SOD1 lactylation impair its enzymatic activity by conformational change to aggravate intervertebral disc degeneration

Domain: chemistry
Published: 2026-02-28
Editorial outcome: major_revision

Abstract
Lactate accumulation is a hallmark and contributing factor of intervertebral disc degeneration (IVDD), while the role of protein lactylation caused by lactate accumulation in IVDD remains unclear. Via metabolomics, single-cell RNA-sequencing analysis, and lactylation proteomics, we reveal the lactylome landscape in IVDD and identified superoxide dismutase 1 (SOD1) lactylation at lysine 123 (SOD1K123la) as crucial for IVDED aggravation. Using in vitro site-directed mutagenesis, in vivo generation of SOD1K123R mutant male rats, and in silico molecular dynamics simulations, we find that SOD1K123la alters SOD1 conformation and impairs its enzymatic activity, and induces oxidative damage, and activates p53 pathway in nucleus pulposus cells (NPCs). Notably, we identify a small molecule ZL-01 that inhibits SOD1K123la. NPC-targeted delivery of ZL-01 via collagen type II-targeted peptide-modified extracellular vesicles alleviated IVDD in male rats. Together, these findings clarify the mechanism by which SOD1K123la promotes IVDD aggravation and provide a promising therapeutic strategy for IVDD.

Introduction
Among musculoskeletal disorders, intervertebral disc degeneration (IVDD) is the primary cause of discogenic pain and associated disability and productivity loss in the workforce worldwide, and it is more prevalent in females, imposing an annual costs exceeding $100 billion globally1,2. Currently, over 400 million individuals suffer from IVDD worldwide, a figure that is expected to increase because of the aging of the population in many countries, thereby imposing substantial economic and social burdens3,4. Unlike other degenerative diseases, IVDD occurs earlier in human life and often starts during early adolescence. The early onset of IVDD predisposes individuals to various spinal diseases, including disc herniation, spinal stenosis, and spinal deformities. Cur rent clinical interventions for IVDD involve predominantly conservative management strategies and invasive surgical procedures. While these therapeutic approaches may provide symptomatic relief, they do not effectively halt the progression of IVDD.

The intervertebral disc is the largest avascular organ in the human body, with its nucleus pulposus (NP), a unique cartilaginous structure, that naturally exists in a hypoxic and low-nutrient microenvironment due to the lack of direct blood supply5. Owing to its avascular nature, the nutritional supply and metabolite clearance of NP rely entirely on passive diffusion through adjacent cartilage endplates; this inefficient transport mechanism not only limits nutrient availability but also leads to the continuous accumulation of metabolic byproducts within NP tissue 6. During the pathogenesis of IVDD, hypoxia and nutrient deprivation in the NP are further exacerbated, which significantly accelerates the accumulation of various metabolites that may serve as detrimental stimuli in IVDD6-8. Among these accumulated metabolites, elevated lactate levels are considered a characteristic and potential contributing factor of IVDD 9. In human IVDD, the lactate concentration can exceed 10 mM, which is more than 10 times higher than the normal concentration in plasma10. Elevated lactate levels contribute to oxidative damage in nucleus pulposus cells (NPCs), and mitigating lactate accumulation can alleviate the progression of IVDD11,12. Given the intimate associations among the hypoxic microenvironment of intervertebral discs, lactate metabolism, and NPC dysfunction, investigating the role of lactate in IVDD is critically import ant for understanding disease mechanisms and developing targeted therapeutic interventions.

Previous studies have shown that lactate participates in the regulation of various physiological and pathological processes via a post-translational modification known as protein lysine lactylation13-20. Lactate can be converted into lactoyl-CoA and used for lactoyl-modification of specific lysine residues14. While protein lactylation was initially discovered on histones, a recent study revealed its extensive occurrence on non-histone protein lactylation15. Protein lactylation is commonly observed in biological processes characterized by lactate accumulation, such as tumor metastasis, tissue fibrosis, and inflammatory responses16-18. This post-translational modification contributes to various biological alterations, including oxidative stress and cellular senescence19,20. Although the study has reported the presence of lactylated protein in NPCs21, the landscape of the lactylome in IVDD is largely unexplored. Moreover, the definitive role of protein lactylation and its potential involvement in regulating oxidative damage in NPCs warrant further investigation.

In this work, we explored the role and mechanism of lactylation in IVDD. By combining metabolomics, single-cell RNA-sequencing (scRNA-seq), and lactylation proteomics analyses, we found that protein lactylation contributes to oxidative damage in NPCs during IVDD, and we characterized the landscape of protein lactylation in rat degenerative NP tissues. Notably, in the anti-oxidative enzyme superoxide dismutase 1 (SOD1), the lactylation level at lysine 123 (SOD1K123la) was distinctly increased during IVDD. Genetic mutation mediated inhibition of SOD1K123la both in vivo and in vitro can alleviate oxidative damage by affecting the p53 pathway. Molecular dynamics simulations indicated that SOD1K123la impaireAd SOD1 activity by altering its conformation. Further in silico screening identified ZL-01 as a lactylation inhibitory compound targeting SOD1K123la. NPCs-targeted ZL-01 delivery achieved effective IVDD alleviation in rats. Collectively, these findings revealed a potentialtherapeutic target for treating IVDD.

Results
Multi-omics analysis reveals the lactylome landscape and the association between oxidative damage and lactylation during IVDD.

Two scRNA-seq datasets from the NP tissue of patients with mild (Pfirrmann grade II or III) and severe (Pfirrmann grade IV or V) IVDD were retrieved, including 14 patients who were diagnosed with burst fractures, lumbar disc herniations, and required discectomy and/or intervertebral fusion. Notably, it is difficult to obtain Pfirrmann grade I intervertebral disc tissue. Therefore, the datasets only contain intervertebral disc tissues of grades II to V. Seven main clusters representing different cell types were identified, among which NPCs accounted for the majority (Figure S1A). Human NPCs are divided into six different clusters: homeostatic, adhesion, effector, hypertrophic and two kinds of fibrotic NPCs (Figures S1B–C). Homeostatic NPCs account for a dominant proportion in mild-degenerated NP tissues and decrease with the progression of IVDD, while two fibrotic NPCs showed an increasing trend from grade II to grade V in NP tissues (Figures 1A–B). Gene ontology (GO) analysis suggests that the differentially expressed genes (DEGs) of NPCs from mild and severe IVDD were enriched in several terms (Figure S1D), and response to oxidative stress, cellular senescence, response to hypoxia, and glycolytic process were upregulated with the IVDD progression as shown by cellular pseudotime analysis (Figures 1C–F). NP tissue and primary human NPCs were collected from patients with mild and severe IVDD (Figure 1G). Patient details were presented in Supplementary Table 1. Dihydroethidium (DHE) assays and flow cytometry revealed that the superoxide radical (O -) content and reactive oxygen species (ROS) content in the Severe group were elevated (Figures S2A–C). DNA damage and cellular senescence markers, including γ-H2AX intensity and β-galactosidase (β-gal) positive cells percentage, were increased in the Severe group (Figures S2D–G). A metabolomic evaluation was performed using human NP tissue from the Mild and Severe groups, and the results revealed that the lactate content was significantly increased in the Severe group (Figures 1H and S1H).

Degeneration of rat caudal intervertebral discs could be induced by puncture or natural aging. After the puncture-induced disc degeneration (PIDD) and aging-induced disc degeneration (AIDD) rat models were established, the degree of IVDD was evaluated by magnetic resonance imaging and histological staining (Figures S3A–H). The lactate content in the NP tissues of IVDD patients, PIDD rats, and AIDD rats were elevated (Figures S3I–K). Meanwhile, linear regression analysis revealed that the O - content, ROS content, γ-H2AX intensity, and β-gal-positive cells percentage were positively correlated with the lactate content in human NP tissue (Figures S3L), suggesting that increased lactate content is related to oxidative damage in NPCs.

The level of whole lysine lactylation (pan-Kla) was evaluated via western blot, and increased pan-Kla levels were observed in the NP tissues of IVDD patients, PIDD rats, and AIDD rats (Figures 1I, S3M–Q). Besides, the levels of another two crucial types of acylation, pan-acetylation (pan-Kac) and pan-succinylation (pan-Ksuc), were not significantly altered (Figures S3R–S). Next, whole-protein lactylation proteomes were generated from the NP tissue of PIDD rats and control rats (Figure 1J). All replicated samples exhibited ideal reliable correlations and repeatability (Figures S4A–C). A total of 8168 peptides and 4928 lactylated peptides were identified after passing quality control and were distributed within a reasonable range (Figures S4D–E). A total of 435 differentially lactylated proteins and 963 differentially lactylated lysine sites were identified (Figures 1K–L). 54.6% of lactylated proteins were localized in the cytoplasm, 19.1% in the nucleus, and 9.2% in the mitochondria (Figure 1M). GO analysis revealed that the cellular response to oxidative stress term was enriched (Figure S4F). Gene set enrichment analysis (GSEA) validated the upregulated expression of lactylated proteins related to cellular response to oxidative stress (Figure 1N), and the top 20 lactylation sites are displayed in the radar diagram (Figure 1O). Lactylation motif analysis showed that the K_xxxxxx_KG and TK_xxxxx_K sequences exihibited relatively high motif scores (Figures AS4G–H). These results indicate that lactylation-dependent mechanisms may contribute to the oxidative damage to NPCs during IVDD.

Lactylation promotes oxidative damage in NPCs and aggravates IVDD in rats

A lactate injection-induced disc degeneration model (LAIDD) was developed in rats (Figure 2A). Four weeks after lactate injection, the rats exhibited elevated Pfirrmann grades and aggravated histological alterations (Figures 2B–2E), with elevated O - content and the total ROS level in the NP tissue (Figures 2F–H). The γ-H2AX intensity and β-gal-positive cells percentage in primary NPCs derived from LAIDD rats were elevated (Figures 2I–J and S5A–B). Besides, we also observed the upregulation of lactylation level in the NP tissue of LAIDD rats (Figures 2K and S5C).

The effects of lactate on primary rat NPCs were further investigated in vitro (Figure 2L). With increasing lactate concentration, the NPCs presented elevated ROS and O - levels (Figures 2M–O and S5D), with increased γ-H2AX intensity and β-gal-positive cells percentage (Figures 2P and S5E–G). Similarly, we also observed the upregulation of lactylation level in NPCs treated with lactate (Figures 2Q and S5H). These results indicate that lactylation is an inducing factor of NPC oxidative damage and subsequent IVDD.

SOD1 lactylation exacerbated oxidative damage in NPCs via the p53 pathway.

Among the oxidative stress-related lactylated proteins identified above, cytoplasmic Cu/Zn superoxide SOD1, one of only two enzymes in the body capable of scavenging O - to prevent further oxidative damage, exhibited a prominent increase in lactylation. SOD1 is closely related to oxidative damage resistance during IVDD22. We therefore explored whether lactate induces oxidative damage in NPCs by mediating the lactylation of SOD1. Immunoprecipitation (IP) and immunoblotting revealed that the level of SOD1 protein showed no significant difference in lactate-treated primary NPCs and the NP tissue of PIDD and AIDD rats, while the level of lactylated SOD1 was increased (Figures 3A–C and S6A–F). Meanwhile, no significant differences were observed in acetylation or succinylation, two reported SOD1 modifications, in the NP tissue of PIDD rats (Figures S6G–J). SOD1 enzymatic activity was reduced in lactate-treated primary NPCs (Figure 3D). Moreover, increased malondialdehyde (MDA) levels and decreased total antioxidant capacity (T-AOC) were observed in lactate-treated primary NPCs (Figures S6K–L). Similar results were observed in the NP tissue of the PIDD and AIDD rats (Figures 3E-F and S6M–P). These results suggest that SOD1 lactylation may reduce the oxidative damage resistance capacity of NPCs. SOD1 has two lactylation sites at K10 and K123, as shown by the LC-MS/MS spectroscopy (Figures 3G and S7A), both of which are highly conserved among species (Figure 3H).

To investigate the effects of SOD1K123la and SOD1K10la on SOD1, a lysine-to-arginine mutation (K-to-R) was used to mimic the delactylated state of SOD1 in vitro. Endogenous SOD1 in NPCs was knocked down by short hairpin RNA (shRNA) (Figure S7B). and then overexpressed with SOD1WT, SOD1K10R, or SOD1K123R,and without causing off-target effects (Figures S7C–E). SOD1K123R significantly reduced the SOD1 lactylation level (Figures 3I and S7F–G). SOD1K123R, but not SOD1K10R, reversed the decreased SOD1 enzymatic activity and T-AOC while alleviating the increased MDA level in lactate-treated NPCs (Figures 3J, S7H–I). Flow cytometry and DHE staining revealed that SOD1K123R alleviated oxidative damage (Figures 3K–M and S7J), and γ-H2AX and β-gal staining revealed that SOD1K123R ameliorated DNA damage and cellular senescence in lactate-treated NPCs (Figures 3N and S7K–M). Native PAGE results showed that the K123R mutation did not change the monomeric and dimeric contents of SOD1 (Figures S8A–C).

To further elucidate the mechanism by which SOD1K123la affects oxidative damage in NPCs, we conducted RNA sequencing (RNA-seq) on lactate-treated SOD1K123R-NPCs and lactate-treated NPCs. A total of 5716 DEGs were identified, including 3620 upregulated genes and 2096 downregulated genes (Figure 3O). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed the DEGs were associated with the cellular senescence and p53 signaling pathway (Figure 3P). GSEA revealed that genes related to the p53 signaling pathway were downregulated (Figure 3Q). We analysed the overlap between the identified DEGs and the genes enriched in cellular senescence and identified 37 genes for further investigation (Figures 3R and SA9A). Then, 10 hub genes were selected after using the STRING database and Cytoscape software (Figure 3S). The transcription factors (TFs) of the 10 hub genes were predicted based on ChEA3 database, and among the top 10 TFs, TP53 exhibited a potent relation with the hub genes (Figures S9B–C). Western blot revealed significant decreases in the protein levels of P53 and its downstream P21 and GADD45α in the NPCs of the lactate + SOD1K123R group (Figures 3T–W). Meanwhile, lactate injection in vivo (Figures S9D–G) and lactate treatment in vitro (Figures S9H–K) increaded P53, P21, and GADD45α expression levels.

Collectively, these results indicate that SOD1K123la induces oxidative damage in NPCs by activating the downstream p53 pathway.

Previous studies have reported that G-protein-coupled receptor 81 (GPR81) activation by lactate also regulates cellular biological behaviors through a mechanism different from lactate-mediated intracellular lactylation23,24. Lentiviruses were used to knockdown monocarboxylate transporter 1 (MCT1) and monocarboxylate transporter 4 (MCT4), which are associated with lactate uptake and lactate efflux, and GPR81 (Figure S10A). MCT1 knockdown reduced the lactate content and MCT4 knockdown elevated the lactate content within NPCs (Figure S10B). In lactate-treated NPCs, MCT1 knockdown alleviated oxidative damage and MCT4 knockdown promoted oxidative damage, whereas GPR81 knockdown resulted in no apparent mitigation (Figures S10C–J), indicating that lactate-induced oxidative damage in NPCs is attributed mainly to the intracellular lactylation.

SOD1K123la aggravated IVDD by impairing SOD1 enzymatic activity.

The specific role of SOD1K123la in IVDD was further investigated in vivo (Figure 4A). SOD1K123R mutant rats were generated by mutating the lysine AAA codon to the arginine AGG codon (Figures 4B–C). SOD1K123R rats did not significantly differ from SOD1WT rats in terms gross phenotype (Figures S11A–K). A PIDD model was then constructed. The magnetic resonance imaging (MRI) results and histological staining revealed that, compared with SOD1WT-PIDD rats, SOD1K123R-PIDD rats presented alleviated degeneration (Figures 4D–G). A SOD1K123la-specific antibody that targeted a modified peptide [VVHE-(lactyl)K-QDDLGKGGC] of SOD1 was constructed and exhibited ideal specificity for the SOD1K123la protein in dot blot assays (Figure 4H), as verified in lactate-treated NPCs with or without SOD1 knockdown by immunofluorescence and western blot (Figures S12A–F). In the NP tissue of SOD1K123R-PIDD rats, the immunofluorescence and western blot results revealed that the SOD1K123R reduced SOD1K123la levels (Figures 4I–J and S13A–B), with declined P53, P21 and GADD45α levels (Figures 4J and S13C–E). The DHE assay results revealed that the O - content was reduced in the NP tissue of SOD1K123R-PIDD rats (Figure 4K), with restored SOD1 enzymatic activity and T-AOC and decreased MDA levels (Figures 4L and S13F–G). Primary NPCs were extracted from SODA1K123R- and SOD1WT-PIDD rats. The ROS content was reduced in SOD1K123R-PIDD rat-derived primary NPCs (Figures 4M–N), and the γ-H2AX intensity and percentage of β-gal-positive cells were decreased (Figures 4O–P and S13H–I). These results suggest that decreasing SOD1K123la levels can alleviate IVDD by increasing SOD1 enzymatic activity and reducing oxidative damage.

To clarify the effect of SOD1K123la on SOD1 enzymatic activity, molecular dynamics simulations were performed (Figure 5A). In the 100 ns simulation, the interaction of the SOD1K123la protein with O - was not stable compared with SOD1WT (Figure 5B). Both the root mean square deviation (RMSD) and radius of gyration (RG) fluctuated significantly after 90 ns of simulation in the SOD1K123la system, indicating decreased system stability (Figures 5C and S14A). Similarly, the solvent accessible surface area (SASA) and root mean square fluctuation (RMSF) characterized the enhanced variation in protein flexibility in the SOD1K123la system (Figures 5D and S14B–C). Compared with SOD1WT, the binding energy between O - and SOD1K123la was lower at 60–100 ns (-251.38 ± 15.66 kJ/mol versus -375.06 ± 18.43 kJ/mol), indicating a weakened enzyme-substrate binding capacity (Figure 5E). This structural shift may result in structural alterations in the active site and the dislocation of copper ions from the active site and hinder the binding of O - and SOD1. Analysis of the secondary structure of SOD1 revealed an elevated coil structure and a reduced sheet structure and turn structure after lactylation, indicating a more disordered protein structure (Figures S14D–F). The docking conformations revealed that residues Glu122 and Arg144 are closer to the SOD1 active site (copper-binding regions) in the SOD1K123la system (Figure 5F). This proximity promoted hydrogen bond formation between Asn140 and lactylated K123, which then caused significant structural alterations in the adjacent region, especially Arg144. The hydrogen bonds between Arg144 and O -, Arg144 and Cys58, and Arg144 and Gly62 were disrupted, and new hydrogen bonds between Arg144 and Lys136 and between Arg144 and His49 formed (Figure 5G). These conformational alterations may constitute the molecular basis for the reduction in the enzymatic activity of lactylated SOD1.

ZL-01 acts as a potential specific inhibitor of SOD1K123la.

The results above indicate that the inhibition of SOD1K123la may alleviate IVDD, suggesting it as a potential therapeutic approach. Previous studies have reported the existence of lactyltransferases, including AARS1, AARS2, GCN5, CBP, P300, TIP60, HBO1, and MYST1. Furthermore, immunoprecipitation-mass spectrometry (IP–MS) analysis revealed variations in the expression of SOD1-interacting proteins before and after lactylation (Figures 5H–I, and S14G). Comparison of the 923 identified proteins with the 8 reported lactyltransferases revealed that AARS1 could interact with lactylated SOD1 in NPCs (Figure 5J). The expression of the above 8 lactyltransferases were knocked down in NPCs (Figure S14H), and immunofluorescence and western blot results revealed that AARS1 and P300 may be involved in SOD1K123la (Figures 5K–Land S14I–J). HDAC1-3 and SIRT1-3 have been shown to be potential delactylases 25, and they were thus overexpressed in NPCs (Figure S14K). Immunofluorescence and western blot results revealed that SIRT1 and HDAC3 overexpression reduced SOD1K123la levels (Figures S14L–O).

To increase the specificity of SOD1K123la inhibition, virtual screening was performed to identify potential compounds with SOD1K123la inhibitory activity. A total of 1.6 million commercially available compounds were screened via the K123 residue of SOD1. The top 1,000 compounds were docked on the basis of total energy (Figure 6A). The top 10 compounds that interacted with the pocket of SOD1 containing the K123 residue (Figures S15A–C). The proper concentrations of the chosen 10 compounds were determined via cell counting kit-8 (CCK-8) assays (Figure S15D). Compound 3 effectively reduced the SOD1K123la level and increased SOD1 activity (Figures S15E–F). The chemical formula of compound 3 is C11H14N4O2 and was named ZL-01. Molecular docking further revealed that ZL-01 binds to Thr40, His44, Lys123, and Asn140 of SOD1 (Figures 6B–C, and S15G). We mutated the aforementioned amino acid residues individually, and microscale thermophoresis (MST) experiments indicated that ZL-01 primarily interacts with SOD1 residues Thr40, His44, and Lys123 (Figure 6D). DHE staining and flow cytometry revealed that ZL-01 alleviated oxidative damage in lactate-treated NPCs (Figures 6E–G and S15H) and ameliorated DNA damage and cellular senescence (Figures 6H–I, 6K–M, and S15I–K). ZL-01 had no significant effect on the acetylation and succinylation of SOD1 in NPCs (Figure S16A–F). In contrast, ZL-01 significantly reduced the pan-lactylation induced by lactate in NPCs, which was mainly manifested at the protein molecular weight around 17 kDa (Figures S16G–I). ZL-01 effectively reduced SOD1K123la levels in lactate-treated NPCs (Figures 6I–J and 6N–O), reversed the decreases in SOD1 enzymatic activity and T-AOC and decreased MDA levels (Figures 6P and S16J–K). Meanwhile, ZL-01 rescued lactate-induced impairment of SOD1 function in SOD1WT NPCs, whereas it resulted in no apparent alteration in SOD1 function in SOD1K123R NPCs. This may have been attributed to the already existing lactylation suppression by SOD1K123R that protects SOD1 function (Figure S16L).

Targeted delivery of ZL-01 reduced oxidative damage and alleviated IVDD.

Different doses of ZL-01 were injected into the NP tissue of PIDD-rats once a week, and assessments of degeneration were performed at 4 weeks post-injury to evaluate the potential therapeutic effects of ZL-01. ZL-01 reduced the histological scores and improve the SOD1 enzymatic activity, but increasing the ZL-01 concentration within a certain range did not significantly increase the therapeutic effect (Figures S17A–C). Moreover, we found that the local concentration of ZL-01 decreased significantly 48 hours after injection (Figure S17D), which indicated its rapid clearance of ZL-01 from degenerated NP tissue.

To improve the drug retention and therapeutic efficacy of ZL-01, we designed a NPC-targeting ZL-01 system based on extracellular vesicles (EVs) (Figure 7A). A collagen type II-targeted peptide (CTP) was genetically modified to the outer surface of the EVs was genetically modified to express a collagen type II-targeted peptide (CTP), yielding CTP-EVs (C-EVs). Transmission electron microscopy showed that both the EVs and C-EVs had a typical disc-like structure (Figure 7B), and highly expressed positive EV markers CD 81 and TSG 101, and weakly expressed negative EV marker calnexin (Figure 7C). Nanoparticle tracking analysis showed that most of the EVs had diameters between 100 and 200 nm (Figures 7D–E). Immunofluorescence staining revealed that compared with GFP-labeled EVs, GFP-labeled C-EVs were more efficiently taken up by NPCs (Figure 7F). ZL-01 was loaded into the C-EVs to obtain ZL-01-loaded C-EVs (C-EVsZ), and sonication at a concentration ratio of 1:6 resulted in better ZL-01 loading efficiency (Figure S18A). Approximately 76% of ZL-01 could be released within 96 h after loading (Figure S18B).

The biocompatibility of the delivery system was comprehensively examined. There were no significant differences in the major indexes of blood routine, liver function, or body weight after the injection of C-EVs, C-EVsZ (1 μM), or C-EVsZ (5 μM) into the intervertebral discs of the rats (Figures S18C–K). No apparent toxicity was observed in hearts, livers, spleens, lungs and kidneys at 4 weeks after drug administration (Figures S18L–Q). C-EVsZ effectively increased local ZL-01 retention (Figures S19A–B). MRI and histological staining revealed that the PIDD rats that received C-EVsZ (5 μM) presented alleviated degenerative alterations (Figures 7G–J). The protein level of SOD1K123la was reduced in the NP tissue of C-EVsZ (5 μM)-treated PIDD rats (Figures 7K–L and S19C–D), with decreased P53, P21, and GADD45α protein levels and the O - content (Figures 7L–M, and S19E–F). Moreover, the SOD1 activity and T-AOC levels in the NP tissue of C-EVsZ (5 μM)-treated PIDD rats increased, and the MDA level decreased (Figures 7N–P). Compared with PIDD model rat-derived NPCs, primary NPCs extracted from C-EVsZ (5 μM)-treated PIDD rats presented lower total ROS levels (Figures 7Q–R) and alleviated DNA damage and cellular senescence (Figures 7S and S19H–J). These results suggest that compared with direct injection of ZL-01, C-EVsZ can maintain drug retention and achieve better therapeutic effects for treating IVDD. A schematic model was drawn to summarize our findings (Figure 8).

Methods
Human NP tissue acquisition

All patient sample collection were reviewed and approved by the Ethics Committee of Xinqiao Hospital, Army Medical University (Approval No. 2023-YD184-01; Approval Date: December 28, 2023). NP tissue samples (solid tissue) from 12 male patients and 12 female patients aged 18–65 years were obtained during intervertebral surgery, following the standard surgical protocols of Xinqiao Hospital, Army Medical University. Sex of the patients was determined based on assigned. Informed consent and clinical data collection protocols were signed prior to surgery. The inclusion criteria were patients with lumbar fracture, lumbar spondylolysis, intervertebral disc herniation, or lumbar spinal stenosis (diagnosed in accordance with the International Classification of Diseases, 11th Revision, ICD-11) and a body mass index between 18 and 24. The exclusion criteria were diabetes, metabolic diseases, severe chronic diseases, hematological diseases, tumors, lumbar infection, or previous lumbar surgery. All patients were alive at the time of NP tissue collection. Immediately after collection from surgical segments, NP tissues were rinsed with sterile 0.9% NaCl solution to remove residual blood and tissue debris. NP tissues free of blood clots or foreign materials were included in the study, with one portion of the tissue was used for extraction of primary NPCs and the other flash-frozen in liquid nitrogen within 10 min. After stabilization, NP tissue specimens were transferred to an ultra-low temperature freezer at -80 °C for long-term preservation, and subsequent experiments were to be completed within one month of freezing. Patient details were provided in Supplementary Table 1.

Experimental animals

All the animal experiments in the present study were approved by the Laboratory Animal Welfare and Ethics Committee of the Army Medical University (Approval No. AMUWEC20235167; Approval Date: December 1, 2023). Given the anatomical-physiological similarity of rat and human intervertebral discs, plus well-established IVDD modeling methods, rats were selected for animal experiments. To avoid hormonal interference, we used only male rats; hence male Sprague-Dawley (SD) rats were purchased from the Chengdu Dashuo Experimental Animal Co., Ltd. The rats were housed in a vivarium with a 12 h light/dark cycle at a temperature of 20–24 °C and a humidity of 40–60%, with unrestricted access to food and water. The animal sample size (α error probability: 0.05; power: 0.8) was determined via the online website http://powerandsamplesize.com/. During the experiment, physical condition monitoring of rats was performed twice weekly, with humane endpoints defined as the occurrence of severe tissue damage or infection. There was no exclusion of animal experimental data in the present study.

Generation of SOD1K123R rats

Male SOD1K123R rats were designed and generated via CRISPR-Cas9 by Cyagen Biosciences, Inc. (Guangzhou, China). The rat Sod1 gene (GenBank accession number: NM_017050.1; Ensembl: EnsRNOG00000002115) is located on rat chromosome 11. Five exons were identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 5 (transcript: NM_017050.1). p.K123 is located in exon 5, and exon 5 was selected as the target site. gRNA (target sequences: gRNA-A1, CCAAGTCATCTTGTTTCTCG-TGG; and gRNA-B1, CCA CGA GAAA CAAGATGACT-TGG) and a donor oligo (with a targeting sequence flanked by 120 bp homologous sequences on both sides) were designed. Cas9, gRNA, and donor oligos were coinjected into fertilized eggs for mutant rat production. The p.K123R mutation (AAA to AGG) in the donor oligo was introduced into exon 5 via homology-directed repair. The pups were genotyped via PCR followed by sequence analysis.

Open field test

An open field test was conducted to evaluate the locomotor activity and anxiety-like behaviour of the rats. All tests were performed in a sound-attenuated, dimly lit room. The apparatus consisted of a square open-field arena (60 cm × 60 cm) made of transparent acrylic, with the floor divided into 25 equal grids. The central zone was defined as the inner grids (9 grids). For each test, a single rat was gently placed in the centre of the arena, and its behaviour was recorded continuously for 5 min. After the test, the arena was thoroughly cleaned to prevent cross-contamination. A video tracking system (Noldus EthoVision XT) automatically extracted the target parameters.

Experimental animal models

A research protocol was formulated prior to the initiation of each segment of the in vivo experiment. A single rat represented an experimental unit. Three different types of rat IVDD models were utilized in this work. Two-month-old male SD rats, weighing between 250 g and 290 g, were randomly assigned to different groups. Before surgery, the rats were effectively anesthetized. Rats were placed in the prone position and their tails were sterilized using iodophor. For the PIDD model69,70, after positioning the rat caudal disc by palpation or X-ray, a 21-gauge needle fitted with a depth limiter was inserted vertically into the disc between caudal vertebrae 5 and 6 at a depth of 5 mm. The needle was rotated 360 degrees and held in place for 30 s and removed vertically. The surgical area was disinfected after operation. Rats in control group did not receive any treatment (Ctrl). For the LAIDD model11, 2 μL of PBS or sodium lactate (2 mM, 6 mM, or 10 mM) was injected into the intervertebral disc between caudal vertebrae 5 and 6 via a 26-gauge needle and a microsyringe (7105KH; Hamilton)71. Rats in the control group received only microsyringe puncture (Sham). For in vivo experiments with ZL-01 or engineered EVs, after puncturing the intervertebral disc following the aforementioned method, 2 μL of PBS, ZL-01 (1 mM, 5 mM, 10 mM or 20 mM) or different kinds of C-EVs was injected into the intervertebral disc between caudal vertebrae 5 and 6 via a 26-gauge needle and a microsyringe. Rats in the control group received only microsyringe puncture (Sham). Each rat was administered with analgesics and antibiotics for 3 days. Four weeks after the operation, imaging data were obtained, and the rats were anesthetized and killed to obtain the intervertebral discs for subsequent experiments. For the AIDD model 72, 20-month-old male rats, weighing between 600 g and 670 g, were used as the natural aging group, and 2-month-old male SD rats weighing between 250 g and 290 g were used as controls (Ctrl). The rats in this group were directly used for imaging data acquisition and subsequent experiments without any treatment. Pfirrmann grade was the primary outcome measure in the rat IVDD model, and histological score, oxidative stress markers, and senescence markers were secondary outcome measures.

Blinding for in vivo experiments

Blinding was intended to reduce bias at different stages of the in vivo experiment, and the personnel aware of the group allocation are described as follows: For randomization, rats to be grouped were numbered, and the number of experimental groups (the number of ranks) was determined. Subsequently, the RAND function and RANK.EQ function in Microsoft Excel were used to assign a unique rank to each rat, and rats with the same rank were assigned to the same group. During group allocation, only experimental designer had full knowledge of the complete group allocation details. Surgical operators only received coded labels and were unaware of the actual intervention types corresponding to each group (sham group, IVDD model group, or treatment group). During the conduct of the experiment, due to differences in intervention methods or body size variations among rats in different groups, surgical operators could not be blinded. Animal feeding and care staff only had access to cage labels with codes and were unaware of the actual group assignments. During outcome assessment and data analysis, the experiment operator first removed all group identifiers from the experimental results and provided the coded data to independent researchers for preliminary analysis. After verifying the accuracy of the data, the research team performed unblinding, matched the codes with the actual groups, and conducted the final result interpretation. The confounders were not controlled in the in vivo experiments.

ScRNA-seq Analysis

ScRNA-seq data from human NP tissue were downloaded from the Gene Expression Omnibus (GEO) database (GSE165722 and GSE251686) and processed using the Seurat software package. Low-quality cellular data with less than 200 or more than 6000 expressed genes were excluded using the Harmony package, and then batch effects were mitigated using the Runharmony function. Subsequently, cells were categorized according to the expression patterns of classical markers. Samples were combined using the UMAP function and projected into 2D space using the first 30 principal components. Cells were then unsupervised clustered using the FindClusters function at a resolution of 0.5. Highly expressed genes relative to other clusters were identified using the FindAllMarkers function with a threshold of log2FC> 0.25 and P Adj < 0.05. The NPCs were clustered using UMAP, unsupervised clustering. The R package clusterProfiler was used to perform GO enrichment analysis, setting GO terms with P and FDR < 0.05 as significantly enriched. The ggplot2 package was used to visualize some of the GO enrichment results. Pseudo-temporal analysis of NPC timings in each sample was performed using the Monocle 2 software package. Pseudotemporal-dependent gene expression was analyzed in an unsupervised manner in all NPCs. The candidate pathways were then scored using the geom_smooth function in Seurat, and the dynamics of the pathway scores over pseudo-time were displayed using the geom_smooth function in the ggplot 2 software package.

Primary NPC isolation and lactate treatment

NP tissues were isolated from patients’ surgical biospecimen or 2-month-old male rats, and digested with 0.2% type II collagenase (C2-BIOC; Sigma‒Aldrich, USA) for approximately 2 h at 37 °C, followed by centrifugation at 400 × g for 5 min. The cell pellet was suspended, and the cells were cultured in DMEM/F12 (BI, Israel) supplemented with 10% fetal bovine serum (C04001500, VivaCell, China) and 1% penicillin/streptomycin (C0222, Beyotime, China) at 37 °C in 5% CO . For lactate treatment, NPCs were treated with different concentrations of sodium lactate for 7 days. The medium was changed every 3 days.

Metabolomic analysis

Metabolomic analysis was conducted by PANOMIX Biomedical Tech Co., Ltd. (Suzhou, China) using an ExionLC UHPLC system and an AB Sciex Triple Quadrupole 6500 plus mass spectrometer (AB Sciex, USA). Fifty milligrams of fresh human NP tissues was mixed with 500 μL of methanol, and then shaken at 1500 rpm, and centrifuged at 12,000 × g for 10 min at 4 °C. A 100 μL supernatant aliquot was combined with a mixed internal standard, dried under nitrogen gas, and reconstituted in 50% acetonitrile. After centrifugation, the supernatant was analyzed using Multiple Reaction Monitoring for quantitative metabolite analysis. Chromatographic peaks were integrated and quantified via standard curves.

Whole-protein lactylation proteomics

The whole-protein lactylation proteome analysis was conducted with the support of PTM Bio, Inc. (Hangzhou, China). Rat NP tissues in lysis buffer (8 M urea, 1% protease inhibitor cocktail, and 3 μM TSA) were sonicated via an ultrasonic processor (Scientz), and digested with trypsin. To enrich the modified peptides, tryptic peptides were incubated overnight at 4 °C with anti-L-lactyllysine antibody-conjugated agarose beads (PTM-1404, PTM Bio). The bound peptides were eluted from the beads with 0.1% trifluoroacetic acid. The eluted fractions were combined and vacuum dried. The peptides were desalted with C18 ZipTips (Millipore). For LC‒MS/MS analysis, the peptides were separated via a nanoElute UHPLC system (Bruker Daltonics) and exposed to a capillary source. The peptides were analyzed via a timsTOF Pro (Bruker Daltonics) mass spectrometer. The resulting MS/MS data were processed via the MaxQuant search engine (v.1.6.15.0). Tandem mass spectra were searched against the human SwissProt database (20422 entries) concatenated with the reverse decoy database, with an FDR < 1%. After centralizing the signal intensity values in different samples, the relative quantitative values of the modified peptides were obtained. The lactylated peptide data were normalized to the respective protein expression levels.

Construction of overexpression and knockdown vectors and transfection

Gene overexpression vectors for rat Sirt1-3, Hdac1-3, Sod1WT (synonymous mutation), Sod1K123R, and Sod1K10R were designed and constructed by Tsingke Biotech (Beijing, China) in pLV4ltr-Puro-CMV-C3 × FLAG plasmids; synonymous Sod1 mutagenesis was performed to avoid shRNA interference. For knockdown, pLKO.1-Puro plasmids with shRNAs targeting Aars1, Aars2, Gcn5, Cbp, P300, Tip60, Hbo1, Myst1, Sod1, Mct1, Mct4, and Gpr81 were created. Lentiviruses were packaged in HEK293T cells (CRL-3216, ATCC) and used to transfect cultured NPCs following the manufacturer’s instructions, with the medium replaced after 12 h. Transfected NPCs were selected with 2 μg/mL puromycin (ST551, Beyotime), and mRNA levels were measured to assess transfection efficiency. Sequences of the shRNA were provided in the Supplementary Table 2.

β-gal staining assay

NPCs received different treatments were prepared for β-gal staining by aspirating culture medium, washing 3 times with pre-warmed PBS, fixing with 4% PFA at room temperature for 15 min, and washing again 3 times with pre-warmed PBS. β-gal staining solution was prepared following the instructions of a β-gal Staining Kit (C0602, Beyotime) and added to fixed NPCs followed by overnight incubation. After incubation, staining solution was aspirated, and cells were washed 3 times with PBS. The percentage of β-gal-positive cells was calculated by examining 300 cells in five microscopic fields. Images were captured with an inverted phase-contrast microscope (IX83, Olympus).

RNA-seq and bioinformatics analyses

RNA-seq was performed by Maiwei Biotechnology Co., Ltd. Total RNA from NPCs was isolated using TRIzol and quantified using a NanoDrop 2000 (NanoDrop, U.S.A.). cDNA libraries were constructed with SuperScript™ II reverse transcriptase. Then, 2 × 150 bp paired-end RNA-seq was performed using the Illumina NovaSeq platform. The significantly DEGs were filtered with the thresholds of a |log2(fold change, FC)| > 2 and a P value < 0.05 using the R package DESeq2. Transcription factor prediction was performed using the STRING database, CHEA3 database or Cytoscape software, and the data were filtered using the cytoHubba plugin.

MST test

Purified SOD1WT and SOD1T40A, SOD1H44A, SOD1K123A, and SOD1N140A mutant proteins were prepared by Tsingke Biotech Co., Ltd. (Beijing, China). pGEX-4T2 plasmids expressing GST-tagged SOD1 variants were transformed into BL21CodonPlus (DE3)-RIL cells, induced with IPTG, lysed, and purified via chromatography and filtration, followed by removal of the GST tag. The purified proteins were labelled with MST fluorescent dye (MO-L011, NanoTemper) per the manufacturer’s protocol. ZL-01 was serially diluted in 50 mM HEPES buffer (0.05% Tween 20; pH 7.4) and incubated with labelled SOD1 for 30 min at room temperature. Affinity was measured using with three replicates, and Kd values were determined using Monolith software.

IP–MS analysis

The SOD1 protein was purified by IP, followed by 12% SDS–PAGE at 125 V for 1 h. The gel slices were rinsed with decolorizing solution for 30 min, reduced, alkylated, and digested with trypsin at 37 °C for 20 h. The peptides were desalted, vacuum-dried, resuspended in 0.1% formic acid, and then analysed using an Easy-nLC 1000 system (Thermo Fisher) eluted with 0.1% aqueous formic acid (mobile phase A) and 0.1% formic acid in 84% acetonitrile (mobile phase B). The column was equilibrated with 95% mobile phase A, and samples were loaded via an autosampler. IP–MS was performed in positive ion mode (scan range, 300–1800 m/z; resolution at 200 m/z, 70,000; AGC, 1e6; max IT, 50 ms; and dynamic exclusion, 30.0 ms), acquiring 20 MS2 spectra per scan with HCD activation (isolation, 2 m/z; resolution at 200 m/z, 17,500; collision energy, 27 eV; and underfill, 0.1%). Data were analysed with Proteome Discoverer 2.5, and the spectra were searched against the UniProt Rattus norvegicus database (93045_20240115) using trypsin, carbamidomethyl C as a fixed modification, protein/methionine oxidation as a variable modification, a maximum of 2 missed cleavages, and high-confidence peptide filtering.

MRI evaluation

A 3.0T animal MRI system (Bruker Pharmascan, Germany) was used to assess the signal and structural changes in rat intervertebral discs via sagittal T2-weighted images. The parameters for the T2-weighted sections were set as follows: TR time, 2000 ms; TE time, 80 ms; incentive time, 2; scan time, 3 min 20 s; fat reduction technology, SPAIR; scan matrix, frequency encoding 368 and phase encoding 288; layer thickness, 2.5 mm; echo chains, 12; and spin echo sequence, TSE. The classic Pfirrmann classification criteria 73 were used to evaluate MRI images of patients. The modified Pfirrmann classification criteria74 were used to evaluate MRI images of rats. To ensure the reliability, the grading was performed by three spinal surgeons (observers A, B, and C). All of them had more than 8 years of experience in spinal imaging diagnosis and received standardized training prior to grading. Each observer was allowed to interpret only 20 sets of images per day to avoid fatigue. During grading, each observer could refer to the classification criteria. In the case of any discrepancy among them, the final decision was based on the majority opinion. The weighted Kappa coefficient was used to assess intra- and inter-observer consistency, and the results were presented in Supplementary Tables 3 and 4.

Histological evaluation

Decalcified rat intervertebral discs or organs were dehydrated, embedded in paraffin, and sliced into 5 μm paraffin sections. The prepared sections were stained with a modified SO & FG Staining Kit (G1371, Solarbio) or a Hematoxylin–Eosin (HE) Staining Kit (G1120, Solarbio) according to the manufacturer’s instructions. Images were captured with an inverted phase-contrast microscope (CKX53, Olympus). The histological grade was calculated following the previous methods 75. The grading was performed by three researchers (observers D, E, and F), and all of them had over 3 years of experience in the establishment and evaluation of the rat caudal vertebra IVDD model and received standardized training prior to grading. Each observer was allowed to interpret only 20 sets of images per day to avoid fatigue. During grading, each observer could refer to the classification criteria. In the case of any discrepancy among them, the final decision was based on the majority opinion. The intra-class correlation coefficient (ICC) was used to assess the intra- and inter-observer consistency, and the results were presented in Supplementary Table 5.

Real-time PCR

Total RNA was extracted from NPCs with TRIzol reagent and reverse transcribed into cDNA via a PrimeScript ERT Kit (RR047A, Takara) according to the manufacturer’s protocol. A PCremix Ex Taq II kit (RR390A, Takara) was used to amplify and detect the relative mRNA expression levels of target genes with a CFX Connect Real-Time System (Bio-Rad). Target gene mRNA expression was normalized to β-actin mRNA expression, and relative mRNA expression was calculated via the 2-ΔΔCt method. Information of the primers used for real-time PCR was provided in Supplementary Table 6.

Immunofluorescence staining assay

NPCs were fixed with 4% paraformaldehyde and permeabilized with immunostaining permeabilization buffer. Tissue sections were deparaffinized, rehydrated, and subjected to antigen retrieval. Samples were blocked with QuickBlock immunostaining buffer and incubated overnight at 4 °C with primary antibodies, including anti-γ-H2AX (AP0687, ABclonal), anti-SOD1 (MA1-105, Thermo Fisher), anti-pan lactylation (PTM-1401RM, PTM BIO), and anti-SOD1K123la (customized, PTM BIO) antibodies. Samples were incubated with fluorescently labeled rabbit and mouse (ab150113, ab150080, Abcam) secondary antibodies for 1 h at 37 °C. Hoechst 33358 was used to counterstain nuclei. Fluorescence images were acquired using a confocal microscope (LSM880, ZEISS, Germany) and quantified with ImageJ.

O - and total ROS detection

Intracellular O₂- production was evaluated using 10 μM DHE (HY-D0079, MCE) and incubating cultured NPCs at 37 °C for 30 min in the dark, after which images were captured with a confocal microscope. The excised NP tissue was homogenized on ice and centrifuged at 4 °C, and the supernatant was treated with 10 μM DHE for 30 min at 37 °C in the dark, after which the fluorescence intensity was measured by a spectrophotometer (M2, Molecular Devices) and normalized to that of protein mass (mg). ROS levels were assessed using a total ROS detection kit (88-5930-74; Invitrogen) and flow cytometry. NPCs and fresh NP tissue were digested into single-cell suspensions stained at 37 °C, and analysed using a flow cytometer (Gallios, Beckman).

Quantification of L-lactate levels

The concentration of L-lactate content in NP tissue and NPCs was assessed using a L-lactate assay kit (ab65331, Abcam). Briefly, NPCs or milled NP tissues were mixed with assay kit buffer while a gradient concentration of lactate standard was prepared. The analytical buffer was added to the above samples and incubated at room temperature for 30 min, then the optical density value at 450 nm was measured using a spectrophotometer, and a standard curve was plotted between the concentration of the lactate standard and the optical density value, and the concentration of lactate in the samples was calculated by obtaining the equation of the standard curve.

Measurement of the MDA level, T-AOC, and SOD1 activity

MDA levels, T-AOC, and SOD1 activity in NPCs were measured using an MDA assay kit (S0131M, Beyotime), a T-AOC assay kit (S0121, Beyotime), and a Superoxide Dismutase Assay Kit (E-BC-K022-M, Elabscience), following the manufacturers’ instructions. Briefly, protein concentration in lysates was quantified, mixed with assay buffers and working enzymes, incubated under specified conditions, and measured for optical density at the respective wavelengths using a spectrophotometer (M2, MOLECULAR).

Molecular dynamics simulations

The SOD1 conformation was sourced from the Protein Data Bank (PDB ID: 1PU0). Homology modeling was performed using Modeller v9.19, with a lactyl group added to the K123 residue via YASARA. Molecular docking of O₂⁻ with SOD1 was conducted using AutoDock 4.2.6. Molecular dynamics simulations were executed with Gromacs 2018.4, employing the Amber14SB force field and TIP3P water model under periodic boundary conditions. Long-range electrostatics were calculated using the particle mesh Ewald method (1.0 nm cutoff), and van der Waals interactions used a 1.0 nm cutoff, with hydrogen bonds constrained by the LINCS algorithm. Temperature was maintained at 300 K via the V-rescale method, and pressure at 1 bar with the Parrinello-Rahman method. SOD1WT and SOD1K123la were energy minimized, equilibrated for 100 ps under NVT at 300 K, and run for 100 ns under NPT conditions with a 2 fs time step, saving trajectories every 10 ps (5,000 snapshots). Analysis was performed by Mode Science and Technology Co., Ltd. (Chengdu, China).

High-throughput virtual screening

Schrödinger Maestro version 11.4 was used for structure-based virtual screening. The X-ray crystal structure of SOD1 (PDB ID: 1PU0) was obtained from the RCSB Protein Data Bank and further processed via the Protein Preparation Wizard module of Schrödinger. A ChemDIV screening library containing 1.6 million compounds was utilized for virtual screening after energy optimization by the LigPrep module of Schrödinger. The optimized compounds were subjected to a virtual screening workflow for molecular docking. All the compounds were screened in Glide HTVS mode, and the top 10% of the scored compounds were obtained to perform second-round Glide Standard Precision docking. The top 10% of the Glide Standard Precision docking results was docked in the third round using Glide Extra Precision. The top 10 compounds with the highest absolute docking scores were selected as candidates for further experimental validation. The 2D and 3D compound‒protein docking modes were created via PyMol. The analysis was performed by TargetMol Chemicals, Inc. (Shanghai, China), and the solid compounds were also purchased from this company. A summary of virtual screening and validation of candidate compounds was provided in Supplementary Table 7.

CCK-8 assay

A CCK-8 reagent (C0038, Beyotime) was employed to evaluate the cytotoxic effects of various compounds on NPCs. NPCs were seeded in 96-well plates at a density of 5×10³ cells per well. Following cell adhesion, 10 candidate compounds were individually added to the respective wells and incubated for 48 hours at final concentrations of 0.5, 1, 2.5, 5, 10, 20, and 50 μM. After replacing the culture medium with fresh complete medium, 10 μL of CCK-8 reagent was pipetted into each well, followed by incubation at 37 °C for 2 hours. A spectrophotometer was utilized to measure the optical density (OD) values at a wavelength of 450 nm.

Western blot, Native PAGE, and IP

For western blot electrophoresis, NP tissues or NPCs were lysed in Western and IP cell lysis buffer (P0013, Beyotime) supplemented with protease inhibitors (P1008, Beyotime) and trichostatin A (Selleck). The protein concentration was determined using a BCA assay kit (P0010s, Beyotime). Proteins were resuspended in SDS–PAGE loading buffer (P0015, Beyotime), boiled, and separated by 10% or 12% SDS–PAGE, then transferred to PVDF membranes (Merck, Germany). Membranes were blocked with QuickBlock Western solution (P0252, Beyotime) and incubated overnight at 4 °C with primary antibodies, including anti-P21 (ab109199, Abcam), anti-GADD45α (ab54210, Abcam), anti-SOD1 (ab13498, Abcam), anti-pan lactylation (PTM-1401RM, PTM), anti-SOD1K123la (customized, PTM), anti-pan acetylation (PTM-101, PTM), anti-pan succinylation (PTM-419, PTM), and anti-β-actin (66009-1-Ig, Proteintech) antibodies. After being washed with TBST, the membranes were incubated with secondary antibodies (SA00001-2 or SA00001-1, Proteintech). Bands were visualized using an ECL kit (1705060, Bio-Rad) and imaged with a ChemiDoc system (Bio-Rad), and the grey values were analysed using ImageJ software and β-actin as the internal control.

For Native PAGE electrophoresis, NPC lysates were prepared using the NativePAGE Sample Preparation Kit (BN2008, Thermo Fisher). After addition of Blue Native PAGE Sample Buffer (P0761, Beyotime), electrophoresis was performed on 4-13% Blue Native PAGE precast gels (P0546S, Beyotime) using cathodic electrophoresis buffer. The other procedures were the same as western blot. For IP electrophoresis, the supernatant was collected, and the lysate was retained as the input group. For the IP group, IgG or anti-SOD1 antibody was conjugated to protein A+G magnetic beads. The IgG-conjugated beads are used to remove nonspecific proteins from the sample that bind to the anti-SOD1 antibody. Proteins in the lysate were precipitated via IgG antibody-bound magnetic beads or SOD1 antibody-bound magnetic beads, and proteins in the input group and the IP group were denatured and separated via SDS‒PAGE loading buffer (P0015, Beyotime). boiled proteins samples were separated by SDS-PAGE, then the coomassie brilliant blue staining (P0003M, Beyotime) was performed to visualize proteins in immunoprecipitated samples. Relative quantification of proteins was then performed via western blot. Uncropped scans of blots were provided in the Source Data file.

Construction of CTP-EGFP-Lamp2b lentivirus and transfection

The Lamp 2b-CTP lentivirus was constructed and synthesized by OBiO Technology Co., Ltd. (Shanghai, China). Briefly, the Lamp2b + EGFP + CTP sequence was constructed, and after digestion by XmaI and EcorI restriction enzymes, Lamp2b-EGFP-CTP and the lentiviral vector psLenti-CMV-MCS-3xFLAG-PGK-Puro-WPRE were purified on gel to construct the CTP-EGFP-Lamp2b lentivirus. The CTP-EGFP-Lamp2b lentivirus was transfected into NPCs after determining the viral titer, and then the stably transfected cells were screened using G418.

Isolation and characterization of EVs, C-EVs and C-EVsZ

NPCs stably expressing CTP-EGFP-Lamp2b were cultured in DMEM/F12 supplemented with 10% exosome-free serum (C3801-0100, Viva Cell, China) and 1% penicillin/streptomycin. C-EVs or EVs were extracted using Total Exosome Isolation Reagent (4478359, Invitrogen, USA). EV particle size was analysed by nanoparticle tracking analysis (ZetaView PMX 110, Particle Metrix, Germany), and their morphology was visualized by transmission electron microscopy (JSM-1400PLUS, JEOL, Japan). EV positive markers CD81, TSG101, and negative marker calnexin were detected by western blot.

ZL-01 was loaded into C-EVs by sonication at ratios of 1:1 (1 mg/ml C-EVs to 1 μM ZL-01), 1:3 (1 mg/ml to 3 μM), and 1:6 (1 mg/ml to 6 μM), with 10 cycles of 30 s of sonication and 30 s in an ice bath, followed by a 2 h incubation at 37 °C to restore EV membrane integrity. ZL-01 concentration was quantified by UV spectrophotometry (Infinite M200 Pro, Tecan, Shanghai, China), and loading efficiency was calculated as the ratio of loaded to total ZL-01. For release testing, 5 ml of C-EVsZ in a dialysis bag was immersed in 25 ml of release medium, with 500 μl sampled at designated times (replaced with fresh medium), and release rate was determined as the ratio of ZL-01 in the medium to the total amount. NPCs labeled with DiD (HY-D1028, MCE) were washed with PBS, mixed with EVs, and incubated at 37 °C. Uptake of DiD-labeled NPCs by EGFP-carrying EVs was observed by confocal microscopy.

High-performance liquid chromatography (HPLC) detection of ZL-01 in the NP tissue of rats

All standards and samples were analysed using an HPLC system (UltiMate 3000 RS, Thermo Fisher, USA) with a triple quadrupole mass spectrometer (TSQ Quantum, Thermo Fisher, USA). Chromatographic separation was performed with a 4.6×150 mm, 5.0 μm column (Welch, China) using a mobile phase of 0.1% formic acid in HPLC-grade water and methanol (20:80, v/v) at a flow rate of 0.8 ml/min being filtered through a 0.22 μm Millipore filter. The calibration samples were ZL-01 standard solutions prepared in methanol at concentrations of 20, 50, 200, 500, 2000, and 5000 nM. NP tissue was treated with methanol and hexane and centrifuged at 12,000 rpm for 10 min at 4 °C, after which the organic layer of the supernatant was collected, the solvent was removed by evaporation with 50% nitrogen, and the residue was diluted with methanol, filtered, and analysed by HPLC.

Quantification and statistical analysis

Statistical assessment and analysis were performed using GraphPad Prism 9 and SPSS v26.0. The Shapiro‒Wilk test was used to test for data normality, and Levene’s test for homogeneity of variance. If the normality test was passed, statistical significance was determined by unpaired, two-tailed Student’s t-test with a confidence level of 95% for comparisons of two groups or one-way analysis of variance (ANOVA) followed by Dunnett’s test for comparisons between multiple experimental groups and a single control group, or Tukey’s test for pairwise comparisons among all groups for comparisons of more than two groups. If the normality test was not passed, statistical significance was determined by the Mann-Whitney test for comparisons of two groups or the Kruskal-Wallis test with Bonferroni-corrected Dunn’s post hoc test for comparisons of more than two groups. Inter- and intra-observer consistency for ordinal variables was verified by weighted Kappa coefficient or intra-class correlation coefficient (ICC). All experiments were performed with at least three individual biological samples and three technical replicates. All error bars represent the mean ± standard deviations derived from at least three independent experiments. Researchers were blinded to the evaluation of experimental outcomes and analysis of the raw data.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript presents a compelling and well-structured study that uncovers a key mechanistic link between SOD1\nlactylation and intervertebral disc degeneration (IVDD). The authors integrate multi-omics approaches, structural biology,\nand in vivo experiments to reveal that lactylation at lysine 123 impairs SOD1 enzymatic activity via conformational changes,\nthereby contributing to oxidative damage and IVDD progression. The study further identifies ZL-01, a small molecule that\ninhibits this specific lactylation, and demonstrates its therapeutic efficacy when delivered via engineered extracellular\nvesicles.\nMajor Strengths:\nThis study presents a highly original and impactful investigation that for the first time delineates the lactylation landscape\n(lactylome) in IVDD and uncovers a key post-translational modification mechanism involving SOD1K123 lactylation. By\nintegrating multi-omics profiling, in vitro and in vivo functional validation, and advanced structural modeling, the authors\nprovide a comprehensive mechanistic explanation of how SOD1K123la impairs enzymatic activity, induces oxidative\ndamage, activates the p53 signaling pathway, and accelerates IVDD progression. Moreover, the identification of a small-\nmolecule inhibitor, ZL-01, through high-throughput virtual screening and its effective targeted delivery via engineered\nextracellular vesicles demonstrate strong translational potential, highlighting a promising therapeutic strategy for treating\nIVDD.\nMajor Concerns:\n1. The level of pan-Kla showed in Figures 1I and Figures S2M-N should provide statistical analysis results. At the same\ntime, the representative image in Figure 1I should be replaced with more representative results.\n2. In the results of Figure 1, why did the authors focus on cellular senescence, oxidative stress, hypoxia, and glycolysis in\nthe single-cell data analysis? Additionally, why was the metabolite lactate highlighted in the metabolomic analysis rather\nthan other metabolites? Please provide justification in the Results or Discussion section to clarify the rationale and enhance\nthe logical flow of the manuscript.\n3. The use of exogenous lactate to induce IVDD may not fully replicate the chronic microenvironmental shifts seen in human\ndisease. How did the author determine the injection concentration of lactate? The authors should discuss this limitation and\njustify its relevance.\n4. Among the many lactylated lysine residues identified in the proteomic dataset, what criteria were used to prioritize SOD1\nfor functional validation? Clarifying this would help justify the depth of investigation focused on a single site.\n5. Although ZL-01 is described as an FDA-approved compound, its precise prior use or safety profile is not discussed.\nClarification of ZL-01’s pharmacokinetics, prior human usage, or potential off-target effects is essential for translational\nclaims.\n6. To demonstrate the specificity of ZL-01 in inhibiting SOD1, comparative experiments on the enzymatic activity regulation\nof SOD1 WT and SOD1 K123R by ZL-01 should be included in Figure 6.\n7. Has ZL-01 been tested for its influence on other known post-translational modifications (e.g., acetylation, succinylation) ?\nHas the effect of ZL-01 on global protein lactylation levels been assessed? While it appears specific to SOD1K123la in this\nstudy, potential broad suppression of lactylation across the proteome might carry unintended biological consequences. A\nglobal Kla Western blot after ZL-01 treatment would be informative.\n8. As lysine residues are often subjected to multiple competing modifications (e.g., acetylation, ubiquitination), do the\nauthors anticipate potential crosstalk between SOD1K123la and other PTMs? This point could be expanded in the\ndiscussion to provide broader mechanistic context.\n9. Some supplementary figures are dense and lack legible axis labels (e.g., S3, S6). High-resolution versions or simplified\nschematics would improve accessibility.\n10. The manuscript would benefit from careful proofreading for minor grammatical inconsistencies and overuse of passive\nvoice.", "reviewer_2": "The study, titled \"SOD1 Lactylation Induces Aberrant Conformational Alteration to Impair Its Enzymatic Activity and\nAggravate Intervertebral Disc Degeneration\", describes the lactylation of superoxide dismutase 1 (SOD1) as a key treatment\ntarget of intervertebral disc degeneration, the primary cause of low back pain. This is an elegant study which consists of in\nvitro, in vivo, and in silico assessments including advanced bioinformatic investigation. As the authors describe, lactate\naccumulation is one of the primary hallmarks of the intervertebral disc, the largest avascular, hypoxic, low-nutrient organ in\nthe human body. The study design is of course complicated; however, the authors kept in mind the data presentation as\nsimple and understandable throughout. Nevertheless, due to the complexity, there are still points to be clarified in the current\nmanuscript, in particular the methodology. Therefore, the reviewer recommends \"major revision\" as the decision to this\nmanuscript review. The reviewer's suggestion includes the following.\nSpecific points\nIntroduction\nAs the intervertebral disc is the largest avascular, hypoxic, low-nutrient organ in the body, depending on the diffusion of\nnutrition and oxygen through the cartilage endplates of the intervertebral disc, the authors should highlight the reason and\nmotivations of why they focused on the accumulation of lactate in the disc in greater details.\nIn p. 4 ll. 73 to 75, despite \"Previous studies\", there is only one reference.\nResults\nIn p. 5 ll. 108 to 110, the detailed information on the datasets for the single cell RNA sequence analysis should be described\nin greater details, including the patient number, age, sex, segment, disease, and surgery type. The dataset of Pfirrmann\ngrades II to III should include many patients with scoliosis whereas that of Pfirrman grades IV to V would come from many\ndegenerative discs. Therefore, in particular genetic backgrounds, the disease difference must be interpreted carefully.\nIn p. 6 ll. 122 to 123, the detailed information is shown in the supplementary table 1. This supplementary tables should also\nbe addressed here within the results section. In addition, surgery type (discectomy, decompression, and fusion surgery)\nshould be included in the supplementary table 1.\nIn pp. 6 to 7 ll. 131 to 135, animal models of annular puncture-induced disc degeneration (PIDD), natural aging-induced disc\ndegeneration (AIDD), and lactate injection-induced disc degeneration (LAIDD) should be clarified in much greater details. In\nPIDD, how did the authors determine the use of 21-G needle? How did the authors measure the accurate insertion of 5 mm?\nDid the authors apply the aspiration of the disc nucleus pulposus? In LAIDD, why did the authors select 26-G needle? The\nreviewer feels the application of 26-G needle can be too big for the local drug delivery to rat tail discs, possibly causing\ntraumatic disc disruption. How did the authors decide the injection volume of 2 μL? Was there no violation of disc tissues? In\nthis injection, how much length of needle insertion was performed? How did the authors identify the disc segment, by X-ray\nfluoroscopy? Please address preliminary data of baseline disc degeneration (radiography, magnetic resonance imaging,\nand histomorphology) and/or background literature evidence in much greater details. Additionally, explain the age (no data\nin LAIDD), sex (all male?), and weight (AIDD rats should be very big) of these animals.\nFor the other injection intervention, was the 26-G needle used?\nIn p. 10 ll. 209 to 211, describe the exclusion of off-target effects of the applied RNA interference using short hairpin RNAs.\nIn p. 11 ll. 239 to 242, despite \"previous studies\", there are no references.\nIn p. 16 l. 347 and thereafter, how much volume of ZL-01 did undergo the intradiscal injection?\nIn this study, despite the additional use of extracellular vesicles (EVs) to aim a prolonged efficiency of ZL-01, why did the\nauthors inject ZL-01-loaded EVs every week? Every week intradiscal injection is very traumatic, potentially inducing disc\ndamage and degenerative changes. Clinically, agents which require the intradiscal injection every week would not be\nacceptable as the treatment drug for intervertebral disc degeneration. Currently, the development of ZL-01 treatment strategy\nis still preliminary, requiring further substantial improvement in the future. This should be described as the present\ntechnological limitation carefully.\nDiscussion\nThere is no section of the study limitation. This study still has many limitations. Please describe the study limitations with the\ncareful interpretation.\nMethods\nIn p. 25 l. 542, add the approval date of the institutional animal care and use committee.\nIn p. 26 l. 555, add the approval date of the institutional review board.\nIn p. 29 ll. 618 to 626, cell experiments would be performed under normoxia. However, intervertebral disc cells live under a\nsignificant hypoxic condition. Therefore, this is a big study limitation. Similarly, the supplementation of 10% fetal bovine\nserum could be too much, potentially providing detrimental effects (Yurube T, et al. Eur Spine J 28(5):993-1004, 2019).\nPlease validate the applied experimental condition and/or discuss as a study limitation.\nIn p. 31 to 32 ll. 673 to 682, explain the Pfirrman grading system in greater details. Who and how many times rated the\ngrade? Unfortunately, Pfirrmann grading is largely subjective. Calculate the inter-observer and intra-observer reliability.\nIn p. 32 ll. 684 to 692, the information on the applied histological grading system is not shown. Provide the information.\nCurrently, the most accepted scoring system is from the Orthopaedic research Society spine section (Lai A, et al. JOR Spine 4(2):e1150, 2021). Hence, present the inter-observer and intra-observer reliability.\nFigures\nIn the figure 1G, what kind of surgical approach was applied? The acquired disc tissues are surprisingly big. However,\ngenerally, in discs with the Pfirrmann grade V, these big disc tissues are strange, as grade-V discs are largely collapsed.\nThus, explain this discrepancy very carefully. This manuscript always has too beautiful data and findings.\nIn the figure 2K, the immunoblot from 6 mM seems to present a higher amount of target protein.", "reviewer_3": "Zhang et al combined metabolomics, sc RNA-seq, and lactylation proteomes to characterize the lactylome landscape of\nAggravate Intervertebral Disc Degeneration. The authors identified the lactylation of SOD1 at lysine 123 as a critical\ndeterminant of the IVDD aggravation. By site-directed mutagenesis, the authors show that lactylation of SOD1 regulates its\nenzymatic activity in vivo and in vitro. Consequently, SOD1K123R stimulated oxidative damage and activated p53. Finally,\nby screening a library of 1.6 million compounds, the authors identified the small molecule ZL-01, which inhibits\nSOD1K123la. Notably, the delivery of ZL-01 to NPCs effectively alleviated IVDD in rats.\nI was asked to review the aspect of this manuscript related to SOD1 specifically. Overall, the manuscript is well-written and\nprovides a novel concept with the lactylation of SOD1. The provided data largely support the conclusions drawn from the\nexperiments. However, before publication, the authors must address the following aspects, which are mostly technical, to\nprovide additional experimental support for their claims.\nSpecific comments:\n1. Fig. 3 A-C. The authors need to provide quantifications of the data. Furthermore, Coomassie or Ponceau staining of the\nSDS-PAGE after IP should be included to demonstrate the successful enrichment of SOD1.\n2. Fig. 3 I. According to the manuscript, the experiment has been performed after depletion of SOD1 by KD, followed by\noverexpression of the WT and mutants, respectively. If K123 is the only residue for lacylation, there should be hardly any\nsignal in the last lane of the blot if endogenous SOD1 is depleted. If the experiment was “only” done by overexpression,\nit needs to be repeated with depletion of endogenous SOD1. For this experiment, quantifications are missing.\n3. While the K123R mutant did not affect the migration in the SDS-PAGE, the dimerization and formation of high-molecular-\nweight species have not been analyzed. Since WT SOD1 also forms HMW species, this aspect should be experimentally\naddressed by analyzing K123R under non-reducing conditions.\n4. The authors should provide at least some basic characterization of the gross phenotype of SOD1K123R mutant rats.\nWhile an in-depth characterization might be out of scope, the overall phenotype (weight changes, any obvious behavioral\nabnormalities, basic histological characterization) should be included.\n5. The specificity of the SOD1K123la antibody should be further validated in SOD1-depleted cells. Since the antibody is one\nof the key tools for the characterization of SOD1K123R mutant rats, the specificity should be “air tight”.\n6. Fig. 4 H -J, Fig. S8 A-C. According to the data provided in these figures, there is hardly any SOD1 lacylation, which is in\ncontrast to the IP data provided in Fig. 3, where already a significant signal under basal conditions is present. What is\nthe explanation for this discrepancy? Quantification of Fig. 4 J is missing.\n7. As a general comment, the statistical analysis needs to be carefully evaluated. In Fig. 6 N-0 (as an example), a two-way\nANOVA has been used, although there is no grouping of conditions; A one-way ANOVA would be appropriate."}, "author_response": ""}, {"round": 2, "reviewer_comments": {"reviewer_1": "I am highly satisfied with the authors' comprehensive and rigorous responses to all my previous comments, as well as the\nsubstantial supplementary experiments and revisions made to the manuscript. The revised version has significantly\nimproved in scientific rigor, logical flow, and completeness, and I recommend the manuscript for acceptance.\nKey Strengths of the Revised Manuscript:\nThe authors have successfully supplemented statistical analyses for pan-Kla levels in Figures 1I and S2M-N, and replaced\nthe representative image in Figure 1I with a more illustrative one (Figure R1). The detailed Western blot data and statistical\nresults effectively support the conclusions regarding pan-Kla expression patterns.\nThe rationale for focusing on cellular senescence, oxidative stress, hypoxia, glycolysis, and lactate has been clearly\nelaborated in the Results and Discussion sections. The integration of GO analysis results and existing literature evidence\nstrongly justifies these research focuses, greatly enhancing the manuscript's logical coherence.\nThe authors have openly discussed the limitations of the lactate-induced IVDD model and the selection basis of injection\nconcentrations, and proposed future validation using large-animal models and organoids. This critical reflection\ndemonstrates the authors' scientific integrity and forward-looking perspective.\nThe criteria for prioritizing SOD1 for functional validation are well-founded, including its high fold change in lactylation, key\nrole in redox homeostasis, and relevance to IVDD. The supplementary discussion on other lactylated antioxidative proteins\nalso provides valuable directions for future research.\nThe correction of ZL-01's FDA approval status and the discussion of its translational limitations (e.g., pharmacokinetics, off-\ntarget effects) are thorough and responsible, laying a solid foundation for subsequent preclinical research.\nThe supplementary experiments on SOD1WT and SOD1K123R enzymatic activity regulation by ZL-01 (Figure R2) and the\nevaluation of ZL-01's effects on other post-translational modifications (Figure R3) effectively verify the specificity of ZL-01,\naddressing the core concern of targeted inhibition.\nThe discussion on potential crosstalk between SOD1K123la and other PTMs enriches the mechanistic depth of the study,\nproviding a broader regulatory network perspective.\nThe revisions to supplementary figures (e.g., adding axis labels, splitting dense figures) and the professional language\nediting by native English speakers (supported by the AJE certificate) have significantly improved the manuscript's readability\nand presentation quality.\nConcluding Remarks:\nThe authors have addressed all my previous concerns in a comprehensive and satisfactory manner. The supplementary\nexperiments are well-designed and executed, providing robust support for the manuscript's conclusions. The revised\nmanuscript meets the standards of scientific publication, and I have no further revisions to suggest. I sincerely congratulate\nthe authors on their excellent work and recommend the manuscript for immediate acceptance.", "reviewer_2": "The authors carefully addressed responses to the reviewer's concerns in the response letter. However, the authors did not\nsignificantly change and improve the manuscript, as compared to the letter. Revisions should be exhaustively reflected in\nthe text.", "reviewer_3": "The authors thoroughly addressed all of my concerns. I have no further comments."}, "author_response": "Reviewer #1:\n1. The level of pan-Kla showed in Figures 1I and Figures S2M-N should provide\nstatistical analysis results. At the same time, the representative image in Figure\n1I should be replaced with more representative results.\nRe: We thank you for your comment. We performed statistical analysis of\npan-Kla levels across the entire manuscript and presented the results in\nFigure R1. Meanwhile, the result in Figure 1I was replaced with more\nrepresentative figure.\nFigure R1. All pan-Kla western blot assays in this research.\n(A-F) The expression level of pan-Kla in the NP tissue from patients, PIDD rats and\nAIDD rats (n = 3 individual samples). (G-H) The expression level of pan-Kla in the NP\ntissue from Sham and LAIDD groups (n = 6 individual samples). (I-J) The expression\nlevel of pan-Kla in lactate-treated primary NPCs (n = 6 individual samples). Data are\npresented as mean ± SD. Statistical significance was calculated with Student’s t test for\ntwo-group comparisons and one-way ANOVA for multiple-group comparisons (*p <\n0.05; ***p < 0.001; ****p < 0.0001; ns stands for no significant change).\n1\n2. In the results of Figure 1, why did the authors focus on cellular senescence,\noxidative stress, hypoxia, and glycolysis in the single-cell data analysis?\nAdditionally, why was the metabolite lactate highlighted in the metabolomic\nanalysis rather than other metabolites? Please provide justification in the\nResults or Discussion section to clarify the rationale and enhance the logical\nflow of the manuscript.\nRe: We thank you for your valuable comments. Our decision to focus\nspecifically on the processes of cellular senescence, oxidative stress,\nhypoxia, and glycolysis was not arbitrary, but rather based on the following\nrationale: Firstly, the GO analysis regarding DEGs between mild and\nsevere degenerated NP samples from the scRNA-seq showed that the terms\n“Response to oxidative stress”, “Cellular senescence”, “Glycolytic\nprocess”, and “Response to hypoxia” were enriched. Secondly, the nucleus\npulposus (NP), as a naturally hypoxic tissue, relies heavily on glycolysis\nfor cellular energy metabolism. During IVDD, endplate sclerosis\nexacerbates pathological hypoxia, disrupting cellular homeostasis. High\nlevels of oxidative stress induce cellular senescence in the disc and\nsuppress mitochondrial function, which in turn shifts cellular energy\nproduction toward glycolysis. Therefore, all these four interconnected\nprocesses drive the progression of IVDD. We have added relevant\ndiscussions in Pages 20 to 21, Lines 433 to 441.\nOur metabolomics results revealed the accumulation of multiple\nmetabolites, including AMP, lactate, 2,3-DPG, FBP, and GAP/DHAP.\nExisting studies have not demonstrated the roles of AMP, 2,3-DPG, FBP,\nor GAP/DHAP in IVDD, osteoarthritis, or degeneration of other similar\n2\ncartilaginous tissues. However, numerous studies have shown a strong\ncorrelation between lactate accumulation and both IVDD and osteoarthritis\n1-3. Therefore, we conducted subsequent studies focusing on lactate. We\nhave added relevant discussions in Page 21, Lines 446 to 452.\n3. The use of exogenous lactate to induce IVDD may not fully replicate the\nchronic microenvironmental shifts seen in human disease. How did the author\ndetermine the injection concentration of lactate? The authors should discuss\nthis limitation and justify its relevance.\nRe: We thank you for your valuable comments. The causes of human\nIVDD are complex and diverse, and we fully acknowledge that intradiscal\nlactate injection in rat discs may not fully recapitulate the complex\nmicroenvironment of human degenerated discs. In human intervertebral\ndiscs, the physiological lactate concentration is below 2 mM, while in\ndegenerated discs, it can reach up to 10 mM 4,5. Accordingly, we designed\nour in vitro and in vivo experiments based on this this physiological and\npathological concentration range. Moreover, the rat model of disc\ndegeneration induced by intradiscal lactate injection has been validated in\na previous study 6. In future studies, we will use more IVDD animal models,\nsuch as large-animal IVDD models, and organoids to further validate the\nmolecular mechanism and therapeutic effects we identified. Therefore, as\nsuggested, we have now included a discussion of this limitation in the\nrevised manuscript (Page 29, Lines 620 to 627).\n4. Among the many lactylated lysine residues identified in the proteomic dataset,\nwhat criteria were used to prioritize SOD1 for functional validation? Clarifying\n3\nthis would help justify the depth of investigation focused on a single site.\nRe: We thank you for your valuable comments. Our study found that\nlactylation occurred in multiple oxidative stress-related proteins. Among\nthem, SOD1 not only showed a relatively high fold change but was also\none of only two enzymes in the body responsible for scavenging\nsuperoxide anions, and plays a crucial role in maintaining redox\nhomeostasis in the intervertebral disc 7,8. In addition to SOD1, other\nantioxidative proteins undergoing lactylation include PPIA, PRDX2,\nNQO1, and ANXA1. We believe their lactylation may also be involved in\nthe regulation of redox homeostasis in the intervertebral disc, though their\nspecific mechanisms of action remain unclear. Future studies will further\nexplore the functions of lactylation on these proteins and their respective\nmodification sites. As suggested, we have also added a description in the\ndiscussion parts (Pages 22 to 24, Lines 477 to 514).\n5. Although ZL-01 is described as an FDA-approved compound, its precise prior\nuse or safety profile is not discussed. Clarification of ZL-01’s pharmacokinetics,\nprior human usage, or potential off-target effects is essential for translational\nclaims.\nRe: We would like to thank you for your careful review. We identified an\ninaccuracy in our original description of the Chem-DIV compound library\nin the manuscript. A small subset of compounds in this library is FDA-\napproved, whereas ZL-01 has not been reported for human use and is\ncurrently not an FDA-approved drug. Consequently, while our study\nsuccessfully identified ZL-01 as a promising candidate with potential\n4\ntherapeutic value for IVDD, its clinical translation and application will\nnecessitate further systematic preclinical evaluation. This evaluation may\ninclude detailed pharmacokinetic studies and comprehensive off-target\neffect assessments. Our current work serves as an initial step in this\ndirection, and our subsequent research will be strategically focused on\nadvancing the translational medical application of ZL-01. Therefore, as\nsuggested, we have now included a discussion of this limitation in the\nrevised manuscript (Page 29, Lines 617 to 620).\n6. To demonstrate the specificity of ZL-01 in inhibiting SOD1, comparative\nexperiments on the enzymatic activity regulation of SOD1 WT and SOD1\nK123R by ZL-01 should be included in Figure 6.\nRe: We thank you for your constructive suggestion. As you suggested, we\ninvestigate the effect of ZL-01 on the enzymatic activity of SOD1WT and\nSOD1K123R. These data showed that ZL-01 rescues lactate-induced\nimpairment of SOD1 function in SOD1WT NPCs, and this is associated\nwith the suppression of SOD1 lactylation by ZL-01. In addition, ZL-01\nresulted in no apparent alteration in SOD1 function in SOD1K123R NPCs,\nwhich may be attributed to the already existing lactylation block by\nSOD1K123R that protects SOD1 function (Figure R2). These results\npartially verify the specificity of ZL-01 in inhibiting SOD1 lactylation.\n5\n****\n100 *\nns\n)n\nie 80\nto\nrp\ng /U 60\n(\ny\ntiv\nitc 40\na\nO\nS\nZL-01 - + - + - + - +\nWT K123R\nFigure R2. SOD1 activity in SOD1WT or SOD1K123R NPCs treated with lactate or\nZL-01. Data are presented as mean ± SD. Statistical significance was calculated with\none-way ANOVA for multiple-group comparisons (*p < 0.05; ****p < 0.0001; ns stands\nfor no significant change).\n7. Has ZL-01 been tested for its influence on other known post-translational\nmodifications (e.g., acetylation, succinylation)? Has the effect of ZL-01 on\nglobal protein lactylation levels been assessed? While it appears specific to\nSOD1K123la in this study, potential broad suppression of lactylation across the\nproteome might carry unintended biological consequences. A global Kla\nWestern blot after ZL-01 treatment would be informative.\nRe: We appreciate your valuable comments. As you suggested, we further\nevaluated the impact of ZL-01 on pan-lactylation, pan-acetylation, and\npan-succinylation. The results demonstrate that ZL-01 does not alter the\nacetylation or succinylation levels of SOD1 (Figures R3A-R3F). ZL-01\nalso had no statistically significant effect on global lactylation (Figures\nR3G-R3H), while the suppression of lactylation by ZL-01 was specifically\nobserved around the 17 kDa region (Figure R3I), which correlated with\nthe molecular position of SOD1, thus supporting the potential specificity\nof ZL-01 in precisely inhibiting SOD1 lactylation.\n(A-F) Quantification and representative western-blots for the SOD1 acetylation level\nand SOD1 succinylation level in lactate and ZL-01 treated NPCs (n = 3 individual\nsamples). (G-I) The expression level of pan-Kla in the NPCs treated with lactate or ZL-\n01 (n = 3 individual samples). Data are presented as mean ± SD. Statistical significance\nwas calculated with one-way ANOVA for multiple-group comparisons (*p < 0.05; **p\n< 0.01; ***p < 0.001; ns stands for no significant change).\n8. As lysine residues are often subjected to multiple competing modifications\n(e.g., acetylation, ubiquitination), do the authors anticipate potential crosstalk\nbetween SOD1K123la and other PTMs? This point could be expanded in the\ndiscussion to provide broader mechanistic context.\nRe: Thank you for raising this valuable comment. Your insight not only\nhighlights the complexity of the PTM regulatory network but also provides\na valuable perspective for our future research. Lysine residues are common\n7\ntargets for lactylation, acetylation, and ubiquitination, leading to potential\nbinding competition. Our study found that K123 lactylation inhibits SOD1\nenzymatic function. Succinylation and acetylation can occur at the K123\nresidue, resulting in the suppression of SOD1 enzymatic activity and the\ninhibition of mitochondrial respiration, respectively 9-11, suggesting that\nthese two modifications at SOD1-K123 may crosstalk with lactylation.\nConsidering that the succinylation and acetylation levels of SOD1 did not\nexhibit significant alterations during IVDD, we surmised that SOD1K123la\nserves as a main regulator of SOD1 enzymatic function during IVDD.\nHowever, the regulatory network of PTMs is complex, and crosstalk indeed\nexists between many PTMs 12-14. Therefore, we do not rule out the\npossibility that SOD1K123la may affect other PTM levels at the K123 site in\nother tissues or other diseases. Investigating the crosstalk between\nSOD1K123la and other PTMs will be of great significance for future research.\nWe have added descriptions addressing this potential issue in the\ndiscussion section (Pages 25 to 26, Lines 541 to 552).\n9. Some supplementary figures are dense and lack legible axis labels (e.g., S3,\nS6). High-resolution versions or simplified schematics would improve\naccessibility.\nRe: Many thanks to you for optimizing our figures. We have added axis\nlabels to Figure S3 and Figure S6. Meanwhile, to reduce the graph density,\nwe have split Figure S2, Figure S5, and Figure S8 each into two or three\nseparate figures.\n8\n10. The manuscript would benefit from careful proofreading for minor\ngrammatical inconsistencies and overuse of passive voice.\nRe: Acknowledging your feedback on the manuscript's language quality,\nwe sincerely apologize for any previous shortcomings. We have\nundertaken a thorough revision focusing on both language and overall\nreadability, and have also further involved native English speakers to\nperform language corrections (Figure R4). We sincerely hope that the\nlanguage quality has been substantially improved.\nFigure R4. Editing certificate from AJE\nReviewer #2\n1. As the intervertebral disc is the largest avascular, hypoxic, low-nutrient organ\n9\nin the body, depending on the diffusion of nutrition and oxygen through the\ncartilage endplates of the intervertebral disc, the authors should highlight the\nreason and motivations of why they focused on the accumulation of lactate in\nthe disc in greater details.\nRe: We thank you for the valuable comments. We have modified our\ndescription of the intervertebral disc microenvironment and lactate\naccumulation in great detail as follows:\n“The intervertebral disc is the largest avascular organ in the human\nbody, with its NP, a unique cartilaginous structure, naturally exists in\na hypoxic and low-nutrient microenvironment due to the absence of direct\nblood supply. During the pathogenesis of IVDD, the hypoxic and nutrient-\ndeprived state of NP is further exacerbated, which significantly accelerates\nthe accumulation of various metabolites that may serve as detrimental\nstimuli in IVDD. Among these accumulated metabolites, elevated lactate\nlevels are considered a characteristic and potential contributing factor in\nIVDD. In human IVDD, the lactate concentration can exceed 10 mM,\nwhich is more than 10 times higher than the normal concentration in\nplasma. Elevated lactate levels contribute to oxidative damage in nucleus\npulposus cells (NPCs), and mitigating lactate accumulation can alleviate\nthe progression of IVDD. Given the intimate link between the\nintervertebral disc’s hypoxic microenvironment, lactate metabolism, and\nNPCs dysfunction, investigating the role of lactate in IVDD is of critical\nimportance for understanding disease mechanisms and developing targeted\ntherapeutic interventions.”\n3 to 4, Line 60 to 87).\n2. In p. 4 ll. 73 to 75, despite \"Previous studies\", there is only one reference.\nRe: Many thanks for your correction to our description. We have added\nreferences to this section (Page 5, Line 90).\n3. In p. 5 ll. 108 to 110, the detailed information on the datasets for the single\ncell RNA sequence analysis should be described in greater details, including\nthe patient number, age, sex, segment, disease, and surgery type. The dataset\nof Pfirrmann grades II to III should include many patients with scoliosis whereas\nthat of Pfirrman grades IV to V would come from many degenerative discs.\nTherefore, in particular genetic backgrounds, the disease difference must be\ninterpreted carefully.\nRe: We thank you for your valuable comments.The two datasets used for\nsingle-cell RNA sequencing analysis were obtained from previously\npublished studies 15,16. GSE165722 includes 8 patients with detailed\ninformation on age, gender, Pfirrmann grade, and primary diagnosis (Table\nR1). The mildly degenerated samples (equivalent to Pfirrmann grades II-\nIII) were derived from patients with vertebral fracture or disc herniation.\nGSE251686 includes 6 patients with available Pfirrmann grades, while\ninformation regarding age and gender is unfortunately not available in the\noriginal article (Table R2). The mildly degenerated samples were obtained\nfrom patients diagnosed with lumbar disc herniation and undergoing\nendoscopic lumbar discectomy. and required discectomy. We have now\nadded relevant descriptions in the manuscript (Page 6, Lines 123 to 129).\nTable R2. Basic information and characteristics for participants in GSE251686.\nRegarding the genetic backgrounds, we acknowledge the concern\nregarding genetic backgrounds that Pfirrmann grades II to III may include\npatients with scoliosis, which is highly associated with genetic mutations\n12\nand gene deficiencies 17. Although our analyzed scRNA-seq datasets and\npatients’ samples did not include data from genetics-related IVDD such as\nscoliosis cases, we hypothesize that the lactylation signature and the\nprofiles of key lactylated proteins may differ in genetics-related IVDD\nscoliosis-associated disc degeneration. Investigating these potential\ndistinctions represents a valuable and compelling direction for future\nresearch. We have acknowledged this point in the limitations section to\nencourage future studies to explore these potential differences (Page 28 to\n29, Lines 611 to 617).\n4. In p. 6 ll. 122 to 123, the detailed information is shown in the supplementary\ntable 1. This supplementary tables should also be addressed here within the\nresults section. In addition, surgery type (discectomy, decompression, and\nfusion surgery) should be included in the supplementary table 1.\nRe: We would like to thank you for your careful review.We have added a\ndescription of the patient information table to the results section (Page 7,\nLines 143 to 144). Meanwhile, we have added relevant data in\nsupplementary Table 1 as you suggested.\nMIS-TLIF: Minimally Invasive Transforaminal Lumbar Interbody Fusion; Endo TLIF:\nEndoscopic Transforaminal Lumbar Interbody Fusion; PELD: Percutaneous\nEndoscopic Lumbar Discectomy; UBE-LIF: Unilateral Biportal Endoscopic Lumbar\nInterbody Fusion.\n5. (1). In p. 6 to 7 ll. 131 to 135, animal models of annular puncture-induced\ndisc degeneration (PIDD), natural aging-induced disc degeneration (AIDD), and\nlactate injection-induced disc degeneration (LAIDD) should be clarified in much\ngreater details. (2). In PIDD, how did the authors determine the use of 21-G\n14\nneedle? How did the authors measure the accurate insertion of 5 mm? Did the\nauthors apply the aspiration of the disc nucleus pulposus? (3). In LAIDD, why\ndid the authors select 26-G needle? The reviewer feels the application of 26-G\nneedle can be too big for the local drug delivery to rat tail discs, possibly causing\ntraumatic disc disruption. (4). How did the authors decide the injection volume\nof 2 μL? Was there no violation of disc tissues? In this injection, how much\nlength of needle insertion was performed? (5). How did the authors identify the\ndisc segment, by X-ray fluoroscopy? (6). Please address preliminary data of\nbaseline disc degeneration (radiography, magnetic resonance imaging,\nand histomorphology) and/or background literature evidence in much greater details.\n(7). Additionally, explain the age (no data in LAIDD), sex (all male?), and weight\n(AIDD rats should be very big) of these animals.\nRe: We acknowledge you for pointing out these previously unelaborated\nareas. We have made the subsequent modifications and additions to\nenhance the clarity and description of the manuscript.\n(1). For the detailed description of the AIDD, PIDD, and LAIDD models:\nDegeneration of rat caudal intervertebral discs can be induced by puncture\nor natural aging 18,19. We have now included a detailed description of the\nthree animal models in the results section (Pages 7 to 8, Lines 153 to 159),\nalong with more comprehensive experimental details in the methods\nsection (Pages 31 to 33, Lines 674 to 700) as follows:\n“Two-month-old male SD rats, weighing between 250 g and 290 g, were\nrandomly assigned to different groups. Before surgery, the rats were\neffectively anesthetized. Rats were placed in the prone position and their\ntails were sterilized with iodophor. For the PIDD model 18, after positioning\nthe rat caudal disc by palpation or X-ray, a 21-G needle fitted with a depth\nlimiter was inserted vertically into the disc between caudal vertebrae 5 and\n6 at a depth of 5 mm. The needle was rotated 360 degrees and held in place\n15\nfor 30 s, and then removed vertically. The surgical area was disinfected\nafter operation. The control group did not receive any treatment. For the\nLAIDD model 6, A previous study has shown that an injection volume of\n2–5 μL is the recommended range for injections into the rat caudal\nintervertebral disc, and the actual volume of the rat NP is estimated to be\n2-3 μL 20. Therefore, we chose 2 μL as the injection volume. Two\nmicroliters of PBS or sodium lactate (2 mM, 6 mM, or 10 mM) was\ninjected into the intervertebral disc between caudal vertebrae 5 and 6 via a\n26-G needle and microsyringe (7105KH; Hamilton). For in vivo\nexperiments with ZL-01 or engineered EVs, after puncturing the\nintervertebral disc following the aforementioned method, 2 μL of PBS, ZL-\n01 (1 mM, 5 mM, 10 mM or 20 mM) or different types of C-EVs were\ninjected into the intervertebral disc between caudal vertebrae 5 and 6 via a\n26-G needle and microsyringe (once a week). Each rat was administered\nwith analgesics and antibiotics for 3 days. Four weeks postoperatively,\nimaging data were collected, and the rats were anesthetized and euthanized\nto obtain the intervertebral discs for subsequent experiments. For the AIDD\nmodel 19, 20-month-old male rats , weighing between 600 g and 670 g,\nwere used as the natural aging group, and 2-month-old male SD rats\nweighing between 250 g and 290 g were used as controls. The rats in this\ngroup were directly used for imaging data acquisition and subsequent\nexperiments without any treatment.”\n16\n(2). For the needle gauge selection and puncture details in the PIDD\nmodel: The PIDD model is a widely used animal model for studying IVDD.\n18-G, 20-G, and 21-G needles are the most commonly used types 20-22,\nwhile needle sizes smaller than 23-G fail to induce consistent and\nsignificant degeneration in rats 23. The purpose of this study was to explore\nthe effect of changes in metabolites on IVDD, which was a slow and\npersistent process. Therefore, we used a 21-G needle for puncture to induce\nprogressive IVDD in rats, which is also consistent with the pathological\nprocess of IVDD in clinical patients.\nThe 5 mm insertion depth refers to the distance from the needle tip to\nthe skin surface of the rat tail vertebrae during percutaneous disc puncture.\nAccording to a previous study 24, a depth limiter was mounted 5 mm from\nthe needle tip to standardize the needle insertion depth during the\nestablishment of the rat tail disc puncture model (Figure R5A). During the\nestablishment of the PIDD model, there were four main steps: positioning\nthe puncture site, puncturing the disc, rotating the needle 360 degrees, and\nmaintaining it in place for 30 seconds. The depth limiter we used\neffectively facilitates fixation and provides sufficient rotation space\n(Figure R5B). No aspiration of NP tissue was performed during this\nprocedure.\n(A) Schematic of a depth limiter for puncture needles. (B) Schematic of PIDD surgical\nprocedure steps\n(3). For the selection of 26-G needle in the LAIDD model: The LAIDD\nmodel was established by intradiscal injection of lactate to induce disc\ndegeneration. A previous study has shown that needle sizes smaller than\n23-G fail to induce consistent and significant degeneration in rats 23, and\nanother study has shown that 26-G does no significant damage the\nintervertebral disc 25. In addition, a previous study using a LAIDD model\nalso used a 26-G for lactate injection. Accordingly, a 26-G needle was used\n(diameter = 0.464 mm), which is thinner than the 21-G needle (diameter =\n0.819 mm) used for establishing the PIDD rat model (Figure R6A). In this\n18\nresearch, we also did not observe significant intervertebral disc\ndegeneration in rats after 4 weeks of injection using a 26-G needle.\nTreatments are commonly administered through a 31-G needle as the outer\ndiameter of the needle is less than 20% of a rat disc height and is the safest\ngauge to avoid promoting further degeneration. We further compared the\neffects of 26-G needle injection versus 21-G needle injection and 31-G\nneedle injection on the intervertebral disc. We injected 2 μL of PBS into\nthe intervertebral discs of rats using 21-G, 26-G, and 31-G needles,\nrespectively. We found that injection with the 21-G needle could induce\nIVDD in rats 4 weeks after injury, which was evidenced by a significant\nincrease in the histological score of the intervertebral disc and a significant\ndecrease in DHI%. In contrast, no significant IVDD was observed when\nintradiscal injection was performed using the 26-G or 31-G needle\n(Figures R6B-E). Therefore, the 26-G needle used in this research did not\ncause apparent IVDD in our observation period.\n(A) Schematic of 21G and 26G needle. (B) Different gauges of needle for puncture. (C)\nRepresentative X-ray images and HE-stained sections of intervertebral discs from rats\nin different experimental groups. (D) Quantitative statistical analysis of intervertebral\ndisc histological scores in rats across different groups. (E) Quantitative statistical\nanalysis of intervertebral disc DHI% in rats across different groups. Scale bar = 500 μm.\nData are presented as mean ± SD. Statistical significance was calculated with one-way\nANOVA for multiple-group comparisons (****p < 0.0001; ns stands for no significant\nchange).\n20\n(4). For the selection of injection volume during intradiscal\nadministration: The 2 μL injection volume was selected based on previous\nliterature. An injection volume of 2-5 μL is the recommended range for\nintradiscal injection 20, and a study by Mao H, et al. indicated that PBS\ninjection with a volume of 2 μL or less does not cause significant IVDD 26.\nTherefore, we chose 2 μL as the injection volume to prevent abnormal\nelevation of intradiscal pressure and subsequent IVDD caused by an\nexcessively large injection volume. The method for intradiscal injection\ndiffers from that used to establish PIDD rats. To minimize invasion of the\nintervertebral disc tissue, the injection needle is immediately stopped when\npiercing the annulus fibrosus and a \"disappearance of resistance\" is felt,\nwith injection performed at this position. The insertion depth of this\nprocedure is approximately 3 mm.\n(5). For the localization of the puncture disc segment: As described in\nprevious studies, the puncture site was determined by a combination of\nmanual palpation and X-ray guidance 27,28. Similar to identifying human\nintervertebral spaces via surface anatomy, the tail vertebrae of SD rats have\nslightly bulging ends and a narrower middle section. By holding the tail\nbetween the thumb and index finger and palpating, the intervertebral disc\nlocation, the junction between two adjacent tail vertebrae, could be\nidentified in most rats (Figure R5B). For the minority of cases where\nmanual localization was uncertain, the tail was securely fixed to a\n21\ngraduated scale. After acquiring both bright-field and X-ray images, we\nmerged the two images. Using image processing software, the\ncorrespondence between the intervertebral spaces and the scale markings\nwas established. Based on the scale markings, the locations of the\nintervertebral spaces were identified on the tail, allowing for accurate\npuncture (Figure R7A). This method proved effective for localizing the\nintervertebral spaces in the tails of SD rats where manual positioning had\nfailed (Figure R7B). We have accordingly clarified this point in the\nmanuscript (Page 32, Lines 678 to 679).\nFigure R7. X-ray guided localization of the punctured intervertebral space.\n(A) Localization of the rat caudal intervertebral disc with X-ray assistance. The upper\nred line indicates the scale position corresponding to the intervertebral disc under X-\nray, while the lower red line shows the intervertebral disc localized according to the\nscale position in the bright-field image. (B) X-ray image confirming the successful\ninsertion of the needle into the intervertebral disc.\n(6). For the preliminary data of baseline disc degeneration induced by\nAIDD animal models are presented in Figures R8A-H below. The PIDD\nmodel was established as described in our previous studies 29-32. The\nLAIDD model was established as reported previously 6. As suggested, we\nhave now provided a more detailed description of these models in the\nnewly revised Figure S3 and manuscript (Pages 7 to 8, Lines 153 to 159).\nFigure R8. Detection of the PIDD model and AIDD model in rats.\n(A-B) MRI and Pfirrmann grade evaluation of Ctrl and PIDD groups (n = 6 individual\nrats). (C-D) SO & FG staining and histological evaluation of Ctrl and PIDD groups (n\n= 6 individual rats), Scale bar = 500 μm. (E-F) MRI and Pfirrmann grade evaluation of\nCtrl and AIDD groups (n = 6 individual rats). (G-H) SO & FG staining and histological\nevaluation of Ctrl and AIDD groups (n = 6 individual rats), Scale bar = 500 μm. Data\nare presented as mean ± SD. Statistical significance was calculated with Student’s t test\nfor two-group comparisons (****p < 0.0001).\n(7). Additionally, we have included information on the age, sex, and body\nweight of all experimental animals in the methods section as shown above\n(Pages 31 to 33, Lines 674 to 700).\n6. For the other injection intervention, was the 26-G needle used?\nRe: We thank you for raising this important methodological point. In the\n23\ntherapeutic delivery section involving CTP-EVZ, we also used a 26-G\nneedle. Notably, we initially attempted to use the reported safest 31-G\nneedle during intradiscal injection. However, we found that the CTP-EVZ\nsolution could not be effectively delivered through this gauge. This\ndifficulty represents the major reported drawback of the 31-G needle, that\nit may present difficulties in the delivery of biomaterials, as parameters\nsuch as viscosity/density and shear may impact the feasibility of material\ndelivery and extrusion through this needle gauge 20. Given that several\nprior studies have successfully employed the 26-G needle for therapeutic\nintradiscal injection 33,34, we adopted the 26-G needle for our own treatment\ndelivery. Futhermore, we acknowledge the limitations in our current\ninjection approach, which are constrained by the use of the rat animal\nmodel. It is challenging to accurately mimic the clinical reality of the\nhuman intradiscal administration in rats due to the limitations of minimally\ninvasive procedures and high-volume injection within the rat intervertebral\ndisc. In future studies, we will use additional IVDD animal models,\nespecially large-animal IVDD models, and organoids to further validate the\nmolecular mechanism and therapeutic effects we identified. Therefore, as\nsuggested, we have now included a discussion of this limitation in the\nrevised manuscript (Page 29, Lines 620 to 627).\n7. In p. 10 ll. 209 to 211, describe the exclusion of off-target effects of the applied\nRe: We thank you for your valuable comments. We selected three\nsequences in the design of the shRNA and finally decided to use the second\nshRNA for subsequent experiments (Figure R9A). To exclude potential\noff-target effects of SOD1 knockdown by shRNA, rescue experiments\nwere conducted. Western blot and enzyme activity analyses demonstrated\nthat the reduced SOD1 protein level and enzymatic activity induced by\nSOD1KD-shRNA could be restored by overexpressing synonymous\nmutation-containing wild-type SOD1 (SOD1WT-plasmid), while the\nnegative control plasmid (NC-plasmid) exerted no such restorative effect\n(Figures R9B-D). The results exhibited that no apparent off-target effect\nwas induced by the shRNA and description are added in Figure S7 and\nPage 11, Lines 237 to 240.\nFigure R9. Specific knockdown of SOD1 by shRNA and exclusion of its off-target\neffects.\n25\n(A) Western blot analysis of SOD1 protein levels in NPCs. NC represents the negative\ncontrol group, and SOD1KD groups (shR-1, shR-2, shR-3) are cells transfected with\ndifferent SOD1-targeting shRNAs. (B-C) Western blot and quantitative analysis of\nSOD1 in NPCs transfected with scramble shRNA, SOD1KD-shRNA, NC-plasmid, or\nSOD1WT-plasmid as indicated. Data are presented as mean ± SD. Statistical\nsignificance was calculated with one-way ANOVA for multiple-group comparisons\n(****p < 0.0001; ns stands for no significant change).\n8. In p. 11 ll. 239 to 242, despite \"previous studies\", there are no references.\nRe: We thank you for your insightful comments. We have added\nreferences for this section (Page 13, Line 273).\n9. In p. 16 l. 347 and thereafter, how much volume of ZL-01 did undergo the\nintradiscal injection?\nRe: We appreciate your comment. To maintain consistency with the\ninjection volume described earlier in the study, 2 μL of ZL-01 was\nadministered at different concentrations (1 μM, 5 μM, 10 μM, 20 μM). It’s\nalso the appropriate volume to avoid abnormal elevation of intradiscal\npressure and subsequent IVDD as mentioned in previous studies 20,26. We\nhave accordingly clarified this point in the manuscript (Page 32, Lines 687\nto 692).\n10. In this study, despite the additional use of extracellular vesicles (EVs) to aim\na prolonged efficiency of ZL-01, why did the authors inject ZL-01-loaded EVs\nevery week? Every week intradiscal injection is very traumatic, potentially\ninducing disc damage and degenerative changes. Clinically, agents which\nrequire the intradiscal injection every week would not be acceptable as the\ntreatment drug for intervertebral disc degeneration. Currently, the development\nof ZL-01 treatment strategy is still preliminary, requiring further substantial\nimprovement in the future. This should be described as the present\ntechnological limitation carefully.\nRe: We are grateful to you for your thoughtful comments. The PIDD\nmodel in rat tails requires a 4-week period to develop progressive\n26\ndegeneration. A single administration after PIDD would minimize potential\nharm. Nevertheless, constrained by the use of the rat animal model,\nintradiscal injection of a high-volume (more than 2 μL) of either ZL-01 or\nZL-01-loaded EVs may further exacerbate IVDD, while increasing the\nconcentration of the ZL-01 or ZL-01-loaded EVs would make it difficult\nto deliver via needle. Therefore, to ensure sufficient drug concentration is\nmaintained within the intervertebral disc, we adopted weekly injections.\nAdditionally, due to the constraints of the rat model, it is challenging to\naccurately mimic the clinical reality of the human intradiscal\nadministration in rats due to the limitations of minimally invasive\nprocedure and high-volumn injection within the rat intervertebral disc.\nIndeed, as you mentioned, currently the development of the ZL-01\ntreatment strategy is still preliminary, requiring substantial further\nrefinement. In future studies, we will use additional IVDD animal models,\nsuch as large-animal IVDD models, and organoids that more closely mimic\nclinical practice to further validate the molecular mechanism and\ntherapeutic effects we identified. We have now included discussion of this\nlimitation in the manuscript (Page 29, Lines 622 to 628).\n11. There is no section of the study limitation. This study still has many\nlimitations. Please describe the study limitations with the careful interpretation.\nRe: We thank you for your valuable comments. We acknowledge the\nlimitations of our current study and have therefore summarized your\n27\nvaluable suggestions above and included a dedicated “Limitations” section\nto explicitly discuss these aspects (Pages 28 to 29, Lines 607 to 627).\n12. In p. 25 l. 542, add the approval date of the institutional animal care and\nuse committee.\nRe: We thank you for your careful review.The animal ethics approval date\nwas December 1, 2023, which we have added to the manuscript (Page 29,\nLine 633).\n13. In p. 26 l. 555, add the approval date of the institutional review board.\nRe: We thank you for your careful review. The ethical approval date for\nthe use of clinical specimens was December 28, 2023, which we have\nadded to the manuscript (Page 30, Line 646).\n14. In p. 29 ll. 618 to 626, cell experiments would be performed under normoxia.\nHowever, intervertebral disc cells live under a significant hypoxic condition.\nTherefore, this is a big study limitation. Similarly, the supplementation of 10%\nfetal bovine serum could be too much, potentially providing detrimental effects\n(Yurube T, et al. Eur Spine J 28(5):993-1004, 2019). Please validate the applied\nexperimental condition and/or discuss as a study limitation.\nRe: We thank you for your valuable comments. We acknowledge that the\nin vitro culture conditions for NPCs used in this experiment cannot\naccurately simulate the hypoxic and nutrient-poor microenvironment\nwithin the intervertebral disc, which may have unknown impacts on the\nexperimental results. However, it is technically challenging to fully\nreplicate these conditions in vitro when applying various treatments. A\nstudy by Yurube T, et al. demonstrated that for intervertebral disc cells,\nserum concentrations below 10% increases cell apoptosis and senescence\n28\n35. Additionally, a previous study from our team have shown that in vitro\nhypoxic and nutrient-deficient conditions induce apoptosis of NPCs 36.\nNumerous studies have demonstrated that normoxic culture conditions\nwith 10% fetal bovine serum can reliably maintain the cellular viability and\nexperimental reproducibility of NPCs in vitro 29-32. Therefore, we adopted\nthis well-established culture system to ensure cell health and\nreproducibility throughout our experiments. But we acknowledge that this\nrepresents a limitation of our current experimental setup, and we have now\nexplicitly addressed this point in the limitations section (Page 28, Lines\n608 to 611).\n15. In p. 31 to 32 ll. 673 to 682, explain the Pfirrman grading system in greater\ndetails. Who and how many times rated the grade? Unfortunately, Pfirrmann\ngrading is largely subjective. Calculate the inter-observer and intra-observer\nreliability.\nRe: We highly appreciate your comments. For patients, we adopted the\nclassic Pfirrmann classification criteria 37, which are listed in Table R3.\nFor rats, we adopted the modified Pfirrmann classification criteria 38, which\nare listed in Table R4. To ensure the reliability, the grading was performed\nby three spinal surgeons from our research team, named Dr. Xian Chang\n(A), Dr. Zhilei Hu (B) and Prof. Minghan Liu (C). All of them had over 8\nyears of experience in spinal imaging diagnosis and received standardized\ntraining prior to grading. All images were independently interpreted by\nthree observers. Each observer was allowed to interpret only 20 sets of\n29\nimages per day to avoid fatigue. During the grading period, every observer\ncould refer to the classification criteria. In case of any discrepancy among\nthem, the final decision was based on the majority opinion.\nReferring to a previous study 37, we used the weighted Kappa coefficient\nto assess consistency. SPSS v27.0 software was used to calculate the Kappa\nvalue, 95% confidence interval (95% CI), and p value. The specific\nprocedure was outlined as follows: 1. The study included 132 sets of MRI\nimages (24 patients and 108 rats). After removing image identifiers, these\nimages were used for blind evaluation. 2.For intra-observer consistency,\nthree observers re-graded all images four weeks later. As shown in Tables\nR5 and R6, the Kappa values of patients were 0.966 (95% CI: 0.901-1.032,\np < 0.001) for A, 0.931 (95% CI: 0.836-1.026, p < 0.001) for B, and 0.966\n(95% CI: 0.901-1.032, p < 0.001) for C, indicating excellent consistency.\nThe Kappa values of rats were 0.966 (95% CI: 0.901-0.981, p < 0.001) for\nA, 0.881 (95% CI: 0.825-0.937, p < 0.001) for B, and 0.935 (95% CI:\n0.893-0.976, p < 0.001) for C, indicating excellent consistency. 3. As\nshown in Tables R5 and R6, for inter-observer consistency in patients, the\nKappa value among the initial scores of A and B was 0.898 (95% CI: 0.785-\n1.011, p < 0.001), A and C was 0.934 (95% CI: 0.842-1.025, p < 0.001),\nand B and C was 0.899 (95% CI: 0.787-1.010, p < 0.001). For inter-\nobserver consistency in rats, the Kappa value among the initial scores of A\nand B was 0.941 (95% CI: 0.902-0.981, p < 0.001), A and C was 0.935 (95%\np < 0.001). These results indicated that both the intra-observer and inter-\nobserver reliability in this study are superior to those reported in the\noriginal Pfirrmann study (Kappa 0.74-0.90) 37. We have added a detailed\ndescription of the Pfirrmann classification and the Tables R5 and R6 to\nthe manuscript (Pages 36 to 37, Lines 778 to 791).\nWe acknowledge the inherent subjectivity in Pfirrmann grading.\nHowever, the marked differences in MRI features between mildly and\nseverely degenerated discs allow for meaningful comparison. By focusing\nour analyses on these two distinct groups, the potential errors introduced\nby grading subjectivity may be mitigated when evaluating degeneration\nseverity or treatment efficacy.\nTable R3. Pfirrmann classification criteria for patients.\nTable R4. Pfirrmann classification criteria for rats.\ntests in patients.\nTable R6. Weighted Kappa coefficients for intra- and inter-observer reliability\ntests in rats.\n16. In p. 32 ll. 684 to 692, the information on the applied histological grading\nsystem is not shown. Provide the information. Currently, the most accepted\nscoring system is from the Orthopaedic research Society spine section (Lai A,\net al. JOR Spine 4(2):e1150, 2021). Hence, present the inter-observer and\nintra-observer reliability.\nRe: We highly appreciate your professional comments. The histological\ngrading system applied in our study was indeed adapted from the work of\nLai A, et al. (JOR Spine 4(2):e1150, 2021) 39, and is presented in Table R7.\nWe have added a detailed description of the histological grading system\nand the Table R7 to the manuscript (Pages 37 to 38, Lines 801 to 812).\nThe grading was performed by three researchers from our research\n32\nteam, named Dr. Chao Liu (D), Dr. Yang Zhang (E) and Prof. Zhenhong Ni\n(F). All of them had over 3 years of experience in the establishment and\nevaluation of the rat caudal vertebra IVDD model and received\nstandardized training prior to grading. All images were independently\ninterpreted by three observers. Each observer was allowed to interpret only\n20 sets of images per day to avoid fatigue. During the grading period, every\nobserver could refer to the classification criteria. In case of any discrepancy\namong them, the final decision was based on the majority opinion.\nReferring to a previous study 38, we used the intra-class correlation\ncoefficient (ICC) to assess consistency. SPSS v27.0 software was used to\ncalculate the ICC value, 95% confidence interval (95% CI), and p value.\nThe inter-observer consistency of scoring results was assessed using a two-\nway random ICC model with absolute agreement and single measure; the\nintra-observer consistency was evaluated using a one-way random ICC\nmodel with single measure. Specific procedures were as follows: 1. A total\nof 144 intervertebral disc histological sections were involved in this study.\nAfter removing identifiers, these sections were subjected to blinded\nevaluation. 2. For intra-observer consistency: Three observers s\nindependently conducted re-evaluations four weeks later, and the results\nwere compared with the initial scores. The ICC values were 0.997 (95%\nCI: 0.996-0.998, p < 0.001) for D, 0.988 (95% CI: 0.983-0.991, p < 0.001)\nfor E, and 0.981 (95% CI: 0.974-0.987, p < 0.001) for F, indicating\n33\nexcellent consistency. 3. For inter-observer consistency: The ICC value of\noverall score was 0.986 (95% CI: 0.975-0.993, p < 0.001), and the scores\nof individual morphological features are presented in Table R8. These\nresults suggested that the inter-and intra-observer subjective rating bias in\nhistological scoring was controllable.\nTable R7. The modified and final consensus degeneration grading system for rat\nintervertebral discs (IVDs).\n17. In the figure 1G, what kind of surgical approach was applied? The acquired\ndisc tissues are surprisingly big. However, generally, in discs with the Pfirrmann\ngrade V, these big disc tissues are strange, as grade-V discs are largely\ncollapsed. Thus, explain this discrepancy very carefully. This manuscript always\nhas too beautiful data and findings.\nRe: We sincerely thank you for your insightful observations. The disc\ntissue sample was obtained from a 61-year-old female patient who\nunderwent minimally invasive transforaminal lumbar interbody fusion\n(MIS-TLIF) at the L5/S1 level. During this procedure, the disc tissue was\nthoroughly removed, resulting in a relatively large amount of tissue\nfragments. When aggregated, these fragments may appear more substantial\nin volume.\nIn contrast, patients with mild degeneration in our institution typically\nundergo percutaneous endoscopic lumbar discectomy (PELD). This\nsurgical approach aims specifically at removing the herniated portion of\nthe nucleus pulposus rather than the entire disc. Consequently, the surgical\n35\nspecimens obtained from these cases are generally much smaller than those\nfrom patients with severe degeneration. This difference in surgical\nmethodology explains why the nucleus pulposus tissue from the Pfirrmann\ngrade V patient appears comparatively more abundant in Figure 1G.\n18. In the figure 2K, the immunoblot from 6 mM seems to present a higher\namount of target protein.\nRe: We thank you for your insightful comments. We have replaced the\nimage with a more representative one from the biological replicates\n(Figures R10), and the original statistical analysis is not altered. The\nresults now demonstrate a progressive increase in the level of pan-\nlactylation modification in the nucleus pulposus tissue with rising\nconcentrations of lactate injection.\nFigure R10. Western blot for pan-Kla in NP tissue of LAIDD rats.\nDuplication of the expression level of pan-Kla in the NP tissue from Sham and LAIDD\ngroups.\n1. Fig. 3 A-C. The authors need to provide quantifications of the data.\nFurthermore, Coomassie or Ponceau staining of the SDS-PAGE after IP should\nbe included to demonstrate the successful enrichment of SOD1.\nRe: We thank you for your valuable comments. We quantified the western\nblot results of IP and added coomassie brilliant blue staining as shown in\nFigures R11A-P. The modified results have been added in Figure 3 and\nFigure S6.\nFigure R11. All immunoprecipitation-western blot images in this study.\n(A-I) Western blot and quantitative analysis of SOD1 protein levels and lactylation\nlevels following different treatments (n = 3 individual samples). (J-M) Western blot and\nquantitative analysis of SOD1 acetylation and succinylation levels following different\n37\ntreatments (n = 3 individual samples or rats). (N-P) Western blot images and\nquantitative analysis of SOD1 protein levels and lactylation levels in lactate-treated\nNPCs expressing SOD1K123R and SOD1K10R mutations (n = 6 individual samples). Data\nare presented as mean ± SD. Statistical significance was determined using Student’s t-\ntest for two-group comparisons and one-way ANOVA for multiple-group comparisons\n(*p < 0.05; ***p < 0.001; ****p < 0.0001; ns stands for no significant difference).\n2. Fig. 3 I. According to the manuscript, the experiment has been performed\nafter depletion of SOD1 by KD, followed by overexpression of the WT and\nmutants, respectively. If K123 is the only residue for lacylation, there should be\nhardly any signal in the last lane of the blot if endogenous SOD1 is depleted. If\nthe experiment was “only” done by overexpression, it needs to be repeated with\ndepletion of endogenous SOD1. For this experiment, quantifications are\nmissing.\nRe: We thank you for your insightful observation. In our original\nlactylomics data (Figures 3G and S7A) and Results sections (Pages 11 to\n12, Lines 232 to 248), we confirmed that K123 is not the only lactylation\nsite on SOD1; the K10 residue also undergoes lactylation but does not\naffect SOD1 function. Since inhibiting lactylation at the K123 site does not\ninhibit lactylation at K10, this likely accounts for the presence of the band\nin the final lane of the corresponding blot. These experiments were\nperformed in a background of endogenous SOD1 knockdown with\noverexpression of the mutant SOD1, and were independently repeated\nthree times. We have now included statistical analysis for these\nexperiments as shown in Figure R11 above.\n3. While the K123R mutant did not affect the migration in the SDS-PAGE, the\ndimerization and formation of high-molecular-weight species have not been\nanalyzed. Since WT SOD1 also forms HMW species, this aspect should be\nexperimentally addressed by analyzing K123R under non-reducing conditions.\nRe: We thank you for your professional comments. The dimeric form is\nthe only form in which SOD1 exerts its antioxidant activity, while the\n38\nmonomeric form of SOD1 exists transiently under physiological conditions.\nHowever, under the pathological conditions of amyotrophic lateral\nsclerosis, the SOD1 protein undergoes mutations that form multimeric\n(high-molecular-weight) species and drive disease progression 40,41. We\nexamined the effect of K123R on SOD1 polymerization by native-PAGE\nwestern blot following previous research 42. The results showed that in the\ncontrol group, most of SOD1 existed as dimers and a few as monomers,\nwhile K123R did not significantly affect the relative amount of SOD1\ndimers. Meanwhile, no significant formation of high-molecular-weight\nspecies of SOD1 was found in any of the three groups.\nFigure R12. Analyzing the influence of K123R to SOD1 under non-reducing\nconditions.\n(A) Image of native PAGE with anti-SOD1 (n = 3 individual samples). (B-C)\nQuantification of monomer or dimer SOD1 in NPCs with SOD1WT or SOD1K123R. Data\nare presented as mean ± SD. Statistical significance was determined using one-way\nANOVA for multiple-group comparisons (*p < 0.05; ***p < 0.001; ****p < 0.0001; ns\nstands for no significant difference).\n4. The authors should provide at least some basic characterization of the gross\nphenotype of SOD1K123R mutant rats. While an in-depth characterization\nmight be out of scope, the overall phenotype (weight changes, any obvious\nbehavioral abnormalities, basic histological characterization) should be\nincluded.\nRe: We thank you for your careful evaluation and valuable comments.We\nmeasured and analyzed the overall phenotype of SOD1K123R mutant rats. In\nthe open field test, there were no significant differences in time spent in the\ncenter, mean speed, and total distance between wild-type rats and\nSOD1K123R mutant rats (Figures R13A-D), these data indicate that the\nanimals did not develop significant motor or neurobehavioral\nabnormalities. Prior to 6 months of age, there was no significant difference\nin body weight between wild-type rats and SOD1K13R mutant rats (Figure\nR13E). The morphology and body ratios of liver, heart, spleen, lungs, and\nkidneys were also not significantly different between the two groups\n(Figures R13F-K). These data indicate there was no evidence of systemic\ninjury or functional impairment, and were supplemented in newly added\nFigure S10 and Page 14, Lines 288 to 290.\n(A-D) The open-field test included representative tracking images of movement in 300s\n(A), time spent in the center (B), mean speed (C), and total distance (D). (E) Body\nweight at 1m-6m of wild-type and SOD1K13R rats (n = 3 individual rats). (F) HE staining\nof heart, liver, spleen, lungs and kidneys of wild-type and SOD1K13R rats. Scale bar =\n200 μm. (G-K) Organ index values of wild-type and SOD1K13R rats. (n = 3 individual\nrats). Data are presented as mean ± SD. Statistical significance was determined using\nStudent’s t-test for two-group comparisons (ns stands for no significant difference).\n5. The specificity of the SOD1K123la antibody should be further validated in\nSOD1-depleted cells. Since the antibody is one of the key tools for the\ncharacterization of SOD1K123R mutant rats, the specificity should be “air tight”.\nRe: We thank you for your constructive suggestions. We examined\nwhether the anti-SOD1K123la antibody could bind to other proteins within\nNPCs by western blot and immunofluorescence. The results showed that\nthere was no protein binding to anti-SOD1K123la antibody in SOD1KD-NPCs,\nand the same results were observed in lactate-treated SOD1KD-NPCs\n(Figures R14A-B). This further demonstrates the specificity of the\nFigure S11 and Page 14, Lines 297 to 299.\nFigure R14. Verification of the specificity of the anti-SOD1K123la antibody.\n(A) Western-blot detection of SOD1K123la levels in SOD1KD -NPCs. (B)\nImmunofluorescence detection of SOD1K123la levels in SOD1KD -NPCs.\n6. Fig. 4 H -J, Fig. S8 A-C. According to the data provided in these figures, there\nis hardly any SOD1 lacylation, which is in contrast to the IP data provided in Fig.\n3, where already a significant signal under basal conditions is present. What is\nthe explanation for this discrepancy? Quantification of Fig. 4 J is missing.\nRe: We thank you for your insightful comment regarding the differential\nbaseline signals in the lactylation detection experiments.\nIn Figure 3, we immunoprecipitated total SOD1 from both control and\nexperimental groups followed by detection of the overall SOD1 lactylation\nlevel using a pan-Kla antibody. In contrast, for Figures 4H-J and Figures\nS8A-C, we employed a site-specific lactylation antibody (SOD1K123la) that\nexclusively recognizes SOD1 lactylated at the K123 residue but not at other\nlysine sites.\nWe have also noted the difference in baseline signals among these\n42\nexperiments. We propose the following explanation, supported by our\nfunctional data demonstrating that lactylation at the K123 site significantly\nimpairs SOD1 enzymatic function. Consequently, the K123 lactylation\nlevel is maintained at a very low level in normal cells or tissues. Lactate\ninduction specifically upregulates K123 lactylation, which correlates with\nthe observed enzymatic impairment (consistent with the results in Figure\n3).\nThe baseline lactylation signal detected in the control groups of Figures\n4H-J and Figures S8A-C may originate from non-functional lactylation\nsites on SOD1, such as K10. Lactylation at these alternative sites may\noccur at basal levels in normal cells without impairing enzymatic function.\nThis fundamental difference in antibody specificity that the pan-Kla\nantibody detecting all lactylation sites versus the SOD1 K123la antibody\ndetecting only the functional K123 site might account for the apparent\ndiscrepancy in baseline lactylation signals between the control groups in\nthese different experimental approaches.\nFor the consideration of figure typesetting, we have included the\nstatistical analysis of Figure 4J in Figures S12C-E.\n7. As a general comment, the statistical analysis needs to be carefully evaluated.\nIn Fig. 6 N-0 (as an example), a two-way ANOVA has been used, although there\nis no grouping of conditions; A one-way ANOVA would be appropriate.\nRe: We highly appreciate your professional comments. We have re-\nevaluated the statistical methods throughout the manuscript and updated\n43\nthe analysis for Figure 4, Figure 6, Figure 7, Figure S14, and Figure S17,\nto one-way analysis of variance (one-way ANOVA). These revisions do not\nalter the original conclusions and provide appropriate support for the\npresentation of our experimental results.\nReference\npathway contributes to ROS-induced osteoarthritis chondrocyte damage. Redox Biol 67,\n102867, doi:10.1016/j.redox.2023.102867 (2023).\nFerroptosis during Intervertebral Disc Degeneration. Adv Sci (Weinh) 12, e2416149,\ndoi:10.1002/advs.202416149 (2025).\nintervertebral disc degeneration via interacting with Akt. Cell Mol Life Sci 81, 24,\ndoi:10.1007/s00018-023-05094-y (2024).\nporcine disc degeneration induced by superficial annular lesion. BMC Musculoskelet Disord 20,\n551, doi:10.1186/s12891-019-2937-x (2019).\noxygen and lactate in intervertebral disc. 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Spine (Phila Pa 1976) 39, E369-378,\ndoi:10.1097/brs.0000000000000194 (2014).\ndisc degeneration in rat models: An initiative of the ORS spine section. JOR Spine 4, e1150,\ndoi:10.1002/jsp2.1150 (2021).\nmetal complexes in SOD1G93A transgenic mice implicates demetalation with pathology. Nat\nCommun 15, 6518, doi:10.1038/s41467-024-50514-7 (2024).\nNeurodegener 14, 21, doi:10.1186/s40035-025-00483-8 (2025).\nnucleolar structure and functions sustaining cell proliferation. Cell Res 31, 664-683,\ndoi:10.1038/s41422-020-00458-6 (2021).\nRe: We sincerely appreciate your time, expertise, and enthusiastic\nrecommendation for immediate acceptance. Your support has been\ninvaluable to the advancement of this work. Thank you for your valuable\ncontribution to the peer review process.\nReviewer #2:\nThe authors carefully addressed responses to the reviewer's concerns in the\nresponse letter. However, the authors did not significantly change and improve\nthe manuscript, as compared to the letter. Revisions should be exhaustively\nreflected in the text.\nRe: Thank you for your careful review and constructive feedback on our\nmanuscript. We have incorporated the additional figures in point-by-point\nresponse into main text and main figures.\nThe contents not included in the manuscript and the corresponding\nreasons are listed below:\n1. Figure R4 is a language polishing certificate, and thus it has not\nbeen added to the manuscript.\n2. Figure R5-R7 were prepared to address your comments regarding\nthe modeling method adopted in our animal experiments. To ensure\nthe reproducibility of the modeling protocol, we have cited the\nrelevant literature for the modeling methods in the manuscript (Page\n32, Line 699) and provided a more detailed description of the\n1\nmethod in the corresponding text (Pages 32-34, Lines 692 to 725).\nTherefore, these figures are not incorporated into the manuscript.\n3. Table R1-R4 and Table R7 are derived from previously published\nliterature. Given that we have cited the original publications of these\ndata in the manuscript, these tables are not included in the revised\nversion.\nYour thoughtful reminder has helped us refine the presentation of our\nwork, and we greatly value your contribution to improving the quality of\nthe manuscript.\nReviewer #3:\nRe: We would like to extend our sincere gratitude for your thorough\nreview of our revised manuscript, and your support has been instrumental\nin moving this work forward.\n2"}]

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