Hydroxychloroquine Protects Against Cyclophosphamide-Induced Premature ovarian failure by Reducing Granulosa Cell Senescence via Regulation of the mtDNA-cGAS Signaling Pathway

Hydroxychloroquine Protects Against Cyclophosphamide-Induced Premature ovarian failure by Reducing Granulosa Cell Senescence via Regulation of the mtDNA-cGAS Signaling Pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Hydroxychloroquine Protects Against Cyclophosphamide-Induced Premature ovarian failure by Reducing Granulosa Cell Senescence via Regulation of the mtDNA-cGAS Signaling Pathway Dehui Su, Ruiqiong Ma, Huina Su, Cheng Tan, Ye Zhu, Yanhua Liu, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8052573/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Mar, 2026 Read the published version in npj Aging → Version 1 posted 10 You are reading this latest preprint version Abstract Cyclophosphamide (CTX) is widely used as a first-line chemotherapeutic agent for various cancers, but it is associated with significant risks of ovarian dysfunction, even progressing to premature ovarian failure (POF), with granulosa cell senescence being a key phenotypic manifestation. hydroxychloroquine (HCQ) has demonstrated anti-senescence properties in the context of age-related diseases, but its efficacy in preventing CTX-induced ovarian damage has not been fully elucidated. We conducted a controlled animal study using a CTX-induced POF model in female C57BL/6 mice, comprising three groups: vehicle control, CTX-only, and CTX + HCQ. Complementary in vitro studies were conducted using the human ovarian granulosa cell line KGN, treated with phosphoramide mustard (PM, the active metabolite of CTX) and HCQ. HCQ partially reversed the CTX-induced decrease in ovarian index, normalized the estrous cycle, reduced follicle depletion, and improved serum hormone levels as well as reproductive outcomes. HCQ reduced the expression of granulosa cell senescence markers and the area of senescence-associated β-galactosidase(SA-β-gal)-positive cells, stabilized mitochondrial membrane potential, decreased reactive oxygen species(ROS) production and mitochondrial DNA(mtDNA) leakage, inhibited cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling and suppressed senescence-associated secretory phenotype(SASP) factors. Collectively, HCQ protects CTX-induced POF by inhibiting activation of the mtDNA-cGAS signaling pathway, thereby alleviating granulosa cell senescence and preserving ovarian function. Biological sciences/Biochemistry Biological sciences/Cancer Biological sciences/Cell biology Biological sciences/Molecular biology Premature ovarian failure Cyclophosphamide Hydroxychloroquine Granulosa cell senescence mtDNA-cGAS signaling pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Cyclophosphamide (CTX) is a widely used alkylating agent in the treatment of breast cancer and various rheumatic immune diseases, including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) [ 1 – 3 ] . Its efficacy in suppressing abnormal cell proliferation and modulating overactive immune responses has been well-established, making it a cornerstone in the management of these conditions [ 4 , 5 ] . However, a significant and clinically concerning side effect of CTX is the induction of premature ovarian failure (POF), which is characterized by the loss of ovarian follicular reserve, menstrual irregularities, and infertility, often occurring in premenopausal women [ 6 – 10 ] . The primary mechanism underlying CTX-induced POF involves direct DNA damage to ovarian cells, particularly oocytes and granulosa cells, through the formation of cross-links in DNA strands, leading to cell cycle arrest, apoptosis, and ultimately follicular depletion [ 7 , 10 , 11 ] . In addition to DNA damage, accumulating evidence indicates that CTX exposure accelerates the senescence of granulosa cells—key somatic cells that support folliculogenesis and steroidogenesis [ 12 – 14 ] . Granulosa cell senescence, marked by increased expression of senescence-associated markers (e.g., p16, p21) and impaired mitochondrial function, further compromises ovarian function by disrupting follicular development and hormone secretion, exacerbating the progression of POF [ 12 , 14 – 17 ] Hydroxychloroquine (HCQ), a derivative of 4-aminoquinoline, is a commonly used drug in rheumatology/immunology and obstetrics/gynecology, and has received increasing attention in recent years [ 18 – 20 ] . Clinical studies have shown that HCQ can improve glucose-lipid metabolism and endocrine function in patients with polycystic ovary syndrome, restore ovarian function, reduce endometriotic lesions and disease risk, increase pregnancy success rates in women with positive antinuclear antibodies, and lower the risks of preeclampsia and mid-to-late pregnancy miscarriage [ 21 – 23 ] . Notably, chloroquine (CQ), a structural analog of HCQ, has been reported to mitigate cellular senescence induced by DNA damage [ 24 ] . Studies have demonstrated that CQ can modulate autophagy, reduce oxidative stress, and alleviate DNA damage responses, thereby delaying senescence in various cell types, including those exposed to genotoxic stress [ 24 – 26 ] . Given the structural and functional similarities between HCQ and CQ, it is plausible that HCQ may exert comparable anti-senescence effects. Against this background, the rational for the present study is to investigate whether hydroxychloroquine can protect against CTX-induced POF. Specifically, we aim to explore whether HCQ can alleviate CTX-mediated DNA damage, suppress granulosa cell senescence, and ultimately preserve ovarian follicular reserve and function. Materials and Methods 1. Study Design and Methodology of Network Meta-Analysis 1.1 Study design We conducted a network meta-analysis of continuous variables under a Bayesian framework, following the analytical framework of the Gemtc R package (based on the Markov Chain Monte Carlo algorithm). This analysis simultaneously compared the effects of four interventions on AMH levels in reproductive-aged women with SLE. By integrating evidence from direct and indirect comparisons, we quantified the effect differences among interventions, ranked their efficacy, and identified the optimal strategy for preserving ovarian reserve (assessed by AMH levels). 1.2 Data sources and search strategy We systematically searched PubMed, Embase, the Cochrane Library, China National Knowledge Infrastructure (CNKI), and Wanfang Data Knowledge Service Platform from database inception to June 2025, with language restrictions limited to Chinese and English. English search terms included: "systemic lupus erythematosus", "SLE", "cyclophosphamide", "hydroxychloroquine", "anti-Müllerian hormone", "AMH", "ovarian reserve". Chinese search terms included: "系统性红斑狼疮" (systemic lupus erythematosus), "SLE", "环磷酰胺" (cyclophosphamide), "羟氯喹" (hydroxychloroquine), "抗苗勒氏管激素" (anti-Müllerian hormone), "AMH", "卵巢储备" (ovarian reserve). The search strategy combined MeSH terms and free-text words using "AND" and "OR" logical operators. 1.3 Inclusion and Exclusion Criteria 1.3.1 Inclusion Criteria (1) Study population: Reproductive-aged women (18–45 years) with SLE who met the diagnostic criteria of the American College of Rheumatology (ACR, 1997) or the ACR/European League Against Rheumatism (EULAR, 2019) [ 27 ] .(2) Interventions: Studies including at least one of the four groups: a. Control group (no CTX or HCQ administration); b. CTX monotherapy group; c. CTX + HCQ combination therapy group; d. HCQ monotherapy group. (3) Study type: Randomized controlled trials (RCTs) or high-quality cohort studies. (4) Outcome measures: Reporting of serum AMH levels (continuous variable) with extractable data on sample size, mean, and standard deviation (SD) for each intervention group. 1.3.2 Exclusion Criteria (1) Non-reproductive-aged participants ( 45 years). (2) Interventions involving drugs known to affect ovarian reserve (e.g., gonadotropin-releasing hormone agonists). (3) Inability to extract AMH data (mean, SD, or sample size). (4) Study types: Reviews, case reports, animal studies, or conference abstracts. 1.4 Data Extraction Data were extracted independently by two researchers using a pre-designed data extraction form. Discrepancies were resolved by consensus or adjudication by a third independent researcher. Extracted information included:(1) Basic study characteristics: First author, publication year, country/region, and study design.(2) Participant characteristics: Total sample size, age (mean ± SD) and SLE duration (mean ± SD) in each intervention group.(3) Intervention details: Drug dosage and treatment duration (weeks/months) for each group.(4) Outcome data: Sample size (n), mean, and SD of AMH levels in each intervention group. 2. Animals and Cell Culture 2.1 Experimental animals Healthy female C57BL/6 mice (6–8 weeks or 10 months of age, specific pathogen-free [SPF]) were purchased from the Animal Core Facility of Peking University Health Science Center. Mice were housed under SPF conditions at 21 ± 2°C, 50% relative humidity, and a 12 h light/dark cycle, with free access to food and water ad libitum. All procedures were approved by the Animal Ethics Committee of Peking University. 2.2 Cell line The human granulosa cell tumor-like cell line KGN (Beina Biotechnology, Beijing, China) was cultured in DMEM/F12 medium (Gibco) supplemented with 10% fetal bovine serum (FBS; ZETA LIFE) and 1% penicillin-streptomycin (Gibco) at 37°C in a humidified atmosphere containing 5% CO₂. Cells were passaged at a 1:3 ratio, and passages 3–10 were used for experiments. 3. Reagents 3.1 Chemicals Cyclophosphamide (CTX; Aladdin, Cat. No.: C126044), hydroxychloroquine (HCQ; Aladdin, Cat. No.: H141480), and phosphoramide mustard (PM; Apexbio, Cat. No.: 135953) were used in this study. 3.2 Antibodies Primary antibodies included AMH (Abcam, Cat. No.: ab313767), FSHR (Proteintech, Cat. No.: 226651AP), ZP3 (Proteintech, Cat. No.: 212791AP), P16 (Abcam, Cat. No.: ab241543), P21 (Santa Cruz, Cat. No.: sc6246), IRF3 (Cell Signaling Technology, Cat. No.: 4302), p-IRF3 (Cell Signaling Technology, Cat. No.: 4947), cytochrome c (Proteintech, Cat. No.: 109931AP), γ-H2AX (Abcam, Cat. No.: ab81299), Ki67 (Abcam, Cat. No.: ab16667), and Tubulin (Proteintech, Cat. No.: 112241AP), all diluted following the manufacturers’ recommended protocols. HRP- or DyLight-conjugated secondary antibodies (Anti-Rabbit; Zhongshan Golden Bridge, Cat. No.: ZB-5301; Anti-Mouse; Zhongshan Golden Bridge, Cat. No.: ZB-2305; Anti-Rabbit; EarthOx, Cat. No.: E032220; Anti-Goat; EarthOx, Cat. No.: E032330) were used for Western blot, immunohistochemistry, and immunofluorescence. 4. Experimental Procedures 4.1 Animal model construction 4.1.1 Natural aging model Ten-month-old female mice were randomly divided into two groups (six mice per group): one group received HCQ (40 mg/kg/day, administered via oral gavage) and the other received saline, for 6 consecutive months. 4.1.2 Cyclophosphamide-induced POF model Six- to eight-week-old female mice were weighed and randomly divided into three groups (twelve mice per group):① Control (CON) group: Daily saline gavage for 1 week before and 4 weeks after CTX injection. ② CTX group: Saline gavage for 1 week, followed by a single intraperitoneal injection of CTX (120 mg/kg), then saline gavage for 4 weeks. ③ CTX + HCQ group: HCQ gavage (40 mg/kg/day) 1 week prior to and 4 weeks after CTX injection. 4.2 Cell senescence model KGN cells were seeded in 10 cm cell culture dishes and cultured to 50% confluence. Prior to treatment, cells were serum-starved in medium containing 0.5% FBS for 12 h, and subsequent treatments were performed as follows:① Control group (CON): Cells were treated with complete medium supplemented with PBS for 48 h, followed by replacement with fresh complete medium supplemented with PBS for an additional 48 h.② Model group (PM): Cells were treated with complete medium supplemented with PBS for 48 h, followed by replacement with fresh complete medium supplemented with PM (1 × 10⁻⁵ mol/L) for an additional 48 h.③ Treatment group (PM + HCQ): Cells were treated with complete medium supplemented with HCQ (1 × 10⁻⁵ mol/L) for 48 h, followed by replacement with fresh complete medium supplemented with both PM (1 × 10⁻⁵ mol/L) and HCQ (1 × 10⁻⁵ mol/L) for an additional 48 h.④ HCQ control group (HCQ): Cells were treated with complete medium supplemented with HCQ (1 × 10⁻⁵ mol/L) for 48 h, followed by replacement with fresh complete medium supplemented with HCQ (1 × 10⁻⁵ mol/L) for an additional 48 h. 4.3 Vaginal cytology and estrous cycle monitoring Vaginal smears were collected daily for 16 consecutive days and classified based on the method described by Liang et al. [ 28 ] : proestrus (characterized by round nucleated epithelial cells), estrus (characterized by cornified squamous epithelial cells), metestrus (characterized by a mixture of nucleated epithelial cells and leukocytes), and diestrus (characterized by predominantly leukocytes). 4.4 Serum hormone measurements Under anesthesia, blood samples were collected from the orbital sinus, allowed to clot at room temperature for 90 min, and then centrifuged at 1800 × g for 10 min to separate serum. Serum estradiol (E₂; Elabscience, Cat. No.: E-OSEL-M0008), anti-Müllerian hormone (AMH; Elabscience, Cat. No.: E-EL-M3015), and follicle-stimulating hormone (FSH; Elabscience, Cat. No.: E-EL-M0511) were measured using ELISA kits. 4.5 Fertility assessment Treated female mice were paired with fertile male mice at a 1:1 ratio. Vaginal plugs were checked daily to confirm successful mating. Litter size and pup survival rate were recorded and compared with those of age-matched control mice. 4.6 Ovarian histology Ovaries were harvested and fixed in 4% paraformaldehyde (PFA) for 12–24 h, then embedded in paraffin and sectioned into 5µm-thick slices. Every fifth section was stained with hematoxylin and eosin (H&E). Follicle classification followed Myers et al. [ 29 ] : primordial (oocyte surrounded by a single layer of flattened granulosa cells), primary (oocyte with a single layer of cuboidal granulosa cells), secondary (oocyte with ≥ 2 layers of cuboidal granulosa cells, no antrum), antral(multiple granulosa cell layers with antral formation), and atretic follicles (degenerating oocyte with pyknotic granulosa cells). Total follicle count = counted follicles × 5. 4.7 Immunofluorescence and immunohistochemistry Sections were deparaffinized, permeabilized with 0.3% Triton X-100 in PBS, blocked with 5% goat serum (in PBS) for 1 h at room temperature, incubated with primary antibodies at 4°C overnight, and subsequently incubated with Alexa Fluor-conjugated (for immunofluorescence) or HRP-conjugated (for immunohistochemistry) secondary antibodies at 37°C for 1 h. Nuclei were counterstained with DAPI or visualized using DAB, and sections were mounted with Vectashield. 4.8 TUNEL assay Sections were deparaffinized, permeabilized with proteinase K (20 µg/mL, dissolved in PBS) for 15–30 min, rinsed with PBS three times, and then incubated with 50 µL TUNEL reaction solution (Beyotime, Cat. No.: C1098) at 37°C for 60 min in the dark. Sections were mounted with anti-fade medium and imaged using a fluorescence microscope. 4.9 Masson’s trichrome staining Sections were deparaffinized and stained sequentially with Weigert’s iron hematoxylin for 10 min, Masson blue for 5 min, followed by fuchsin, 1% phosphomolybdic acid, and aniline blue; finally, sections were mounted with neutral balsam. 4.10 SA-β-Gal staining Granulosa cells or ovarian sections were stained using the SA-β-Gal staining kit (Beyotime, Cat. No.: C0602) following the manufacturers’ recommended protocols. Prior to staining, ovarian sections were equilibrated to room temperature. 4.11 RNA sequencing (RNA-seq) Ovarian granulosa cells were isolated from mice, and total RNA was extracted for sequencing on an Illumina HiSeq platform. Differential gene expression analysis and pathway enrichment analysis were performed using the DESeq2 and clusterProfiler R packages, respectively. 4.12 Cell viability assay (CCK-8) KGN cells were seeded in 96-well cell culture plates at a density of 5×10³–1×10⁴ cells per well and treated with PM or HCQ for 12 h. After treatment, 10 µL of CCK-8 solution (YEASEN, Cat. No.: 40203ES76) was added to each well, and the plates were incubated at 37°C for 2 h. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated using the formula: (OD value of treatment group / OD value of control group) × 100%. 4.13 Mitochondrial membrane potential (JC-1) According to the kit instructions (Beyotime, Cat. No.: C2006), JC-1 working solution was prepared as follows: 37.5 µL of 200× JC-1 stock solution + 6 mL of ultrapure water + 1.5 mL of 5× JC-1 buffer. Cells were washed with PBS three times, incubated with 500 µL of JC-1 working solution at 37°C for 20 min, then washed with cold JC-1 buffer three times, and finally imaged using a fluorescence microscope. 4.14 Reactive oxygen species (ROS) assay According to the kit instructions (Beyotime, Cat. No.: S0033S), cells were incubated with 10 µM DCFH-DA (dissolved in serum-free medium) at 37°C for 20 min, washed three times with PBS, and imaged under a fluorescence microscope with excitation wavelength at 488 nm and emission wavelength at 525 nm. 4.15 EdU proliferation assay Cells seeded in 6-well cell culture plates were incubated with EdU solution (2× stock solution, diluted to a final concentration of 10 µM) for 4 h. After incubation, cells were fixed in 4% PFA for 15–20 min, permeabilized with 0.3% Triton X-100 in PBS for 10 min, stained with Click reaction solution (Beyotime, Cat. No.: C0078S) for 30 min at room temperature, counterstained with Hoechst 33342 (for nuclear staining) for 5 min, and finally imaged using a fluorescence microscope. 4.16 Cytoplasmic mtDNA quantification Cells were divided into two aliquots. One aliquot was lysed in DNA extraction buffer (5 mM EDTA, pH 8.0; 0.2% SDS; 100 mM Tris-HCl, pH 8.5; 200 mM NaCl; 100 µg/mL proteinase K) to extract total DNA (for normalizing total mtDNA content). The other aliquot was treated with 25 mg/mL digitonin buffer (dissolved in PBS) and centrifuged at 12,000 × g for 10 min at 4°C to isolate the cytoplasmic fraction. mtDNA in the cytoplasmic fraction and nuclear DNA (nDNA) were extracted using a DNA extraction kit (TIANGEN, Cat. No.: DP210831) and quantified via quantitative real-time PCR (qPCR). 4.17 Western blot (WB) KGN cells were lysed in RIPA lysis buffer (20 mM HEPES, 150 mM NaCl, 1% CA630, 0.1% SDS, 2 mM EDTA, 1% sodium deoxycholate) supplemented with protease and phosphatase inhibitor cocktail (Roche). Protein concentration was determined using a BCA protein assay kit (Beyotime, Cat. No.: P0012). Proteins (30–50 µg per lane) were denatured at 98°C for 12 min, separated by 10% or 15% SDS-PAGE gels, transferred onto PVDF membranes (activated with methanol), blocked with 5% non-fat milk (dissolved in TBST) for 1 h at room temperature, incubated with primary antibodies at 4°C overnight, and then probed with HRP-conjugated secondary antibodies (diluted 1:4000 in TBST). Protein bands were visualized using the Amersham ImageQuant™ 800 system and quantified using ImageJ software (Version 1.8.0). 4.18 PCR According to the kit instructions (RNA extraction kit: Apexbio, Cat. No.: K1072; reverse transcription kit: Novoprotein, Cat. No.: E096-01A), total RNA was extracted from cell lysates using the chloroform-isopropanol method (chloroform extraction followed by isopropanol precipitation). The concentration and purity of total RNA were assessed using a NanoDrop spectrophotometer (A260/A280 ratio 1.8–2.0 was considered pure). Total RNA was reverse transcribed into complementary DNA (cDNA) using Oligo(dT)₂₃VN primers and dNTP mixture. Quantitative real-time PCR (qPCR) amplification was performed using 2× SuperMix and specific primers on the ABI 7500 Fast Real-Time PCR System. 4.19 Transmission Electron Microscopy (TEM) for Mitochondrial Ultrastructure Analysis Target tissues or cells were processed as follows: tissues were cut into fragments < 1 mm³ in volume, while cells were harvested following relevant treatments. Samples were fixed in 2.5% glutaraldehyde for 2 h, followed by three rinses with 0.1 mol/L phosphate-buffered saline (PBS). They were then transferred to 1% osmium tetroxide for 2 h, and rinsed three times with 0.1 mol/L PBS again. Samples underwent gradient ethanol dehydration at 4°C. Post-dehydration, they were sequentially incubated in pure acetone + embedding medium at room temperature for 4 h, the same mixture overnight at room temperature, and finally in pure embedding medium at 37°C for 3 h to complete embedding. Embedded blocks were sectioned into ultrathin slices (~ 50 nm thickness) using an ultramicrotome. After double staining with 3% uranyl acetate followed by lead citrate, sections were imaged under a TEM to visualize mitochondrial ultrastructure. 5. Statistical Analysis 5.1 Statistical Analysis Methods for Network Meta-Analysis Statistical analyses for network meta-analysis were performed using R software (Version 3.6.2; RStudio, Version 1.2.1335), JAGS software (Version 4.3.0, based on the MCMC algorithm), and the gemtc R package. Data, including study identifiers, intervention groups (Control, CTX, CTX + HCQ, HCQ), sample sizes, and AMH-related metrics (mean and SD), were formatted into a standardized CSV file to construct the network analysis dataset. A consistency random-effects model was constructed, and MCMC iterations were executed via JAGS (4 chains, 20,000 burn-in iterations, 5,000 sampling iterations). Using the Control group as the reference, mean differences (MD) and 95% credible intervals (CrI) of AMH levels were calculated for each intervention group. Finally, the surface under the cumulative ranking curve (SUCRA) values were used to quantify the protective efficacy of each intervention on ovarian reserve (higher values indicating superior efficacy). 5.2 Experimental Data Experimental data are presented as mean ± standard deviation (SD). The normality of data distribution was tested using the Shapiro–Wilk test, and the homogeneity of variance was tested using the Bartlett test (for normal distribution) or Brown–Forsythe test (for non-normal distribution). Two-group comparisons were performed using Student’s t-test (two-tailed), and multi-group comparisons were performed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. A P value < 0.05 was considered statistically significant. All statistical analyses were conducted using GraphPad Prism software (Version 9.20). Results Result 1: Network meta-analysis suggests that hydroxychloroquine protect against cyclophosphamide-induced POF. To investigate the protective effects of CTX combined with HCQ on ovarian function, we conducted a network meta-analysis to compare the efficacy among different CTX/HCQ treatment groups (Fig. 1 A). The study distribution across comparison pairs is illustrated in the network plot (Fig. 1 B). Cumulative ranking probability suggests that compared to CTX alone, the combination of CTX and HCQ reduces the risk of POF. (Fig. 1 C). Based on this finding, we proposed a hypothesis that HCQ can protect against CTX-induced POF. Result 2: HCQ protects against CTX-induced POF in mice To verify whether HCQ can protect against CTX-induced POI, a murine model of CTX-induced POF was established, followed by HCQ administration as described in Materials and Methods (Fig. 2 A). It was observed that CTX induced significant estrous cycle irregularities and body weight loss in mice, whereas HCQ significantly ameliorated these abnormalities (Fig. 2 B–C, Supplementary Fig. S1A–B). Additionally, HCQ treatment effectively mitigated the CTX-induced declines in serum AMH and E2 levels (Fig. 2 D–F). Administration of CTX significantly decreased ovarian and uterine weights, as well as their respective organ indices; and this effect was abrogated by HCQ (Fig. 2 G–I, Supplementary Fig. S1C–E). At the histomorphology level, CTX treatment markedly downregulated the expression of follicle-stimulating hormone receptor (FSHR) and AMH in ovarian granulosa cells, and HCQ treatment effectively restored their expression (Fig. 2 J–M). Notably, CTX caused a significant increase in ovarian fibrosis, which was prevented by HCQ treatment (Supplementary Fig. S1F–G). Taken together, HCQ treatment effectively protects against CTX-induced ovarian damage. Result 3: HCQ preserves CTX-induced decline in ovarian reserve function Given the close association between ovarian reserve function and follicle number, we further evaluated ovarian follicle counts of different developmental stages in CTX-induced mice with or without HCQ treatment. CTX depleted ovarian follicles at all developmental stages and elevated the number of atretic follicles; conversely, HCQ conferred significant protection against these changes (Fig. 3 A–D, Supplementary Fig. S1H–K). Additionally, CTX increased the ratio of growing follicles to primordial follicles, suggesting that CTX may induce excessive ovarian follicle activation—an effect also mitigated by HCQ (Fig. 3 E, Supplementary Fig. S1L). Furthermore, CTX caused disruption of the zona pellucida in ovarian follicles, which was ameliorated by HCQ treatment—indicating that CTX impairs oocyte quality [ 30 , 31 ] , while HCQ preserves oocyte structural integrity (Fig. 3 F). Moreover, CTX led to a significant decrease in pup survival rate and a trend toward a reduced offspring number, both of which were mitigated by HCQ treatment. (Fig. 3 G–I). Result 4: HCQ reduces granulosa cell senescence and DNA damage in CTX-induced POF mouse model Given that cellular senescence is a key mechanism underlying CTX-induced decline in ovarian reserve function [ 32 ] , we further evaluated the protective effect of HCQ on cellular senescence in the CTX-induced POF model. Senescence-associated β-galactosidase (SA-β-gal) staining revealed that CTX significantly increased the senescence of ovarian granulosa cells, while HCQ exerted a mitigating effect on this senescent change (Fig. 4 A–B). Immunohistochemical staining for P53, P21, and P16 revealed that CTX significantly upregulated the expression of these senescence markers in ovarian granulosa cells, whereas HCQ downregulated their expression levels (Fig. 4 C–H). Furthermore, immunohistochemical staining for γ-H2AX demonstrated that nuclear γ-H2AX expression was significantly increased in the CTX group, and this increase was abrogated by HCQ (Fig. 4 I–J). TUNEL staining showed a marked increase in TUNEL-positive ovarian granulosa cells in the CTX group, and this phenomenon was also suppressed by HCQ (Supplementary Fig. S2A–B). Notably, Ki67 expression in ovarian granulosa cells was significantly decreased in the CTX group, while HCQ maintained its normal expression (Fig. 4 K–L). Result 5: HCQ suppresses PM-induced granulosa cell senescence and DNA damage in vitro To further validate the protective effect of HCQ against cyclophosphamide (CTX)-induced senescence, we conducted in vitro experiments using a granulosa cell line. Western blot (WB) and quantitative real-time PCR (qPCR) analyses revealed that phosphoramide mustard (PM)—the active metabolite of CTX—significantly upregulated the protein and mRNA expression levels of P53, P21, and P16, whereas HCQ reversed this upregulation (Fig. 5 A–F). SA-β-gal staining showed a significant increase in the area of SA-β-gal-positive cells in the PM group, and HCQ treatment markedly reduced PM-induced granulosa cell senescence (Fig. 5 G–H). Notably, we also assessed factors associated with the senescence-associated secretory phenotype (SASP). Measurements of secretory factors in cell culture supernatants demonstrated that the levels of IL-6, IL-8, TGF-β, and TNF-α in the PM group were significantly higher than those in the Control (Con) group, and HCQ abrogated these PM-induced elevations (Fig. 5 I–L). Analysis of SASP-related gene expression indicated that the mRNA expressions of IL-6, IL-8, IL-11, IL-33, CCL2, CXCL3, MMP3, TGF-β, and TNF-α were all significantly upregulated in the PM group, while HCQ inhibited such upregulation (Fig. 5 M–N). Furthermore, the PM group exhibited a significant reduction in EdU-positive cells and overall cell viability, along with a marked increase in γ-H2AX expression, all of which were counteracted by HCQ treatment (Supplementary Fig. S2C–G). Result 6: Transcriptomic analysis identifies mitochondrial and cGAS-STING pathways as potential targets of HCQ in antagonizing CTX-induced granulosa cell senescence To investigate the potential mechanism by which HCQ counteracts CTX-induced ovarian granulosa cell senescence, we performed transcriptome sequencing on granulosa cells isolated from CTX-treated mice, with or without HCQ co-treatment (Fig. 6 A). We focused on genes whose expression was significantly dysregulated by CTX but reversed by HCQ co-treatment (Fig. 6 B).. Results showed that these differentially expressed genes were primarily enriched in the oxidative phosphorylation pathway and multiple aging-related disease terms (Fig. 6 C). Notably, the activation of the oxidative phosphorylation pathway exhibited a significant positive correlation with CTX treatment and a significant negative correlation with HCQ intervention (Fig. 6 D–E). Further analysis revealed that several differentially expressed genes were enriched in the mitochondrial DNA (mtDNA) stress signature and cGAS-STING signaling pathway—pathways that lie downstream of the oxidative phosphorylation pathway. These findings suggest that the mtDNA-cGAS signaling pathway may be a key target through which HCQ antagonizes CTX-induced senescence of ovarian granulosa cells. Result 7: HCQ preserves mitochondrial function and inhibits cGAS-STING pathway activation Then, we investigated whether HCQ protects ovarian granulosa cells from CTX-induced senescence via the mtDNA–cGAS pathway. PM treatment elicited more pronounced mitochondrial dysfunction—characterized by severe membrane structure loss and cristae disappearance under electron microscopy—whereas mitochondrial morphology was comparable between PM + HCQ and control groups (Fig. 7 A). Cytochrome c localization revealed that CTX (and PM in vitro) treatments increased cytoplasmic cytochrome c expression in granulosa cells in vitro and in vivo , whereas HCQ abrogated these effects (Fig. 7 B–C, Supplementary Fig. S3F–G). In vitro, we evaluated intracellular reactive oxygen species (ROS) levels, mitochondrial membrane potential stability, and mitochondrial status. Results showed that PM significantly increased intracellular ROS levels in granulosa cells and reduced mitochondrial membrane potential stability, while HCQ prevented these alterations (Supplementary Fig. S3A–D). Additionally, analysis of free mitochondrial DNA (mtDNA) in the cytoplasm to assess cytoplasmic mtDNA leakage revealed that PM treatment significantly increased mtDNA release into the cytoplasm, whereas HCQ treatment reduced this mtDNA leakage (Fig. 7 D–E, Supplementary Fig. S3E). Further analysis of cGAS pathway activation downstream of mtDNA showed that CTX treatment significantly upregulated the phosphorylation of IRF3 (p-IRF3) in mouse ovarian granulosa cells, and HCQ inhibited this CTX-induced phosphorylation of IRF3 (Fig. 7 F–G). In vitro experiments corroborated this finding, demonstrating that HCQ prevented PM-induced upregulation of cGAS pathway-related molecules (Supplementary Fig. S3F–K). Result 8: HCQ delays physiological aging in the ovaries and other organs. Given that HCQ exerts significant ovarian protective effects in CTX-induced POF mice, and its congener chloroquine (CQ) has been demonstrated to possess anti-aging properties [ 25 ] , we further established a naturally aged female mouse model to evaluate the protective role of HCQ in physiological ovarian aging (Fig. 8 A). Compared with the naturally aged control group, the HCQ-treated group showed a marked increase in relative ovarian volume (Fig. 8 B), a significant elevation in ovarian index (Fig. 8 C), and improved regularity of the estrous cycle (Fig. 8 D). H&E staining staining demonstrated that the HCQ-treated group exhibited a significant increase in total follicle number compared to control group (Fig. 8 E–F). Immunohistochemical analysis demonstrated that HCQ significantly upregulated anti-Müllerian hormone (AMH) expression in the ovary (Fig. 8 G–H), indicating its role in preserving follicle reserve. We further explored HCQ's role in delaying senescence in other organs. H&E staining revealed that the lungs, livers, and kidneys of HCQ-treated mice all exhibited delayed aging phenotypes (Fig. 8 I–K). Additionally, compared with naturally aged control mice, the HCQ-treated group exhibited less severe hair thinning and alopecia (Fig. 8 L), with a significant reduction in the relative proportion of hair loss area (Fig. 8 M). Discussion HCQ is an FDA-approved, widely used clinical agent with a well-established safety profile [ 33 ] , and has long been employed in rheumatology, immunology, and obstetric/gynecologic practice [ 21 , 23 , 34 – 36 ] . Notably, A trend from our network meta-analysis indicated a potential benefit of CTX + HCQ combination therapy in maintaining AMH levels among patients with SLE, which merits further investigation into repurposing HCQ for protection against CTX-induced POI. Our subsequent research in a mouse model confirmed that HCQ protects against CTX-induced POI, demonstrating that it specifically alleviates cellular senescence in ovarian granulosa cells. By employing transcriptomic sequencing on ovarian granulosa cells from a mouse model, we identified that the preservation of mitochondrial function and inhibition of the cGAS-STING signaling pathway represent the mechanisms through which HCQ protects against CTX-induced POI, which we subsequently validated. Furthermore, we demonstrated that long-term HCQ administration delays multi-organ aging in a naturally aged mouse model. Granulosa cells are core supportive cells for follicular development and steroid hormone synthesis, and their senescence serves as a key pathological driver of CTX-induced POF [ 13 , 37 – 42 ] . Senescent granulosa cells not only lose proliferative capacity and reduce secretory function but also induce senescence in surrounding normal cells via the SASP, thereby exacerbating the overall aging process of ovarian tissue [ 16 , 43 – 46 ] . Over recent years, accumulating studies have established mitochondrial dysfunction as a major driver of granulosa cell senescence and POF pathogenesis [ 47 – 49 ] , while the specific signaling pathways underlying this process remain elusive. Our study demonstrated that HCQ restores stability of mitochondrial membrane potential, reduces ROS levels, lowers cytoplasmic free mtDNA levels, and subsequently inhibits activation of the cGAS pathway and downstream SASP factors. Our proposed mechanism aligns with prior observations that chloroquine-derived agents suppress DNA sensing pathwaysand innate immune activation [ 50 – 52 ] .Our study further advances this understanding: HCQ functions as a regulator of mitochondrial homeostasis, thereby effectively preventing the abnormal cytoplasmic accumulation of mtDNA and subsequently suppressing the DNA-sensing cGAS pathway. Furthermore, our study identifies a broader anti-aging potential for HCQ, as it mitigates both ovarian dysfunction and systemic aging in naturally aging mice—a finding that corroborates prior reports that chloroquine-derived agents delay aging and extend lifespan by modulating DNA damage repair and inflammatory responses [ 24 , 26 ] . Given the established role of the cGAS-STING pathway in multi-organ degeneration, HCQ's potential to delay natural aging by modulating this pathway is a possibility that merits further investigation [ 53 , 54 ] . While our study provides the first evidence confirming that HCQ protects against CTX-induced POI and its underlying mechanism, the findings must be interpreted with consideration of its limitations. First, the translatability of our findings from murine models to clinical practice requires careful consideration of species differences and the optimization of dosing regimens. Second, the precise molecular target of HCQ within the mitochondria that initiates this protective cascade remains to be elucidated. Finally, while HCQ showed promise in delaying multi-organ physiological aging, the potential risks of long-term administration and the universality of the proposed mechanism across different organs merit further in-depth investigation. further investigation is needed to elucidate the precise molecular targets of HCQ at the mitochondrial level and validating its efficacy in patients through clinical trials, while closely monitoring its long-term safety. Declarations Ethics Statement This study was approved by Peking University People's Hospital (Ethics Approval Number: 2025PHE008). Author Contributions D.S. and H.S. prepared figures 1-6. R.M. prepared figure 8. C.T. and X.Y. wrote the main manuscript text. Y.Z., Y.L., X.J., X.S., T.L., M.Z. and S.L. prepared figure 7. All authors reviewed the manuscript. Funding statement This work was funded by the Youth Science Foundation Project of the National Natural Science Foundation of China (Grant No. 82401901) and the National Key R&D Program of China (Grants 2023YFC2706000 and 2023YFC2706002). Acknowledgements We would like to thank the staff of the National Key Laboratory of Obstetrics and Gynecology (Peking University People's Hospital) and the National Key Laboratory of Pathogenic Biology (Peking University) for their support in providing experimental equipment, consumables, and technical assistance related to research facilities. Data availability Data will be available upon request from the corresponding author. References Gu H C, Wang L F, Zhang Y W, Zhuo Y Q, Zhang Z H, et al. Human urine stem cells protect against cyclophosphamide-induced premature ovarian failure by inhibiting SLC1A4-mediated outflux of intracellular serine in ovarian granulosa cells [J]. Cell Mol Biol Lett, 2025, 30(1): 21. Fu Q, Wu C, Dai M, Wang S, Xu J, et al. Leflunomide versus azathioprine for maintenance therapy of lupus nephritis: a prospective, multicentre, randomised trial and long-term follow-up [J]. Ann Rheum Dis, 2022, 81(11): 1549-55. Arnaud L, Ruiz-Irastorza G, Aranow C, Bernatsky S, Dall'Era M, et al. ERN ReCONNET-SLICC-SLEuro expert consensus on the therapeutic management of rare systemic lupus erythematosus manifestations [J]. Lancet Rheumatol, 2025, 7(7): e505-e18. Yue Y, Yang H J, Zhang T, Li C, Kim M J, et al. Porcine Brain Enzyme Hydrolysate Enhances Immune Function and Antioxidant Defense via Modulation of Gut Microbiota in a Cyclophosphamide-Induced Immunodeficiency Model [J]. Antioxidants (Basel), 2024, 13(4). Highley M S, Landuyt B, Prenen H, Harper P GDe Bruijn E A. The Nitrogen Mustards [J]. Pharmacol Rev, 2022, 74(3): 552-99. Pascuali N, Scotti L, Di Pietro M, Oubiña G, Bas D, et al. Ceramide-1-phosphate has protective properties against cyclophosphamide-induced ovarian damage in a mice model of premature ovarian failure [J]. Hum Reprod, 2018, 33(5): 844-59. Luan Y, Yu S Y, Abazarikia A, Dong RKim S Y. TAp63 determines the fate of oocytes against DNA damage [J]. Sci Adv, 2022, 8(51): eade1846. Stern E, Ben-Ami M, Gruber N, Toren A, Caspi S, et al. Hypothalamic-pituitary-gonadal function, pubertal development, and fertility outcomes in male and female medulloblastoma survivors: a single-center experience [J]. Neuro Oncol, 2023, 25(7): 1345-54. Pluma A, Hamroun S, Rüegg L, Cecchi I, Kramer M, et al. Antirheumatic drugs in reproduction, pregnancy, and lactation: a systematic literature review informing the 2024 update of the EULAR recommendations [J]. Ann Rheum Dis, 2025, 84(9): 1561-90. Spears N, Lopes F, Stefansdottir A, Rossi V, De Felici M, et al. Ovarian damage from chemotherapy and current approaches to its protection [J]. Hum Reprod Update, 2019, 25(6): 673-93. Tuppi M, Kehrloesser S, Coutandin D W, Rossi V, Luh L M, et al. Oocyte DNA damage quality control requires consecutive interplay of CHK2 and CK1 to activate p63 [J]. Nat Struct Mol Biol, 2018, 25(3): 261-9. Li X, Ye H, Su T, Hu C, Huang Y, et al. Immunity and reproduction protective effects of Chitosan Oligosaccharides in Cyclophosphamide/Busulfan-induced premature ovarian failure model mice [J]. Front Immunol, 2023, 14: 1185921. Xiong Y, Liu T, Wang S, Chi H, Chen C, et al. Cyclophosphamide promotes the proliferation inhibition of mouse ovarian granulosa cells and premature ovarian failure by activating the lncRNA-Meg3-p53-p66Shc pathway [J]. Gene, 2017, 596: 1-8. Hense J D, Isola J V V, Garcia D N, Magalhães L S, Masternak M M, et al. The role of cellular senescence in ovarian aging [J]. NPJ Aging, 2024, 10(1): 35. Wang H. The role of granulosa cells in oocyte development and aging: Mechanisms and therapeutic opportunities [J]. Semin Cell Dev Biol, 2025, 171: 103614. Pan P, Cao S, Gao H, Qu X, Ma Y, et al. Immp2l gene knockout induces granulosa cell senescence by activation of cGAS-STING pathway via TFAM-mediated mtDNA leakage [J]. Int J Biol Macromol, 2025, 307(Pt 4): 142368. Li J, Yu Q, Huang H, Deng W, Cao X, et al. Human chorionic plate-derived mesenchymal stem cells transplantation restores ovarian function in a chemotherapy-induced mouse model of premature ovarian failure [J]. Stem Cell Res Ther, 2018, 9(1): 81. Nguyen N V, Sandström A, Svenungsson E, Dominicus A, Arkema E V, et al. Exposure to hydroxychloroquine in early pregnancy and incidence of pre-eclampsia and pre-term delivery in patients with systemic lupus erythematosus in Sweden: a nationwide population-based cohort study [J]. Lancet Rheumatol, 2025, 7(10): e687-e96. Rector A, Marić I, Chaichian Y, Chakravarty E, Cantu M, et al. Hydroxychloroquine in Lupus Pregnancy and Risk of Preeclampsia [J]. Arthritis Rheumatol, 2024, 76(6): 919-27. Schrezenmeier EDörner T. Mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology [J]. Nat Rev Rheumatol, 2020, 16(3): 155-66. Wang R, Qi S, Wang Q, Lang J, Pang C, et al. Hydroxychloroquine enhances insulin sensitivity and ameliorates abnormal lipid metabolism in obese women with polycystic ovary syndrome [J]. BMC Endocr Disord, 2025, 25(1): 2. Ruiz A, Rockfield S, Taran N, Haller E, Engelman R W, et al. Effect of hydroxychloroquine and characterization of autophagy in a mouse model of endometriosis [J]. Cell Death Dis, 2016, 7(1): e2059. Chen F Y, Chen S W, Chen X, Huang J Y, Ye Z, et al. Hydroxychloroquine might reduce risk of incident endometriosis in patients with systemic lupus erythematosus: A retrospective population-based cohort study [J]. Lupus, 2021, 30(10): 1609-16. Qian M, Liu Z, Peng L, Tang X, Meng F, et al. Boosting ATM activity alleviates aging and extends lifespan in a mouse model of progeria [J]. Elife, 2018, 7. Doeppner T R, Coman C, Burdusel D, Ancuta D L, Brockmeier U, et al. Long-term treatment with chloroquine increases lifespan in middle-aged male mice possibly via autophagy modulation, proteasome inhibition and glycogen metabolism [J]. Aging (Albany NY), 2022, 14(10): 4195-210. Li W, Zou Z, Cai Y, Yang K, Wang S, et al. Low-dose chloroquine treatment extends the lifespan of aged rats [J]. Protein Cell, 2022, 13(6): 454-61. Aringer M, Costenbader K, Dörner TJohnson S R. Advances in SLE classification criteria [J]. J Autoimmun, 2022, 132: 102845. Liang X, Yan Z, Ma W, Qian Y, Zou X, et al. Peroxiredoxin 4 protects against ovarian ageing by ameliorating D-galactose-induced oxidative damage in mice [J]. Cell Death Dis, 2020, 11(12): 1053. Myers M, Britt K L, Wreford N G, Ebling F JKerr J B. Methods for quantifying follicular numbers within the mouse ovary [J]. Reproduction, 2004, 127(5): 569-80. Yang P, Chen T, Liu Y, Hou Z, Wu K, et al. The critical role of ZP genes in female infertility characterized by empty follicle syndrome and oocyte degeneration [J]. Fertil Steril, 2021, 115(5): 1259-69. Wassarman P MLitscher E S. Female fertility and the zona pellucida [J]. Elife, 2022, 11. Guo Y, Xue L, Tang W, Xiong J, Chen D, et al. Ovarian microenvironment: challenges and opportunities in protecting against chemotherapy-associated ovarian damage [J]. Hum Reprod Update, 2024, 30(5): 614-47. Rosenbaum J T, Costenbader K H, Desmarais J, Ginzler E M, Fett N, et al. American College of Rheumatology, American Academy of Dermatology, Rheumatologic Dermatology Society, and American Academy of Ophthalmology 2020 Joint Statement on Hydroxychloroquine Use With Respect to Retinal Toxicity [J]. Arthritis Rheumatol, 2021, 73(6): 908-11. Rajput R, Upadhyay P, Rajput S, Saini SKharab S. Effect of hydroxychloroquine on beta cell function, insulin resistance, and inflammatory markers in type 2 diabetes patients uncontrolled on glimepiride and metformin therapy [J]. Int J Diabetes Dev Ctries, 2023: 1-6. Siegel C HSammaritano L R. Systemic Lupus Erythematosus: A Review [J]. Jama, 2024, 331(17): 1480-91. Gao R, Deng W, Meng C, Cheng K, Zeng X, et al. Combined treatment of prednisone and hydroxychloroquine may improve outcomes of frozen embryo transfer in antinuclear antibody-positive patients undergoing IVF/ICSI treatment [J]. Lupus, 2021, 30(14): 2213-20. Mo J, Hu H, Li P, Ye Y, Chen W, et al. Human hair follicle-derived mesenchymal stem cells improve ovarian function in cyclophosphamide-induced POF mice [J]. Stem Cell Res Ther, 2025, 16(1): 67. Rosario R, Stewart H L, Spears N, Telfer E EAnderson R A. Anti-Mullerian hormone attenuates both cyclophosphamide-induced damage and PI3K signalling activation, while rapamycin attenuates only PI3K signalling activation, in human ovarian cortex in vitro [J]. Hum Reprod, 2024, 39(2): 382-92. Ding C, Li H, Wang Y, Wang F, Wu H, et al. Different therapeutic effects of cells derived from human amniotic membrane on premature ovarian aging depend on distinct cellular biological characteristics [J]. Stem Cell Res Ther, 2017, 8(1): 173. Xu Z, Takahashi N, Harada M, Kunitomi C, Kusamoto A, et al. The Role of Cellular Senescence in Cyclophosphamide-Induced Primary Ovarian Insufficiency [J]. Int J Mol Sci, 2023, 24(24). Liu T, Liu Y, Huang Y, Chen J, Yu Z, et al. miR-15b induces premature ovarian failure in mice via inhibition of α-Klotho expression in ovarian granulosa cells [J]. Free Radic Biol Med, 2019, 141: 383-92. Li Q, Shen H, Liu A, Yu L, Wang L, et al. FTO-mediated m6A modification elevates the MFN2 mRNA stability to suppress ovarian granulosa cell senescence [J]. Int J Biol Macromol, 2025, 307(Pt 4): 142181. Luo Q, Yin N, Zhang L, Yuan W, Zhao W, et al. Role of SDF-1/CXCR4 and cytokines in the development of ovary injury in chemotherapy drug induced premature ovarian failure mice [J]. Life Sci, 2017, 179: 103-9. Zhou C, Li D, He J, Luo T, Liu Y, et al. TRIM28-Mediated Excessive Oxidative Stress Induces Cellular Senescence in Granulosa Cells and Contributes to Premature Ovarian Insufficiency In Vitro and In Vivo [J]. Antioxidants (Basel), 2024, 13(3). Lu H, Jing Y, Zhang C, Ma S, Zhang W, et al. Aging hallmarks of the primate ovary revealed by spatiotemporal transcriptomics [J]. Protein Cell, 2024, 15(5): 364-84. Devrukhkar P R, Watson M A, Soygur B, Wu F, Bons J, et al. A Comprehensive Multiomics Signature of Doxorubicin-Induced Cellular Senescence in the Postmenopausal Human Ovary [J]. Aging Cell, 2025, 24(8): e70111. Miwa S, Kashyap S, Chini Evon Zglinicki T. Mitochondrial dysfunction in cell senescence and aging [J]. J Clin Invest, 2022, 132(13). Dong T, Li N, Wang H, Zhu H, Gao Y, et al. Stress granule clearance mediated by V-ATPase-interacting protein NCOA7 mitigates ovarian aging [J]. Nat Aging, 2025, 5(8): 1548-67. Smits M A J, Schomakers B V, van Weeghel M, Wever E J M, Wüst R C I, et al. Human ovarian aging is characterized by oxidative damage and mitochondrial dysfunction [J]. Hum Reprod, 2023, 38(11): 2208-20. Bonam S R, Muller S, Bayry JKlionsky D J. Autophagy as an emerging target for COVID-19: lessons from an old friend, chloroquine [J]. Autophagy, 2020, 16(12): 2260-6. Xu YWan W. Lysosomal control of the cGAS-STING signaling [J]. Trends Cell Biol, 2024, 34(8): 622-5. An J, Woodward J J, Lai W, Minie M, Sun X, et al. Inhibition of Cyclic GMP-AMP Synthase Using a Novel Antimalarial Drug Derivative in Trex1-Deficient Mice [J]. Arthritis Rheumatol, 2018, 70(11): 1807-19. Gulen M F, Samson N, Keller A, Schwabenland M, Liu C, et al. cGAS-STING drives ageing-related inflammation and neurodegeneration [J]. Nature, 2023, 620(7973): 374-80. Sladitschek-Martens H L, Guarnieri A, Brumana G, Zanconato F, Battilana G, et al. YAP/TAZ activity in stromal cells prevents ageing by controlling cGAS-STING [J]. Nature, 2022, 607(7920): 790-8. Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigures.docx Cite Share Download PDF Status: Published Journal Publication published 17 Mar, 2026 Read the published version in npj Aging → Version 1 posted Editorial decision: Revision requested 11 Dec, 2025 Reviews received at journal 03 Dec, 2025 Reviews received at journal 22 Nov, 2025 Reviewers agreed at journal 21 Nov, 2025 Reviewers agreed at journal 17 Nov, 2025 Reviewers agreed at journal 14 Nov, 2025 Reviewers invited by journal 14 Nov, 2025 Editor assigned by journal 14 Nov, 2025 Submission checks completed at journal 12 Nov, 2025 First submitted to journal 06 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8052573","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":549088960,"identity":"0cb80f0a-2f55-466e-817a-498ef18fee63","order_by":0,"name":"Dehui Su","email":"","orcid":"","institution":"Peking University People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Dehui","middleName":"","lastName":"Su","suffix":""},{"id":549088963,"identity":"7fddc376-f815-4513-bdef-2e3b18c8e07e","order_by":1,"name":"Ruiqiong Ma","email":"","orcid":"","institution":"Peking University People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ruiqiong","middleName":"","lastName":"Ma","suffix":""},{"id":549088964,"identity":"20a3a804-1d96-478d-a32c-d7a0d7698de1","order_by":2,"name":"Huina Su","email":"","orcid":"","institution":"Peking University People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Huina","middleName":"","lastName":"Su","suffix":""},{"id":549088965,"identity":"b1c502cb-d5d2-4b44-be8d-2b9278b6f17c","order_by":3,"name":"Cheng Tan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYDACCRiDvYEZRDE2EKnFgIGB5wDJWiQSiNTCP7v52MMvDH/kzSXfHjbmYbCR3XCA+dkDvJbcOZZuLMNgYLhzdl5yMg9DmvGGA2zmBvi0GEjkmElLMBgwbridY3yYh+Fw4oYDPGwS+LXkfwNpsd9w8wxIy39itOSwSX5gMEjccIPHGOiwA4S1SNxIM5NmMDBO3nAmL9lwjkGy8czDbGZ4tfDPSH4m+aNCznbD8bOHJd5U2Mn2HW9+hlcLCDDzgEOIhwEcPwzMhNQDAeMPBpiWUTAKRsEoGAVYAABd50KBrWsK9AAAAABJRU5ErkJggg==","orcid":"","institution":"Peking University People's Hospital","correspondingAuthor":true,"prefix":"","firstName":"Cheng","middleName":"","lastName":"Tan","suffix":""},{"id":549088966,"identity":"f71f1cc1-8867-4ac7-babe-a7f402b6ff57","order_by":4,"name":"Ye Zhu","email":"","orcid":"","institution":"Peking University People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ye","middleName":"","lastName":"Zhu","suffix":""},{"id":549088967,"identity":"2d664a55-31a4-48fe-8476-d9bfb8b42050","order_by":5,"name":"Yanhua Liu","email":"","orcid":"","institution":"Peking University People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yanhua","middleName":"","lastName":"Liu","suffix":""},{"id":549088970,"identity":"bfd9b183-9284-4a60-9136-49a2001f2070","order_by":6,"name":"Xiaolin Jiang","email":"","orcid":"","institution":"Peking University People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaolin","middleName":"","lastName":"Jiang","suffix":""},{"id":549088972,"identity":"ecf69755-c82b-4539-b8c9-5cbe4133d024","order_by":7,"name":"Xiaowan Sun","email":"","orcid":"","institution":"Peking University People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaowan","middleName":"","lastName":"Sun","suffix":""},{"id":549088974,"identity":"9210a188-a5f2-41a7-9bfa-db3fb8106ff0","order_by":8,"name":"Tonghao Lei","email":"","orcid":"","institution":"Peking University People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Tonghao","middleName":"","lastName":"Lei","suffix":""},{"id":549088976,"identity":"1fdc019b-2b37-4c90-8cff-bd249d4a6f0d","order_by":9,"name":"Mengyu Zhang","email":"","orcid":"","institution":"Peking University People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Mengyu","middleName":"","lastName":"Zhang","suffix":""},{"id":549088977,"identity":"e19a868f-73af-41b3-a38a-c154f54d7c53","order_by":10,"name":"Shengrui Lin","email":"","orcid":"","institution":"Peking University People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Shengrui","middleName":"","lastName":"Lin","suffix":""},{"id":549088979,"identity":"8611c781-8f2e-496e-bb23-41b91db284b4","order_by":11,"name":"Xin Yang","email":"","orcid":"","institution":"Peking University People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2025-11-07 03:38:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8052573/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8052573/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41514-026-00359-9","type":"published","date":"2026-03-17T15:58:47+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":96914924,"identity":"b7771bab-a097-4414-a808-a87155f333b1","added_by":"auto","created_at":"2025-11-27 14:06:35","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":20235053,"visible":true,"origin":"","legend":"","description":"","filename":"FigurelegendHydroxychloroquineProtectsAgainstCyclophosphamideInducedPrematureovarianfailurebyReducingGranulosaCellSenescenceviaRegulationofthemtDNAcGASSignalingPathway.docx","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/a46864612ad3dd49e594137f.docx"},{"id":96914410,"identity":"e3d86270-a7be-4d8e-8732-0ad3d4f32cf6","added_by":"auto","created_at":"2025-11-27 14:05:53","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":12088,"visible":true,"origin":"","legend":"","description":"","filename":"ae34133830014683b939f6a4699936a4.json","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/1f2a8b5801406dc0ace31e18.json"},{"id":96915014,"identity":"1e442c56-fbab-42af-8b78-b92ffbb6fae2","added_by":"auto","created_at":"2025-11-27 14:06:44","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":142835,"visible":true,"origin":"","legend":"","description":"","filename":"ae34133830014683b939f6a4699936a41enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/0972b33ec16005ecb9727c3d.xml"},{"id":96757331,"identity":"9af98b3f-099d-4154-a847-38e93d0e8571","added_by":"auto","created_at":"2025-11-25 18:11:02","extension":"jpeg","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":192322,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/af66bba7259b23d5622767ee.jpeg"},{"id":96914387,"identity":"fd8e8b38-eb9d-4ac4-8304-72a8a9d15c26","added_by":"auto","created_at":"2025-11-27 14:05:51","extension":"jpeg","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1409640,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage10.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/f9c1fecaae9ad216f727d2ca.jpeg"},{"id":96757343,"identity":"cc799b5b-6cb6-40b6-b4c6-f49f1d630d2e","added_by":"auto","created_at":"2025-11-25 18:11:02","extension":"jpeg","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1621693,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage11.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/ee8b4cb94e58f063c0040208.jpeg"},{"id":96915384,"identity":"657086c5-1824-4937-8c5c-6aa289f678d1","added_by":"auto","created_at":"2025-11-27 14:07:11","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":95787,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage12.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/33bd88708178e885cb378473.jpeg"},{"id":96914889,"identity":"5739ba5e-9fdf-420b-a6a1-efff22b32f03","added_by":"auto","created_at":"2025-11-27 14:06:33","extension":"jpeg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2778768,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/16474d408b2de3a7ae63acdb.jpeg"},{"id":96914996,"identity":"67e438e6-beb7-4af3-9611-0b9effa74a0d","added_by":"auto","created_at":"2025-11-27 14:06:43","extension":"jpeg","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1727508,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/645001f2904dc35e2786c06b.jpeg"},{"id":96757355,"identity":"d020c67d-d8c8-4e3b-8d45-3f70c77b73c0","added_by":"auto","created_at":"2025-11-25 18:11:02","extension":"jpeg","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4373494,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/b85cfba1a50fa48c3f3b4bcd.jpeg"},{"id":96913877,"identity":"d6ec05ed-579d-4bdc-a89d-77ed02a72372","added_by":"auto","created_at":"2025-11-27 14:04:36","extension":"jpeg","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1183874,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/6c4216bf2dcc3056c8fba1e8.jpeg"},{"id":96757347,"identity":"9dd98449-a821-4309-822d-3a4fb53778a6","added_by":"auto","created_at":"2025-11-25 18:11:02","extension":"jpeg","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":457216,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/e992511d2a104e5bdcea91ef.jpeg"},{"id":96757346,"identity":"323a340f-45d0-4928-b956-7c5cc285e851","added_by":"auto","created_at":"2025-11-25 18:11:02","extension":"jpeg","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2811936,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/28520e2dae4af1a8bc28ec75.jpeg"},{"id":96757350,"identity":"80a26e7d-fe8b-46f0-a642-abb69904d72a","added_by":"auto","created_at":"2025-11-25 18:11:02","extension":"jpeg","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2876450,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/7c95eac222a32ab34fe96e82.jpeg"},{"id":96913823,"identity":"27c8537c-f1c4-49fb-841c-3d667447eb6a","added_by":"auto","created_at":"2025-11-27 14:04:25","extension":"jpeg","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1069732,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/6472b62908b54b9af1a2ea03.jpeg"},{"id":96914392,"identity":"156b2dcb-2b61-4651-b239-14db46ab4a88","added_by":"auto","created_at":"2025-11-27 14:05:52","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":36158,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/8008f05b7b430a9cefc630cc.png"},{"id":96915243,"identity":"b506ea2f-4f86-43ab-b834-f512d85d3401","added_by":"auto","created_at":"2025-11-27 14:07:01","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":249198,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/e1dba5911a3c0b1816942932.png"},{"id":96915960,"identity":"39a4fdf1-f1e3-4ba0-b7e6-9f32b7fceaa5","added_by":"auto","created_at":"2025-11-27 14:07:50","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":225101,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/265ffac459a57022c80ee546.png"},{"id":96757352,"identity":"ee03f0d8-37d2-4012-951e-08f03bbec611","added_by":"auto","created_at":"2025-11-25 18:11:02","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":13610,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/e422df2f2fb046de47be4077.png"},{"id":96914407,"identity":"6ad6a6df-0976-4b22-9a33-12fb7e3ca7f5","added_by":"auto","created_at":"2025-11-27 14:05:52","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":448170,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/1d61c31179341865a2032a56.png"},{"id":96757361,"identity":"eebb0617-ddae-44ed-b617-53c114fd6863","added_by":"auto","created_at":"2025-11-25 18:11:02","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":269762,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/f719b1870ef6669f91d42807.png"},{"id":96915011,"identity":"f94eccc9-5926-4f81-8895-a9f630d8afed","added_by":"auto","created_at":"2025-11-27 14:06:43","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":665524,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/7f0b4ce01c80ea2232c4ecf9.png"},{"id":96914393,"identity":"78d6b62b-ac5e-4260-8785-36436580df47","added_by":"auto","created_at":"2025-11-27 14:05:52","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":172549,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/47299feab94f3d2125e14251.png"},{"id":96757358,"identity":"308a6d05-120d-4e96-a430-0eb515ff18d1","added_by":"auto","created_at":"2025-11-25 18:11:02","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":78420,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/72598afc741749a7b9356392.png"},{"id":96757357,"identity":"abcd134f-ab8b-4aff-9336-89f7422f1d7f","added_by":"auto","created_at":"2025-11-25 18:11:02","extension":"png","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":494132,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/9c7e3128e80e7bebccf1bbed.png"},{"id":96757362,"identity":"7f0be0a0-44f4-4a7e-ba7c-d8f2654a511c","added_by":"auto","created_at":"2025-11-25 18:11:02","extension":"png","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":428883,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/993d41b8b0fdf783a1023ad9.png"},{"id":96757360,"identity":"9675ea07-d0d0-4a21-ac5d-0e8d786f1d28","added_by":"auto","created_at":"2025-11-25 18:11:02","extension":"png","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":177062,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/225462d1b628026a2025c166.png"},{"id":96757364,"identity":"d01bbb55-4922-46fc-8036-8ba1a89bbcd8","added_by":"auto","created_at":"2025-11-25 18:11:02","extension":"xml","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":145481,"visible":true,"origin":"","legend":"","description":"","filename":"ae34133830014683b939f6a4699936a41structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/efe44b7c12090f3932cf00af.xml"},{"id":96757366,"identity":"30953122-2f18-4cc6-835b-300ade0ecd9a","added_by":"auto","created_at":"2025-11-25 18:11:02","extension":"html","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":154952,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/a673f2fecb3f97a10f0c60f2.html"},{"id":96757329,"identity":"fde7fe1c-fee6-4b47-b314-41ce0784c9bb","added_by":"auto","created_at":"2025-11-25 18:11:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":36158,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eNetwork meta-analysis of the protective effects of CTX combined with HCQ on ovarian function (A) PRISMA flow diagram depicting the process of identifying, screening, and including studies. (B) Network plot illustrating comparisons among control (Con), cyclophosphamide (CTX), hydroxychloroquine (HCQ), and cyclophosphamide plus hydroxychloroquine (CTX+HCQ), which displays direct or indirect comparative relationships among all treatment groups. (C) Probability of each rank position for each treatment.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/9a94009f149dfbe21a383eab.png"},{"id":96757328,"identity":"02906b6a-b67f-4960-b448-02de0dc36683","added_by":"auto","created_at":"2025-11-25 18:11:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":448170,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eHCQ protects against CTX-induced POF in mice (A) Schematic diagram of the experimental procedure: mice were gavaged with 40 mg/kg HCQ or normal saline (NS) daily, and intraperitoneally injected with 120 mg/kg CTX or NS on day 7, followed by vaginal smear testing until day 35 when mice were sacrificed for sample collection (ovaries, uterus, and blood). (B) Representative estrous cycles in control (Con), CTX, and CTX+HCQ groups (estrus (E), metestrus (M), diestrus (D), proestrus (P)). (C) Proportions of regular and irregular estrous cycles in each group (n=12, ∗P \u0026lt; 0.05, ∗∗P \u0026lt; 0.01). (D–F) Serum concentrations of follicle-stimulating hormone (FSH, D), anti-Müllerian hormone (AMH, E), and estradiol (E2, F) in each group (n=6, ∗P \u0026lt; 0.05, ∗∗P \u0026lt; 0.01). (G) Ovarian weight in each group (n=12, ∗∗P \u0026lt; 0.01).(H) Representative photographs showing the gross morphology of ovaries from each treatment group (rular provided for reference). (I) Ovarian index (ovarian weight/body weight) in each group (n=12, ∗∗P \u0026lt; 0.01). (J) Representative immunofluorescence staining of follicle-stimulating hormone receptor (Fshr, green) in ovaries, with DAPI staining for nuclei (blue; red arrows indicate Fshr-positive signals; scale bar = 100μm). (K) Representative immunohistochemical staining of anti-Müllerian hormone (Amh, brown) in ovaries (scale bars: 50μm for low-magnification images, 20μm for high-magnification insets). (L) Relative expression of Fshr based on integrated optical density (IOD) from immunofluorescence (n=6, ∗∗P \u0026lt; 0.01). (M) Relative expression of Amh based on IOD from immunohistochemistry. (n=6, ∗P \u0026lt; 0.05).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/7fdc116f2c91bb6ea6178641.png"},{"id":96914337,"identity":"d298a4cf-6035-4e08-9e22-29225ac0a8d3","added_by":"auto","created_at":"2025-11-27 14:05:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":269762,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eHCQ preserves CTX-induced decline in ovarian reserve function (A) Representative hematoxylin and eosin (HE) staining of ovaries in control (Con), CTX, and CTX+HCQ groups (scale bar = 200μm for left images, 50μm for right magnified images; red arrows indicate follicles). (B) Statistics of total follicle number in ovaries (n=6; ∗∗P \u0026lt; 0.01).(C) Statistics of antral follicle number in ovaries (n=6; ∗P \u0026lt; 0.05, ∗∗P \u0026lt; 0.01).(D) Statistics of primordial follicle ratio (n=6; ∗∗P \u0026lt; 0.01). (E) Ratio of early growing follicles to primordial follicles (Early growing follicles = number of primary follicles + number of secondary follicles) (n=6; ∗∗P \u0026lt; 0.01). (F) Representative immunofluorescence staining of zona pellucida 3 (Zp3, green fluorescence) in ovaries, with DAPI staining for cell nuclei (blue; red arrows indicate Zp3-positive signals; scale bar = 50μm). (G) Representative photographs showing the grossmorphology of offspring (pups) in each group. (H) Statistics of survival rate of newborn mice (n=6; ∗P \u0026lt; 0.05). (I) Statistics of the number of newborn mice in each group (comparison between CTX group and Con group, CTX+HCQ group, n=6, P=0.0768)\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/5f0d17baf75d0577998fdb4b.png"},{"id":96915098,"identity":"f3514ac4-b610-4949-a2ba-9603cc298ae8","added_by":"auto","created_at":"2025-11-27 14:06:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":665524,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eHCQ reduces granulosa cell senescence and DNA damage in CTX-induced POF mouse ovaries (A) Representative hematoxylin and eosin (HE) staining and senescence-associated β-galactosidase (SA-β-gal) staining of ovaries in control (Con), CTX, and CTX+HCQ groups (scale bar = 200μm for HE staining; magnified insets in SA-β-gal staining show positive signals). (B) Quantitative analysis of SA-β-gal-positive area (n=6; ∗∗P \u0026lt; 0.01).(C–J) Representative immunohistochemical staining and quantitative analysis of relative expression of p53 (C-D), p21 (E-F), p16 (G-H), and γ-H2A.x (I-J) in ovaries (scale bars: 100μm for C, I; 50μm for E, G; insets show magnified fields) (n=6; ∗∗P \u0026lt; 0.01). (K–L) Representative immunohistochemical staining and quantitative analysis of relative expression of Ki67(K-L) in ovaries (scale bar = 200μm; insets show magnified fields) (n=6; ∗P \u0026lt; 0.05).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/6e9d733dcb92775652adc973.png"},{"id":96915785,"identity":"40d66dd0-3e23-44b7-87b5-bab3912b44b0","added_by":"auto","created_at":"2025-11-27 14:07:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":172549,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eHCQ suppresses PM-induced granulosa cell senescence, SASP secretion, and DNA damage in vitro (A) Representative western blot analysis of P21 and P16 proteins in the control group (Con), PM-treated group, PM+HCQ-treated group, and HCQ-treated group (Tubulin served as the internal reference).(B–C) Quantitative analysis of relative protein expression of P21 (B) and P16 (C) (n=3; ∗P \u0026lt; 0.05, ∗∗P \u0026lt; 0.01).(D–F) qPCR analysis of relative mRNA expression of P53 (D), p21 (E), and P16 (F) (n=3; ∗P \u0026lt; 0.05, ∗∗P \u0026lt; 0.01).(G) Representative senescence-associated β-galactosidase (SA-β-gal) staining of cells in each group (Upper scale bar: 200μm; Lower scale bar: 100μm; blue indicates positive signals).(H) Quantitative analysis of SA-β-gal-positive areas (n=6; ∗P \u0026lt; 0.05, ∗∗P \u0026lt; 0.01).(I–L) ELISA detection of the concentrations of IL-6 (I), IL-8 (J), TGF-β (K), and TNF-α (L) in cell culture supernatants (n=3; ∗P \u0026lt; 0.05, ∗∗P \u0026lt; 0.01).(M–N) qPCR analysis of relative mRNA expression of senescence-associated secretory phenotype (SASP) factors (IL-6, IL-11, IL-33, CCL2, and CXCL3 in M; TNF, TGFB1, MMP3, and IL-8 in N) (n=3; ∗P \u0026lt; 0.05, ∗∗P \u0026lt; 0.01).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/341700d869f68b70c19cfe4e.png"},{"id":96757335,"identity":"677e0d8a-dd47-433c-a5af-bcc0c6e1ee10","added_by":"auto","created_at":"2025-11-25 18:11:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":78420,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eTranscriptomic analysis identifies mitochondrial and cGAS-STING pathways as potential targets of HCQ in antagonizing CTX-induced granulosa cell senescence (A) Schematic diagram of the experimental procedure: Mice were treated with intraperitoneal injection (i.p) and intragastric administration (i.g) [normal saline (NS, i.p) + NS (i.g), CTX (i.p) + HCQ (i.g), CTX (i.p) + NS (i.g)], after which ovarian samples were collected for bulk RNA sequencing (Bulk RNA-seq). (B) Venn diagram of differentially expressed genes (DEGs), showing the overlap of DEGs between \"CTX group vs control group (Con) (up/downregulated)\" and \"CTX+HCQ group vs CTX group (up/downregulated)\" (percentages in parentheses represent the proportion of DEGs in the corresponding comparison group). (C) Bubble plot of KEGG pathway enrichment for DEGs, displaying the top 10 enriched pathways (dot color represents P-value, dot size represents the number of enriched genes, and RichFactor denotes the enrichment factor). (D) Gene Set Enrichment Analysis (GSEA) showing the enrichment profile of the \"Oxidative phosphorylation\" pathway in the \"CTX group (positively correlated with the Con group)\" (green curve = enrichment profile, black vertical lines = hit genes, horizontal axis = rank order of the ordered gene set). (E) GSEA showing the enrichment profile of the \"Oxidative phosphorylation\" pathway in the \"CTX+HCQ group (positively correlated with the CTX group)\".(F) Expression heatmap of mitochondrial DNA (mtDNA) stress signature genes (color gradients indicate relative changes in gene expression levels). (G) Expression heatmap of cGAS-STING signaling pathway-related genes (color gradients indicate relative changes in gene expression levels).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/147d6a9462c1882a7f5b19ef.png"},{"id":96757337,"identity":"17950109-982a-4d6d-90b0-ac911b807fde","added_by":"auto","created_at":"2025-11-25 18:11:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":494132,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eHCQ preserves mitochondrial function and inhibits cGAS-STING pathway activation (A) Representative image of cell mitochondrial electron microscopy. (B) Representative immunohistochemical staining of cytochrome c (Cyto-c) in the ovaries of the control group (Con), CTX group, and CTX+HCQ group (scale bar = 50μm; insets show magnified views). (C) Quantitative analysis of the relative expression level of Cyto-c (based on Integrated Optical Density [IOD], ×10⁵) (n=6; ∗P \u0026lt; 0.05, ∗∗P \u0026lt; 0.01). (D) Schematic diagram of the experimental procedure for detecting free mitochondrial DNA (mtDNA) in the cytosol. (E) qPCR analysis of mtDNA expression in each group (Con, PM, PM+HCQ, HCQ) (n=3; ∗∗P \u0026lt; 0.01). (F) Quantitative analysis of the relative expression level of phosphorylated interferon regulatory factor 3 (p-Irf3) in the ovaries of each group (based on IOD, ×10⁵) (n=6; ∗P \u0026lt; 0.05, ∗∗P \u0026lt; 0.01). (G) Representative immunofluorescence staining of p-Irf3 in the ovary (DAPI stains nuclei blue, p-Irf3 is shown in red, Merge = merged images; green arrows indicate positive signals; scale bar = 150μm).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/c75487be74dc8c8d9c8fc7c4.png"},{"id":96757340,"identity":"c0ac3ab9-518e-4e5e-9483-85f42f9d7a20","added_by":"auto","created_at":"2025-11-25 18:11:02","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":428883,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eHCQ protects ovarian function and systemic homeostasis in naturally aged mice (A) Schematic diagram of the experimental procedure: Mice received daily intragastric administration of 40 mg/kg HCQ or normal saline (NS). During the experiment, vaginal smear testing was performed; at the end of the experiment, mice were euthanized and samples (ovaries, uteri, hair, lungs, livers, and kidneys) were collected. (B) Representative photographs showing the gross morphology of ovaries in the control group (Con) and HCQ-treated group (scale bar for indicating ovary size). (C) Quantitative analysis of the ovarian index (ovary weight/body weight)\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003e(n=6; ∗P \u0026lt; 0.05). (D) Proportion of regular and irregular estrous cycles in each group. (E) Representative hematoxylin-eosin (HE) staining of ovaries (Upper scale bar = 200μm, Lower scale bar = 50μm; insets show magnified views). (F) Statistics of total follicle number per ovary (n=6; ∗P \u0026lt; 0.05). (G) Representative immunohistochemical staining of anti-Müllerian hormone (Amh) in ovaries (Upper scale bar = 200μm, Lower scale bar = 50μm; insets show magnified views). (H) Quantitative analysis of the relative expression level of Amh (based on Integrated Optical Density [IOD], ×10⁵) (n=6; ∗P \u0026lt; 0.05, ∗∗P \u0026lt; 0.01). (I–K) Representative HE staining of the lung (I), liver (J), and kidney (K) (Scale bars: 200μm for I, J, K; inset scale bars = 100μm for I, J, K). (L) Representative photographs showing the gross morphology of hair in the control group and HCQ-treated group. (M) Quantitative analysis of hair loss area percentage (n=6; ∗∗P \u0026lt; 0.01).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/ee1a24b5858f32b899d192eb.png"},{"id":105223432,"identity":"2769d712-e00a-48cb-aa8f-3bb2c7701dc7","added_by":"auto","created_at":"2026-03-23 16:06:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4726580,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/7822db3e-dd41-46b0-a17f-522d2e560500.pdf"},{"id":96914344,"identity":"bbb2cfd2-af45-41c0-b679-cba22a78519b","added_by":"auto","created_at":"2025-11-27 14:05:47","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3681642,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-8052573/v1/ce05087f3d4983cb83fad31f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Hydroxychloroquine Protects Against Cyclophosphamide-Induced Premature ovarian failure by Reducing Granulosa Cell Senescence via Regulation of the mtDNA-cGAS Signaling Pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCyclophosphamide (CTX) is a widely used alkylating agent in the treatment of breast cancer and various rheumatic immune diseases, including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA)\u003csup\u003e[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Its efficacy in suppressing abnormal cell proliferation and modulating overactive immune responses has been well-established, making it a cornerstone in the management of these conditions\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eHowever, a significant and clinically concerning side effect of CTX is the induction of premature ovarian failure (POF), which is characterized by the loss of ovarian follicular reserve, menstrual irregularities, and infertility, often occurring in premenopausal women\u003csup\u003e[\u003cspan additionalcitationids=\"CR7 CR8 CR9\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. The primary mechanism underlying CTX-induced POF involves direct DNA damage to ovarian cells, particularly oocytes and granulosa cells, through the formation of cross-links in DNA strands, leading to cell cycle arrest, apoptosis, and ultimately follicular depletion\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn addition to DNA damage, accumulating evidence indicates that CTX exposure accelerates the senescence of granulosa cells\u0026mdash;key somatic cells that support folliculogenesis and steroidogenesis\u003csup\u003e[\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Granulosa cell senescence, marked by increased expression of senescence-associated markers (e.g., p16, p21) and impaired mitochondrial function, further compromises ovarian function by disrupting follicular development and hormone secretion, exacerbating the progression of POF\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eHydroxychloroquine (HCQ), a derivative of 4-aminoquinoline, is a commonly used drug in rheumatology/immunology and obstetrics/gynecology, and has received increasing attention in recent years \u003csup\u003e[\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Clinical studies have shown that HCQ can improve glucose-lipid metabolism and endocrine function in patients with polycystic ovary syndrome, restore ovarian function, reduce endometriotic lesions and disease risk, increase pregnancy success rates in women with positive antinuclear antibodies, and lower the risks of preeclampsia and mid-to-late pregnancy miscarriage\u003csup\u003e[\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eNotably, chloroquine (CQ), a structural analog of HCQ, has been reported to mitigate cellular senescence induced by DNA damage\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Studies have demonstrated that CQ can modulate autophagy, reduce oxidative stress, and alleviate DNA damage responses, thereby delaying senescence in various cell types, including those exposed to genotoxic stress\u003csup\u003e[\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Given the structural and functional similarities between HCQ and CQ, it is plausible that HCQ may exert comparable anti-senescence effects.\u003c/p\u003e\u003cp\u003eAgainst this background, the rational for the present study is to investigate whether hydroxychloroquine can protect against CTX-induced POF. Specifically, we aim to explore whether HCQ can alleviate CTX-mediated DNA damage, suppress granulosa cell senescence, and ultimately preserve ovarian follicular reserve and function.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\n\u003ch3\u003e1. Study Design and Methodology of Network Meta-Analysis\u003c/h3\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e1.1 Study design\u003c/h2\u003e\u003cp\u003eWe conducted a network meta-analysis of continuous variables under a Bayesian framework, following the analytical framework of the Gemtc R package (based on the Markov Chain Monte Carlo algorithm). This analysis simultaneously compared the effects of four interventions on AMH levels in reproductive-aged women with SLE. By integrating evidence from direct and indirect comparisons, we quantified the effect differences among interventions, ranked their efficacy, and identified the optimal strategy for preserving ovarian reserve (assessed by AMH levels).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e1.2 Data sources and search strategy\u003c/h2\u003e\u003cp\u003eWe systematically searched PubMed, Embase, the Cochrane Library, China National Knowledge Infrastructure (CNKI), and Wanfang Data Knowledge Service Platform from database inception to June 2025, with language restrictions limited to Chinese and English. English search terms included: \"systemic lupus erythematosus\", \"SLE\", \"cyclophosphamide\", \"hydroxychloroquine\", \"anti-M\u0026uuml;llerian hormone\", \"AMH\", \"ovarian reserve\". Chinese search terms included: \"系统性红斑狼疮\" (systemic lupus erythematosus), \"SLE\", \"环磷酰胺\" (cyclophosphamide), \"羟氯喹\" (hydroxychloroquine), \"抗苗勒氏管激素\" (anti-M\u0026uuml;llerian hormone), \"AMH\", \"卵巢储备\" (ovarian reserve). The search strategy combined MeSH terms and free-text words using \"AND\" and \"OR\" logical operators.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e1.3 Inclusion and Exclusion Criteria\u003c/h2\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e1.3.1 Inclusion Criteria\u003c/h2\u003e\u003cp\u003e(1) Study population: Reproductive-aged women (18\u0026ndash;45 years) with SLE who met the diagnostic criteria of the American College of Rheumatology (ACR, 1997) or the ACR/European League Against Rheumatism (EULAR, 2019)\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e.(2) Interventions: Studies including at least one of the four groups: a. Control group (no CTX or HCQ administration); b. CTX monotherapy group; c. CTX\u0026thinsp;+\u0026thinsp;HCQ combination therapy group; d. HCQ monotherapy group. (3) Study type: Randomized controlled trials (RCTs) or high-quality cohort studies. (4) Outcome measures: Reporting of serum AMH levels (continuous variable) with extractable data on sample size, mean, and standard deviation (SD) for each intervention group.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e1.3.2 Exclusion Criteria\u003c/h2\u003e\u003cp\u003e(1) Non-reproductive-aged participants (\u0026lt;\u0026thinsp;18 or \u0026gt;\u0026thinsp;45 years). (2) Interventions involving drugs known to affect ovarian reserve (e.g., gonadotropin-releasing hormone agonists). (3) Inability to extract AMH data (mean, SD, or sample size). (4) Study types: Reviews, case reports, animal studies, or conference abstracts.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e1.4 Data Extraction\u003c/h2\u003e\u003cp\u003eData were extracted independently by two researchers using a pre-designed data extraction form. Discrepancies were resolved by consensus or adjudication by a third independent researcher. Extracted information included:(1) Basic study characteristics: First author, publication year, country/region, and study design.(2) Participant characteristics: Total sample size, age (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) and SLE duration (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) in each intervention group.(3) Intervention details: Drug dosage and treatment duration (weeks/months) for each group.(4) Outcome data: Sample size (n), mean, and SD of AMH levels in each intervention group.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003e2. Animals and Cell Culture\u003c/h3\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Experimental animals\u003c/h2\u003e\u003cp\u003eHealthy female C57BL/6 mice (6\u0026ndash;8 weeks or 10 months of age, specific pathogen-free [SPF]) were purchased from the Animal Core Facility of Peking University Health Science Center. Mice were housed under SPF conditions at 21\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 50% relative humidity, and a 12 h light/dark cycle, with free access to food and water ad libitum. All procedures were approved by the Animal Ethics Committee of Peking University.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Cell line\u003c/h2\u003e\u003cp\u003eThe human granulosa cell tumor-like cell line KGN (Beina Biotechnology, Beijing, China) was cultured in DMEM/F12 medium (Gibco) supplemented with 10% fetal bovine serum (FBS; ZETA LIFE) and 1% penicillin-streptomycin (Gibco) at 37\u0026deg;C in a humidified atmosphere containing 5% CO₂. Cells were passaged at a 1:3 ratio, and passages 3\u0026ndash;10 were used for experiments.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003e3. Reagents\u003c/h3\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Chemicals\u003c/h2\u003e\u003cp\u003eCyclophosphamide (CTX; Aladdin, Cat. No.: C126044), hydroxychloroquine (HCQ; Aladdin, Cat. No.: H141480), and phosphoramide mustard (PM; Apexbio, Cat. No.: 135953) were used in this study.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Antibodies\u003c/h2\u003e\u003cp\u003ePrimary antibodies included AMH (Abcam, Cat. No.: ab313767), FSHR (Proteintech, Cat. No.: 226651AP), ZP3 (Proteintech, Cat. No.: 212791AP), P16 (Abcam, Cat. No.: ab241543), P21 (Santa Cruz, Cat. No.: sc6246), IRF3 (Cell Signaling Technology, Cat. No.: 4302), p-IRF3 (Cell Signaling Technology, Cat. No.: 4947), cytochrome c (Proteintech, Cat. No.: 109931AP), γ-H2AX (Abcam, Cat. No.: ab81299), Ki67 (Abcam, Cat. No.: ab16667), and Tubulin (Proteintech, Cat. No.: 112241AP), all diluted following the manufacturers\u0026rsquo; recommended protocols. HRP- or DyLight-conjugated secondary antibodies (Anti-Rabbit; Zhongshan Golden Bridge, Cat. No.: ZB-5301; Anti-Mouse; Zhongshan Golden Bridge, Cat. No.: ZB-2305; Anti-Rabbit; EarthOx, Cat. No.: E032220; Anti-Goat; EarthOx, Cat. No.: E032330) were used for Western blot, immunohistochemistry, and immunofluorescence.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003e4. Experimental Procedures\u003c/h3\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e4.1 Animal model construction\u003c/h2\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003ch2\u003e4.1.1 Natural aging model\u003c/h2\u003e\u003cp\u003eTen-month-old female mice were randomly divided into two groups (six mice per group): one group received HCQ (40 mg/kg/day, administered via oral gavage) and the other received saline, for 6 consecutive months.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\u003ch2\u003e4.1.2 Cyclophosphamide-induced POF model\u003c/h2\u003e\u003cp\u003eSix- to eight-week-old female mice were weighed and randomly divided into three groups (twelve mice per group):① Control (CON) group: Daily saline gavage for 1 week before and 4 weeks after CTX injection. ② CTX group: Saline gavage for 1 week, followed by a single intraperitoneal injection of CTX (120 mg/kg), then saline gavage for 4 weeks. ③ CTX\u0026thinsp;+\u0026thinsp;HCQ group: HCQ gavage (40 mg/kg/day) 1 week prior to and 4 weeks after CTX injection.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e4.2 Cell senescence model\u003c/h2\u003e\u003cp\u003eKGN cells were seeded in 10 cm cell culture dishes and cultured to 50% confluence. Prior to treatment, cells were serum-starved in medium containing 0.5% FBS for 12 h, and subsequent treatments were performed as follows:① Control group (CON): Cells were treated with complete medium supplemented with PBS for 48 h, followed by replacement with fresh complete medium supplemented with PBS for an additional 48 h.② Model group (PM): Cells were treated with complete medium supplemented with PBS for 48 h, followed by replacement with fresh complete medium supplemented with PM (1 \u0026times; 10⁻⁵ mol/L) for an additional 48 h.③ Treatment group (PM\u0026thinsp;+\u0026thinsp;HCQ): Cells were treated with complete medium supplemented with HCQ (1 \u0026times; 10⁻⁵ mol/L) for 48 h, followed by replacement with fresh complete medium supplemented with both PM (1 \u0026times; 10⁻⁵ mol/L) and HCQ (1 \u0026times; 10⁻⁵ mol/L) for an additional 48 h.④ HCQ control group (HCQ): Cells were treated with complete medium supplemented with HCQ (1 \u0026times; 10⁻⁵ mol/L) for 48 h, followed by replacement with fresh complete medium supplemented with HCQ (1 \u0026times; 10⁻⁵ mol/L) for an additional 48 h.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e4.3 Vaginal cytology and estrous cycle monitoring\u003c/h2\u003e\u003cp\u003eVaginal smears were collected daily for 16 consecutive days and classified based on the method described by Liang et al.\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e: proestrus (characterized by round nucleated epithelial cells), estrus (characterized by cornified squamous epithelial cells), metestrus (characterized by a mixture of nucleated epithelial cells and leukocytes), and diestrus (characterized by predominantly leukocytes).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e4.4 Serum hormone measurements\u003c/h2\u003e\u003cp\u003eUnder anesthesia, blood samples were collected from the orbital sinus, allowed to clot at room temperature for 90 min, and then centrifuged at 1800 \u0026times; g for 10 min to separate serum. Serum estradiol (E₂; Elabscience, Cat. No.: E-OSEL-M0008), anti-M\u0026uuml;llerian hormone (AMH; Elabscience, Cat. No.: E-EL-M3015), and follicle-stimulating hormone (FSH; Elabscience, Cat. No.: E-EL-M0511) were measured using ELISA kits.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e4.5 Fertility assessment\u003c/h2\u003e\u003cp\u003eTreated female mice were paired with fertile male mice at a 1:1 ratio. Vaginal plugs were checked daily to confirm successful mating. Litter size and pup survival rate were recorded and compared with those of age-matched control mice.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e4.6 Ovarian histology\u003c/h2\u003e\u003cp\u003eOvaries were harvested and fixed in 4% paraformaldehyde (PFA) for 12\u0026ndash;24 h, then embedded in paraffin and sectioned into 5\u0026micro;m-thick slices. Every fifth section was stained with hematoxylin and eosin (H\u0026amp;E). Follicle classification followed Myers et al.\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e: primordial (oocyte surrounded by a single layer of flattened granulosa cells), primary (oocyte with a single layer of cuboidal granulosa cells), secondary (oocyte with \u0026ge;\u0026thinsp;2 layers of cuboidal granulosa cells, no antrum), antral(multiple granulosa cell layers with antral formation), and atretic follicles (degenerating oocyte with pyknotic granulosa cells). Total follicle count\u0026thinsp;=\u0026thinsp;counted follicles \u0026times; 5.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e4.7 Immunofluorescence and immunohistochemistry\u003c/h2\u003e\u003cp\u003eSections were deparaffinized, permeabilized with 0.3% Triton X-100 in PBS, blocked with 5% goat serum (in PBS) for 1 h at room temperature, incubated with primary antibodies at 4\u0026deg;C overnight, and subsequently incubated with Alexa Fluor-conjugated (for immunofluorescence) or HRP-conjugated (for immunohistochemistry) secondary antibodies at 37\u0026deg;C for 1 h. Nuclei were counterstained with DAPI or visualized using DAB, and sections were mounted with Vectashield.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e4.8 TUNEL assay\u003c/h2\u003e\u003cp\u003eSections were deparaffinized, permeabilized with proteinase K (20 \u0026micro;g/mL, dissolved in PBS) for 15\u0026ndash;30 min, rinsed with PBS three times, and then incubated with 50 \u0026micro;L TUNEL reaction solution (Beyotime, Cat. No.: C1098) at 37\u0026deg;C for 60 min in the dark. Sections were mounted with anti-fade medium and imaged using a fluorescence microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003e4.9 Masson\u0026rsquo;s trichrome staining\u003c/h2\u003e\u003cp\u003eSections were deparaffinized and stained sequentially with Weigert\u0026rsquo;s iron hematoxylin for 10 min, Masson blue for 5 min, followed by fuchsin, 1% phosphomolybdic acid, and aniline blue; finally, sections were mounted with neutral balsam.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e4.10 SA-β-Gal staining\u003c/h2\u003e\u003cp\u003eGranulosa cells or ovarian sections were stained using the SA-β-Gal staining kit (Beyotime, Cat. No.: C0602) following the manufacturers\u0026rsquo; recommended protocols. Prior to staining, ovarian sections were equilibrated to room temperature.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003e4.11 RNA sequencing (RNA-seq)\u003c/h2\u003e\u003cp\u003eOvarian granulosa cells were isolated from mice, and total RNA was extracted for sequencing on an Illumina HiSeq platform. Differential gene expression analysis and pathway enrichment analysis were performed using the DESeq2 and clusterProfiler R packages, respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec30\" class=\"Section2\"\u003e\u003ch2\u003e4.12 Cell viability assay (CCK-8)\u003c/h2\u003e\u003cp\u003eKGN cells were seeded in 96-well cell culture plates at a density of 5\u0026times;10\u0026sup3;\u0026ndash;1\u0026times;10⁴ cells per well and treated with PM or HCQ for 12 h. After treatment, 10 \u0026micro;L of CCK-8 solution (YEASEN, Cat. No.: 40203ES76) was added to each well, and the plates were incubated at 37\u0026deg;C for 2 h. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated using the formula: (OD value of treatment group / OD value of control group) \u0026times; 100%.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\u003ch2\u003e4.13 Mitochondrial membrane potential (JC-1)\u003c/h2\u003e\u003cp\u003eAccording to the kit instructions (Beyotime, Cat. No.: C2006), JC-1 working solution was prepared as follows: 37.5 \u0026micro;L of 200\u0026times; JC-1 stock solution\u0026thinsp;+\u0026thinsp;6 mL of ultrapure water\u0026thinsp;+\u0026thinsp;1.5 mL of 5\u0026times; JC-1 buffer. Cells were washed with PBS three times, incubated with 500 \u0026micro;L of JC-1 working solution at 37\u0026deg;C for 20 min, then washed with cold JC-1 buffer three times, and finally imaged using a fluorescence microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec32\" class=\"Section2\"\u003e\u003ch2\u003e4.14 Reactive oxygen species (ROS) assay\u003c/h2\u003e\u003cp\u003eAccording to the kit instructions (Beyotime, Cat. No.: S0033S), cells were incubated with 10 \u0026micro;M DCFH-DA (dissolved in serum-free medium) at 37\u0026deg;C for 20 min, washed three times with PBS, and imaged under a fluorescence microscope with excitation wavelength at 488 nm and emission wavelength at 525 nm.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec33\" class=\"Section2\"\u003e\u003ch2\u003e4.15 EdU proliferation assay\u003c/h2\u003e\u003cp\u003eCells seeded in 6-well cell culture plates were incubated with EdU solution (2\u0026times; stock solution, diluted to a final concentration of 10 \u0026micro;M) for 4 h. After incubation, cells were fixed in 4% PFA for 15\u0026ndash;20 min, permeabilized with 0.3% Triton X-100 in PBS for 10 min, stained with Click reaction solution (Beyotime, Cat. No.: C0078S) for 30 min at room temperature, counterstained with Hoechst 33342 (for nuclear staining) for 5 min, and finally imaged using a fluorescence microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec34\" class=\"Section2\"\u003e\u003ch2\u003e4.16 Cytoplasmic mtDNA quantification\u003c/h2\u003e\u003cp\u003eCells were divided into two aliquots. One aliquot was lysed in DNA extraction buffer (5 mM EDTA, pH 8.0; 0.2% SDS; 100 mM Tris-HCl, pH 8.5; 200 mM NaCl; 100 \u0026micro;g/mL proteinase K) to extract total DNA (for normalizing total mtDNA content). The other aliquot was treated with 25 mg/mL digitonin buffer (dissolved in PBS) and centrifuged at 12,000 \u0026times; g for 10 min at 4\u0026deg;C to isolate the cytoplasmic fraction. mtDNA in the cytoplasmic fraction and nuclear DNA (nDNA) were extracted using a DNA extraction kit (TIANGEN, Cat. No.: DP210831) and quantified via quantitative real-time PCR (qPCR).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec35\" class=\"Section2\"\u003e\u003ch2\u003e4.17 Western blot (WB)\u003c/h2\u003e\u003cp\u003eKGN cells were lysed in RIPA lysis buffer (20 mM HEPES, 150 mM NaCl, 1% CA630, 0.1% SDS, 2 mM EDTA, 1% sodium deoxycholate) supplemented with protease and phosphatase inhibitor cocktail (Roche). Protein concentration was determined using a BCA protein assay kit (Beyotime, Cat. No.: P0012). Proteins (30\u0026ndash;50 \u0026micro;g per lane) were denatured at 98\u0026deg;C for 12 min, separated by 10% or 15% SDS-PAGE gels, transferred onto PVDF membranes (activated with methanol), blocked with 5% non-fat milk (dissolved in TBST) for 1 h at room temperature, incubated with primary antibodies at 4\u0026deg;C overnight, and then probed with HRP-conjugated secondary antibodies (diluted 1:4000 in TBST). Protein bands were visualized using the Amersham ImageQuant\u0026trade; 800 system and quantified using ImageJ software (Version 1.8.0).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec36\" class=\"Section2\"\u003e\u003ch2\u003e4.18 PCR\u003c/h2\u003e\u003cp\u003eAccording to the kit instructions (RNA extraction kit: Apexbio, Cat. No.: K1072; reverse transcription kit: Novoprotein, Cat. No.: E096-01A), total RNA was extracted from cell lysates using the chloroform-isopropanol method (chloroform extraction followed by isopropanol precipitation). The concentration and purity of total RNA were assessed using a NanoDrop spectrophotometer (A260/A280 ratio 1.8\u0026ndash;2.0 was considered pure). Total RNA was reverse transcribed into complementary DNA (cDNA) using Oligo(dT)₂₃VN primers and dNTP mixture. Quantitative real-time PCR (qPCR) amplification was performed using 2\u0026times; SuperMix and specific primers on the ABI 7500 Fast Real-Time PCR System.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec37\" class=\"Section2\"\u003e\u003ch2\u003e4.19 Transmission Electron Microscopy (TEM) for Mitochondrial Ultrastructure Analysis\u003c/h2\u003e\u003cp\u003eTarget tissues or cells were processed as follows: tissues were cut into fragments\u0026thinsp;\u0026lt;\u0026thinsp;1 mm\u0026sup3; in volume, while cells were harvested following relevant treatments. Samples were fixed in 2.5% glutaraldehyde for 2 h, followed by three rinses with 0.1 mol/L phosphate-buffered saline (PBS). They were then transferred to 1% osmium tetroxide for 2 h, and rinsed three times with 0.1 mol/L PBS again. Samples underwent gradient ethanol dehydration at 4\u0026deg;C. Post-dehydration, they were sequentially incubated in pure acetone\u0026thinsp;+\u0026thinsp;embedding medium at room temperature for 4 h, the same mixture overnight at room temperature, and finally in pure embedding medium at 37\u0026deg;C for 3 h to complete embedding. Embedded blocks were sectioned into ultrathin slices (~\u0026thinsp;50 nm thickness) using an ultramicrotome. After double staining with 3% uranyl acetate followed by lead citrate, sections were imaged under a TEM to visualize mitochondrial ultrastructure.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003e5. Statistical Analysis\u003c/h3\u003e\n\u003cdiv id=\"Sec39\" class=\"Section2\"\u003e\u003ch2\u003e5.1 Statistical Analysis Methods for Network Meta-Analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses for network meta-analysis were performed using R software (Version 3.6.2; RStudio, Version 1.2.1335), JAGS software (Version 4.3.0, based on the MCMC algorithm), and the gemtc R package. Data, including study identifiers, intervention groups (Control, CTX, CTX\u0026thinsp;+\u0026thinsp;HCQ, HCQ), sample sizes, and AMH-related metrics (mean and SD), were formatted into a standardized CSV file to construct the network analysis dataset. A consistency random-effects model was constructed, and MCMC iterations were executed via JAGS (4 chains, 20,000 burn-in iterations, 5,000 sampling iterations). Using the Control group as the reference, mean differences (MD) and 95% credible intervals (CrI) of AMH levels were calculated for each intervention group. Finally, the surface under the cumulative ranking curve (SUCRA) values were used to quantify the protective efficacy of each intervention on ovarian reserve (higher values indicating superior efficacy).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec40\" class=\"Section2\"\u003e\u003ch2\u003e5.2 Experimental Data\u003c/h2\u003e\u003cp\u003eExperimental data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). The normality of data distribution was tested using the Shapiro\u0026ndash;Wilk test, and the homogeneity of variance was tested using the Bartlett test (for normal distribution) or Brown\u0026ndash;Forsythe test (for non-normal distribution). Two-group comparisons were performed using Student\u0026rsquo;s t-test (two-tailed), and multi-group comparisons were performed using one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s post hoc test. A P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. All statistical analyses were conducted using GraphPad Prism software (Version 9.20).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eResult 1: Network meta-analysis suggests that hydroxychloroquine protect against cyclophosphamide-induced POF.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the protective effects of CTX combined with HCQ on ovarian function, we conducted a network meta-analysis to compare the efficacy among different CTX/HCQ treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The study distribution across comparison pairs is illustrated in the network plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Cumulative ranking probability suggests that compared to CTX alone, the combination of CTX and HCQ reduces the risk of POF. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Based on this finding, we proposed a hypothesis that HCQ can protect against CTX-induced POF.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eResult 2: HCQ protects against CTX-induced POF in mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo verify whether HCQ can protect against CTX-induced POI, a murine model of CTX-induced POF was established, followed by HCQ administration as described in Materials and Methods (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). It was observed that CTX induced significant estrous cycle irregularities and body weight loss in mice, whereas HCQ significantly ameliorated these abnormalities (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u0026ndash;C, Supplementary Fig. S1A\u0026ndash;B). Additionally, HCQ treatment effectively mitigated the CTX-induced declines in serum AMH and E2 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u0026ndash;F). Administration of CTX significantly decreased ovarian and uterine weights, as well as their respective organ indices; and this effect was abrogated by HCQ (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG\u0026ndash;I, Supplementary Fig. S1C\u0026ndash;E). At the histomorphology level, CTX treatment markedly downregulated the expression of follicle-stimulating hormone receptor (FSHR) and AMH in ovarian granulosa cells, and HCQ treatment effectively restored their expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ\u0026ndash;M). Notably, CTX caused a significant increase in ovarian fibrosis, which was prevented by HCQ treatment (Supplementary Fig. S1F\u0026ndash;G). Taken together, HCQ treatment effectively protects against CTX-induced ovarian damage.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eResult 3: HCQ preserves CTX-induced decline in ovarian reserve function\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGiven the close association between ovarian reserve function and follicle number, we further evaluated ovarian follicle counts of different developmental stages in CTX-induced mice with or without HCQ treatment. CTX depleted ovarian follicles at all developmental stages and elevated the number of atretic follicles; conversely, HCQ conferred significant protection against these changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;D, Supplementary Fig. S1H\u0026ndash;K). Additionally, CTX increased the ratio of growing follicles to primordial follicles, suggesting that CTX may induce excessive ovarian follicle activation\u0026mdash;an effect also mitigated by HCQ (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, Supplementary Fig. S1L). Furthermore, CTX caused disruption of the zona pellucida in ovarian follicles, which was ameliorated by HCQ treatment\u0026mdash;indicating that CTX impairs oocyte quality\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e, while HCQ preserves oocyte structural integrity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Moreover, CTX led to a significant decrease in pup survival rate and a trend toward a reduced offspring number, both of which were mitigated by HCQ treatment. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG\u0026ndash;I).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eResult 4: HCQ reduces granulosa cell senescence and DNA damage in CTX-induced POF mouse model\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGiven that cellular senescence is a key mechanism underlying CTX-induced decline in ovarian reserve function\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e, we further evaluated the protective effect of HCQ on cellular senescence in the CTX-induced POF model. Senescence-associated β-galactosidase (SA-β-gal) staining revealed that CTX significantly increased the senescence of ovarian granulosa cells, while HCQ exerted a mitigating effect on this senescent change (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026ndash;B). Immunohistochemical staining for P53, P21, and P16 revealed that CTX significantly upregulated the expression of these senescence markers in ovarian granulosa cells, whereas HCQ downregulated their expression levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u0026ndash;H). Furthermore, immunohistochemical staining for γ-H2AX demonstrated that nuclear γ-H2AX expression was significantly increased in the CTX group, and this increase was abrogated by HCQ (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI\u0026ndash;J). TUNEL staining showed a marked increase in TUNEL-positive ovarian granulosa cells in the CTX group, and this phenomenon was also suppressed by HCQ (Supplementary Fig. S2A\u0026ndash;B). Notably, Ki67 expression in ovarian granulosa cells was significantly decreased in the CTX group, while HCQ maintained its normal expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK\u0026ndash;L).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eResult 5: HCQ suppresses PM-induced granulosa cell senescence and DNA damage in vitro\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo further validate the protective effect of HCQ against cyclophosphamide (CTX)-induced senescence, we conducted in vitro experiments using a granulosa cell line. Western blot (WB) and quantitative real-time PCR (qPCR) analyses revealed that phosphoramide mustard (PM)\u0026mdash;the active metabolite of CTX\u0026mdash;significantly upregulated the protein and mRNA expression levels of P53, P21, and P16, whereas HCQ reversed this upregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026ndash;F). SA-β-gal staining showed a significant increase in the area of SA-β-gal-positive cells in the PM group, and HCQ treatment markedly reduced PM-induced granulosa cell senescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG\u0026ndash;H). Notably, we also assessed factors associated with the senescence-associated secretory phenotype (SASP). Measurements of secretory factors in cell culture supernatants demonstrated that the levels of IL-6, IL-8, TGF-β, and TNF-α in the PM group were significantly higher than those in the Control (Con) group, and HCQ abrogated these PM-induced elevations (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI\u0026ndash;L). Analysis of SASP-related gene expression indicated that the mRNA expressions of IL-6, IL-8, IL-11, IL-33, CCL2, CXCL3, MMP3, TGF-β, and TNF-α were all significantly upregulated in the PM group, while HCQ inhibited such upregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eM\u0026ndash;N). Furthermore, the PM group exhibited a significant reduction in EdU-positive cells and overall cell viability, along with a marked increase in γ-H2AX expression, all of which were counteracted by HCQ treatment (Supplementary Fig. S2C\u0026ndash;G).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eResult 6: Transcriptomic analysis identifies mitochondrial and cGAS-STING pathways as potential targets of HCQ in antagonizing CTX-induced granulosa cell senescence\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the potential mechanism by which HCQ counteracts CTX-induced ovarian granulosa cell senescence, we performed transcriptome sequencing on granulosa cells isolated from CTX-treated mice, with or without HCQ co-treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). We focused on genes whose expression was significantly dysregulated by CTX but reversed by HCQ co-treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).. Results showed that these differentially expressed genes were primarily enriched in the oxidative phosphorylation pathway and multiple aging-related disease terms (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Notably, the activation of the oxidative phosphorylation pathway exhibited a significant positive correlation with CTX treatment and a significant negative correlation with HCQ intervention (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD\u0026ndash;E). Further analysis revealed that several differentially expressed genes were enriched in the mitochondrial DNA (mtDNA) stress signature and cGAS-STING signaling pathway\u0026mdash;pathways that lie downstream of the oxidative phosphorylation pathway. These findings suggest that the mtDNA-cGAS signaling pathway may be a key target through which HCQ antagonizes CTX-induced senescence of ovarian granulosa cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eResult 7: HCQ preserves mitochondrial function and inhibits cGAS-STING pathway activation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThen, we investigated whether HCQ protects ovarian granulosa cells from CTX-induced senescence via the mtDNA\u0026ndash;cGAS pathway. PM treatment elicited more pronounced mitochondrial dysfunction\u0026mdash;characterized by severe membrane structure loss and cristae disappearance under electron microscopy\u0026mdash;whereas mitochondrial morphology was comparable between PM\u0026thinsp;+\u0026thinsp;HCQ and control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Cytochrome c localization revealed that CTX (and PM in vitro) treatments increased cytoplasmic cytochrome c expression in granulosa cells \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, whereas HCQ abrogated these effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB\u0026ndash;C, Supplementary Fig. S3F\u0026ndash;G). In vitro, we evaluated intracellular reactive oxygen species (ROS) levels, mitochondrial membrane potential stability, and mitochondrial status. Results showed that PM significantly increased intracellular ROS levels in granulosa cells and reduced mitochondrial membrane potential stability, while HCQ prevented these alterations (Supplementary Fig. S3A\u0026ndash;D). Additionally, analysis of free mitochondrial DNA (mtDNA) in the cytoplasm to assess cytoplasmic mtDNA leakage revealed that PM treatment significantly increased mtDNA release into the cytoplasm, whereas HCQ treatment reduced this mtDNA leakage (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD\u0026ndash;E, Supplementary Fig. S3E). Further analysis of cGAS pathway activation downstream of mtDNA showed that CTX treatment significantly upregulated the phosphorylation of IRF3 (p-IRF3) in mouse ovarian granulosa cells, and HCQ inhibited this CTX-induced phosphorylation of IRF3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF\u0026ndash;G). In vitro experiments corroborated this finding, demonstrating that HCQ prevented PM-induced upregulation of cGAS pathway-related molecules (Supplementary Fig. S3F\u0026ndash;K).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eResult 8: HCQ delays physiological aging in the ovaries and other organs.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGiven that HCQ exerts significant ovarian protective effects in CTX-induced POF mice, and its congener chloroquine (CQ) has been demonstrated to possess anti-aging properties\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e, we further established a naturally aged female mouse model to evaluate the protective role of HCQ in physiological ovarian aging (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Compared with the naturally aged control group, the HCQ-treated group showed a marked increase in relative ovarian volume (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB), a significant elevation in ovarian index (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC), and improved regularity of the estrous cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). H\u0026amp;E staining staining demonstrated that the HCQ-treated group exhibited a significant increase in total follicle number compared to control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE\u0026ndash;F). Immunohistochemical analysis demonstrated that HCQ significantly upregulated anti-M\u0026uuml;llerian hormone (AMH) expression in the ovary (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG\u0026ndash;H), indicating its role in preserving follicle reserve.\u003c/p\u003e\u003cp\u003eWe further explored HCQ's role in delaying senescence in other organs. H\u0026amp;E staining revealed that the lungs, livers, and kidneys of HCQ-treated mice all exhibited delayed aging phenotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eI\u0026ndash;K). Additionally, compared with naturally aged control mice, the HCQ-treated group exhibited less severe hair thinning and alopecia (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eL), with a significant reduction in the relative proportion of hair loss area (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eM).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHCQ is an FDA-approved, widely used clinical agent with a well-established safety profile\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e, and has long been employed in rheumatology, immunology, and obstetric/gynecologic practice\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. Notably, A trend from our network meta-analysis indicated a potential benefit of CTX\u0026thinsp;+\u0026thinsp;HCQ combination therapy in maintaining AMH levels among patients with SLE, which merits further investigation into repurposing HCQ for protection against CTX-induced POI. Our subsequent research in a mouse model confirmed that HCQ protects against CTX-induced POI, demonstrating that it specifically alleviates cellular senescence in ovarian granulosa cells. By employing transcriptomic sequencing on ovarian granulosa cells from a mouse model, we identified that the preservation of mitochondrial function and inhibition of the cGAS-STING signaling pathway represent the mechanisms through which HCQ protects against CTX-induced POI, which we subsequently validated. Furthermore, we demonstrated that long-term HCQ administration delays multi-organ aging in a naturally aged mouse model.\u003c/p\u003e\u003cp\u003eGranulosa cells are core supportive cells for follicular development and steroid hormone synthesis, and their senescence serves as a key pathological driver of CTX-induced POF \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan additionalcitationids=\"CR38 CR39 CR40 CR41\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. Senescent granulosa cells not only lose proliferative capacity and reduce secretory function but also induce senescence in surrounding normal cells via the SASP, thereby exacerbating the overall aging process of ovarian tissue\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan additionalcitationids=\"CR44 CR45\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. Over recent years, accumulating studies have established mitochondrial dysfunction as a major driver of granulosa cell senescence and POF pathogenesis\u003csup\u003e[\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e, while the specific signaling pathways underlying this process remain elusive. Our study demonstrated that HCQ restores stability of mitochondrial membrane potential, reduces ROS levels, lowers cytoplasmic free mtDNA levels, and subsequently inhibits activation of the cGAS pathway and downstream SASP factors. Our proposed mechanism aligns with prior observations that chloroquine-derived agents suppress DNA sensing pathwaysand innate immune activation \u003csup\u003e[\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e.Our study further advances this understanding: HCQ functions as a regulator of mitochondrial homeostasis, thereby effectively preventing the abnormal cytoplasmic accumulation of mtDNA and subsequently suppressing the DNA-sensing cGAS pathway.\u003c/p\u003e\u003cp\u003eFurthermore, our study identifies a broader anti-aging potential for HCQ, as it mitigates both ovarian dysfunction and systemic aging in naturally aging mice\u0026mdash;a finding that corroborates prior reports that chloroquine-derived agents delay aging and extend lifespan by modulating DNA damage repair and inflammatory responses\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Given the established role of the cGAS-STING pathway in multi-organ degeneration, HCQ's potential to delay natural aging by modulating this pathway is a possibility that merits further investigation\u003csup\u003e[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWhile our study provides the first evidence confirming that HCQ protects against CTX-induced POI and its underlying mechanism, the findings must be interpreted with consideration of its limitations. First, the translatability of our findings from murine models to clinical practice requires careful consideration of species differences and the optimization of dosing regimens. Second, the precise molecular target of HCQ within the mitochondria that initiates this protective cascade remains to be elucidated. Finally, while HCQ showed promise in delaying multi-organ physiological aging, the potential risks of long-term administration and the universality of the proposed mechanism across different organs merit further in-depth investigation. further investigation is needed to elucidate the precise molecular targets of HCQ at the mitochondrial level and validating its efficacy in patients through clinical trials, while closely monitoring its long-term safety.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by Peking University People\u0026apos;s Hospital (Ethics Approval Number: 2025PHE008).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eD.S. and H.S. prepared figures 1-6. R.M. prepared figure 8. C.T. and X.Y. wrote the main manuscript text. Y.Z., Y.L., X.J., X.S., T.L., M.Z. and S.L. prepared figure 7. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the Youth Science Foundation Project of the National Natural Science Foundation of China (Grant No. 82401901) and the National Key R\u0026amp;D Program of China (Grants 2023YFC2706000 and 2023YFC2706002).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the staff of the National Key Laboratory of Obstetrics and Gynecology (Peking University People\u0026apos;s Hospital) and the National Key Laboratory of Pathogenic Biology (Peking University) for their support in providing experimental equipment, consumables, and technical assistance related to research facilities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be available upon request from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGu H C, Wang L F, Zhang Y W, Zhuo Y Q, Zhang Z H, et al. Human urine stem cells protect against cyclophosphamide-induced premature ovarian failure by inhibiting SLC1A4-mediated outflux of intracellular serine in ovarian granulosa cells [J]. Cell Mol Biol Lett, 2025, 30(1): 21.\u003c/li\u003e\n\u003cli\u003eFu Q, Wu C, Dai M, Wang S, Xu J, et al. Leflunomide versus azathioprine for maintenance therapy of lupus nephritis: a prospective, multicentre, randomised trial and long-term follow-up [J]. Ann Rheum Dis, 2022, 81(11): 1549-55.\u003c/li\u003e\n\u003cli\u003eArnaud L, Ruiz-Irastorza G, Aranow C, Bernatsky S, Dall\u0026apos;Era M, et al. ERN ReCONNET-SLICC-SLEuro expert consensus on the therapeutic management of rare systemic lupus erythematosus manifestations [J]. Lancet Rheumatol, 2025, 7(7): e505-e18.\u003c/li\u003e\n\u003cli\u003eYue Y, Yang H J, Zhang T, Li C, Kim M J, et al. Porcine Brain Enzyme Hydrolysate Enhances Immune Function and Antioxidant Defense via Modulation of Gut Microbiota in a Cyclophosphamide-Induced Immunodeficiency Model [J]. Antioxidants (Basel), 2024, 13(4).\u003c/li\u003e\n\u003cli\u003eHighley M S, Landuyt B, Prenen H, Harper P GDe Bruijn E A. The Nitrogen Mustards [J]. Pharmacol Rev, 2022, 74(3): 552-99.\u003c/li\u003e\n\u003cli\u003ePascuali N, Scotti L, Di Pietro M, Oubi\u0026ntilde;a G, Bas D, et al. Ceramide-1-phosphate has protective properties against cyclophosphamide-induced ovarian damage in a mice model of premature ovarian failure [J]. Hum Reprod, 2018, 33(5): 844-59.\u003c/li\u003e\n\u003cli\u003eLuan Y, Yu S Y, Abazarikia A, Dong RKim S Y. TAp63 determines the fate of oocytes against DNA damage [J]. Sci Adv, 2022, 8(51): eade1846.\u003c/li\u003e\n\u003cli\u003eStern E, Ben-Ami M, Gruber N, Toren A, Caspi S, et al. Hypothalamic-pituitary-gonadal function, pubertal development, and fertility outcomes in male and female medulloblastoma survivors: a single-center experience [J]. Neuro Oncol, 2023, 25(7): 1345-54.\u003c/li\u003e\n\u003cli\u003ePluma A, Hamroun S, R\u0026uuml;egg L, Cecchi I, Kramer M, et al. Antirheumatic drugs in reproduction, pregnancy, and lactation: a systematic literature review informing the 2024 update of the EULAR recommendations [J]. Ann Rheum Dis, 2025, 84(9): 1561-90.\u003c/li\u003e\n\u003cli\u003eSpears N, Lopes F, Stefansdottir A, Rossi V, De Felici M, et al. Ovarian damage from chemotherapy and current approaches to its protection [J]. Hum Reprod Update, 2019, 25(6): 673-93.\u003c/li\u003e\n\u003cli\u003eTuppi M, Kehrloesser S, Coutandin D W, Rossi V, Luh L M, et al. Oocyte DNA damage quality control requires consecutive interplay of CHK2 and CK1 to activate p63 [J]. Nat Struct Mol Biol, 2018, 25(3): 261-9.\u003c/li\u003e\n\u003cli\u003eLi X, Ye H, Su T, Hu C, Huang Y, et al. Immunity and reproduction protective effects of Chitosan Oligosaccharides in Cyclophosphamide/Busulfan-induced premature ovarian failure model mice [J]. Front Immunol, 2023, 14: 1185921.\u003c/li\u003e\n\u003cli\u003eXiong Y, Liu T, Wang S, Chi H, Chen C, et al. Cyclophosphamide promotes the proliferation inhibition of mouse ovarian granulosa cells and premature ovarian failure by activating the lncRNA-Meg3-p53-p66Shc pathway [J]. Gene, 2017, 596: 1-8.\u003c/li\u003e\n\u003cli\u003eHense J D, Isola J V V, Garcia D N, Magalh\u0026atilde;es L S, Masternak M M, et al. The role of cellular senescence in ovarian aging [J]. NPJ Aging, 2024, 10(1): 35.\u003c/li\u003e\n\u003cli\u003eWang H. The role of granulosa cells in oocyte development and aging: Mechanisms and therapeutic opportunities [J]. Semin Cell Dev Biol, 2025, 171: 103614.\u003c/li\u003e\n\u003cli\u003ePan P, Cao S, Gao H, Qu X, Ma Y, et al. Immp2l gene knockout induces granulosa cell senescence by activation of cGAS-STING pathway via TFAM-mediated mtDNA leakage [J]. Int J Biol Macromol, 2025, 307(Pt 4): 142368.\u003c/li\u003e\n\u003cli\u003eLi J, Yu Q, Huang H, Deng W, Cao X, et al. Human chorionic plate-derived mesenchymal stem cells transplantation restores ovarian function in a chemotherapy-induced mouse model of premature ovarian failure [J]. Stem Cell Res Ther, 2018, 9(1): 81.\u003c/li\u003e\n\u003cli\u003eNguyen N V, Sandstr\u0026ouml;m A, Svenungsson E, Dominicus A, Arkema E V, et al. Exposure to hydroxychloroquine in early pregnancy and incidence of pre-eclampsia and pre-term delivery in patients with systemic lupus erythematosus in Sweden: a nationwide population-based cohort study [J]. Lancet Rheumatol, 2025, 7(10): e687-e96.\u003c/li\u003e\n\u003cli\u003eRector A, Marić I, Chaichian Y, Chakravarty E, Cantu M, et al. Hydroxychloroquine in Lupus Pregnancy and Risk of Preeclampsia [J]. Arthritis Rheumatol, 2024, 76(6): 919-27.\u003c/li\u003e\n\u003cli\u003eSchrezenmeier ED\u0026ouml;rner T. Mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology [J]. Nat Rev Rheumatol, 2020, 16(3): 155-66.\u003c/li\u003e\n\u003cli\u003eWang R, Qi S, Wang Q, Lang J, Pang C, et al. Hydroxychloroquine enhances insulin sensitivity and ameliorates abnormal lipid metabolism in obese women with polycystic ovary syndrome [J]. BMC Endocr Disord, 2025, 25(1): 2.\u003c/li\u003e\n\u003cli\u003eRuiz A, Rockfield S, Taran N, Haller E, Engelman R W, et al. Effect of hydroxychloroquine and characterization of autophagy in a mouse model of endometriosis [J]. Cell Death Dis, 2016, 7(1): e2059.\u003c/li\u003e\n\u003cli\u003eChen F Y, Chen S W, Chen X, Huang J Y, Ye Z, et al. Hydroxychloroquine might reduce risk of incident endometriosis in patients with systemic lupus erythematosus: A retrospective population-based cohort study [J]. Lupus, 2021, 30(10): 1609-16.\u003c/li\u003e\n\u003cli\u003eQian M, Liu Z, Peng L, Tang X, Meng F, et al. Boosting ATM activity alleviates aging and extends lifespan in a mouse model of progeria [J]. Elife, 2018, 7.\u003c/li\u003e\n\u003cli\u003eDoeppner T R, Coman C, Burdusel D, Ancuta D L, Brockmeier U, et al. Long-term treatment with chloroquine increases lifespan in middle-aged male mice possibly via autophagy modulation, proteasome inhibition and glycogen metabolism [J]. Aging (Albany NY), 2022, 14(10): 4195-210.\u003c/li\u003e\n\u003cli\u003eLi W, Zou Z, Cai Y, Yang K, Wang S, et al. Low-dose chloroquine treatment extends the lifespan of aged rats [J]. Protein Cell, 2022, 13(6): 454-61.\u003c/li\u003e\n\u003cli\u003eAringer M, Costenbader K, D\u0026ouml;rner TJohnson S R. Advances in SLE classification criteria [J]. J Autoimmun, 2022, 132: 102845.\u003c/li\u003e\n\u003cli\u003eLiang X, Yan Z, Ma W, Qian Y, Zou X, et al. Peroxiredoxin 4 protects against ovarian ageing by ameliorating D-galactose-induced oxidative damage in mice [J]. Cell Death Dis, 2020, 11(12): 1053.\u003c/li\u003e\n\u003cli\u003eMyers M, Britt K L, Wreford N G, Ebling F JKerr J B. Methods for quantifying follicular numbers within the mouse ovary [J]. Reproduction, 2004, 127(5): 569-80.\u003c/li\u003e\n\u003cli\u003eYang P, Chen T, Liu Y, Hou Z, Wu K, et al. The critical role of ZP genes in female infertility characterized by empty follicle syndrome and oocyte degeneration [J]. Fertil Steril, 2021, 115(5): 1259-69.\u003c/li\u003e\n\u003cli\u003eWassarman P MLitscher E S. Female fertility and the zona pellucida [J]. Elife, 2022, 11.\u003c/li\u003e\n\u003cli\u003eGuo Y, Xue L, Tang W, Xiong J, Chen D, et al. Ovarian microenvironment: challenges and opportunities in protecting against chemotherapy-associated ovarian damage [J]. Hum Reprod Update, 2024, 30(5): 614-47.\u003c/li\u003e\n\u003cli\u003eRosenbaum J T, Costenbader K H, Desmarais J, Ginzler E M, Fett N, et al. American College of Rheumatology, American Academy of Dermatology, Rheumatologic Dermatology Society, and American Academy of Ophthalmology 2020 Joint Statement on Hydroxychloroquine Use With Respect to Retinal Toxicity [J]. Arthritis Rheumatol, 2021, 73(6): 908-11.\u003c/li\u003e\n\u003cli\u003eRajput R, Upadhyay P, Rajput S, Saini SKharab S. Effect of hydroxychloroquine on beta cell function, insulin resistance, and inflammatory markers in type 2 diabetes patients uncontrolled on glimepiride and metformin therapy [J]. Int J Diabetes Dev Ctries, 2023: 1-6.\u003c/li\u003e\n\u003cli\u003eSiegel C HSammaritano L R. Systemic Lupus Erythematosus: A Review [J]. Jama, 2024, 331(17): 1480-91.\u003c/li\u003e\n\u003cli\u003eGao R, Deng W, Meng C, Cheng K, Zeng X, et al. Combined treatment of prednisone and hydroxychloroquine may improve outcomes of frozen embryo transfer in antinuclear antibody-positive patients undergoing IVF/ICSI treatment [J]. Lupus, 2021, 30(14): 2213-20.\u003c/li\u003e\n\u003cli\u003eMo J, Hu H, Li P, Ye Y, Chen W, et al. Human hair follicle-derived mesenchymal stem cells improve ovarian function in cyclophosphamide-induced POF mice [J]. Stem Cell Res Ther, 2025, 16(1): 67.\u003c/li\u003e\n\u003cli\u003eRosario R, Stewart H L, Spears N, Telfer E EAnderson R A. Anti-Mullerian hormone attenuates both cyclophosphamide-induced damage and PI3K signalling activation, while rapamycin attenuates only PI3K signalling activation, in human ovarian cortex in vitro [J]. Hum Reprod, 2024, 39(2): 382-92.\u003c/li\u003e\n\u003cli\u003eDing C, Li H, Wang Y, Wang F, Wu H, et al. Different therapeutic effects of cells derived from human amniotic membrane on premature ovarian aging depend on distinct cellular biological characteristics [J]. Stem Cell Res Ther, 2017, 8(1): 173.\u003c/li\u003e\n\u003cli\u003eXu Z, Takahashi N, Harada M, Kunitomi C, Kusamoto A, et al. The Role of Cellular Senescence in Cyclophosphamide-Induced Primary Ovarian Insufficiency [J]. Int J Mol Sci, 2023, 24(24).\u003c/li\u003e\n\u003cli\u003eLiu T, Liu Y, Huang Y, Chen J, Yu Z, et al. miR-15b induces premature ovarian failure in mice via inhibition of \u0026alpha;-Klotho expression in ovarian granulosa cells [J]. Free Radic Biol Med, 2019, 141: 383-92.\u003c/li\u003e\n\u003cli\u003eLi Q, Shen H, Liu A, Yu L, Wang L, et al. FTO-mediated m6A modification elevates the MFN2 mRNA stability to suppress ovarian granulosa cell senescence [J]. Int J Biol Macromol, 2025, 307(Pt 4): 142181.\u003c/li\u003e\n\u003cli\u003eLuo Q, Yin N, Zhang L, Yuan W, Zhao W, et al. Role of SDF-1/CXCR4 and cytokines in the development of ovary injury in chemotherapy drug induced premature ovarian failure mice [J]. Life Sci, 2017, 179: 103-9.\u003c/li\u003e\n\u003cli\u003eZhou C, Li D, He J, Luo T, Liu Y, et al. TRIM28-Mediated Excessive Oxidative Stress Induces Cellular Senescence in Granulosa Cells and Contributes to Premature Ovarian Insufficiency In Vitro and In Vivo [J]. Antioxidants (Basel), 2024, 13(3).\u003c/li\u003e\n\u003cli\u003eLu H, Jing Y, Zhang C, Ma S, Zhang W, et al. Aging hallmarks of the primate ovary revealed by spatiotemporal transcriptomics [J]. Protein Cell, 2024, 15(5): 364-84.\u003c/li\u003e\n\u003cli\u003eDevrukhkar P R, Watson M A, Soygur B, Wu F, Bons J, et al. A Comprehensive Multiomics Signature of Doxorubicin-Induced Cellular Senescence in the Postmenopausal Human Ovary [J]. Aging Cell, 2025, 24(8): e70111.\u003c/li\u003e\n\u003cli\u003eMiwa S, Kashyap S, Chini Evon Zglinicki T. Mitochondrial dysfunction in cell senescence and aging [J]. J Clin Invest, 2022, 132(13).\u003c/li\u003e\n\u003cli\u003eDong T, Li N, Wang H, Zhu H, Gao Y, et al. Stress granule clearance mediated by V-ATPase-interacting protein NCOA7 mitigates ovarian aging [J]. Nat Aging, 2025, 5(8): 1548-67.\u003c/li\u003e\n\u003cli\u003eSmits M A J, Schomakers B V, van Weeghel M, Wever E J M, W\u0026uuml;st R C I, et al. Human ovarian aging is characterized by oxidative damage and mitochondrial dysfunction [J]. Hum Reprod, 2023, 38(11): 2208-20.\u003c/li\u003e\n\u003cli\u003eBonam S R, Muller S, Bayry JKlionsky D J. Autophagy as an emerging target for COVID-19: lessons from an old friend, chloroquine [J]. Autophagy, 2020, 16(12): 2260-6.\u003c/li\u003e\n\u003cli\u003eXu YWan W. Lysosomal control of the cGAS-STING signaling [J]. Trends Cell Biol, 2024, 34(8): 622-5.\u003c/li\u003e\n\u003cli\u003eAn J, Woodward J J, Lai W, Minie M, Sun X, et al. Inhibition of Cyclic GMP-AMP Synthase Using a Novel Antimalarial Drug Derivative in Trex1-Deficient Mice [J]. Arthritis Rheumatol, 2018, 70(11): 1807-19.\u003c/li\u003e\n\u003cli\u003eGulen M F, Samson N, Keller A, Schwabenland M, Liu C, et al. cGAS-STING drives ageing-related inflammation and neurodegeneration [J]. Nature, 2023, 620(7973): 374-80.\u003c/li\u003e\n\u003cli\u003eSladitschek-Martens H L, Guarnieri A, Brumana G, Zanconato F, Battilana G, et al. YAP/TAZ activity in stromal cells prevents ageing by controlling cGAS-STING [J]. Nature, 2022, 607(7920): 790-8.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-aging","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Aging](https://www.nature.com/npjamd/)","snPcode":"41514","submissionUrl":"https://submission.springernature.com/new-submission/41514/3","title":"npj Aging","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Premature ovarian failure, Cyclophosphamide, Hydroxychloroquine, Granulosa cell senescence, mtDNA-cGAS signaling pathway","lastPublishedDoi":"10.21203/rs.3.rs-8052573/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8052573/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCyclophosphamide (CTX) is widely used as a first-line chemotherapeutic agent for various cancers, but it is associated with significant risks of ovarian dysfunction, even progressing to premature ovarian failure (POF), with granulosa cell senescence being a key phenotypic manifestation. hydroxychloroquine (HCQ) has demonstrated anti-senescence properties in the context of age-related diseases, but its efficacy in preventing CTX-induced ovarian damage has not been fully elucidated. We conducted a controlled animal study using a CTX-induced POF model in female C57BL/6 mice, comprising three groups: vehicle control, CTX-only, and CTX\u0026thinsp;+\u0026thinsp;HCQ. Complementary in vitro studies were conducted using the human ovarian granulosa cell line KGN, treated with phosphoramide mustard (PM, the active metabolite of CTX) and HCQ. HCQ partially reversed the CTX-induced decrease in ovarian index, normalized the estrous cycle, reduced follicle depletion, and improved serum hormone levels as well as reproductive outcomes. HCQ reduced the expression of granulosa cell senescence markers and the area of senescence-associated β-galactosidase(SA-β-gal)-positive cells, stabilized mitochondrial membrane potential, decreased reactive oxygen species(ROS) production and mitochondrial DNA(mtDNA) leakage, inhibited cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling and suppressed senescence-associated secretory phenotype(SASP) factors. Collectively, HCQ protects CTX-induced POF by inhibiting activation of the mtDNA-cGAS signaling pathway, thereby alleviating granulosa cell senescence and preserving ovarian function.\u003c/p\u003e","manuscriptTitle":"Hydroxychloroquine Protects Against Cyclophosphamide-Induced Premature ovarian failure by Reducing Granulosa Cell Senescence via Regulation of the mtDNA-cGAS Signaling Pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-25 18:10:57","doi":"10.21203/rs.3.rs-8052573/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-11T14:23:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-03T11:45:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-22T09:29:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"228077382904068509051568144150528359353","date":"2025-11-21T22:07:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"98350900764716658866418756545670590298","date":"2025-11-17T18:37:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"84157816178051353721976185339502998794","date":"2025-11-14T15:32:37+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-14T15:24:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-14T14:22:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-12T10:56:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Aging","date":"2025-11-07T03:24:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-aging","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Aging](https://www.nature.com/npjamd/)","snPcode":"41514","submissionUrl":"https://submission.springernature.com/new-submission/41514/3","title":"npj Aging","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9656f06e-8dc1-447d-83a1-05ed92738522","owner":[],"postedDate":"November 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":58428793,"name":"Biological sciences/Biochemistry"},{"id":58428794,"name":"Biological sciences/Cancer"},{"id":58428795,"name":"Biological sciences/Cell biology"},{"id":58428796,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2026-03-23T16:03:00+00:00","versionOfRecord":{"articleIdentity":"rs-8052573","link":"https://doi.org/10.1038/s41514-026-00359-9","journal":{"identity":"npj-aging","isVorOnly":false,"title":"npj Aging"},"publishedOn":"2026-03-17 15:58:47","publishedOnDateReadable":"March 17th, 2026"},"versionCreatedAt":"2025-11-25 18:10:57","video":"","vorDoi":"10.1038/s41514-026-00359-9","vorDoiUrl":"https://doi.org/10.1038/s41514-026-00359-9","workflowStages":[]},"version":"v1","identity":"rs-8052573","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8052573","identity":"rs-8052573","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}