BFGF Mitigates CTX-Induced Ovarian Cytotoxicity via SERPINE1/HIF-1 and Nrf-2/HO-1 Signaling Pathways

BFGF Mitigates CTX-Induced Ovarian Cytotoxicity via SERPINE1/HIF-1 and Nrf-2/HO-1 Signaling Pathways | 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 Research Article BFGF Mitigates CTX-Induced Ovarian Cytotoxicity via SERPINE1/HIF-1 and Nrf-2/HO-1 Signaling Pathways Yao Li, Lin Zhang, Hongqing Lv, Chenxuan Mei, Min Hu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6396471/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Jul, 2025 Read the published version in Journal of Ovarian Research → Version 1 posted 9 You are reading this latest preprint version Abstract Chemotherapy-induced ovarian damage is a significant concern for female cancer patients, often resulting in infertility, hormonal imbalance, and premature ovarian failure. Cyclophosphamide (CTX), a widely used chemotherapeutic agent, is highly effective against tumors but causes severe ovarian cytotoxicity. This study explores the protective effects of basic fibroblast growth factor (BFGF) and its underlying mechanisms in mitigating CTX-induced ovarian damage. BFGF treatment significantly enhanced cell viability, reduced apoptosis, and restored mitochondrial membrane potential in CTX-treated ovarian cells. Transcriptomic analysis revealed that BFGF activated the SERPINE1/HIF-1 signaling pathway, promoting angiogenesis, reducing apoptotic signaling, and enhancing cell cycle progression by upregulating Cyclin D1 and CDK4. Additionally, BFGF activated the Nrf-2/HO-1 pathway, boosting cellular defenses against oxidative stress and mitigating mitochondrial dysfunction. Functional studies confirmed that the inhibition of SERPINE1 or BFGF abrogated these protective effects, underscoring the critical roles of these pathways. These findings demonstrate that BFGF protects ovarian cells from CTX-induced damage by modulating the SERPINE1/HIF-1 and Nrf-2/HO-1 pathways, reducing apoptosis, and enhancing cell survival, providing a promising foundation for developing BFGF-based therapies to preserve ovarian function and fertility in female cancer patients undergoing chemotherapy. Chemotherapy-induced ovarian damage Basic fibroblast growth factor SERPINE1/HIF-1 signaling pathway Nrf-2/HO-1 pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Chemotherapy is a cornerstone of modern cancer treatment, offering significant survival benefits and disease control for patients with various malignancies. However, its therapeutic effects often come at the cost of severe side effects, as most chemotherapeutic agents lack selectivity for tumor cells and inadvertently damage healthy tissues. Among these side effects, ovarian toxicity is particularly concerning for young female patients, as it results in infertility, hormonal imbalances, and premature ovarian failure[ 1 ]. These complications not only affect physical health but also have profound psychological and social implications, making ovarian protection a critical aspect of survivorship care in oncology. Cyclophosphamide (CTX), a widely used alkylating agent in chemotherapy, is notorious for its high ovarian toxicity, as it directly induces apoptosis in ovarian follicles, damages stromal vascular structures, and reduces ovarian reserve[ 2 , 3 ]. Its mechanisms of toxicity include DNA damage, oxidative stress, and disruption of the ovarian microenvironment, leading to irreversible declines in reproductive and endocrine function[ 4 ]. Despite advancements in fertility preservation techniques, existing strategies have significant limitations and are not suitable for all patients. Hormonal suppression with gonadotropin-releasing hormone analogs (GnRHa) can reduce ovarian damage but does not fully prevent follicular loss or ensure fertility preservation[ 5 ]. Surgical approaches, such as oocyte and ovarian tissue cryopreservation, provide alternative options but come with risks and logistical challenges[ 6 , 7 ]. For instance, ovarian tissue cryopreservation carries a potential risk of reintroducing malignant cells during transplantation, and oocyte cryopreservation requires ovarian stimulation, which can delay cancer treatment and is unsuitable for patients with estrogen-sensitive tumors[ 8 ]. Moreover, both methods are costly and not universally accessible. These challenges highlight the urgent need for novel, non-invasive interventions that can protect ovarian function during chemotherapy while being safe and readily implementable. Basic fibroblast growth factor (BFGF) has emerged as a promising candidate for addressing chemotherapy-induced ovarian toxicity. A member of the fibroblast growth factor family, BFGF is a multifunctional growth factor known for its roles in angiogenesis, tissue repair, and cellular proliferation[ 9 ]. In the context of ovarian biology, BFGF has been shown to promote follicular growth, enhance granulosa cell proliferation, and stimulate estradiol production[ 10 , 11 ]. Furthermore, studies have demonstrated that BFGF can mitigate tissue damage in other organ systems, such as the heart and skeletal muscle, by promoting vascular repair and reducing fibrosis[ 12 , 13 ]]. Importantly, BFGF has been reported to improve ovarian function in models of age-related dysfunction, suggesting its potential for reversing ovarian damage caused by chemotherapy[ 14 ]. Its ability to regulate key molecular pathways involved in stress response and tissue regeneration makes BFGF a compelling candidate for ovarian protection. By expanding on these observations, the present study aims to comprehensively investigate the role of BFGF in ameliorating CTX-induced ovarian cytotoxicity and to elucidate the molecular mechanisms underlying its protective effects. Through in vitro and in vivo experiments, we explore how BFGF activates the SERPINE1/HIF-1 signaling pathway to promote ovarian repair and improve overall reproductive health. This research seeks to provide a scientific basis for the development of BFGF-based therapeutic strategies to preserve ovarian function and fertility in female cancer patients, addressing a critical unmet need in the field of oncofertility. In this study, we hypothesized that BFGF mitigates CTX-induced ovarian damage by modulating key molecular pathways involved in cell survival and angiogenesis. Our preliminary findings demonstrated that BFGF treatment improved ovarian structure and function in a CTX-induced ovarian insufficiency model. Specifically, BFGF upregulated the expression of SERPINE1 and HIF-1, two critical components of a signaling pathway known to regulate cellular responses to stress, promote angiogenesis, and enhance tissue repair. In addition, BFGF reduced apoptosis and restored mitochondrial function, further supporting its protective role. Building on these observations, the present study investigates the role of the SERPINE1/HIF-1 signaling pathway in mediating the protective effects of BFGF against CTX-induced ovarian cytotoxicity. We conducted a series of in vitro and in vivo experiments to explore the molecular mechanisms underlying BFGF's actions and to evaluate its potential as a therapeutic agent for preserving ovarian function and fertility in women undergoing chemotherapy. By elucidating the mechanisms of BFGF action, this research aims to provide a foundation for the development of targeted therapies to improve the quality of life for cancer survivors. Materials and Methods Materials and Reagents DMEM/F12 Medium: Gibco, Thermo Fisher Scientific (Catalog No. 11330032);Fetal Bovine Serum (FBS): Gibco, Thermo Fisher Scientific (Catalog No. 10099141)༛Penicillin-Streptomycin: Gibco, Thermo Fisher Scientific (Catalog No. 15140122)༛Cyclophosphamide (CTX): Sigma-Aldrich (Catalog No. C0768)༛Basic Fibroblast Growth Factor (BFGF): PeproTech (Catalog No. 100-18B)༛BFGF Inhibitor: R&D Systems (Catalog No. 7183-FB)༛CCK-8 Cell Viability Assay Kit: Dojindo Laboratories (Catalog No. CK04)༛Annexin V-FITC/PI Apoptosis Detection Kit: BD Biosciences (Catalog No. 556547)༛JC-1 Mitochondrial Membrane Potential Assay Kit: Abcam (Catalog No. ab113850)༛TUNEL Assay Kit: Roche (Catalog No. 11684795910)༛RIPA Lysis Buffer: Thermo Fisher Scientific (Catalog No. 89901)༛Protease and Phosphatase Inhibitor Cocktail: Sigma-Aldrich (Catalog No. P5726)༛PVDF Membranes: Millipore (Catalog No. IPVH00010)༛Enhanced Chemiluminescence (ECL) Detection Kit: Thermo Fisher Scientific (Catalog No. 32106)༛SERPINE1 Antibody: Abcam (Catalog No. ab125687)༛HIF-1α Antibody: Cell Signaling Technology (Catalog No. 3716)༛Nrf-2 Antibody: Abcam (Catalog No. ab137550)༛HO-1 Antibody: Cell Signaling Technology (Catalog No. 43966)༛Cyclin D1 Antibody: Cell Signaling Technology (Catalog No. 2978)༛CDK4 Antibody: Cell Signaling Technology (Catalog No. 12790)༛TGF-β Antibody: Abcam (Catalog No. ab92486)༛FSH Antibody: Abcam (Catalog No. ab151564)༛ER Antibody: Abcam (Catalog No. ab16660)༛β-actin Antibody: Cell Signaling Technology (Catalog No. 4970)༛HRP-Conjugated Secondary Antibodies: Cell Signaling Technology (Catalog No. 7074 for anti-rabbit, 7076 for anti-mouse)༛TRIzol Reagent: Thermo Fisher Scientific (Catalog No. 15596026)༛cDNA Synthesis Kit: Thermo Fisher Scientific (Catalog No. 4368814)༛SYBR Green PCR Master Mix: Thermo Fisher Scientific (Catalog No. A25742)༛Triton X-100: Sigma-Aldrich (Catalog No. T8787)༛Bovine Serum Albumin (BSA): Sigma-Aldrich (Catalog No. A9418)༛DAPI Stain: Thermo Fisher Scientific (Catalog No. D1306)༛Fluorescent Secondary Antibodies: Thermo Fisher Scientific (Catalog Nos. A-11001 and A-11008)༛Illumina TruSeq RNA Sample Preparation Kit: Illumina (Catalog No. RS-122-2001)༛Agilent RNA 6000 Nano Kit: Agilent Technologies (Catalog No. 5067 − 1511)。 Cell Culture and Treatment Ovarian granulosa cells were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a humidified atmosphere containing 5% CO₂ at 37°C. Cells were divided into the following groups: (1) control group, (2) CTX group (treated with cyclophosphamide at a concentration of 50 µM), (3) CTX + BFGF group (co-treated with 50 µM CTX and 10 ng/mL BFGF), (4) CTX + BFGF inhibitor group, (5) CTX + SERPINE1 overexpression group, and (6) CTX + SERPINE1 inhibitor group. Treatment durations were set according to experimental requirements, ranging from 24 to 120 hours. Cell Viability Assay Cell viability was assessed using the CCK-8 assay. Briefly, cells were seeded into 96-well plates at a density of 5 × 10³ cells per well and treated according to their respective groups. After incubation, 10 µL of CCK-8 reagent was added to each well, and plates were incubated for an additional 2 hours at 37°C. Absorbance was measured at 450 nm using a microplate reader, and cell viability was calculated as a percentage relative to the control group. Apoptosis Analysis Apoptosis was analyzed using flow cytometry and TUNEL staining. For flow cytometry, cells were harvested, washed with PBS, and stained with Annexin V-FITC and PI for 15 minutes at room temperature. Apoptotic cells were quantified using a flow cytometer. For TUNEL staining, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and incubated with the TUNEL reaction mixture. Stained cells were observed under a fluorescence microscope, and apoptotic cells were quantified. Mitochondrial Membrane Potential (MMP) Analysis MMP was assessed using JC-1 staining. Cells were incubated with JC-1 dye (5 µg/mL) at 37°C for 20 minutes, washed with PBS, and observed under a fluorescence microscope. The ratio of red fluorescence (JC-1 aggregates, indicating intact MMP) to green fluorescence (JC-1 monomers, indicating depolarized MMP) was calculated to assess mitochondrial health. Transcriptomic Sequencing and Analysis To investigate the molecular mechanisms underlying the protective effects of BFGF against CTX-induced ovarian damage, transcriptomic sequencing was performed on ovarian cells treated with CTX alone and CTX + BFGF. Total RNA was extracted from the samples using the TRIzol reagent, and RNA integrity was assessed using an Agilent 2100 Bioanalyzer. RNA with an RNA Integrity Number (RIN) > 7.0 was used for sequencing. RNA-seq libraries were prepared using the Illumina TruSeq RNA Sample Preparation Kit, following the manufacturer’s protocol. Sequencing was performed on the Illumina HiSeq 2500 platform, generating paired-end reads of 150 bp. Raw sequencing data were processed to remove low-quality reads and adapters using FastQC and Trimmomatic. High-quality clean reads were aligned to the reference human genome (GRCh38) using HISAT2. Differentially expressed genes (DEGs) were identified using DESeq2 with |log2 fold change| > 1 and p-value < 0.05 as thresholds. Volcano plots were generated to visualize upregulated and downregulated DEGs. Hierarchical clustering analysis and heatmaps were constructed to illustrate gene expression patterns among different groups. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed using the clusterProfiler package in R to identify biological processes, cellular components, molecular functions, and pathways associated with the DEGs. The HIF-1 signaling pathway and oxidative stress-related pathways were highlighted based on their significant enrichment in the BFGF-treated group. Visualization of pathway enrichment was presented using bubble plots. These analyses provided insights into the molecular pathways activated by BFGF to counteract CTX-induced ovarian damage. Western Blot Analysis Western Blot Analysis Protein expression was analyzed by Western blot. Cells were lysed using RIPA buffer containing protease and phosphatase inhibitors. Equal amounts of protein were separated by SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked with 5% non-fat milk and incubated overnight at 4°C with primary antibodies against SERPINE1, HIF-1α, Nrf-2, HO-1, Cyclin D1, CDK4, TGF-β, FSH, ER, and β-actin. After washing, membranes were incubated with HRP-conjugated secondary antibodies. Protein bands were visualized using ECL reagents and quantified using ImageJ software. Quantitative Real-Time PCR (qRT-PCR) Total RNA was extracted using TRIzol reagent, and cDNA was synthesized using a reverse transcription kit. qRT-PCR was performed using SYBR Green PCR Master Mix on a real-time PCR system. Primers for SERPINE1, HIF-1α, Nrf-2, HO-1, Cyclin D1, and CDK4 were designed and validated. Relative gene expression was calculated using the 2^−ΔΔCt method, with β-actin as the internal control. Colony Formation Assay Cells were seeded into 6-well plates at a density of 500 cells per well and treated according to their respective groups. After 10 days of incubation, colonies were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. Colonies containing more than 50 cells were counted manually under a microscope. Immunofluorescence Staining Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% BSA. Primary antibodies against Nrf-2 and HO-1 were applied overnight at 4°C. After washing, cells were incubated with fluorescently labeled secondary antibodies and counterstained with DAPI. Images were captured using a fluorescence microscope, and fluorescence intensity was quantified using ImageJ. Transcriptomic Analysis Total RNA from CTX and CTX + BFGF groups was extracted and subjected to high-throughput RNA sequencing. Differentially expressed genes (DEGs) were identified based on |log2 fold change| > 1 and p < 0.05. KEGG pathway enrichment and GO functional analyses were performed to identify enriched biological pathways and processes. Heatmaps and volcano plots were generated to visualize expression profiles and pathway activation. Statistical Analysis All experiments were performed in triplicate, and data are presented as mean ± SEM. Statistical analysis was conducted using GraphPad Prism. Differences between groups were analyzed using one-way ANOVA followed by Tukey’s post hoc test. A p-value < 0.05 was considered statistically significant. Results BFGF mitigates CTX-induced ovarian cytotoxicity by improving cell viability and reducing apoptosis To investigate the protective effects of BFGF against CTX-induced ovarian damage, we assessed cell viability, nuclear morphology, apoptotic rates, and the expression of regulatory proteins. The cell viability assay (Fig. 1 A) showed a significant dose-dependent reduction in proliferation in the CTX-treated group, consistent with the cytotoxic effects of CTX on ovarian cells. In contrast, BFGF co-treatment significantly improved cell viability, with higher concentrations producing more pronounced protective effects. To further explore the impact of BFGF, nuclear morphology was analyzed using DAPI staining (Fig. 1 B). CTX treatment induced typical apoptotic features, including nuclear condensation and chromatin aggregation. These changes were significantly mitigated in the BFGF-treated group, suggesting that BFGF reduces CTX-induced apoptosis. Additionally, Western blot analysis was performed to examine the expression of apoptosis-related regulatory proteins, including TGF-β, FSH, and ER (Fig. 1 C-F). CTX treatment resulted in a notable disruption in the expression levels of these proteins, while BFGF treatment restored their levels closer to those of the control group. These findings indicate that BFGF effectively modulates key ovarian signaling pathways to counteract the effects of CTX. These results demonstrate that BFGF mitigates CTX-induced ovarian cytotoxicity by enhancing cell viability, reducing apoptosis, and regulating the expression of critical ovarian regulatory proteins, highlighting its potential role as a therapeutic agent to preserve ovarian function during chemotherapy. BFGF reduces CTX-induced apoptosis by stabilizing mitochondrial membrane potential Following the observation that BFGF enhances cell viability and reduces apoptosis in CTX-treated ovarian cells, we further investigated its effects on mitochondrial function, a key regulator of apoptosis. Mitochondrial membrane potential (MMP) was assessed using JC-1 staining, which differentiates between polarized (red fluorescence) and depolarized (green fluorescence) mitochondria. As shown in Fig. 2 A, CTX treatment significantly increased green fluorescence intensity, indicating a marked loss of MMP and mitochondrial dysfunction. In contrast, BFGF treatment restored MMP in a dose-dependent manner, as evidenced by a shift toward increased red fluorescence, suggesting improved mitochondrial stability[ 15 , 16 ]. To confirm the anti-apoptotic effects of BFGF, TUNEL staining was performed to visualize DNA fragmentation, a hallmark of late-stage apoptosis (Fig. 2 B). CTX treatment resulted in a significant increase in TUNEL-positive cells, indicative of extensive apoptotic damage. However, BFGF treatment dramatically reduced the number of TUNEL-positive cells, further demonstrating its protective effect against CTX-induced apoptosis. Notably, the protective effect of BFGF was partially inhibited by the addition of a BFGF antagonist, confirming the specificity of its action. These results indicate that BFGF protects ovarian cells from CTX-induced apoptosis by stabilizing mitochondrial function and preventing downstream apoptotic signaling. This suggests that the anti-apoptotic effects of BFGF are closely associated with its ability to maintain mitochondrial integrity, reinforcing its therapeutic potential in mitigating chemotherapy-induced ovarian damage. BFGF activates the SERPINE1/HIF-1 signaling pathway to mitigate CTX-induced ovarian damage To further explore the mechanisms underlying the protective effects of BFGF, transcriptomic analysis was performed to identify differentially expressed genes (DEGs) in ovarian cells treated with CTX and CTX + BFGF (Fig. 3 ). The volcano plot (Fig. 3 A) revealed significant differences in gene expression between the two groups, with several genes upregulated or downregulated in response to BFGF treatment. Heatmap analysis (Fig. 3 B) demonstrated distinct clustering of DEGs, indicating that BFGF treatment substantially altered the gene expression profile in CTX-treated ovarian cells. KEGG pathway enrichment analysis (Fig. 3 C) highlighted the HIF-1 signaling pathway as one of the most significantly enriched pathways in the BFGF-treated group, suggesting its involvement in mediating the protective effects of BFGF. Additionally, GO functional enrichment analysis (Fig. 3 D) revealed that BFGF influenced processes related to apoptosis regulation and cellular survival, further supporting its role in mitigating CTX-induced ovarian cytotoxicity. To validate these findings, we assessed the expression of key proteins involved in the SERPINE1/HIF-1 pathway. Western blot analysis (Fig. 4 A) showed that CTX treatment significantly downregulated SERPINE1 and HIF-1α protein levels, whereas BFGF treatment restored their expression to near-normal levels. The protective effects of BFGF were further confirmed through qRT-PCR analysis (Figs. 4 F and 4 G), which revealed a similar pattern of mRNA expression. Importantly, the addition of a BFGF inhibitor attenuated these effects, indicating the specificity of BFGF in activating the SERPINE1/HIF-1 pathway. Furthermore, we examined the downstream effects of BFGF-mediated activation of this pathway on cell cycle regulation. Western blot results (Fig. 4 A) indicated that CTX treatment reduced the expression of Cyclin D1 and CDK4, key regulators of cell cycle progression, while BFGF treatment significantly restored their expression levels. This was further supported by qRT-PCR analysis (Figs. 4 H and 4 I), which showed a consistent increase in Cyclin D1 and CDK4 mRNA expression following BFGF treatment. Notably, the BFGF inhibitor reversed these effects, underscoring the critical role of the SERPINE1/HIF-1 signaling pathway in mediating BFGF’s protective effects. These findings demonstrate that BFGF alleviates CTX-induced ovarian damage by activating the SERPINE1/HIF-1 signaling pathway, restoring cell cycle regulation, and enhancing ovarian cell survival. These results highlight the therapeutic potential of BFGF in protecting ovarian function during chemotherapy. BFGF exerts its protective effects through the activation of SERPINE1 To further confirm the role of SERPINE1 in BFGF-mediated protection against CTX-induced ovarian damage, we evaluated cell growth, colony formation, and the expression of key signaling proteins under conditions of SERPINE1 overexpression (OE) and inhibition. Cell growth analysis (Fig. 5 A) revealed that CTX significantly suppressed cell proliferation compared to the control group. However, treatment with BFGF or SERPINE1 overexpression restored cell growth to near-normal levels, demonstrating a strong protective effect. In contrast, the addition of a SERPINE1 inhibitor abrogated this protective effect, further confirming the involvement of SERPINE1 in mediating BFGF's actions. Colony formation assays (Fig. 5 B) supported these findings, showing that CTX-treated cells exhibited a marked reduction in colony-forming ability. This impairment was reversed by both BFGF treatment and SERPINE1 overexpression, with the number of colonies significantly increasing. Conversely, SERPINE1 inhibition led to a substantial reduction in colony formation, highlighting the critical role of this protein in ovarian cell survival and proliferation. Western blot analysis (Fig. 5 C) demonstrated that CTX reduced the expression of SERPINE1 and its downstream targets, including HIF-1α, Cyclin D1, and CDK4. BFGF treatment or SERPINE1 overexpression restored the expression levels of these proteins, while SERPINE1 inhibition reversed these effects. Quantitative analysis of SERPINE1 and HIF-1α expression (Figs. 5 D–E) confirmed these trends, indicating that the activation of SERPINE1 is essential for BFGF's protective mechanism. Additionally, the expression levels of Cyclin D1 and CDK4, key regulators of cell cycle progression, were significantly upregulated by BFGF and SERPINE1 overexpression, as shown in Figs. 5 F–G. In contrast, SERPINE1 inhibition suppressed their expression, further supporting the role of SERPINE1 in promoting cell survival and proliferation. The results demonstrate that the protective effects of BFGF against CTX-induced ovarian cytotoxicity are mediated through the activation of SERPINE1, which regulates key downstream targets such as HIF-1α, Cyclin D1, and CDK4. This highlights the pivotal role of the SERPINE1 signaling axis in preserving ovarian function during chemotherapy. BFGF activates the Nrf-2/HO-1 signaling pathway to protect against CTX-induced oxidative damage To investigate whether the Nrf-2/HO-1 signaling pathway contributes to the protective effects of BFGF against CTX-induced ovarian damage, we examined the expression and localization of Nrf-2 and HO-1 using immunofluorescence and Western blot analyses. Immunofluorescence staining revealed that CTX treatment significantly decreased the nuclear localization and fluorescence intensity of Nrf-2 and HO-1, indicative of impaired oxidative stress response (Figs. 6 A–B). In contrast, BFGF treatment restored the nuclear localization and expression of both proteins, suggesting that BFGF activates the Nrf-2/HO-1 signaling pathway. Notably, the addition of a BFGF inhibitor attenuated these effects, confirming the specificity of BFGF's action. Western blot analysis further corroborated these findings (Fig. 6 C). CTX treatment led to a marked reduction in Nrf-2 and HO-1 protein expression compared to the control group. However, BFGF treatment significantly upregulated the expression of both proteins, restoring their levels closer to normal. Quantitative analysis (Figs. 6 D–E) demonstrated that BFGF increased the relative expression of Nrf-2 and HO-1, while the presence of a BFGF inhibitor reversed these effects, further highlighting the importance of this pathway in mediating BFGF’s protective effects. These results indicate that BFGF protects ovarian cells from CTX-induced oxidative damage by activating the Nrf-2/HO-1 signaling pathway. By promoting the expression and activity of Nrf-2 and HO-1, BFGF enhances the cellular defense against oxidative stress, thereby mitigating mitochondrial dysfunction and reducing apoptosis. This underscores the critical role of oxidative stress regulation in the therapeutic potential of BFGF for preserving ovarian function during chemotherapy. Discussion Chemotherapy-induced ovarian toxicity remains a significant challenge in the treatment of female cancer patients, particularly those of reproductive age. CTX, a widely used chemotherapeutic agent, is effective against tumors but causes severe damage to ovarian cells, resulting in reduced ovarian reserve, impaired reproductive function, and premature ovarian failure[ 10 – 12 ]. In this study, we demonstrate that BFGF effectively protects against CTX-induced ovarian damage through multiple mechanisms, highlighting its therapeutic potential for ovarian preservation. Our findings indicate that BFGF enhances cell viability and reduces apoptosis in ovarian cells exposed to CTX. These effects are associated with the stabilization of mitochondrial membrane potential, a key determinant of cell survival. Mitochondrial dysfunction is a central feature of chemotherapy-induced cell damage, and BFGF’s ability to maintain mitochondrial stability suggests its direct role in preventing CTX-induced apoptosis[ 14 , 17 ]. By preserving mitochondrial function, BFGF not only promotes cell survival but also supports the maintenance of the ovarian microenvironment, which is essential for normal ovarian function and follicular development. Further mechanistic studies revealed that BFGF activates the SERPINE1/HIF-1 signaling pathway, which plays a pivotal role in cellular response to stress, angiogenesis, and tissue repair[ 18 ]. The upregulation of SERPINE1 and HIF-1α by BFGF suggests that this pathway mediates its protective effects on ovarian cells. Functional analyses confirmed that activation of this pathway restores cell proliferation and reduces apoptosis, emphasizing the critical role of SERPINE1 in BFGF-mediated ovarian protection. These results highlight the therapeutic potential of targeting the SERPINE1/HIF-1 axis to counteract ovarian damage caused by chemotherapy. In addition, BFGF was found to activate the Nrf-2/HO-1 signaling pathway, which regulates cellular defense against oxidative stress. Oxidative stress is a major contributor to CTX-induced ovarian damage, leading to increased apoptosis and mitochondrial dysfunction. By promoting the nuclear localization of Nrf-2 and upregulating HO-1 expression[ 19 , 20 ], BFGF enhances the antioxidative capacity of ovarian cells, mitigating oxidative damage and preserving cell viability. This dual role of BFGF in regulating oxidative stress and angiogenesis underscores its multifaceted nature and therapeutic potential. Importantly, the addition of inhibitors targeting SERPINE1 or BFGF significantly attenuated its protective effects, further validating the specificity of these signaling pathways in mediating BFGF's actions. These findings suggest that the SERPINE1/HIF-1 and Nrf-2/HO-1 pathways are critical mediators of ovarian protection and represent promising therapeutic targets for mitigating chemotherapy-induced ovarian damage. Although this study provides compelling evidence for the efficacy of BFGF in protecting ovarian cells, further research is needed to address its clinical applicability. Future studies should focus on optimizing the dosing, delivery methods, and long-term safety of BFGF. Additionally, combining BFGF with existing ovarian protection strategies, such as hormonal therapies or antioxidants, may enhance its effectiveness and broaden its clinical use. In conclusion, this study demonstrates that BFGF mitigates CTX-induced ovarian damage by activating the SERPINE1/HIF-1 and Nrf-2/HO-1 signaling pathways, reducing apoptosis, and enhancing cell survival. These findings provide a strong foundation for the development of BFGF-based therapeutic approaches to preserve ovarian function and fertility in female cancer patients undergoing chemotherapy, addressing a critical unmet need in oncofertility care. Declarations Acknowledgements Not applicable. Funding This work was partly supported by Key Science and Technology Plan Project of Jinhua City (2023-3-143). Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Consent for publication Not applicable. Competing interests Not applicable. Author Contribution Y.L was primarily responsible for the design and execution of the experiments, data analysis, and original draft preparation. L.Z contributed to data curation, visualization, and manuscript revision. H.L provided experimental resources, assisted in data acquisition, and coordinated laboratory activities. C.M conducted the transcriptomic sequencing and performed bioinformatics analyses. M. H conceived and supervised the overall project, acquired funding, and critically revised the manuscript for important intellectual content. All authors read and approved the final version of the manuscript. 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Cite Share Download PDF Status: Published Journal Publication published 12 Jul, 2025 Read the published version in Journal of Ovarian Research → Version 1 posted Editorial decision: Revision requested 26 May, 2025 Reviews received at journal 14 May, 2025 Reviewers agreed at journal 12 May, 2025 Reviews received at journal 22 Apr, 2025 Reviewers agreed at journal 22 Apr, 2025 Reviewers invited by journal 14 Apr, 2025 Editor assigned by journal 09 Apr, 2025 Submission checks completed at journal 08 Apr, 2025 First submitted to journal 07 Apr, 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. 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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-6396471","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":446535803,"identity":"3c873066-d623-4803-b0c2-26b4880e6ebf","order_by":0,"name":"Yao Li","email":"","orcid":"","institution":"Jinhua Maternity and Child Health Care Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yao","middleName":"","lastName":"Li","suffix":""},{"id":446535804,"identity":"94108aea-29ca-4de2-8494-8d7d536ac512","order_by":1,"name":"Lin Zhang","email":"","orcid":"","institution":"Jinhua Maternity and Child Health Care Hospital","correspondingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Zhang","suffix":""},{"id":446535805,"identity":"d5f50f5a-686b-46cb-b6bb-774f6afd81c8","order_by":2,"name":"Hongqing Lv","email":"","orcid":"","institution":"Jinhua Maternity and Child Health Care Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hongqing","middleName":"","lastName":"Lv","suffix":""},{"id":446535806,"identity":"b5a2047f-84c5-4ce0-bf3e-87b796c2a3bb","order_by":3,"name":"Chenxuan Mei","email":"","orcid":"","institution":"Jinhua Municipal Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chenxuan","middleName":"","lastName":"Mei","suffix":""},{"id":446535807,"identity":"9d116e05-3728-484d-8dcc-2af27604a6a3","order_by":4,"name":"Min Hu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYBACAyA+wFAhIcfP3nzwQUJFDbFazlgYS/YcSzZ4cOYYcVoYGFsqEjfc8DGTfNjCTFiLuUR24uHCBgljyRlsaRWJDWwM/O3dCXi1WM7I3XB45g6gX6Sbj91I3CHDIHHm7Ab8DrsB1MJ7BmjLnGNpNxLPsDEYSOQSo6VNAuiXHLOCxDZmErUwEKflzNsNh3lADgMGskTCmWM8hP1yPHfzZ56KOnBUfvxRUSPH396LXwsG4CFN+SgYBaNgFIwCrAAAaGxR1hvQEekAAAAASUVORK5CYII=","orcid":"","institution":"Jinhua Maternity and Child Health Care Hospital","correspondingAuthor":true,"prefix":"","firstName":"Min","middleName":"","lastName":"Hu","suffix":""}],"badges":[],"createdAt":"2025-04-07 17:23:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6396471/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6396471/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13048-025-01736-w","type":"published","date":"2025-07-12T15:57:17+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82097952,"identity":"458c11fa-db3b-4ddb-a5b0-7195f1f3c404","added_by":"auto","created_at":"2025-05-06 17:59:32","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":94483,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCTX induces dose-dependent ovarian cytotoxicity by inhibiting cell proliferation, increasing apoptosis, and altering the expression of ovarian regulatory proteins.\u003c/strong\u003e (A) Cell proliferation rate in ovarian granulosa cells treated with increasing concentrations of cyclophosphamide (CTX: 0–7 mM) for 24 hours, measured by CCK-8 assay. CTX inhibited cell proliferation in a dose-dependent manner. (B) TUNEL staining of ovarian cells treated with 0.5, 1.0, and 1.5 mM CTX. DAPI staining was used to label nuclei. CTX treatment increased the number of TUNEL-positive apoptotic cells in a concentration-dependent manner. (C) Western blot analysis of TGF-β, FSH, and ER protein levels in cells treated with different concentrations of CTX. GAPDH was used as a loading control. (D–F) Quantitative analysis of TGF-β (D), FSH (E), and ER. (F) protein expression levels based on Western blot data. Data are presented as mean ± SEM (n = 3). **p \u0026lt; 0.01 vs. control group, \u003csup\u003e##\u003c/sup\u003ep \u0026lt; 0.01 vs. CTX group.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6396471/v1/44684f4bcecd3ac5f60396ce.jpeg"},{"id":82099600,"identity":"48ad7390-1b9b-48d4-b142-a3665c8207b1","added_by":"auto","created_at":"2025-05-06 18:23:32","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":136893,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtective effects of BFGF against CTX-induced cell damage.\u003c/strong\u003e (A) Cell viability analysis after pretreatment with different concentrations of BFGF (5-35 nM) followed by CTX exposure. Data are presented as percentage mean ± SEM (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ****p \u0026lt; 0.0001). (B) TUNEL assay showing the effects of BFGF on CTX-induced apoptosis. DAPI staining shows nuclei, and green fluorescence indicates TUNEL-positive apoptotic cells. Low (L, 5nM), medium (M, 10nM), and high (H, 20nM) doses of BFGF reduced apoptosis, while the BFGF inhibitor increased apoptosis. (C) JC-1 staining showing changes in mitochondrial membrane potential. Green fluorescence represents JC-1 monomers, and red fluorescence represents JC-1 aggregates. BFGF restored mitochondrial membrane potential, while the BFGF inhibitor exacerbated mitochondrial damage. Low (L, 5nM), medium (M, 10nM), and high (H, 20nM).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6396471/v1/f60a4a377357ab719cd8a1c4.jpeg"},{"id":82099601,"identity":"1b820161-5581-446c-83a6-2eda620f617a","added_by":"auto","created_at":"2025-05-06 18:23:32","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":73770,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptomic analysis results.\u003c/strong\u003e (A) Volcano plot showing the differential gene expression analysis results. Red and blue dots represent significantly upregulated and downregulated differentially expressed genes (DEGs), respectively, while gray dots indicate genes with no significant changes. (B) Heatmap showing the expression patterns of significantly differentially expressed genes across two sample groups. The color intensity indicates gene expression levels (red for high expression, blue for low expression). (C) Bubble plot of KEGG pathway enrichment analysis for the differentially expressed genes. (D) Bubble plot of GO functional enrichment analysis for the differentially expressed genes, including three categories: biological processes (BP), cellular components (CC), and molecular functions (MF). The color and size of the bubbles represent p-values and the number of differentially expressed genes, respectively.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6396471/v1/c4343243fa916361fac4eb1c.jpeg"},{"id":82098221,"identity":"96880a0f-a147-4df7-941e-1d8c726bec2a","added_by":"auto","created_at":"2025-05-06 18:07:32","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":115542,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRegulation of key proteins and genes by BFGF under CTX-induced cell damage. \u003c/strong\u003e(A) Western blot analysis of SERPINE1, HIF-1α, Cyclin D1, and CDK4 protein levels, with β-actin as a loading control. (B-E) Quantification of SERPINE1 (B), HIF-1α (C), Cyclin D1 (D), and CDK4 (E) protein expression levels. Data are presented as mean ± SEM (*p \u0026lt; 0.05, **p \u0026lt; 0.01 vs. control group; #p \u0026lt; 0.05, ##p \u0026lt; 0.01 vs. CTX+BFGF group). (F-I) qRT-PCR analysis of SERPINE1 (F), HIF-1α (G), Cyclin D1 (H), and CDK4 (I) mRNA expression levels. BFGF significantly increased protein and mRNA levels of these genes, while the BFGF inhibitor suppressed these effects.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6396471/v1/490d88256458dd231bfc57d2.jpeg"},{"id":82097948,"identity":"c642b094-98bf-40cc-9c07-d9c7f0b8b682","added_by":"auto","created_at":"2025-05-06 17:59:32","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":91826,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSERPINE1 mediates the protective effects of BFGF against CTX-induced cell damage. \u003c/strong\u003e(A) Cell growth curves showing proliferation rates under different treatment conditions. CTX treatment suppressed cell growth, which was restored by BFGF or SERPINE1 overexpression. SERPINE1 inhibition further suppressed cell proliferation. (B) Colony formation assay showing the effects of BFGF and SERPINE1 on cell proliferative capacity. Both BFGF and SERPINE1 overexpression enhanced colony formation, while SERPINE1 inhibition reduced colony numbers. (C) Western blot analysis of SERPINE1, HIF-1α, Cyclin D1, and CDK4 protein levels, with β-actin as a loading control. (D-G) Quantification of SERPINE1 (D), HIF-1α (E), Cyclin D1 (F), and CDK4 (G) protein levels. BFGF and SERPINE1 overexpression increased protein levels, while SERPINE1 inhibition reduced their expression (**p \u0026lt; 0.01 vs. control group; #p \u0026lt; 0.05, ##p \u0026lt; 0.01 vs. CTX+BFGF group).\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6396471/v1/4e9e2182112d8ba7f1070a1e.jpeg"},{"id":82097953,"identity":"7463239c-58b1-4e08-aa5e-e664db98979c","added_by":"auto","created_at":"2025-05-06 17:59:32","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":107224,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActivation of the Nrf-2/HO-1 signaling pathway by BFGF in CTX-induced cell damage. \u003c/strong\u003e(A) Immunofluorescence staining of HO-1 showing its expression and localization under different treatments. DAPI staining shows nuclei, and green fluorescence indicates HO-1. BFGF significantly increased HO-1 expression, while the BFGF inhibitor reduced it. (B) Immunofluorescence staining of Nrf-2 showing its expression and localization. DAPI staining shows nuclei, and green fluorescence indicates Nrf-2. BFGF restored Nrf-2 expression, while the BFGF inhibitor suppressed this restoration. (C) Western blot analysis of Nrf-2 and HO-1 protein levels, with β-actin as a loading control. (D, E) Quantification of Nrf-2 (D) and HO-1 (E) protein expression levels. CTX treatment significantly reduced Nrf-2 and HO-1 levels, while BFGF restored their expression (**p \u0026lt; 0.01 vs. control group). Low (L, 5nM), and high (H, 20nM).\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6396471/v1/763499175082448e50cfad20.jpeg"},{"id":86699448,"identity":"a2665776-9f81-4220-827d-8581a3b6cd07","added_by":"auto","created_at":"2025-07-14 16:09:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1568528,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6396471/v1/e7dbce9d-5571-440d-8d9d-16bb4e3e2e9b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"BFGF Mitigates CTX-Induced Ovarian Cytotoxicity via SERPINE1/HIF-1 and Nrf-2/HO-1 Signaling Pathways","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChemotherapy is a cornerstone of modern cancer treatment, offering significant survival benefits and disease control for patients with various malignancies. However, its therapeutic effects often come at the cost of severe side effects, as most chemotherapeutic agents lack selectivity for tumor cells and inadvertently damage healthy tissues. Among these side effects, ovarian toxicity is particularly concerning for young female patients, as it results in infertility, hormonal imbalances, and premature ovarian failure[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. These complications not only affect physical health but also have profound psychological and social implications, making ovarian protection a critical aspect of survivorship care in oncology. Cyclophosphamide (CTX), a widely used alkylating agent in chemotherapy, is notorious for its high ovarian toxicity, as it directly induces apoptosis in ovarian follicles, damages stromal vascular structures, and reduces ovarian reserve[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Its mechanisms of toxicity include DNA damage, oxidative stress, and disruption of the ovarian microenvironment, leading to irreversible declines in reproductive and endocrine function[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite advancements in fertility preservation techniques, existing strategies have significant limitations and are not suitable for all patients. Hormonal suppression with gonadotropin-releasing hormone analogs (GnRHa) can reduce ovarian damage but does not fully prevent follicular loss or ensure fertility preservation[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Surgical approaches, such as oocyte and ovarian tissue cryopreservation, provide alternative options but come with risks and logistical challenges[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. For instance, ovarian tissue cryopreservation carries a potential risk of reintroducing malignant cells during transplantation, and oocyte cryopreservation requires ovarian stimulation, which can delay cancer treatment and is unsuitable for patients with estrogen-sensitive tumors[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Moreover, both methods are costly and not universally accessible. These challenges highlight the urgent need for novel, non-invasive interventions that can protect ovarian function during chemotherapy while being safe and readily implementable.\u003c/p\u003e \u003cp\u003eBasic fibroblast growth factor (BFGF) has emerged as a promising candidate for addressing chemotherapy-induced ovarian toxicity. A member of the fibroblast growth factor family, BFGF is a multifunctional growth factor known for its roles in angiogenesis, tissue repair, and cellular proliferation[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In the context of ovarian biology, BFGF has been shown to promote follicular growth, enhance granulosa cell proliferation, and stimulate estradiol production[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Furthermore, studies have demonstrated that BFGF can mitigate tissue damage in other organ systems, such as the heart and skeletal muscle, by promoting vascular repair and reducing fibrosis[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]]. Importantly, BFGF has been reported to improve ovarian function in models of age-related dysfunction, suggesting its potential for reversing ovarian damage caused by chemotherapy[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Its ability to regulate key molecular pathways involved in stress response and tissue regeneration makes BFGF a compelling candidate for ovarian protection.\u003c/p\u003e \u003cp\u003eBy expanding on these observations, the present study aims to comprehensively investigate the role of BFGF in ameliorating CTX-induced ovarian cytotoxicity and to elucidate the molecular mechanisms underlying its protective effects. Through in vitro and in vivo experiments, we explore how BFGF activates the SERPINE1/HIF-1 signaling pathway to promote ovarian repair and improve overall reproductive health. This research seeks to provide a scientific basis for the development of BFGF-based therapeutic strategies to preserve ovarian function and fertility in female cancer patients, addressing a critical unmet need in the field of oncofertility.\u003c/p\u003e \u003cp\u003eIn this study, we hypothesized that BFGF mitigates CTX-induced ovarian damage by modulating key molecular pathways involved in cell survival and angiogenesis. Our preliminary findings demonstrated that BFGF treatment improved ovarian structure and function in a CTX-induced ovarian insufficiency model. Specifically, BFGF upregulated the expression of SERPINE1 and HIF-1, two critical components of a signaling pathway known to regulate cellular responses to stress, promote angiogenesis, and enhance tissue repair. In addition, BFGF reduced apoptosis and restored mitochondrial function, further supporting its protective role.\u003c/p\u003e \u003cp\u003eBuilding on these observations, the present study investigates the role of the SERPINE1/HIF-1 signaling pathway in mediating the protective effects of BFGF against CTX-induced ovarian cytotoxicity. We conducted a series of in vitro and in vivo experiments to explore the molecular mechanisms underlying BFGF's actions and to evaluate its potential as a therapeutic agent for preserving ovarian function and fertility in women undergoing chemotherapy. By elucidating the mechanisms of BFGF action, this research aims to provide a foundation for the development of targeted therapies to improve the quality of life for cancer survivors.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials and Reagents\u003c/h2\u003e \u003cp\u003eDMEM/F12 Medium: Gibco, Thermo Fisher Scientific (Catalog No. 11330032);Fetal Bovine Serum (FBS): Gibco, Thermo Fisher Scientific (Catalog No. 10099141)༛Penicillin-Streptomycin: Gibco, Thermo Fisher Scientific (Catalog No. 15140122)༛Cyclophosphamide (CTX): Sigma-Aldrich (Catalog No. C0768)༛Basic Fibroblast Growth Factor (BFGF): PeproTech (Catalog No. 100-18B)༛BFGF Inhibitor: R\u0026amp;D Systems (Catalog No. 7183-FB)༛CCK-8 Cell Viability Assay Kit: Dojindo Laboratories (Catalog No. CK04)༛Annexin V-FITC/PI Apoptosis Detection Kit: BD Biosciences (Catalog No. 556547)༛JC-1 Mitochondrial Membrane Potential Assay Kit: Abcam (Catalog No. ab113850)༛TUNEL Assay Kit: Roche (Catalog No. 11684795910)༛RIPA Lysis Buffer: Thermo Fisher Scientific (Catalog No. 89901)༛Protease and Phosphatase Inhibitor Cocktail: Sigma-Aldrich (Catalog No. P5726)༛PVDF Membranes: Millipore (Catalog No. IPVH00010)༛Enhanced Chemiluminescence (ECL) Detection Kit: Thermo Fisher Scientific (Catalog No. 32106)༛SERPINE1 Antibody: Abcam (Catalog No. ab125687)༛HIF-1α Antibody: Cell Signaling Technology (Catalog No. 3716)༛Nrf-2 Antibody: Abcam (Catalog No. ab137550)༛HO-1 Antibody: Cell Signaling Technology (Catalog No. 43966)༛Cyclin D1 Antibody: Cell Signaling Technology (Catalog No. 2978)༛CDK4 Antibody: Cell Signaling Technology (Catalog No. 12790)༛TGF-β Antibody: Abcam (Catalog No. ab92486)༛FSH Antibody: Abcam (Catalog No. ab151564)༛ER Antibody: Abcam (Catalog No. ab16660)༛β-actin Antibody: Cell Signaling Technology (Catalog No. 4970)༛HRP-Conjugated Secondary Antibodies: Cell Signaling Technology (Catalog No. 7074 for anti-rabbit, 7076 for anti-mouse)༛TRIzol Reagent: Thermo Fisher Scientific (Catalog No. 15596026)༛cDNA Synthesis Kit: Thermo Fisher Scientific (Catalog No. 4368814)༛SYBR Green PCR Master Mix: Thermo Fisher Scientific (Catalog No. A25742)༛Triton X-100: Sigma-Aldrich (Catalog No. T8787)༛Bovine Serum Albumin (BSA): Sigma-Aldrich (Catalog No. A9418)༛DAPI Stain: Thermo Fisher Scientific (Catalog No. D1306)༛Fluorescent Secondary Antibodies: Thermo Fisher Scientific (Catalog Nos. A-11001 and A-11008)༛Illumina TruSeq RNA Sample Preparation Kit: Illumina (Catalog No. RS-122-2001)༛Agilent RNA 6000 Nano Kit: Agilent Technologies (Catalog No. 5067\u0026thinsp;\u0026minus;\u0026thinsp;1511)。\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell Culture and Treatment\u003c/h3\u003e\n\u003cp\u003eOvarian granulosa cells were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a humidified atmosphere containing 5% CO₂ at 37\u0026deg;C. Cells were divided into the following groups: (1) control group, (2) CTX group (treated with cyclophosphamide at a concentration of 50 \u0026micro;M), (3) CTX\u0026thinsp;+\u0026thinsp;BFGF group (co-treated with 50 \u0026micro;M CTX and 10 ng/mL BFGF), (4) CTX\u0026thinsp;+\u0026thinsp;BFGF inhibitor group, (5) CTX\u0026thinsp;+\u0026thinsp;SERPINE1 overexpression group, and (6) CTX\u0026thinsp;+\u0026thinsp;SERPINE1 inhibitor group. Treatment durations were set according to experimental requirements, ranging from 24 to 120 hours.\u003c/p\u003e\n\u003ch3\u003eCell Viability Assay\u003c/h3\u003e\n\u003cp\u003eCell viability was assessed using the CCK-8 assay. Briefly, cells were seeded into 96-well plates at a density of 5 \u0026times; 10\u0026sup3; cells per well and treated according to their respective groups. After incubation, 10 \u0026micro;L of CCK-8 reagent was added to each well, and plates were incubated for an additional 2 hours at 37\u0026deg;C. Absorbance was measured at 450 nm using a microplate reader, and cell viability was calculated as a percentage relative to the control group.\u003c/p\u003e\n\u003ch3\u003eApoptosis Analysis\u003c/h3\u003e\n\u003cp\u003eApoptosis was analyzed using flow cytometry and TUNEL staining. For flow cytometry, cells were harvested, washed with PBS, and stained with Annexin V-FITC and PI for 15 minutes at room temperature. Apoptotic cells were quantified using a flow cytometer. For TUNEL staining, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and incubated with the TUNEL reaction mixture. Stained cells were observed under a fluorescence microscope, and apoptotic cells were quantified.\u003c/p\u003e\n\u003ch3\u003eMitochondrial Membrane Potential (MMP) Analysis\u003c/h3\u003e\n\u003cp\u003eMMP was assessed using JC-1 staining. Cells were incubated with JC-1 dye (5 \u0026micro;g/mL) at 37\u0026deg;C for 20 minutes, washed with PBS, and observed under a fluorescence microscope. The ratio of red fluorescence (JC-1 aggregates, indicating intact MMP) to green fluorescence (JC-1 monomers, indicating depolarized MMP) was calculated to assess mitochondrial health.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptomic Sequencing and Analysis\u003c/h2\u003e \u003cp\u003eTo investigate the molecular mechanisms underlying the protective effects of BFGF against CTX-induced ovarian damage, transcriptomic sequencing was performed on ovarian cells treated with CTX alone and CTX\u0026thinsp;+\u0026thinsp;BFGF. Total RNA was extracted from the samples using the TRIzol reagent, and RNA integrity was assessed using an Agilent 2100 Bioanalyzer. RNA with an RNA Integrity Number (RIN)\u0026thinsp;\u0026gt;\u0026thinsp;7.0 was used for sequencing. RNA-seq libraries were prepared using the Illumina TruSeq RNA Sample Preparation Kit, following the manufacturer\u0026rsquo;s protocol. Sequencing was performed on the Illumina HiSeq 2500 platform, generating paired-end reads of 150 bp. Raw sequencing data were processed to remove low-quality reads and adapters using FastQC and Trimmomatic. High-quality clean reads were aligned to the reference human genome (GRCh38) using HISAT2. Differentially expressed genes (DEGs) were identified using DESeq2 with |log2 fold change| \u0026gt; 1 and p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 as thresholds. Volcano plots were generated to visualize upregulated and downregulated DEGs. Hierarchical clustering analysis and heatmaps were constructed to illustrate gene expression patterns among different groups. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed using the clusterProfiler package in R to identify biological processes, cellular components, molecular functions, and pathways associated with the DEGs. The HIF-1 signaling pathway and oxidative stress-related pathways were highlighted based on their significant enrichment in the BFGF-treated group. Visualization of pathway enrichment was presented using bubble plots. These analyses provided insights into the molecular pathways activated by BFGF to counteract CTX-induced ovarian damage.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWestern Blot Analysis\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eWestern Blot Analysis\u003c/div\u003e \u003cp\u003eProtein expression was analyzed by Western blot. Cells were lysed using RIPA buffer containing protease and phosphatase inhibitors. Equal amounts of protein were separated by SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked with 5% non-fat milk and incubated overnight at 4\u0026deg;C with primary antibodies against SERPINE1, HIF-1α, Nrf-2, HO-1, Cyclin D1, CDK4, TGF-β, FSH, ER, and β-actin. After washing, membranes were incubated with HRP-conjugated secondary antibodies. Protein bands were visualized using ECL reagents and quantified using ImageJ software.\u003c/p\u003e\n\u003ch3\u003eQuantitative Real-Time PCR (qRT-PCR)\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted using TRIzol reagent, and cDNA was synthesized using a reverse transcription kit. qRT-PCR was performed using SYBR Green PCR Master Mix on a real-time PCR system. Primers for SERPINE1, HIF-1α, Nrf-2, HO-1, Cyclin D1, and CDK4 were designed and validated. Relative gene expression was calculated using the 2^\u0026minus;ΔΔCt method, with β-actin as the internal control.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eColony Formation Assay\u003c/h2\u003e \u003cp\u003eCells were seeded into 6-well plates at a density of 500 cells per well and treated according to their respective groups. After 10 days of incubation, colonies were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. Colonies containing more than 50 cells were counted manually under a microscope.\u003c/p\u003e \u003cp\u003eImmunofluorescence Staining\u003c/p\u003e \u003cp\u003eCells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% BSA. Primary antibodies against Nrf-2 and HO-1 were applied overnight at 4\u0026deg;C. After washing, cells were incubated with fluorescently labeled secondary antibodies and counterstained with DAPI. Images were captured using a fluorescence microscope, and fluorescence intensity was quantified using ImageJ.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptomic Analysis\u003c/h2\u003e \u003cp\u003eTotal RNA from CTX and CTX\u0026thinsp;+\u0026thinsp;BFGF groups was extracted and subjected to high-throughput RNA sequencing. Differentially expressed genes (DEGs) were identified based on |log2 fold change| \u0026gt; 1 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. KEGG pathway enrichment and GO functional analyses were performed to identify enriched biological pathways and processes. Heatmaps and volcano plots were generated to visualize expression profiles and pathway activation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll experiments were performed in triplicate, and data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistical analysis was conducted using GraphPad Prism. Differences between groups were analyzed using one-way ANOVA followed by Tukey\u0026rsquo;s post hoc test. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eBFGF mitigates CTX-induced ovarian cytotoxicity by improving cell viability and reducing apoptosis\u003c/h2\u003e \u003cp\u003eTo investigate the protective effects of BFGF against CTX-induced ovarian damage, we assessed cell viability, nuclear morphology, apoptotic rates, and the expression of regulatory proteins. The cell viability assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) showed a significant dose-dependent reduction in proliferation in the CTX-treated group, consistent with the cytotoxic effects of CTX on ovarian cells. In contrast, BFGF co-treatment significantly improved cell viability, with higher concentrations producing more pronounced protective effects. To further explore the impact of BFGF, nuclear morphology was analyzed using DAPI staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). CTX treatment induced typical apoptotic features, including nuclear condensation and chromatin aggregation. These changes were significantly mitigated in the BFGF-treated group, suggesting that BFGF reduces CTX-induced apoptosis.\u003c/p\u003e \u003cp\u003eAdditionally, Western blot analysis was performed to examine the expression of apoptosis-related regulatory proteins, including TGF-β, FSH, and ER (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-F). CTX treatment resulted in a notable disruption in the expression levels of these proteins, while BFGF treatment restored their levels closer to those of the control group. These findings indicate that BFGF effectively modulates key ovarian signaling pathways to counteract the effects of CTX. These results demonstrate that BFGF mitigates CTX-induced ovarian cytotoxicity by enhancing cell viability, reducing apoptosis, and regulating the expression of critical ovarian regulatory proteins, highlighting its potential role as a therapeutic agent to preserve ovarian function during chemotherapy.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eBFGF reduces CTX-induced apoptosis by stabilizing mitochondrial membrane potential\u003c/h2\u003e \u003cp\u003eFollowing the observation that BFGF enhances cell viability and reduces apoptosis in CTX-treated ovarian cells, we further investigated its effects on mitochondrial function, a key regulator of apoptosis. Mitochondrial membrane potential (MMP) was assessed using JC-1 staining, which differentiates between polarized (red fluorescence) and depolarized (green fluorescence) mitochondria. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, CTX treatment significantly increased green fluorescence intensity, indicating a marked loss of MMP and mitochondrial dysfunction. In contrast, BFGF treatment restored MMP in a dose-dependent manner, as evidenced by a shift toward increased red fluorescence, suggesting improved mitochondrial stability[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo confirm the anti-apoptotic effects of BFGF, TUNEL staining was performed to visualize DNA fragmentation, a hallmark of late-stage apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). CTX treatment resulted in a significant increase in TUNEL-positive cells, indicative of extensive apoptotic damage. However, BFGF treatment dramatically reduced the number of TUNEL-positive cells, further demonstrating its protective effect against CTX-induced apoptosis. Notably, the protective effect of BFGF was partially inhibited by the addition of a BFGF antagonist, confirming the specificity of its action.\u003c/p\u003e \u003cp\u003eThese results indicate that BFGF protects ovarian cells from CTX-induced apoptosis by stabilizing mitochondrial function and preventing downstream apoptotic signaling. This suggests that the anti-apoptotic effects of BFGF are closely associated with its ability to maintain mitochondrial integrity, reinforcing its therapeutic potential in mitigating chemotherapy-induced ovarian damage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eBFGF activates the SERPINE1/HIF-1 signaling pathway to mitigate CTX-induced ovarian damage\u003c/h2\u003e \u003cp\u003eTo further explore the mechanisms underlying the protective effects of BFGF, transcriptomic analysis was performed to identify differentially expressed genes (DEGs) in ovarian cells treated with CTX and CTX\u0026thinsp;+\u0026thinsp;BFGF (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The volcano plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) revealed significant differences in gene expression between the two groups, with several genes upregulated or downregulated in response to BFGF treatment. Heatmap analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) demonstrated distinct clustering of DEGs, indicating that BFGF treatment substantially altered the gene expression profile in CTX-treated ovarian cells. KEGG pathway enrichment analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) highlighted the HIF-1 signaling pathway as one of the most significantly enriched pathways in the BFGF-treated group, suggesting its involvement in mediating the protective effects of BFGF. Additionally, GO functional enrichment analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) revealed that BFGF influenced processes related to apoptosis regulation and cellular survival, further supporting its role in mitigating CTX-induced ovarian cytotoxicity.\u003c/p\u003e \u003cp\u003eTo validate these findings, we assessed the expression of key proteins involved in the SERPINE1/HIF-1 pathway. Western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) showed that CTX treatment significantly downregulated SERPINE1 and HIF-1α protein levels, whereas BFGF treatment restored their expression to near-normal levels. The protective effects of BFGF were further confirmed through qRT-PCR analysis (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG), which revealed a similar pattern of mRNA expression. Importantly, the addition of a BFGF inhibitor attenuated these effects, indicating the specificity of BFGF in activating the SERPINE1/HIF-1 pathway.\u003c/p\u003e \u003cp\u003eFurthermore, we examined the downstream effects of BFGF-mediated activation of this pathway on cell cycle regulation. Western blot results (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) indicated that CTX treatment reduced the expression of Cyclin D1 and CDK4, key regulators of cell cycle progression, while BFGF treatment significantly restored their expression levels. This was further supported by qRT-PCR analysis (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI), which showed a consistent increase in Cyclin D1 and CDK4 mRNA expression following BFGF treatment. Notably, the BFGF inhibitor reversed these effects, underscoring the critical role of the SERPINE1/HIF-1 signaling pathway in mediating BFGF\u0026rsquo;s protective effects.\u003c/p\u003e \u003cp\u003eThese findings demonstrate that BFGF alleviates CTX-induced ovarian damage by activating the SERPINE1/HIF-1 signaling pathway, restoring cell cycle regulation, and enhancing ovarian cell survival. These results highlight the therapeutic potential of BFGF in protecting ovarian function during chemotherapy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eBFGF exerts its protective effects through the activation of SERPINE1\u003c/h2\u003e \u003cp\u003eTo further confirm the role of SERPINE1 in BFGF-mediated protection against CTX-induced ovarian damage, we evaluated cell growth, colony formation, and the expression of key signaling proteins under conditions of SERPINE1 overexpression (OE) and inhibition. Cell growth analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) revealed that CTX significantly suppressed cell proliferation compared to the control group. However, treatment with BFGF or SERPINE1 overexpression restored cell growth to near-normal levels, demonstrating a strong protective effect. In contrast, the addition of a SERPINE1 inhibitor abrogated this protective effect, further confirming the involvement of SERPINE1 in mediating BFGF's actions. Colony formation assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) supported these findings, showing that CTX-treated cells exhibited a marked reduction in colony-forming ability. This impairment was reversed by both BFGF treatment and SERPINE1 overexpression, with the number of colonies significantly increasing. Conversely, SERPINE1 inhibition led to a substantial reduction in colony formation, highlighting the critical role of this protein in ovarian cell survival and proliferation.\u003c/p\u003e \u003cp\u003eWestern blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) demonstrated that CTX reduced the expression of SERPINE1 and its downstream targets, including HIF-1α, Cyclin D1, and CDK4. BFGF treatment or SERPINE1 overexpression restored the expression levels of these proteins, while SERPINE1 inhibition reversed these effects. Quantitative analysis of SERPINE1 and HIF-1α expression (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD\u0026ndash;E) confirmed these trends, indicating that the activation of SERPINE1 is essential for BFGF's protective mechanism. Additionally, the expression levels of Cyclin D1 and CDK4, key regulators of cell cycle progression, were significantly upregulated by BFGF and SERPINE1 overexpression, as shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF\u0026ndash;G. In contrast, SERPINE1 inhibition suppressed their expression, further supporting the role of SERPINE1 in promoting cell survival and proliferation.\u003c/p\u003e \u003cp\u003eThe results demonstrate that the protective effects of BFGF against CTX-induced ovarian cytotoxicity are mediated through the activation of SERPINE1, which regulates key downstream targets such as HIF-1α, Cyclin D1, and CDK4. This highlights the pivotal role of the SERPINE1 signaling axis in preserving ovarian function during chemotherapy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eBFGF activates the Nrf-2/HO-1 signaling pathway to protect against CTX-induced oxidative damage\u003c/h2\u003e \u003cp\u003eTo investigate whether the Nrf-2/HO-1 signaling pathway contributes to the protective effects of BFGF against CTX-induced ovarian damage, we examined the expression and localization of Nrf-2 and HO-1 using immunofluorescence and Western blot analyses. Immunofluorescence staining revealed that CTX treatment significantly decreased the nuclear localization and fluorescence intensity of Nrf-2 and HO-1, indicative of impaired oxidative stress response (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u0026ndash;B). In contrast, BFGF treatment restored the nuclear localization and expression of both proteins, suggesting that BFGF activates the Nrf-2/HO-1 signaling pathway. Notably, the addition of a BFGF inhibitor attenuated these effects, confirming the specificity of BFGF's action.\u003c/p\u003e \u003cp\u003eWestern blot analysis further corroborated these findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). CTX treatment led to a marked reduction in Nrf-2 and HO-1 protein expression compared to the control group. However, BFGF treatment significantly upregulated the expression of both proteins, restoring their levels closer to normal. Quantitative analysis (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD\u0026ndash;E) demonstrated that BFGF increased the relative expression of Nrf-2 and HO-1, while the presence of a BFGF inhibitor reversed these effects, further highlighting the importance of this pathway in mediating BFGF\u0026rsquo;s protective effects.\u003c/p\u003e \u003cp\u003eThese results indicate that BFGF protects ovarian cells from CTX-induced oxidative damage by activating the Nrf-2/HO-1 signaling pathway. By promoting the expression and activity of Nrf-2 and HO-1, BFGF enhances the cellular defense against oxidative stress, thereby mitigating mitochondrial dysfunction and reducing apoptosis. This underscores the critical role of oxidative stress regulation in the therapeutic potential of BFGF for preserving ovarian function during chemotherapy.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eChemotherapy-induced ovarian toxicity remains a significant challenge in the treatment of female cancer patients, particularly those of reproductive age. CTX, a widely used chemotherapeutic agent, is effective against tumors but causes severe damage to ovarian cells, resulting in reduced ovarian reserve, impaired reproductive function, and premature ovarian failure[\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In this study, we demonstrate that BFGF effectively protects against CTX-induced ovarian damage through multiple mechanisms, highlighting its therapeutic potential for ovarian preservation.\u003c/p\u003e \u003cp\u003eOur findings indicate that BFGF enhances cell viability and reduces apoptosis in ovarian cells exposed to CTX. These effects are associated with the stabilization of mitochondrial membrane potential, a key determinant of cell survival. Mitochondrial dysfunction is a central feature of chemotherapy-induced cell damage, and BFGF\u0026rsquo;s ability to maintain mitochondrial stability suggests its direct role in preventing CTX-induced apoptosis[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. By preserving mitochondrial function, BFGF not only promotes cell survival but also supports the maintenance of the ovarian microenvironment, which is essential for normal ovarian function and follicular development.\u003c/p\u003e \u003cp\u003eFurther mechanistic studies revealed that BFGF activates the SERPINE1/HIF-1 signaling pathway, which plays a pivotal role in cellular response to stress, angiogenesis, and tissue repair[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The upregulation of SERPINE1 and HIF-1α by BFGF suggests that this pathway mediates its protective effects on ovarian cells. Functional analyses confirmed that activation of this pathway restores cell proliferation and reduces apoptosis, emphasizing the critical role of SERPINE1 in BFGF-mediated ovarian protection. These results highlight the therapeutic potential of targeting the SERPINE1/HIF-1 axis to counteract ovarian damage caused by chemotherapy.\u003c/p\u003e \u003cp\u003eIn addition, BFGF was found to activate the Nrf-2/HO-1 signaling pathway, which regulates cellular defense against oxidative stress. Oxidative stress is a major contributor to CTX-induced ovarian damage, leading to increased apoptosis and mitochondrial dysfunction. By promoting the nuclear localization of Nrf-2 and upregulating HO-1 expression[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], BFGF enhances the antioxidative capacity of ovarian cells, mitigating oxidative damage and preserving cell viability. This dual role of BFGF in regulating oxidative stress and angiogenesis underscores its multifaceted nature and therapeutic potential.\u003c/p\u003e \u003cp\u003eImportantly, the addition of inhibitors targeting SERPINE1 or BFGF significantly attenuated its protective effects, further validating the specificity of these signaling pathways in mediating BFGF's actions. These findings suggest that the SERPINE1/HIF-1 and Nrf-2/HO-1 pathways are critical mediators of ovarian protection and represent promising therapeutic targets for mitigating chemotherapy-induced ovarian damage.\u003c/p\u003e \u003cp\u003eAlthough this study provides compelling evidence for the efficacy of BFGF in protecting ovarian cells, further research is needed to address its clinical applicability. Future studies should focus on optimizing the dosing, delivery methods, and long-term safety of BFGF. Additionally, combining BFGF with existing ovarian protection strategies, such as hormonal therapies or antioxidants, may enhance its effectiveness and broaden its clinical use.\u003c/p\u003e \u003cp\u003eIn conclusion, this study demonstrates that BFGF mitigates CTX-induced ovarian damage by activating the SERPINE1/HIF-1 and Nrf-2/HO-1 signaling pathways, reducing apoptosis, and enhancing cell survival. These findings provide a strong foundation for the development of BFGF-based therapeutic approaches to preserve ovarian function and fertility in female cancer patients undergoing chemotherapy, addressing a critical unmet need in oncofertility care.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was partly supported by Key Science and Technology Plan Project of Jinhua City (2023-3-143).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eY.L was primarily responsible for the design and execution of the experiments, data analysis, and original draft preparation. L.Z contributed to data curation, visualization, and manuscript revision. H.L provided experimental resources, assisted in data acquisition, and coordinated laboratory activities. C.M conducted the transcriptomic sequencing and performed bioinformatics analyses. M. H conceived and supervised the overall project, acquired funding, and critically revised the manuscript for important intellectual content. All authors read and approved the final version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCho H-W, Lee S, Min K-J, Hong JH, Song JY, Lee JK, Lee NW, Kim T. Advances in the treatment and prevention of chemotherapy-induced ovarian toxicity. Int J Mol Sci. 2020;21(20):7792.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZohni K, Lopez L, Mander P, Szaraz P, Filice M, Wyse BA, Garcia M, Gat I, Glass K, Gauthier-Fisher A. 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Antioxidants. 2022;11(4):782.\u003c/span\u003e\u003c/li\u003e\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":"journal-of-ovarian-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jovr","sideBox":"Learn more about [Journal of Ovarian Research](http://ovarianresearch.biomedcentral.com)","snPcode":"13048","submissionUrl":"https://submission.nature.com/new-submission/13048/3","title":"Journal of Ovarian Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Chemotherapy-induced ovarian damage, Basic fibroblast growth factor, SERPINE1/HIF-1 signaling pathway, Nrf-2/HO-1 pathway","lastPublishedDoi":"10.21203/rs.3.rs-6396471/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6396471/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChemotherapy-induced ovarian damage is a significant concern for female cancer patients, often resulting in infertility, hormonal imbalance, and premature ovarian failure. Cyclophosphamide (CTX), a widely used chemotherapeutic agent, is highly effective against tumors but causes severe ovarian cytotoxicity. This study explores the protective effects of basic fibroblast growth factor (BFGF) and its underlying mechanisms in mitigating CTX-induced ovarian damage. BFGF treatment significantly enhanced cell viability, reduced apoptosis, and restored mitochondrial membrane potential in CTX-treated ovarian cells. Transcriptomic analysis revealed that BFGF activated the SERPINE1/HIF-1 signaling pathway, promoting angiogenesis, reducing apoptotic signaling, and enhancing cell cycle progression by upregulating Cyclin D1 and CDK4. Additionally, BFGF activated the Nrf-2/HO-1 pathway, boosting cellular defenses against oxidative stress and mitigating mitochondrial dysfunction. Functional studies confirmed that the inhibition of SERPINE1 or BFGF abrogated these protective effects, underscoring the critical roles of these pathways. These findings demonstrate that BFGF protects ovarian cells from CTX-induced damage by modulating the SERPINE1/HIF-1 and Nrf-2/HO-1 pathways, reducing apoptosis, and enhancing cell survival, providing a promising foundation for developing BFGF-based therapies to preserve ovarian function and fertility in female cancer patients undergoing chemotherapy.\u003c/p\u003e","manuscriptTitle":"BFGF Mitigates CTX-Induced Ovarian Cytotoxicity via SERPINE1/HIF-1 and Nrf-2/HO-1 Signaling Pathways","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-06 17:59:28","doi":"10.21203/rs.3.rs-6396471/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-26T13:51:03+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-14T12:42:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"129471753540531394069528843434960266894","date":"2025-05-12T04:31:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-22T19:32:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"337590607873845587622081177211681828716","date":"2025-04-22T17:53:08+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-15T03:19:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-09T14:34:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-08T14:39:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Ovarian Research","date":"2025-04-07T17:12:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-ovarian-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jovr","sideBox":"Learn more about [Journal of Ovarian Research](http://ovarianresearch.biomedcentral.com)","snPcode":"13048","submissionUrl":"https://submission.nature.com/new-submission/13048/3","title":"Journal of Ovarian Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9662f7c6-0a58-4680-8c83-b9ffbbf47754","owner":[],"postedDate":"May 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-14T16:04:41+00:00","versionOfRecord":{"articleIdentity":"rs-6396471","link":"https://doi.org/10.1186/s13048-025-01736-w","journal":{"identity":"journal-of-ovarian-research","isVorOnly":false,"title":"Journal of Ovarian Research"},"publishedOn":"2025-07-12 15:57:17","publishedOnDateReadable":"July 12th, 2025"},"versionCreatedAt":"2025-05-06 17:59:28","video":"","vorDoi":"10.1186/s13048-025-01736-w","vorDoiUrl":"https://doi.org/10.1186/s13048-025-01736-w","workflowStages":[]},"version":"v1","identity":"rs-6396471","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6396471","identity":"rs-6396471","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}