The natural flavonoid dihydromyricetin targets senescent cells via PRDX2 and alleviates age-related diseases

Domain: chemistry
Published: 2026-03-06
Editorial outcome: major_revision

Abstract
Aging is a primary risk factor for chronic diseases, with cellular senescence as an effective target to delay, prevent or alleviate age-related disorders. Here we report in vitro screening outputs from a natural medicinal agent library, wherein dihydromyricetin, a natural flavonoid, showed senotherapeutic potential. Dihydromyricetin protects senescent fibroblasts against further DNA damage and attenuates the senescence-associated secretory phenotype, acting as a senomorphic agent. Proteomics suggests that dihydromyricetin promotes nuclear translocation of peroxiredoxin 2 (PRDX2) to facilitate DNA repair in senescent cells. In prematurely aged mice, dihydromyricetin administration mitigates tissue aging and age-related physiological decline. In anticancer regimens, dihydromyricetin improves outcomes of chemotherapy. However, dihydromyricetin demonstrates senolytic activity against senescent microglial cells, whose basal PRDX2 expression remains low, by impairing mitochondrial function to promote apoptosis. In mice developing Alzheimer's disease, dihydromyricetin eliminates senescent microglial cells from amyloid β-protein plaques and alleviates neurodegenerative symptoms. Together, our study proposes dihydromyricetin as a natural senotherapeutic agent for mitigating age-related morbidities, including but not limited to cancers and Alzheimer's disease.

Introduction
Aging is characterized by time-dependent declines in the functional integrity of multiple organs, which underly the pathogenesis of multiple age-related disorders 1. Insights into mechanisms that allow aging to promote diseases are critical for effectively combating adverse consequences of aging. As a hallmark of organismal aging, cellular senescence appears to have evolved to have protective functions in vivo, occurring in response to macromolecular damage, including those caused by replicative exhaustion (replicative senescence, RS), oncogenic activation (oncogene-induced senescence, OIS) and genotoxic therapeutic agents (therapy-induced senescence, TIS) 2. Senescent cells undergo profound changes ranging from transcriptional, epigenetic and morphological to metabolic alterations, and exhibit a distinct secretome, the senescence-associated secretory phenotype (SASP) 3, 4. Senescence-associated stable cell cycle arrest underpins a number of pathophysiological conditions, but the vast majority of age-related disorders are attributable to the capacity of senescent cells to influence their microenvironment through development of the SASP 4.

Activation of senescent cell anti-apoptotic pathways (SCAPs), can effectively prevent these cells from undergoing programmed cell death, despite the accumulation of damage to DNA and other macromolecules 5. SCAPs and associated pathways activated in senescent cells present therapeutic targets for development of senotherapeutic agents, including drugs that induce selective elimination of senescent cells (senolytics) or suppress release of tissue-damaging, pro-inflammatory SASP factors (senomorphics) 6. Advances in understanding aging and senescence have provided a basis for developing senolytics and senomorphics, which hold the potential to intervene against various age-related pathologies and to achieve a healthy longevity 7. There have been challenges in developing senolytic drugs that are safe, efficacious, and have lasting effects. Efforts to translate such agents into clinical application have begun recently.

Senomorphic treatments are an alternative therapeutic approach for targeting senescent cells through limiting detrimental effects of the SASP without directly eliminating senescent cells. Early senomorphics were mainly discovered by serendipity, including metformin and rapamycin 8. Many natural products, particularly polyphenols, exhibit antioxidant and anti-inflammatory activities, and potentially suppress senescence-associated phenotypes by attenuating the pro-inflammatory SASP. Several naturally occurring flavonoids including apigenin and kaempferol can suppress SASP factors in bleomycin-induced senescent BJ fibroblasts 9. Studies focusing on synthetic flavones suggested that hydroxyl substitutions at C-2',3',4',5 and 7 are critical for inhibiting the SASP 9.

Alzheimer’s disease (AD) is a neurodegenerative disorder associated with presence of amyloid-β (Aβ)-containing plaques and tau-containing neurofibrillary tangles 10. As the most prevalent cause of dementia, AD affects over 35 million people worldwide 11. Development of pharmacological agents for AD has been slow and expensive, with an overall rate of treatment failure greater than 99% 12. To date, although there are FDA-approved anti-amyloid therapies, optimal strategies to prevent or delay the onset or slow the progression of AD remain highly desired. AD drug discovery entails target identification, followed by high-throughput screening and lead optimization of new compounds. Advanced age has been proposed as the greatest risk factor for AD, with the link between age and AD becoming increasingly clear. Cells displaying senescent features are identified in brains of AD patients and animals, implying a causal role of senescence in AD pathogenesis 13-15.

Dihydromyricetin (DMY) is a natural flavonoid with biological activities including potential protection against brain aging-related dysfunction by modulating oxidative stress and inflammation-related senescence of hippocampal neurons 16. DMY can alleviate asthma, osteoporosis, kidney injury, nephrotoxicity, atherosclerosis, myocardial infarction, depressive disorders and behavioral deficits 17-19. Although these effects imply a role for DMY in disease prevention and treatment, insights into key mechanisms of DMY in maintaining health and curtailing pathologies remain limited.

In this study, we screened a natural medicinal agent (NMA) library and identified candidate phytochemicals including DMY as being potential senotherapeutic agents. We found that DMY, a flavonoid compound, has senomorphic effects in assays involving stromal cell lines such as fibroblasts and endothelial cells. In contrast, DMY exhibited a senolytic potential against microglial cells, suggesting it has pleiotropic senotherapeutic effects. DMY combined with DNA damaging chemotherapy reduced tumor sizes and improved treatment efficacy. In preclinical studies involving a 5xFAD mouse model, DMY reduced Aβ accumulation and improved cognitive ability. We propose that DMY may be a phytochemical senotherapeutic agent with the potential for delaying age-related dysfunction and mitigating age-related diseases.

Results
Drug screening reveals DMY as a senotherapeutic agent

A handful of medicinal herbs and bioactive components derived from plant sources have shown geroprotective properties and therapeutic potential 7, 20. To identify novel senotherapeutic compounds targeting senescent cells, we conducted unbiased drug screening of a library composed of 50 natural medicinal agents (NMAs), most of which are phytochemical compounds per se (Supplementary Table 1). In this study, we employed a primary normal human prostate stromal cell line, PSC27, as a cell-based model which comprises predominantly fibroblasts and exhibits fibroblast characteristics 21. PSC27 develops a typical pro-inflammatory SASP upon exposure to stresses such as genotoxic chemotherapy and ionizing radiation 21, 22. To induce cellular senescence, we treated cells with pre-optimized sub-lethal doses of either bleomycin (BLEO, 50 μg/ml) or ionizing radiation (RAD, 10 Gy), and observed increased staining positivity of senescence-associated β-galactosidase (SA-β-Gal), decreased BrdU incorporation and elevated DNA damage response (DDR) foci 8-10 days afterwards (Supplementary Fig. 1a-c). Similar effects on human umbilical vein endothelial cells (HUVECs) and human breast fibroblasts (HBF1203) were observed, including increased SA-β-Gal staining positivity (Supplementary Fig. 1d, e). Thus, these cells appear to be an effective model for studying cellular senescence.

We established an in vitro screening strategy to assess the effects of individual phytochemical agents on two parameters: senescent cell expression profile (at 50 μM) and senescent cell viability (at 100 μM). Procyanidin C1 (PCC1) served as a positive senolytic control (Supplementary Fig. 2a, b). Our preliminary data indicated that a number of these agents were able to downregulate the SASP when used at 50 μM (Supplementary Fig. 2c, d). However, thorough screening of the NMA library failed to identify any senolytic agents that can selectively eliminate senescent cells, as was the case for PCC1 (100 μM) (Supplementary Fig. 2b-f). Our results not only imply the scarcity of naturally available senolytics but also indicate challenges in expanding the arsenal of this specific subclass of senotherapeutics. Although no new senolytic compounds were identified, several NMA components exhibited senomorphic activity, including dihydromyricetin (DMY), warranting further investigation (Supplementary Fig. 2c, d and Supplementary Fig. 3a-c).

When DMY was used at higher concentrations such as 400 μM, we observed significantly reduced survival of PSC27 cells, no matter if cells were senescent or not, suggesting that this concentration of DMY is cytotoxic (Fig. 1a, b). Thus, DMY may not be an ideal senolytic agent for fibroblasts. However, DMY exhibited senomorphic activity at 100 μM, as evidenced by attenuated SASP factor expression (Fig. 1c, d). Despite this, it preserves other senescence features such as nuclear membrane degradation and cell cycle arrest (Supplementary Fig. 5a-e). RNA sequencing (RNA-seq) data showed that DMY comprehensively modulated gene expression in senescent cells, as it upregulated 1152 and downregulated 2044 genes, respectively, while 4059 and 1959 genes were upregulated and downregulated, respectively, during cellular senescence (Supplementary Fig. 4a, b). Heatmap, volcano plot and GSEA analyses revealed significant gene expression alterations induced by DMY, including changes in the SASP signature (Fig. 1e-h). DMY predominantly affected pathways related to DNA damage- and telomere stress-induced senescence, as well as the NF-κB pathway, with most of these changes being reversed by DMY treatment (Supplementary Fig. 4c). Gene Ontology (GO) profiling indicated that the most affected biological processes, cellular components, and molecular functions were associated with the pro-inflammatory or secretory phenotype of senescent cells, while Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis suggested that the PI3K-Akt and MAPK signaling pathways were among the most affected axes by DMY (Fig. 1i).

To test further the data from BLEO-induced senescence, we examined the effect of ionizing radiation (RAD) and observed a similar pattern (Supplementary Fig. 4d). Furthermore, we compared DMY's efficacy to established SASP inhibitors, such as the flavonoid rutin and the small molecule compound SB203580 23, 24, and observed comparable results (Supplementary Fig. 4e). Next we employed HUVEC and HBF1203 cells to further assess effects of DMY and found that DMY caused concentration-dependent inhibition of the SASP (Supplementary Fig. 4f-k). The results further indicate that DMY has efficacy as a broad-spectrum senomorphic agent. Notably, we observed a significant reduction of reactive oxygen species (ROS) levels following DMY treatment (Fig. 1j), consistent with its antioxidant activity 25. As one of the most abundant natural flavonoids in plants such as vine tea, DMY has demonstrated a wide range of pharmacological effects 25. In addition to the general properties of flavonoids, DMY exhibits potential anti-diabetes, anti-tumor, cardioprotection, hepatoprotection, neuroprotection and dermatoprotection activities 26-28. However, the geroprotective potential and functional mechanism of DMY, particularly its role in modulating senescent cells and their bioactivities including SASP regulation, remain hitherto underexplored.

DMY exhibits senolytic activity against microglial cells

Given that DMY acts as an effective senomorphic agent for certain cell types such as fibroblasts and endothelial cells, we asked whether it exhibits distinct activities, such as a senolytic properties in other cell lineages. We noted that DMY significantly reduced SA-β-Gal positivity and the number of senescent HMC3 cells, an established human microglia cell line, starting at 50 μM (Fig. 2a, b). Immunoblots showed that DMY promoted apoptosis of HMC3 cells, as evidenced by increased cleavage of caspase 3 and elevated expression of both PMAIP1 (NOXA), BBC3 (PUMA) and BAX, key apoptosis regulators, while BCL2 levels remained unchanged (Fig. 2c). In contrast to the 3,196 significantly expression-altered genes induced by DMY in senescent PSC27 cells, RNA-seq analysis identified 634 upregulated and 461 downregulated genes in DMY-treated senescent HMC3 cells (fold change > 2, P < 0.05) (Supplementary Fig. 6a, b). These GSEA data suggest that HMC3 cells exhibit activities including DNA damage response and secretion during cellular senescence, resembling those of PSC27 cells (Supplementary Fig. 6c-g). However, HMC3 cells did not exhibit a typical SASP upon senescence induction (Fig. 2d). Consistent with GSEA results indicating DMY-induced programmed cell death (Fig. 2e), volcano plot analysis revealed significant upregulation of the apoptosis-regulatory genes NOXA and PUMA (Fig. 2f). We next mapped the significantly changed transcripts to the GO and KEGG databases comprising Entrez Gene, HPRD and UniProt accession identifiers 29-31. Analysis indicated that the most prevalent biological processes (BP) altered by DMY were the ERK1/ERK2 cascade and aging, while molecular functions (MF) were primarily related to cytokine/chemokine activity and oxidoreductase activity (Fig. 2g). Additionally, flow cytometry analysis after Annexin V-FITC/PI double staining showed that DMY significantly induced apoptosis in senescent HMC3 cells (Fig. 2h). JC-1 staining revealed that DMY decreased mitochondrial membrane potential (MMP) in senescent HMC3 cells, as evidenced by reduced J-aggregate levels (red fluorescence intensity) (Fig. 2i). Notably, in contrast to PSC27 cells, fluorescent probe DCFH-DA staining showed that DMY significantly increased ROS levels in senescent HMC3 cells (Fig. 2j). Collectively, these results indicate that DMY has senolytic activity in microglial cells, mediated largely through apoptosis induction, MMP decline and ROS bioproduction.

Given the potential senolytic effects of DMY on microglial cells, we extended our assays to other glial cell types, including oligodendrocytes, astrocytes and murine microglia. Notably, DMY demonstrated senolytic activity against the murine microglial line BV2 (Supplementary Fig. 6g, h), resembling the effect observed in human microglial line HMC3. Notably, senescent MO3.13 oligodendrocytes exhibited apoptotic responses starting at 100 μM DMY, but their non-senescent counterparts were similarly affected at the same concentration (Supplementary Fig. 6i, g). Similarly, senescent SVG p12 astrocytes underwent apoptosis at a DMY concentration of 200 μM, but their non-senescent counterparts responded at the same concentration (Supplementary Fig. 6k, l). Since both senescent and non-senescent cells exhibited similar concentration-dependent responses, our results suggest that DMY cannot serve as an ideal senolytic agent for oligodendrocytes or astrocytes.

DMY directly binds to PRDX2 as a target in senescent cells

Given the efficacy of DMY in suppressing SASP secretion across certain cell types upon senescence while promoting apoptosis in microglial cells upon senescence, we queried underlying mechanisms of the senomorphic and senolytic activities of DMY. To this end, we employed two methodologies: human proteome microarray and pull-down, the latter combined with liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) assays to identify potential intracellular factors that directly interact with DMY. First, we chose to use biotin-labeled DMY (DMY-Biotin) to assess its binding affinity to recombinant proteins coated on the surface of HuProtTM 20K human protein microarrays (Fig. 3a and Supplementary Fig. 7a). We reasoned that proteins binding to DMY-Biotin may represent direct targets of DMY. After applying DMY-Biotin to the HuProtTM 20K human protein microarray, we identified 395 proteins with significant specific binding (Fig. 3b, c and Supplementary Fig. 7b), the majority of which were associated with antioxidant and oxidoreducatase activity according to GO and KEGG analyses (Supplementary Fig. 7c). Additionally, we applied pull-down by incubating DMY-Biotin with lysates from two cell lines, PSC27 and HMC3, which were induced to become senescent, then performed LC-MS/MS analyses to further identify potential target proteins (Fig. 3d). Venn and volcano plot analyses identified a total of 130 potential target proteins shared between senescent PSC27 and HMC3 cells (Fig. 3e, f). Interestingly, GO and KEGG analyses revealed a distinct DMY-binding pattern in PSC27 cells, namely DMY-pull-down proteins primarily associated with DNA damage repair and nucleotide excision repair pathways (Supplementary Fig. 7d). Conversely, in HMC3 cells these proteins exhibited intensive enrichment of oxidative phosphorylation and mitochondrial functions (Supplementary Fig. 7e). Venn diagram analyses integrating data from human protein microarray alongside pull-down and LC-MS/MS techniques revealed that only 4 proteins, namely APIP, PRDX2, CHD4 and MMS19, can likely interact with DMY-Biotin (Fig. 3g). Silver staining suggested that each of these DMY-pull-down proteins has a molecular weight of approximately 52, 43, 35 and 22 KDa, respectively (Fig. 3h). We reasoned that DMY directly binds to PRDX2 (22 KDa), with the interaction subsequently confirmed by immunoblots (Supplementary Fig. 7f).

Despite the above findings, the interaction between PRDX2 and DMY warrants further validation. Molecular docking simulations suggested DEMY has the potential to interact with the Phe49, Cys51, Arg91, Gly94 and Gly95 residues of PRDX2 (Fig. 3i). Human protein microarray disclosed that all PRDX isoforms (PRDX1-4) can interact with DMY, wherein PRDX2 produced the highest signal-to-noise ratio (SNR) (Supplementary Fig. 7g). Detailed analysis via surface plasmon resonance (SPR) demonstrated that recombinant human PRDX2 interacts with DMY with prominent affinity, indicated by a dissociation constant (KD) of 3.88 μM (Fig. 3j). In contrast, recombinant human PRDX1 and PRDX6 had diminished SNR in protein microarrays, yielding dissociation constants of 24.51 μM and 34.45 μM, respectively (Supplementary Fig. 7h, i). A cellular thermal shift assay (CETSA) was performed using a temperature gradient (35°C to 65°C), along with increasing concentrations of DMY at 55°C. The results indicated concentration-dependent thermal stabilization of PRDX2 upon DMY binding (Fig. 3k and Supplementary Fig. 7j). To further examine binding dynamics, we performed drug affinity responsive target stability (DARTS) with DMY (0-400 μM) in the presence of pronase, which revealed a significant concentration-dependent increase in PRDX2 stability (Fig. 3l). Collectively, our data suggest that DMY, a naturally derived flavonoid, binds to PRDX2 protein with high affinity. Thus, PRDX2 appears to be a valid molecular target of DMY.

To validate PRDX2 as the target of DMY as to its senotherapeutic properties, we employed shRNAs to downregulate PRDX2 expression in fibroblasts and microglia (Supplementary Fig. 8a, b). PRDX2 deficiency in senescent PSC27 fibroblast cells significantly decreased cell viability upon DMY treatment (Supplementary Fig. 8c, e). PRDX2 depletion in HMC3 microglial cells caused reduced cell viability even at lower DMY concentrations (Supplementary Fig. 8d, f). To the contrary, overexpression of PRDX2 in HMC3 cells abrogated DMY's capacity in eliminating senescent microglial cells (Supplementary Fig. 8g-i). We further found reduced ROS accumulation in senescent microglial cells treated with DMY after PRDX2 overexpression (Supplementary Fig. 8j). Together, these results validate PRDX2 as a major target of DMY to exert its senolytic effect.

DMY is senomorphic by promoting PRDX2 nuclear translocation

Previous studies reported that DMY activates SIRT1 in the context of diseases including AD and myocardial injury 32, 33. However, our immunoblot analysis revealed no significant effect on SIRT1 expression in senescent PSC27 cells. Notably, DMY significantly suppressed the ATM/p38/AKT/mTOR signaling axis, which regulates SASP production, while showing no effect on p16INK4a and p21CIP1 expression in senescent cells (Fig. 4a). Thus, DMY effectively inhibited SASP expression of senescent cells without altering cellular senescence per se (Supplementary Fig. 5). Notably, immunofluorescence staining indicated that DMY significantly reduced the signal of γH2AX, a key marker of DDR, indicating its potential capacity to repair DNA damage in senescent cells (Fig. 4b). Alternatively, data from comet assay and transduction of a pLCN DSB repair reporter (DRR) suggest that DMY increased the DDR capacity of senescent cells (Fig. 4c, d). To elucidate the PRDX2-mediated biological effects of DMY, we chose to first assess the expression level of PRDX2 during cellular senescence. Previous studies reported that PRDX2, an antioxidant peroxidase, is upregulated in senescent cells 34. To the contrary, we found that upon senescence, expression of PRDX2 and its family members (PRDX1-6) exhibited no significant changes at either the transcriptional or protein level (Supplementary Fig. 7a, b). Although DMY significantly inhibited expression of the SASP in senescent cells, it did not generate a notable impact on PRDX2 expression (Supplementary Fig. 7c, d).

Subsequently, we performed PRDX2-based co-immunoprecipitation (Co-IP) in DMY-treated senescent PSC27 cells to identify PRDX2-interacting proteins by mass spectrometry (Fig. 4e). The heatmap analysis showed that proteins with significant binding affinity to PRDX2 were predominantly nuclear-localized in senescent cells (Fig. 4f). Volcano plot analysis revealed that PRDX2-interacting proteins included XRCC5, XRCC6, GIT1 and GIT2, which have been implicated in nuclear DNA damage repair 35-37 (Fig. 4g, h), are basically in line with our observation that DMY may facilitate DDR in senescent cells. GO analysis indicated that proteins interacting with PRDX2 are functionally associated with nucleic acid binding, while KEGG analysis further identified non-homologous end-joining as the major pathway regulated by these proteins in senescent cells (Supplementary Fig. 9e). Co-IP and immunoblot assays further confirmed interactions of PRDX2 with XRCC5, GIT1, GIT2 and ATM in DMY-treated senescent cells (Fig. 4i and Supplementary Fig. 9f). Additionally, nuclear-cytoplasmic fractionation followed by immunoblotting indicated that DMY induced PRDX2 nuclear translocation and decreased the nuclear level of p65 in senescent cells (Fig. 4j), suggesting that PRDX2 enters nuclei to facilitate the DDR, while p65 exits the nucleus and suppresses the SASP. Immunofluorescence staining of PRDX2 confirmed basal nuclear PRDX2 localization during cellular senescence, which was markedly intensified after DMY treatment (Fig. 4k and Supplementary Fig. 9g). Furthermore, qRT-PCR analysis indicated that Conoidin A, a PRDX1/2 inhibitor, significantly inhibited the SASP, with effects being similar to those of DMY treatment in senescent PSC27 cells (Fig. 4l and Supplementary Fig. 9h, i). Together, these findings indicated that binding of DMY to PRDX2 promotes its nuclear translocation and interaction with DNA repair proteins, thereby enhancing DNA repair efficacy in senescent cells.

DMY is senolytic by inducing ROS-mediated microglial apoptosis

We next queried the molecular mechanism underlying the senolytic activity of DMY in HMC3 cells. Similar with PSC27 cells, DMY treatment did not affect PRDX2 expression in senescent HMC3 cells (Supplementary Fig. 10a). Notably, HMC3 cells exhibited a significantly lower basal level of PRDX2 than other cell types, including fibroblasts and endothelial cells (Supplementary Fig. 10b). Previous studies reported that inhibition of the oxidoreductase activity of PRDX2 can induce cancer cell apoptosis 38. Molecular docking simulations suggested an interaction of DMY with Cys51 of PRDX2 (Fig. 3i). This residue, together with Cys172, forms the enzyme's catalytic site with oxidoreductase activity 39. As an oxidoreductase, PRDX2 catalyzes H2O2 reduction to water in the presence of NADPH 38. To verify the inhibitory effect of DMY on PRDX2 enzymatic activity, we applied an Amplex Red coupled spectrophotometric method as described previously 38. Our results indicated that enhancing the concentration of PRDX2 reduced H2O2 levels (Supplementary Fig. 10c). Importantly, DMY was found to inhibit the activity of recombinant human PRDX2 (Fig. 5a), mimicking the effect of Conoidin A, a PRDX2 inhibitor (Fig. 5b).

We found that Conoidin A exhibits senolytic activity, as evidenced by significantly reduced in cell viability of BLEO-induced senescent HMC3 cells (Supplementary Fig. 10d, e). To elucidate the molecular mechanism by which DMY induces HMC3 cell apoptosis through inhibition of PRDX2’s oxidoreductase activity, we conducted quantitative proteomics to compare the effects of DMY and Conoidin A (Fig. 5c). Quantitative proteomics revealed that DMY modified the expression of 3,771 proteins (2,510 upregulated, 1,261 downregulated), while Conoidin A modified 3,347 changes (2,122 upregulated, 1,225 downregulated) in senescent HMC3 cells (Fig. 5d). Venn analysis showed 2,886 common protein alterations, comprising 76.5% (DMY) and 86.2% (Conoidin A) of differentially expressed proteins (Supplementary Fig. 10f). Hierarchical clustering heatmap and principal component analysis (PCA) indicated nearly identical protein expression patterns between DMY- and Conoidin A-treated cells, suggesting that DMY exerts pharmacological effects in a way similar to Conoidin A in senescent HMC3 cells (Supplementary Fig. 10g, h). GSEA analysis identified enriched pathways in DMY-treated senescent HMC3 cells, including oxidative phosphorylation, mitochondrial complex I/III assembly, and ROS/RNS production (Fig. 5e and Supplementary Fig. 10i). GO analyses indicated that the most significantly affected biological processes (BP) were apoptotic signaling, mitochondrial respiratory chain complex assembly, and apoptotic mitochondrial changes, while KEGG analysis highlighted reactive oxygen species (ROS), oxidative phosphorylation, apoptosis, and p53 signaling as major pathways altered by DMY (Supplementary Fig. 10j). Volcano plots revealed upregulation of ROS-associated proteins, including UQCC1, UQCRB, NDUFC1, and ATP6V1H, following DMY and Conoidin A treatment in senescent HMC3 cells (Fig. 5f, g). Since the mitochondrial electron transport chain (ETC) is a primary source of ROS 40, we assessed mitochondrial function via oxygen consumption rate (OCR) measurement through seahorse assays. Senescent HMC3 cells displayed diminished basal respiration and ATP production as compared with non-senescent cells. Treatment with DMY further decreased basal respiration and ATP production in senescent HMC3 cells, indicating DMY-induced mitochondrial dysfunction (Fig. 5h, i). Moreover, DMY was observed to reduce the activity of mitochondrial complex III and complex V, indicating compromised ETC function (Fig. 5j). Taken together, our data suggest that DMY interacts with PRDX2, inhibiting its oxidoreductase activity, disrupting redox homeostasis and impairing ETC function. These changes ultimately resulted in elevated ROS-mediated apoptosis in senescent HMC3 cells.

Subsequently, nuclear-cytoplasmic fractionation followed by immunoblotting and immunofluorescence staining showed that unlike in PSC27 cells, PRDX2 significantly localized to nuclei in senescent HMC3 cells. Moreover, DMY treatment retained PRDX2 in the cytoplasm in a concentration-dependent manner, suggesting that PRDX2 activity blockade inhibits PRDX2’s nuclear translocation (Fig. 5k, l and Supplementary Fig. 10k). Since DMY increased ROS levels in senescent cells, we next employed a ROS scavenger N-acetyl-cysteine (NAC) to determine whether ROS mediated DMY's effects. DCFH-DA-based fluorescence staining confirmed that NAC reversed the increased ROS levels in DMY-treated senescent HMC3 cells (Fig. 5m). JC-1 staining indicated that NAC restored mitochondrial membrane potential (MMP) in DMY-treated senescent HMC3 cells (Supplementary Fig. 10l). SA-β-Gal staining and CCK-8 assays suggested that NAC restored cell viability in DMY-treated senescent HMC3 cells (Fig. 5n, o). Collectively, these results indicate that DMY induces apoptosis in senescent HMC3 cells via a ROS-dependent mechanism, which is subject to reversal by ROS scavenging.

DMY mitigates tissue aging and age-related physical dysfunction

Senescent cells, along with their SASP, can be found in various tissues and organs across a range of pathophysiological states, most of which are implicated in aging changes and age-associated decline of physical health. To further examine the impact of DMY on senescent cells in organismal aging in vivo, we challenged mice with whole body irradiation. Three-month-old wild type (WT) mice were subjected to WBI at a sublethal dose of 5 Gy to induce substantial senescence within tissues. Following this, the mice received either a placebo or DMY treatment (20 mg/kg via intraperitoneal injection) twice a week for 3 months, and senescence biomarkers and physical function were subsequently tested (Fig. 6a). Mice that underwent WBI display physical changes, including significant greying of the fur. However, this condition was attenuated by DMY administration (Supplementary Fig. 11a).

In WBI-induced prematurely aged mice, there were considerably accumulated senescent cells in various tissues, as reflected by a significant increase in SA-β-Gal positivity within spleen tissues subjected to WBI (Fig. 6b, d). Notably, the data indicated that treatment with DMY led to a substantial reduction of the percentage of SA-β-Gal-positive cells, while concomitantly enhancing the structural integrity of the spleen tissues in prematurely aged mice when compared to the placebo group (Fig. 6b, d). Further examination uncovered the presence of senescent cells in several vital organs, as evidenced by elevated SA-β-Gal positivity in the kidney, liver, and pancreas tissues of these mice following WBI exposure. In contrast to the placebo-treated mice, a significant decrease in the percentage of SA-β-Gal-positive cells was observed in the DMY-treated mice (Fig. 6c, e and Supplementary Fig. 11b, c). Immunofluorescence staining provided compelling evidence that DMY treatment markedly reduced the expression of senescence markers, such as p16Ink4a, and canonical SASP markers such as IL6, in the spleen tissues of WBI-induced aged mice when compared to the placebo (Fig. 6f, g).

Consistent with immunofluorescence staining results in spleen tissues, DMY treatment in WBI-induced aged mice resulted in a significantly downregulated expression of senescence markers, including CDK inhibitors p16Ink4a and p21Cip1, as well as some but not all proinflammatory factors, as determined by RT-qPCR analysis (Fig. 6h). Unlike the spleen tissues, we observed no substantial alteration in the immunofluorescence staining for p16Ink4a and IL6 in kidney tissues of WBI-induced aged mice in the DMY treatment group as compared with the placebo, suggesting a heterogeneity of senescent cells (Supplementary Fig. 11d, e). Nevertheless, the transcript expression levels of the senescence biomarker p16Ink4a and a variety of proinflammatory cytokines were significantly reduced in the kidney, liver and pancreas tissues of WBI-induced aged mice following DMY treatment compared to the placebo group (Supplementary Fig. 11f-h). Collectively, these results indicated that DMY reduced the accumulation of senescent cells and downregulated the expression of senescence markers in WBI-induced prematurely aged mice.

Next, we evaluated the potential of DMY administration to enhance the physical functions of WBI-induced prematurely aged mice. In comparison to aged mice that received placebo treatment, animals treated with DMY demonstrated marked recovery of short-term memory, as indicated by results from Y-maze assessments (Fig. 6i). Furthermore, DMY-treated aged mice showed a notable increase in grip strength (Fig. 6j) and exhibited superior performance in Rotarod test (Fig. 6k). These findings suggest that DMY administration is an effective intervention to mitigate physical impairments and avert cognitive decline associated with premature aging, ultimately enhancing overall health condition.

DMY combined with chemotherapy promotes tumor regression

Given the efficacy of DMY in restraining SASP expression across certain cell types upon senescence while promoting apoptosis in senescent microglial cells, we queried its effects on tumor progression. Former studies demonstrated that senescent stromal cells secrete multiple soluble factors or metabolites to support tumor progression 22, 24, 41, 42. We were promoted to determine whether DMY treatment of stromal cells can affect the potential of their conditioned medium (CM) in altering malignant phenotypes of cancer cells. To this end, PSC27 stromal cells were treated with BLEO to induce senescence, with or without DMY. After a 3-d incubation, CM derived from PSC27 cells were collected and used to treat multiple prostate cancer (PCa) cell lines, including PC3, DU145, MR12 and LNCaP (Supplementary Fig. 12a). Consistent with previous findings, exposure to senescent PSC27 cell-derived CM substantially increased the proliferative capacity of PCa cells (Supplementary Fig. 12b). The proliferative advantage was accompanied by enhanced migratory and invasive activities (Supplementary Fig. 12c, d). However, DMY pretreatment of stomal cells significantly reduced these pro-tumorigenic effects of stromal cell-derived CM (Supplementary Fig. 12b-d).

Since DMY downregulates SASP expression in senescent stromal cells and inhibits cancer cell malignancy in vitro, we sought to investigate its potential therapeutic effects on tumors in vivo. To simulate clinical settings, we established xenograft tumors by co-injecting PSC27 sublines with PC3 cells at an optimized ratio of 1:4 into NOD/SCID mice through tissue recombination. After confirming successful engraftment 2 weeks post-implantation, intervention regimen was initiated with weekly administrations of placebo, MIT and/or DMY starting at weeks 3, 5 and 7, continuing through an 8-week therapeutic period. Tumor sizes were measured at the end of the 8th week (Fig. 7a). Although DMY monotherapy showed modest antitumor effects, MIT treatment induced significant tumor regression, validating its chemotherapeutic efficacy. Importantly, combination therapy with DMY and MIT resulted in enhanced tumor suppression compared to MIT alone (Fig. 7b). To assess senescence induction in the tumor microenvironment, we analyzed xenograft tissues from PC3/PSC27 recombinants. Stromal cells in MIT-treated tumors displayed significant upregulation of multiple SASP components (IL6, IL8, IL1α, MMP3, GM-CSF, IL-1β, ANGPTL4 and MMP1) along with increased expression of senescence markers p16INK4a and p21CIP1, confirming chemotherapy-induced senescence in vivo (Fig. 7c, d and Supplementary Fig. 13a). Histopathological examination revealed a substantial increase of SA-β-Gal-positivity following MIT treatment (Fig. 7e). Notably, while DMY co-administration did not affect the chemotherapy-induced senescence burden (Fig. 7e), it effectively attenuated SASP factor expression, as demonstrated by transcriptomic analysis (Supplementary Fig. 13b). These findings suggest that DMY specifically targets the SASP without interfering with the senescence program per se.

Subsequently, we investigated the molecular mechanisms through which DMY combined with MIT contributes to tumor regression. To elucidate these pathways, we performed immunohistochemical analysis of cleaved caspase-3, an established apoptotic marker, in tumor xenograft models. Our data demonstrated that while DMY monotherapy exhibited limited cytotoxic activity, its combination with MIT substantially potentiated apoptotic cells (Fig. 7f). Further immunohistochemical examination revealed that DMY effectively counteracted the tumor-promoting effects of chemotherapy-induced SASP, as evidenced by reduced expression of IL6 and IL1β (Fig. 7g and Supplementary Fig. 13c, d). Quantitative analysis of serum SASP components through ELISA showed that MIT treatment markedly increased circulating levels of AREG and IL6, which were significantly attenuated by DMY co-administration (Fig. 7h). These results provide compelling evidence that DMY may serve as a valuable agent in combinatorial anticancer regimens, offering potential clinical benefits by modulating therapy-induced senescence. The observed pharmacological effects underscore the translational potential of DMY in improving therapeutic outcomes.

DMY eliminates senescent microglial cells and prevents cognitive decline

Given the effectiveness of DMY in eliminating senescent HMC3 cell lines in vitro, it is tempting to determine whether DMY administration would eliminate Aβ plaque-associated senescent microglia and ameliorate cognitive dysfunction in 5×FAD mice, a transgenic model of AD. We designed an experimental protocol in which either placebo or 50 mg/kg DMY was administered once per day for 7 days to 12-month-old 5×FAD mice, before Aβ load, senescence biomarker expression and senescent microglia were subsequently determined (Fig. 8a). Our data indicated that this short-term DMY treatment had no significant impact on Aβ load. Instead, it significantly reduced the number of microglial cells associated with Aβ plaques, as well as the level of p16Ink4a (Fig. 8b, d). Immunofluorescence staining further demonstrated that acute DMY administration triggered cellular apoptosis, as evidenced by a significant increase in cleaved caspase 3 associated with Aβ plaques (Fig. 8c, d). We noticed that there was no significant alteration in the expression of GNFAP and NeuN in 5×FAD mice treated with DMY compared with the placebo group (Fig. 8d and Supplementary Fig. 14a, b), indicating that DMY treatment had no significant effect on the survival of astrocytes and neurons associated with Aβ plaques. Taken together, the data implied a target-specific effect of acute DMY treatment on senescent microglial cells associated with Aβ plaques, while largely sparing astrocytes and neurons.

Previous studies showed that DMY administration prevents Aβ deposition and cognitive decline in APP/PS1 mice 43. However, none of these studies focused on the role of DMY administration in the interaction between cellular senescence and Aβ pathology. We next assessed whether elimination of senescent microglial cells through long-term intermittent senolytic treatment with DMY can affect SA-β-Gal activity and Aβ pathology in 5×FAD mice. Beginning at 3 months of age, female 5×FAD mice were treated with either placebo or DMY twice per week for 3 months. Spatial learning and memory were evaluated by testing the 5×FAD mice in the Y-maze at 6 months of age, whereas animals were euthanized at 6.5 months, with their brains processed for histological and biochemical analyses (Fig. 8e). Aβ plaque-associated SA-β-Gal activity in the cortex and hippocampus was diminished in the DMY-treated 5×FAD mice compared to the placebo-treated littermates (Fig. 8f, g and Supplementary Fig. 14c, d). Unlike the acute DMY treatment, there was a marked reduction in Aβ plaque load in the cortex and hippocampus of DMY-treated 5×FAD mice compared to placebo-treated (Fig. 8f, g and Supplementary Fig. 14c, d). Moreover, DMY administration reduced levels of Aβ1-42 and Aβ1-40 in serum, suggesting potential effects of the DMY senolytic treatment on Aβ production and/or clearance (Fig. 8h). In line with a reduction of IL6 level in response to DMY treatment (Fig. 8f, g and Supplementary Fig. 14c, d), the transcript level of the senescence biomarker p16Ink4a and various proinflammatory cytokines (TNF-α, IL6, IL1β) was significantly lower in the cortex and hippocampus of DMY-treated AD mice compared to those treated with placebo (Fig. 8i and Supplementary Fig. 14e). We also observed that SA-β-Gal positivity associated with activation of microglial and astrocytes were significantly reduced in the cortex and hippocampus of DMY-treated 5×FAD mice compared to placebo-treated (Supplementary Fig. 14f, g). Unsurprisingly, as compared with placebo-treated 5×FAD mice, those treated with DMY over the long-term performed significantly better in Y-maze test, grip strength assay and rotarod test (Fig. 8j-l). These data suggested that elimination of senescent microglial cells through DMY administration is sufficient to restore tissue homeostasis and physiological function, alleviating Aβ pathology and cognitive impairment in AD mice.

Methods
Cell culture

The primary normal human prostate stromal cell line PSC27 and the breast fibroblast cell line HBF1203 were provided by Dr. Peter Nelson (Fred Hutchinson Cancer Center) and maintained in a complete stromal medium as described previously 54. Specifically, PSC27 derived from a normal human prostate tissue, and is composed of mainly fibroblasts, with a minor proportion of non-fibroblastic cells such as myofibroblasts and endothelial cells. The human umbilical vein endothelial cell line HUVEC was acquired from ATCC (PCS-100-013) and cultured in DMEM medium, supplemented with 10% fetal bovine serum (FBS). The prostate cancer epithelial cell lines PC3, DU145 and LNCaP were from ATCC (CRL-1435, HTB-81 and CRL-1740, respectively) and routinely cultured in RPMI 1640 medium supplemented with 10% FBS. The prostate cancer epithelial line M12 was from Dr. Stephen Plymate (University of Washington). Furthermore, human brain tissue-derived cell lines HMC3, MO3.13 and SVG p12, and murine microglial cell line BV2 were from the National Collection of Authenticated Cell Cultures (China) (GNHu70, SCSP-5420, SCSP-5424 and SCSP-5208, respectively) and were regularly cultured in DMEM medium supplemented with 10% FBS. All cell lines underwent routine test for mycoplasma contamination and were authenticated using Short Tandem Repeat assays.

Cell treatments

Cells were cultured until reaching approximately 80% confluence (CTRL) under the condition of 5% CO2 at 37°C, and subsequently treated with 50 μg/ml bleomycin for a duration of 6-12 hr. Following treatment, cells were briefly washed with phosphate buffered saline (PBS) and maintained for a period of 7-10 d to allow for cellular senescence, before subject to various analyses. The senotherapeutic agents screened in this study were commercially acquired from TargetMol L8200-Anti-aging Compound Library. To identify potential senotherapeutics, natural candidates (totally 50 in the NMA library) were tested each at 100 μM on the survival of 5.0 × 103 non-senescent and senescent cells for 3 d (senolytics). Subsequently, appraisal of the effects of remaining agents (50 in the library, each applied at 50 μM) was performed to test the ability to inhibit the SASP (senomorphics). For those demonstrating significant potential as effective and safe senotherapeutics, further assessments were followed. The phytochemical agent DMY was tested across a concentration gradient 0 μM to 800 μM, with 100 μM identified as a fairly effective concentration for senomorphic activity in PSC27 cells, with 100 μM (starting from 50 μM) as an effective concentration for senolytic activity in HMC3 cells. Additionally, the small molecule inhibitor SB203580 targeting p38MAPK was utilized at a concentration of 20 μM rutin, while the PRDX2 inhibitor Conoidin A was applied at concentrations 1 μM to 4 μM to treat senescent cells in culture prior to cell lysis and expression assays.

HPLC-QTOF-MS/MS analysis

The standard high-performance liquid chromatography (HPLC) coupled with quadrupole time-of-flight tandem mass spectrometry (HPLC-QTOF-MS/MS) was performed on a Nexera X2 LC-40 system (SHIMADZU), integrated with the AB SCIEX TripleTOF 6600 LC/MS/MS apparatus (SCIEX). The data were processed using Analyst TF software (version 1.7.1). Briefly, the chromatographic separation was conducted on an Accucore C30 column (2.6 μm, 250 × 2.1 mm, Thermo Fisher) at room temperature. The mobile phase consisted of 1% acetic acid in water (designated as solvent A) and 100% methanol (designated as solvent B). A multistep linear gradient was employed, beginning at 5% solvent B and progressing to 15% B within 5 min, reaching 35% B at 30 min, and 70% B at 45 min, sustaining 70% B until 49 min, followed by a transition to 100% B at 50 min. The system was held at 100% B for an additional 10 min before returning to 5% B by the 60-min mark. During both the initial and final gradient stages, the column underwent equilibration and maintenance or washing with 5% B for a duration of 10 min. The flow rate was maintained at 0.2 ml/min, with a sample injection volume of 10 μl. Detection was performed via the TripleTOF 6600 qTOF mass spectrometer (AB SCIEX), which utilized an electrospray ionization (ESI) interface in negative ion mode. The operating parameters of the ESI source included a capillary voltage of -4000 V, a drying gas flow rate of 60 (arbitrary units), nebulization gas pressure set at 60 psi, a capillary temperature of 650°C and a collision energy of 30 eV. Mass spectra were recorded over a mass-to-charge ratio (m/z) range of 50 to 1200, with an acquisition rate of 3 spectra per second. Data acquisition and subsequent analysis were conducted using Analyst TF software (AB SCIEX, version 1.7.1). The number of biological replicates is provided in the legend per figure.

Human proteome microarray

DMY coupled with biotin (DMY-Biotin) was synthesized and identified by NMR, which was performed by Wayen Biotechnologies (Shanghai, China). D-Biotin was used as a control. The human proteome microarray was also sourced and performed by Wayen, wherein the HuProtTM 20K microarray (CDI Laboratories, USA) consists of 20,240 full-length human proteins. Microarrays were immersed in pre-cooled blocking buffer (5% BSA and 0.1% Tween 20 in PBST) for 5 min and treated with blocking buffer at room temperature for 1.5 hr with gentle agitation in dark. DMY-Biotin and D-Biotin were then diluted to a concentration of 10 μM in the blocking buffer and applied to the previously blocked proteome microarray, and incubated at room temperature for 1 hr. Microarrays were washed thrice with PBST, each for 5 min. Microarrays were then treated with a 0.1% Cy5-Streptavidin solution for 20 min at room temperature, followed by 3 additional washes with PBST, each lasting 5 min. The microarrays were subsequently scanned with a GenePix 4000B microarray scanner (Axon Instruments, USA) to visualize and record the results. Data analysis was performed utilizing GenePixTM Pro v6.0 software (Axon Instruments, USA).

DMY-Biotin-target protein pull-down and LC-MS/MS analysis

Pull-down experiments along with LC-MS/MS analyses were performed by Wayen Biotechnologies (Shanghai, China). Briefly, proteins were extracted from senescent HMC3 and PSC27 cells using immunoprecipitation-specific lysis buffer containing protease inhibitors (1:100). For pull-down, a PierceTM Spin Column was prepared by adding TBS, which was subsequently immobilized and labeled with 5 mM DMY-Biotin or D-Biotin (as a control) overnight at 4°C. To block the PierceTM Spin Column, a biotin blocking solution was added at room temperature. Following this, 1 mg of the extracted protein was incubated with the PierceTM Spin Column at 4°C for 2 hr, after which the column was washed 3 times with a wash buffer. After reduction in 10 mM DTT, alkylation in 40 mM IAA and protease digestion with a trypsin solution (Promega, 1:50), the pull-down proteins were extracted using 50 mM NH4HCO3, before peptide solution was transferred to Solid Phase Extraction Cartridge (MonoSpin C18, GL Sciences) for desalting and clean-up.

Samples obtained above were analyzed by a Orbitrap Fusion Lumos mass spectrometer equipped with a Nanospray Flex source (Thermo Fisher Scientific, USA). Sample was separated by a C18 column (75 μm × 20 cm) packed with Reprosil-Pur C18-AQ, 1.9 mm resin on a nanoflow HPLC Easy-nLC 1000 system (Thermo Fisher Scientific). Buffer A comprising 0.1% (v/v) formic acid in H2O and Buffer B comprising 0.1% (v/v) formic acid in 80% acetonitrile. The separation was achieved using gradient elution program at 300 nl/min at 50°C: 1%-5% B in 4 min; 5%-26% B in 42 min; 26%-40% B in 10 min; 40%–100% B in 4 min; 100% B in 5 min. MS scan was performed in positive ion mode with spray voltage at 1,900 V, with ion transfer tube temperature at 320°C. The acquisition of MS data was conducted by Data Dependent Acquisition (DDA) mode. The MS1 full scan (350-1800) was set at a resolution of 60,000 @ m/z 200, automatic gain control (AGC) target 4e5 and maximum IT 50 ms. Isolation window was set at 0.7 m/z. MS2 scans were generated by HCD fragmentation at a resolution of 15,000 @ m/z 200, AGC target 1e5 and maximum IT 22 ms. The normalized collision energy (NCE) was set at NCE 30%, and the dynamic exclusion time was 30 sec. The Sequest HT search algorithm in Proteome Discoverer 2.4 was used to search MS/MS spectra against a composite database comprised of all the UniProt human database (swissprot_human_20423_20240516.fasta). Minimum cutoff for peptide length was set at 6 amino acids, and maximum permissible missed cleavage was set at 2. Maximal FDR for peptide spectral match, proteins and site was set to 0.01.

Co-IP and LC-MS/MS analysis

PSC27 cells were induced senescent by exposure to 50 μg/ml BLEO for 12 hr before treated with 100 μM DMY or vehicle. Following this, cells were harvested using immunoprecipitation (IP) lysis buffer (Beyotime, P0013J) supplemented with protease and phosphatase inhibitors (1:100), and then incubated overnight at 4°C with a PRDX2 antibody (1:100, Abclonal, A1919) or IgG antibody (negative control). The resulting cell lysates were incubated with Protein A/G Magnetic Beads at 4°C for 2 hr. The magnetic beads were subjected to 5 washes with the IP lysis buffer. Proteins captured by the beads were subsequently analyzed using silver staining or Western blot. Alternatively, immunoprecipitated proteins were separated from magnetic beads using 8 M urea and subsequently analyzed by LC-MS/MS. Peptides were resolved in 0.1% formic acid, with the BCA protein quantification kit employed to measure peptide concentration. Samples were separated utilizing an analytic column (75 μm × 15 cm, 3 μm, 100Å) (C18, Thermo Fisher Scientific) on a nanoflow HPLC Easy-nLC 1200 system (Thermo Fisher Scientific, USA), using a 120 min LC gradient at 300 nl/min. Buffer A comprising 0.1% (v/v) formic acid in H2O and Buffer B comprising 0.1% (v/v) formic acid in 80% acetonitrile. The solution gradient was as follows: 1%-5% B in 6 min; 5%-26% B in 88 min; 26%-40% B in 22 min; 40%–100% B in 5 min; 90% B in 5 min. MS analyses were conducted on a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific). MS scan was performed in positive ion mode with spray voltage at 1,900 V, with ion transfer tube temperature at 320°C. Xcalibur software was used to acquire profile spectrum data in data-dependent acquisition pattern (DDA). The MS1 full scan was set at a resolution of 60,000 @ m/z 200, AGC target 3e6 and maximum IT 50 ms by orbitrap mass analyzer (350-1800 m/z), followed by ‘top 15’ MS2 scans generated by HCD fragmentation at a resolution of 15,000 @ m/z 200, AGC target 1e5 and maximum IT 30 ms. Isolation window was set at 1.6 m/z. The normalized collision energy (NCE) was set at NCE 28%, with the dynamic exclusion time as 45 sec. Precursors with charge 1, 7, 8 and > 8 were excluded from MS2 analysis. All raw mass spectrometric data were analyzed using MaxQuant 2.0.3.0 against the human Swiss-Prot database comprising 20,423 sequences (2024). Carbamidomethyl of cysteine was selected as a fixed modification. Oxidized methionine, protein N-term acetylation, lysine acetylation, asparagine and glutamine (NQ) deamidation were selected as variable modifications. Enzyme specificity was set as trypsin. The tolerances of first search and main search (Andromeda search engine) for peptides were set at 20 ppm and 6 ppm, respectively. Minimum cutoff for peptide length was set at 7 amino acids, with maximum permissible missed cleavage set at 2. Maximal FDR for peptide spectral match, proteins and site was set to 0.01. A minimum of 2 sequence-unique peptides was required for identification. Feature matching between runs was done with a retention time window of 2 min, with the label-free quantitation (LFQ) function enabled. The MaxQuant peptide and protein quantification results from the “peptides.txt” and “proteinGroups.txt” files were imported into Perseus software (version 1.5.1.6) for further analysis. Statistical significance between the groups was evaluated using Student's t tests (P < 0.05), Proteins were defined as differentially expressed if the ratios were ≥ 1.5 or ≤ 0.67 in the experiment group compared with control.

Quantitative proteomics

Quantitative proteomics were performed by Wayen Biotechnologies (Shanghai, China). Approximately 200 μg total protein from cells was digested. After desalting and cleanup, peptides were analyzed by a Orbitrap Astral mass spectrometer equipped with a Nanospray Flex source (Thermo Fisher Scientific, USA). Samples were loaded and separated by a C18 column of ES906 (PepMap TM Neo UHPLC 150 μM X 15 cm, 2 μM) (Thermo Fisher Scientific). Mobile phase A: 0.1% (v/v) formic acid in water; Mobile phase B: 0.1% (v/v) formic acid in acetonitrile/water (80:20, v/v). The separation was achieved using gradient elution program: 96% mobile phase A at 2.5 µl/min for 0-4 min, 75% mobile phase A at 2.5 µl/min for 4-5.8 min, 65% mobile phase A at 2.5 µl/min for 5.8-6.2 min, 1% mobile phase A at 2.5 µl/min for 6.2-6.9 min. Separated peptides proceeded directly to MS for DIA analysis after ionizing by Easy-spray ion (ESI) source. The ion spray voltage was set to 2.0 kV, with the ion transfer tube temperature maintained at 290°C. Raw files obtained from Astral mass spectrometer were analyzed by DIA NN, and the database was Swissprot human protein database (20,423 entries, 2024). Quantitative analysis was performed at the MS2 level using peak areas. Peptide abundance was calculated by summing the peak areas of their respective MS2 fragment ions, with protein abundance calculated by summing the abundance of their respective peptides. Upon Student’s t-test analysis, differential proteins with P value < 0.05 and fold change > 1.2 were selected. Numerical proteom datasets are organized as Supplementary Data 1-4.

SPR assay

The binding affinity of DMY to recombinant human PRDX2, PRDX1 and PRDX6 was assessed at 22°C utilizing the Biacore T200 instrument equipped with CM5 sensor chips (GE Healthcare, BR100530). Briefly, recombinant human protein in acetate acid buffer (pH 4.5) was covalently immobilized on the sensor chips following activation by 40 mM EDC and 10 mM NHS in aqueous solution. Various concentrations of DMY, ranging from 1.563 μM to 50 μM, were prepared with running buffer (PBS, 5% DMSO and 0.5% Tween 20). The DMY compound at different concentrations was injected simultaneously, injection of DMY solution was performed for 60 sec, with a dissociation time of 60 sec in each binding cycle. Data processing and analysis were performed using Biacore 4000 and Biacore T200 evaluation software (GE healthcare).

CETSA assay

PSC27 cells were collected, and frozen-thawed 3 times using liquid nitrogen. The lysates were diluted with PBS containing protease inhibitor cocktail and divided into 2 aliquots, with 1 aliquot being treated with DMY (200 μM) and the other aliquot as control (DMSO). Each lysate was incubated with DMY or DMSO for 30 min at room temperature, then equally aliquoted into several parts in EP tubes and heated individually at different temperatures (35°C to 65°C) for 15 min. The precipitated proteins were separated from the soluble fraction by centrifugation at 14,000 × g at 4°C for 10 min, and boiled at 95°C for 10 min. Samples were subsequently used for immunoblot analysis. For the different drug concentration gradient CETSA experiments, PSC27 cell lysates were collected and aliquoted into several parts in EP tubes, then incubated with different DMY concentrations (0-400 μM) for 30 min at room temperature. Samples were heated at 55°C for 15 min before soluble fractions were isolated and analyzed by Western blot.

DARTS assay

PSC27 cells were collected and total protein was lysed on ice with RIPA buffer containing protease inhibitors. Lysates were then treated with different DMY concentrations (0-400 μM) at room temperature. After incubation for 1 hr at room temperature, 6 μg/ml pronase was added into the lysates for a further 10 min at 37°C. Reactions were stopped by 1×SDS-PAGE loading buffer and samples were analyzed by Western blot.

PRDX2 activity measurement

The activity of recombinant human PRDX2 protein was determined as previously described 38. PRDX2 exhibits the ability to reduce hydrogen peroxide (H2O2). Briefly, recombinant human PRDX2 proteins were either utilized alone or mixed with different concentrations of DMY, and incubated in a solution of 50 mM HEPES-HCl (pH 7.0) containing 1 mM DTT at room temperature for 10 min. The enzymatic reaction commenced with the introduction of 5 μl H2O2 (final concentration 100 μM) and incubated for 5 min. The remaining concentration of H2O2 was quantified, serving as an indicator of inhibitory effects on PRDX2's catalytic function. Conoidin A was used as a positive control.

Animals and drug treatments

All research complied with relevant ethical regulations. All animal experiments were performed in compliance with NIH Guide for the Care and Use of Laboratory Animals (National Academies Press, 2011) and the ARRIVE guidelines, and were approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences. Mice were maintained in a specific pathogen-free (SPF) facility, with a controlled ambient temperature ranging from 22 to 25°C and humidity set at 50% ± 10%. The animals were subjected to a 12-hr light-dark cycle, given drinking water and fed standard chow diet ad libitum.

Ionizing radiation

To induce in vivo senescence, C57BL/6J mice (Model Animal Research Center of Nanjing University) were treated twice at 3-4 months of age with a sub-lethal dose of whole-body irradiation (WBI) at 5 Gy. Following a one-week recovery period, the mice received intraperitoneal injections of 20 mg/kg DMY or a placebo, administered twice every week for 3 consecutive months. Functional assays were performed after the final injection, after which mice were euthanized by manual cervical dislocation or carbon dioxide inhalation. Tissues were collected for RNA extraction and frozen in OCT solution for subsequent cryo-sectioning, SA-β-Gal staining, immunohistochemistry and immunofluorescence analyses. All mice involved in the experiments were randomly assigned to either control or treatment groups, with both male and female animals recruited.

Chemotherapy

NOD-obese diabetic and severe combined immunodeficiency (NOD-SCID) mice (males, Model Animal Research Center of Nanjing University) aged 6 to 8 weeks were housed in a specific pathogen-free (SPF) environment, in compliance with the animal care guidelines established by the SINH. Each experimental group consisted of 10 mice, with tumor xenografts being established subcutaneously on the hind flank following anesthesia induced by isoflurane inhalation. Human prostate stromal cells (PSC27) were mixed with cancer cells (PC3) in a ratio of 1:4, specifically 250,000 stromal cells mixed with 1,000,000 cancer cells, to create tissue recombinants prior to in vivo implantation, as previously described 22. Following a 2-week period, mice were randomly assigned to various treatment groups for preclinical interventions. Treatments included MIT (0.2 mg/kg) alone, DMY (20 mg/kg) alone, a combination of MIT and DMY, or a placebo control, all administered via subcutaneous injection on the first day of the 3rd, 5th and 7th weeks, culminating in a total of 3 cycles over the entire 8-week treatment regimen. Tumor progression was monitored weekly, with tumor volume (v) evaluated and calculated using the formula: v = L(π/6) × ((l+w+h)/3)³, where length (l), width (w), and height (h) of tumors were measured. The maximal tumour size/burden permitted by the ethics committee is 3.375 cm3, and the maximal tumour size/burden was not exceeded. Animals were euthanized by manual cervical dislocation or carbon dioxide inhalation, with tumors freshly excised and either snap-frozen or fixed for preparation of formalin-fixed paraffin-embedded (FFPE) samples. These prepared sections were subsequently utilized for SA-β-Gal staining, immunohistochemistry and immunofluorescence staining.

AD mouse model and treatment

The 5×FAD transgenic mice were provided by Dr. Jiawei Zhou (Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences). These mice were identified by PCR-based genotyping and used for pharmacological analysis at 3 months of age. Genotyping primers: APP forward 5’-AGGACTGACCACTCGACCAG-3’, reverse 5’-CGGGGGTCTAGTTCTGCAT-3’; PS1 forward 5’-AATAGAGAACGGCAGGAGCA-3’, reverse 5’-GCCATGAGGGCACTAATCAT-3’. A cohort of 14 3-month-old female 5×FAD transgenic mice was randomly assigned to receive either intraperitoneal injections of 20 mg/kg DMY or a placebo, administered twice weekly for 3 consecutive months. In a separate experiment, ten 12-month-old female 5×FAD transgenic mice were randomly assigned to undergo acute short-term treatment, receiving a single daily intraperitoneal injection of 20 mg/kg DMY or placebo for a consecutive 7 d period prior to tissue collection. Upon completion of functional assays, mice were anesthetized using isoflurane and subsequently subjected to transcardial perfusion with PBS followed by 4% paraformaldehyde in PBS. The fixed and sucrose-cryoprotected brain tissues were sectioned into 10 μm sections for various staining.

Physical function measurements

All measurements were performed at least 5 days after the last dose of DMY treatment.

Y-maze test

The Y-maze spontaneous alternation was employed to evaluate short-term spatial memory using an opaque Perspex Y-maze (31 cm long, 5 cm wide and 10 cm high walls). Each arm had markers of different colors as distinct visual cues. Each animal was placed in turn in arm A of the Y-maze and allowed to explore freely for 5 min, with arm entries, alternations and walking distance recorded visually to calculate the percentage of the alternation behavior with the following formula: Alternation % = [Number of Alternations/ (Total number of arm entries - 2)] x 100. Spontaneous alternation (%) was defined as a successive entry into 3 different arms, on overlapping triplet sets.

Grip strength test

The forelimb grip strength (N-F/kg) was evaluated by employing a grip strength meter (Shanghai XinRuan). To ensure the accuracy and reliability of results, the test was conducted over 10 trials. In each trial, mice were carefully positioned. Upon completion of all trials, results were averaged to obtain a comprehensive assessment of the forelimb grip strength.

Rotarod test

The Rotarod test was utilized to assess motor coordination and balance with an advanced accelerating RotaRod system (Shanghai XinRua). Mice were trained on the RotaRod for a consecutive 2 d at a speed of 4 rpm for a duration of 200 sec. Subsequently, testing was conducted on the 3rd, 4th, 5th and 6th days. On the test day, mice were placed onto the RotaRod at an initial speed of 4 rpm/min. The rotating speed was gradually increased from 4 to 40 rpm/min over a 5 min interval. To accurately monitor the performance of the mice, a timer was employed to precisely record the moment when each mouse either dropped off the RotaRod or clung to the rod and completed a full passive rotation.

Statistics and reproducibility

Statistical analyses were predominantly conducted using GraphPad Prism version 8.0. A two-tailed unpaired Student’s t-test with Welch’s correction was used to assess statistically significant differences between 2 groups. One-way ANOVA with Tukey’s post-hoc comparison was used for multiple comparisons. Investigators were blinded to the allocation during the majority of experiments and outcome assessments. Animals were assigned to experimental groups based on their baseline body weight to ensure comparable body weights across groups, with randomization occurring only within groups. The determination of sample size was derived from prior experiments, so that statistical power analysis was not performed. No data were excluded from the analyses. Values are presented as mean ± SD unless otherwise indicated, with P < 0.05 considered significant. All data in this study were derived from independent biological replicates. Representative images (e.g., micrographs, blots) are shown from experiments that were repeated independently with comparable outcomes. Sample sizes (n) refer to biologically independent replicates (e.g., independent mice, cell cultures or tissue samples) unless otherwise indicated.

Data availability

Data supporting the plots within this paper and other findings of this study are available as source data. The RNA-seq data generated in the present study have been deposited in the NCBI Gene Expression Omnibus (GEO) database under accession code GSE190280 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE190280] (human stromal cells), GSE275834 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE275834] (human microglial cells) and GSE291050 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE291050] (human stromal cells in mouse xenografts during preclinical treatment) . The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository iProX (project ID no. IPX0011774000) with the ProteomeXchange dataset identifier PXD063165 [https://proteomecentral.proteomexchange.org/ui?pxid=PXD063165]. Source Data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors present a wealth of data in vitro and in vivo on potential senolytic and senomorphic properties of DMY. Mostly the data are good quality and advance the field. However, I do have some conners to be addressed prior to publication.\n1. As a model to explain these results, the authors state “When DMY was used against senescent fibroblasts, DMY interacts with PRDX2 and promotes its nuclear translocation to facilitate DDR and reduce ROS levels. However, when DMY was employed to treat senescent microglial cells, it interacts with PRDX2 and prevents it from nuclear translocation, causing elevated redox imbalance and resulting in cell apoptosis.” This model requires validation by siRNA knock down to confirm that PRDX2 is the target of DMY. In senescent fibroblasts, I would predict that PRDX2 knock down promotes accumulation of nuclear ROS and abrogates the effect of DMY. In microglial cells, I would predict that PRDX2 knock down would phenocopy DMY. It is essential that PRDX2 is validated as a DMY target by siRNA knock down of the target.\n2. The authors say that PSC27 is “mainly” fibroblasts. What does mainly mean? Is the line not clonal. Please clarify.\n3. In Figure 1, suppression of SASP can be more convincingly shown. For example, generate a heatmap from a panel of SASP genes, e.g. SenMayo. Does this confirm that many/most of these SASP genes are downregulated by DMY?\n4. The authors make broad generalizations from a small number of cell lines, for example “DMY exhibits senolytic activity against microglial cells” based on only 1 cell line, HMC3. To make cell type generalizations, results should be shown in at least 3 cell lines from that cell type.\n5. Given the various senolytic vs senomorphic effects in vitro, how are these processes defined in vivo? To really confirm senolytic (not senomorphic) activity in vivo, the authors would have to measure markers of cell death, not just fewer senescent cells. Have the authors done this?\n6. The authors state “binding of DMY to PRDX2 promotes its nuclear translocation and interaction with DNA repair proteins, thereby enhancing DNA repair efficacy in senescent cells.” To show enhanced DNA repair, the authors should measure DNA damage and repair directly, e.g. by Comet assay and DNA repair reporters.\n7. Similarly, to show impaired ETC function, the authors should perform mitochondria Seahorse assays.\n8. Antibodies to mouse p16 for tissue IHC/IF require validation on a knock out or tissues cleared for p16+ cells by INK-ATTAC. P16 antibodies are notoriously bad in the mouse.", "reviewer_2": "This study compellingly provides a comprehensive and well-structured demonstration of the senotherapeutic potential of dihydromyricetin (DMY), highlighting both its senomorphic and senolytic properties. Senotherapeutics, including agents that selectively eliminate senescent cells (senolytics) and those that suppress the senescence-associated secretory phenotype (SASP) without inducing cell death (senomorphics), represent a promising avenue for combating age-related diseases and promoting healthy aging. While DMY has previously been recognized for its roles in disease prevention and treatment, the authors rigorously elucidate its mechanisms of action in the context of aging. Notably, DMY exhibits senomorphic effects in stromal cell types such as fibroblasts and endothelial cells, by enhancing DNA repair via PRDX2 and suppressing SASP. We commend the author’s rigorous validation of PRDX2 interaction by multiple orthogonal methods, including thermal protein profiling, SPR, affinity MS, protein array, DARTS, and computational modeling. DMY exerted senolytic activity in microglial cells through ROS-mediated apoptosis. Furthermore, this study showed DMY mitigated tissue aging, enhanced chemotherapy efficacy, and reduced Alzheimer-like pathology by clearing senescent microglia. In a preclinical model, DMY was shown to improve cognitive function in mice, supporting its potential translational relevance. Overall, the findings support DMY's potential to combat aging-associated diseases, including cancer and neurodegeneration.\nXu et al. present a rigorous and well-executed study proposing dihydromyricetin (DMY) as a senotherapeutic agent with both senomorphic and senolytic properties—an important advancement toward the clinical translation of senescence-targeting therapies for age-related diseases. The manuscript is well written, well-structured, and supported by comprehensive set of in vivo and in vitro experiments and supplementary data. With some revisions addressing the minor comments outlined below, this work will make a valuable and much needed contribution to the journal and community.\nPlease address the following comments/questions/concerns:\n• Please make proteomic data/results available in a public repository such as MassIVE, PRIDE, etc.\n• Can the authors rule out alternative factors to basal PRDX2 levels in determining the cell-type specificity of the DMY mechanism? This is a reasonable proposed mechanism but if alternate possibilities cannot be ruled out, it would be worth stating it. E.g. the existence of a glia-specific protein that inhibits translocation of PRDX2.\n• DMY was a significant hit in two cell lines and well validated. It would be more compelling if tested for senotherapuetic activity in a few primary cell types. It will be of interest also to know the cell type specificity in different cells (perhaps in future studies).\n• Figure 1 - The RNAseq heatmap in Fig 1 highlights the top 50 differentially expressed genes, including seven representative SASP factors (CCL3, SERPINA5, SAA1, CCL22, WNT3A, CSF3 and SELP), which are different from the targets selected for qPCR/western blot validation. If the qPCR/WB markers (IL6, IL8, IL1β, MMP3, and GAPDH) were also seen to change in the RNAseq (outside the top 50), it may strengthen the data to show or state that.\n• Figure 3e: please clarify what was used as non-specific binding control condition in the results or caption. If D-Bio was not conducted for both HMC3 and PSC27 cells, what is the condition represented in the venn?\n• Figure 3g: I believe the left side of the venn has a typo and should read 391- so that a total targets correspond to 395 that were identified in Fig 3b.\n• Fig 5e – suggest checking for accuracy - enrichment of ROS production is not significant by the pvalue/FDR shown, so it does not seem to align with the claim that the pathway is enriched.\n• Can the authors speculate as to why DMY seems to activate PRDx2 translocation in senescent cells but not non-senescent, which seem to have a decrease upon DMY treatment, according to the western blot in Fig 4h. Is it consistent with the proposed mechanism?\n• It may be of interest in future experiments to utilize siRNA or KO of PRDX2 in cells in the context of DMY treatment, to verify the mechanism\n• May be useful to provide:\n1. A reference at line 49-54 for sentence “As a hallmark of organismal aging…………………. genotoxic therapeutic agents (therapy induced senescence, TIS)\n2. A reference at line 646-649- The primary normal human prostate stromal cell line PSC27……as described previously. Please reference the previous study.\n• Details of cell culture conditions- like %CO2, O2, and temperature\n• LC–MS Protocol: The description of the LC–MS workflow is currently incomplete and would be improved by expanding and reorganizing. The authors appear to have conducted three separate experiments:\n1. DMY-biotin target pulldown followed by LC–MS analysis using an Orbitrap Lumos with an Easy-nLC 1000 system.\n2. Quantitative proteomics (outsourced), and\n3. Co-IP followed by LC–MS/MS.\nHowever, it is difficult to determine which datasets correspond to which mass spectrometer, aside from the pulldown assay. In particular, the use of the Sciex TripleTOF 6600 system is not attributed to any specific experiment. A clearer delineation of which instrument was used for each part of the study would greatly enhance transparency and reproducibility.\n• Sample Preparation and Data Acquisition Details: The manuscript lacks a clear description of the sample preparation protocol for each of the LC–MS/MS experiments, such as the trypsin digestion protocol (conditions for reduction, alkylation, trypsin) and whether desalting was performed. Additionally, the MS1 and MS2 data acquisition parameters for the Sciex TripleTOF 6600 mass spectrometers and Orbitrap Fusion Lumos mass spectrometer are not provided. Information regarding the data processing and analysis parameters using Analyst TF software and Protein Discoverer is also missing. Also- please state how protein quantification was performed – e.g. is abundance based on the peak areas? Was data normalized during quantification? Including these details is essential for reproducibility and proper evaluation of the proteomics workflow. Including these details is essential for reproducibility and proper evaluation of the proteomics workflow.\n• Negative ion mode – was the acquisition really performed in negative ion mode? It may be worth rechecking; this is uncommon for proteomics.\n• Quantitative proteomics- This section required detailed methodology as described above or perhaps can be merged/included into the earlier section.\n• At line 837, please correct “D-Biontin” to “D-Biotin”.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "The manuscript by Xu and colleagues present a data-dense study proposing that the natural flavonoid, dihydromyricetin (DMY), as a senotherapeutic with applications in accelerated aging, cancer, and Alzheimer’s disease (AD). While the breadth of the claims is ambitious, the evidence supporting these conclusions is not sufficiently compelling at this stage. A more focused approach, perhaps concentrating on one disease area with deeper experimental validation, would strengthen the manuscript. In particular, the data supporting senolytic activity and improvements in AD mouse models require substantial additional substantiation. A major concern is the interpretation of DMY as a senotherapeutic and not merely restoring lysosomal function as previously reported.\nMajor Concerns\n1. Definition and Characterization of Senescence\no To classify a compound as a senomorphic agent, evidence is needed for sustained senescent cell phenotypes (e.g., cell cycle arrest via canonical CDK inhibitors, markers of cellular stress/damage) in the absence of inflammatory secretions.\no The data presented do not comprehensively evaluate senescence phenotypes. A broader panel of senescence markers should be included, as suggested in recent reviews.\no Reliance on SA-β-gal as the primary marker is not sufficient. Secreted analytes provide some supporting evidence, but additional markers, particularly for cell cycle arrest and mitochondrial dysfunction, are necessary given the proposed mechanism of action.\n2. Alternative Interpretation – Lysosomal Function Restoration\no A major concern is that the observed results may reflect restoration of lysosomal function rather than a true senotherapeutic effect. A recent study (Liu, X. et al., Arch Toxicol, 2025; https://doi.org/10.1007/s00204-025-04030-2) demonstrated that DMY restores lysosomal activity. Given that lysosomal functon(SA-β-gal) is used as the primary readout across these studies, the data here may similarly reflect lysosomal modulation rather than elimination or modulation of senescent cells.\no Stronger evidence is required to distinguish senotherapeutic activity from lysosomal restoration. Without this, the central claim of the manuscript is not supported.\n3. Alzheimer’s Disease Model and Interpretation\no The abstract claim that “DMY 38 eliminated senescent microglial cells from amyloid β plaques, alleviating Aβ accumulation, neuroinflammation and cognitive decline” is not well supported by the experimental data.\no Additional characterization of amyloid pathology is needed, including analyses of soluble versus insoluble Aβ species (e.g., Aβ1–40 and Aβ1–42 by ELISA).\no Figure 8F is confounded by plaque-associated lysosomal activity. A pH 4.0 control (versus pH 6.0) and analysis of tissue from mice with low or no plaque burden would strengthen interpretation.\no Immunofluorescence data with p16 appear non-specific (e.g., plaque-associated staining). Additional confirmation of senescence markers is needed—why was p16 not included in Fig. 8H, and p21 used instead?\n4. Rigor and Transparency of Experimental Design\no Key methodological details are missing, including sample sizes, number of biological and technical replicates, randomization procedures, order and spacing of behavioral assays, and how data were handled. These details are essential for assessing rigor and reproducibility.\n5. Scope of Senolytic Activity\no The manuscript acknowledges that DMY is not effective against senescent oligodendrocytes or astrocytes. Given that these cell types contribute to AD pathology, the authors should discuss how this limitation affects the therapeutic potential of DMY in AD.\nAccuracy of Statements\n• Lines 87–88: The statement “To date, AD has no approved disease-modifying therapies” is not correct. FDA-approved anti-amyloid therapies exist and are considered disease-modifying.\n• Lines 93–96: The claim that senescent cells have not been implicated in AD pathogenesis is inaccurate. Several publications (Musi et al PMID: 30126037, Bussian et al. PMID: 30232451; Zhang et al. PMID: 30936558 and others) demonstrate a causal role.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response to reviewers’ comments\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors present a wealth of data in vitro and in vivo on potential senolytic and\nsenomorphic properties of DMY. Mostly the data are good quality and advance the field.\nHowever, I do have some conners to be addressed prior to publication.\n1. As a model to explain these results, the authors state “When DMY was used against\nsenescent fibroblasts, DMY interacts with PRDX2 and promotes its nuclear translocation\nto facilitate DDR and reduce ROS levels. However, when DMY was employed to treat\nsenescent microglial cells, it interacts with PRDX2 and prevents it from nuclear\ntranslocation, causing elevated redox imbalance and resulting in cell apoptosis.” This\nmodel requires validation by siRNA knock down to confirm that PRDX2 is the target of\nDMY. In senescent fibroblasts, I would predict that PRDX2 knock down promotes\naccumulation of nuclear ROS and abrogates the effect of DMY. In microglial cells, I would\npredict that PRDX2 knock down would phenocopy DMY. It is essential that PRDX2 is\nvalidated as a DMY target by siRNA knock down of the target.\nResponse:\nWe appreciate the reviewer’s positive comments on our study. We have validated\nby shRNA-mediated knockdown (among the 3 shRNAs we designed, #1 and #3\nexhibited more effective knockdown of PRDX2 (Supplementary Fig. 8a, b)).\nExperimental results indicated that PRDX2 knockdown abrogated the effect of\nDMY in senescent fibroblasts (Supplementary Fig. 8c, e). In microglial cells,\nhowever, PRDX2 knockdown phenocopied DMY treatment (Supplementary Fig. d,\nf). Given that the basal expression level of PRDX2 in PSC27 cells is substantially\nhigher than that in HMC3 cells, we overexpressed PRDX2 in HMC3 cells\n(Supplementary Fig. 8g). Following PRDX2 overexpression in HMC3, we observed\nthat the effect of DMY in clearing senescent microglia was basically abrogated\n1\n(Supplementary Fig. 8h, i). Concurrently, we detected reactive oxygen species\n(ROS) and found that the accumulation of ROS in senescent microglia treated with\nDMY was reduced after PRDX2 overexpression (Supplementary Fig. 8j). Thus,\nPRDX2 is validated as a molecular target of DMY in our study. Please also refer to\nthe new contents in Page 11.\n2. The authors say that PSC27 is “mainly” fibroblasts. What does mainly mean? Is the line\nnot clonal. Please clarify.\nResponse:\nGood question. We have clarified in the manuscript that PSC27 is a primary cell\nline derived from human prostate stromal cells. Of note, this line compromises\npredominantly fibroblasts and exhibits fibroblast characteristics (refer to the text in\nPage 5). It is not clonal.\n3. In Figure 1, suppression of SASP can be more convincingly shown. For example,\ngenerate a heatmap from a panel of SASP genes, e.g. SenMayo. Does this confirm that\nmany/most of these SASP genes are downregulated by DMY?\nResponse:\nThis does make sense and we appreciate the reviewer’s comments. To address,\nwe have generated a heatmap (Fig. 1e), which displays the expression change of\na panel of senescence/SASP-associated genes, including those organized from\nthe SenMayo geneset (PMID: 35974106). Our data suggest that many, although\nnot all, canonical SenMayo genes are significantly (P < 0.05) downregulated\nfollowing DMY treatment. Please refer to the updated Fig. 1e (legend in Page 43).\n4. The authors make broad generalizations from a small number of cell lines, for example\n“DMY exhibits senolytic activity against microglial cells” based on only 1 cell line, HMC3.\nTo make cell type generalizations, results should be shown in at least 3 cell lines from that\n2\ncell type.\nResponse:\nWe do appreciate above advice. After searching microglial cell lines available in the\nscientific community, however, we found there was a limited number of such lines,\nincluding human microglial cell line HMC3 and murine microglial cell line BV2. To\nvalidate the efficacy of DMY across different cell lines, we provided additional data\nto substantiate the effects of DMY on microglial cells, particularly BV2 cells. Our\nresults indicate that, similar to findings we observed in human HMC3 cells, DMY\nexhibits senolytic activity in mouse BV2 cells (Supplementary Fig. 6g, h). Together,\nit is evident that DMY has the efficacy in clearing senescent microglial cells derived\nfrom both human and mouse brain tissues (refer to new text across Pages 8 and\n9).\n5. Given the various senolytic vs senomorphic effects in vitro, how are these processes\ndefined in vivo? To really confirm senolytic (not senomorphic) activity in vivo, the authors\nwould have to measure markers of cell death, not just fewer senescent cells. Have the\nauthors done this?\nResponse:\nGood point. We have assessed the tissue level change of markers indicative of cell\ndeath in vivo, including immunofluorescence staining of cleaved caspase 3 (Fig.\n8c, d). This is in addition to measurement of the reduced number of senescent cells,\nthus confirming the senolytic activity of DMY. Please see Pages 19-20.\n6. The authors state “binding of DMY to PRDX2 promotes its nuclear translocation and\ninteraction with DNA repair proteins, thereby enhancing DNA repair efficacy in senescent\ncells.” To show enhanced DNA repair, the authors should measure DNA damage and repair\ndirectly, e.g. by Comet assay and DNA repair reporters.\nExcellent suggestions. We have confirmed the ability of DMY to enhance DNA\nrepair capacity of senescent cells through direct detection of DNA damage extent\nas well as repair efficiency using comet assay and DNA repair reporter systems.\nData from these assays have been displayed in Fig. 4b, c. The corresponding\nexperimental details are now included in the methods section. Please refer to\nPages 11 and 28-29.\n7. Similarly, to show impaired ETC function, the authors should perform mitochondria\nSeahorse assays.\nResponse:\nThis is a good point. We have now conducted seahorse assays to assess\nmitochondrial function by measuring oxygen consumption rate (OCR). As\ncompared with proliferating cells, senescent HMC3 exhibited decreased basal\nrespiration and ATP production capacity. Notably, DMY treatment further reduced\nthe basal respiration and ATP production ability of senescent HMC3 cells,\nsuggesting that DMY caused mitochondrial dysfunction in these cells. Additionally,\nwe further found that DMY decreased the activities of mitochondrial complex III and\ncomplex V. These results together indicated that DMY impaired ETC function of\nsenescent cells. The new data are incorporated into Fig. 5h-j, with relevant\nexperimental procedures detailed in the methods section. See Pages 14 and 30.\n8. Antibodies to mouse p16 for tissue IHC/IF require validation on a knock out or tissues\ncleared for p16+ cells by INK-ATTAC. P16 antibodies are notoriously bad in the mouse.\nResponse:\nWe agree with the reviewer on above advice and do appreciate it. Indeed, we also\nfound that p16 antibodies performed poorly in mouse tissues for IHC/IF assays. In\n4\nthis study, we tested p16 antibodies from multiple manufacturers, with one of them\n(Abcam, cat. no. ab241543) appearing to be basically acceptable for use. We\nnoticed that regions stained with this p16 antibody largely overlapped with the\npositive regions of SA-β-Gal staining for IF/IHC staining (below Supplemental Data\nFig. 1a, b). Additionally, to rule out the possibility of non-specific antibody\nadsorption resulting from positive SA-β-Gal staining, we co-stained with antibodies\nsuch as those against Aβ , IBA1 and GFAP alongside SA-β-Gal staining. IHC\n1-42\nassays demonstrated that antibodies including those against Aβ , IBA1 and\n1-42\nGFAP exhibited good specificity (Fig. 8h; below Supplemental Data Fig. 1c).\nAlternatively, we utilized an established senescence mouse model, p16-3MR\n(PMID: 25499914), which allows to similarly clear p16+ cells (below Supplemental\nData Fig. 1d) (we apologize that we do not have INK-ATTAC model). We crossed\np16-3MR with 5×FAD mice to generate p16-3MR/5×FAD mice. Brain tissue\nsections from these mice revealed mRFP signals upon fluorescence microscopic\nexamination. Although p16+ cells exhibit mRFP expression and their area basically\nresembled SA-β-Gal+ area in this model (below Supplemental Data Fig. 1e), we\nnoted that inherent mRFP signals in these mice was weak and could only be\ndetected in the absence of experimental treatment. Upon co-staining with other\nproteins, such as Aβ , fluorescence of these proteins obscured the inherent\n1-42\nmRFP in p16-3MR/5×FAD mice. We then employed GCV to eliminate p16+ cells\nfrom mouse tissues and performed immunofluorescence staining (below\nSupplemental Data Fig. 1f). The staining outcomes were comparable to those from\nDMY treatment to clear senescent microglia from mouse brain tissues (Fig. 8b).\nThus, we have largely validated the specificity and reliability of a p16 antibody\n(Abcam) for mouse tissue staining with IHC. Despite this, we acknowledge that the\nperformance of this antibody is not superior, and higher quality staining of p16 in\nmouse tissues remains a technical challenge.\nThis study compellingly provides a comprehensive and well-structured demonstration of the\nsenotherapeutic potential of dihydromyricetin (DMY), highlighting both its senomorphic\nand senolytic properties. Senotherapeutics, including agents that selectively eliminate\nsenescent cells (senolytics) and those that suppress the senescence-associated secretory\nphenotype (SASP) without inducing cell death (senomorphics), represent a promising\navenue for combating age-related diseases and promoting healthy aging. While DMY has\npreviously been recognized for its roles in disease prevention and treatment, the authors\nrigorously elucidate its mechanisms of action in the context of aging. Notably, DMY exhibits\nsenomorphic effects in stromal cell types such as fibroblasts and endothelial cells, by\nenhancing DNA repair via PRDX2 and suppressing SASP. We commend the author’s\nrigorous validation of PRDX2 interaction by multiple orthogonal methods, including\nthermal protein profiling, SPR, affinity MS, protein array, DARTS, and computational\nmodeling. DMY exerted senolytic activity in microglial cells through ROS-mediated\n6\napoptosis. Furthermore, this study showed DMY mitigated tissue aging, enhanced\nchemotherapy efficacy, and reduced Alzheimer-like pathology by clearing senescent\nmicroglia. In a preclinical model, DMY was shown to improve cognitive function in mice,\nsupporting its potential translational relevance. Overall, the findings support DMY's\npotential to combat aging-associated diseases, including cancer and neurodegeneration.\nXu et al. present a rigorous and well-executed study proposing dihydromyricetin (DMY) as\na senotherapeutic agent with both senomorphic and senolytic properties—an important\nadvancement toward the clinical translation of senescence-targeting therapies for age-\nrelated diseases. The manuscript is well written, well-structured, and supported by\ncomprehensive set of in vivo and in vitro experiments and supplementary data. With some\nrevisions addressing the minor comments outlined below, this work will make a valuable\nand much needed contribution to the journal and community.\nPlease address the following comments/questions/concerns:\nPlease make proteomic data/results available in a public repository such as MassIVE,\nPRIDE, etc.\nResponse:\nOur proteomic data/results are available in ProteomeXchange Consortium,\ndeposited via the PRIDE partner repository iProX with the dataset identifier\nPXD063165.\nCan the authors rule out alternative factors to basal PRDX2 levels in determining the cell-\ntype specificity of the DMY mechanism? This is a reasonable proposed mechanism but if\nalternate possibilities cannot be ruled out, it would be worth stating it. E.g. the existence of\na glia-specific protein that inhibits translocation of PRDX2.\nResponse:\nand choose to address this concern by incorporating a discussion in the manuscript.\nWhile our proposed mechanism is supported by the data presented so far, we admit\nthe possibility of additional senotherapeutic mechanisms of DMY, which may\nthereby enhance the robustness of our study. To tackle this issue, we performed\nfurther experiments using shRNAs to reduce PRDX2 expression. The experimental\nresults validated that the basal PRDX2 level determines how DMY exerts its\nsenotherapeutic effects. Detailed findings are available in Supplementary Fig. 8.\nThe expression level of PRDX2 in a designated cell type is critical to dictate its\ncellular response to DMY treatment, determining whether DMY suppresses SASP\nexpression or triggers apoptosis of senescent cells. Please refer to the new text in\nPage 11.\nDMY was a significant hit in two cell lines and well validated. It would be more compelling\nif tested for senotherapuetic activity in a few primary cell types. It will be of interest also to\nknow the cell type specificity in different cells (perhaps in future studies).\nResponse:\nIn our study, we have observed senotherapeutic effects of DMY in several primary\ncell types, including fibroblasts, glial cells and vascular endothelial cells. Future\ninvestigations could allow to explore the impact of DMY on additional cell types. To\naddress this, we have conducted extra experiments with a mouse-derived\nmicroglial cell line (BV2) to evaluate the senotherapeutic activity of DMY. The\nrelevant data have been organized into Supplementary Fig. 6g-h, which largely\nconfirms the efficacy of DMY in clearing certain types of senescent cells such as\nmicroglial cells. Please see Pages 8-9.\nFigure 1 - The RNAseq heatmap in Fig 1 highlights the top 50 differentially expressed genes,\nincluding seven representative SASP factors (CCL3, SERPINA5, SAA1, CCL22, WNT3A,\nCSF3 and SELP), which are different from the targets selected for qPCR/western blot\n8\nvalidation. If the qPCR/WB markers (IL6, IL8, IL1β, MMP3, and GAPDH) were also seen\nto change in the RNAseq (outside the top 50), it may strengthen the data to show or state\nthat.\nResponse:\nWe appreciate this suggestion. To answer, we have generated a heatmap (Fig. 1e)\nbased on a panel of senescence/SASP-associated genes, including those from the\nSenMayo gene set (PMID: 35974106). This heatmap demonstrates that many, if\nnot all, SASP genes are significantly downregulated following DMY treatment.\nFigure 3e: please clarify what was used as non-specific binding control condition in the\nresults or caption. If D-Bio was not conducted for both HMC3 and PSC27 cells, what is the\ncondition represented in the venn?\nResponse:\nWe have revised the figure legend of Fig. 3e to clarify that the non-specific binding\ncontrol involved a sample treated with D-Bio, utilizing a biotinylated control in\nsenescent PSC27 cells. This fact is now mentioned in the updated figure caption.\nWe admit that the use of D-Bio from PSC27 cells for DMY-Biotin in HMC3 cells as\na control condition has somehow inherent limitations.\nFigure 3g: I believe the left side of the venn has a typo and should read 391- so that a total\ntargets correspond to 395 that were identified in Fig 3b.\nResponse:\nThanks for your kind reminder. We have corrected the typo in Fig. 3g to present the\naccurate number of 391. Please see the updated figure.\nFig 5e – suggest checking for accuracy - enrichment of ROS production is not significant\n9\nby the pvalue/FDR shown, so it does not seem to align with the claim that the pathway is\nenriched.\nResponse:\nWe appreciate your question. We have looked at the data of Fig. 5e and conducted\nGSEA. GSEA revealed significant enrichment of gene sets associated with\nmitochondrial function, particularly 'Oxidative phosphorylation' (NES = 2.052, FDR\n= 0.100), 'Mitochondrial complex III assembly' (NES = 1.802, FDR = 0.090), and\n'ROS and RNS production' (NES = 1.646, FDR = 0.167). Typically, |NES| > 1.5 and\nFDR < 0.25 are considered significant. In comparison to the 'ROS production' gene\nsets, the 'Oxidative phosphorylation' gene sets demonstrated greater significance.\nTherefore, Fig. 5e has been revised to accurately reflect the significance of\noxidative phosphorylation, in line with the recommendations of two other reviewers\nto supplement mitochondrial function experiments (Fig. 5h-j). Specifically, DMY\ncaused mitochondrial dysfunction in senescent microglia, which was consistent\nwith the functional results we observed, including decreased oxygen consumption\nrate (OCR) (Fig. 5h, i), reduced activities of complexes III and V (Fig. 5j) and\nincreased ROS production (Fig. 2j). Please refer to the new text in Page 14.\nCan the authors speculate as to why DMY seems to activate PRDx2 translocation in\nsenescent cells but not non-senescent, which seem to have a decrease upon DMY treatment,\naccording to the western blot in Fig 4h. Is it consistent with the proposed mechanism?\nResponse:\nIn this study, we hypothesize that DNA damage response (DDR) triggers cells to\nrecruit PRDX2 to the nucleus, despite its primary cytoplasmic role as a Redox\nprotein. Previous studies have shown that PRDX2 can be mobilized to double-\nstrand breaks (DSB) upon DNA damage repair (PMID: 21148313, PMID:\n30657231). It is thus plausible that PRDX2 is only translocated to the nucleus in\n10\nsenescent cells, as non-senescent cells lack DDR. In our study, DMY treatment\nfacilitated DNA damage repair and interacted with PRDX2, suggesting that in\nsenescent cells, binding of DMY to PRDX2 may enhance its nuclear translocation\nto facilitate DNA damage repair.\nWe improved the experiment by repeating the WB and updated the image in\nFig. 4j. It is now clear that DMY does not significantly change nuclear translocation\nof PRDX2 in non-senescent cells.\nIt may be of interest in future experiments to utilize siRNA or KO of PRDX2 in cells in the\ncontext of DMY treatment, to verify the mechanism.\nResponse:\nTo address this point, we did new experiments and organized the data into new\nSupplementary Fig. 8. Given that the induction time for cellular senescence ranges\nfrom 7 to 10 days, we used shRNA to reduce the expression of PRDX2 in both\nfibroblasts and microglia. We first confirmed the knockdown effect of PRDX2 at the\nprotein level with WB. Among the 3 shRNAs tested, no. 1 and no. 3 exhibited\neffective knockdown of PRDX2 expression (Supplementary Fig. 8a, b).\nConsequently, these 2 shRNAs were selected for subsequent experiments. In\nPSC27 line, the survival rate of senescent cells significantly decreased following\nPRDX2 knockdown by shRNA upon DMY treatment (Supplementary Fig. 8d, f).\nSimilarly, in microglial HMC3 cells, PRDX2 knockdown via shRNA resulted in a\nreduced cell survival rate even under treatment with a lower concentration of DMY\n(Supplementary Fig. 8e, g). Given that the basal expression level of PRDX2 in\nPSC27 cells is much higher than that in HMC3 cells, we overexpressed PRDX2\nsolely in HMC3 cells (Supplementary Fig. 8c). Following PRDX2 overexpression,\nwe observed that the effect of DMY in clearing senescent microglia was abrogated\n(Supplementary Fig. 8h, i). These results confirm that PRDX2 is indeed a target of\nDMY, as the knockdown of PRDX2 in fibroblasts leads to reduced cell survival, a\n11\nphenomenon similarly observed in microglial HMC3 cells. In microglial cells,\nPRDX2 knockdown repeated the effects of DMY treatment, while PRDX2\noverexpression negated the effects of DMY in clearing senescent microglia. These\nresults together further supported our data.\nMay be useful to provide:\n1. A reference at line 49-54 for sentence “As a hallmark of organismal aging………………….\ngenotoxic therapeutic agents (therapy induced senescence, TIS)\n2. A reference at line 646-649- The primary normal human prostate stromal cell line\nPSC27……as described previously. Please reference the previous study.\nResponse:\nWe appreciate these suggestions. We have added the requested references at\noriginal lines 49-54 and 646-649 to support the individual statements. Please see\nthe text of revised manuscript. Please see the upper part of Page 3.\n• Details of cell culture conditions- like %CO2, O2, and temperature\nResponse:\nNow we have included a detailed description of the cell culture conditions,\nincluding %CO2 and temperature, in the cell culture of methods section.\n• LC–MS Protocol: The description of the LC–MS workflow is currently incomplete and\nwould be improved by expanding and reorganizing. The authors appear to have conducted\nthree separate experiments:\n1. DMY-biotin target pulldown followed by LC–MS analysis using an Orbitrap Lumos with\nan Easy-nLC 1000 system.\n2. Quantitative proteomics (outsourced), and\n3. Co-IP followed by LC–MS/MS.\naside from the pulldown assay. In particular, the use of the Sciex TripleTOF 6600 system is\nnot attributed to any specific experiment. A clearer delineation of which instrument was\nused for each part of the study would greatly enhance transparency and reproducibility.\nResponse:\nDMY-biotin target pull-down followed by LC-MS analysis corresponds to Easy-nLC\n1000 system and Orbitrap Fusion Lumos mass spectrometer. Quantitative\nproteomics corresponds to Orbitrap Astral. Co-IP assay coupled with LC-MS/MS\ncorresponds to Orbitrap Fusion Lumos mass spectrometer.\nWe have also revised the LC-MS and quantitative proteomics protocol section\nto provide more details. Please see Pages 31-32.\nSample Preparation and Data Acquisition Details: The manuscript lacks a clear\ndescription of the sample preparation protocol for each of the LC–MS/MS experiments,\nsuch as the trypsin digestion protocol (conditions for reduction, alkylation, trypsin) and\nwhether desalting was performed. Additionally, the MS1 and MS2 data acquisition\nparameters for the Sciex TripleTOF 6600 mass spectrometers and Orbitrap Fusion Lumos\nmass spectrometer are not provided. Information regarding the data processing and\nanalysis parameters using Analyst TF software and Protein Discoverer is also missing.\nAlso- please state how protein quantification was performed – e.g. is abundance based on\nthe peak areas? Was data normalized during quantification? Including these details is\nessential for reproducibility and proper evaluation of the proteomics workflow. Including\nthese details is essential for reproducibility and proper evaluation of the proteomics\nworkflow.\nResponse:\nWe have provided a comprehensive description of the sample preparation protocol\n13\nfor each LC-MS/MS and quantitative proteomics experiment, including desalting\nprocedures and MS acquisition parameters for both mass spectrometers. Please\nsee Pages 31-32.\nNegative ion mode – was the acquisition really performed in negative ion mode? It may be\nworth rechecking; this is uncommon for proteomics.\nResponse:\nNegative ion mode only performed on high resolution MS identification of DMY,\nwhile all other proteomic experiment was acquired in positive ion mode.\nQuantitative proteomics- This section required detailed methodology as described above or\nperhaps can be merged/included into the earlier section.\nResponse:\nThanks for the constructive advice. Now we have provided further details for\nquantitative proteomics as requested by the reviewer to enhance clarity and\nreproducibility. Please see Page 32.\nAt line 837, please correct “D-Biontin” to “D-Biotin”.\nResponse:\nYes, we have corrected the typo from \"D-Biontin\" to \"D-Biotin\" in the revised\nmanuscript. Please see the lower part of Page 30.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nOK, we understand. Our response to this co-reviewer should be already provided\nwherever appropriate.\nReviewer #4 (Remarks to the Author):\nThe manuscript by Xu and colleagues present a data-dense study proposing that the natural\nflavonoid, dihydromyricetin (DMY), as a senotherapeutic with applications in accelerated\naging, cancer, and Alzheimer’s disease (AD). While the breadth of the claims is ambitious,\nthe evidence supporting these conclusions is not sufficiently compelling at this stage. A\nmore focused approach, perhaps concentrating on one disease area with deeper\nexperimental validation, would strengthen the manuscript. In particular, the data\nsupporting senolytic activity and improvements in AD mouse models require substantial\nadditional substantiation. A major concern is the interpretation of DMY as a\nsenotherapeutic and not merely restoring lysosomal function as previously reported.\nResponse:\nWe appreciate the constructive comments of this reviewer, which do help improve\nthe overall profile of our manuscript.\nMajor Concerns\n1. Definition and Characterization of Senescence\no To classify a compound as a senomorphic agent, evidence is needed for sustained\nsenescent cell phenotypes (e.g., cell cycle arrest via canonical CDK inhibitors, markers of\ncellular stress/damage) in the absence of inflammatory secretions.\no The data presented do not comprehensively evaluate senescence phenotypes. A broader\npanel of senescence markers should be included, as suggested in recent reviews.\no Reliance on SA-β-gal as the primary marker is not sufficient. Secreted analytes provide\n15\nsome supporting evidence, but additional markers, particularly for cell cycle arrest and\nmitochondrial dysfunction, are necessary given the proposed mechanism of action.\nResponse:\nThese are good points and we do appreciate. In the revised manuscript, our\nanalysis has been expanded to cover a broader panel of senescence markers,\nincluding additional indicators of cell cycle arrest and mitochondrial dysfunction.\nThese findings are now incorporated into current Fig. 5 and Supplementary Fig. 5.\nWhen classifying DMY as a senomorphic agent, we mainly refer to the capacity\nof DMY in restraining the expression of a wide spectrum of the SASP (Fig. 1c-h)\nand associated regulatory pathway (Fig. 4a). However, as is in line with and\nsupportive of the reviewer’s inputs, we noticed that senescent PSC27 cells\nmaintained a number of senescence phenotypes even after DMY treatment, such\nas sustained SA-β-gal positivity (Fig. 1a) and cell cycle arrest (Supplementary Fig.\n5c-e), nuclear enlargement and nuclear membrane disappearance (Supplementary\nFig. 5a). However, upon assessment of other senescence markers, we found that\nDMY reduced ROS production level (Fig. 1j) and DNA damage foci (Fig. 4a, 4b),\nas DMY facilitated DNA damage repair in senescent PSC27 cells (Fig. 4b-i).\nDespite these effects, DMY treatment did not significantly impact mitochondrial\nfunction in senescent fibroblasts (PSC27 cells) (Supplementary Fig. 5g-h). In\ncontrast, DMY it did impair mitochondrial function in senescent microglial cells (Fig.\n5), causing apoptosis of senescent HMC3 cells (Fig. 2). Collectively, these results\nsupport our conclusion that DMY exhibits senomorphic activity in certain cell types\nparticularly fibroblasts.\n2. Alternative Interpretation – Lysosomal Function Restoration\no A major concern is that the observed results may reflect restoration of lysosomal function\nthan a true senotherapeutic effect. A recent study (Liu, X. et al., Arch Toxicol, 2025;\nhttps://doi.org/10.1007/s00204-025-04030-2) demonstrated that DMY restores lysosomal\n16\nactivity. Given that lysosomal functon(SA-β-gal) is used as the primary readout across these\nstudies, the data here may similarly reflect lysosomal modulation rather than elimination\nor modulation of senescent cells.\no Stronger evidence is required to distinguish senotherapeutic activity from lysosomal\nrestoration. Without this, the central claim of the manuscript is not supported.\nResponse:\nWe recognize these as excellent questions. To address, we chose to use\nLysoTracker fluorescent probe to assess lysosomal function and morphology, and\nDQ-BSA to measure lysosomal enzyme activity and degradation extent. In\nsenescent fibroblasts such as PSC27 cells, there was an increase in the number\nof lysosomes but a decrease in lysosomal function. Treatment with DMY did not\nsignificantly change lysosomal function in senescent PSC27 cells (Supplementary\nData Fig. 2a, 2b). Similarly, senescent microglia HMC3 cells exhibited an increase\nin lysosome number and a decline in lysosomal function. In contrast, however, upon\ntreatment with DMY, senescent HMC3 cells underwent apoptosis, leading to a\nreduction in lysosome number (Supplementary Data Fig. 2c, 2d). DMY failed to\nrestore lysosomal function in either senescent PSC27 or senescent HMC3 cells.\nUpon careful reading of the literature (Liu, X. et al., Arch Toxicol, 2025), we\nspeculate that the discrepancy may be attributed to the cellular state, specifically\nsenescence. Although DMY was effective in restoring lysosomal function under\ncertain conditions, such as bortezomib-induced Schwann cell damage, it did not\nreverse cellular senescence. As is well known, once cells are induced to senesce,\nit is difficult and challenging to reverse. To the contrary, prolonged maintenance of\nsenescence phenotype is usually observed, including impaired lysosomal function.\nTo distinguish between senotherapeutic effects and restoration of lysosomal\nfunction, we conducted further analyses to elucidate the senotherapeutic properties\nof DMY. DMY exhibits senomorphic activity against senescent fibroblasts by\n17\nsuppressing SASP expression while maintaining persistent senescence-related\nphenotypes (Supplementary Fig. 5). Furthermore, DMY demonstrates senolytic\nactivity against senescent microglial cells by impairing mitochondrial function (Fig.\n5), a property that eventually leads to apoptosis.\n3. Alzheimer’s Disease Model and Interpretation\no The abstract claim that “DMY 38 eliminated senescent microglial cells from amyloid β\nplaques, alleviating Aβ accumulation, neuroinflammation and cognitive decline” is not well\nsupported by the experimental data.\nResponse:\nWe do recognize this issue and highly appreciate the constructive comment to\n18\nstrengthen our abstract, which is supposed to reflect our experimental results.\nPrevious studies have reported the role of DMY in alleviating Aβ accumulation,\nneuroinflammation, and cognitive decline (PMID: 29869390, PMID: 35758423,\nPMID: 24728903). Therefore, our study focuses on demonstrating the DMY’s\nsenolytic activity against senescent microglia both in vitro and in vivo, thereby\nalleviating Aβ accumulation. We now have revised our abstract and improved the\nrelevant discussion to more accurately report critical experimental findings that are\nrelevant to DMY's effects on senescent microglial cells. Please see these updates\nin the text.\no Additional characterization of amyloid pathology is needed, including analyses of soluble\nversus insoluble Aβ species (e.g., Aβ1–40 and Aβ1–42 by ELISA).\nResponse:\nWe appreciate above advice. Relevant assays to analyze amyloid pathology with\nELISA by measurement of Aβ and Aβ levels in the serum were performed.\n1-42 1-40\nExperimental data have been updated in Fig. 8h.\no Figure 8F is confounded by plaque-associated lysosomal activity. A pH 4.0 control\n(versus pH 6.0) and analysis of tissue from mice with low or no plaque burden would\nstrengthen interpretation.\nResponse:\nAn excellent input. We conducted additional experiments to stain β-Gal (pH 4.0)\nand SA-β-Gal (pH 6.0) with tissues of both WT and 5×FAD mice (Supplemental\nData Fig. 3a). We also employed 6-month-old and 24-month-old WT mice as\ncontrols, which appeared to have no plaque burden, for the staining of β-Gal and\nSA-β-Gal, respectively, followed by IHC staining for Aβ (Supplemental Data Fig.\n1-42\n3b). The results suggested enhanced plaque-associated lysosomal activity in the\nbrain tissue of 5×FAD mice, as evidenced by the overlap of β-Gal and SA-β-Gal\n19\nwith plaques. However, this observation may also suggest the accumulation of\nsenescent cells at plaque-specific sites, causing increased SA-β-Gal staining due\nto the elevated number of lysosomes in senescent cells. Additionally, in 5×FAD\nmice treated with DMY, β-Gal remained positive in plaque-free regions, while\nplaques exclusively co-localized with SA-β-gal. Together, our data support that\nDMY administration reduced the extent of Aβ accumulation and the number of\nsenescent cells.\no Immunofluorescence data with p16 appear non-specific (e.g., plaque-associated staining).\nAdditional confirmation of senescence markers is needed—why was p16 not included in\nFig. 8H, and p21 used instead?\nResponse:\nnon-specific, data from our assays suggest that certain p16 antibody such as the\none\nof the one from Abcam (cat. no. ab241543) is acceptable for use. We first examined p16\nantibodies using IF and IHC assays in 5×FAD mice tissue, and found p16 staining\nlargely overlapped with the positive regions of SA-β-Gal staining for IF/IHC staining\n(Supplemental Data Fig. 1a, 1b). Additionally, to eliminate the possibility of non-\nspecific antibody adsorption resulting from positive SA-β-Gal staining, we co-\nstained with antibodies such as Aβ , IBA1, and GFAP alongside SA-β-Gal\n1-42\nstaining. IHC assays demonstrated that antibodies like Aβ , IBA1, and GFAP\n1-42\nexhibited good specificity (Supplemental Data Fig. 1c).\nAdditionally, we crossed p16-3MR (PMID: 25499914) with 5×FAD mice to\ngenerate p16-3MR/5×FAD mice (Supplementary Data Fig. 1d). p16+ cells\nexhibiting red fluorescence (mRFP) were observed, and their morphology\nresembled the positive results of SA-β-Gal in this model (Supplemental Data Fig.\n1e). Therefore, wo chose p16 rather than p21 as a senescence marker. We\nemployed GCV to eliminate p16+ cells from mouse tissues and performed IF\nstaining (Supplemental Data Fig. 1f). The staining outcomes were comparable to\nthose observed with DMY treatment for the clearance of senescent microglia from\nmouse brain tissues, which is incorporated into the manuscript (Fig. 8b).\n4. Rigor and Transparency of Experimental Design\no Key methodological details are missing, including sample sizes, number of biological and\ntechnical replicates, randomization procedures, order and spacing of behavioral assays,\nand how data were handled. These details are essential for assessing rigor and\nreproducibility.\nResponse:\nThanks for these comments. In the revised manuscript, we have incorporated\ncomprehensive methodological details, including sample sizes, the number of\n21\nbiological and technical replicates, randomization procedures, and specifics\nregarding the order and spacing of behavioral assays, within the figure legends and\nmethods sections. Relevant information is now clearly provided to enhance the\nrigor and reproducibility of our study.\n5. Scope of Senolytic Activity\no The manuscript acknowledges that DMY is not effective against senescent\noligodendrocytes or astrocytes. Given that these cell types contribute to AD pathology, the\nauthors should discuss how this limitation affects the therapeutic potential of DMY in AD.\nResponse:\nWe appreciate this comment. We have provided a short discussion regarding the\nlimitations of DMY’s effectiveness against senescent oligodendrocytes and\nastrocytes, highlighting restrained implications for its therapeutic potential in\nAlzheimer’s disease. Please see the text in Page 24.\nAccuracy of Statements\n• Lines 87–88: The statement “To date, AD has no approved disease-modifying therapies”\nis not correct. FDA-approved anti-amyloid therapies exist and are considered disease-\nmodifying.\nResponse:\nWe appreciate the advice regarding the statement on FDA-approved therapies for\nAD. We have revised this statement to accurately reflect the current landscape of\ndisease-modifying therapies. Please see Page 4.\n• Lines 93–96: The claim that senescent cells have not been implicated in AD pathogenesis\nis inaccurate. Several publications (Musi et al PMID: 30126037, Bussian et al. PMID:\n30232451; Zhang et al. PMID: 30936558 and others) demonstrate a causal role.\nResponse:\ndemonstrate a causal role of senescent cells in AD pathogenesis, we have now\nrevised the manuscript to incorporate relevant literatures, which support the causal\nrole of senescent cells in AD. These efforts can further improve the quality of our\nmanuscript. Please see the relevant text in Page 4.\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse:\nWe highly appreciate the positive comments of this reviewer on our work and\naccurate recognition of the strength of our study.\nThere are a few points that would benefit from further clarification and elaboration:\nMajor concerns:\n1. SA-β-gal assay relies on the enzymatic activity of β-galactosidase. 5xFAD mouse tissues\nwere fixed by transcardial perfusion and sucrose-cryopreserved, which likely compromised\nthe protein activity and potentially invalidated the assay results. This assay should be\nperformed on fresh frozen tissue immediately after harvest. Moreover, as listed in the\noriginal method publication (PMID: 7568133), staining with a pH 4.0 solution should be\nincluded as a positive technical control to confirm the preservation of β-galactosidase\nactivity.\nResponse:\nWe appreciate these suggestions, and agree with the reviewer on the application\nof SA-β-Gal staining with fresh frozen tissues immediately post-collection. We\nwould share the following experimental details and technical tricks: (1) Prompt\ncollection of fresh brain tissues from 5xFAD mice immediately post-dissection,\nfollowed by embedding and storage at -80 °C within 12 hours, results in successful\npositive SA-β-gal staining after overnight incubation. This method is suitable for\ntissue SA-β-gal staining. (2) Immediate collection and embedding of fresh brain\ntissue from 5xFAD mice, when stored at -80 °C for 12-24 hours, necessitates 2-4\ndays of staining to achieve satisfactory results. Prolonged storage beyond 24 hours\nresults in staining failure, regardless of the staining duration. (3) Cardiac perfusion\nfixation of mouse brain tissue pre-dissection, followed by embedding in OCT and\nstorage at -80 °C for up to 2 years, enables successful positive SA-β-gal staining\nafter overnight incubation. This method has been implemented in our experiments.\n(4) SA-β-gal staining fails with fresh perfused fixed tissue for paraffin sections.\nMoreover, the SA-β-gal staining kit manual (Beyotime, C0602) recommends fixing\nfresh cells or tissues with the fixative for 20 minutes before staining. Finally, we\nwould share that fixation with 4% paraformaldehyde does not impair SA-β-gal\nactivity; instead, it prolongs preservation of tissue SA-β-gal at -80 °C and improves\nthe flexibility of experiments.\nIn addition, we agree with β-gal staining with a pH 4.0 solution as a positive\ntechnical control is essential and have performed this experiment. Please refer to\nthe results presented in Supplemental Data Fig. 3a (in this author response letter).\n24\n2. Figs 1e and 2d, to demonstrate whether DMY modulates the SASP milieu, which is\ncomposed of a large number of secreted factors, the authors should consider re-plotting\nthese 2 panels as heatmaps displaying only SASP-related genes. The SenMayo gene set\n(PMID: 35974106), a curated and validated list of senescence/SASP-associated genes,\nwould serve as an appropriate reference for selecting these factors.\nResponse:\nWe appreciate the reviewer’s insightful comments and kind advice. We have\ngenerated a heatmap (Fig. 1e and Fig. 2d) based on a number of\nsenescence/SASP-related genes in the SenMayo geneset (PMID: 35974106),\ndemonstrating comprehensive downregulation of multiple SASP genes post-DMY\ntreatment.\n3. Fig 4a, p16INK4a and p21CIP are cyclin-dependent kinase inhibitors, which mediate cell\ncycle arrest and apoptosis resistance, 2 of the 3 features of senescent cell. Staining/blotting\nfor additional apoptosis and cell cycle markers will provide supportive evidence that DMY\nexhibits senomorphic properties by inhibit SASP production without altering the other 2\nsenescence processes.\nResponse:\nThese are very good suggestions. We have performed additional experiments to\nexamine apoptosis and cell cycle arrest-associated markers. Experimental results,\nwhich are now presented in Supplementary Fig. 5 of the revised manuscript, further\nsupport that DMY inhibits SASP production while preserving other characteristics\nof senescence. Please refer also to the text in Page 6.\n4. In Figures 4 and 5, the authors propose that DMY exhibits differential effects on\nsenescent cell populations based on their basal levels of PRDX2 expression. To directly\nsupport this conclusion, the authors should experimentally modulating PRDX2 levels—\neither by downregulating or overexpressing PRDX2 using appropriate molecular tools—in\ndifferent senescent cell lines and then assess whether these manipulations alter the cellular\nresponse to DMY treatment.\nResponse:\nYes, we have now performed these experiments, with the results organized in\nSupplementary Fig. 8 of the revised manuscript. We used shRNAs to reduce\nPRDX2 expression in both fibroblasts and microglia. Among the 3 shRNAs\nexamined, #1 and #3 exhibited effective knockdown of PRDX2 (Supplementary Fig.\n8a, b), thus these shRNAs were selected for subsequent experiments. In\nfibroblast PSC27 cells, the survival rate of senescent PSC27 cells significantly decreased\nfollowing PRDX2 knockdown by shRNA during DMY treatment (Supplementary Fig.\n8c, 8e). Similarly, in microglial HMC3 cells, PRDX2 knockdown by shRNA resulted\nin a reduced cell survival rate even under treatment with a lower concentration of\nDMY (Supplementary Fig. 8d, 8f). Given that the basal expression level of PRDX2\nin PSC27 cells is substantially higher than that in HMC3 cells, we overexpressed\nPRDX2 in HMC3 cells (Supplementary Fig. 8g), and found that the effect of DMY\nin clearing senescent microglia was abrogated (Supplementary Fig. 8h, i). These\nresults suggest that PRDX2 is a target of DMY, as PRDX2 knockdown in fibroblasts\ncauses reduced cell survival, a phenomenon similarly observed in microglial HMC3\ncells. In microglial cells, PRDX2 knockdown repeated the effects of DMY treatment,\nwhile PRDX2 overexpression negated the effects of DMY in clearing senescent\nmicroglial cells. These results together further support our conclusion.\n5. Figure 8: the ‘hit-and-run’ intermittent dosing is a typical approach for senolytic\nadministration. The potential side effect of DMY on other brain cell types should be\nexamined after the long-course treatment, rather than after treating the 5xFAD mice with\nDMY once per day for 7 days. Oligodendrocytes should be characterized as well, as they\nundergo apoptosis upon exposure to DMY in vitro and are known to respond to amyloid\nplaques (PMID: 38819990).\nResponse:\nWe appreciate above suggestions and concur to use intermittent administration for\nsenolytic therapy. In our study, we implemented two distinct DMY administration\nprotocols. The first involved daily administration of DMY for 7 days to 5xFAD mice\nto investigate the specific target sites of DMY (Fig. 8a-d). The second regimen\nfollowed a twice/week administration of DMY over a 3-month period. This long-\ncourse DMY treatment strategy allowed evaluation of both the efficacy and\npotential side-effects of DMY (Fig. 8e-l), a common approach in senolytic therapy.\nAdditionally, we have also performed supplementary assays to evaluate the\nimpact of DMY treatment on oligodendrocytes. Our findings suggest that while\nDMY exerts effects on senescent microglial cells, it is not senolytic for\noligodendrocytes (Supplementary Fig. 6i, 6j). Moreover, DMY administration has\nno significant effect on oligodendrocytes in 5xFAD mice (Supplemental Data Fig.\n4).\nMinor concerns:\n1. Human gene symbols should be uppercase and italicized to distinguish them from\ncorresponding protein names.\nWe have revised the manuscript to display human gene symbols in an italicized\ncapital form, distinguishing them from corresponding protein names (standardized\nform). Please refer to updated Fig. 1c and Supplementary Fig. 4d, h, k as examples\nfor human gene symbols.\n2. The Western blot data presented in the figures should be accompanied by densitometric\nquantification, with the number of technical and biological replicates stated in the figure\nlegend.\nResponse:\nWe appreciate the kind suggestions. We have performed densitometric\nquantification for all Western blot data (not shown, due to space limitation),\nand\nspecified the number of technical and biological replicates in the figure legends as\nrequested.\n3. Line 340, please specify what “rh” refers to in “rhPRDX2”.\nResponse:\nWe have clarified that “rh” refers to “recombinant human” in the revised manuscript.\nPlease refer to the text in Page 10.\nAgain, we highly appreciate all the valuable inputs from the reviewers who have\nhelped improve the overall profile of our manuscript and strengthen the conclusions.\nSincerely,\nDr. Yu Sun\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author)\nThe authors have comprehensively and conscientiously addressed my concerns. The MS is\nin principle acceptable for publication in Nat Comms. However, I think there are some text\nchanges to be made.\n1. For PSC27 cells, rather than cite Peter Nelson in methods, give all available details re.\nthese cells. You say they are \"primary\" - if so, they should undergo replicative senescence in\nculture. You say they are \"predominantly fibroblasts\". Does this mean there are other non-\nfibroblastic cells present? These ambiguities together with the apparent lack of information\nfrom a reputable cell repository like ATCC suggests to me that they are not well understood\nor characterized. The authors should be clear about this in the methods. To me, they do not\nsound like the ideal cell model.\nResponse:\nExcellent points, and we appreciate the reviewer for raising above questions. PSC27\ncell line was originally established as a primary normal human prostate stromal cell\nline. It comprises predominantly fibroblasts, with a minor proportion of non-\nfibroblastic cells such as myofibroblasts and endothelial cells. Although it is not\ncommercially provided by reputable cell repositories like ATCC, several labs are\nusing it for senescence-related studies and senotherapeutic investigations. We have\naddressed the details in methods now. Please see “Cell culture” subsection of\nmethods.\n2. Re. the p16 abs, the authors have gone some way to confirming specificity, however, the\nauthors should state as a limitation of the MS the caveats re. specificity of these abs.\nResponse:\nThanks for the reviewer’s reminder. We chose to state the issue in the “Immunoblot,\nBrdU staining and immunofluorescence staining” subsection of Supplementary\nMethods. Please see the relevant statement.\nReviewer #2 (Remarks to the Author)\nOverall this is a very impressive and robust paper, and authors have provided detailed\nexplanations and additional experiments in response to most comments. However, several\nimportant points remain and should be addressed in the revised manuscript. In particular,\naccessible links and accession numbers (please provide both) to the raw proteomic data,\nreports of the proteomic results in the supplement, and detailed clarification around the\nMS/proteomic methodology are crucial for transparency and reproducibility.\nOnce these issues are addressed, we believe all other concerns were adequately addressed\nand the paper is suitable for publication.\n1) Dataset with identifier PXD063165 cannot be found either in iProX or in any\nProteomeXchange affiliated repository we searched. Please verify the identifier and update\nit – and provide a link to access it. The reviewers must have a chance to review these.\nResponse:\nWe kindly appreciate the reviewer for asking this question. Indeed, we submitted\nthe proteome data in April, 2025 to iProX to share the raw datasets publicly. The\nplatform of iProX has received our submission and approved the project\n“IPX0011774000” (”https://www.iprox.cn/page/project.html?id=IPX0011774000”,\nincluding 3 sub-projects IPX0011774001, IPX0011774002 and IPX0011774003）.\nWith a simultaneously allocated PXD number (PXD063165), the data are also\naccessible at the following link ：\nhttp://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD063165, and\nprojected to be released to public on 2026.12.31. However, if the manuscript is\naccepted for publication, we will be obligated to release the datasets even earlier.\n2) Provide the numerical proteomic datasets as supplementary tables – including the\nproteins, annotations, peak areas, fold changes, and statistics.\nResponse:\nNow we have prepared the numerical proteomic datasets as additional\nsupplementary files (Supplementary Tables 3-6), including those associated with\nseveral different proteomic analysis approaches. Thanks for the reviewer’s advice,\nand the data are readily available.\n3) Incomplete details for DMY-Biotin-target protein pull-down and subsequent MS analysis.\nThe revised method section still lacks essential experimental parameters needed for\nreproducibility. Please provide,\na) Composition of IP- specific buffers including concentration of protease inhibitors have\nused.\nb) NH4HCO3 concentration used for pull-down protein extraction.\nc) Vendor/ source of the solid phase extraction cartridges.\nd) Nanospray Flex ion source settings\ne) Solvent compositions\nf) LC gradient details, including whether the column temperature controlled.\ng) Trypsin concentration used for protein digestion\nh) DDA acquisition parameters (resolution, AGC target, Max IT, scan range, dynamic\nexclusion, etc.)\ni) Specify please the version of “SEQUEST”. Whether it was standalone or used by other\nplatforms like Proteome Discoverer, etc.\nResponse:\nWell, we appreciate the reviewer’s constructive comments. Please find the technical\ndetails as necessary. Although the section of methods is requested by the journal to\nbe no more than 3000 words, we managed to provide relevant details as sufficient\nas possible (see below).\n********************************************************************************************\n“DMY-Biotin-target pull-down and LC-MS/MS analysis\nPull-down experiments along with LC-MS/MS analyses were performed by Wayen\nBiotechnologies (Shanghai, China). Briefly, proteins were extracted from senescent\nHMC3 and PSC27 cells using immunoprecipitation-specific lysis buffer containing\nprotease inhibitors (1:100). For pull-down, a PierceTM Spin Column was prepared by\nadding TBS, which was subsequently immobilized and labeled with 5 mM DMY-\nBiotin or D-Biotin (as a control) overnight at 4°C. To block the PierceTM Spin Column,\na biotin blocking solution was added at room temperature. Following this, 1 mg of\nthe extracted protein was incubated with the PierceTM Spin Column at 4°C for 2 hr,\nafter which the column was washed 3 times with a wash buffer. After reduction in 10\nmM DTT, alkylation in 40 mM IAA and protease digestion with a trypsin solution\n(Promega, 1:50), the pull-down proteins were extracted using 50 mM NH HCO ,\n4 3\nbefore peptide solution was transferred to Solid Phase Extraction Cartridge\n(MonoSpin C18, GL Sciences) for desalting and clean-up.\nSamples were then analyzed by a Orbitrap Fusion Lumos mass spectrometer\n(Thermo Fisher Scientific, USA) equipped with a Nanospray Flex source. Sample\nwas separated by a C18 column (75 μm × 20 cm) packed with Reprosil-Pur C18-AQ,\n1.9 mm resin on a nanoflow HPLC Easy-nLC 1000 system (Thermo Fisher\nScientific). Buffer A comprising 0.1% (v/v) formic acid in H O and Buffer B\n2\ncomprising 0.1% (v/v) formic acid in 80% acetonitrile. The separation was achieved\nusing gradient elution program at 300 nl/min at 50°C: 1%-5% B in 4 min; 5%-26% B\nin 42 min; 26%-40% B in 10 min; 40%-100% B in 4 min; 100% B in 5 min. MS scan\nwas performed in positive ion mode with spray voltage at 1,900 V, with ion transfer\ntube temperature at 320°C. The acquisition of MS data was conducted by Data\nDependent Acquisition mode (DDA). The MS1 full scan (350-1800) was set at a\nresolution of 60,000 @ m/z 200, AGC target 4e5 and maximum IT 50 ms, Isolation\nwindow was set at 0.7 m/z. MS2 scans were generated by HCD fragmentation at a\nresolution of 15,000 @ m/z 200, AGC target 1e5 and maximum IT 22 ms. The\nnormalized collision energy (NCE) was set at NCE 30%, and the dynamic exclusion\ntime was 30 sec. The Sequest HT search algorithm in Proteome Discoverer 2.4 was\nused to search MS/MS spectra against a composite database comprised of all the\nUniProt human database (swissprot_human_20423_20240516.fasta). Minimum\ncutoff for peptide length was set at 7 amino acids, and maximum permissible missed\ncleavage was set at 2. Maximal FDR for peptide spectral match, proteins and site\nwas set to 0.01.”\nOf note, in our study, raw data were searched using the SEQUEST algorithm,\nwhich was integrated into Proteome Discoverer platform. In this case, SEQUEST\nwas not standalone, it was integrated in the Proteome Discoverer.\n4) Although author addressed this in the response that Orbitrap Fusion Lumos was used for\nCo-IP - MS analysis. This information does not appear in the revised manuscript. Please\ninclude it explicitly in the manuscript.\nResponse:\nThe relevant information is now provided in the revised manuscript, please see the\nsection of Co-IP and LC-MS/MS analysis in Methods.\n5) Concern in quantitative proteomics workflow:\na) Please provide mobile phase A and B.\nb) A 6.9 min total gradient using 15 cm column is exceptionally (unusually) fast! Since this\nis ‘quantitative’ proteomics, it’s important to specify the DIA method details such as the DIA\nwindow scheme (width and # per cycle), injection time, and the datapoints per peak achieved.\nc) The reported 2.5 µl/ min flow rate exceeds the manufacturer’s (Thermo) maximum of 2000\nnl/ min for EASY-nLCTM 1200 system (https://documents.thermofisher.com/TFS-\nAssets/CMD/Specification-Sheets/PS-64582-LC-EASY-nLC-1200-PS64582-EN.pdf). Please\nclarify/recheck this.\nd) Please specify the version of DIA NN and the parameters for analysis using DIA NN.\nResponse:\nAll the relevant information is now mentioned in the manuscript, please see the\nsection of Quantitative Proteomics. Specifically, Mobile phase A: 0.1% (v/v) formic\nacid in water, Mobile phase B: 0.1% (v/v) formic acid in acetonitrile/water (80:20, v/v).\nWe double checked the instrument/device used in quantitative proteomics and found\nthe correct description should be C18 analytical column of ES906 (PepMap TM Neo\nUHPLC 150 μM × 15 cm, 2 μM), while the EASY-nLCTM 1200 system (Thermo,\nUSA) was not used at all (mistakenly incorporated into the text in an earlier version\nof the manuscript). In our study, a 2.5 µl/min flow rate on the C18 analytical column\nof ES906 worked just fine, and we did not have problems. We thank the reviewer for\nhelping correct the error anyway.\n6) Kindly specify which mass spectrometer has been used for DMY identification in negative\nion mode and provide the corresponding instrument settings (if not specified in the\nmanuscript).\nResponse:\nNegative ion mode was only performed on HPLC-QTOF-MS/MS identification of\nDMY, while all other proteomic experiments were carried out in positive ion mode.\nWe have now specified details and provided the instrument settings as following:\n“Detection was performed via the TripleTOF 6600 qTOF mass spectrometer (AB\nSCIEX), which utilized an electrospray ionization (ESI) interface in negative ion\nmode…” in the HPLC-QTOF-MS/MS analysis section of Methods.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nReviewer #4 (Remarks to the Author):\nThe authors have addressed all concerns - no further comments. Congratulations on a well-\nconducted, thorough study.\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse:\nWe are grateful to all the reviewers’ dedicated review of this manuscript.\nFinally, due to the space limitation of the Methods section (3000 words), we chose\nto move some contents of Methods to the Supplementary Information.\nAgain, we highly appreciate all the valuable inputs from the reviewers who have\nhelped improve the overall profile of our manuscript and strengthen the conclusions.\nSincerely,\nDr. Yu Sun\nNature communication 2025 Xu et al\nIn this manuscript, the authors investigated the modulatory effect of dihydromyricetin\n(DMY), a natural flavonoid hit from a natural medicinal library, on cellular senescence. The\nstudy employs a comprehensive approach, utilizing multiple cell lines and mouse models, to\nexplore the context-dependent senolytic and senomorphic properties of DMY. Overall, the\nexperimental design is robust, and the data presented are largely supportive of the authors’\nconclusions.\nThere are a few points that would benefit from further clarification and elaboration:\nMajor concerns:\n1. SA-β-gal assay relies on the enzymatic activity of β-galactosidase. 5xFAD mouse tissues\nwere fixed by transcardial perfusion and sucrose-cryopreserved, which likely\ncompromised the protein activity and potentially invalidated the assay results. This assay\nshould be performed on fresh frozen tissue immediately after harvest. Moreover, as listed\nin the original method publication (PMID: 7568133), staining with a pH 4.0 solution\nshould be included as a positive technical control to confirm the preservation of β-\ngalactosidase activity.\n2. Figs 1e and 2d, to demonstrate whether DMY modulates the SASP milieu, which is\ncomposed of a large number of secreted factors, the authors should consider re-plotting\nthese 2 panels as heatmaps displaying only SASP-related genes. The SenMayo gene set\n(PMID: 35974106), a curated and validated list of senescence/SASP-associated genes,\nwould serve as an appropriate reference for selecting these factors.\n3. Fig 4a, p16INK4a and p21CIP are cyclin-dependent kinase inhibitors, which mediate cell\ncycle arrest and apoptosis resistance, 2 of the 3 features of senescent cell.\nStaining/blotting for additional apoptosis and cell cycle markers will provide supportive\nevidence that DMY exhibits senomorphic properties by inhibit SASP production without\naltering the other 2 senescence processes.\n4. In Figures 4 and 5, the authors propose that DMY exhibits differential effects on\nsenescent cell populations based on their basal levels of PRDX2 expression. To directly\nsupport this conclusion, the authors should experimentally modulating PRDX2 levels—\neither by downregulating or overexpressing PRDX2 using appropriate molecular tools—\nin different senescent cell lines and then assess whether these manipulations alter the\ncellular response to DMY treatment.\n5. Figure 8: the ‘hit-and-run’ intermittent dosing is a typical approach for senolytic\nadministration. The potential side effect of DMY on other brain cell types should be\nexamined after the long-course treatment, rather than after treating the 5xFAD mice with\nDMY once per day for 7 days. Oligodendrocytes should be characterized as well, as they\nundergo apoptosis upon exposure to DMY in vitro and are known to respond to amyloid\nplaques (PMID: 38819990).\nMinor concerns:\n1. Human gene symbols should be uppercase and italicized to distinguish them from\ncorresponding protein names.\n2. The Western blot data presented in the figures should be accompanied by densitometric\nquantification, with the number of technical and biological replicates stated in the figure\nlegend.\n3. Line 340, please specify what “rh” refers to in “rhPRDX2”."}]

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