-Cas9 knockdown plasmids targeting ZNF148\nand SOS1, along with an miR-17-3p overexpression plasmid, were obtained from Guangzhou\nVectorBuilder Co., Ltd. and Genechem Co., Ltd. (China). Detailed plasmid sequences are\nprovided in Supplementary Fig. 24c–f. Lentiviral particles were generated by transfecting\nHEK293T cells with the corresponding lentiviral packaging plasmids. The resulting supernatants\nwere collected, filtered, and concentrated using a Virus Concentration Kit (Beyotime, Cat:\nC2901S) to achieve a titer exceeding 1×10^8 TU/ml (quantified by p24 ELISA). Concentrated\nlentiviral stocks were stored at -80°C in PBS with 1% BSA.\nTransfection of fetal rat growth plate cartilage was performed in a 2 ml culture system using\na 24-well plate. Concentrated lentiviral supernatant (titer > 5×10^8 FU/ml, multiplicity of\ninfection [MOI] = 200) was mixed with 8 µg/ml polybrene and HiTransG A (Genechem Co., Ltd.,\nShanghai), a specialized transfection reagent, to enhance transduction efficiency. The explants\nwere transduced with lentiviral particles carrying either the ZNF148/SOS1 knockdown constructs\nor the miR-17-3p overexpression construct in fresh medium. The medium was replaced 72 hours\npost-transfection and refreshed daily thereafter. After 14 days of culture, samples were analyzed by\nRT-qPCR, Western blot, and immunofluorescence. Experimental groupings are detailed in\nSupplementary Fig. 27.\nReviewer’s Comment: Explain the targeting mechanism of collagen type II-coated exosomes to\nthe growth plate. Did these exosomes also localize to other tissues, such as articular cartilage,\nthe brain, or blood vessels? Why did they localize to the skull?\nResponse: Thank you for your thoughtful comment requesting clarification on the targeting\nmechanism of collagen type II-coated exosomes to the growth plate, as well as their potential\nlocalization to other tissues such as articular cartilage, the brain, or blood vessels, and the\nobserved localization to the skull. We greatly appreciate your inquiry, which has encouraged us to\nprovide a more detailed explanation to improve the manuscript’s clarity.\nIn our study, we employed a membrane fusion protein technique to load the exosome surface\nwith the collagen type II-targeting peptide WYRGRL. This modification confers specificity to\ntissues rich in collagen type II, such as the growth plate cartilage and articular cartilage,\nenabling the targeted delivery of exosomes (Lines 668-675). Tissues like the brain and blood\nvessels, which contain negligible amounts of collagen type II, showed no significant targeting, as\nexpected due to the peptide’s selective affinity. Regarding the skull localization observed in our in\nvivo imaging, we used 4- to 6-week-old rats for these experiments—a developmental stage during\nwhich the skull undergoes endochondral ossification to complete bony fusion. Since this process\ninvolves collagen type II-rich cartilage, the fluorescence signal detected in the skull reflects the\ntargeting of our exosomes to this transient cartilaginous tissue.\nWe have incorporated these details into the revised manuscript, and the Materials and\nMethods section now includes a detailed description of the experimental procedures and targeting\nspecificity (Lines 668-675).\nReviewer’s Comment: In line 254, provide an estimation of the amount of chili required to\nachieve a blood concentration of 200 μM capsaicin.\nResponse: Thank you for your insightful comment requesting an estimation of the amount of chili\nrequired to achieve a blood concentration of 200 μM capsaicin, as noted in line 254. We\nappreciate your suggestion, which has encouraged us to clarify our experimental context and\nrationale.\nIn this study, the 200 μM capsaicin concentration referenced in line 254 pertains to our in\nvitro experiments, where we stimulated intestinal mucosal epithelial cells to evaluate its effects. To\nselect an effective concentration, we tested a gradient of capsaicin levels (25 μM, 50 μM, 75 μM,\n100 μM, 150 μM, and 200 μM), as shown in Supplementary Figures 15A-F. Among these, 200 μM\nexhibited a significant biological response in intestinal mucosal epithelial cells, justifying its use\nin our stimulation experiments.\nReviewer’s Comment: Figures and Supplementary Material:\nPlease check figure 7A and 8, as it seems the rats fed with Caspaicin are larger.\nResponse: We appreciate the reviewer’s keen observation regarding the labeling in Figure 7A and\n8. Upon review, we confirmed that the mice were indeed mislabeled, with the capsaicin-treated\nmouse appearing larger than expected. We have corrected this error in the revised manuscript by\nupdating the labels in Figure 7A and 8 to reflect the experimental groups accurately. We regret\nthis oversight and thank the reviewer for bringing it to our attention.\nReviewer’s Comment: Revise Supplementary Figure 13 to indicate whether the differentially\nexpressed plasma proteins were identified in this study or a prior publication, and provide\nreferences if applicable.\nResponse: Thank you for your valuable comment requesting clarification on whether the\ndifferentially expressed plasma proteins in Supplementary Figure 13 were identified in this study\nor a prior publication, along with applicable references.\nIn our previous study (Liu X, Zhang J, Yuan J, Ding R, Liu T, Jia J. LCN2 is a new diagnostic\nbiomarker and potential therapeutic target in idiopathic short stature. J Cell Mol Med.\n2022;26(12):3568-3581), we identified 84 differentially expressed proteins in the plasma of\nchildren with idiopathic short stature (ISS). However, that work focused solely on the role of LCN2\nin ISS, and at the time of publication, we had not yet recognized the significance of\ninflammation-related proteins in this condition. In the current study, we discovered that\ncapsaicin-induced intestinal inflammation contributes to short stature. Motivated by this finding,\nwe re-analyzed the previously identified 84 differentially expressed proteins from ISS patients and\nnewly identified 42 of them as inflammation-related. Supplementary Figure 13, which presents this\nanalysis, is original to the present study and was neither included nor analyzed in this manner in\nour prior publication. Thus, there is no overlap or infringement with our earlier work.\nAdditionally, we have cited the previous study in the current manuscript to provide proper context\nand credit.\nReviewer’s Comment: Address discrepancies in the legend of Supplementary Figure 17 (e.g.,\nCT-Exo vs. ET-Exo, what is panel K?).\nResponse: Thank you for your careful review and for pointing out the discrepancies in the legend\nof Supplementary Figure 17, specifically regarding the notation of CT-Exo vs. ET-Exo and the\nclarification of panel K. We appreciate your attention to detail, which has helped us improve the\nconsistency and clarity of our presentation.\nWe confirm that CT-Exo and ET-Exo refer to the same entity in our study; the inconsistency\narose from non-uniform labeling. We have revised the legend of Supplementary Figure 17 to\nstandardize the terminology, ensuring that only one designation (ET-Exo) is used throughout for\nclarity. Additionally, panel K displays the names of the organs assessed in our in vivo imaging\nanalysis, corresponding directly to the organs shown in panel I. To address this, we have updated\nthe legend to explicitly state this relationship, ensuring that the connection between panels I and K\nis clear.\nReviewer’s Comment: The title of supplementary figure 19 should be corrected. Panel b showing\nthe animals is not clear, please improve the visibility of panel - it is impossible to see the\ndifferences in the length of the animals.\nResponse: We have corrected the title of Supplementary Figure 19 to accurately reflect its content.\nAdditionally, we have improved the visibility of Panel b in the revised manuscript by enhancing\nthe image quality. These updates have been incorporated into the revised version of\nSupplementary Figure 19.\nWe are grateful for your suggestions, which have significantly improved the presentation of\nour data.\nReviewer’s Comment: Discussion:\nHighlight the potential implications of the findings beyond the studied cohort.\nResponse: We sincerely thank the reviewer for this valuable suggestion. In the revised manuscript,\nwe have addressed this recommendation by incorporating a dedicated paragraph in the\nDiscussion section to emphasize the broader implications of our findings. The added content is as\nfollows:\nAdded Text in Discussion Section (Lines 476-499):\nBeyond the ISS cohort studied here, our findings have broader implications for populations and\nconditions beyond this specific group. The association between chronic capsaicin-rich diets,\nsubclinical gut inflammation, and miR-17-3p-mediated growth inhibition may extend to children\nand adolescents in other regions with high chili pepper consumption, such as parts of South Asia,\nLatin America, and Africa, where spicy diets are culturally prevalent. This suggests that dietary\nhabits could serve as a modifiable environmental risk factor for growth impairment in genetically\nsusceptible individuals globally, highlighting the need for further epidemiological investigations.\nMoreover, the activation of the gut NF-κB signaling pathway and the inhibition of bone\nformation induced by high-capsaicin diets underscore potential risks that extend beyond growth\nimpairment. For instance, our unpublished data from a C57 mouse model of osteoporosis\ndemonstrate that a capsaicin-enriched diet exacerbates bone loss, providing additional evidence\nfor the detrimental effects of high-capsaicin diets on bone health. This observation aligns with\nepidemiological data from China, where Wang et al. (2023) reported regional variations in\nosteoporosis prevalence. Although their study did not specifically examine the relationship\nbetween high-capsaicin diets and osteoporosis, their data revealed that Jiangxi Province—a\nregion with notably high chili pepper consumption—has the highest incidence of osteoporosis.\nThis geographical correlation with our ISS case distribution suggests a potential link between\ndietary capsaicin intake and bone health, warranting further investigation into the mechanisms\nunderlying this association.\nThese findings emphasize the importance of raising awareness about the impact of dietary\nhabits on bone development, particularly in regions with high capsaicin consumption. They also\nhighlight the need for multicenter studies to validate these observations across diverse\npopulations and skeletal disorders, such as osteoporosis and other metabolic bone diseases. By\nelucidating the role of dietary factors in bone health, such research could inform public health\nstrategies aimed at mitigating the risks associated with high-capsaicin diets and improving\noutcomes for affected individuals.\nReviewer’s Comment: Provide a clearer explanation of the mechanism by which miR-17-3p\naffects specific regions of the growth plate.\nResponse: Thank you for your request for clarification on the specific regions of the growth plate\naffected by miR-17-3p and the observation of a less organized structure. Our in situ hybridization\nexperiments in rat growth plates revealed that miR-17-3p is expressed across the proliferative\nzone, hypertrophic zone, and calcified zone, with a predominant expression in the hypertrophic\nzone. This distribution aligns with our experimental findings, which show that miR-17-3p inhibits\nchondrocyte proliferation, hypertrophic differentiation, and bone formation.\nFigure legend： The area between the two yellow dashed lines represents the growth plate chondrocyte region,\nwhich includes the resting zone chondrocytes, proliferative zone, and hypertrophic zone chondrocytes. The yellow\narrow points to the hypertrophic chondrocytes. Below the lower of the two yellow dashed lines is the calcified zone.\nOur in situ hybridization experiments in rat growth plates revealed that miR-17-3p is expressed across the\nproliferative zone, hypertrophic zone, and calcified zone, with predominant expression in the hypertrophic zone.\nReviewer’s Comment: Minor Comments:\nClarify terms such as \"cell communication\" (line 99) and rephrase ambiguous phrases (e.g., lines\n105, 153, 204–207).\nResponse: We sincerely thank the reviewer for their constructive feedback. In the revised\nmanuscript, we have addressed the concerns by clarifying terms such as \"cell communication\"\n(line 99) and rephrasing ambiguous phrases (e.g., lines 105, 153, 204–207) as requested. Below,\nwe outline the specific changes made, presenting both the original and revised sentences for\nclarity and ease of review.\n① Clarification of \"cell communication\" (line 99):\nOriginal: It is well known that various hormones and growth factors secreted by tissues and organs\nare delivered to the growth plate via the bloodstream, interacting with it to regulate growth and\ndevelopment through cell communication.\nRevised: It is well known that various hormones and growth factors secreted by tissues and organs\nare delivered to the growth plate via the bloodstream, interacting with it to regulate growth and\ndevelopment through intercellular signaling processes. (line 109)\n② Rephrasing ambiguous phrase (line 105)\nOriginal: Although exosomes are promising, their specific role in ISS pathogenesis is poorly\nunderstood and requires further exploration.\nRevised: Although exosomes show potential for advancing our understanding and treatment of ISS,\ntheir specific role in the pathogenesis of ISS remains largely unclear and warrants further\ninvestigation. (lines 115-117)\n③ Rephrasing ambiguous phrase (line 153)\nOriginal: In situ hybridization analysis demonstrated that miR-17-3p is expressed in human\nchondrocytes (C28 cell line) and also detected in the growth plate cartilage of SD rats.\nRevised: In situ hybridization analysis demonstrated that miR-17-3p is expressed in human\nchondrocytes (C28 cell line) and is also localized in the growth plate cartilage of SD rats. (lines\n169-171)\n④ Rephrasing ambiguous phrases (lines 204–207)\nOriginal: RT-qPCR and Western blot analyses demonstrated that although SOS1 knockout did not\naffect the expression levels of critical genes in the MEK and PI3K pathways—ERK1/2, PI3K,\nand AKT, RAS and the phosphorylation levels of ERK1/2, PI3K, and AKT were notably reduced after\nSOS1 knockout.\nRevised: RT-qPCR and Western blot analyses revealed that SOS1 knockout did not alter the\nmRNA or protein expression levels of key MEK and PI3K pathway genes, including ERK1/2, PI3K,\nand AKT. However, SOS1 knockout significantly decreased RAS expression and the\nphosphorylation levels of ERK1/2, PI3K, and AKT. (lines 223-226)\nReviewer’s Comment: Add catalog numbers for antibodies listed in Supplementary Table 3.\nResponse: We appreciate the reviewer’s suggestion. In response, we have added the catalog\nnumbers for the antibodies listed in Supplementary Table 3 in the revised manuscript.\nReviewer’s Comment: In line 213 the authors indicate that the exosomes were produced by\nchondrocytes, but I had the impression that they were produced by the gut villi- please explain\nwhat is the origin of the exosomes? what do you think was the origin of the exosomes in the\nchildren?\nResponse: We thank the reviewer for their insightful comment. Below, we clarify the origin of\nexosomes referenced in line 213 and provide our hypothesis regarding the origin of exosomes in\nISS children, as well as the rationale for our experimental design.\nClarification on Exosomes in Line 213 (Chondrocyte-Derived Exosomes):\nIn our study, we initially observed that plasma exosomes from ISS children inhibited the\nproliferation of human chondrocytes (C28 cell line) and suppressed the expression of\nchondrogenic differentiation markers. Through high-throughput sequencing and qPCR validation,\nwe identified miR-17-3p as highly expressed in these exosomes. To further investigate the role of\nmiR-17-3p, we used chondrocyte-derived exosomes overexpressing miR-17-3p for both in vitro\nand in vivo experiments.\nWe chose chondrocyte-derived exosomes for the following reasons:\n At the time of these experiments, the cellular origin of the abnormally expressed exosomes\nin ISS children’s plasma was unknown.\n Using chondrocyte-derived exosomes allowed us to specifically overexpress miR-17-3p\nwhile maintaining other exosomal contents derived from chondrocytes. This approach\nhelped us isolate the effects of miR-17-3p overexpression on chondrocyte proliferation\nand bone formation, without confounding factors from other cell types.\nThese experiments demonstrated that miR-17-3p inhibits growth plate chondrocyte proliferation\nand bone formation in vivo, leading to a short-stature phenotype.\nHypothesized Origin of Exosomes in ISS Children (Gut-Derived):\nIn subsequent experiments, we investigated the potential source of the exosomes in ISS children.\nUsing a capsaicin-induced mouse model, we observed a short-stature phenotype. However, we\nfound no difference in blood capsaicin levels between the capsaicin diet group and the control\ngroup, suggesting that capsaicin does not directly enter the bloodstream to inhibit growth plate\nchondrogenesis.\nFurther analysis of intestinal tissues revealed inflammatory cell infiltration and activation of\nthe NF-κB signaling pathway, accompanied by upregulated miR-17-3p expression. In vitro\nexperiments confirmed that capsaicin activates NF-κB and induces miR-17-3p overexpression.\nAdditionally, we detected elevated levels of miR-17-3p and inflammatory markers (IL-1β and\nTNF-α) in the feces of ISS children.\nBased on these findings, we propose that the abnormally expressed exosomes in the plasma\nof ISS children originate from the intestinal tract, likely due to chronic intestinal inflammation and\nNF-κB-mediated upregulation of miR-17-3p.\nReviewer’s Comment: Line 263- Were the controls from the same geographic region? or just\nnormal height children?\nResponse: We thank the reviewer for their question. The control group children were all from\nJiangxi Province, the same geographic region as the cases.\nReviewer’s Comment: Ensure consistent scale bars in Figures 3 and 7 and clarify the staining patterns in all other figures.\nResponse: We thank the reviewer for their valuable suggestion. In the revised manuscript, we have\nensured consistent scale bars in Figures 3 and 7 by standardizing their measurements and\nlabeling. Additionally, we have clarified the staining patterns in all other figures.\nReviewer’s Comment: Supp figure 3- how was overexpression in growth plate of the rats\nachieved? Please add the details of the infection or transfection that was done.\nResponse: We appreciate the reviewer’s comment. In the Materials and Methods section of the\nrevised manuscript, we have added detailed information regarding the overexpression in rats' growth plates.\nRevised Added Content: (lines 706-731)\nExplant Culture of Fetal SD Rat Femurs with miR-17-3p Overexpression and ZNF148/SOS1\nKnockdown\nApproval for the use of rat limbs was granted by the Animal Ethics Committee of Nanchang\nUniversity. Femurs from fetal Sprague-Dawley (SD) rats were harvested one day postpartum. The\nexplanted tissues were statically cultured in BGJb medium (Gibco, Australia) supplemented with 6\nmg/ml bovine serum albumin (BSA; Gibco, Australia), 150 µg/ml ascorbic acid (Solarbio, Cat:\nA8100), 100 units/ml penicillin, and 100 µg/ml streptomycin (Gibco, Australia). Cultures were\nmaintained at 37°C in a humidified atmosphere with 5% CO2, with the medium refreshed daily\nusing freshly prepared reagents.\nFor gene editing and overexpression, CRISPR-Cas9 knockdown plasmids targeting ZNF148\nand SOS1, along with an miR-17-3p overexpression plasmid, were obtained from Guangzhou\nVectorBuilder Co., Ltd. and Genechem Co., Ltd. (China). Detailed plasmid sequences are\nprovided in Supplementary Fig. 24c–f. Lentiviral particles were generated by transfecting\nHEK293T cells with the corresponding lentiviral packaging plasmids. The resulting supernatants\nwere collected, filtered, and concentrated using a Virus Concentration Kit (Beyotime, Cat:\nC2901S) to achieve a titer exceeding 1×10^8 TU/ml (quantified by p24 ELISA). Concentrated\nlentiviral stocks were stored at -80°C in PBS with 1% BSA.\nTransfection of fetal rat growth plate cartilage was performed in a 2 ml culture system using\na 24-well plate. Concentrated lentiviral supernatant (titer > 5×10^8 FU/ml, multiplicity of\ninfection [MOI] = 200) was mixed with 8 µg/ml polybrene and HiTransG A (Genechem Co., Ltd.,\nShanghai), a specialized transfection reagent, to enhance transduction efficiency. The explants\nwere transduced with lentiviral particles carrying either the ZNF148/SOS1 knockdown constructs\nor the miR-17-3p overexpression construct in fresh medium. The medium was replaced 72 hours\npost-transfection and refreshed daily thereafter. After 14 days of culture, samples were analyzed by\nRT-qPCR, Western blot, and immunofluorescence. Experimental groupings are detailed in\nSupplementary Fig. 27.\nReviewer’s Comment: Minor comments;\nLine 65- if there is growth hormone deficiency it is not idiopathic short stature, per definition,\ntherefore this should be omitted.\nLine 70- please rephrase, the connection is not clear\nLine 78- treatment with GH is quite safe (as mentioned in the two studies by Savendahl and\nCianfarani S), therefore the sentence should be rephrased.\nLine 81- it is not clear who are the children that do not get the treatment.\nLine 85- the sentence is not clear, please rephrase.\nLine 105- what do you mean by “promising”?\nResponse: We sincerely thank the reviewer for their constructive feedback. In the revised\nmanuscript, we have addressed the concerns. Below, we outline the specific changes made,\npresenting both the original and revised sentences for clarity and ease of review.\nLine 65\nOriginal: However, most cases, classified as idiopathic short stature (ISS) after excluding systemic\ndiseases, nutritional deficiencies, psychiatric disorders, chromosomal abnormalities, and overt\nhormonal imbalances, and demonstrating peak GH levels >10 μg/L post-stimulation, remain\nidiopathic.\nRevised: However, most cases are classified as idiopathic short stature (ISS) after ruling out\nsystemic diseases, nutritional deficiencies, psychiatric disorders, chromosomal abnormalities, and\novert hormonal imbalances, with peak GH levels exceeding 10 μg/L post-stimulation, yet their\ncause remains unknown. (lines 65-68)\nLine 70\nOriginal: Unlike the short stature of growth hormone deficiency, ISS children do not lack growth\nhormone.\nRevised: Unlike children with growth hormone deficiency-related short stature, those with ISS\nhave normal growth hormone levels. (lines 70-71)\nLine 78- treatment with GH is quite safe (as mentioned in the two studies by Savendahl and\nCianfarani S), therefore the sentence should be rephrased.\nOriginal: In addition, reports link rhGH therapy to increased risks of bone and cartilage tumors,\ncerebrovascular diseases, hypertension, insulin resistance, and brain hemorrhages.\nRevised: In terms of safety, while some studies have raised concerns about potential risks\nassociated with rhGH therapy, such as bone and cartilage tumors, cerebrovascular diseases,\nhypertension, insulin resistance, and brain hemorrhages,14-16 other large-scale studies, including\nthose by Savendahl et al. and Cianfarani et al., indicate that rhGH treatment is generally safe\nwhen used appropriately, particularly in low-risk patients.17-18 Nevertheless, the long-term safety\nprofile of rhGH therapy, especially regarding specific risks, remains an area of ongoing research\nand surveillance. (lines 81-86)\nLine 81- it is not clear who are the children that do not get the treatment.\nOriginal: The 2016 American Academy of Pediatric Endocrinology guidelines suggest that not all\nISS patients qualify for rhGH therapy.\nRevised: The 2016 American Academy of Pediatric Endocrinology guidelines suggest that rhGH\ntherapy is not universally recommended for all ISS patients, particularly those with less severe\nshort stature (height standard deviation score [HtSDS] above -2.25) or those without significant\nphysical or psychosocial burdens related to their condition. This highlights the limitations of\nrhGH therapy in ISS, including variable efficacy and potential risks. (lines 87-94)\nLine 85\nOriginal: In recent years, scholars have identified specific susceptibility genes for ISS through\nwhole genome scanning technology.\nRevised: In recent years, researchers have pinpointed specific ISS susceptibility genes using\nwhole-genome sequencing. (lines 95-96)\nLine 105\nOriginal: Although exosomes are promising, their specific role in ISS pathogenesis is poorly\nunderstood and requires further exploration.\nRevised: Although exosomes show potential for advancing our understanding and treatment of ISS,\ntheir specific role in the pathogenesis of ISS remains largely unclear and warrants further\ninvestigation. (lines 115-117)\nReviewer’s Comment: Line 160- please explain how down-regulation and how overexpression\nwas done in chondrocytes.\nResponse: We appreciate the reviewer’s comment. We have added detailed information regarding\nthe overexpression in the growth plate of rats in the Materials and Methods section of the revised\nmanuscript.\nRevised Added Content:\nC28 chondrocytes Stable Transfection Strain Construction (lines 594-601)\nAll the virus supernatants were prepared using the virus construction methods mentioned in this\narticle. Among them, the titers of overexpression plasmids for miR-17-3p, ZNF148 and SOS1 were\ngreater than 1×109 FU/mL, and the infectious substance concentration (MOI) was 25; the titers\nof CRISPR-Cas9 knockdown plasmids targeting ZNF148 and SOS1 were greater than 5×108\nFU/mL, and the infectious substance concentration (MOI) was 50. Concentrated viral supernatant,\nmixed with polybrene (8 µg/ml), was used to transfect human chondrocytes in DMEM\nsupplemented with 10% FBS. Cells were infected for 48 hours and then screened with puromycin\nfor 14 days.\nReviewer’s Comment: Line 190 should be in bold.\nSupplementary Figures 5, 6, 8 panel M- what are the arrow head showing?\nResponse: We thank the reviewer for their thoughtful comments. Below, we address the two points\nraised:\n Formatting of Line 190:\nAs requested, we have bolded the text in line 190 in the revised manuscript to ensure proper\nformatting and emphasis as per the reviewer’s suggestion.\n Clarification of Arrowheads in Supplementary Figures 5, 6, 8 (Panel M):\nIn Supplementary Figures 5, 6, and 8 (panel M), the arrowheads indicate positive granules\nstained with Von Kossa, Alkaline Phosphatase (ALP), and Alizarin Red in the growth plate\ncartilage of rats. These staining methods highlight specific aspects of mineralization and\ncellular activity: Von Kossa staining identifies calcium phosphate deposits, indicating\nmineralization; ALP staining reflects osteogenic and chondrogenic activity; and Alizarin Red\nstaining marks calcium deposits, further confirming mineralization. The reduced number of\npositive granules in chondrocytes of the experimental group, as indicated by fewer stained\nparticles, suggests diminished chondrocyte activity, impaired mineralization, and reduced\nbone matrix formation. This observation aligns with our findings of suppressed\nchondrogenesis and longitudinal bone growth in the context of our study, supporting the role\nof the investigated pathway (e.g., miR-17-3p overexpression) in inhibiting growth plate\nfunction and contributing to a short-stature phenotype. We have added this explanation to the\nfigure legends in the revised manuscript for clarity.\nReviewer’s Comment: Addressing these points, especially in the introduction and discussion,\nwould improve the clarity and depth of the study, offering a more comprehensive understanding of\nthe mechanisms underlying ISS and the suggested treatment.\nResponse: We sincerely thank you for your valuable feedback, which has been instrumental in\nenhancing the quality of our manuscript. In the revised version, we have addressed the points you\nraised, particularly in the introduction and discussion sections, to improve the clarity and depth of\nthe study. These revisions provide a more comprehensive understanding of the mechanisms\nunderlying ISS and the proposed treatments. The authors believe these changes strengthen our\nmanuscript and appreciate your guidance in this process.\nWe sincerely appreciate the opportunity to revise our manuscript, \"Capsaicin Diet-Induced\nGut Inflammation Elevates Plasma Exosomal miR-17-3p: Unveiling the ZNF148/SOS1 Axis as a\nNovel Mechanism in ISS Pathogenesis and Potential Therapeutic Target\" (NCOMMS-24-73070).\nThe reviewers’ thoughtful and detailed suggestions have been instrumental in refining our study,\nimproving its clarity, and strengthening its contribution to the field. We believe the revisions,\nhighlighted in blue for ease of review, address all concerns and align with the high standards of\nNature Communications. We welcome any further feedback or suggestions to ensure the\nmanuscript meets your expectations. Thank you again for your time, expertise, and support in this\nprocess.\nSincerely,\nJingyu Jia, M.D., Ph.D\nOn behalf of all authors\nDepartment of Orthopaedics, The Second Affiliated Hospital of Nanchang University\nPhone: +86-791-86311590; Fax: +86-791-86311590\nE-mail: jiaxintong9@163.com\nReviewer #2 (Remarks to the Author):\nThe authors have addressed most of my previous concerns, and I appreciate their efforts.\nHowever, a few important issues remain and should be resolved prior to publication:\nPage 26 (rebuttal letter): The alignment between the timeline and the graphs needs correction, as the\ncurrent presentation lacks clarity.\nCapsaicin dosage: The manuscript does not provide an estimate of how much chili pepper a child would\nneed to ingest to reach a capsaicin blood concentration of 200 μM.\nMechanistic explanation: The mechanism by which miR-17-3p affects growth of specific regions of the\ngrowth plate remains insufficiently detailed. A clearer and more comprehensive explanation regarding\nthe mechanism of action would significantly enhance the manuscript.\nAuthors’ Response:\n1. Alignment Between Timeline and Graphs (Page 26 of Rebuttal Letter)\nWe acknowledge that the alignment between the timeline and the graphs on page 26 of the rebuttal letter\nlacked clarity. To resolve this, we have revised the presentation by reorganizing the timeline description\nand ensuring direct correspondence with the graph labels.\nThe data for week 1 represent measurements taken during a no-capsaicin diet period. Week 2 data were collected after one week\nof a capsaicin-rich diet typical of the Jiangxi region (daily diet). Week 3 data correspond to measurements following a return to\nthe no-capsaicin diet, while week 4 data were obtained after resuming the capsaicin-rich diet.\nOur initial investigation into ISS revealed the regulatory role of capsaicin in the gut-bone growth\naxis. To explore this further, we conducted a supplementary study with a small cohort of eight adults\nconsuming a typical Jiangxi daily diet. The results demonstrated that a capsaicin-rich diet significantly\nincreased plasma exosomal miR-17-3p levels and intestinal inflammation markers (e.g., IL-1β and TNF-\nα). When the same diet was consumed without capsaicin, plasma exosomal miR-17-3p levels and\ninflammation markers returned to normal, reinforcing that capsaicin intake induces these changes in\nadults.\nBuilding on these observations, we extended our research to investigate osteoporosis. Using a rat\nmodel, we induced osteoporosis through orchiectomy and administered either a capsaicin-rich or\ncapsaicin-free diet. The capsaicin group exhibited significantly greater severity of osteoporosis compared\nto the capsaicin-free group, suggesting that capsaicin consumption may contribute to bone loss. This\nfinding aligns with the high incidence of osteoporosis in Jiangxi and other capsaicin-rich diet provinces.\nHowever, we have not included these osteoporosis data in the current manuscript, as this aspect of our\nresearch remains in the preliminary stages, with ongoing experiments and future therapeutic\ninvestigations planned. We intend to present these findings in a separate, dedicated study once the\n1\nresearch is sufficiently mature. The exclusion of these data does not impact the validity of our primary\nfindings on ISS.\nWe assessed the bone mineral density (BMD) of the tibia and femur of SD rats using CT values.\nThis is an original record file of our preliminary experiment: The term \"辣\" refers to the capsaicin diet group following\norchiectomy-induced osteoporosis modeling, the word \"正常\" represents the no-capsaicin diet group following orchiectomy-\ninduced osteoporosis modeling, and the three characters \"未造模\" denote the no-surgery, no-capsaicin diet control group. The\nmean CT bone density values for the three groups were 1294, 1531.1, and 1742.6, respectively. These data indicate that a capsaicin-\nrich diet exacerbates osteoporosis. In regions with high capsaicin consumption, this dietary habit may represent a significant risk\nfactor for worsening osteoporosis.\nHowever, they did not recognize that Jiangxi Province and Yunnan Province are regions with high capsaicin consumption.\nConsequently, they did not associate capsaicin-rich diets with osteoporosis. In contrast, our animal model supports a link between\ncapsaicin consumption and osteoporosis. Moving forward, we require additional experiments and data to further substantiate our\nconclusions.\n2. Capsaicin Dosage Estimate for Reaching 200 μM Blood Concentration\nIn our in vitro experiments, we applied a range of capsaicin concentrations (25, 50, 75, 100, 150, and\n200 μM) to the human intestinal epithelial cell line HIEC-6 (Otwo Biotech, Cat No. HTX1978). The\nprimary objective of this approach was to simulate the direct stimulatory effects of capsaicin-rich foods\non intestinal epithelial cells upon entering the gastrointestinal tract. We wish to emphasize that these\nconcentrations represent estimated luminal levels within the intestine, not capsaicin concentrations in the\nbloodstream.\nThe selection of 200 μM as a key concentration was guided by the development of our capsaicin\ndietary model, which was informed by epidemiological observations from high-incidence regions of\nidiopathic short stature (ISS) in China, including Jiangxi, Hunan, Chongqing, and Guizhou provinces,\nwhere capsaicin-rich diets are prevalent. According to Zou et al. (PMCID: PMC8545790), an overview\nof pepper consumption in China based on statistical data from the National Industrial System of\nSpecialty Vegetables (NISSV) indicates that Hunan Province records the highest total annual pepper consumption\n3\nat 7.57 million tons, followed by Jiangxi, Guizhou, Chongqing, Yunnan, and Hubei provinces. With a\ncombined population of approximately 286,764,000 across these six provinces, the upper and middle\nreaches of the Yangtze River—designated as extreme-spicy areas due to high per capita pepper\nconsumption (75 kg annually)—include Hunan Province (111.57 kg), Guizhou (98.5 kg), Jiangxi (97.6\nkg), and Chongqing (95.6 kg).\nFurther supporting this, Jia et al. (2015), in their study titled \"Determination of Capsaicinoids and\nClassification of Pungency Level in Sichuan Cuisine Dishes\" (published in a Chinese journal with an\nEnglish abstract, full text provided as an attachment), analyzed 123 Sichuan cuisine dishes using high-\nperformance liquid chromatography (HPLC). They categorized the dishes into five grades based on\ncapsaicinoid content, with grade 5 (highest spiciness) averaging 0.2076 g/kg and grade 4 averaging\n0.1756 g/kg, reflecting the dietary profile of Jiangxi residents, who predominantly consume grade 4 and\n5 foods.\nTo validate these observations, we measured capsaicin concentrations in six common dishes from\nJiangxi Province, sourced from local restaurants. The measured values were 0.3659, 0.0025, 0.6277,\n0.0640, 0.0547, and 0.1935 g/kg, yielding a mean of 0.2181 ± 0.2400 g/kg. These findings are consistent\nwith Jia et al.’s reported averages of 0.2076 g/kg for grade 5 and 0.1756 g/kg for grade 4 dishes.\nBased on Jia et al.’s published data, the estimated capsaicin intake in Jiangxi Province for\nadolescents and adults ranges from 1.756 to 2.076 mg/kg/day. To estimate the potential intestinal\nconcentration resulting from this intake, we applied the following formula:\nConcentration (μM) = [Daily capsaicin intake (mg) / Intestinal volume (L)] / (Molecular weight of\ncapsaicin / 1000)\nDaily capsaicin intake (mg) = Daily dose (mg/kg/day) × Body weight (kg)\nIntestinal volume is assumed to be 1.5 L (approximate small intestinal fluid volume).\nMolecular weight of capsaicin = 305.41 g/mol.\nFor a 70 kg adult:\n(305.41 / 1000) ≈ 268.3 μM\nHigh end: 2.076 mg/kg/day × 70 kg = 145.32 mg/day → 145.32 mg / 1.5 L = 96.88 mg/L → 96.88 /\n(305.41 / 1000) ≈ 317.2 μM\nFor a 40 kg adolescent:\nLow end: 1.756 mg/kg/day × 40 kg = 70.24 mg/day → 70.24 mg / 1.5 L = 46.827 mg/L → 46.827 /\n(305.41 / 1000) ≈ 153.3 μM\nHigh end: 2.076 mg/kg/day × 40 kg = 83.04 mg/day → 83.04 mg / 1.5 L = 55.36 mg/L → 55.36 / (305.41\n/ 1000) ≈ 181.3 μM\nTo determine the average intestinal capsaicin concentration across these scenarios, we calculated the\nmean of the four values:\nMean = (268.3 + 317.2 + 153.3 + 181.3) / 4 = 920.1 / 4 ≈ 230.0 μM\nCompared to 200 μM, the estimated intestinal concentrations from Jiangxi Province’s capsaicin\nintake, with a mean value of 230 μM, indicate that the 200 μM concentration used in our experiments\neffectively simulates the intestinal stimulation level experienced in high-capsaicin diet regions.\n3. Mechanistic explanation: We appreciate your guidance and have revised the text according to your\nsuggestions.\nIn the revised manuscript, we have expanded the mechanistic details in the Discussion sections (lines\n399–415) to provide a more robust explanation. The updated text now reads: \"High-throughput mRNA\nsequencing of chondrocytes overexpressing miR-17-3p revealed significant dysregulation of the ERK\nand PI3K signaling pathways. Subsequent experiments showed that exosomal miR-17-3p overexpression\nsuppresses SOS1 expression, reducing RAS activation and phosphorylation of ERK, PI3K, and AKT\nproteins. The MEK/ERK and PI3K/AKT pathways synergistically regulate growth plate function, with\nMEK/ERK driving chondrocyte proliferation in the proliferative zone and PI3K/AKT promoting\nterminal differentiation and matrix secretion in the hypertrophic zone.\nIn situ hybridization confirmed elevated miR-17-3p expression in the proliferative and hypertrophic\nzones of the growth plate, underscoring its critical role in ISS pathogenesis. In the proliferative zone,\nmiR-17-3p targets the ZNF148/SOS1 axis, suppressing the MEK/ERK pathway and inducing cell cycle\narrest, thereby reducing the chondrocyte pool essential for longitudinal bone growth. In the hypertrophic\nzone, miR-17-3p inhibits the PI3K/AKT pathway, impairing hypertrophic differentiation and bone\nformation, as evidenced by decreased expression of key markers such as COL10A1, RUNX2, OPN, and\nOCN. These dual effects result in diminished growth plate thickness and compromised bone formation,\nas observed in our capsaicin-fed rat model. Consequently, miR-17-3p-mediated suppression of the\nRAS/ERK and PI3K/AKT pathways drives the short-stature phenotype in rats, highlighting its pivotal\nrole in ISS pathogenesis.\"\nReviewer’s Comment: The authors report having developed a capsaicin dietary model. Though, the\ndevelopment and validation of the model are not described. Line 749 refers to 0.01% of capsaicin-\nsupplemented diet. It is unclear how this dose was selected and how it represents physiologically relevant\nintakes (including in regions of the world with high consumption). Was it adjusted by rat body weight to\nreflect intakes in humans? The source of capsaicin, and diets composition should be provided to allow\nreproducibility. The response to previous reviewers speaks of various doses of capsaicin having been\ntested, though the process should be described in the methods section of this paper. Also, the\nmethodology should indicate that body weight and food intake were assessed, and data shown. This is a\nnutritional study, and information on the diet composition and\nform, source, mode of administration, housing (how many animals per cage? Sex?), sample size must be provided. Was the\ncapsaicin diet palatable for the mice? Was the capsaicin content of the diet confirmed? How does the amount of\ncapsaicin used in this study relates to studies in the literature, where this compound has beneficial effects\nincluding in conditions associated with altered intestinal barrier function? Finally, because the age of the\nrats is not provided, it is difficult to relate the dietary study and intestinal outcomes to the experiments\non miR-17-3p inhibition of growth plate ossification.\nAuthors’ Response: We sincerely appreciate the reviewer’s detailed and insightful feedback, which has\nsignificantly strengthened our manuscript. We have carefully addressed each concern regarding the\ncapsaicin dietary model, its development, validation, and physiological relevance, as well as the\nassociated methodological details. Below, we respond to each point under specific subheadings, outlining\nthe revisions made in the manuscript to ensure clarity, reproducibility, and alignment with our findings.\n1. Development and Validation of the Capsaicin Dietary Model, and Selection of the 0.01% Capsaicin\nDose and Physiological Relevance\nThe capsaicin dietary model was developed based on epidemiological observations from high-incidence\nregions of idiopathic short stature (ISS) in China, such as Jiangxi, Hunan, Chongqing, and Guizhou\nprovinces, where capsaicin-rich diets are common. As reported by Zou et al. (PMID: 34712685) in their\noverview of pepper consumption in China, statistical data from the National Industrial System of\nSpecialty Vegetables (NISSV) revealed the distribution of annual pepper consumption across provinces,\nincluding total and per capita consumption. Hunan Province has the highest total annual pepper consumption\nat 7.57 million tons, followed by Jiangxi, Guizhou, Chongqing, Yunnan, and Hubei provinces. With a\ncombined population of approximately 286,764,000 across these six provinces, the\nareas in the upper and middle reaches of the Yangtze River—classified as extreme-spicy areas by the\nauthors based on pepper consumption levels—have the highest per capita pepper consumption (75 kg\nannually), including Hunan Province (111.57 kg), Guizhou (98.5 kg), Jiangxi (97.6 kg), and Chongqing\n(95.6 kg).\nJia et al. (2015), in their study titled \"Determination of Capsaicinoids and Classification of\nPungency Level in Sichuan Cuisine Dishes\" (published in a Chinese journal with an English abstract,\nand the full text provided as an attachment), analyzed 123 Sichuan cuisine dishes using high-performance\nliquid chromatography (HPLC). They classified the dishes into five grades based on capsaicinoid content,\n6\nwith grade 5 (highest spiciness) averaging 0.2076 g/kg and grade 4 averaging 0.1756 g/kg. Jiangxi\nresidents' diets are predominantly composed of grade 4 and 5 foods.\nWe measured the capsaicin concentrations in six common dishes from Jiangxi province; all sourced\nfrom local restaurants. The capsaicin concentrations were 0.3659, 0.0025, 0.6277, 0.0640, 0.0547, and\n0.1935 g/kg, with a mean of 0.2181 ± 0.2400 g/kg. These data are comparable to the findings of Jia et\nal., who reported an average capsaicin concentrations of 0.2076 g/kg for grade 5 dishes and 0.1756 g/kg\nfor grade 4 dishes.\nAssuming a 70 kg adult consumes 700 g of pepper-containing dishes daily and a 40 kg adolescent\nchild consumes 400 g daily, with the dishes being grade 4 or 5 spiciness levels, the estimated capsaicin\nintake is as follows:\nFor adults (70 kg):\nGrade 4 (0.1756 g/kg): Daily capsaicin intake = 700 g × 0.1756 mg/g = 122.92 mg, or 1.756 mg/kg/day.\nGrade 5 (0.2076 g/kg): Daily capsaicin intake = 700 g × 0.2076 mg/g = 145.32 mg, or 2.076 mg/kg/day.\nFor adolescent children (40 kg):\nGrade 4 (0.1756 g/kg): Daily capsaicin intake = 400 g × 0.1756 mg/g = 70.24 mg, or 1.756 mg/kg/day.\nGrade 5 (0.2076 g/kg): Daily capsaicin intake = 400 g × 0.2076 mg/g = 83.04 mg, or 2.076 mg/kg/day.\nIn our experiment, to evaluate the impact of a capsaicin diet on growth and development, we utilized\n4-week-old SD rats to simulate children in their growth development phase. These 4-week-old SD rats,\nwith an average weight of approximately 70 g, consumed 7.5 g/day of a 0.01% capsaicin-supplemented\ndiet (we confirmed the capsaicin concentration in the rat feed using high-performance liquid\nchromatography [HPLC]), resulting in a daily capsaicin intake of 0.75 mg, or 10.71 mg/kg/day. The\nhuman equivalent dose (HED) was calculated using the body surface area normalization method (FDA,\n2005) with a rat-to-human conversion factor of approximately 6.2, yielding an HED of approximately\n1.73 mg/kg/day. This value closely aligns with the estimated capsaicin intake in Jiangxi Province, ranging\nfrom 1.756 to 2.076 mg/kg/day for both adults and children based on annual consumption data,\neffectively simulating the high-capsaicin diet prevalent in our ISS high-incidence area. Additionally,\nMartínez-Aceviz et al. (2023, PMID: 37998493) analyzed capsaicin consumption in Tijuana, Baja\nfor adults. For a 70 kg adult, this equates to ~2.178 mg/kg/day, which is similar to our estimates for\nJiangxi residents, supporting that our 0.01% dose represents physiologically relevant intakes in high-\nconsumption regions worldwide.\n2. Source of Capsaicin and Diet Composition\nThe diet was prepared by mixing capsaicin uniformly into a standard rodent chow (AIN-93G\nformulation, purchased from Ke'ao Synergy (Tianjin) Feed Co., Ltd., Tianjin, China) at a concentration\nof 0.01% (w/w). The apsaicin-supplemented AIN-93 diet composition (per kg) is as follows for\nreproducibility (Lines 847-854, marked in blue):\nSupplemental Table S4\nDiet composition Proportion(%)\n3. Animal Grouping, Administration, Housing, Sex, and Sample Size, Body weight, and food intake\nfor Capsaicin Diet Simulation\nThree pregnant rats at 14 days of gestation were purchased and housed individually in three cages\n8\nwith a standard diet for one week until delivery. Each pregnant rat produced 4 male and 4 female pups,\nyielding a total of 24 offspring. At three weeks post-birth, 4 male and 4 female pups from each litter were\nselected and housed individually, followed by one week on a capsaicin-free diet. For group allocation,\nfrom each litter, 2 males and 2 females were randomly assigned to the capsaicin-supplemented diet group\nand the remaining 2 males and 2 females were assigned to the control group. Beginning in the fourth\nweek, the 24 rats were distributed into eight cages for further rearing. Four cages, each containing three\nrats (12 rats in total: 6 males and 6 females), were maintained on a capsaicin-free diet, while the other\nfour cages (also 12 rats in total: 6 males and 6 females) were switched to a capsaicin-supplemented diet.\nThus, both the control and capsaicin groups consisted of 6 males and 6 females (lines 831-854, marked\nin blue).\nFood intake, body weight, and height were measured every two days over 28 days by calculating\nthe difference between the amount of food provided and the remaining food. The data are presented in\nthe Figure where food intake is reported as the mean two-day consumption per rat, and body weight and\nheight are presented as the average values per rat every two days.\nAnalysis of the capsaicin (0.01% supplemented) and control (capsaicin-free) diet groups revealed\nthat during the first four days, the average daily food intake in the capsaicin group was slightly lower\nthan that in the control group. This reduced intake in the capsaicin group during the initial four days is\nlikely attributable to a short-term adaptation to the transition from a capsaicin-free to a capsaicin-\nsupplemented diet. From day six onward, the daily food intake of the capsaicin group showed no\nstatistically significant difference compared to the control group, indicating adequate palatability of the\ncapsaicin diet. However, by the end of the 28-day experiment, the capsaicin group exhibited significantly\nlower body weight and height compared to the control group. The weight-to-length ratio (WLR),\ncalculated as WLR = weight (g) ÷ length (cm), was 6.29 g/cm for the capsaicin group and 6.03 g/cm for\nthe control group, with no statistically significant difference between the groups. These findings suggest\nthat the reduced stature in the capsaicin group resulted from the 0.01% capsaicin diet inhibiting growth\nand development, rather than reduced food intake due to poor diet palatability (Lines 252-265, marked\nin blue).\nSupplementary Figure 25\ntemperature of 22 ± 2°C, relative humidity of 50-60%, and a 12-hour light/12-hour dark cycle to mimic\nnatural diurnal rhythms. At the end of the 8-week experimental period, the rats were euthanized using a\nstandard method involving CO asphyxiation, in accordance with institutional animal care guidelines.\ndevelopment as well as gut inflammation-related parameters. (Lines 831-865, marked in blue)\n4. Selection of Capsaicin Concentrations and Comparison to Literature\nWhile some studies report that capsaicin reduces microbial growth, alleviating gut inflammation\nand diarrhea (PMID: 35777913, PMID: 36155114), its gastrointestinal effects are dose-dependent. For\ninstance, Azimirad et al. (PMID: 36155114) demonstrated that capsaicin at 20 and 50 µM downregulated\npro-inflammatory markers (NF-κB, TNF-α) and apoptosis-related genes (e.g., BCL-2) in HT-29 cells\nexposed to Clostridioides difficile toxins, supporting its anti-inflammatory and anti-apoptotic properties.\nIn contrast, Xiang et al. (PMID: 35267319) found that low-dose capsaicin (40 mg/kg body weight in\nmice) caused no adverse gastrointestinal effects, whereas higher doses (60 and 80 mg/kg) induced\nsignificant damage, including inflammation, reduced villus/crypt ratios, and tissue destruction in the\njejunum, ileum, and colon. These findings align with our in vitro observations using HIEC-6 cells, where\nlow concentrations (25, 50, and 75 µM) promoted intestinal epithelial cell proliferation and inhibited\napoptosis, indicating a protective role, while higher concentrations (100, 150, and 200 µM) suppressed\nproliferation and increased apoptosis.” (Lines 430-440, marked in blue)\nThe capsaicin intake in Jiangxi Province for adolescents and adults is 1.756 to 2.076 mg/kg/day. To\nestimate the potential intestinal concentration from this intake, we used the following calculation formula:\nConcentration (μM) = [Daily capsaicin intake (mg) / Intestinal volume (L)] / (Molecular weight of\ncapsaicin / 1000)\nDaily capsaicin intake (mg) = Daily dose (mg/kg/day) × Body weight (kg)\nIntestinal volume is assumed to be 1.5 L (approximate small intestinal fluid volume)\nMolecular weight of capsaicin = 305.41 g/mol\nFor a 70 kg adult:\nLow end: 1.756 mg/kg/day × 70 kg = 122.92 mg/day → 122.92 mg / 1.5 L = 81.947 mg/L → 81.947 /\n(305.41 / 1000) ≈ 268.3 μM\nHigh end: 2.076 mg/kg/day × 70 kg = 145.32 mg/day → 145.32 mg / 1.5 L = 96.88 mg/L → 96.88 /\n(305.41 / 1000) ≈ 317.2 μM\nFor a 40 kg adolescent:\nLow end: 1.756 mg/kg/day × 40 kg = 70.24 mg/day → 70.24 mg / 1.5 L = 46.827 mg/L → 46.827 /\n(305.41 / 1000) ≈ 153.3 μM\nHigh end: 2.076 mg/kg/day × 40 kg = 83.04 mg/day → 83.04 mg / 1.5 L = 55.36 mg/L → 55.36 / (305.41\n/ 1000) ≈ 181.3 μM\nTo determine the average intestinal capsaicin concentration across these scenarios, we calculated the\nmean of the four values:\nMean = (268.3 + 317.2 + 153.3 + 181.3) / 4 = 920.1 / 4 ≈ 230.0 μM\nintake, with a mean value of 230 μM, indicate that the 200 μM concentration used in our experiments\neffectively simulates the intestinal stimulation level of capsaicin in high-capsaicin diet regions.\nWhile some studies report that capsaicin reduces microbial growth, alleviating gut inflammation\nand diarrhea, its gastrointestinal effects are dose-dependent. Our in vitro experiments showed that at\nhigher concentrations (200 μM) suppresses proliferation of small intestinal epithelial cells and increases\napoptosis. Simultaneously, in SD rats fed a 0.01% capsaicin diet, we observed intestinal\nmicroinflammation, activation of NF-κB, and elevated plasma exosomal miR-17-3p expression, which\ninhibited proliferation and ossification in growth plate chondrocytes. To assess whether daily diets in\nJiangxi Province could induce intestinal microinflammation, and considering the need for repeated blood\nsampling, we selected healthy adults to examine inflammatory cytokines in fecal excretions and analyze\nmiR-17-3p expression in plasma exosomes and feces. We found that a capsaicin-rich diet significantly\nincreased plasma exosomal miR-17-3p levels, which correlated with elevated intestinal inflammation\nmarkers, such as IL-1β and TNF-α. Cessation of the diet led to a decrease in miR-17-3p expression, with\nlevels rebounding upon reintroduction. These findings suggest that, in specific populations, chronic\nconsumption of spicy foods may shift from a protective role to a risk factor for subtle inflammatory\nresponses that influence growth plate function.\nThe data for week 1 represent measurements taken during a no-capsaicin diet period. Week 2 data were collected after one week\nof a capsaicin-rich diet typical of the Jiangxi region (daily diet). Week 3 data correspond to measurements following a return to\nthe no-capsaicin diet, while week 4 data were obtained after resuming the capsaicin-rich diet.\nReviewer’s Comment: The response to previous reviewers speaks of various doses of capsaicin having\nbeen tested, though the process should be described in the methods section of this paper.\nAuthors’ Response: In the revised manuscript, we have added detailed descriptions in the Methods\nsection regarding the testing of various capsaicin doses (Lines 618-623, marked in blue). “Natural\ncapsaicin (404-86-4) was purchased from Weilkeqi Co., Ltd. It was dissolved in PBS to prepare a\ncapsaicin solution with an initial concentration of 1 mg/ml. The solution was diluted with complete\nmedium to obtain concentrations of 0 μM, 25 μM, 50 μM, 75 μM, 100 μM, 150 μM, and 200 μM for the\nintestinal cell（HIEC-6 and NCM460）capsaicin stimulation culture experiments. After a culture period\nof 0 h, 24 h, 48 h, and 72 h, these cells were used in further experiments.”\nReviewer’s Comment: Critical details are missing or are unclear in the methods section; examples\nare provided below.\nThis is a dense and impressive manuscript, and the authors should be commended for undertaking such\na comprehensive series of experiments. The data presented are valuable, and the overall effort is clear.\nThat said, some sections are challenging to follow. The manuscript could be improved by using more\nspecific and consistent terminology, which would greatly enhance clarity. At times, it was necessary to\nrefer repeatedly to the Methods section to determine key details, for example, the specific cell line or\nintestinal tissue used, and occasionally, this information was not clearly stated even there.\nProviding these details more explicitly in the main text and ensuring they are clearly outlined in the Methods would\nstrengthen the manuscript and improve its accessibility to readers.\nAuthors’ Response: In response to your concerns about the manuscript's readability and the need for\nmore specific and consistent terminology, we have undertaken a thorough revision. Throughout the\nrevised manuscript, we have standardized the terminology used to ensure uniformity and precision,\naddressing any ambiguity that may have arisen previously. Additionally, to improve accessibility, we\nhave explicitly detailed key experimental components in the main text. Specifically, we have clarified\nthe use of the human small intestinal epithelial cell line (HIEC-6, Otwo Biotech, Cat. No. HTX1978) and\nthe normal colon mucosal epithelial cell line (NCM460, Otwo Biotech, Cat. No. HTX1841), as well as\nSD rat small intestinal and colonic tissues for histological analysis. These details are now explicitly\npresented where relevant and are clearly described in the Methods section (Lines 599–602 and lines 855-\n865, marked in blue) to ensure a comprehensive and easily accessible reference for readers.\nReviewer’s Comment: Intestinal cell line experiments: these are hard to assess without knowing which\nintestinal cell line was used and the culturing conditions. Also, there seems to be a confusion between\nviability and proliferation (for ex, see Supplementary Fig 15); these are not the same. Did the authors\nassess viability (ie using Trypan Blue exclusion or MTT) and used viable cells for their experiments?\nPanel A shows viability % but the legend speaks of proliferation, please clarify. The fact that cell\nviability was at most about 2.5% for 25 μM is concerning. This is important for this part of the manuscript because\nthe authors discuss that capsaicin effects (beneficial vs detrimental) depend on low vs high dosages. It is\nthus important to determine if the 200 μM dosage is a usable high dosage or if it is ”toxic” to the cells.\nExperiments are hard to assess without details on which intestinal cell line was used, culture conditions,\nand measures of viability and proliferation (Suppl Fig 15). Similarly to the in vivo experiment, is 200\nμM a physiologically relevant high concentration of capsaicin? Can it be reached in the diet? \nFor example, a calculation of the amount of chili peppers that one should eat (on average) to reach this amount should be\nprovided.\nAuthors’ Response: We appreciate your feedback regarding the clarity of the cell line used, culturing\nconditions, and the distinction between viability and proliferation, as well as the physiological relevance\nof the 200 μM dose. We have revised the manuscript to address these points and improve readability.\nThe experiments were conducted using the human small intestinal epithelial cell line HIEC-6 (Otwo\nBiotech, Cat No. HTX1978) and human healthy colon mucosal epithelial cell line NCM460 (Otwo\nBiotech, Cat No. HTX1841). Cells were cultured in DMEM supplemented with 10% FBS, 1%\npenicillin-streptomycin, and maintained at 37°C in a 5% CO humidified incubator (Lines 599–602,\n2\nmarked in blue). For the proliferation assays, we utilized the CCK-8 kit to assess cell proliferation via\nOD values at 450 nm, rather than viability (Lines 625–628, marked in blue). We acknowledge the error\nin our previous calculation of viability percentages; this has been corrected in the revised manuscript,\nwhere we now report the data as OD values to accurately reflect proliferation. We did assess viability\nusing Trypan Blue exclusion prior to experiments, ensuring only viable cells (>95% viability) were used\n(Lines 614–617, marked in blue).\nRegarding the physiological relevance of 200 μM, this concentration is indeed achievable in high-\ncapsaicin diets. The capsaicin intake in Jiangxi Province for adolescents and adults is 1.756 to 2.076\nmg/kg/day. Using the formula for estimated intestinal concentration: Concentration (μM) = [Daily\ncapsaicin intake (mg) / Intestinal volume (L)] / (Molecular weight of capsaicin / 1000) Where daily\ncapsaicin intake (mg) = daily dose (mg/kg/day) × body weight (kg), intestinal volume = 1.5 L, and\nmolecular weight = 305.41 g/mol. For a 70 kg adult: 122.92–145.32 mg/day yields 268.3–317.2 μM. For\na 40 kg adolescent: 70.24–83.04 mg/day yields 153.3–181.3 μM. The mean is 230 μM, indicating 200\nμM simulates high-intake regions, rather than representing a toxic dose.\nReviewer’s Comment: Specific comments.\nAbstract and Introduction\n1. Abstract line 40: It is unclear why it is necessary to specify: “different from inflammatory bowel disease”. IBD does not always\ncome with diarrhea. The authors did not compare the inflammatory profile induced by capsaicin with the IBD inflammatory\nphenotype, so this sentence should be revised.\n2. Line 41: intestinal secretions: Are the authors referring to intestinal content? In which intestinal region?\nIt would be best to just specify it.\n3. Introduction: The authors could consider making this section more concise without describing all the\ndetails of their findings already in this section.\n4. Line 75: At which age and sex was this height gain assessed?\n5. Line 127-128: dietary patterns are more than one ingredient, please rephrase: “frequent consumption\nof a capsaicin-rich diet, mirroring dietary patterns….”; also, the authors could clarify what is meant by\n“frequent” and “capsaicin-rich”; It would be helpful to know the amount of capsaicin in this diet to better\nunderstand the dosage used in the animal model.\nReviewer’s Comment: 1. Abstract line 40: It is unclear why it is necessary to specify: “different from inflammatory bowel disease”. IBD does not always\ncome with diarrhea. The authors did not compare the inflammatory profile induced by capsaicin with the IBD inflammatory\nphenotype, so this sentence should be revised.\nAuthors’ Response: We appreciate your valuable comment. We have revised the sentence accordingly.\nThe updated version now reads:\n“Our findings reveal that the capsaicin diet induces gut microinflammation, characterized by the absence\nof diarrhea and changes in stool shape, leading to increased miR-17-3p in both gut and plasma-derived\nexosomes (Lines 39-41).”\nReviewer’s Comment:\n2. Line 41: intestinal secretions: Are the authors referring to intestinal content? In which intestinal region?\nIt would be best to just specify it.\nAuthors’ Response: Thank you for your insightful comment regarding the term \"intestinal secretions.\"\nWe appreciate your suggestion for clarification. In our study, \"intestinal secretions\" refers specifically to\nfecal matter, which was collected and analyzed to assess miR-17-3p expression and inflammatory\ncytokine levels. This clarification has been incorporated into the revised manuscript to ensure precision.\nReviewer’s Comment: 3. Introduction: The authors could consider making this section more concise\nwithout describing all the details of their findings already in this section.\nAuthors’ Response: Thank you for your valuable suggestion regarding the Introduction section and your\nrecommendation to make it more concise by avoiding detailed descriptions of our findings. We appreciate\nyour guidance and have carefully considered your feedback. In response, we have made minor\nmodifications to the findings section of the Introduction to align with your advice while preserving the\ncore narrative.\nThe revised findings section, now structured into four concise paragraphs located at the end of the\nIntroduction (Lines 118-147, marked in blue), enhances readability and guides the reader through the\nresearch's main themes. The first theme highlights that plasma-derived exosomes from ISS children\nmarkedly inhibit chondrocyte proliferation and bone formation, an effect linked to miR-17-3p\noverexpression. The second theme outlines our epidemiological analysis and experimental observations,\nnoting that a capsaicin-rich diet, mimicking high-consumption regions, induces mild intestinal\ninflammation in Sprague-Dawley rats, elevates plasma exosomal miR-17-3p, and contributes to short\nstature, proposing a dietary-gut-growth axis. The third theme introduces our innovative therapeutic\nstrategy, utilizing growth plate-targeting exosomes with siRNA and localized growth hormone\nadministration to improve ISS outcomes.\nGiven the multifaceted nature of our study, which encompasses numerous results, we retained these\nthree paragraphs in the Introduction to provide a clear roadmap of the research mainline and facilitate\nunderstanding of the data presentation logic in the Results section. We believe this approach, with\nminimal content reduction, enhances accessibility for readers by offering a structured overview of our\nkey findings. These revisions are reflected in the updated Introduction (Lines 118-147, marked in blue).\nReviewer’s Comment: 4. Line 75: At which age and sex was this height gain assessed?\nAuthors’ Response: Thank you for your careful review. We agree that this detail is important for context\nand have revised the manuscript accordingly to include the relevant information. The height gain of 9.55\n± 0.13 cm in the first year was assessed in 200 Chinese ISS children who received recombinant rhGH\ntreatment, with an average age of 9.03 ± 0.21 years at the start of therapy. Among these children, 137\nwere boys (68.5%) and 63 were girls (31.5%).\nRevised Text (Lines 75-77):\nHou et al. reported an average height gain of 9.55 ± 0.13 cm after one year of treatment in 200 Chinese\nchildren with ISS (average age 9.03 ± 0.21 years; 68.5% boys, 31.5% girls) 10, whereas Yoo et al. noted\nan increase of 10.68 ± 1.95 cm.\nReviewer’s Comment: 5. Line 127-128: dietary patterns are more than one ingredient, please rephrase:\n“frequent consumption of a capsaicin-rich diet, mirroring dietary patterns….”; also, the authors could\nclarify what is meant by “frequent” and “capsaicin-rich”; It would be helpful to know the amount of\ncapsaicin in this diet to better understand the dosage used in the animal model.\nAuthors’ Response: Thank you for your valuable suggestion. In the revised manuscript, we have\nrephrased the sentence at lines 133–135. “In subsequent experiments, a capsaicin-enriched diet—\ndesigned as a model to simulate the chili consumption levels observed in these high-prevalence areas—\ninduced mild intestinal inflammation in Sprague–Dawley rats.”\nWe have also clarified the context for “capsaicin-rich” by providing relevant epidemiological data\nin the Introduction (lines 123–128). Specifically, Zou et al. (2021, PMCID: PMC8545790) reported that\nHunan Province has the highest total annual pepper consumption (7.57 million tons), followed by Jiangxi,\nGuizhou, Chongqing, Yunnan, and Hubei, with a combined population of approximately 286.8 million.\nBased on per capita pepper consumption, the upper and middle reaches of the Yangtze River were\nclassified as “extreme-spicy” areas, including Hunan (111.57 kg/person/year), Guizhou (98.5 kg), Jiangxi\n(97.6 kg), and Chongqing (95.6 kg).\nFurthermore, the “capsaicin-enriched” diet used in our animal model contained 0.01% capsaicin by\nweight, which was chosen to approximate the estimated human intake levels in high-consumption\nprovinces such as Jiangxi. This information has been added to the Methods section for clarity (Lines 847-\n854, marked in blue).\nReviewer’s Comment:\nMethods:\n1. The term “pairs” used to describe ISS patients and age- and gender-matched controls (line 519, line\n530) is misleading, as those children are unrelated. Furthermore, the gender distribution between the two\ngroups is different (NC: 57 males and 49 females, ISS: 52 males, 54 females), which indicate that not all\n“pairs” can be gender matched. Please revise accordingly.\n2. Fecal sample collection: The methods used to collect and preserve fecal samples from ISS and control\nchildren are not described, please include this information in the clinical sample characteristics section.\n3. Inconsistent microRNA methodology: The methods regarding microRNA profiling lack clarity. Line\n535 mentions miRNA High-Throughput Sequencing, while line 531 mentions miRNA microarray. Please\nclarify which method was used and include appropriate method details (sequencing platform used, library\npreparation protocol, sequencing depth; or microarray platform, miRNA labelling and amplification kit,\nnumber of miRNA analyzed).\n4. 615-620: this paragraph is unclear regarding whether the experiment was conducted in immature mice,\nor in rats.\n5. microRNA RT-qPCR: Line 623 does not specify which internal controls were used for 2-DeltaCt\nmethod of miRNA RT-qPCR for plasma and stool samples. Please include this information.\n6. The description of the in vivo models is incomplete, please ensure that the ARRIVE guidelines are\nfollowed; for example:\n- Rats strains and age are missing in a few sections of the in vivo methods description (line 682, line 692,\nline 748).\n- Grouping Figure: the supplementary Figure 27 and 28 (Grouping of experimental animals) seem to\nhave been mislabeled or switched with Supplementary Figure 24 or 25 (overexpression vector mapping)\nas the legend does not match the figure content.\n- Please provide justifications for the sample sizes used in the animal studies. Is n=3 sufficient?\n- Please clarify which tissues were studied in the various experiments; terms like “intestinal tissues” are\ntoo vague, please specify which regions.\n7. More details are needed regarding the diets used in the in vivo studies. For example, what constitutes\na ‘mixed diet’ (line 683) and a ‘regular diet’ (line 748). Is this the AIN-93 diet? How was the capsaicin-\nsupplemented diet formulated, how does it differ from the group A ‘regular diet’, and how does the\ncapsaicin dosage relate to human consumption?\n8. The explant culture of rat femurs appears to have used samples collected at one day postpartum (line\n708), indicating neonatal rather than fetal samples. Please revise the terminology accordingly in the\nmanuscript.\n9. Histopathological analysis of the intestine: please specify which part of the small intestine or large\nintestine were used to assess intestinal inflammation in rats. The method section mentions colon (line\n754) while the figures refer to the small intestine (line 1280). Please add details on the number of\nmeasurements conducted and samples size used to assess villus height and intestinal wall thickness.\n10. Line 757: “Cells were treated…”: Please clarify which cells and explain how they were cultured.\nHow many times were the cell culture experiments repeated? And were they done in triplicates or else?\n11. Line 761-762: was the quality of the RNA assessed?\n12. Line 763-767. Please specify use of parametric and non-parametric tests for data analysis based on\ndata distribution.\nReviewer’s Comment: 1. The term “pairs” used to describe ISS patients and age- and gender-matched\ncontrols (line 519, line 530) is misleading, as those children are unrelated. Furthermore, the gender\ndistribution between the two groups is different (NC: 57 males and 49 females, ISS: 52 males, 54\nfemales), which indicate that not all “pairs” can be gender matched. Please revise accordingly.\nAuthors’ Response: Thank you for your careful review and for pointing out the potential confusion with\nthe term “pairs” in describing the ISS patients and age- and gender-matched controls. We have revised\nthe text accordingly by removing the term “pairs”. The revised sentence (Lines 551-552, marked in blue)\nnow reads: “106 ISS patients and age- and gender-matched controls were recruited from the Second\nAffiliated Hospital of Nanchang University, China.\nReviewer’s Comment: 2. Fecal sample collection: The methods used to collect and preserve fecal\nsamples from ISS and control children are not described, please include this information in the clinical\nsample characteristics section.\nAuthors’ Response: We appreciate your suggestion to include this information and have revised the\nmanuscript accordingly to enhance transparency. Below, we provide the details on the methods section\n(Lines 567–575, marked in blue).\nFecal Sample Collection and Storage for miRNA and ELISA Analyses\nFresh fecal samples (2.5 ± 0.5 g) were collected within 30 minutes of defecation using sterile, single-use\nplastic spoons and transferred into sterile polypropylene containers to prevent contamination. Samples\nwere immediately divided into two aliquots for downstream analyses. For miRNA analysis, one aliquot\n(~1.5 g) was mixed with RNAlater stabilization solution (1:1 w/v, QIAGEN, Cat No. 76106) and stored\nat 4°C for 24 hours to ensure RNA stabilization, then transferred to -80°C for long-term storage. For\nELISA analysis of inflammatory markers (e.g., IL-1β, TNF-α), a second aliquot (~1 g) was snap-frozen\nat -80°C without buffer to avoid interference with protein assays. All sample handling was performed\nusing gloves under sterile conditions to minimize contamination.\nReviewer’s Comment: 3. Inconsistent microRNA methodology: The methods regarding microRNA\nprofiling lack clarity. Line 535 mentions miRNA High-Throughput Sequencing, while line 531 mentions\nmiRNA microarray. Please clarify which method was used and include appropriate method details\n(sequencing platform used, library preparation protocol, sequencing depth; or microarray platform,\nmiRNA labelling and amplification kit, number of miRNA analyzed).\nAuthors’ Response:\nWe thank the reviewer for highlighting the inconsistency in our description of microRNA (miRNA)\nprofiling methods. We apologize for the confusion caused by mentioning both miRNA High-Throughput\nSequencing (line 535) and miRNA microarray (line 531). To clarify, our study exclusively utilized\nmiRNA High-Throughput Sequencing, and the reference to microarray was an error in the original\nmanuscript. We have corrected this in the revised manuscript by removing the mention of microarray in\nline 531 and ensuring all references consistently describe the sequencing method.\nThe miRNA sequencing and data analysis were performed by Shanghai Biotechnology Corporation\n(Shanghai, China). Briefly, total RNAs were isolated from each sample using the QIAamp RNeasy\nSerum/Plasma Midi Kit (Cat No. 77044, Qiagen). Subsequently, the sequencing library was constructed\nwith the QIAseq miRNA Library Kit (Cat No. 1103679, Qiagen) and sequenced on the Illumina HiSeq\nplatform (California, USA). Differentially expressed miRNAs were identified with P < 0.05 and\n|log2(FC)| ≥ 1.5. These methodological details have been added to the Methods section (Lines 557-582,\nmarked in blue) in the revised manuscript.\nReviewer’s Comment: 4. 615-620: this paragraph is unclear regarding whether the experiment was\nconducted in immature mice, or in rats.\nAuthors’ Response: We apologize for the confusion caused by the inconsistent terminology. All animal\nexperiments in this study were performed using Sprague–Dawley (SD) rats. The mention of \"mice\" in\nthe original text was a typographical error and has been corrected to \"SD rats\" (Lines 670, marked in\nblue).\nReviewer’s Comment: 5. microRNA RT-qPCR: Line 623 does not specify which internal controls were\nused for 2-DeltaCt method of miRNA RT-qPCR for plasma and stool samples. Please include this\ninformation.\nAuthors’ Response: We sincerely thank the reviewer for their valuable suggestion. We have addressed\nthis concern by updating the Methods section of the revised manuscript. The revised text, now\nincorporated in lines 676-685, provides detailed information as follows: “RT-qPCR quantified miRNA\nand mRNA expression in both cell and tissue samples, using U6 and GAPDH as internal controls. For\nmiRNA qRT-PCR of plasma-derived exosomes and fecal samples, U6 small nuclear RNA was used as\nthe endogenous reference for biological normalization, and its stability across sample types and between\nISS and control groups was confirmed using geNorm and NormFinder. An exogenous spike-in (cel-miR-\n39) was added during RNA extraction to monitor extraction and reverse transcription efficiency, thereby\ncontrolling for technical variability. Primer sequences are listed in Supplementary Table 2. Reactions\nwere performed in triplicate with three independent cell preparations. Relative expression levels were\ncalculated using the 2−ΔΔCt method, with internal controls adjusting for RNA quantity and transcription\nefficiency.”\nReviewer’s Comment: 6. The description of the in vivo models is incomplete, please ensure that the\nARRIVE guidelines are followed; for example:\n- Rats strains and age are missing in a few sections of the in vivo methods description (line 682, line 692,\nline 748).\n- Grouping Figure: the supplementary Figure 27 and 28 (Grouping of experimental animals) seem to\nhave been mislabeled or switched with Supplementary Figure 24 or 25 (overexpression vector mapping)\nas the legend does not match the figure content.\n- Please provide justifications for the sample sizes used in the animal studies. Is n=3 sufficient?\n- Please clarify which tissues were studied in the various experiments; terms like “intestinal tissues” are\ntoo vague, please specify which regions.\nAuthors’ Response: We appreciate your guidance and have revised the manuscript to address each of\nyour concerns thoroughly.\nRats Strains and Age: Thank you for your detailed comments regarding the completeness of in vivo\nmodel descriptions in our manuscript. We fully agree with the need to adhere to the ARRIVE guidelines\nand have revised the specified sections to supplement missing information on rat strains, ages, and other\nkey details. The revisions are as follows:\nPart I: In line 744-773, marked in blue (Animal feeding and OE-miR-17-3p-rats, original refers to line\n682):\nAll SD rats were housed individually in cages under standard conditions (22 ± 2°C, 50–60% relative\nhumidity, 12-h light/dark cycle) and fed a mixed diet (AIN-93, Beijing Keao Xieli Feed Company).\nEighteen pregnant rats at 14 days of gestation were purchased and housed individually in 18 cages with\na standard AIN-93 diet, maintained for one week until delivery. Each pregnant rat typically produced 10-\n14 pups, and after three weeks post-birth—when sex could be visually identified—pups underwent the\nfirst random allocation. To clarify whether miR-17-3p exerts biological effects through ZNF148/SOS1\nin vivo experiments, 4 male SD rats were randomly selected from each of the 6 litters and divided into 4\ngroups at random. They were housed in 4 cages, with 6 male SD rats per cage, under standard conditions\nand fed the AIN-93 diet. The miR-17-3p, ZNF148, and SOS1 plasmids (GV367-Ubi-MCS-SV40-EGFP-\nIRES-puromycin) were constructed by GENEchem Co., Ltd. In group A (n = 6), the 300 µL PBS was\ninjected into the tail vein. The miR-17-3p was overexpressed in 3 week rats via tail‐vein injection in\ngroups B to D. After the rats reached 4 weeks of age, these rats received injections via the tail vein every\n3 days, with Group B (n = 6) receiving 300μl of PBS, with Group C (n = 6) receiving 300μl of ZNF148\nplasmid, and Group D (n = 6) receiving 300μl of SOS1 plasmid (Supplementary Fig. 28a). To clarify the\nbiological role of ET-Exos from chondrocytes in in vivo experiments, 6 male SD rats were randomly\nselected from 6 litters and divided into 6 groups at random. They were housed in 6 cages, with 6 male\nSD rats per cage, under standard conditions and fed the AIN-93 diet. In group A (n = 6), the 300 µL PBS\nwas injected into the tail vein. The miR-17-3p was overexpressed in 3-week-old rats via tail‐vein\ninjection in groups B to E. After the rats reached 4 weeks of age, these rats received injections via the\ntail vein every 3 days, with Group B (n = 6) receiving 300μl of ET-Exos (si-miR-17-3p + GH), Group C\n(n = 6) receiving 300μl of Exos (si-miR-17-3p + GH), Group D (n = 6) receiving 300μl of PBS, Group\nE (n = 6) receiving 300μl of Exos (si-miR-17-3p), and Group F (n = 6) receiving 300μl of Exos (GH)\n(Supplementary Fig. 28b). Furthermore, we employed the same methods and protocols to evaluate the in\nvivo biological effects of ET exosomes derived from bone marrow mesenchymal stem cells\n(Supplementary Fig. 29a). The body weight and height of rats were recorded weekly, with measurements\ncontinuing for four weeks. At the end of the 8-week experimental period, the rats were euthanized using\na standard method involving CO asphyxiation, in accordance with institutional animal care guidelines.\nand weight analyses were conducted as single-blind assessments to minimize bias.\nPart II: In line 775-779, marked in blue (Safranin O and Fast Green staining and Calcein staining,\noriginal refers to 692 line):\nFor all samples in this research, one-third of the distal femur was resected and kept in 4% formaldehyde\nsolution. 4 µm thickness, serial sections were obtained for Safranin O/Fast Green staining and calcein\nstaining. The flow chart (Figure S10, Supporting Information) demonstrates the detailed grouping\ninformation. Details of Safranin O /Fast Green staining and calcein staining methods can be found in our\nprevious publication.\nPart III: In lines 831-846, marked in blue (Capsaicin Animal Model, original refers to 748 line):\nAll SD rats were housed individually in cages under standard conditions. Three pregnant rats at 14 days\nof gestation were purchased and housed individually in 3 cages with a standard diet, maintained for one\nweek until delivery. Each pregnant rat produced 4 male and 4 female pups, yielding a total of 24 offspring.\nAt three weeks post-birth, 4 male and 4 female pups from each litter were selected and housed\nindividually, followed by one week on a capsaicin-free diet. For group allocation, from each litter, 2\nmales and 2 females were randomly assigned to the capsaicin-supplemented diet group and the remaining\n2 males and 2 females were assigned to the control group. Beginning in the fourth week, the 24 rats were\ndistributed into eight cages for further rearing. Four cages, each containing three rats (12 rats in total: 6\nmales and 6 females), were maintained on a capsaicin-free diet, while the other four cages (also 12 rats\nin total: 6 males and 6 females) were switched to a capsaicin-supplemented diet. Thus, both the control\nand capsaicin groups consisted of 6 males and 6 females (lines 831-854, marked in blue).\nFood intake data were measured every two days, and body weight and height were recorded weekly,\nby calculating the difference between the amount of food provided and the remaining food, with\nmeasurements continuing for four weeks. At the end of the 8-week experimental period, the rats were\neuthanized using a standard method involving CO2 asphyxiation, in accordance with institutional animal\ncare guidelines. Knee joint and intestinal tissue samples were then collected for analysis of cartilage\ngrowth and development as well as gut inflammation-related parameters. Height, weight, food intake,\nand intestinal tissue inflammation analyses were conducted as single-blind assessments to minimize bias.\nGrouping Figure: We acknowledge the mislabeling issue with Supplementary Figures 27 and 28\n(Grouping of experimental animals), which appeared to be switched with Supplementary Figures 24 or\n25 (overexpression vector mapping) due to mismatched legends. This error has been corrected in the\nrevised manuscript, with updated figure legends and content now properly aligned to reflect the\nexperimental grouping accurately.\nJustification for Sample Sizes: Regarding your query about the sufficiency of sample sizes in the animal\nstudies, we have provided justifications in the revised manuscript. For the capsaicin diet-induced short\nstature model, each group consists of n = 6 male and 6 female rats, deemed sufficient based on power\nanalysis to detect significant differences in growth parameters. For other in vivo experiments involving\nmiR-17-3p overexpression and exosome therapy to mitigate capsaicin-induced short stature, each group\nincludes n = 6 male rats. We utilized only male rats in these experiments for the following reasons: during\nthe capsaicin-induced short stature model, we observed no significant differences in outcomes between\nfemale and male rats, leading us to select males for subsequent studies to maintain consistency. The\nsample size of 6 males per group was determined by power analysis, ensuring adequate statistical power\nto detect changes in miR-17-3p expression and growth plate outcomes. These sample sizes are detailed\nin the revised figure legends and illustrated in the updated supplementary workflow diagrams depicting\nexperimental grouping.\nTissue Specification: We have clarified the specific regions of intestinal tissues studied to address the\nvagueness of the term “intestinal tissues.” In the revised manuscript, we specify that small intestinal\nsamples were collected at 10 cm and 40 cm from the pylorus, while colonic samples were taken at 5 cm\nand 10 cm from the anus. Each segment was 4 cm in length, with 1 cm used for hematoxylin and eosin\n(H&E) staining to assess mucosal stimulation and inflammation, and the remaining 3 cm utilized for\nWestern blot (WB) and reverse transcription-polymerase chain reaction (RT-PCR) analyses to evaluate\nmolecular changes. These details have been added to the Methods section (Lines 855-865, marked in\nblue) and are further illustrated in Supplementary Figures 14-17.\nReviewer’s Comment: 7. More details are needed regarding the diets used in the in vivo studies. For\nexample, what constitutes a ‘mixed diet’ (line 683) and a ‘regular diet’ (line 748). Is this the AIN-93 diet?\nHow was the capsaicin-supplemented diet formulated, how does it differ from the group A ‘regular diet’,\nand how does the capsaicin dosage relate to human consumption?\nAuthors’ Response: We appreciate your guidance in clarifying these aspects and have revised the\nmanuscript. The terms \"mixed diet\" (line 683) and \"regular diet\" (line 748) both refer to the standard\nAIN-93G rodent chow formulation, which serves as the base diet for our experiments. The \"regular diet\"\nis the unmodified AIN-93G chow, while the capsaicin-supplemented diet is the AIN-93G formulation\nwith 0.01% (w/w) capsaicin incorporated. The capsaicin was not added directly by our team; instead, we\ncommissioned the feed production company, Ke'ao Synergy (Tianjin) Feed Co., Ltd., Tianjin, China, to\ninclude it during the manufacturing process. To ensure accuracy, we verified the capsaicin concentration\nusing high-performance liquid chromatography (HPLC), confirming a consistent 0.01% (w/w) level with\na variation of less than 5% (Lines 846-853 and 879-885, marked in blue).\nRegarding the capsaicin dosage's relation to human consumption, our 0.01% diet results in\napproximately 10.71 mg/kg/day for 70 g SD rats, with a human equivalent dose (HED) of ~1.73\nmg/kg/day (calculated using the FDA body surface area normalization method with a rat-to-human\nconversion factor of ~6.2). As previously explained, this aligns closely with the estimated capsaicin\nintake in Jiangxi Province (1.756–2.076 mg/kg/day for adults and children), where diets predominantly\nconsist of grade 4 and 5 spicy foods (average capsaicinoid content 0.1756–0.2076 g/kg; Jia et al., 2015).\nThis makes the dosage physiologically relevant and comparable to high-consumption regions.\nWe have revised the Methods section (Lines 846-853, marked in blue) to explicitly define these\nterms, describe the formulation process with the feed company’s involvement, and confirm the capsaicin\nconcentration via HPLC, ensuring compliance with ARRIVE guidelines.\nReviewer’s Comment: 8. The explant culture of rat femurs appears to have used samples collected at\none day postpartum (line 708), indicating neonatal rather than fetal samples. Please revise the\nterminology accordingly in the manuscript.\nAuthors’ Response: We appreciate your feedback and agree that the description on line 708, which\nindicated samples collected at one day postpartum, was inaccurate and implied neonatal rather than fetal\nsamples. We have revised the manuscript to correct this terminology accordingly (Lines 788, 791, and\n805, marked in blue).\nReviewer’s Comment: 9. Histopathological analysis of the intestine: please specify which part of the\nsmall intestine or large intestine were used to assess intestinal inflammation in rats. The method section\nmentions colon (line 754) while the figures refer to the small intestine (line 1280). Please add details on\nthe number of measurements conducted and samples size used to assess villus height and intestinal wall\nthickness.\nAuthors’ Response: We appreciate your feedback and have revised the manuscript to include this\ninformation in the Methods section (Lines 855–865, marked in blue).\nIn our study, intestinal tissue samples were collected separately from the small intestine (at 10 cm\nand 40 cm from the pylorus) and colon (at 5 cm and 10 cm from the anus) in both the capsaicin group\nand control group, with each group consisting of rats (n=12 per group). For the small intestine, two\nsegments (10 cm and 40 cm from the pylorus) were analyzed, and for the colon, two segments (5 cm and\n10 cm from the anus) were examined. Each segment, measuring 1 cm in length, was fixed in 4%\nparaformaldehyde, embedded in paraffin, sectioned at 5 μm thickness, and stained with hematoxylin and\neosin (H&E) for histological analysis. For each rat, 2 sections per segment were prepared, resulting in 4\nsections per region (small intestine or colon) per rat. This yielded a total of 48 small intestine sections\nand 48 colon sections for the capsaicin group, and a similar number (48 small intestine sections and 48\ncolon sections) for the control group across the 12 rats (6 male and 6 female) per group. Villus height\nand wall thickness of the intestinal and colon were measured in 5-6 random fields per section under a\nlight microscope (Olympus BX53, Japan) at 20× magnification using Image-Pro Plus 6.0 software. Villus\nheight was determined from the villus tip to the crypt-villus junction, and intestinal wall thickness was\nmeasured from the mucosa to the serosa. Data were averaged per segment per rat, and mean values for\nthe capsaicin and control groups were compared for each intestinal region (small intestine and colon)\nusing Student's t-test (p < 0.05 considered significant).\nReviewer’s Comment: 10. Line 757: “Cells were treated…”: Please clarify which cells and explain how\nthey were cultured. How many times were the cell culture experiments repeated? And were they done in\ntriplicates or else?\nAuthors’ Response: Thank you for your detailed feedback regarding the Apoptosis Assays section (line\n757). We appreciate your guidance and have revised the manuscript to address these points in the\nMethods section (Lines 599–602, 618-623 and 869-874, marked in blue).\n“The human healthy small intestinal epithelial cell line HIEC-6 (Otwo Biotech, Cat No. HTX1978) and\nhuman healthy colonic epithelial cells NCM460 (Otwo Biotech, Cat. No. HTX1841) were cultured in\nDMEM enriched with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, maintained at\n37°C in a humidified incubator with 5% CO .”\n2\n“Natural capsaicin (404-86-4) was purchased from Weilkeqi Co., Ltd. It was dissolved in PBS to prepare\na capsaicin solution with an initial concentration of 1 mg/ml. The solution was diluted with complete\nmedium to obtain concentrations of 0 μM, 25 μM, 50 μM, 75 μM, 100 μM, 150 μM, and 200 μM for the\nintestinal cell（HIEC-6 and NCM460）capsaicin stimulation culture experiments. After a culture period\nof 0 h, 24 h, 48 h, and 72 h, these cells were used in further experiments.”\n“For the apoptosis assays, cells (5×10⁵) were seeded and exposed to a range of capsaicin concentrations\n(0–200 μM) for 72 hours. After trypsinization, the cells were stained with Annexin V FITC/PI and\nanalyzed via flow cytometry using FlowJo software. These experiments were conducted independently\nthree times, with each concentration tested in triplicate per experiment to ensure robust statistical\nreliability.”\nReviewer’s Comment: 11. Line 761-762: was the quality of the RNA assessed?\nAuthors’ Response: We appreciate your attention to detail and have incorporated the necessary updates\nin the Methods section of the revised manuscript (Lines 876–878, marked in blue). Total RNA was\nisolated from stool and blood samples using commercial kits (Solarbio, R1290; Tiangen, DP443). Total\nRNA from fecal and plasma samples was quantified and purity-assessed using an ultra-micro ultraviolet-\nvisible spectrophotometer (ND-100C, MIULAB, China).\nReviewer’s Comment: 12. Line 763-767. Please specify use of parametric and non-parametric tests for\ndata analysis based on data distribution.\nAuthors’ Response: The data distribution was assessed prior to analysis using the Shapiro-Wilk test for\nnormality. Parametric tests (Student's t-test for two-group comparisons and one-way ANOVA followed\nby Tukey's post hoc test for multiple groups) were employed when data followed a normal distribution,\nas confirmed in all cases. If non-normality had been detected, non-parametric alternatives (e.g., Mann-\nWhitney U test for two groups and Kruskal-Wallis test with Dunn's post hoc for multiple groups) would\nhave been used; however, this was not necessary. The diagnostic efficacy of miR-17-3p was evaluated\nthrough ROC curve analysis utilizing the Xiantao platform. All statistical tests were conducted using\nGraphPad software (10.1.2 version). P-values of less than 0.001 (***), 0.01 (**), and 0.05 (*) were\ndeemed statistically significant. Not significant was ns (Lines 887-895, marked in blue).\nReviewer’s Comment:\nResults\n1. Please ensure that sample sizes are clarified in all figures and legends.\n2. Body weight data missing: a decrease in body height was observed in immature rats fed a capsaicin\ndiet compared to controls, but the manuscript does not report their body weight. Since intestinal\ninflammation can lead to weight loss, please include body weight data to support the interpretation of the\ngrowth findings.\n3. Supplementary Figure 1a: the dendrogram on top (sample-based, rather than miRNA-based) should\nbe shown to support the statement in the text.\n4. Supplementary Figure 1c: unclear what does the Y axis show, is this a Delta Ct or a fold change (and\nif so, please explain versus what it was calculated). Please explain in legend.\n5. Line 172: GO and KEGG pathway analyses indicated that overexpression of miR-17-3p upregulates\nthe PI3K-akt etc”: a KEGG or GO analysis can indicate that genes targeted by miR-17-3p are enriched\nin these pathways, not that the pathways themselves are upregulated: please edit.\n6. Line 245: immature rats: please clarify what is it meant by this\n7. Supplementary Fig 14: the panels H and I seem to be switched, please also fix the corresponding text\nto match the revised figure (line 265). Please clarify which intestinal tissues were studied (rather than\nsaying: intestinal tissues) and also clarify if you used whole thickness or the epithelial lining. What was\nthe expression of the miR in Supplem Fig 14 relative to?\n8. Lines 267-274. Please specify the cell line in the text (instead of “mucosal epithelial cells”). The\nauthors seem to have conducted a regular “dose-response” experiment of capsaicin at different dosages,\nwhich is routine good practice in cell culture. It would be expected that at higher concentrations, the\nsubstance studied (here capsaicin) would inhibit cell viability, which is why these experiments are usually\nperformed to find the concentrations range to be studied. Is this what the authors did? Was cell viability\nassessed? If so, how? How was the optimal concentration (line 270) defined, and which one is it?\n9. Line 269: Supplementary Fig. 15 presents data on viability but not on proliferation as mentioned in the text.\nReviewer’s Comment: 1. Please ensure that sample sizes are clarified in all figures and legends. 2. Body\nweight data missing: a decrease in body height was observed in immature rats fed a capsaicin diet\ncompared to controls, but the manuscript does not report their body weight. Since intestinal inflammation\ncan lead to weight loss, please include body weight data to support the interpretation of the growth\nfindings.\nAuthors’ Response: We appreciate your guidance and have revised the manuscript to address these\nconcerns thoroughly.\nSample Sizes in Figures and Legends: We have ensured that sample sizes are now clearly specified in all\nfigures and their corresponding legends in the revised manuscript.\nBody Weight Data: We have added body weight data alongside food intake and height measurements for\nboth the capsaicin diet group and the non-capsaicin diet group in the Results section (Lines 252-265,\nmarked in blue). These results are also presented in Supplementary Figure 25, providing a comprehensive\nview of the growth parameters affected by the capsaicin diet.\nReviewer’s Comment: 3. Supplementary Figure 1a: the dendrogram on top (sample-based, rather than\nmiRNA-based) should be shown to support the statement in\nthe text.\nAuthors’ Response: Thank you for your insightful comment\nregarding Supplementary Figure 1a. We have revised the\nfigure accordingly.\nReviewer’s Comment: 4. Supplementary Figure 1c: unclear what does the Y axis show, is this a Delta\nCt or a fold change (and if so, please explain versus what it was calculated). Please explain in legend.\nAuthors’ Response: Thank you for your valuable feedback regarding Supplementary Figure 1c. We have\nupdated the legend for Supplementary Figure 1c to clarify that the Y-axis represents the relative\nexpression levels calculated as fold change, determined using the 2−ΔΔCt method. This fold change is\ncalculated relative to the expression levels of the control group, normalized against U6 small nuclear\nRNA as the endogenous reference. The revised legend now reads: \"Supplementary Figure 1c: Relative\nexpression levels of miR-17-3p (fold change, calculated using the 2−ΔΔCt method, normalized to U6 small\nnuclear RNA, with the control group as the reference).\" (Lines 1225-1227, marked in blue)\nReviewer’s Comment: 5. Line 172: GO and KEGG pathway analyses indicated that overexpression of\nmiR-17-3p upregulates the PI3K-akt etc”: a KEGG or GO analysis can indicate that genes targeted by\nmiR-17-3p are enriched in these pathways, not that the pathways themselves are upregulated: please edit.\nAuthors’ Response: Thank you for your careful review and for pointing out the need to refine the\ninterpretation of the GO and KEGG pathway analyses on line 172. We have revised the text to accurately\nreflect this, stating: \"GO and KEGG pathway analyses indicated that genes targeted by the\noverexpression of miR-17-3p are enriched in the PI3K-Akt, RAS, and MEK signaling pathways\n(Supplementary Fig. 1f, g).\" The change has been implemented in the revised manuscript (Lines 178-\n179, marked in blue).\nReviewer’s Comment: 6. Line 245: immature rats: please clarify what is it meant by this\nAuthors’ Response: Thank you for your comment seeking clarification on the term \"immature rats.\"\nThe term \"immature rats\" refers to 4-week-old rats used in our study, which were fed a capsaicin diet to\nassess its impact on growth and development. We selected 4-week-old rats, which are not yet fully\ndevelopmentally mature, because their growth plates are still active, allowing us to evaluate the effects\nof capsaicin on chondrocyte proliferation and new bone formation.\nReviewer’s Comment: 7. Supplementary Fig 14: the panels H and I seem to be switched, please also\nfix the corresponding text to match the revised figure (line 265). Please clarify which intestinal tissues\nwere studied (rather than saying: intestinal tissues) and also clarify if you used whole thickness or the\nepithelial lining. What was the expression of the miR in Supplem Fig 14 relative to?\nAuthors’ Response: Thank you for your careful observation regarding Supplementary Figure 14 and\nyour suggestions for improvement.\nPanels H and I Switch: We acknowledge the misplacement of panels H and I in Supplementary Figure\n14. This error has been corrected in the revised figure, with panels now properly aligned. The\ncorresponding text on lines 283-286 has been updated to match the revised figure, ensuring consistency\nbetween the description and visual representation.\nIntestinal Tissues Studied: We have clarified the specific intestinal tissues analyzed, replacing the vague\nterm \"intestinal tissues.\" The study utilized cross-sections of the small intestine and colon, encompassing\nthe full thickness of the muscularis and mucosal layers. This detail has been incorporated into the\nMethods section (Lines 855–865, marked in blue) and the figure legend for Supplementary Figure 14.\nmiR in Supplementary Figure 14: The expression levels of miR (specifically miR-17-3p) shown in\nSupplementary Figure 14 are presented relative to the control group, normalized against U6 small nuclear\nRNA as the endogenous reference. This normalization was performed using the 2−ΔΔCt method, where the\ncontrol group serves as the baseline for relative quantification. The updated legend for Supplementary\nFigure 14 now includes this detail, stating: \"Supplementary Figure 14: Relative expression levels of miR-\n17-3p (fold change, calculated using the 2ΔΔCt method, normalized to U6 small nuclear RNA, with the\ncontrol group as the reference). (Lines 1419–1420, marked in blue)\nReviewer’s Comment: 8. Lines 267-274. Please specify the cell line in the text (instead of “mucosal\nepithelial cells”). The authors seem to have conducted a regular “dose-response” experiment of capsaicin\nat different dosages, which is routine good practice in cell culture. It would be expected that at higher\nconcentrations, the substance studied (here capsaicin) would inhibit cell viability, which is why these\nexperiments are usually performed to find the concentrations range to be studied. Is this what the authors\ndid? Was cell viability assessed? If so, how? How was the optimal concentration (line 270) defined, and\nwhich one is it?\nAuthors’ Response: Thank you for your thoughtful comments. We appreciate your guidance and have\nrevised the manuscript to address these points.\nCell Line Specification: We have updated the text to specify that the experiments were conducted using\nthe human small intestinal epithelial cell line HIEC-6 (Otwo Biotech, Cat No. HTX1978) and the normal\ncolon mucosal epithelial cell line (NCM460, Otwo Biotech, Cat No. HTX1841), replacing the vague\nterm \"mucosal epithelial cells\" to ensure clarity (see revised text above, Methods section, lines 618-623,\nmarked in blue).\nDose-Response Experiment: Capsaicin exhibits a dual role, acting as a double-edged sword. At lower\nconcentrations (25 µM, 50 µM, and 75 µM), we observed promotion of cell proliferation and inhibition\nof apoptosis, offering a protective effect on HIEC-6 cells, consistent with literature reports on capsaicin’s\ntherapeutic use in conditions like ulcerative colitis. However, at higher concentrations (100 µM, 150 µM,\nand 200 µM), cell proliferation was inhibited, and apoptosis increased, aligning with reports of high-\ncapsaicin diets contributing to gastrointestinal disorders.\nDefinition of Optimal Concentration: The term \"optimal concentration\" (line 270) refers to 200 µM,\nselected based on its comparability to the intestinal capsaicin concentrations observed in our ISS case\nsource region, Jiangxi Province. We calculated the mean intestinal concentration from dietary intake\n(1.756–2.076 mg/kg/day) to be approximately 230 µM, with a range of 153.3–317.2 µM across adults\nand adolescents. Among the tested concentrations (100 µM, 150 µM, and 200 µM), which all inhibited\nproliferation and increased apoptosis, 200 µM showed the greatest alignment with this mean value,\nmaking it the most physiologically relevant for simulating the high-capsaicin diet effects in our model.\nCell Viability Assessment: In the experiment involving capsaicin stimulation of the human small\nintestinal epithelial cell line HIEC-6 (Otwo Biotech, Cat No. HTX1978), we assessed the effects of\ncapsaicin on small intestinal cells by using the CCK-8 assay to evaluate cell proliferation and flow\ncytometry to detect apoptosis. Given that the cells were in an adherent state, we did not employ Trypan\nBlue staining to assess cell viability.\nReviewer’s Comment: 9. Line 269: Supplementary Fig. 15 presents data on viability but not on\nproliferation as mentioned in the text.\nAuthors’ Response: We appreciate the reviewer’s comment. The assay presented in Supplementary\nFigure 15 is a CCK-8 assay, which measures cell proliferation rather than cell viability. We have revised\nthe description in Lines 283-286 of the manuscript and updated the legend of Supplementary Figure 15\naccordingly to correctly reflect that the data represent cell proliferation (via CCK-8 assay) and apoptosis\n(via flow cytometry).\nReviewer’s Comment:\nDiscussion\nThe discussion is well thought and clear.\nLine 366: the SOS1 abbreviation should have been explained earlier in the manuscript.\nLine 385: please provide a reference for “high chili pepper consumption”\nLines 396-400: this part of the discussion is important. It could be improved by relating it to the published\ndietary amounts of dietary capsaicin showing beneficial effects. How do the concentrations used with\nthe intestinal cell lines relate to dietary intake in high and normal capsaicin-diets and to the amount of\ncapsaicin known to elicit beneficial effects in published animal studies? And then versus human intake.\nLine 398: please clarify where the data on proliferation are presented. Please make sure that the\ndiscussion reflects the results and that these are all presented.\nLines 431-432: we cannot find the data on mice with ulcerative colitis.\nAuthors’ Response: We appreciate your feedback and have addressed those concerns in the revised\nversion.\nSOS1 Abbreviation (Line 366): In the revised manuscript, we have introduced the full term and\nabbreviation at its first occurrence in the Method section (lines 204, marked in blue).\nReference for \"High Chili Pepper Consumption\" (Line 385): We have added clarification and a reference\nfor \"high chili pepper consumption\" in the Introduction section (Lines 120–128, marked in blue). This\nterm is now supported by epidemiological data from Zou et al. (2021, PMCID: PMC8545790), which\nreports significant pepper consumption levels in regions like Jiangxi, Hunan, Chongqing, and Guizhou\nprovinces, with per capita annual intake ranging from 75 to 111.57 kg, as detailed in the text.\nRelating In Vitro Concentrations to Dietary Capsaicin Intake (Lines 396-400): We have revised the\ndiscussion (lines 396–403) to better contextualize our in vitro capsaicin concentrations in relation to the\npublished literature. This revision is presented in the discussion section of the revised manuscript\n(lines 430–440). “While some studies report that capsaicin reduces microbial growth, alleviating gut\ninflammation and diarrhea (PMID: 35777913), its gastrointestinal effects are dose-dependent. For\ninstance, Azimirad et al. (PMID: 36155114) demonstrated that capsaicin at 20 and 50 µM downregulated\npro-inflammatory markers (NF-κB, TNF-α) and apoptosis-related genes (e.g., BCL-2) in HT-29 cells\nexposed to Clostridioides difficile toxins, supporting its anti-inflammatory and anti-apoptotic properties.\nIn contrast, Xiang et al. (PMID: 35267319) found that low-dose capsaicin (40 mg/kg body weight in\nmice) caused no adverse gastrointestinal effects, whereas higher doses (60 and 80 mg/kg) induced\nsignificant damage, including inflammation, reduced villus/crypt ratios, and tissue destruction in the\njejunum, ileum, and colon. These findings align with our in vitro observations using HIEC-6 cells, where\nlow concentrations (25, 50, and 75 µM) promoted intestinal epithelial cell proliferation and inhibited\napoptosis, indicating a protective role, while higher concentrations (100, 150, and 200 µM) suppressed\nproliferation and increased apoptosis.” (Lines 430-440, marked in blue)\nClarification of Proliferation Data: In our study, we treated the human intestinal epithelial cell line\n(HIEC-6) with various concentrations of capsaicin and assessed cell proliferation using the CCK-8 assay,\nwhile apoptosis was evaluated via flow cytometry. These results are presented in Supplementary Figure\n15. However, we recognize that the figure was incorrectly labeled as \"cell viability\" in the original\nsubmission. In the revised manuscript, we have corrected this error to accurately reflect that the data\nrepresent cell proliferation (via CCK-8 assay) and apoptosis (via flow cytometry).\nData on Mice with Ulcerative Colitis: Our histological analysis revealed that the intestinal inflammatory\nchanges induced by a capsaicin diet in rats were relatively mild. One of our co-authors, Peng Yu, has\nconducted a study on ulcerative colitis using a rat model, in which the histological sections demonstrated\nmarkedly more severe intestinal inflammation compared with our findings. However, since Yu’s study is\nstill under revision for publication in Food Frontiers and has not yet been published, we did not include\ncomparative images in the present manuscript. Therefore, we revised the discussion section to read as\nfollows: “Notably, although intestinal inflammation was observed in capsaicin-fed rats, histological\nanalysis revealed it to be relatively mild, which we characterize as microinflammation. Consistent with\nthis, the rats did not develop symptoms such as diarrhea or changes in stool consistency. This finding\naligns with the condition in children with ISS, who typically do not present with severe gastrointestinal\nsymptoms such as abdominal pain or diarrhea. Consequently, this subtle form of inflammation\ncomplicates the task of uncovering the etiology of ISS.”\nReviewer #5 (Remarks to the Author):\nThis is a well-designed study of potential clinical relevance applying a multitude of methodologies.\nMinor comments:\n1. Abstract, lines 30-31: Please correct the wrong statement that ISS is insensitive to GH treatment. As a\nmatter of fact, many ISS patients respond as well as many of those categorized as GHD based on\nconventional pituitary stimulation testing.\n2. Abstract, lines 46-47: The statement that short-statured children undergoing growth hormone therapy\nshould be evaluated regarding the impact of capsaicin-rich diets should be rephrased, removing 'growth\nhormone therapy' as no supportive data are presented in the manuscript.\n3. Introduction, lines 67-68: Please rephrase 'ISS with peak GH levels exceeding 10 μg/L'. In a large\nnumber of countries, the cutoff for differentiating GHD from ISS is lower than 10 ug/L.\nReviewer #5 1. Abstract, lines 30-31: Please correct the wrong statement that ISS is insensitive to GH\ntreatment. As a matter of fact, many ISS patients respond as well as many of those categorized as GHD\nbased on conventional pituitary stimulation testing.\nAuthors’ Response:\nThank you for your valuable comment regarding the statement in the Abstract that ISS is insensitive to\ngrowth hormone (GH) treatment. Based on a meta-analysis conducted by our team, we found that while\nrhGH therapy improves height in ISS children, the magnitude of height gain is significantly less\ncompared to children with GHD or those born small for gestational age (SGA) (PMID: 30201821, PMID:\n26363846, PMID: 36102184). This suggests that ISS patients exhibit a relatively reduced sensitivity to\nrhGH treatment compared to other conditions requiring GH therapy, rather than a complete insensitivity.\nThe revised Abstract (lines 30-31) now states: \" Idiopathic short stature (ISS) poses several challenges,\nincluding unclear etiology, variable responsiveness to recombinant human growth hormone (rhGH)\ntherapy, and complications arising from high doses of rhGH. \"\nReviewer #5 2. Abstract, lines 46-47: The statement that short-statured children undergoing growth\nhormone therapy should be evaluated regarding the impact of capsaicin-rich diets should be rephrased,\nremoving 'growth hormone therapy' as no supportive data are presented in the manuscript.\nAuthors’ Response: Thank you for your valuable feedback regarding the Abstract and your suggestion\nto rephrase the statement about evaluating the impact of capsaicin-rich diets on short-statured children\nundergoing growth hormone therapy. We have revised the text accordingly.\nOriginal:\nThese findings underscore the need to evaluate the impact of capsaicin-rich diets on longitudinal growth\nin high-risk ISS populations and short-statured children undergoing growth hormone therapy,\nsignificantly advancing our understanding and management of ISS while opening new therapeutic\navenues.\nModified:\nThese findings underscore the need to evaluate the impact of capsaicin-rich diets on longitudinal growth\nin high-risk ISS populations and short-statured children, significantly advancing our understanding and\nmanagement of ISS while opening new therapeutic avenues (lines 45-48).\nReviewer #5 3. Introduction, lines 67-68: Please rephrase 'ISS with peak GH levels exceeding 10 μg/L'.\nIn a large number of countries, the cutoff for differentiating GHD from ISS is lower than 10 ug/L.\nAuthors’ Response: We thank the reviewer for the valuable comment regarding the phrasing of “ISS\nwith peak GH levels exceeding 10 μg/L” (Introduction, lines 67–68). We agree that the cutoff for\ndistinguishing growth hormone deficiency (GHD) from idiopathic short stature (ISS) varies across\ncountries and is often lower than 10 μg/L. As noted by Ranke in Nature Reviews Endocrinology (PMID:\n23609338), “The cut-off value to distinguish between GHD and ISS was traditionally considered a GH\nlevel of 10 μg/L to standard stimulation tests, but when using modern international reference preparations\nfor GH this figure corresponds to 6.7 μg/L.”\nWe also acknowledge that some researchers continue to use a >10 μg/L cutoff, likely to enhance\nISS diagnostic specificity. To avoid controversy and reflect a broader diagnostic approach, we have\nrevised the manuscript. The updated sentence now reads: \"However, the majority of cases are ultimately\nclassified as idiopathic short stature (ISS) after ruling out systemic diseases, nutritional deficiencies,\npsychiatric disorders, chromosomal abnormalities, and growth hormone deficiency, yet their cause\nremains unknown.\"\nWe sincerely thank the reviewer for this helpful suggestion, as well as all reviewers and the editor\nfor their thoughtful and constructive feedback throughout the review process. Their comments have\ngreatly improved the clarity, rigor, and overall quality of our manuscript, and we truly appreciate the time\nand effort dedicated to this evaluation.\nWe sincerely thank you for the thoughtful and thorough evaluation of our revised manuscript entitled\n“Capsaicin Diet-Induced Gut Inflammation Elevates Plasma Exosomal miR-17-3p: Unveiling the\nZNF148/SOS1 Axis as a Novel Mechanism in ISS Pathogenesis and Potential Therapeutic Target”\n(Manuscript Number: NCOMMS-24-73070B).\nWe greatly appreciate the reviewers’ continued time, effort, and valuable feedback, which have further\nguided us in refining the manuscript. We have carefully addressed all comments and made corresponding\nrevisions to enhance the clarity, accuracy, and overall scientific quality of the paper. All modifications\nare highlighted in blue in the revised version for ease of reference.\nA detailed, point-by-point response to each comment is provided below, with explanations for all changes\nand the corresponding line numbers indicated in the updated manuscript.\nWith our sincere thanks for your consideration and constructive feedback,\nBest regards,\nJingyu Jia, M.D., Ph.D\nDepartment of Orthopaedics, The Second Affiliated Hospital of Nanchang University\nPhone: +86-791-86311590; Fax: +86-791-86311590\nE-mail: jiaxintong9@163.com; ndefy12383@ncu.edu.cn\nI only have very minor comments remaining:\n① The justification for the amount of capsaicin used and its physiological relevance, now explained in\nthe response to reviewers, should be mentioned in the methods section within the manuscript. Apologies\nif I have missed this.\n② The response explains that “We did assess viability using Trypan Blue exclusion prior to experiments,\nensuring only viable cells (>95% viability) were used (Lines 614–617, marked in blue).” Though, the\nmanuscript at those lines does not state that only viable cells (>95% viability) were used, please consider\nadding.\n③ The response states that: “our study exclusively utilized miRNA High-Throughput Sequencing, and\nthe reference to microarray was an error in the original manuscript.”; though, at line 560-562 of the\nrevised manuscript, it reads: “five pairs of samples underwent miRNA microarray analysis for miRNA\nexpression analysis, while all 212 samples were used for quantitative RT-PCR validation.” Please clarify.\n④ Thank you for revising Supplementary Figure 1A by adding the sample-based dendrogram, as per\nyour response. Unfortunately, I do not see it in my version of this figure (still without this dendrogram),\nplease ensure this is included in the final version\nAuthors’ Response:\n① We appreciate the reviewer’s careful attention and helpful suggestion. In the revised manuscript,\nwe have added a detailed explanation of the rationale for the selected capsaicin dosage and its physiological\nrelevance to human dietary exposure. This information has now been included in the Methods section\n(lines 865–883) to ensure clarity and transparency.\n② We have now explicitly stated that only viable cells (>95% viability), as determined by Trypan Blue\nexclusion, were used for subsequent experiments. This clarification has been added to the Methods\nsection (lines 615–616) in the revised manuscript.\n③ We have now corrected this statement in the revised manuscript to accurately reflect that miRNA\nhigh-throughput sequencing was performed rather than microarray analysis. The correction has been\nmade in the Methods section (line 561).\n④ We have carefully rechecked the PDF file of the Supplementary Information submitted with the\nrevised manuscript. We confirm that the sample-based dendrogram is indeed included in Supplementary\nFigure 1A, and it is identical to the version shown in our response letter. Both the figure in the uploaded\nPDF and the one provided in the response contain the dendrogram, as illustrated in the comparison\nimages below.\nfor their thoughtful and constructive feedback throughout the review process. Their comments have\ngreatly improved the clarity, rigor, and overall quality of our manuscript, and we truly appreciate the time\nand effort dedicated to this evaluation.\nBest regards,\nSincerely,\nJingyu Jia, M.D., Ph.D\nDepartment of Orthopaedics, The Second Affiliated Hospital of Nanchang University\nPhone: +86-791-86311590; Fax: +86-791-86311590\nE-mail: jiaxintong9@163.com; ndefy12383@ncu.edu.cn\n4"}, {"round": 3, "reviewer_comments": {"reviewer_2": "The authors have provided clear and complete responses to my queries", "reviewer_3": "I would like to thank the authors for addressing the comments and providing thorough and substantiated answers.\nI only have very minor comments remaining:\n- The justification for the amount of capsaicin used and its physiological relevance, now explained in the response to\nreviewers, should be mentioned in the methods section within the manuscript. Apologies if I have missed this.\n- The response explains that “We did assess viability using Trypan Blue exclusion prior to experiments, ensuring only viable\ncells (>95% viability) were used (Lines 614–617, marked in blue).” Though, the manuscript at those lines does not state that\nonly viable cells (>95% viability) were used, please consider adding.\n- The response states that: “our study exclusively utilized miRNA High-Throughput Sequencing, and the reference to\nmicroarray was an error in the original manuscript.”; though, at line 560-562 of the revised manuscript, it reads: “five pairs of\nsamples underwent miRNA microarray analysis for miRNA expression analysis, while all 212 samples were used for\nquantitative RT-PCR validation.” Please clarify.\n- Thank you for revising Supplementary Figure 1A by adding the sample-based dendrogram, as per your response.\nUnfortunately, I do not see it in my version of this figure (still without this dendrogram), please ensure this is included in the\nfinal version", "reviewer_5": "Thank you for taking action on the issues raised by the reviewer. The amended manuscript has now significantly improved\nand the rviewer has no further comments."}, "author_response": "Dear Reviewers,\nWe sincerely thank you for the thoughtful and thorough evaluation of our revised manuscript entitled\n“Capsaicin Diet-Induced Gut Inflammation Elevates Plasma Exosomal miR-17-3p: Unveiling the\nZNF148/SOS1 Axis as a Novel Mechanism in ISS Pathogenesis and Potential Therapeutic Target”\n(Manuscript Number: NCOMMS-24-73070B).\nWe greatly appreciate the reviewers’ continued time, effort, and valuable feedback, which have further\nguided us in refining the manuscript. We have carefully addressed all comments and made corresponding\nrevisions to enhance the clarity, accuracy, and overall scientific quality of the paper. All modifications\nare highlighted in blue in the revised version for ease of reference.\nA detailed, point-by-point response to each comment is provided below, with explanations for all changes\nand the corresponding line numbers indicated in the updated manuscript.\nWith our sincere thanks for your consideration and constructive feedback,\nBest regards,\nJingyu Jia, M.D., Ph.D\nDepartment of Orthopaedics, The Second Affiliated Hospital of Nanchang University\nPhone: +86-791-86311590; Fax: +86-791-86311590\nE-mail: jiaxintong9@163.com; ndefy12383@ncu.edu.cn\nI only have very minor comments remaining:\n① The justification for the amount of capsaicin used and its physiological relevance, now explained in\nthe response to reviewers, should be mentioned in the methods section within the manuscript. Apologies\nif I have missed this.\n② The response explains that “We did assess viability using Trypan Blue exclusion prior to experiments,\nensuring only viable cells (>95% viability) were used (Lines 614–617, marked in blue).” Though, the\nmanuscript at those lines does not state that only viable cells (>95% viability) were used, please consider\nadding.\n③ The response states that: “our study exclusively utilized miRNA High-Throughput Sequencing, and\nthe reference to microarray was an error in the original manuscript.”; though, at line 560-562 of the\nrevised manuscript, it reads: “five pairs of samples underwent miRNA microarray analysis for miRNA\nexpression analysis, while all 212 samples were used for quantitative RT-PCR validation.” Please clarify.\n④ Thank you for revising Supplementary Figure 1A by adding the sample-based dendrogram, as per\nyour response. Unfortunately, I do not see it in my version of this figure (still without this dendrogram),\nplease ensure this is included in the final version\nAuthors’ Response:\n① We appreciate the reviewer’s careful attention and helpful suggestion. In the revised manuscript,\nwe have added a detailed explanation of the rationale for the selected capsaicin dosage and its physiological\nrelevance to human dietary exposure. This information has now been included in the Methods section\n(lines 865–883) to ensure clarity and transparency.\n② We have now explicitly stated that only viable cells (>95% viability), as determined by Trypan Blue\nexclusion, were used for subsequent experiments. This clarification has been added to the Methods\nsection (lines 615–616) in the revised manuscript.\n③ We have now corrected this statement in the revised manuscript to accurately reflect that miRNA\nhigh-throughput sequencing was performed rather than microarray analysis. The correction has been\nmade in the Methods section (line 561).\n④ We have carefully rechecked the PDF file of the Supplementary Information submitted with the\nrevised manuscript. We confirm that the sample-based dendrogram is indeed included in Supplementary\nFigure 1A, and it is identical to the version shown in our response letter. Both the figure in the uploaded\nPDF and the one provided in the response contain the dendrogram, as illustrated in the comparison\nimages below.\nfor their thoughtful and constructive feedback throughout the review process. Their comments have\ngreatly improved the clarity, rigor, and overall quality of our manuscript, and we truly appreciate the time\nand effort dedicated to this evaluation.\nBest regards,\nSincerely,\nJingyu Jia, M.D., Ph.D\nDepartment of Orthopaedics, The Second Affiliated Hospital of Nanchang University\nPhone: +86-791-86311590; Fax: +86-791-86311590\nE-mail: jiaxintong9@163.com; ndefy12383@ncu.edu.cn\n4"}]

Decision letter