The mechanism of Ningxin-Tongyu-Zishen formula regulating proBDNF/mBDNF balance through PAI-1/tPA signaling pathway in the treatment of premature ovarian insufficiency

The mechanism of Ningxin-Tongyu-Zishen formula regulating proBDNF/mBDNF balance through PAI-1/tPA signaling pathway in the treatment of premature ovarian insufficiency | 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 The mechanism of Ningxin-Tongyu-Zishen formula regulating proBDNF/mBDNF balance through PAI-1/tPA signaling pathway in the treatment of premature ovarian insufficiency Jiawen Ma, Chaofan Zhu, Lifang Xie, Shuaiqi An, Zaiyang Zhang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6810873/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Background Premature ovarian insufficiency (POI) is a refractory gynecological endocrine disorder. Ningxin-Tongyu-Zishen formula (NTZF), developed based on the "simultaneous heart-kidney treatment" principle, demonstrates efficacy in treating POI, potentially through regulating proBDNF/mBDNF balance. This study aimed to elucidate the molecular mechanism by which NTZF treats POI via proBDNF/mBDNF modulation. Methods POI rat models were established using cyclophosphamide (CTX). The therapeutic effects of NTZF were evaluated by analyzing estrous cycles, ovarian indices, follicular development, serum sex hormone levels (FSH, E 2 , AMH), and ovarian granulosa cells (OGCs) apoptosis. Following immunofluorescence staining to localize BDNF receptors, proBDNF/mBDNF protein expression was quantified in brain and ovarian tissues. CTX's active metabolite, Phosphoramide mustard (PM), was used to induce KGN cell damage. The regulatory effect of NTZF on proBDNF/mBDNF was investigated and compared with recombinant mBDNF protein. tPA and PAI-1 was screened, and their interactions with NTZF were analyzed. mRNA and protein expression of tPA, PAI-1, and tPA-PAI-1 complexes were assessed via q-PCR and Western Blot. Results NTZF composition was characterized and shown to improve ovarian function in POI rats. Its mechanism involves correcting proBDNF/mBDNF imbalance in both brain and ovarian tissues. NTZF achieved this correction through the PAI-1/tPA signaling pathway, thereby inhibiting apoptosis in damaged KGN cells. Conclusion Our findings demonstrate that NTZF inhibits PAI-1, reduces tPA-PAI-1 complex synthesis, and enhances tPA-mediated proteolytic conversion of proBDNF to mBDNF. This restores proBDNF/mBDNF balance, suppresses OGCs apoptosis, and ultimately ameliorates POI. premature ovarian insufficiency Ningxin-Tongyu-Zishen formula proBDNF mBDNF PAI-1 tPA Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Premature ovarian insufficiency (POI) is a refractory gynecological endocrine disorder. The clinical manifestations include irregular menstruation, elevated follicle-stimulating hormone (FSH > 25 U/L), and decreased estrogen (E 2 ) levels in women under 40 years of age [ 1 ]. The incidence of POI is approximately 3.5%, with an increasing trend among younger populations [ 2 ]. Early menopausal symptoms in POI patients, such as hot flashes, night sweats, osteoporosis, and vaginal dryness, severely affect their quality of life. If untreated, POI may progress to premature ovarian failure (POF), manifesting as irreversible amenorrhea and complete loss of fertility. Due to its complex etiology [ 3 ], effective treatments for POI remain limited. Hormone replacement therapy (HRT) can regulate menstrual cycles and alleviate hypoestrogenic symptoms but fails to effectively delay ovarian functional decline [ 4 ]. Therefore, novel therapeutic strategies for POI are urgently needed. Ningxin-Tongyu-Zishen formula (NTZF) is a clinically validated prescription based on the "simultaneous heart-kidney treatment" principle, derived from Yijing Decoction in Fu Qing Zhu's Obstetrics and Gynecology and Tianwang Buxin Pill in Jiao Zhu Fu Ren Liang Fang . This formula comprises ten traditional Chinese medicinal components, including Rehmanniae Radix Praeparata (shudihuang), Testudinis Carapax et Plastrum (guiban), Codonopsis Radix (dangshen), Cuscutae Semen (tusizi), Angelicae Sinensis Radix (danggui), Ziziphi Spinosae Semen (suanzaoren), Moutan Cortex (mudanpi), Paeoniae Radix Alba (baishao), Dioscoreae Rhizoma (shanyao), and Bupleuri Radix (chaihu). Clinical studies indicate that NTZF not only significantly reduces FSH and luteinizing hormone (LH) levels in POI patients but also alleviates "heart depression" symptoms [ 5 ]. Our previous experiments confirmed that NTZF increases primordial follicle proportions, improves serum sex hormone levels, and delays ovarian aging in D-galactose-induced POI mice [ 6 ]. However, the mechanism underlying NTZF’s "simultaneous heart-kidney treatment" efficacy remains unclear from a modern scientific perspective. Brain-derived neurotrophic factor (BDNF) may mediate NTZF’s regulatory effects on the "heart-kidney". According to traditional Chinese medicine (TCM) theory, "heart depression" primarily manifests as mood and sleep disturbances, including irritability, insomnia, and dreaminess. As a neurotrophic factor in the central nervous system, BDNF not only regulates emotional states but also correlates with insomnia [ 7 ]. Notably, BDNF levels fluctuate with sex hormone variations during the menstrual cycle [ 8 ] and exhibit synchronized changes in the brain and serum [ 9 ]. Furthermore, BDNF acts as an ovarian secretory factor localized in follicular granulosa and cumulus cells, regulating follicular development via autocrine and paracrine mechanisms [ 10 ]. Recent studies highlight the distinct roles of BDNF isoforms—proBDNF and mature BDNF (mBDNF)—in cellular processes: proBDNF binding to p75 NTR induces apoptosis, whereas mBDNF binding to TrkB promotes proliferation [ 11 ]. These findings suggest that BDNF dysregulation, particularly proBDNF/mBDNF imbalance, may contribute to POI pathogenesis. This study aimed to investigate proBDNF/mBDNF balance alterations in the brain and ovaries of POI rats and identify key proteolytic enzymes modulating this balance. We further elucidate the molecular mechanism by which NTZF regulates proBDNF/mBDNF equilibrium to treat POI, thereby providing a scientific basis for its "simultaneous heart-kidney treatment" theory. Materials and methods NTZF and EV preparation NTZF is composed of ten kinds of Chinese medicinal materials, which are Rehmanniae Radix Praeparata, Testudinis Carapax et Plastrum, Codonopsis Radix, Cuscutae Semen, Angelicae Sinensis Radix, Ziziphi Spinosae Semen, Moutan Cortex, Paeoniae Radix Alba, Dioscoreae Rhizoma, and Bupleuri Radix. The above medicinal materials were purchased from the Third Affiliated Hospital of Zhejiang Chinese Medical University, and then mixed at a weight ratio of 10:4:4:4:5:4:3:4:5:2. NTZF has been confirmed by HPLC, and its main chemical constituents include adenosine, gallic acid, 5-hydroxymethyl-2-furaldehyde+, magnoflorine, caffeic acid, (+)-catechin, chlorogenic acid, paeoniflorin, ferulic acid, quercetin, isorhamnetin, and linoleic acid. The decoction of NTZF and estradiol valerate (EV, J20171038, Bayer, Germany) suspension was based on previous studies [ 6 ]. Construction of POI rat models Forty specific pathogen-free (SPF) female Sprague-Dawley (SD) rats (6–7 weeks old, 200 ± 10g) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. During the 10 days of adaptive feeding, vaginal exfoliated cells were collected daily between 8:30 − 9:30 AM. Rats with regular estrous cycles were randomly allocated into five groups (n = 8): Control, Model, EV, Low-dose NTZF (L-NTZF), and High-dose NTZF (H-NTZF). For POI model establishment, cyclophosphamide (CTX) was intraperitoneally injected at 100 mg/kg on D11 (excluding Control). From D12 to D25, non-Control groups received daily intraperitoneal injections of CTX (8mg/kg). Throughout D11-25, Control group rats were administered equivalent volumes of normal saline (NS) via the same route. Oral gavage administration commenced on D33: Control and Model groups received NS (3mL/100g), EV group received EV suspension (3.7µg/mL), while L-NTZF and H-NTZF groups received NTZF decoctions (0.47g/mL and 0.94g/mL) for 28 days. Following 12-hours fasting, rats were anesthetized with Zoletil-50 (20mg/kg). Some ovarian tissues were either fixed in 4% paraformaldehyde (PFA), and the others were stored at -80°C with serum and brain tissues. All procedures were approved by Animal Ethical and Welfare Committee of Zhejiang Chinese Medical University (IACUC-20240603-17). Supplementary Fig. 1 depicted the animal experimental protocol. Cell culture In this study, KGN cells (iCell-h298) were chosen as the subject of investigation in order to examine the mechanisms related to ovarian granulosa cells (OGCs). These cells are considered ideal for studying reproductive dysfunction and potential treatments [ 12 ]. KGN cells were cultured in DMEM/F12 (PM150312, Procell) containing 10% fetal bovine serum (FBS, 164210-50, Procell) and 0.01% penicillin-streptomycin solution (PB180120, Procell). KGN cells were subcultured once they had reached 80–90% confluence. Cell treatment Phosphoramide mustard (PM) is one of the active metabolites of CTX and the main component of ovarian toxicity [ 13 ]. Therefore, PM is used as a modeling drug in cell experiments. KGN cells were seeded in a cell culture plate for 12h, and then treated with DMEM/F12 without 10% FBS for 2h. Then, PM (HY-137316A, MCE) with final concentrations of 100, 150, 200, 250µM were added to each group. After 24h or 48h, 100µL working solution containing 10% cell counting kit-8 (CCK-8, BMU106, Abbkine) was added to each well and cultured in a cell incubator at 37°C for 1h. KGN cell viability was calculated by OD450 value to determine PM concentration and intervention duration. When PM was used for culture, NTZF (final concentrations were 0.26, 1.06, and 4.22µg/µL) was added to the medium to determine the appropriate concentration. At the same time, the final intervention concentrations of the recombinant mBDNF protein (450-02, Pepro Tech) were 0.1, 1, and 10ng/mL, while the final intervention concentrations of the PAI-1 agonist Anecortave acetate (AA, HY-116868, MCE) were 12.5, 25, 50, and 100µM to determine the appropriate concentration. Analysis of ovarian index Body mass of rats was measured prior to euthanasia. During specimen collection, bilateral ovarian weight was quantified gravimetrically. Ovarian index of rats was calculated using the following formula: Ovarian Index (%) = [Bilateral Ovarian Weight (g)/ Body Mass (g)] × 100%. Estrous cycle detection Vaginal cytology was performed in rats during D51-D60 using exfoliative cell sampling. Specimens were stained with Wright-Giemsa Stain (R20659, Yuanye). Estrous cycle phases (proestrus, estrus, metestrus, diestrus) were classified based on the relative proportions of nucleated epithelial cells, cornified epithelial cells, and leukocytes [ 14 ]. Enzyme-linked immunosorbent assay (Elisa) According to the instructions in the Elisa kit of Jiangsu Meimian Industrial Co., Ltd. (China), the serum levels of FSH (MM-70867R1), E 2 (MM-0575R1) and anti-Müllerian hormone (AMH, MM-0219R1) in rats were detected. In addition, the levels of proBDNF (JL48660) and mBDNF (JL15984) in KGN cell culture medium were detected in accordance with Shanghai Future Industry Co., Ltd. (China) specifications. Hematoxylin-eosin (HE) staining Fresh ovarian tissues were fixed overnight in 4% PFA. After dehydration, the tissues were embedded into blocks using paraffin. The tissues were sliced into 5µm pieces, which were attached to the slides for HE staining. TUNEL staining Ovarian tissue sections were dewaxed, and then incubated with 20µg/mL proteinase K solution at 37℃ for 30min. The endogenous peroxidase in the sections was inactivated by 3% H 2 O 2 , and then labeled by TUNEL cell apoptosis detection kit (C1098, Beyotime). Image J software was used to analyze the average optical density (AOD) of primordial follicles and growing follicles in ovarian tissue sections. Cell crystal violet staining After KGN cells were fixed with 4% PFA for 30min, an appropriate amount of crystal violet staining solution (C0121, Beyotime) was added and incubated in dark for 10min. After washing with PBS, the cells were photographed and recorded under an inverted microscope. Cell proliferation detection At the end of KGN cell culture, EdU was added and incubated for 3 h to label the cells. After fixation with 4% PFA for 15min, the cells were incubated with a working solution prepared according to the instructions of BeyoClick™ EdU-555 Cell Proliferation Detection Kit (C0075S, Beyotime). After adding anti-fluorescence quencher containing DAPI (MA0222, Meilunbio), the images were photographed and analyzed at 454nm and 555 nm using a fluorescence microscope. Immunofluorescence (IF) staining IF staining of ovarian tissues and KGN cells was performed using a fluorescent duplex-labeled high-sensitivity signal amplification kit (HKI0000-2S, Haoke) according to manufacturer protocols. Tissue sections were dewaxed and processed through sequential antigen retrieval, permeabilization, and endogenous peroxidase inactivation steps, followed by blocking with 3%BSA for 30min. Primary antibodies and universal secondary antibodies were applied sequentially with appropriate incubation periods. After the secondary antibody incubation, the steps starting from antigen retrieval were repeated again. DAPI was added to label the nucleus, and then anti-fluorescence quenching sealing agent was added. KGN cells were fixed in 4%PFA prior to identical staining procedures. Imaging was conducted using Leica inverted fluorescence microscope (DMi8). Primary antibody specifications are detailed in Supplementary Table 1. Quantitative real-time PCR (q-PCR) SevenFast® total RNA extraction kit for Cells (SM130, Seven, Beijing, China ) was used to isolate total RNA from KGN cells. After reverse transcription into cDNA, q-PCR was performed using the SYBR Green premix Pro Taq HS qPCR kit (AG11718) purchased from ACCURATE BIOTECHNOLOGY (HUNAN) CO., LTD (ChangSha, China). Using β-actin as an internal reference gene, 2 −△△Ct was calculated to analyze the relative mRNA expression of the target gene. The primer sequence is shown in Supplementary Table 2. Western Blot The total protein content of ovarian tissue and KGN cells was detected by BCA protein concentration assay kit (P0011, Beyotime). The protein extracts were separated by SDS-PAGE gels, and then transferred to PVDF membranes (Millipore). The PVDF membranes were blocked with 3% skim milk at room temperature for 1h, and then incubated with primary antibody overnight at 4℃. After incubation with secondary antibody at room temperature for 1h, imagings were performed using ECL developers (BMU102, Abbkine). Antibody information is shown in Supplementary Table 3. Statistical analysis The data of this study were expressed as mean ± SEM. Graphpad Prism 8.0 software was used for statistical analysis and statistical drawing. One-way analysis of variance (ANOVA) was used to compare the differences between different groups. p < 0.05 indicated that the difference was statistically significant. Result NTZF improves ovarian function in POI rats In this study, estrous cycle changes in rats were assessed by analyzing vaginal exfoliated cell morphology during the late treatment phase (D51-D60, Fig. 1 A). Notably, model group rats persistently remained in diestrus, indicating abnormal cyclicity. NTZF treatment effectively ameliorated this estrous cycle disruption in POI rats. The ovarian index, a key indicator of reproductive health [ 15 ], was significantly increased by NTZF treatment in POI rats (Fig. 1 B). Given POI's characteristic endocrine dysfunction, we measured serum levels of FSH, E 2 , and AMH. Model group rats exhibited markedly elevated FSH levels alongside reduced E 2 and AMH levels, mirroring clinical POI hormone profiles. NTZF administration reversed these abnormalities (Fig. 1 C-E), demonstrating its capacity to restore hormonal balance. To evaluate follicular development, we quantified follicular stage proportions (Fig. 1 F), with specific focus on primordial and atretic follicles (Fig. 1 G-H). The model group showed reduced primordial follicle counts and increased atresia, both of which were counteracted by NTZF treatment. TUNEL staining revealed OGCs apoptosis levels (Fig. 1 I). Both primordial and growing follicles in model rats exhibited significantly elevated apoptosis, which was mitigated by NTZF intervention (Fig. 1 J-K). In conclusion, NTZF inhibits ovarian granulosa cell apoptosis and improves ovarian function in POI rats. NTZF restores proBDNF/mBDNF homeostasis in POI rats IF analysis revealed p75 NTR and TrkB receptor localization in OGCs of primordial, growing, and mature follicles (Fig. 2 A), indicating that proBDNF and mBDNF regulate follicular maturation through receptor binding in rat ovaries. Serum proBDNF and total BDNF levels were quantified via Elisa. POI rats exhibited elevated proBDNF levels and an increased proBDNF/total BDNF ratio. Notably, high-dose NTZF treatment significantly reduced serum proBDNF levels, while both low-does NTZF and high-dose NTZF treatments decreased the proBDNF proportion relative to total BDNF (Fig. 2 B). Western Blot analysis demonstrated an elevated proBDNF/mBDNF ratio in both brain and ovarian tissues of POI rats, confirming systemic imbalance. NTZF administration effectively corrected this dysregulation (Fig. 2 C-D). Collectively, these findings demonstrate that NTZF not only reduces circulating proBDNF dominance but also restores proBDNF/mBDNF equilibrium in brain and ovarian tissues. proBDNF/mBDNF imbalance induces apoptosis in KGN cells To investigate proBDNF/mBDNF dysregulation in injured OGCs and maintain experimental consistency between in vivo and in vitro models, we utilized PM to induce cellular damage. Screening identified 150µM PM treatment for 48 hours as the optimal condition, significantly reducing KGN cell viability and altering cellular morphology (Fig. 3 A-B). Under this regimen, EdU-positive KGN cells were markedly diminished, while mRNA and protein expression of apoptotic markers (p53, p21, and bax/bcl2) were upregulated (Fig. 3 C-H). These parameters defined subsequent in vitro experimental conditions. IF analysis localized TrkB and p75 NTR receptors in KGN cells (Fig. 3 I). Damaged cells exhibited reduced TrkB fluorescence intensity and enhanced p75 NTR signal (Fig. 3 J-K), correlating with mRNA/protein expression patterns (Fig. 3 L, 3 O-P). These findings suggest predominant activation of apoptosis-related pathways in damaged KGN cells. Notably, despite elevated BDNF mRNA levels, damaged cells demonstrated increased proBDNF/mBDNF secretion ratios and heightened intracellular proBDNF/mBDNF protein expression (Fig. 3 L-N). Collectively, these results establish proBDNF/mBDNF imbalance as a critical mediator of KGN cell apoptosis. NTZF restores proBDNF/mBDNF balance to mitigate apoptosis in damaged KGN cells Five NTZF concentrations (4.22µg/µL, 1.06µg/µL, 0.26µg/µL, 0.07µg/µL, 0.02µg/µL) were tested and showed no cytotoxicity to KGN cells (Fig. 4 A). Among these, 1.06µg/µL NTZF most effectively restored cell viability in damaged KGN cells (Fig. 4 B). Recombinant mBDNF protein (1ng/mL) exhibited comparable efficacy to NTZF in rescuing viability (Fig. 4 D-E). Western Blot analysis further confirmed that NTZF corrected proBDNF/mBDNF imbalance in damaged KGN cells, mirroring the effects of recombinant mBDNF protein (Fig. 4 F). Notably, both NTZF and recombinant mBDNF interventions partially restored proliferation levels (Fig. 4 G) and significantly suppressed mRNA/protein expression of apoptotic markers (p53, p21, and bax/bcl-2) (Fig. 4 H-M). These comparative analyses validate that NTZF inhibits apoptosis in damaged KGN cells by normalizing proBDNF/mBDNF equilibrium. tPA mediates proBDNF proteolytic cleavage to mBDNF in KGN cells In damaged KGN cells, mRNA and protein expression of proteolytic enzymes regulating proBDNF/mBDNF balance—including Furin, matrix metallopeptidase2 (MMP2), and matrix metallopeptidase9 (MMP9)—were significantly upregulated, whereas tissue plasminogen activator (tPA) exhibited discordant changes: despite increased tPA mRNA levels, its protein expression decreased (Fig. 5 A-E). Given the known inhibitory interaction between plasminogen activator inhibitor-1 (PAI-1) and tPA, we further analyzed PAI-1 expression. Both PAI-1 mRNA and protein levels were markedly elevated (Fig. 5 F-G), accompanied by increased tPA-PAI-1 complex formation (Fig. 5 H). These findings suggest that PAI-1 overexpression suppresses tPA activity, potentially impairing proBDNF-to-mBDNF conversion in damaged KGN cells. NTZF restores proBDNF/mBDNF balance via PAI-1/tPA signaling to suppress apoptosis PAI-1 agonist—anecortave acetate (AA) dose-dependently reduced viability in damaged KGN cells, with 50µM AA causing significant impairment compared to the model group (Fig. 6 A). NTZF treatment reversed this viability loss (Fig. 6 B) and counteracted AA-induced proliferation inhibition (Fig. 6 C-D). Furthermore, NTZF attenuated AA-driven upregulation of apoptotic markers (p53, p21, and bax/bcl2) at both mRNA and protein levels (Fig. 6 E-J). Building on prior findings, NTZF also rescued AA-exacerbated proBDNF/mBDNF imbalance in damaged cells (Fig. 6 K). Western Blot analysis confirmed that NTZF suppresses PAI-1 expression, reduces tPA-PAI-1 complex synthesis, and restores tPA activity (Fig. 6 L-Q). Discussion POI is recorded in the classic books of gynecology in TCM as "menstruation stopped when not old yet." The kidney stores essence and dominates reproduction. And the rise and fall of kidney qi directly affects ovarian function. Current research in TCM generally recognizes that tonifying the kidney is the core treatment for POI, and substantial evidence supports the efficacy of this approach [ 16 , 17 ]. In clinical practice, the team observed that POI patients often exhibit symptoms of "depression", such as irritability, low mood, insomnia, or excessive dreaming [ 5 ]. Therefore, it is proposed that "heart depression disturbing the kidney leading to depletion of kidney essence"—constitutes the core pathogenesis of POI. Treatment should follow the principle of "simultaneous heart-kidney treatment". The NTZF, derived from classical formulas, primarily includes Rehmanniae Radix Praeparata, which targets the heart and kidney meridians. This herb not only nourishes heart blood but also replenishes essence and strengthens bone marrow. Testudinis Carapax et Plastrum and Cuscutae Semen, entering the kidney meridian, synergize with rehmannia to tonify both yin and yang. Codonopsis Radix, Dioscoreae Rhizoma and Angelicae Sinensis Radix, classified under the spleen meridian, enhance qi and blood while supporting Rehmanniae Radix Praeparata's effects. Bupleuri Radix and Paeoniae Radix Alba nourish yin and blood, soften the liver, and alleviate depression. Ziziphi Spinosae Semen and Moutan Cortex clear heart heat, relieve restlessness, and calm the mind. Given the "heart depression" manifestations observed in POI patients, this study aims to validate the therapeutic mechanism of NTZF for POI. Consequently, the selected POI model must concurrently exhibit "heart depression" characteristics. Literature review revealed that CTX-induced model mice demonstrate significant depression-like behaviors [ 18 ]. Building on the CTX-induced POI rat model, studies have demonstrated that NTZF restores prolonged estrous cycles to normal duration, promotes follicular development, and reduces the proportion of follicular atresia. Furthermore, NTZF significantly reverses elevated serum FSH levels while restoring diminished E 2 and AMH levels in POI rats. These findings confirm NTZF's efficacy in protecting ovarian function and mitigating persistent ovarian decline in POI, thereby establishing a foundation for subsequent mechanistic investigations. The components of NTZF have been analyzed via HPLC, with key compounds clearly identified, including adenosine, gallic acid, 5-hydroxymethyl-2-furaldehyde+, magnoflorine, caffeic acid, and others. Through these components, NTZF has demonstrated efficacy in delaying ovarian function decline in POI mice [ 6 ]. However, the mechanism underlying NTZF's therapeutic effect on POI via "heart and kidney regulation" remains to be fully elucidated within modern scientific frameworks. The role of BDNF offers a potential breakthrough in deciphering this mechanism. As a member of the neurotrophic factor family, BDNF promotes neuronal development, synaptic connectivity, and modulation of mood-related disorders. In corticosterone-induced murine depression models, chronic corticosterone elevation triggers excessive lysosomal degradation of neuronal BDNF, leading to depressive behaviors [ 19 ]. Additionally, in humans, physical activity elevates BDNF expression in the brains of depressed patients, regulates emotion-associated neural pathways, and alleviates depressive symptoms [ 20 ]. Thus, BDNF serves as a critical mediator of emotional regulation and a pivotal factor in addressing "heart depression". Increasing evidence highlights the critical role of BDNF in the female reproductive system. Studies indicate that circulating BDNF concentrations in amenorrheic and postmenopausal women are significantly lower than those in fertile women during the follicular phase, with BDNF levels correlating closely with fluctuations in sex hormone levels during the menstrual cycle [ 21 ]. BDNF is also recognized as an ovarian secretory factor, expressed in granulosa cells at various developmental stages. It regulates OGCs function through autocrine and paracrine mechanisms. Notably, knockout of BDNF in mice reduces postnatal OGCs proliferation, leading to impaired follicular formation [ 22 ]. Furthermore, exosomal miR-10a-5p has been shown to target BDNF, inhibit the TrkB/Akt/mTOR signaling pathway, and exacerbate disease progression in a POF rabbit model [ 23 ]. These findings collectively underscore BDNF's pivotal association with female ovarian function. Building on BDNF's dual role in emotion regulation and ovarian function, subsequent studies explored BDNF-related pathways. The two BDNF subtypes, proBDNF and mBDNF, exhibit opposing biological functions by binding to p75 NTR and TrkB receptors, respectively. Researchers demonstrated that intraventricular injection of proBDNF adeno-associated virus in rats subjected to unpredictable chronic mild stress exacerbated depression-like behaviors [ 24 ]. Concurrently, studies have shown that increasing the expression of mBDNF and p-TrkB in the amygdala can reduce stress hormone levels, alleviate anxiety, and negative emotions induced by alcohol use [ 25 ]. These findings underscore the importance of maintaining a dynamic balance between proBDNF and mBDNF for normal physiological function. Preliminary studies confirm that p75 NTR and TrkB receptors are expressed in ovarian tissue, particularly in OGCs, supporting BDNF's role in ovarian regulation. Under NTZF treatment, the proBDNF/mBDNF imbalance was rectified in both brain and ovarian tissues. To further elucidate NTZF's regulatory mechanism, the team compared recombinant mBDNF protein with NTZF. Results revealed that both recombinant mBDNF protein and NTZF effectively restored proBDNF/mBDNF equilibrium in damaged KGN cells. Furthermore, correcting this imbalance enhanced KGN cell proliferation and suppressed apoptosis. The above results clarify the regulatory mechanism of NTZF to correct the imbalance of proBDNF/mBDNF. The study further investigated the intermediate mechanisms through which NTZF corrects the proBDNF/mBDNF imbalance. Following initial gene transcription and translation, proBDNF is synthesized in the Golgi apparatus. Subsequent cleavage of proBDNF into mBDNF is mediated by enzymes such as Furin [ 26 ], extracellular tPA [ 27 ], and MMPs—specifically MMP-2 and MMP-9 [ 28 ]. These proteolytic enzymes are pivotal in regulating the proBDNF/mBDNF equilibrium. This study identified tPA as a critical factor influencing the proBDNF/mBDNF balance in damaged KGN cells. As a plasminogen activator, tPA converts plasminogen into plasmin. Plasmin then facilitates the proteolytic cleavage of proBDNF to release mBDNF. Why is tPA's function of activating plasminogen inhibited? It is well known that PAI-1 and tPA are key regulatory factors that antagonize each other in the fibrinolytic system, jointly maintaining the dynamic balance of coagulation and fibrinolysis [ 29 ]. PAI-1 inhibits tPA activity by binding to it and forming tPA-PAI-1. Studies have demonstrated that injecting active PAI-1 into the rat hippocampus upregulates the proBDNF/mBDNF ratio by inhibiting tPA [ 30 ]. This study confirmed that in damaged KGN cells, elevated PAI-1 transcription levels promote PAI-1 protein production, further increasing tPA-PAI-1 formation. This process suppresses tPA activity, disrupts the proBDNF/mBDNF balance, and ultimately triggers KGN cell apoptosis. To verify whether NTZF regulates the proBDNF/mBDNF balance via the PAI-1/tPA pathway, the PAI-1 agonist—Anecortave acetate (AA) was introduced into cell experiments to induce KGN cells injury. Results showed that NTZF reversed the increased apoptosis levels in damaged KGN cells following PAI-1 activation. Additionally, NTZF reduced the proBDNF/mBDNF ratio and restored equilibrium in damaged KGN cells. Western Blot analyses at both animal and cellular levels confirmed that NTZF significantly decreased PAI-1 protein expression and reduced tPA-PAI-1 synthesis, thereby enhancing tPA activity. In general, NTZF inhibits PAI-1, reduces tPA-PAI-1 complex synthesis, and promotes tPA-mediated proteolysis of proBDNF to generate mBDNF, thereby correcting the proBDNF/mBDNF imbalance in cells. This mechanism ultimately suppresses OGCs apoptosis and ameliorates POI (Fig. 7 ). However, this study has limitations. While it primarily focused on elucidating NTZF’s role in restoring ovarian proBDNF/mBDNF balance, only preliminary evidence was provided for its cerebral effects in alleviating "heart depression". Therefore, future research should integrate behavioral science, gene editing, and other methodologies to systematically investigate NTZF’s therapeutic effects on brain tissue. Such efforts will enable a more comprehensive and scientifically rigorous explanation of NTZF’s "heart-kidney treatment" principle in POI management. Conclusion In summary, this study investigated the therapeutic effects of NTZF on CTX-induced POI rat models. The results demonstrated that NTZF exerted therapeutic benefits through multiple mechanisms: regulating abnormal sex hormone levels, promoting follicular development, reducing follicular atresia, and improving ovarian function in POI rats. Furthermore, we elucidated the therapeutic mechanism of NTZF via its modulation of the PAI-1/tPA signaling pathway, which corrects the proBDNF/mBDNF imbalance and inhibits OGCs apoptosis. Notably, our study provides preliminary evidence that NTZF regulates cerebral proBDNF/mBDNF balance to alleviate "heart depression" manifestations in POI. These findings not only offer novel mechanistic insights into the "simultaneous heart-kidney treatment" principle for POI but also position NTZF as a promising multi-target therapeutic agent for POI management. Abbreviations POI premature ovarian insufficiency NTZF Ningxin-Tongyu-Zishen formula CTX Cyclophosphamide FSH follicle-stimulating hormone E 2 estrogen AMH anti-Müllerian hormone LH luteinizing hormone OGCs ovarian granulosa cells PM phosphoramide mustard POF premature ovarian failure HRT hormone replacement therapy BDNF brain-derived neurotrophic factor TCM traditional Chinese medicine SPF specific pathogen-free SD Sprague-Dawley NS normal saline PFA Paraformaldehyde Elisa enzyme-linked immunosorbent assay HE hematoxylin-eosin AOD average optical density IF Immunofluorescence ANOVA one-way analysis of variance MMP2 matrix metallopeptidase2 MMP9 matrix metallopeptidase9 tPA tissue plasminogen activator Declarations Ethics approval and consent to participate Not applicable. Clinical trial number Not applicable. Consent for publication Not applicable. Availability of data and materials All data and materials are available in the manuscript, further inquiries can be directed to the corresponding author. Competing interests The authors declare that there are no conflicts of interest. Funding This work was supported by the Zhejiang Province Traditional Chinese Medicine Modernization Project [grant numbers: 2022ZX011]. Authors' contributions Jiawen Ma, Yizhou Zhang conceived and designed the experiments. Jiawen Ma, Chaofan Zhu, Lifang Xie performed the experiments and drafted the manuscript. Shuaiqi An analyzed the data. Zaiyang Zhang, Keying Wang helped in performing the analysis with constructive discussions. Yizhou Zhang provided financial support. All authors reviewed the manuscript. Acknowledgments We appreciate experimental support from the Medical Research Center, Academy of Chinese Medical Sciences, Zhejiang Chinese Medical University. Supplementary materials The supplementary materials related to this article are shown in the annex. References Panay N, Anderson RA, Bennie A, Cedars M, Davies M, Ee C, et al. Evidence-based guideline: Premature Ovarian Insufficiency. Hum Reprod Open. 2024(4):hoae065. Li M, Zhu Y, Wei J, Chen L, Chen S, Lai D. The global prevalence of premature ovarian insufficiency: a systematic review and meta-analysis. Climacteric. 2023;26(2):95-102. Touraine P, Chabbert-Buffet N, Plu-Bureau G, Duranteau L, Sinclair AH, Tucker EJ. Premature ovarian insufficiency. Nat Rev Dis Primers. 2024;10(1):63. Kuang X, Tang Y, Xu H, Ji M, Lai D. The Evaluation of Ovarian Function Recovery Following Treatment of Primary Ovarian Insufficiency: A Systematic Review. Front Endocrinol (Lausanne). 2022;13:855992. Yan X, Zhang Z, Ma J, Zhang Y. Ningxin Tongyu Zishen Tang in Treatment of Premature Ovarian Insufficiency: A Retrospective Control Study. Clinical Complementary Medicine and Pharmacology. 2023;3(1):100067. 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Zarza-Rebollo JA, López-Isac E, Rivera M, Gómez-Hernández L, Pérez-Gutiérrez AM, Molina E. The relationship between BDNF and physical activity on depression. Prog Neuropsychopharmacol Biol Psychiatry. 2024;134:111033. Chow R, Wessels JM, Foster WG. Brain-derived neurotrophic factor (BDNF) expression and function in the mammalian reproductive Tract. Hum Reprod Update. 2020;26(4):545-64. Ojeda SR, Romero C, Tapia V, Dissen GA. Neurotrophic and cell-cell dependent control of early follicular development. Mol Cell Endocrinol. 2000;163(1-2):67-71. Bao Z, Li J, Cai J, Yao S, Yang N, Yang J, et al. Plasma-derived exosome miR-10a-5p promotes premature ovarian failure by target BDNF via the TrkB/Akt/mTOR signaling pathway. Int J Biol Macromol. 2024;277(Pt 1):134195. Bai YY, Ruan CS, Yang CR, Li JY, Kang ZL, Zhou L, et al. ProBDNF Signaling Regulates Depression-Like Behaviors in Rodents under Chronic Stress. Neuropsychopharmacology. 2016;41(12):2882-92. Seo SY, Bang SK, Kang SY, Cho SJ, Choi KH, Ryu YH. Acupuncture Alleviates Anxiety and 22-kHz Ultrasonic Vocalizations in Rats Subjected to Repeated Alcohol Administration by Modulating the Brain-Derived Neurotrophic Factor/Corticotropin-Releasing Hormone Signaling Pathway. Int J Mol Sci. 2021;22(8):4037. Wang M, Xie Y, Qin D. Proteolytic cleavage of proBDNF to mBDNF in neuropsychiatric and neurodegenerative diseases. Brain Res Bull. 2021;166:172-84. Yesilkaya UH, Gica S, Menekseoglu PO, Tasdemir BG, Cirakli Z, Karamustafalioglu N. Can the Imbalance between Neurotrophic and Apoptotic Proteins Be the "Beware the Ides of March" for Unaffected Relatives of Schizophrenia Patients? .Mol Neurobiol. 2022;59(12):7413-22. Dorandish S, Atali S, Ray R, Al Khashali H, Coleman KL, Guthrie J, et al. Differences in the Relative Abundance of ProBDNF and Mature BDNF in A549 and H1299 Human Lung Cancer Cell Media. Int J Mol Sci. 2021;22(13):7059. Tsantarliotou MP, Lavrentiadou SN, Psalla DA, Margaritis IE, Kritsepi MG, Zervos IA, et al. Suppression of plasminogen activator inhibitor-1 (PAI-1) activity by crocin ameliorates lipopolysaccharide-induced thrombosis in rats. Food Chem Toxicol. 2019;125:190-7. Zhang F, Luo J, Zhu X. Ketamine ameliorates depressive-like behaviors by tPA-mediated conversion of proBDNF to mBDNF in the hippocampus of stressed rats. Psychiatry Res. 2018;269:646-51. Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx Blotimages.zip Graphicalabstract.tif supplementaryfile.docx supplementaryfileimages.zip Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 03 Jul, 2025 Reviews received at journal 03 Jul, 2025 Reviews received at journal 30 Jun, 2025 Reviews received at journal 24 Jun, 2025 Reviewers agreed at journal 23 Jun, 2025 Reviewers agreed at journal 21 Jun, 2025 Reviewers agreed at journal 20 Jun, 2025 Reviewers invited by journal 17 Jun, 2025 Editor assigned by journal 11 Jun, 2025 Submission checks completed at journal 10 Jun, 2025 First submitted to journal 03 Jun, 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. 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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-6810873","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":472863398,"identity":"1698e18c-e6bf-4d60-8c3e-2fd6cffa011e","order_by":0,"name":"Jiawen Ma","email":"","orcid":"","institution":"Zhejiang Chinese Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jiawen","middleName":"","lastName":"Ma","suffix":""},{"id":472863399,"identity":"d5009831-61ed-4884-ad8e-4fd6054f7d2f","order_by":1,"name":"Chaofan Zhu","email":"","orcid":"","institution":"Zhejiang Chinese Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chaofan","middleName":"","lastName":"Zhu","suffix":""},{"id":472863400,"identity":"341e5044-971f-4fe2-b916-43cb04187240","order_by":2,"name":"Lifang Xie","email":"","orcid":"","institution":"Zhejiang Chinese Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lifang","middleName":"","lastName":"Xie","suffix":""},{"id":472863403,"identity":"9d4f429f-259b-4be3-823f-66f79f616d7a","order_by":3,"name":"Shuaiqi An","email":"","orcid":"","institution":"Zhejiang Chinese Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shuaiqi","middleName":"","lastName":"An","suffix":""},{"id":472863404,"identity":"fbef3c7e-d2bf-48ad-a74a-f09e9c59a8b3","order_by":4,"name":"Zaiyang Zhang","email":"","orcid":"","institution":"Zhejiang Chinese Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zaiyang","middleName":"","lastName":"Zhang","suffix":""},{"id":472863405,"identity":"f8c249c3-bcbe-44a0-93cb-f29fd06cb591","order_by":5,"name":"Keying Wang","email":"","orcid":"","institution":"Zhejiang Chinese Medical University","correspondingAuthor":false,"prefix":"","firstName":"Keying","middleName":"","lastName":"Wang","suffix":""},{"id":472863406,"identity":"19b367c7-3a95-4c06-9342-0f4c4db1383e","order_by":6,"name":"Yizhou Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCklEQVRIie3QMUvDQBTA8XccXJfE+YWg8SO8EmgVhH6VE8Es10komQQJvCk4BxT9FnVNKcQlOgs6VITOdVAyiJiIk5C0o+D9hzcc7ze8A7DZ/mL4MwlALDR97qhekm9MJK3iPNxyCr0JgYYoLyvzwys0u50iuEiWz2+8Nxr2UvJdfooYDEAVT1uJuCyG4Taj3E9LCl1ejhnucpGWj61Eoh74HqOiB0NHLssxi3MtBbcThdF7Q5yGzGsSKelQJ3HQDLxXRqxJ/ywr51qpNQTRTHy4R6KyOIFVfNxnR+lZ1y1BFk29anI6otvkptJ0EATXL7NFFbeT7y9w1a+XvHO/TlQf61ZsNpvtX/cFFgFU2np8b8UAAAAASUVORK5CYII=","orcid":"","institution":"Zhejiang Chinese Medical University","correspondingAuthor":true,"prefix":"","firstName":"Yizhou","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-06-03 11:39:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6810873/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6810873/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84989408,"identity":"06c62900-3bd8-416c-bd61-3adf6362be3c","added_by":"auto","created_at":"2025-06-19 14:54:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1004996,"visible":true,"origin":"","legend":"\u003cp\u003eNTZF improved ovarian function in POI rats. (A) Representative images of vaginal exfoliated cells and estrous cycle phase distribution in each group, where abbreviations denote: NEC = nucleated epithelial cells, CEC = cornified epithelial cells, L = leukocytes. (B) Ovarian index across groups. (C-E) Serum levels of FSH, E\u003csub\u003e2\u003c/sub\u003e, and AMH. (F-H) Representative H\u0026amp;E staining of follicles at various developmental stages, and quantitative analysis of primordial follicle and atretic follicle proportions. (I–K) Representative TUNEL staining of follicles,and mean optical density (AOD) of TUNEL-positive signals in primordial and growing follicles (n=5: *\u003cem\u003ep\u003c/em\u003e＜0.05, **\u003cem\u003ep\u003c/em\u003e＜0.01, ***\u003cem\u003ep\u003c/em\u003e＜0.001, versus Control group; #\u003cem\u003ep\u003c/em\u003e＜0.05, ##\u003cem\u003ep\u003c/em\u003e＜0.01, ###\u003cem\u003ep\u003c/em\u003e＜0.001, versus Model group).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6810873/v1/8ff7336fff67413d99977106.png"},{"id":84990613,"identity":"a8490431-e575-42d9-81be-2517f2a457f3","added_by":"auto","created_at":"2025-06-19 15:10:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":572592,"visible":true,"origin":"","legend":"\u003cp\u003eNTZF restored proBDNF/mBDNF balance in brain tissue, serum, and ovarian tissue of POI rats. (A) Localization of TrkB and p75\u003csup\u003eNTR\u003c/sup\u003e in rat ovaries. (B) Serum proBDNF levels and proBDNF proportion relative to total BDNF. (C-D) Changes in proBDNF/mBDNF protein expression in brain and ovarian tissues (n=5: *\u003cem\u003ep\u003c/em\u003e＜0.05, **\u003cem\u003ep\u003c/em\u003e＜0.01, ***\u003cem\u003ep\u003c/em\u003e＜0.001, versus Control group; #\u003cem\u003ep\u003c/em\u003e＜0.05, ##\u003cem\u003ep\u003c/em\u003e＜0.01, ###\u003cem\u003ep\u003c/em\u003e＜0.001, versus Model group).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6810873/v1/d4c9c857dffb38c36f53adda.png"},{"id":84989421,"identity":"1fce4d39-eafc-461f-9317-0a446da12fb3","added_by":"auto","created_at":"2025-06-19 14:54:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":304930,"visible":true,"origin":"","legend":"\u003cp\u003eproBDNF/mBDNF imbalance in KGN cells induces apoptosis. (A) CCK-8 assay assessed KGN cells viability under varying PM concentrations and treatment durations. (B) Morphological alterations in KGN cells following 48h PM treatment. (C) EdU assay quantified cell proliferation changes. (D-H) Apoptosis-related markers: mRNA and protein levels analyzed by q-PCR and Western Blot. (I-K) IF staining for TrkB and p75\u003csup\u003eNTR\u003c/sup\u003e localization and expression. (L) q-PCR analysis of BDNF and its receptor mRNA expression. (M) Elisa measured proBDNF/mBDNF levels in cell culture medium. (N-P) Western Blot detection of proBDNF/mBDNF and receptor protein expression (n=3: *\u003cem\u003ep\u003c/em\u003e＜0.05, **\u003cem\u003ep\u003c/em\u003e＜0.01, ***\u003cem\u003ep\u003c/em\u003e＜0.001, versus Control group).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6810873/v1/2a1c798361cd0d5c030ad26a.png"},{"id":84989422,"identity":"47189fb8-cb53-4fb2-ae50-426611054b7f","added_by":"auto","created_at":"2025-06-19 14:54:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":323144,"visible":true,"origin":"","legend":"\u003cp\u003eNTZF inhibits apoptosis of damaged KGN cells by restoring proBDNF/mBDNF balance. (A) CCK-8 assay assessed KGN cell viability under varying NTZF concentrations. (B) Optimal NTZF intervention concentration screened via CCK-8 in damaged KGN cells. (C) Recombinant mBDNF protein molecular weight (~14kDa). (D) CCK-8 assay assessed recombinant mBDNF protein concentration optimization in damaged KGN cells. (E) NTZF’s effect on viability of damaged KGN cells. (F) NTZF-mediated modulation of proBDNF/mBDNF protein expression. (G) EdU assay quantified NTZF’s pro-proliferative effects in damaged KGN cells. (H-M) Apoptosis-related markers: mRNA and protein levels analyzed by q-PCR and Western Blot (n=3: *\u003cem\u003ep\u003c/em\u003e＜0.05, **\u003cem\u003ep\u003c/em\u003e＜0.01, ***\u003cem\u003ep\u003c/em\u003e＜0.001, versus Control group; #\u003cem\u003ep\u003c/em\u003e＜0.05, ##\u003cem\u003ep\u003c/em\u003e＜0.01, ###\u003cem\u003ep\u003c/em\u003e＜0.001, versus Model group; ^\u003cem\u003ep\u003c/em\u003e＜0.05, ^^\u003cem\u003ep\u003c/em\u003e＜0.01, ^^^\u003cem\u003ep\u003c/em\u003e＜0.001, versus mBDNF group).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6810873/v1/c977e43c6c5366057fb92109.png"},{"id":84989414,"identity":"15fa7c38-94f6-45ef-9865-bc96b1c305fa","added_by":"auto","created_at":"2025-06-19 14:54:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":194263,"visible":true,"origin":"","legend":"\u003cp\u003etPA is a key regulator of proBDNF/mBDNF balance in damaged KGN cells. (A-E) Proteolytic enzyme expression associated with proBDNF/mBDNF balance was analyzed by q-PCR and Western Blot. (F) q-PCR analysis of tPA and PAI-1 mRNA levels in damaged KGN cells. (G-H) Western Blot detection of PAI-1 and tPA-PAI-1 protein expression (n=3: *\u003cem\u003ep\u003c/em\u003e＜0.05, **\u003cem\u003ep\u003c/em\u003e＜0.01, ***\u003cem\u003ep\u003c/em\u003e＜0.001, versus Control group).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6810873/v1/5ee98e57332717441994e058.png"},{"id":84989425,"identity":"3c320445-45f9-422a-b912-80721d3c9cb2","added_by":"auto","created_at":"2025-06-19 14:54:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":420836,"visible":true,"origin":"","legend":"\u003cp\u003eNTZF inhibits apoptosis of damaged KGN cells by restoring proBDNF/mBDNF balance via the PAI-1/tPA signaling pathway. (A) CCK-8 assay optimized Anecortave acetate (AA) concentration for intervention in damaged KGN cells. (B) Viability of damaged KGN cells treated with NTZF and AA. (C-D) EdU assay quantified proliferative effects of NTZF and AA. (E-J) Apoptosis-related markers: mRNA and protein levels analyzed by q-PCR and Western Blot. (K) NTZF and AA effects on proBDNF/mBDNF protein expression in damaged KGN cells. (L-N) Western Blot analysis of PAI-1, tPA, and tPA-PAI-1 protein expression in damaged KGN cells (n=3: *\u003cem\u003ep\u003c/em\u003e＜0.05, **\u003cem\u003ep\u003c/em\u003e＜0.01, ***\u003cem\u003ep\u003c/em\u003e＜0.001, versus Control group; #\u003cem\u003ep\u003c/em\u003e＜0.05, ##\u003cem\u003ep\u003c/em\u003e＜0.01, ###\u003cem\u003ep\u003c/em\u003e＜0.001, versus Model group; ^\u003cem\u003ep\u003c/em\u003e＜0.05, ^^\u003cem\u003ep\u003c/em\u003e＜0.01, ^^^\u003cem\u003ep\u003c/em\u003e＜0.001, versus mBDNF group). (O-Q) Western Blot analysis of PAI-1, tPA, and tPA-PAI-1 protein expression in ovaries of POI rats (n=5: *\u003cem\u003ep\u003c/em\u003e＜0.05, **\u003cem\u003ep\u003c/em\u003e＜0.01, ***\u003cem\u003ep\u003c/em\u003e＜0.001, versus Control group; #\u003cem\u003ep\u003c/em\u003e＜0.05, ##\u003cem\u003ep\u003c/em\u003e＜0.01, ###\u003cem\u003ep\u003c/em\u003e＜0.001, versus Model group).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6810873/v1/1f74a6c0791c1ffa859e3778.png"},{"id":84989417,"identity":"83d867e8-338b-4186-87e7-f82e0fe297fb","added_by":"auto","created_at":"2025-06-19 14:54:32","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":171396,"visible":true,"origin":"","legend":"\u003cp\u003eOverview of the mechanism by which NTZF corrects proBDNF/mBDNF imbalance in OGCs. The figure (ID:IWAASccac2) was created by Figdraw (www.figdraw.com).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6810873/v1/9a72775ee818692ab8f325c0.png"},{"id":84991166,"identity":"16ad9be7-3826-43cf-8061-e363aee096cc","added_by":"auto","created_at":"2025-06-19 15:18:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3616445,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6810873/v1/19d5d2f9-ac0e-46fc-997c-b2982a63843a.pdf"},{"id":84989405,"identity":"b3a35d93-07ca-40be-93c1-b5c140a13546","added_by":"auto","created_at":"2025-06-19 14:54:32","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":242670,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6810873/v1/b2deaf3f386b89a79cf116da.docx"},{"id":84989409,"identity":"81f66c3a-34c3-475f-bd64-4b48c02ec86c","added_by":"auto","created_at":"2025-06-19 14:54:32","extension":"zip","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11203558,"visible":true,"origin":"","legend":"","description":"","filename":"Blotimages.zip","url":"https://assets-eu.researchsquare.com/files/rs-6810873/v1/6555d2477f899c4bac7d9fab.zip"},{"id":84989415,"identity":"738e645e-d76f-4e91-9fd8-075f9dea1129","added_by":"auto","created_at":"2025-06-19 14:54:32","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3771236,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-6810873/v1/1bc8a69b89150d54cf0ce561.tif"},{"id":84989433,"identity":"8842d608-5005-40c4-95cf-dfe7f305bdc1","added_by":"auto","created_at":"2025-06-19 14:54:33","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":5373822,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-6810873/v1/a5b1327bc5bff22f7412b1be.docx"},{"id":84989672,"identity":"aac773db-9280-45c7-80f0-236b07471918","added_by":"auto","created_at":"2025-06-19 15:02:32","extension":"zip","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":11218373,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfileimages.zip","url":"https://assets-eu.researchsquare.com/files/rs-6810873/v1/fbdcbced8e9ccd3ba29c0e22.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"The mechanism of Ningxin-Tongyu-Zishen formula regulating proBDNF/mBDNF balance through PAI-1/tPA signaling pathway in the treatment of premature ovarian insufficiency","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePremature ovarian insufficiency (POI) is a refractory gynecological endocrine disorder. The clinical manifestations include irregular menstruation, elevated follicle-stimulating hormone (FSH\u0026thinsp;\u0026gt;\u0026thinsp;25 U/L), and decreased estrogen (E\u003csub\u003e2\u003c/sub\u003e) levels in women under 40 years of age [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The incidence of POI is approximately 3.5%, with an increasing trend among younger populations [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Early menopausal symptoms in POI patients, such as hot flashes, night sweats, osteoporosis, and vaginal dryness, severely affect their quality of life. If untreated, POI may progress to premature ovarian failure (POF), manifesting as irreversible amenorrhea and complete loss of fertility. Due to its complex etiology [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], effective treatments for POI remain limited. Hormone replacement therapy (HRT) can regulate menstrual cycles and alleviate hypoestrogenic symptoms but fails to effectively delay ovarian functional decline [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Therefore, novel therapeutic strategies for POI are urgently needed.\u003c/p\u003e \u003cp\u003eNingxin-Tongyu-Zishen formula (NTZF) is a clinically validated prescription based on the \"simultaneous heart-kidney treatment\" principle, derived from Yijing Decoction in \u003cem\u003eFu Qing Zhu's Obstetrics and Gynecology\u003c/em\u003e and Tianwang Buxin Pill in \u003cem\u003eJiao Zhu Fu Ren Liang Fang\u003c/em\u003e. This formula comprises ten traditional Chinese medicinal components, including Rehmanniae Radix Praeparata (shudihuang), Testudinis Carapax et Plastrum (guiban), Codonopsis Radix (dangshen), Cuscutae Semen (tusizi), Angelicae Sinensis Radix (danggui), Ziziphi Spinosae Semen (suanzaoren), Moutan Cortex (mudanpi), Paeoniae Radix Alba (baishao), Dioscoreae Rhizoma (shanyao), and Bupleuri Radix (chaihu). Clinical studies indicate that NTZF not only significantly reduces FSH and luteinizing hormone (LH) levels in POI patients but also alleviates \"heart depression\" symptoms [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Our previous experiments confirmed that NTZF increases primordial follicle proportions, improves serum sex hormone levels, and delays ovarian aging in D-galactose-induced POI mice [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, the mechanism underlying NTZF\u0026rsquo;s \"simultaneous heart-kidney treatment\" efficacy remains unclear from a modern scientific perspective.\u003c/p\u003e \u003cp\u003eBrain-derived neurotrophic factor (BDNF) may mediate NTZF\u0026rsquo;s regulatory effects on the \"heart-kidney\". According to traditional Chinese medicine (TCM) theory, \"heart depression\" primarily manifests as mood and sleep disturbances, including irritability, insomnia, and dreaminess. As a neurotrophic factor in the central nervous system, BDNF not only regulates emotional states but also correlates with insomnia [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Notably, BDNF levels fluctuate with sex hormone variations during the menstrual cycle [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and exhibit synchronized changes in the brain and serum [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Furthermore, BDNF acts as an ovarian secretory factor localized in follicular granulosa and cumulus cells, regulating follicular development via autocrine and paracrine mechanisms [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Recent studies highlight the distinct roles of BDNF isoforms\u0026mdash;proBDNF and mature BDNF (mBDNF)\u0026mdash;in cellular processes: proBDNF binding to p75\u003csup\u003eNTR\u003c/sup\u003e induces apoptosis, whereas mBDNF binding to TrkB promotes proliferation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These findings suggest that BDNF dysregulation, particularly proBDNF/mBDNF imbalance, may contribute to POI pathogenesis.\u003c/p\u003e \u003cp\u003eThis study aimed to investigate proBDNF/mBDNF balance alterations in the brain and ovaries of POI rats and identify key proteolytic enzymes modulating this balance. We further elucidate the molecular mechanism by which NTZF regulates proBDNF/mBDNF equilibrium to treat POI, thereby providing a scientific basis for its \"simultaneous heart-kidney treatment\" theory.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eNTZF and EV preparation\u003c/p\u003e \u003cp\u003eNTZF is composed of ten kinds of Chinese medicinal materials, which are Rehmanniae Radix Praeparata, Testudinis Carapax et Plastrum, Codonopsis Radix, Cuscutae Semen, Angelicae Sinensis Radix, Ziziphi Spinosae Semen, Moutan Cortex, Paeoniae Radix Alba, Dioscoreae Rhizoma, and Bupleuri Radix. The above medicinal materials were purchased from the Third Affiliated Hospital of Zhejiang Chinese Medical University, and then mixed at a weight ratio of 10:4:4:4:5:4:3:4:5:2. NTZF has been confirmed by HPLC, and its main chemical constituents include adenosine, gallic acid, 5-hydroxymethyl-2-furaldehyde+, magnoflorine, caffeic acid, (+)-catechin, chlorogenic acid, paeoniflorin, ferulic acid, quercetin, isorhamnetin, and linoleic acid. The decoction of NTZF and estradiol valerate (EV, J20171038, Bayer, Germany) suspension was based on previous studies [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eConstruction of POI rat models\u003c/p\u003e \u003cp\u003eForty specific pathogen-free (SPF) female Sprague-Dawley (SD) rats (6\u0026ndash;7 weeks old, 200\u0026thinsp;\u0026plusmn;\u0026thinsp;10g) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. During the 10 days of adaptive feeding, vaginal exfoliated cells were collected daily between 8:30\u0026thinsp;\u0026minus;\u0026thinsp;9:30 AM. Rats with regular estrous cycles were randomly allocated into five groups (n\u0026thinsp;=\u0026thinsp;8): Control, Model, EV, Low-dose NTZF (L-NTZF), and High-dose NTZF (H-NTZF). For POI model establishment, cyclophosphamide (CTX) was intraperitoneally injected at 100 mg/kg on D11 (excluding Control). From D12 to D25, non-Control groups received daily intraperitoneal injections of CTX (8mg/kg). Throughout D11-25, Control group rats were administered equivalent volumes of normal saline (NS) via the same route. Oral gavage administration commenced on D33: Control and Model groups received NS (3mL/100g), EV group received EV suspension (3.7\u0026micro;g/mL), while L-NTZF and H-NTZF groups received NTZF decoctions (0.47g/mL and 0.94g/mL) for 28 days. Following 12-hours fasting, rats were anesthetized with Zoletil-50 (20mg/kg). Some ovarian tissues were either fixed in 4% paraformaldehyde (PFA), and the others were stored at -80\u0026deg;C with serum and brain tissues. All procedures were approved by Animal Ethical and Welfare Committee of Zhejiang Chinese Medical University (IACUC-20240603-17). Supplementary Fig.\u0026nbsp;1 depicted the animal experimental protocol.\u003c/p\u003e \u003cp\u003eCell culture\u003c/p\u003e \u003cp\u003eIn this study, KGN cells (iCell-h298) were chosen as the subject of investigation in order to examine the mechanisms related to ovarian granulosa cells (OGCs). These cells are considered ideal for studying reproductive dysfunction and potential treatments [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. KGN cells were cultured in DMEM/F12 (PM150312, Procell) containing 10% fetal bovine serum (FBS, 164210-50, Procell) and 0.01% penicillin-streptomycin solution (PB180120, Procell). KGN cells were subcultured once they had reached 80\u0026ndash;90% confluence.\u003c/p\u003e \u003cp\u003eCell treatment\u003c/p\u003e \u003cp\u003ePhosphoramide mustard (PM) is one of the active metabolites of CTX and the main component of ovarian toxicity [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Therefore, PM is used as a modeling drug in cell experiments. KGN cells were seeded in a cell culture plate for 12h, and then treated with DMEM/F12 without 10% FBS for 2h. Then, PM (HY-137316A, MCE) with final concentrations of 100, 150, 200, 250\u0026micro;M were added to each group. After 24h or 48h, 100\u0026micro;L working solution containing 10% cell counting kit-8 (CCK-8, BMU106, Abbkine) was added to each well and cultured in a cell incubator at 37\u0026deg;C for 1h. KGN cell viability was calculated by OD450 value to determine PM concentration and intervention duration. When PM was used for culture, NTZF (final concentrations were 0.26, 1.06, and 4.22\u0026micro;g/\u0026micro;L) was added to the medium to determine the appropriate concentration. At the same time, the final intervention concentrations of the recombinant mBDNF protein (450-02, Pepro Tech) were 0.1, 1, and 10ng/mL, while the final intervention concentrations of the PAI-1 agonist Anecortave acetate (AA, HY-116868, MCE) were 12.5, 25, 50, and 100\u0026micro;M to determine the appropriate concentration.\u003c/p\u003e \u003cp\u003eAnalysis of ovarian index\u003c/p\u003e \u003cp\u003eBody mass of rats was measured prior to euthanasia. During specimen collection, bilateral ovarian weight was quantified gravimetrically. Ovarian index of rats was calculated using the following formula: Ovarian Index (%) = [Bilateral Ovarian Weight (g)/ Body Mass (g)] \u0026times; 100%.\u003c/p\u003e \u003cp\u003eEstrous cycle detection\u003c/p\u003e \u003cp\u003eVaginal cytology was performed in rats during D51-D60 using exfoliative cell sampling. Specimens were stained with Wright-Giemsa Stain (R20659, Yuanye). Estrous cycle phases (proestrus, estrus, metestrus, diestrus) were classified based on the relative proportions of nucleated epithelial cells, cornified epithelial cells, and leukocytes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEnzyme-linked immunosorbent assay (Elisa)\u003c/p\u003e \u003cp\u003eAccording to the instructions in the Elisa kit of Jiangsu Meimian Industrial Co., Ltd. (China), the serum levels of FSH (MM-70867R1), E\u003csub\u003e2\u003c/sub\u003e (MM-0575R1) and anti-M\u0026uuml;llerian hormone (AMH, MM-0219R1) in rats were detected. In addition, the levels of proBDNF (JL48660) and mBDNF (JL15984) in KGN cell culture medium were detected in accordance with Shanghai Future Industry Co., Ltd. (China) specifications.\u003c/p\u003e \u003cp\u003eHematoxylin-eosin (HE) staining\u003c/p\u003e \u003cp\u003eFresh ovarian tissues were fixed overnight in 4% PFA. After dehydration, the tissues were embedded into blocks using paraffin. The tissues were sliced into 5\u0026micro;m pieces, which were attached to the slides for HE staining.\u003c/p\u003e \u003cp\u003eTUNEL staining\u003c/p\u003e \u003cp\u003eOvarian tissue sections were dewaxed, and then incubated with 20\u0026micro;g/mL proteinase K solution at 37℃ for 30min. The endogenous peroxidase in the sections was inactivated by 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and then labeled by TUNEL cell apoptosis detection kit (C1098, Beyotime). Image J software was used to analyze the average optical density (AOD) of primordial follicles and growing follicles in ovarian tissue sections.\u003c/p\u003e \u003cp\u003eCell crystal violet staining\u003c/p\u003e \u003cp\u003eAfter KGN cells were fixed with 4% PFA for 30min, an appropriate amount of crystal violet staining solution (C0121, Beyotime) was added and incubated in dark for 10min. After washing with PBS, the cells were photographed and recorded under an inverted microscope.\u003c/p\u003e \u003cp\u003eCell proliferation detection\u003c/p\u003e \u003cp\u003eAt the end of KGN cell culture, EdU was added and incubated for 3 h to label the cells. After fixation with 4% PFA for 15min, the cells were incubated with a working solution prepared according to the instructions of BeyoClick\u0026trade; EdU-555 Cell Proliferation Detection Kit (C0075S, Beyotime). After adding anti-fluorescence quencher containing DAPI (MA0222, Meilunbio), the images were photographed and analyzed at 454nm and 555 nm using a fluorescence microscope.\u003c/p\u003e \u003cp\u003eImmunofluorescence (IF) staining\u003c/p\u003e \u003cp\u003eIF staining of ovarian tissues and KGN cells was performed using a fluorescent duplex-labeled high-sensitivity signal amplification kit (HKI0000-2S, Haoke) according to manufacturer protocols. Tissue sections were dewaxed and processed through sequential antigen retrieval, permeabilization, and endogenous peroxidase inactivation steps, followed by blocking with 3%BSA for 30min. Primary antibodies and universal secondary antibodies were applied sequentially with appropriate incubation periods. After the secondary antibody incubation, the steps starting from antigen retrieval were repeated again. DAPI was added to label the nucleus, and then anti-fluorescence quenching sealing agent was added. KGN cells were fixed in 4%PFA prior to identical staining procedures. Imaging was conducted using Leica inverted fluorescence microscope (DMi8). Primary antibody specifications are detailed in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003cp\u003eQuantitative real-time PCR (q-PCR)\u003c/p\u003e \u003cp\u003eSevenFast\u0026reg; total RNA extraction kit for Cells (SM130, Seven, Beijing, China ) was used to isolate total RNA from KGN cells. After reverse transcription into cDNA, q-PCR was performed using the SYBR Green premix Pro Taq HS qPCR kit (AG11718) purchased from ACCURATE BIOTECHNOLOGY (HUNAN) CO., LTD (ChangSha, China). Using β-actin as an internal reference gene, 2\u003csup\u003e\u0026minus;△△Ct\u003c/sup\u003e was calculated to analyze the relative mRNA expression of the target gene. The primer sequence is shown in Supplementary Table\u0026nbsp;2.\u003c/p\u003e \u003cp\u003eWestern Blot\u003c/p\u003e \u003cp\u003eThe total protein content of ovarian tissue and KGN cells was detected by BCA protein concentration assay kit (P0011, Beyotime). The protein extracts were separated by SDS-PAGE gels, and then transferred to PVDF membranes (Millipore). The PVDF membranes were blocked with 3% skim milk at room temperature for 1h, and then incubated with primary antibody overnight at 4℃. After incubation with secondary antibody at room temperature for 1h, imagings were performed using ECL developers (BMU102, Abbkine). Antibody information is shown in Supplementary Table\u0026nbsp;3.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data of this study were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Graphpad Prism 8.0 software was used for statistical analysis and statistical drawing. One-way analysis of variance (ANOVA) was used to compare the differences between different groups. \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicated that the difference was statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Result","content":"\u003cp\u003eNTZF improves ovarian function in POI rats\u003c/p\u003e \u003cp\u003eIn this study, estrous cycle changes in rats were assessed by analyzing vaginal exfoliated cell morphology during the late treatment phase (D51-D60, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Notably, model group rats persistently remained in diestrus, indicating abnormal cyclicity. NTZF treatment effectively ameliorated this estrous cycle disruption in POI rats.\u003c/p\u003e \u003cp\u003eThe ovarian index, a key indicator of reproductive health [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], was significantly increased by NTZF treatment in POI rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eGiven POI's characteristic endocrine dysfunction, we measured serum levels of FSH, E\u003csub\u003e2\u003c/sub\u003e, and AMH. Model group rats exhibited markedly elevated FSH levels alongside reduced E\u003csub\u003e2\u003c/sub\u003e and AMH levels, mirroring clinical POI hormone profiles. NTZF administration reversed these abnormalities (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-E), demonstrating its capacity to restore hormonal balance.\u003c/p\u003e \u003cp\u003eTo evaluate follicular development, we quantified follicular stage proportions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), with specific focus on primordial and atretic follicles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG-H). The model group showed reduced primordial follicle counts and increased atresia, both of which were counteracted by NTZF treatment.\u003c/p\u003e \u003cp\u003eTUNEL staining revealed OGCs apoptosis levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). Both primordial and growing follicles in model rats exhibited significantly elevated apoptosis, which was mitigated by NTZF intervention (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ-K).\u003c/p\u003e \u003cp\u003eIn conclusion, NTZF inhibits ovarian granulosa cell apoptosis and improves ovarian function in POI rats.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNTZF restores proBDNF/mBDNF homeostasis in POI rats\u003c/p\u003e \u003cp\u003eIF analysis revealed p75\u003csup\u003eNTR\u003c/sup\u003e and TrkB receptor localization in OGCs of primordial, growing, and mature follicles (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), indicating that proBDNF and mBDNF regulate follicular maturation through receptor binding in rat ovaries.\u003c/p\u003e \u003cp\u003eSerum proBDNF and total BDNF levels were quantified via Elisa. POI rats exhibited elevated proBDNF levels and an increased proBDNF/total BDNF ratio. Notably, high-dose NTZF treatment significantly reduced serum proBDNF levels, while both low-does NTZF and high-dose NTZF treatments decreased the proBDNF proportion relative to total BDNF (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eWestern Blot analysis demonstrated an elevated proBDNF/mBDNF ratio in both brain and ovarian tissues of POI rats, confirming systemic imbalance. NTZF administration effectively corrected this dysregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D).\u003c/p\u003e \u003cp\u003eCollectively, these findings demonstrate that NTZF not only reduces circulating proBDNF dominance but also restores proBDNF/mBDNF equilibrium in brain and ovarian tissues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eproBDNF/mBDNF imbalance induces apoptosis in KGN cells\u003c/p\u003e \u003cp\u003eTo investigate proBDNF/mBDNF dysregulation in injured OGCs and maintain experimental consistency between in vivo and in vitro models, we utilized PM to induce cellular damage. Screening identified 150\u0026micro;M PM treatment for 48 hours as the optimal condition, significantly reducing KGN cell viability and altering cellular morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B). Under this regimen, EdU-positive KGN cells were markedly diminished, while mRNA and protein expression of apoptotic markers (p53, p21, and bax/bcl2) were upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-H). These parameters defined subsequent in vitro experimental conditions.\u003c/p\u003e \u003cp\u003eIF analysis localized TrkB and p75\u003csup\u003eNTR\u003c/sup\u003e receptors in KGN cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). Damaged cells exhibited reduced TrkB fluorescence intensity and enhanced p75\u003csup\u003eNTR\u003c/sup\u003e signal (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ-K), correlating with mRNA/protein expression patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eO-P). These findings suggest predominant activation of apoptosis-related pathways in damaged KGN cells. Notably, despite elevated BDNF mRNA levels, damaged cells demonstrated increased proBDNF/mBDNF secretion ratios and heightened intracellular proBDNF/mBDNF protein expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL-N). Collectively, these results establish proBDNF/mBDNF imbalance as a critical mediator of KGN cell apoptosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNTZF restores proBDNF/mBDNF balance to mitigate apoptosis in damaged KGN cells\u003c/p\u003e \u003cp\u003eFive NTZF concentrations (4.22\u0026micro;g/\u0026micro;L, 1.06\u0026micro;g/\u0026micro;L, 0.26\u0026micro;g/\u0026micro;L, 0.07\u0026micro;g/\u0026micro;L, 0.02\u0026micro;g/\u0026micro;L) were tested and showed no cytotoxicity to KGN cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Among these, 1.06\u0026micro;g/\u0026micro;L NTZF most effectively restored cell viability in damaged KGN cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Recombinant mBDNF protein (1ng/mL) exhibited comparable efficacy to NTZF in rescuing viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-E). Western Blot analysis further confirmed that NTZF corrected proBDNF/mBDNF imbalance in damaged KGN cells, mirroring the effects of recombinant mBDNF protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Notably, both NTZF and recombinant mBDNF interventions partially restored proliferation levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG) and significantly suppressed mRNA/protein expression of apoptotic markers (p53, p21, and bax/bcl-2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH-M). These comparative analyses validate that NTZF inhibits apoptosis in damaged KGN cells by normalizing proBDNF/mBDNF equilibrium.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003etPA mediates proBDNF proteolytic cleavage to mBDNF in KGN cells\u003c/p\u003e \u003cp\u003eIn damaged KGN cells, mRNA and protein expression of proteolytic enzymes regulating proBDNF/mBDNF balance\u0026mdash;including Furin, matrix metallopeptidase2 (MMP2), and matrix metallopeptidase9 (MMP9)\u0026mdash;were significantly upregulated, whereas tissue plasminogen activator (tPA) exhibited discordant changes: despite increased tPA mRNA levels, its protein expression decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-E). Given the known inhibitory interaction between plasminogen activator inhibitor-1 (PAI-1) and tPA, we further analyzed PAI-1 expression. Both PAI-1 mRNA and protein levels were markedly elevated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF-G), accompanied by increased tPA-PAI-1 complex formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). These findings suggest that PAI-1 overexpression suppresses tPA activity, potentially impairing proBDNF-to-mBDNF conversion in damaged KGN cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNTZF restores proBDNF/mBDNF balance via PAI-1/tPA signaling to suppress apoptosis\u003c/p\u003e \u003cp\u003ePAI-1 agonist\u0026mdash;anecortave acetate (AA) dose-dependently reduced viability in damaged KGN cells, with 50\u0026micro;M AA causing significant impairment compared to the model group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). NTZF treatment reversed this viability loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) and counteracted AA-induced proliferation inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-D). Furthermore, NTZF attenuated AA-driven upregulation of apoptotic markers (p53, p21, and bax/bcl2) at both mRNA and protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-J). Building on prior findings, NTZF also rescued AA-exacerbated proBDNF/mBDNF imbalance in damaged cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK). Western Blot analysis confirmed that NTZF suppresses PAI-1 expression, reduces tPA-PAI-1 complex synthesis, and restores tPA activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL-Q).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePOI is recorded in the classic books of gynecology in TCM as \"menstruation stopped when not old yet.\" The kidney stores essence and dominates reproduction. And the rise and fall of kidney qi directly affects ovarian function. Current research in TCM generally recognizes that tonifying the kidney is the core treatment for POI, and substantial evidence supports the efficacy of this approach [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In clinical practice, the team observed that POI patients often exhibit symptoms of \"depression\", such as irritability, low mood, insomnia, or excessive dreaming [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Therefore, it is proposed that \"heart depression disturbing the kidney leading to depletion of kidney essence\"\u0026mdash;constitutes the core pathogenesis of POI. Treatment should follow the principle of \"simultaneous heart-kidney treatment\". The NTZF, derived from classical formulas, primarily includes Rehmanniae Radix Praeparata, which targets the heart and kidney meridians. This herb not only nourishes heart blood but also replenishes essence and strengthens bone marrow. Testudinis Carapax et Plastrum and Cuscutae Semen, entering the kidney meridian, synergize with rehmannia to tonify both yin and yang. Codonopsis Radix, Dioscoreae Rhizoma and Angelicae Sinensis Radix, classified under the spleen meridian, enhance qi and blood while supporting Rehmanniae Radix Praeparata's effects. Bupleuri Radix and Paeoniae Radix Alba nourish yin and blood, soften the liver, and alleviate depression. Ziziphi Spinosae Semen and Moutan Cortex clear heart heat, relieve restlessness, and calm the mind.\u003c/p\u003e \u003cp\u003eGiven the \"heart depression\" manifestations observed in POI patients, this study aims to validate the therapeutic mechanism of NTZF for POI. Consequently, the selected POI model must concurrently exhibit \"heart depression\" characteristics. Literature review revealed that CTX-induced model mice demonstrate significant depression-like behaviors [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Building on the CTX-induced POI rat model, studies have demonstrated that NTZF restores prolonged estrous cycles to normal duration, promotes follicular development, and reduces the proportion of follicular atresia. Furthermore, NTZF significantly reverses elevated serum FSH levels while restoring diminished E\u003csub\u003e2\u003c/sub\u003e and AMH levels in POI rats. These findings confirm NTZF's efficacy in protecting ovarian function and mitigating persistent ovarian decline in POI, thereby establishing a foundation for subsequent mechanistic investigations.\u003c/p\u003e \u003cp\u003eThe components of NTZF have been analyzed via HPLC, with key compounds clearly identified, including adenosine, gallic acid, 5-hydroxymethyl-2-furaldehyde+, magnoflorine, caffeic acid, and others. Through these components, NTZF has demonstrated efficacy in delaying ovarian function decline in POI mice [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, the mechanism underlying NTZF's therapeutic effect on POI via \"heart and kidney regulation\" remains to be fully elucidated within modern scientific frameworks. The role of BDNF offers a potential breakthrough in deciphering this mechanism. As a member of the neurotrophic factor family, BDNF promotes neuronal development, synaptic connectivity, and modulation of mood-related disorders. In corticosterone-induced murine depression models, chronic corticosterone elevation triggers excessive lysosomal degradation of neuronal BDNF, leading to depressive behaviors [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Additionally, in humans, physical activity elevates BDNF expression in the brains of depressed patients, regulates emotion-associated neural pathways, and alleviates depressive symptoms [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Thus, BDNF serves as a critical mediator of emotional regulation and a pivotal factor in addressing \"heart depression\".\u003c/p\u003e \u003cp\u003eIncreasing evidence highlights the critical role of BDNF in the female reproductive system. Studies indicate that circulating BDNF concentrations in amenorrheic and postmenopausal women are significantly lower than those in fertile women during the follicular phase, with BDNF levels correlating closely with fluctuations in sex hormone levels during the menstrual cycle [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. BDNF is also recognized as an ovarian secretory factor, expressed in granulosa cells at various developmental stages. It regulates OGCs function through autocrine and paracrine mechanisms. Notably, knockout of BDNF in mice reduces postnatal OGCs proliferation, leading to impaired follicular formation [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Furthermore, exosomal miR-10a-5p has been shown to target BDNF, inhibit the TrkB/Akt/mTOR signaling pathway, and exacerbate disease progression in a POF rabbit model [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. These findings collectively underscore BDNF's pivotal association with female ovarian function.\u003c/p\u003e \u003cp\u003eBuilding on BDNF's dual role in emotion regulation and ovarian function, subsequent studies explored BDNF-related pathways. The two BDNF subtypes, proBDNF and mBDNF, exhibit opposing biological functions by binding to p75\u003csup\u003eNTR\u003c/sup\u003e and TrkB receptors, respectively. Researchers demonstrated that intraventricular injection of proBDNF adeno-associated virus in rats subjected to unpredictable chronic mild stress exacerbated depression-like behaviors [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Concurrently, studies have shown that increasing the expression of mBDNF and p-TrkB in the amygdala can reduce stress hormone levels, alleviate anxiety, and negative emotions induced by alcohol use [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. These findings underscore the importance of maintaining a dynamic balance between proBDNF and mBDNF for normal physiological function. Preliminary studies confirm that p75\u003csup\u003eNTR\u003c/sup\u003e and TrkB receptors are expressed in ovarian tissue, particularly in OGCs, supporting BDNF's role in ovarian regulation. Under NTZF treatment, the proBDNF/mBDNF imbalance was rectified in both brain and ovarian tissues. To further elucidate NTZF's regulatory mechanism, the team compared recombinant mBDNF protein with NTZF. Results revealed that both recombinant mBDNF protein and NTZF effectively restored proBDNF/mBDNF equilibrium in damaged KGN cells. Furthermore, correcting this imbalance enhanced KGN cell proliferation and suppressed apoptosis. The above results clarify the regulatory mechanism of NTZF to correct the imbalance of proBDNF/mBDNF.\u003c/p\u003e \u003cp\u003eThe study further investigated the intermediate mechanisms through which NTZF corrects the proBDNF/mBDNF imbalance. Following initial gene transcription and translation, proBDNF is synthesized in the Golgi apparatus. Subsequent cleavage of proBDNF into mBDNF is mediated by enzymes such as Furin [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], extracellular tPA [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], and MMPs\u0026mdash;specifically MMP-2 and MMP-9 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. These proteolytic enzymes are pivotal in regulating the proBDNF/mBDNF equilibrium. This study identified tPA as a critical factor influencing the proBDNF/mBDNF balance in damaged KGN cells. As a plasminogen activator, tPA converts plasminogen into plasmin. Plasmin then facilitates the proteolytic cleavage of proBDNF to release mBDNF.\u003c/p\u003e \u003cp\u003eWhy is tPA's function of activating plasminogen inhibited? It is well known that PAI-1 and tPA are key regulatory factors that antagonize each other in the fibrinolytic system, jointly maintaining the dynamic balance of coagulation and fibrinolysis [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. PAI-1 inhibits tPA activity by binding to it and forming tPA-PAI-1. Studies have demonstrated that injecting active PAI-1 into the rat hippocampus upregulates the proBDNF/mBDNF ratio by inhibiting tPA [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. This study confirmed that in damaged KGN cells, elevated PAI-1 transcription levels promote PAI-1 protein production, further increasing tPA-PAI-1 formation. This process suppresses tPA activity, disrupts the proBDNF/mBDNF balance, and ultimately triggers KGN cell apoptosis. To verify whether NTZF regulates the proBDNF/mBDNF balance via the PAI-1/tPA pathway, the PAI-1 agonist\u0026mdash;Anecortave acetate (AA) was introduced into cell experiments to induce KGN cells injury. Results showed that NTZF reversed the increased apoptosis levels in damaged KGN cells following PAI-1 activation. Additionally, NTZF reduced the proBDNF/mBDNF ratio and restored equilibrium in damaged KGN cells. Western Blot analyses at both animal and cellular levels confirmed that NTZF significantly decreased PAI-1 protein expression and reduced tPA-PAI-1 synthesis, thereby enhancing tPA activity.\u003c/p\u003e \u003cp\u003eIn general, NTZF inhibits PAI-1, reduces tPA-PAI-1 complex synthesis, and promotes tPA-mediated proteolysis of proBDNF to generate mBDNF, thereby correcting the proBDNF/mBDNF imbalance in cells. This mechanism ultimately suppresses OGCs apoptosis and ameliorates POI (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). However, this study has limitations. While it primarily focused on elucidating NTZF\u0026rsquo;s role in restoring ovarian proBDNF/mBDNF balance, only preliminary evidence was provided for its cerebral effects in alleviating \"heart depression\". Therefore, future research should integrate behavioral science, gene editing, and other methodologies to systematically investigate NTZF\u0026rsquo;s therapeutic effects on brain tissue. Such efforts will enable a more comprehensive and scientifically rigorous explanation of NTZF\u0026rsquo;s \"heart-kidney treatment\" principle in POI management.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, this study investigated the therapeutic effects of NTZF on CTX-induced POI rat models. The results demonstrated that NTZF exerted therapeutic benefits through multiple mechanisms: regulating abnormal sex hormone levels, promoting follicular development, reducing follicular atresia, and improving ovarian function in POI rats. Furthermore, we elucidated the therapeutic mechanism of NTZF via its modulation of the PAI-1/tPA signaling pathway, which corrects the proBDNF/mBDNF imbalance and inhibits OGCs apoptosis. Notably, our study provides preliminary evidence that NTZF regulates cerebral proBDNF/mBDNF balance to alleviate \"heart depression\" manifestations in POI. These findings not only offer novel mechanistic insights into the \"simultaneous heart-kidney treatment\" principle for POI but also position NTZF as a promising multi-target therapeutic agent for POI management.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePOI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003epremature ovarian insufficiency\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNTZF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNingxin-Tongyu-Zishen formula\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCTX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCyclophosphamide\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFSH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003efollicle-stimulating hormone\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eE\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eestrogen\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAMH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eanti-M\u0026uuml;llerian hormone\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eluteinizing hormone\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eOGCs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eovarian granulosa cells\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ephosphoramide mustard\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePOF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003epremature ovarian failure\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHRT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ehormone replacement therapy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eBDNF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ebrain-derived neurotrophic factor\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTCM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003etraditional Chinese medicine\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSPF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003especific pathogen-free\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSprague-Dawley\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003enormal saline\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePFA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eParaformaldehyde\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eElisa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eenzyme-linked immunosorbent assay\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ehematoxylin-eosin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAOD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eaverage optical density\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eIF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eImmunofluorescence\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eANOVA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eone-way analysis of variance\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMMP2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ematrix metallopeptidase2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMMP9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ematrix metallopeptidase9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003etPA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003etissue plasminogen activator\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\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\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data and materials are available in the manuscript, further inquiries can be directed to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Zhejiang Province Traditional Chinese Medicine Modernization Project [grant numbers: 2022ZX011].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJiawen Ma, Yizhou Zhang conceived and designed the experiments. Jiawen Ma, Chaofan Zhu, Lifang Xie performed the experiments and drafted the manuscript. Shuaiqi An analyzed the data. Zaiyang Zhang, Keying Wang helped in performing the analysis with constructive discussions. Yizhou Zhang provided financial support. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe appreciate experimental support from the Medical Research Center, Academy of Chinese Medical Sciences, Zhejiang Chinese Medical University.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe supplementary materials related to this article are shown in the annex.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePanay N, Anderson RA, Bennie A, Cedars M, Davies M, Ee C, et al. Evidence-based guideline: Premature Ovarian Insufficiency. Hum Reprod Open. 2024(4):hoae065.\u003c/li\u003e\n\u003cli\u003eLi M, Zhu Y, Wei J, Chen L, Chen S, Lai D. The global prevalence of premature ovarian insufficiency: a systematic review and meta-analysis. Climacteric. 2023;26(2):95-102.\u003c/li\u003e\n\u003cli\u003eTouraine P, Chabbert-Buffet N, Plu-Bureau G, Duranteau L, Sinclair AH, Tucker EJ. Premature ovarian insufficiency. Nat Rev Dis Primers. 2024;10(1):63.\u003c/li\u003e\n\u003cli\u003eKuang X, Tang Y, Xu H, Ji M, Lai D. The Evaluation of Ovarian Function Recovery Following Treatment of Primary Ovarian Insufficiency: A Systematic Review. Front Endocrinol (Lausanne). 2022;13:855992.\u003c/li\u003e\n\u003cli\u003eYan X, Zhang Z, Ma J, Zhang Y. Ningxin Tongyu Zishen Tang in Treatment of Premature Ovarian Insufficiency: A Retrospective Control Study. Clinical Complementary Medicine and Pharmacology. 2023;3(1):100067.\u003c/li\u003e\n\u003cli\u003eMa JW, Xiong ZY, Cai XC, Li X, Ren SY, An SQ, et al. Ningxin-Tongyu-Zishen formula alleviates the senescence of granulosa cells on D-galactose-induced premature ovarian insufficiency mice. Aging (Albany NY). 2024;16(5):4541-62.\u003c/li\u003e\n\u003cli\u003eDitmer M, Gabryelska A, Turkiewicz S, Sochal M. Investigating the Role of BDNF in Insomnia: Current Insights. Nat Sci Sleep. 2023;15:1045-60.\u003c/li\u003e\n\u003cli\u003eCzyzyk A, Filipowicz D, Podfigurna A, Ptas P, Piestrzynska M, Smolarczyk R, et al. Brain-derived neurotrophic factor (BDNF) plasma concentration in patients diagnosed with premature ovarian insufficiency (POI). Gynecol Endocrinol. 2017;33(5):413-7.\u003c/li\u003e\n\u003cli\u003eYi X, Yang Y, Zhao Z, Xu M, Zhang Y, Sheng Y, et al. Serum mBDNF and ProBDNF Expression Levels as Diagnosis Clue for Early Stage Parkinson\u0026apos;s Disease. Front Neurol. 2021;12:680765.\u003c/li\u003e\n\u003cli\u003eAnderson RA, Bayne RA, Gardner J, De Sousa PA. Brain-derived neurotrophic factor is a regulator of human oocyte maturation and early embryo development. Fertil Steril. 2010;93(5):1394-406.\u003c/li\u003e\n\u003cli\u003eZhang Y, Chen D, Wang D, Wang L, Weng Y, Wang H, et al. Moderate Aerobic Exercise Regulates Follicular Dysfunction by Initiating Brain-Derived Neurotrophic Factor (BDNF)-Mediated Anti-Apoptotic Signaling Pathways in Polycystic Ovary Syndrome. J Clin Med. 2022;11(19):5584.\u003c/li\u003e\n\u003cli\u003eYang L, He Z, Hu L, Tang H, Geng Y, Tan Q, et al. Ti(3)C(2) nanosheet-induced autophagy derails ovarian functions. J Nanobiotechnology. 2024;22(1):242.\u003c/li\u003e\n\u003cli\u003eClark KL, Keating AF. Ataxia-telangiectasia mutated coordinates the ovarian DNA repair and atresia-initiating response to phosphoramide mustard. Biol Reprod. 2020;102(1):248-60.\u003c/li\u003e\n\u003cli\u003eSano K, Matsuda S, Tohyama S, Komura D, Shimizu E, Sutoh C. Deep learning-based classification of the mouse estrous cycle stages. Sci Rep. 2020;10(1):11714.\u003c/li\u003e\n\u003cli\u003eLiu M, Zhang D, Zhou X, Duan J, Hu Y, Zhang W, et al. Cell-free fat extract improves ovarian function and fertility in mice with premature ovarian insufficiency. Stem Cell Res Ther. 2022;13(1):320.\u003c/li\u003e\n\u003cli\u003eChen S, Lu Y, Chen Y, Xu J, Chen L, Zhao W, et al. The effect of Bu Shen Huo Xue Tang on autoimmune premature ovarian insufficiency via Modulation of the Nrf2/Keap1 signaling pathway in mice. J Ethnopharmacol. 2021;273:113996.\u003c/li\u003e\n\u003cli\u003eHuang Y, Hu R, Liu Z, Geng Y, Li F, Song Y, et al. Bushen Huoxue recipe ameliorates ovarian function via promoting BMSCs proliferation and homing to ovaries in POI mice. Phytomedicine. 2024;129:155630.\u003c/li\u003e\n\u003cli\u003eChen JL, Zhou X, Liu BL, Wei XH, Ding HL, Lin ZJ, et al. Normalization of magnesium deficiency attenuated mechanical allodynia, depressive-like behaviors, and memory deficits associated with cyclophosphamide-induced cystitis by inhibiting TNF-\u0026alpha;/NF-\u0026kappa;B signaling in female rats. J Neuroinflammation. 2020;17(1):99.\u003c/li\u003e\n\u003cli\u003eZhang K, Wang F, Zhai M, He M, Hu Y, Feng L, et al. Hyperactive neuronal autophagy depletes BDNF and impairs adult hippocampal neurogenesis in a corticosterone-induced mouse model of depression. Theranostics. 2023;13(3):1059-75.\u003c/li\u003e\n\u003cli\u003eZarza-Rebollo JA, L\u0026oacute;pez-Isac E, Rivera M, G\u0026oacute;mez-Hern\u0026aacute;ndez L, P\u0026eacute;rez-Guti\u0026eacute;rrez AM, Molina E. The relationship between BDNF and physical activity on depression. Prog Neuropsychopharmacol Biol Psychiatry. 2024;134:111033.\u003c/li\u003e\n\u003cli\u003eChow R, Wessels JM, Foster WG. Brain-derived neurotrophic factor (BDNF) expression and function in the mammalian reproductive Tract. Hum Reprod Update. 2020;26(4):545-64.\u003c/li\u003e\n\u003cli\u003eOjeda SR, Romero C, Tapia V, Dissen GA. Neurotrophic and cell-cell dependent control of early follicular development. Mol Cell Endocrinol. 2000;163(1-2):67-71.\u003c/li\u003e\n\u003cli\u003eBao Z, Li J, Cai J, Yao S, Yang N, Yang J, et al. Plasma-derived exosome miR-10a-5p promotes premature ovarian failure by target BDNF via the TrkB/Akt/mTOR signaling pathway. Int J Biol Macromol. 2024;277(Pt 1):134195.\u003c/li\u003e\n\u003cli\u003eBai YY, Ruan CS, Yang CR, Li JY, Kang ZL, Zhou L, et al. ProBDNF Signaling Regulates Depression-Like Behaviors in Rodents under Chronic Stress. Neuropsychopharmacology. 2016;41(12):2882-92.\u003c/li\u003e\n\u003cli\u003eSeo SY, Bang SK, Kang SY, Cho SJ, Choi KH, Ryu YH. Acupuncture Alleviates Anxiety and 22-kHz Ultrasonic Vocalizations in Rats Subjected to Repeated Alcohol Administration by Modulating the Brain-Derived Neurotrophic Factor/Corticotropin-Releasing Hormone Signaling Pathway. Int J Mol Sci. 2021;22(8):4037.\u003c/li\u003e\n\u003cli\u003eWang M, Xie Y, Qin D. Proteolytic cleavage of proBDNF to mBDNF in neuropsychiatric and neurodegenerative diseases. Brain Res Bull. 2021;166:172-84.\u003c/li\u003e\n\u003cli\u003eYesilkaya UH, Gica S, Menekseoglu PO, Tasdemir BG, Cirakli Z, Karamustafalioglu N. Can the Imbalance between Neurotrophic and Apoptotic Proteins Be the \u0026quot;Beware the Ides of March\u0026quot; for Unaffected Relatives of Schizophrenia Patients? .Mol Neurobiol. 2022;59(12):7413-22.\u003c/li\u003e\n\u003cli\u003eDorandish S, Atali S, Ray R, Al Khashali H, Coleman KL, Guthrie J, et al. Differences in the Relative Abundance of ProBDNF and Mature BDNF in A549 and H1299 Human Lung Cancer Cell Media. Int J Mol Sci. 2021;22(13):7059.\u003c/li\u003e\n\u003cli\u003eTsantarliotou MP, Lavrentiadou SN, Psalla DA, Margaritis IE, Kritsepi MG, Zervos IA, et al. Suppression of plasminogen activator inhibitor-1 (PAI-1) activity by crocin ameliorates lipopolysaccharide-induced thrombosis in rats. Food Chem Toxicol. 2019;125:190-7.\u003c/li\u003e\n\u003cli\u003eZhang F, Luo J, Zhu X. Ketamine ameliorates depressive-like behaviors by tPA-mediated conversion of proBDNF to mBDNF in the hippocampus of stressed rats. Psychiatry Res. 2018;269:646-51.\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":"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":"premature ovarian insufficiency, Ningxin-Tongyu-Zishen formula, proBDNF, mBDNF, PAI-1, tPA","lastPublishedDoi":"10.21203/rs.3.rs-6810873/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6810873/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePremature ovarian insufficiency (POI) is a refractory gynecological endocrine disorder. Ningxin-Tongyu-Zishen formula (NTZF), developed based on the \"simultaneous heart-kidney treatment\" principle, demonstrates efficacy in treating POI, potentially through regulating proBDNF/mBDNF balance. This study aimed to elucidate the molecular mechanism by which NTZF treats POI via proBDNF/mBDNF modulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePOI rat models were established using cyclophosphamide (CTX). The therapeutic effects of NTZF were evaluated by analyzing estrous cycles, ovarian indices, follicular development, serum sex hormone levels (FSH, E\u003csub\u003e2\u003c/sub\u003e, AMH), and ovarian granulosa cells (OGCs) apoptosis. Following immunofluorescence staining to localize BDNF receptors, proBDNF/mBDNF protein expression was quantified in brain and ovarian tissues. CTX's active metabolite, Phosphoramide mustard (PM), was used to induce KGN cell damage. The regulatory effect of NTZF on proBDNF/mBDNF was investigated and compared with recombinant mBDNF protein. tPA and PAI-1 was screened, and their interactions with NTZF were analyzed. mRNA and protein expression of tPA, PAI-1, and tPA-PAI-1 complexes were assessed via q-PCR and Western Blot.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNTZF composition was characterized and shown to improve ovarian function in POI rats. Its mechanism involves correcting proBDNF/mBDNF imbalance in both brain and ovarian tissues. NTZF achieved this correction through the PAI-1/tPA signaling pathway, thereby inhibiting apoptosis in damaged KGN cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur findings demonstrate that NTZF inhibits PAI-1, reduces tPA-PAI-1 complex synthesis, and enhances tPA-mediated proteolytic conversion of proBDNF to mBDNF. This restores proBDNF/mBDNF balance, suppresses OGCs apoptosis, and ultimately ameliorates POI.\u003c/p\u003e","manuscriptTitle":"The mechanism of Ningxin-Tongyu-Zishen formula regulating proBDNF/mBDNF balance through PAI-1/tPA signaling pathway in the treatment of premature ovarian insufficiency","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-19 14:54:26","doi":"10.21203/rs.3.rs-6810873/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-03T16:00:11+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-03T15:35:20+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-30T13:58:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-24T05:24:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"103229906958106071752275735071653515506","date":"2025-06-23T05:43:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4171912930878180783156864765531179789","date":"2025-06-21T09:25:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"167050242793025667584491543357454503271","date":"2025-06-20T14:41:17+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-17T15:52:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-12T00:59:10+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-10T11:40:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Ovarian Research","date":"2025-06-03T11:28:28+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":"7e94b258-06e2-46b5-a5fc-7fc6910d4173","owner":[],"postedDate":"June 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-07-28T21:08:10+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-19 14:54:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6810873","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6810873","identity":"rs-6810873","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}