Bushen Huatan formula alleviates polycystic ovary syndrome in rats by activating the PI3K/Akt pathway to inhibit GSDMD-mediated pyroptosis and mitochondrial damage

Abstract

Background

Previous studies suggest that the traditional Chinese medicinal formula Bushen Huatan Fang (BSHT) possesses the capacity to modulate hormonal levels in patients with endocrine and metabolic disorders, indicating its potential clinical utility. This study aims to elucidate the specific mechanisms through which BSHT exerts therapeutic effects in a rat model of polycystic ovary syndrome (PCOS).

Methods

A PCOS model was established in female Sprague-Dawley rats using a high-fat diet and letrozole. Rats were randomly assigned to control, model, positive control (metformin), and BSHT low dose (BSHT-L), medium dose (BSHT-M), and high dose (BSHT-H) groups, with interventions administered for 31 days. Endocrine markers (follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), anti-Müllerian hormone (AMH), insulin resistance (fasting blood glucose (FBG), fasting insulin (INS), homeostasis model assessment of insulin resistance (HOMA-IR)), and ovarian function were assessed by enzyme-linked immunosorbent assay (ELISA), histopathology, and immunohistochemistry. Inflammatory cytokines (interleukin-1 beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α)), lactate dehydrogenase (LDH), mitochondrial function (reactive oxygen species (ROS), mitochondrial membrane potential (MMP), ATPase activity), and pyroptosis markers (generating its GSDMD-N, cleaved caspase-1, IL-1β) were measured by Western blot. Network pharmacology predicted BSHT’s active compounds and targets, and PI3K/Akt pathway modulation was evaluated through protein expression analysis. These methods were used to investigate BSHT’s effects on inflammation, mitochondrial dysfunction, and cellular damage in PCOS.

Results

BSHT effectively ameliorated metabolic, endocrine, and ovarian abnormalities in PCOS rats. It controlled weight gain, reversed endocrine dysfunction (FSH, LH, E2, LH/FSH), and improved ovarian morphology, including increased corpora lutea and reduced cystic follicles. BSHT also improved insulin resistance, as indicated by decreased FBG, FINS, and HOMA-IR levels, and upregulated GLUT4 expression. It restored ovarian reserve, reduced inflammatory markers (IL-1β, IL-6, TNF-α), and improved mitochondrial function by enhancing ATP activity and reducing ROS levels. Furthermore, BSHT activated the PI3K/Akt pathway, significantly reducing pyroptosis and mitochondrial damage, with effects further enhanced by LY294002.

Conclusion

This study shows that BSHT dose-dependently activates the PI3K/Akt pathway, inhibits GSDMD-mediated pyroptosis, and improves mitochondrial dysfunction in PCOS. Its regulation of inflammation, oxidative stress, metabolism, and ovarian function suggests a promising multi-target therapeutic strategy and provides insights for future clinical applications.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13048-025-01887-w.

Introduction

Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder among women of reproductive age and one of the leading causes of female infertility [1]. The etiology of PCOS is multifaceted, resulting in considerable clinical heterogeneity among affected individuals [2]. This complexity arises from the interplay of genetic predisposition, environmental influences, and metabolic dysfunctions, though its precise pathogenic mechanisms remain incompletely elucidated [3]. The core pathological features of PCOS include hyperandrogenism, ovulatory dysfunction, and polycystic ovarian morphology. These abnormalities manifest clinically as menstrual irregularities, hyperandrogenic symptoms, and metabolic derangements such as insulin resistance [4]. These symptoms not only compromise patients’ immediate quality of life but, if unresolved, substantially increase the long-term risk of type 2 diabetes, cardiovascular disease, and endometrial cancer, thereby posing a serious threat to women’s long-term health [5]. Current clinical management is primarily symptom-oriented and tailored to individual fertility needs, involving lifestyle modification, pharmacotherapy, and, in some cases, surgical interventions [6]. While these approaches may alleviate clinical symptoms and improve quality of life, they fall short of offering a definitive cure, often necessitating lifelong treatment and disease management [7]. Therefore, unraveling the underlying pathophysiology of PCOS and developing more efficacious therapeutic strategies has become an urgent medical imperative to improve patient prognosis and quality of life.

Recent studies have revealed that excessive activation of pyroptosis and mitochondrial dysfunction are central to the pathophysiological progression of PCOS and are intricately linked in a vicious cycle that exacerbates disease development [8]. Pyroptosis is a form of programmed cell death initiated by inflammasome activation, whereby inflammatory signals induce maturation of pro-IL-1β into active IL-1β and activate caspase-1 [9]. Activated caspase-1 cleaves gasdermin D (GSDMD), generating its N-terminal fragment (GSDMD-N), which forms pores in the plasma membrane. This results in cell swelling, membrane rupture, and release of proinflammatory cytokines, fueling a robust inflammatory response. When dysregulated, this process aggravates the chronic inflammation seen in PCOS [10]. In parallel, mitochondrial dysfunction—evident by decreased mitochondrial membrane potential (MMP), increased reactive oxygen species (ROS), and aberrant opening of the mitochondrial permeability transition pore (mPTP)—impairs energy metabolism and cellular homeostasis, contributing to cellular dysfunction [11, 12]. Critically, these two pathological processes are not independent but mutually reinforcing: mitochondrial damage releases contents such as cytochrome c, which serve as danger signals that activate inflammasomes and initiate pyroptosis. Conversely, cytokines like IL-1β released during pyroptosis exacerbate oxidative stress, further impairing mitochondrial function [13, 14]. Thus, disrupting this pyroptosis–mitochondrial dysfunction axis may offer a novel therapeutic approach for PCOS.

Recent studies have identified the PI3K/Akt signaling pathway as a critical regulatory axis for cell survival, with its activation potentially disrupting the vicious cycle between pyroptosis and mitochondrial damage—thus offering a novel therapeutic avenue for PCOS intervention [15, 16]. Research indicates that activation of this pathway can downregulate the proinflammatory transcription factor NF-κB, reduce the expression of the pyroptosis executioner protein GSDMD, and prevent the membrane pore formation by its cleaved GSDMD-N [17, 18]. Moreover, the PI3K/Akt pathway can phosphorylate components of the NLRP3 inflammasome or inhibit its assembly, thereby blocking caspase-1 activation and the maturation of pro-IL-1β into active IL-1β, effectively suppressing the initiation of pyroptosis [18, 19]. In addition to these anti-inflammatory effects, Akt activation promotes the expression of the anti-apoptotic protein Bcl-2 while inhibiting the mitochondrial translocation of the pro-apoptotic protein Bax, thereby stabilizing mitochondrial outer membrane permeability. Akt also activates FOXO transcription factors, enhancing the expression of antioxidant enzymes such as superoxide dismutase 2 (SOD2), thus reducing intracellular ROS accumulation [20, 21]. Notably, phosphorylation-dependent inactivation of Akt further leads to reduced activity of glycogen synthase kinase-3β (GSK-3β), which in turn inhibits abnormal opening of the mPTP, preventing cytochrome c release and preserving mitochondrial energy homeostasis [22]. Bushen Huatan Formula (BSHT), a multi-component traditional Chinese herbal compound used clinically in the treatment of PCOS, has demonstrated potential multi-target regulatory effects. Evidence suggests that BSHT may exert its therapeutic benefits via activation of the PI3K/Akt pathway [23]. For instance, prior studies have reported that BSHT activates the IRS-1/PI3K/Akt axis to suppress hyperinsulinemia-driven androgen synthesis in the ovaries, thereby ameliorating insulin resistance and restoring hormonal balance [24]. However, the detailed molecular mechanism through which BSHT exerts its effects on PCOS remains unclear.

Based on these insights, we propose a central hypothesis: BSHT improves the PCOS phenotype in rats by activating the PI3K/Akt signaling pathway, thereby inhibiting GSDMD cleavage and membrane pore formation, suppressing inflammatory cytokine release, restoring MMP, and reducing abnormal mPTP opening. Together, these effects may disrupt the pyroptosis–mitochondrial dysfunction cycle. To test this hypothesis, we established a PCOS model in rats via a high-fat diet combined with letrozole administration. Our experimental findings demonstrate that BSHT treatment significantly activates the ovarian PI3K/Akt pathway, concurrently inhibits GSDMD cleavage and inflammatory cytokine release, and restores mitochondrial function. These results substantiate the mechanistic role of BSHT in directly inhibiting GSDMD-mediated pyroptosis and repairing mitochondrial damage via PI3K/Akt signaling, thereby providing an experimental basis for the development of novel targeted therapies for PCOS.

Materials and methods

Experimental design

Healthy 5-week-old female Sprague-Dawley rats were housed under controlled conditions with a 12-hour light/dark cycle, ambient temperature maintained at 22–25 °C, and relative humidity of 70%. Vaginal smears were conducted daily for 5–10 consecutive days to assess estrous cyclicity. Rats exhibiting two consecutive normal estrous cycles (lasting 4–5 days) were selected for inclusion in the study. The PCOS model was established following previously published protocols [25]. Rats in the experimental groups were fed a high-fat diet and administered letrozole (1 mg/kg/day) via oral gavage for 21 consecutive days. The letrozole suspension was prepared by dissolving letrozole in 0.5% carboxymethylcellulose sodium (CMC-Na).

Part I – BSHT Dose-Response Study: A total of 30 successfully modeled PCOS rats were randomly divided into five groups (n = 6 per group): Control group: Received distilled water via oral gavage. Model group: Received distilled water via oral gavage. Positive control group: Received metformin (300 mg/kg, Cat No. HY-B0627, MedChemExpress, USA) via oral gavage. BSHT low-dose group: Received BSHT at 11.1 g/kg via oral gavage. BSHT medium-dose group: BSHT at 22.2 g/kg via oral gavage. BSHT high-dose group: BSHT at 44.4 g/kg via oral gavage. All treatments were administered at a uniform volume of 5 mL/kg once daily for 31 consecutive days.

Part II – Mechanistic Verification with PI3K/Akt Inhibition: Another cohort of successfully modeled rats was randomly assigned to four groups: Control group: Received distilled water via oral gavage. Model group: Received distilled water via oral gavage. BSHT high-dose group: Received BSHT at 44.4 g/kg via oral gavage. BSHT + LY294002 group: Received BSHT (44.4 g/kg via oral gavage) in combination with the PI3K/Akt pathway inhibitor LY294002 (20 mg/kg, administered via intraperitoneal injection). All treatments in this part also lasted 31 days. BSHT Composition: The BSHT decoction consisted of the following herbal components (doses per formulation): Cuscuta chinensis (30 g), Rubus chingii (30 g), Morus alba (30 g), Psoralea corylifolia (10 g), Cornu Cervi Degelatinatum (10 g), Placenta Hominis (10 g), Poria cocos (20 g), Pericarpium Citri Reticulatae (15 g), Plantago asiatica (15 g), and Cistanche deserticola (15 g).

Body weights of rats were recorded every two days throughout the intervention period. At the end of the experiment, all rats were fasted for 12 h, then euthanized humanely. Blood was collected via cardiac puncture, and serum was immediately snap-frozen in liquid nitrogen for storage. Ovarian tissues were dissected and divided for storage at −80 °C or fixed in 10% formalin for histological analysis. All procedures adhered to the Guidelines for the Care and Use of Laboratory Animals issued by the Ministry of Health of the People’s Republic of China. Biomedical Ethics Committee of Chongqing Three Gorges Medical College (SXYZ-A-2305-0003).

Assessment of endocrine Dysregulation, ovarian reserve and insulin resistance Markers, and fertility restoration in rats

To evaluate endocrine status and ovarian reserve function, serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), and anti-Müllerian hormone (AMH) were measured using rat-specific ELISA kits, including FSH (Cat No. ml059034, Mlbio, China), LH (Cat No. ml059034, Mlbio, China), E2 (Cat No. ml103526, Mlbio, China), and AMH (Cat No. JL12462, JONLNBIO, China). All assays were performed in strict accordance with the manufacturers’ instructions, and optical density values were determined using a microplate reader.

For the assessment of insulin resistance, fasting blood glucose (FBG) levels were measured in each experimental group using a Roche Accu-Chek glucometer, while fasting serum insulin (INS) concentrations were quantified with a rat insulin ELISA kit (Cat No. ml302840, Mlbio, China). The homeostatic model assessment of insulin resistance (HOMA-IR) was subsequently calculated using the formula: HOMA-IR = (FBG × INS)/22.5, where FBG was expressed in mmol/L and INS in mIU/L.

To further verify molecular alterations associated with glucose metabolism and lipid regulation, Western blot analysis was performed. Total protein was extracted from rat tissue samples, quantified by BCA assay, and separated via SDS-PAGE, followed by transfer to PVDF membranes. After blocking, membranes were incubated overnight at 4 °C with primary antibodies against glucose transporter type 4 (GLUT4; Cat No. 21048-1-AP, Proteintech, USA) and fatty acid-binding protein 4 (FABP4; Cat No. 12802-1-AP, Proteintech, USA). Membranes were then incubated with HRP-conjugated secondary antibodies, and protein bands were visualized using enhanced chemiluminescence (ECL). Densitometric analysis was performed to quantify relative protein expression levels.

Histopathological examination

Ovarian and visceral tissues were fixed in 10% neutral buffered formalin, paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H&E; Cat No. G1120, Solarbio, China) following the manufacturer’s protocol. For immunohistochemistry, sections from lung, liver, spleen, kidney, and heart underwent dewaxing, antigen retrieval, peroxidase and serum blocking, incubation with primary and secondary antibodies, and visualization using the Enhanced DAB Substrate Kit (Cat No. DA1016, Solarbio, China) with hematoxylin counterstaining. Nuclei appeared blue, cytoplasm pink to red in H&E, and positive IHC signals were observed as brown-yellow. At least three microscopic fields were examined per sample.

The primary antibodies used included Esr1 (Cat No. EM1708-51, HUABIO, China), Esr2 (Cat No. 340379, Zenbio, China), Fshr (Cat No. ER1909-08, HUABIO, China), BMP15 (Cat No. ET7110-03, HUABIO, China), and LHCGR (Cat No. BS-6431R, Biossm, China). Esr1 was detected using a goat anti-mouse IgG H&L (HRP) secondary antibody (Cat No. 511103, Zenbio, China), while the other primary antibodies (Esr2, Fshr, BMP15, and LHCGR) were detected with a goat anti-rabbit IgG H&L (HRP) secondary antibody (Cat No. 511203, Zenbio, China).

Measurement of inflammatory Cytokines, cellular injury Indicators, and mitochondrial dysfunction markers

To evaluate systemic inflammatory responses, serum and ovarian levels of proinflammatory cytokines were quantified using enzyme-linked immunosorbent assay (ELISA) kits, including Rat IL-1β ELISA Kit (Cat No. EK301B, MultiSciences, China), Rat IL-6 ELISA Kit (Cat No. EK306, MultiSciences, China), and Rat TNF-α ELISA Kit (Cat No. EK382, MultiSciences, China). All assays were performed strictly following the manufacturers’ instructions. Samples and standards were assayed in duplicate, and absorbance was measured at 450 nm using a microplate reader. Cytokine concentrations were calculated based on standard curves. Cellular injury and metabolic activity were assessed by measuring total lactate dehydrogenase (LDH) activity in serum using a Total LDH Assay Kit (Cat No. P0395S, Beyotime, China). Absorbance was determined at 490 nm, and LDH activity was used as an indicator of cell membrane integrity and tissue damage.

To further evaluate mitochondrial function, activities of Ca²⁺-ATP, Ca²⁺-Mg²⁺-ATP, and Na⁺-K⁺-ATP were measured using an ATPase Assay Kit (Cat No. A016-1, Nanjing Jiancheng Bioengineering Institute, China). The principle of the assay is based on the generation of inorganic phosphate (Pi), with absorbance measured at 636 nm and enzyme activity calculated from the standard curve. Mitochondrial dysfunction was further characterized by multiple indicators. mPTP opening was assessed using an MPTP Assay Kit (Cat No. C2009S, Beyotime, China), and changes in fluorescence intensity were used to reflect mitochondrial permeability. ROS levels were determined using a ROS Assay Kit (Cat No. S0035S, Beyotime, China) with the DCFH-DA probe, where fluorescence intensity is positively correlated with intracellular ROS content. MMP was measured by the JC-1 method using a MMP Assay Kit (Cat No. C2001S, Beyotime, China). The red/green fluorescence ratio was observed under a fluorescence microscope or flow cytometer, with decreased membrane potential indicated by reduced red fluorescence and enhanced green fluorescence.

Detection of the inhibitory role of BSHT in pyroptosis associated with PCOS

Western blotting was performed as described above. Primary antibodies included GSDMD-N (Cat No. 20770-1-AP, Proteintech, USA), cleaved caspase-1 (Cat No. 22915-1-AP, Proteintech, USA), and IL-1β (Cat No. A20527, ABclonal, China) for ovarian tissue, with β-actin as the loading control; and GSDMD-N (as above), ACO2 (Cat No. 11134-1-AP, Proteintech, USA), and cytochrome C (Cat No. ET1610-16, HUABIO, China) for mitochondrial fractions, with HSP60 as the loading control.

Network Pharmacology-Based target prediction

Active compounds in the BSHT formula were screened via TCMSP and BATMAN databases using OB ≥ 30% and DL ≥ 0.18, yielding ingredients from ten herbs. Compound structures were validated through PubChem, and potential targets were predicted using SwissTargetPrediction. Disease-related targets for PCOS were retrieved from GeneCards, OMIM, and DisGeNET, and overlapping targets were identified with Venny 2.1. A Herb–Compound–Target network was constructed in Cytoscape 3.7.2, and Protein–Protein Interaction (PPI) analysis was performed using STRING (Homo sapiens, confidence > 0.40). Core targets were defined as Degree ≥ 100. Functional annotation and pathway enrichment of intersecting targets were conducted using DAVID (GO; KEGG; P < 0.05, q < 0.05), and results were visualized with the Bioinformatics platform.

Investigation of BSHT regulation on the PI3K/AKT pathway in PCOS models

Western blotting was used to detect p-PI3K, PI3K, p-AKT, and AKT expression. After cell lysis and BCA quantification, 30 µg protein was separated by SDS-PAGE and transferred to membranes. After blocking, primary antibodies were incubated: p-PI3K (Cat No. HA721672, HUABIO, China), PI3K (Cat No. HA721672, HUABIO, China), p-AKT (Cat No. ET1607-73, HUABIO, China)), AKT (Cat No. ET1607-73, HUABIO, China), β-Actin. HRP-conjugated goat anti-rabbit IgG was used as secondary antibody. Signals were detected by ECL and analyzed with ImageJ.

Investigation of BSHT-Mediated modulation of pyroptosis and mitochondrial dysfunction through the PI3K/Akt pathway in PCOS

Western blotting was performed as described above. Primary antibodies included p-PI3K, PI3K, p-AKT, and AKT for PI3K/AKT signaling pathway proteins; GSDMD-N (Cat No. 20770-1-AP, Proteintech, China), C-caspase-1, and IL-1β for pyroptosis-related proteins; Beclin-1 (Cat No. 11306-1-AP, Proteintech, China), ACO2, and Cytochrome C for autophagy and mitochondrial markers; with β-Actin used as the loading control.

Signals were detected by ECL and analyzed with ImageJ. Simultaneously, cytokine levels of IL-1β, IL-6, TNF-α, total LDH, MDA, and SOD were measured using commercial kits. HRP-conjugated goat anti-rabbit IgG was used as secondary antibody. Signals were detected by ECL and analyzed with ImageJ.

Results

Therapeutic effects of BSHT on PCOS rats

To investigate the comprehensive therapeutic effects of BSHT on PCOS, a systematic evaluation was performed from the aspects of body weight changes, endocrine markers, and ovarian morphological function. PCOS is often associated with typical metabolic abnormalities, especially weight changes; thus, we monitored the body weight of rats in all groups during the treatment period. The results showed (Fig. 1A) that the weight gain trend was effectively controlled under the intervention of BSHT at various doses and in the positive drug group, compared to the model group. To further validate the effect of BSHT on weight changes and exclude potential drug toxicity, we performed HE staining on the five major organs of the rats. The results showed (Figure S1) that no significant lesions were observed in the tissues, indicating that the effects of BSHT on body weight changes were not due to drug toxicity. This initial finding suggests that BSHT has a positive therapeutic effect in mitigating the weight gain induced by PCOS.

Fig. 1.

Comprehensive Therapeutic Effects of BSHT on PCOS. A Body weight changes in rats. B FSH in serum. C LH in serum. D LH/FSH in serum. E E2 in serum. F Mature follicle numbers. G Growing follicle numbers. H Cystic follicle numbers. I Corpus luteum numbers. J Ovarian index. K Ovarian pathological slices of rats. L FBG. M FINS. N HOMA-IR O AMH. Scale bar: 25 μm. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, BSHT group vs. Model group, Metformin group vs. Model group, #p < 0.05 ##p < 0.01, ###p < 0.001, Model group vs. Control group.) (n = 6)

PCOS is also often accompanied by endocrine disorders, and we thus assessed the levels of FSH, LH, and LH/FSH in the serum of the rats. The results indicated (Fig. 1B-E) that following BSHT treatment, abnormalities in FSH, LH, E2, and LH/FSH were reversed, with the BSHT-H group showing the most significant reversal of these abnormalities (P < 0.01), approaching the positive drug group. This suggests that BSHT can effectively reverse endocrine disorders and ovarian reserve dysfunction associated with PCOS.

In addition, ovarian morphological dysfunction is another significant feature of PCOS. To evaluate the impact of BSHT on follicular development disorders in PCOS, we conducted histological observations of the ovaries and statistical analysis of follicles at various developmental stages. The results showed (Fig. 1F-I) that compared to the model group, although there was no significant difference in the number of mature follicles after BSHT intervention, the number of growing follicles increased significantly, the number of cystic follicles decreased markedly, and the number of corpora lutea increased significantly. The BSHT-H group exhibited the best effect in reversing these abnormalities (P < 0.01), suggesting that BSHT can effectively alleviate follicular developmental disorders and promote the restoration of ovulation function.

We also evaluated changes in ovarian indices, which showed similar results (Fig. 1J). BSHT demonstrated a dose-dependent reversal of ovarian index abnormalities caused by PCOS, with the BSHT-H group showing the best reversal effect, approaching that of the positive drug (P < 0.01), indicating that BSHT has a beneficial effect on PCOS-related ovarian morphological changes. Ovarian histopathological examination further confirmed these findings (Fig. 1K), showing structural improvements following BSHT treatment. In the BSHT-L group, follicular morphology restoration and reduced inflammatory infiltration were observed, with more significant improvements in the BSHT-M and BSHT-H groups. The ovarian structure in the BSHT-H group was almost restored to the level of the control group, approaching that of the positive drug.

Taken together, these results demonstrate that BSHT effectively ameliorates metabolic, endocrine, and ovarian morphological abnormalities in PCOS rats, highlighting its therapeutic potential for the management of PCOS.

BSHT improves insulin resistance in PCOS rats

PCOS is often associated with insulin resistance (IR), and thus, we further investigated the effect of BSHT on insulin resistance. The results showed (Fig. 1L-N) that in the PCOS model, FBG, FINS, and HOMA-IR levels were significantly elevated. After the introduction of BSHT, a significant reversal of these abnormal indicators was observed, with the high-dose group showing the best effect, similar to that of the positive drug. These findings indicate that BSHT can effectively overcome the glucose metabolism abnormalities and insulin resistance associated with PCOS.

At the molecular mechanism level, our WB experiments revealed corresponding results (Figure S2A), showing that BSHT significantly upregulated the expression of GLUT4 in a dose-dependent manner, while downregulating the expression of FABP4. The BSHT-H group exhibited the most pronounced regulatory effect. These results collectively suggest that BSHT not only effectively improves glucose metabolism abnormalities and insulin resistance in PCOS, but its action is likely mediated through the regulation of key targets such as GLUT4 and FABP4, which in turn influence glucose uptake and lipid metabolism pathways. This provides important molecular evidence for the multi-target therapeutic mechanism of BSHT.

BSHT restores fertility in PCOS model rats

Based on the improvement of ovarian and serum markers by BSHT, we further assessed its effect on fertility recovery in PCOS rats. We first examined ovarian reserve function, and the results showed (Fig. 1O) that the abnormal upregulation of AMH, an important marker of ovarian reserve, induced by PCOS, was significantly suppressed in a dose-dependent manner by BSHT. The recovery effect in the BSHT-H group was comparable to that of the positive drug, suggesting that BSHT effectively improves ovarian reserve function.

To further investigate, we performed immunohistochemical analysis to evaluate the expression of oocyte growth-related genes (Esr1, Esr2), oocyte maturation-related genes (Bmp15, Lhcgr), as well as follicular development and steroidogenesis-related genes (Fshr) in the ovaries. The results indicated (Figure S2B) that in PCOS rats, Esr1, Esr2, Bmp15, and Fshr were abnormally downregulated, while Lhcgr was abnormally upregulated. However, after BSHT treatment, the abnormal expression of these genes was significantly reversed, similarly to the positive drug group. The reversal effect in the BSHT-H group was comparable to that of the positive drug. These results suggest that BSHT may improve the ovarian microenvironment for follicular development by regulating the expression of local key genes in the ovaries, thus providing a basis for the recovery of ovarian function and fertility.

BSHT improves inflammatory damage in PCOS rats

BSHT effectively alleviated the inflammation associated with PCOS. The levels of inflammatory factors IL-1β, IL-6, and TNF-α in the serum of the model group were significantly higher than those in the control group (P < 0.001) (Figs. 2A-C). The positive drug group and the medium and high-dose BSHT groups were able to significantly reduce the serum levels of these inflammatory factors (P < 0.001). However, except for the BSHT low-dose group, which showed no significant change in serum IL-1β levels, the BSHT low-dose group still demonstrated a significant reduction in the levels of IL-6 and TNF-α (P < 0.01). In the ovarian tissue of the rats, BSHT exhibited similar effects (Figs. 2C-F). Both the positive drug group and all BSHT dose groups were able to effectively reverse the increase in IL-1β, IL-6, and TNF-α levels caused by the model group (P < 0.05). Additionally, in ovarian tissue (Fig. 2G), PCOS also led to a significant increase in total LDH activity, and both the positive drug and BSHT treatments significantly reduced total LDH activity (P < 0.001). Furthermore, in both rat serum and ovarian tissue, the inhibitory effect of BSHT on these inflammatory factors increased with the dose, with the high-dose group showing effects comparable to those of the positive drug group. These results indicate that BSHT has a beneficial impact on improving inflammatory damage in PCOS rats, providing a foundation for treating inflammation-related pathological changes in PCOS.

Fig. 2.

The effect of BSHT on inflammatory damage in PCOS rats. A-C Serum inflammatory factor levels, A IL-6, B TNF-α, C IL-1β. D-F Inflammatory factor levels in ovarian tissue, D IL-6, E TNF-α, F IL-1β. G Serum LDH (lactate dehydrogenase) level, a marker of cell damage. (*p< 0.05, **p < 0.01, ***p < 0.001 BSHT group vs. Model group, Metformin group vs. Model group, #p < 0.05 ##p < 0.01, ###p < 0.001, Model group vs. Control group.) (n=6)

BSHT improves mitochondrial function in PCOS rats

Further analysis of mitochondrial function in rats revealed significant changes. Enzyme activity assays (Figs. 3A-C) showed that the mitochondrial Ca²⁺-ATP, Ca²⁺-Mg²⁺-ATP, and Na⁺-K⁺-ATP enzyme activities in the model group were significantly lower than those in the control group (P < 0.01). The positive drug group and the medium and high-dose BSHT groups exhibited significantly higher mitochondrial Ca²⁺-ATP enzyme activity compared to the model group (P < 0.05). MMP measurements (Fig. 3D) showed that, compared to the control group, the model group had a significant decrease in mitochondrial membrane potential (P < 0.001). Both the positive drug group and BSHT treatments significantly restored mitochondrial membrane potential (P < 0.05). ROS levels in mitochondria (Fig. 3E) were significantly higher in the model group than in the control group (P < 0.001). In contrast, the positive drug group and the medium and high-dose BSHT groups showed a significant decrease in ROS levels compared to the model group (P < 0.01). The extent of mPTP opening (Fig. 3F) in the model group showed a similar trend. Additionally, BSHT exhibited a dose-dependent effect, with the high-dose BSHT group showing the most pronounced improvement. In conclusion, these results demonstrate that BSHT provides significant protection and improvement of mitochondrial function in PCOS rats.

Fig. 3.

The effect of BSHT on mitochondrial function in PCOS rats. A-C Mitochondrial enzyme activity, A Ca2+-ATPase activity, B Ca2+-Mg2+-ATPase activity, C K+-Na+-ATPase activity. D Mitochondrial membrane potential (MMP); E Reactive oxygen species (ROS) levels; F Mitochondrial permeability transition pore (mPTP) opening level. (*p < 0.05, **p < 0.01, ***p < 0.001 BSHT group vs. Model group, Metformin group vs. Model group, #p < 0.05 ##p < 0.01, ###p < 0.001 Model group vs. Control group.) (n = 6)

BSHT improves ovarian cell pyroptosis and mitochondrial damage

Building upon the observed effects of BSHT in improving inflammation and mitochondrial function in PCOS rats, this study further analyzed its impact on ovarian cell pyroptosis and the expression of mitochondrial function-related proteins. Expression of ovarian cell pyroptosis-related proteins (Fig. 4A), including GSDMD-N, C-caspase-1, and IL-1β, was significantly elevated in the PCOS model (P < 0.001). After treatment with the positive drug and the medium and high-dose BSHT groups, there was a significant reversal of the expression of these pyroptosis-related proteins (P < 0.05). The reversal effect of BSHT was dose-dependent, with the high-dose group showing the most significant effect, approaching that of the positive drug.

Fig. 4.

The effect of BSHT on ovarian pyroptosis and mitochondrial damage. A Western blot analysis and quantification of protein expression of GSDMD-N, C-caspase-1, and IL-1β; B Western blot analysis and quantification of protein expression of ACO2, GSDMD-N, and Cytochrome C. (*p < 0.05, **p < 0.01, ***p < 0.001 BSHT group vs. Model group, Metformin group vs. Model group, #p < 0.05 ##p < 0.01, ###p < 0.001 Model group vs. Control group.) (n=6)

Similarly, mitochondrial-related protein expression (Fig. 4B) followed a similar trend. In the PCOS pathological model, the expression of mitochondrial damage-related proteins ACO2, GSDMD-N, and Cytochrome C was abnormally elevated (P < 0.001). Both the positive drug group and BSHT were able to reduce the expression of these mitochondrial damage-related proteins to some extent. The low-dose BSHT group showed a less pronounced effect, but the medium and high-dose BSHT groups, along with the positive drug group, significantly downregulated the abnormal expression of these mitochondrial damage-related proteins (P < 0.05). Moreover, BSHT’s ability to downregulate mitochondrial damage-related protein expression showed a dose-dependent effect, with the high-dose group demonstrating the most significant improvement, comparable to the positive drug.Notably, these changes were consistent with the alterations in ovarian function and inflammatory factors induced by PCOS.

In conclusion, BSHT significantly inhibited the expression of ovarian cell pyroptosis-related proteins in the PCOS model rats, alleviated the degree of cell pyroptosis, improved mitochondrial function, reduced the expression of mitochondrial damage-related proteins, and protected the integrity and function of mitochondria, with a dose-dependent effect.

Regulation of the PI3K/AKT pathway by BSHT improves PCOS

Further analysis was conducted to explore the mechanisms through which BSHT improves PCOS. The active ingredient-target genes of BSHT include 450 genes, while PCOS-related genes number 6,009, with 551 common intersecting target genes (Fig. 5A). The Traditional Chinese Medicine (TCM)-compound-disease-target network (Fig. 5B) shows that BSHT’s herbal components affect multiple targets through various active ingredients, thereby influencing potential therapeutic mechanisms related to disease pathways. The Protein-Protein Interaction (PPI) network diagram (Fig. 5C) suggests that key targets for BSHT in the treatment of PCOS may include AKT1, TP53, TNF, IL6, and SRC.Enrichment analysis of the intersecting targets revealed (Fig. 5D) a total of 1,854 GO terms, encompassing three main categories: cellular component (CC), biological process (BP), and molecular function (MF). In CC, 178 terms were identified, primarily localized in the plasma membrane, cytosol, cytoplasm, receptor complex, dendrites, cell surface, perinuclear region of the cytoplasm, neuronal cell body, nucleoplasm, and protein-containing complexes. In BP, 1,351 terms were identified, with primary involvement in processes such as protein phosphorylation, phosphorylation, response to xenobiotic stimulus, peptidyl-serine phosphorylation, negative regulation of apoptotic processes, signal transduction, positive regulation of gene expression, response to hypoxia, protein autophosphorylation, and positive regulation of the MAPK cascade. In MF, 325 terms were identified, focusing on ATP binding, protein serine/threonine kinase activity, protein serine kinase activity, protein kinase activity, protein tyrosine kinase activity, identical protein binding, protein binding, kinase activity, enzyme binding, and nuclear receptor activity.

KEGG pathway enrichment (Fig. 5E) highlighted several signal pathways related to BSHT treatment of PCOS, with cancer-related pathways and the PI3K-Akt signaling pathway being among those with the highest number of enriched genes. This suggests that these pathways may be central to BSHT’s intervention in PCOS. Verification of the PI3K/AKT pathway-related protein expression (Fig. 5F) showed that in the ovarian tissue of rats in the PCOS model group, the expressions of p-PI3K and p-AKT were significantly downregulated (P < 0.05). After treatment with the positive drug and BSHT, the expressions of p-PI3K and p-AKT were significantly increased (P < 0.05). Additionally, BSHT showed a dose-dependent increase in p-PI3K and p-AKT protein expression, with levels higher than those in the control group for p-PI3K expression. The total protein levels of PI3K and AKT in rat ovarian tissue exhibited relatively minor changes across the groups, with no significant differences between the model and control groups. However, compared to the model group, the positive drug group and all BSHT dose groups showed an increase in PI3K expression. In conclusion, based on network pharmacology analysis and Western blotting experiments, BSHT promotes the phosphorylation and activation of the PI3K/Akt pathway in a dose-dependent manner, revealing its multi-target molecular mechanism in improving PCOS.

Fig. 5.

Network pharmacology analysis and PI3K/Akt pathway validation for BSHT in treating PCOS. A Venn diagram of compounds, diseases, and shared targets. B Compound-disease-target network. C Protein-protein interaction (PPI) network of shared compound-disease targets. D GO functional enrichment bubble chart. E KEGG pathway enrichment bubble chart. F Western blot analysis and quantification of PI3K/Akt pathway protein expression. (*p < 0.05, **p < 0.01, ***p < 0.001 BSHT group vs. Model group, Metformin group vs. Model group, #p < 0.05 ##p < 0.01, ###p < 0.001 Model group vs. Control group.) (n = 6)

BSHT regulates cell pyroptosis and mitochondrial damage in PCOS through the PI3K/Akt pathway

An analysis was conducted to investigate the effects of BSHT on cell pyroptosis and mitochondrial damage in PCOS rats through the regulation of the PI3K/Akt pathway. Inflammatory factor levels (Fig. 6A) revealed that serum levels of L-1β, IL-6, and TNF-α were abnormally elevated in the PCOS model group (P < 0.001). After high-dose BSHT treatment, these inflammatory factors were significantly reversed (P < 0.01). However, when the PI3K/Akt pathway inhibitor LY294002 was combined with BSHT treatment, the effect of BSHT on reversing L-1β, IL-6, and TNF-α levels was significantly weakened (P < 0.05). WB analysis of pyroptosis pathway-related protein expression (Fig. 6G) also showed a similar trend. In the PCOS pathological environment, the expressions of pyroptosis-related proteins GSDMD-N, C-caspase-1, and IL-1β were significantly increased (P < 0.001). High-dose BSHT significantly reduced the expression of these pyroptosis-related proteins caused by the PCOS pathological environment (P < 0.01). However, when the PI3K/Akt pathway inhibitor LY294002 was combined with BSHT treatment, the downregulation effect of BSHT on pyroptosis-related proteins was significantly inhibited (P < 0.05).

Fig. 6.

The effect of BSHT combined with the PI3K/Akt pathway inhibitor on inflammation and oxidative stress in PCOS rats, and the exploration of underlying mechanisms. A-C Serum inflammatory factor level detection: IL-1β A, IL-6 B, TNF-α C; D-F Detection of oxidative stress indicators: LDH D, MDA E, SOD F levels; G Western blot analysis and quantification of inflammatory-related protein expression; H Western blot analysis and quantification of mitochondrial function-related protein expression. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, BSHT group vs. Model group, BSHT group vs. Model group, BSHT group vs. BSHT + LY294002, Model group vs. Control group.) (n = 6)

Levels of mitochondrial damage-related factors such as LDH, MDA, and SOD (Fig. 6D) showed significant increases in the PCOS pathological environment (P < 0.001). BSHT was able to significantly reduce the abnormal increase of these mitochondrial damage-related factors caused by PCOS (P < 0.01). However, when combined with the PI3K/Akt pathway inhibitor LY294002, BSHT’s ability to restore mitochondrial damage-related factors was significantly suppressed (P < 0.05). WB analysis of mitochondrial function-related protein expression (Fig. 6H) further confirmed BSHT’s role in mitochondrial damage repair. The results showed that BSHT significantly reversed the abnormal elevation of mitochondrial damage-related proteins ACO2, Beclin-1, and Cytochrome C induced by the PCOS pathological environment (P < 0.01). However, when combined with LY294002 and BSHT treatment, the reversal of the pathological state was significantly suppressed (P < 0.05).

In conclusion, BSHT regulates the PI3K/Akt pathway, significantly reducing the levels of inflammatory factors and oxidative stress in PCOS model rats, inhibiting cell pyroptosis and mitochondrial damage. The combined use of LY294002 further underscores the necessity of PI3K/Akt pathway activation for BSHT’s therapeutic effect, providing deeper experimental evidence for its application in the treatment of PCOS.

Discussion

PCOS is a complex and common endocrine and metabolic disorder affecting women of reproductive age. It is characterized by hyperandrogenism, ovulatory dysfunction, and polycystic ovarian morphology, often accompanied by insulin resistance, chronic low-grade inflammation, and oxidative stress [26, 27]. In recent years, the roles of inflammation and mitochondrial dysfunction in the pathogenesis of PCOS have increasingly attracted attention [28]. To comprehensively simulate the pathological phenotypes mentioned above, this study employed a rat model of PCOS induced by a combination of letrozole and a high-fat diet. Letrozole, a potent non-steroidal aromatase inhibitor, specifically blocks the conversion of androgens to estrogens, significantly elevating serum androgen levels, and stably induces hyperandrogenism, an imbalance in the LH/FSH ratio, and polycystic ovarian changes (increased cystic follicles and stromal hyperplasia), which are core reproductive endocrine abnormalities [29]. The high-fat diet (45%−60% fat as energy) synergistically triggers insulin resistance and lipid metabolism disorders, compensating for the metabolic phenotype deficiencies seen in the single letrozole model [30]. This combined approach has been verified in research to highly replicate the reproductive-metabolic dual pathological features of clinical PCOS [25].Building on this model, we systematically evaluated the therapeutic effects of BSHT on PCOS rats and delved into its potential molecular mechanisms.

PCOS is a complex disorder characterized by both metabolic and reproductive abnormalities, including insulin resistance, endocrine imbalance, and ovarian morphological and functional defects, ultimately leading to reduced fertility [31, 32]. Therefore, therapeutic strategies should comprehensively target metabolic, endocrine, and ovarian dysfunction. In the present study, we systematically evaluated the effects of BSHT in a PCOS rat model and demonstrated significant efficacy across multiple dimensions. At the metabolic level, BSHT not only effectively suppressed abnormal weight gain but also markedly improved insulin resistance, accompanied by upregulation of GLUT4 and downregulation of FABP4. These findings suggest that BSHT may restore insulin signaling through modulation of glucose transport and lipid metabolism, consistent with clinical observations of impaired metabolic pathways in PCOS patients [33]. Endocrinologically, BSHT significantly normalized abnormal levels of LH, FSH, E2, and the LH/FSH ratio, indicating that it may act through regulation of the hypothalamic–pituitary–ovarian (HPO) axis to correct hormonal imbalance and thereby promote ovulation, in line with reports of Chinese herbal formulas exerting dual regulatory effects on endocrine homeostasis [34]. Regarding ovarian function, BSHT increased the numbers of growing follicles and corpora lutea, reduced cystic follicles, and improved ovarian index and histological structure, suggesting an enhanced follicular microenvironment and alleviation of follicular arrest [35]. Moreover, BSHT significantly reduced abnormally elevated AMH levels and reversed aberrant expression of key folliculogenesis- and steroidogenesis-related genes (Esr1, Esr2, Bmp15, Fshr, and Lhcgr), indicating potential restoration of ovarian reserve and fertility through modulation of local ovarian signaling networks [36]. Importantly, unlike conventional single-target agents, the traditional Chinese herbal formula BSHT exerts multi-target and multi-level regulatory effects, providing a more comprehensive therapeutic profile [23, 37]. Collectively, this study highlights the ability of BSHT to ameliorate metabolic, endocrine, and ovarian dysfunction in PCOS rats and underscores its potential translational value as a novel therapeutic approach for PCOS management.

Related studies have revealed that the vicious cycle of pyroptosis and mitochondrial damage in PCOS constitutes the core pathological basis of the disease’s progression. This is manifested by the abnormal elevation of pro-inflammatory factors, direct damage to ovarian tissue, activation of the NF-κB pathway, and the induction of mitochondrial oxidative stress and energy metabolism collapse. The resulting mitochondrial dysfunction, leading to reduced ATP synthesis, membrane potential collapse, and Cytochrome C release, further activates the caspase-1 pathway, triggering pyroptosis. This amplifies the inflammatory cascade, forming a feedback loop of “mitochondrial damage-pyroptosis ”[8, 38]. A key aspect of this process is that mitochondrial ROS induce pyroptosis through the activation of the NLRP3 inflammasome, while IL-1β released during pyroptosis feedback inhibits the electron transport chain activity, exacerbating the damage cycle [9, 39]. As an effective clinical treatment for PCOS, BSHT has shown good efficacy in improving reproductive endocrine disorders and metabolic abnormalities [40]. Based on this pathological mechanism, this study further explored the therapeutic effects of BSHT in intervening with this vicious cycle. The results indicated that after treatment with different doses of BSHT, the formulation dose-dependently significantly reduced the levels of IL-1β, IL-6, and TNF-α in the pyroptosis-inflammation axis. Additionally, BSHT downregulated the expression of GSDMD-N and C-caspase-1, as well as the expression of pyroptosis execution proteins GSDMD-N and activated C-caspase-1. On the mitochondrial functional level, BSHT also enhanced key ATP enzyme activity, restored mitochondrial membrane potential, reduced ROS production, and upregulated ACO2 expression while effectively correcting the mislocalization of Cytochrome C. In conclusion, this study reveals that BSHT can effectively intervene in the vicious cycle of pyroptosis and mitochondrial damage in PCOS. On the one hand, it suppresses the execution proteins of pyroptosis, and on the other hand, it repairs mitochondrial integrity to effectively intervene in this cycle, thereby achieving better therapeutic effects in PCOS.

Based on the potential of BSHT to effectively intervene in the “pyroptosis-mitochondrial damage” vicious cycle in the treatment of PCOS, this study systematically explores its mechanism of action by integrating network pharmacology, molecular validation, and pathway inhibition strategies. The results of the network pharmacology analysis indicate that AKT1 is the most critical target of BSHT in the intervention of PCOS. As a key effector molecule downstream of the PI3K/Akt signaling pathway, AKT1’s phosphorylation state directly regulates the overall activity of this pathway[42], which shifts the research focus to the PI3K/Akt pathway. Subsequent GO and KEGG enrichment analyses further emphasize the central and pivotal role of the PI3K/Akt pathway within the disease regulatory network. Although this pathway has been extensively studied in cancer research, its functional abnormalities have also been identified as one of the core pathogenic mechanisms in PCOS. Studies have shown direct and close associations between the inhibition of PI3K/Akt pathway activity and the core clinical pathological features of PCOS[23,43]. Notably, key active components in BSHT, such as β-sitosterol, kaempferol, and quercetin, have also been confirmed to be associated with the PI3K/Akt pathway through their anti-inflammatory, antioxidant, and metabolic regulation effects: β-sitosterol promotes granulosa cell proliferation and inhibits apoptosis; kaempferol maintains follicle survival rate and promotes oocyte meiosis; and quercetin improves hormonal imbalance and metabolic disorders in PCOS patients[43, 44]. Our findings further suggest that BSHT can effectively and specifically activate the PI3K/Akt pathway, regulate the GSDMD-mediated pyroptosis process, and improve mitochondrial function damage, consistent with the regulatory role of the PI3K/Akt pathway in pyroptosis and mitochondrial damage reported in the literature[45], thus elucidating the mechanism by which BSHT improves the pathological state of PCOS.

Compared with conventional pharmacological agents, the greatest advantage of BSHT lies in its multi-target and multi-pathway regulatory effects. Conventional Western medicines generally act on a single aspect, such as inducing ovulation or regulating endocrine function, but they cannot fully address the multidimensional pathological alterations of PCOS, which include metabolic disorders, hormonal imbalance, and ovarian dysfunction [41]. In contrast, BSHT, composed of multiple active constituents, can simultaneously modulate inflammation, oxidative stress, mitochondrial function, glucose and lipid metabolism, and the HPO axis, thereby providing a more comprehensive therapeutic effect. However, this study also has limitations. The PCOS model was established by letrozole combined with a high-fat diet, which can simulate the reproductive-metabolic dual phenotype of PCOS but cannot fully replicate the complexity and heterogeneity of human PCOS. Thus, the clinical extrapolation of the study results is limited, and further validation in human-derived models (such as PCOS patient-derived organoids) is needed. Moreover, although we identified the PI3K/Akt pathway as a likely mediator, BSHT is a complex formulation containing multiple bioactive compounds. The precise contributions and potential interactions of these individual components to the overall therapeutic effect remain undefined. The relatively short observation period further restricts evaluation of long-term efficacy and safety, and pharmacokinetic characteristics, optimal dosing strategies, and the potential for integration with existing therapies were not explored. These limitations underscore the need for further mechanistic studies and large-scale clinical trials to validate the therapeutic potential of BSHT in women with PCOS.

Conclusion

This study confirms, using a PCOS animal model, that BSHT can dose-dependently activate the PI3K/Akt pathway, leading to the effective inhibition of GSDMD-dependent pyroptosis and concurrent improvement of mitochondrial dysfunction. The improvement in pyroptosis and mitochondrial dysfunction induced by BSHT through the PI3K/Akt pathway is likely closely associated with its regulation of key inflammatory factors or the inhibition of the inflammatory signaling cascade, highlighting inflammation as a crucial bridge connecting pathway activation with improvements in cellular dysfunction. Furthermore, this study suggests that BSHT’s multifaceted mechanism, which includes modulation of oxidative stress, glucose and lipid metabolism, and ovarian function, may offer a comprehensive therapeutic approach for managing the complex reproductive and metabolic disturbances in PCOS. These results not only offer important insights into the pathogenesis of PCOS and the identification of novel therapeutic targets but also provide a rationale for further investigation into multi-target herbal formulations as a potential strategy for managing the complex pathophysiology of PCOS.

Authors’ contributions

Qian Xiong and Jing Yang contributed equally to this work. Qian Xiong: Conceptualization, Funding acquisition, Methodology, Writing-original draft. Jing Yang: Data curation, Formal analysis, Investigation, Visualization. Qingyan Liu: Resources, Validation. Penglong Yu and Mengyue Shen: Investigation, Methodology. Jiao Liang: Supervision, Project administration, Writing-review & editing. All authors reviewed and approved the final manuscript.

Funding

This study was supported by Scientific and Technological Research Program of Chongqing Municipal Education Commission (grant number QN202202719) to Qian Xiong.

Data availability

All the original data of this study are available from the corresponding author upon request.

Declarations

Ethics approval and consent to participate

The animal study was reviewed and approved by Biomedical Ethics Committee of Chongqing Three Gorges Medical College (SXYZ-A-2305-0003).

Competing interests

The authors declare no competing interests.