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

Abstract

Background

Premature ovarian insufficiency (POI) is a refractory gynecological endocrine disorder. Ningxin-Tongyu-Zishen formula (NTZF), developed based on the ‘simultaneous heart-kidney regulation’ principle, exhibits therapeutic 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, E₂, 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. The active metabolite of CTX, phosphoramide mustard (PM), was employed to induce damage in KGN cells. 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 attenuates PAI-1 expression, diminishes tPA-PAI-1 complex formation, and potentiates tPA-dependent proteolysis of proBDNF into mBDNF, thereby restoring their balance. This restores proBDNF/mBDNF balance, suppresses OGCs apoptosis, and ultimately ameliorates POI.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13048-025-01769-1.

Graphical abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s13048-025-01769-1.

Introductin

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 (E₂) 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 patients with POI, 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 regulation’ principle, derived from Yijing Decoction in Fu Qing Zhu’s Obstetrics and Gynecology and Tianwang Buxin Pill in Jiao Zhu Fu Ren Liang Fang. NTZF, as a traditional Chinese medicine formula, demonstrates its core advantage through synergistic multi-component interactions, achieving holistic regulation via the compatibility principles of ‘sovereign (Jun), minister (Chen), assistant (Zuo), and courier (Shi)’. 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 patients with POI 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]. Preliminary clinical trials and animal studies suggest that NTZF may serve as an adjuvant therapeutic option for patients with POI who are unable or unwilling to undergo HRT. However, the mechanism underlying NTZF’s ‘simultaneous heart-kidney regulation’ 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 regulation’ 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 gallic acid, quercetin, adenosine, 5-hydroxymethyl-2-furaldehyde+, magnoflorine, caffeic acid, (+)-catechin, chlorogenic acid, paeoniflorin, ferulic acid, 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 ± 10 g) 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). The rats were housed under controlled conditions with an ambient temperature of 20–25 ℃, relative humidity of 50–65%, and 12 h light/dark cycles, with ad libitum access to food and water. For POI model establishment, cyclophosphamide (CTX, MB1315, meilunbio, China) was intraperitoneally injected at 100 mg/kg on D11 (excluding Control). From D12 to D25, non-Control groups received daily intraperitoneal injections of CTX (8 mg/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 (3 mL/100 g), EV group received EV suspension (3.7 µg/mL), while L-NTZF and H-NTZF groups received NTZF decoctions (0.47 g/mL and 0.94 g/mL) for 28 days. Following 12 h fasting, rats were anesthetized with Zoletil-50 (20 mg/kg). Some ovarian tissues were either fixed in 4% paraformaldehyde (PFA, G1101, Servicebio, China), 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 (h298, iCell, China) 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, China) 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 modelling drug in cell experiments. KGN cells were seeded in a cell culture plate for 12 h, and then treated with DMEM/F12 without 10% FBS for 2 h. Then, PM (HY-137316 A, MCE, USA) with final concentrations of 100, 150, 200, 250 µM were added to each group. After 24–48 h, 100 µL working solution containing 10% cell counting kit-8 (CCK-8, BMU106, Abbkine, China) was added to each well and cultured in a cell incubator at 37 °C for 1 h. 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 µg/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, USA) were 0.1, 1, and 10 ng/mL, while the final intervention concentrations of the PAI-1 agonist Anecortave acetate (AA, HY-116868, MCE) were 12.5, 25, 50, and 100 µM 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, China). 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), E₂ (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 (H&E) 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 5 μm pieces, which were attached to the slides for HE staining.

TUNEL staining

Ovarian tissue sections were dewaxed, and then incubated with 20 µg/mL proteinase K solution at 37 ℃ for 30 min. The endogenous peroxidase in the sections was inactivated by 3% H₂O₂, and then labeled by TUNEL cell apoptosis detection kit (C1098, Beyotime, China). 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 30 min, an appropriate amount of crystal violet staining solution (C0121, Beyotime) was added and incubated in dark for 10 min. 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 15 min, 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 454 nm 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 (HKI0000-2 S, Haoke, China) 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 30 min. 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, Leica, Germany). 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 (ISEQ00010, Millipore, Germany). The PVDF membranes were blocked with 3% skim milk at room temperature for 1 h, and then incubated with primary antibody overnight at 4 ℃. After incubation with secondary antibody at room temperature for 1 h, imagings were performed using ECL developers (BMU102, Abbkine). Antibody information is shown in Supplementary Table 3.

Lentivirus transfection

Three distinct tPA-targeting shRNA lentiviral particles were constructed by Nanjing Corues Biotechnology Co., Ltd. And specific sequences provided in Supplementary Table 4. First, optimal viral titers and puromycin selection concentrations were determined. Concentrated lentiviral particles were then co-cultured with KGN cells for 24 h followed by puromycin selection. Transfection efficiency and cellular morphology were subsequently evaluated by Leica inverted fluorescence microscope (DMi8, Leica, Germany).

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 significantly ameliorates ovarian dysfunction 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. 1A). Notably, model group rats persistently remained in diestrus, indicating abnormal cyclicity. NTZF treatment effectively ameliorated this estrous cycle disruption in POI rats.

Fig. 1.

NTZF 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, E₂, and AMH. (F-H) Representative H&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: *p<0.05, **p<0.01, ***p<0.001, versus Control group; #p<0.05, ##p<0.01, ###p<0.001, versus Model group)

The ovarian index, a key indicator of reproductive health [15], was significantly increased by NTZF treatment in POI rats (Fig. 1B).

Given the characteristic endocrine dysfunction associated with POI, we quantified serum concentrations of FSH, E₂, and AMH. Model group rats exhibited markedly elevated FSH levels alongside reduced E₂ and AMH levels, mirroring clinical POI hormone profiles. NTZF administration reversed these abnormalities (Fig. 1C-E), demonstrating its capacity to restore hormonal balance.

To evaluate follicular development, we quantified follicular stage proportions (Fig. 1F), with specific focus on primordial and atretic follicles (Fig. 1G-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. 1I). Both primordial and growing follicles in model rats exhibited significantly elevated apoptosis, which was mitigated by NTZF intervention (Fig. 1J-K).

In conclusion, NTZF inhibits OGCs 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. 2A), indicating that proBDNF and mBDNF regulate follicular maturation through receptor binding in rat ovaries.

Fig. 2.

NTZF restored proBDNF/mBDNF balance in brain tissue, serum, and ovarian tissue of POI rats. (A) Localization of TrkB and p75^NTR 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: *p<0.05, **p<0.01, ***p<0.001, versus Control group; #p<0.05, ##p<0.01, ###p<0.001, versus Model group)

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. 2B).

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. 2C-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 damaged OGCs and maintain experimental consistency between in vivo and in vitro models, we utilized PM to induce cellular damage. Screening identified 150 µM PM treatment for 48 h as the optimal condition, significantly reducing KGN cell viability and altering cellular morphology (Fig. 3A-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. 3C-H). These parameters defined subsequent in vitro experimental conditions.

Fig. 3.

proBDNF/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 48 h 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^NTR 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: *p<0.05, **p<0.01, ***p<0.001, versus Control group)

IF analysis localized TrkB and p75^NTR receptors in KGN cells (Fig. 3I). Damaged cells exhibited reduced TrkB fluorescence intensity and enhanced p75^NTR signal (Fig. 3J-K), correlating with mRNA/protein expression patterns (Fig. 3L and 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. 3L-N). Collectively, these results establish proBDNF/mBDNF imbalance as a critical mediator of KGN cells apoptosis.

NTZF restores proBDNF/mBDNF balance to mitigate apoptosis in damaged KGN cells

Five NTZF concentrations (4.22 µg/µL, 1.06 µg/µL, 0.26 µg/µL, 0.07 µg/µL, 0.02 µg/µL) were tested and showed no cytotoxicity to KGN cells (Fig. 4A). Among these, 1.06 µg/µL NTZF most effectively restored cell viability in damaged KGN cells (Fig. 4B). Recombinant mBDNF protein (1 ng/mL) exhibited comparable efficacy to NTZF in rescuing viability (Fig. 4D-E). Western Blot analysis further confirmed that NTZF corrected proBDNF/mBDNF imbalance in damaged KGN cells, mirroring the effects of recombinant mBDNF protein (Fig. 4F). Notably, both NTZF and recombinant mBDNF interventions partially restored proliferation levels (Fig. 4G) and significantly suppressed mRNA/protein expression of apoptotic markers (p53, p21, and bax/bcl-2) (Fig. 4H-M). These comparative analyses validate that NTZF inhibits apoptosis in damaged KGN cells by normalizing proBDNF/mBDNF balance.

Fig. 4.

NTZF 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 (∼ 14 kDa). (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: *p<0.05, **p<0.01, ***p<0.001, versus Control group; #p<0.05, ##p<0.01, ###p<0.001, versus Model group; ^p<0.05, ^^p<0.01, ^^^p<0.001, versus mBDNF group)

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. 5A-E). To investigate the functional significance of tPA, we attempted to establish stable tPA-knockdown cell lines using shRNA lentiviral transduction. However, we observed that tPA silencing with different targeting sequences resulted in loss of cell adhesion, proliferation arrest, and abnormal cellular morphology (Supplementary Fig. 2). 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. 5F-G), accompanied by increased tPA-PAI-1 complex formation (Fig. 5H). These findings suggest that PAI-1 overexpression suppresses tPA activity, potentially impairing proBDNF to mBDNF conversion in damaged KGN cells.

Fig. 5.

tPA 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: *p<0.05, **p<0.01, ***p<0.001, versus Control group)

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 50 µM AA causing significant impairment compared to the model group (Fig. 6A). NTZF treatment reversed this viability loss (Fig. 6B) and counteracted AA-induced proliferation inhibition (Fig. 6C-D). Furthermore, NTZF attenuated AA-driven upregulation of apoptotic markers (p53, p21, and bax/bcl2) at both mRNA and protein levels (Fig. 6E-J). Building on prior findings, NTZF also rescued AA-exacerbated proBDNF/mBDNF imbalance in damaged cells (Fig. 6K). Western Blot analysis confirmed that NTZF suppresses PAI-1 expression, reduces tPA-PAI-1 complex synthesis, and restores tPA activity (Fig. 6L-Q).

Fig. 6.

NTZF 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: *p<0.05, **p<0.01, ***p<0.001, versus Control group; #p<0.05, ##p<0.01, ###p<0.001, versus Model group; ^p<0.05, ^^p<0.01, ^^^p<0.001, versus AA group). ( O-Q) Western Blot analysis of PAI-1, tPA, and tPA-PAI-1 protein expression in ovaries of POI rats (n = 5: *p<0.05, **p<0.01, ***p<0.001, versus Control group; #p<0.05, ##p<0.01, ###p<0.001, versus Model group)

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 patients with POI 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 regulation’. 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. Classified within the spleen meridian, Codonopsis Radix, Dioscoreae Rhizoma and Angelicae Sinensis Radix potentiate qi and blood replenishment while synergistically augmenting the therapeutic actions of Rehmanniae Radix Praeparata. 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.

To validate the efficacy of NTZF, this study preliminarily proposes that the POI model employed should encompass both features associated with ‘heart depression’ and the essential phenotypes required for POI models. Literature review revealed that CTX-induced model mice demonstrate significant depression-like behaviours [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 E₂ and AMH levels in POI rats. Collectively, these data substantiate the therapeutic potential of NTZF in protecting ovarian function and mitigating persistent ovarian decline in POI, thereby establishing a foundation for subsequent mechanistic investigations.

The mechanism by which NTZF treats POI through the principle of ‘simultaneous heart-kidney regulation’ requires an elucidation through modern scientific approaches, and the involvement of BDNF provides 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 behaviours [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 substantiate the critical role of BDNF in modulating 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 behaviours [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]. Our findings further confirm that tPA deficiency impairs cell adhesion, suppresses proliferation, and even induces morphological alterations in KGN cells, underscoring the critical role of tPA in these cells. Moreover, 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.

Building on our previous findings, PAI-1 may be a critical target for NTZF in the intervention of POI. HPLC analysis confirmed that NTZF contains well-defined components, including gallic acid, quercetin, adenosine, 5-hydroxymethyl-2-furaldehyde, magnoflorine, and caffeic acid [6]. In prior published work, we demonstrated that gallic acid—one of the primary constituents of NTZF—protects injured human umbilical vein endothelial cells by down regulation of PAI-1 and up regulation of tPA [31]. Additionally, quercetin has also been shown to inhibit PAI-1 [32]. Based on this evidence, we hypothesise that NTZF may regulate the proBDNF/mBDNF balance via the PAI-1/tPA signalling pathway. To test this hypothesis, we introduced the PAI-1 agonist—Anecortave acetate (AA) in cellular experiments to intervene in damaged KGN cells. 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 attenuates PAI-1 expression, diminishes tPA-PAI-1 complex formation, and potentiates tPA-dependent proteolysis of proBDNF into mBDNF, thereby restoring their balance. This mechanism ultimately suppresses OGCs apoptosis and ameliorates POI (Fig. 7). Notwithstanding these findings, several limitations warrant consideration. This study has principally elucidated the role of NTZF in restoring proBDNF/mBDNF balance within ovarian tissue, but the evidence supporting its capacity to rectify cerebral proBDNF/mBDNF imbalance and thereby ameliorate ‘heart depression’ remains preliminary in nature. Therefore, future research should integrate behavioural 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 ‘simultaneous heart-kidney regulation’ principle in POI management.

Fig. 7.

Overview of the mechanism by which NTZF corrects proBDNF/mBDNF imbalance in OGCs. The figure (ID: IWAASccac2) was created by Figdraw (www.figdraw.com)

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 regulation’ principle for POI but also position NTZF as a promising multi-target therapeutic agent for POI management.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

POI

Premature ovarian insufficiency

NTZF

Ningxin-Tongyu-Zishen formula

CTX

Cyclophosphamide

FSH

Follicle-stimulating hormone

E₂

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

Author 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.

Funding

This work was supported by the Zhejiang Province Traditional Chinese Medicine Modernization Project [grant numbers: 2022ZX011].

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Clinical trial number

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.