Wuzi Yanzong Pill ameliorates diminished ovarian reserve in mice by inhibiting granulosa cell apoptosis via the TGF-β1/PI3K/Akt/mTOR pathway

doi:10.12122/j.issn.1673-4254.2026.09.17

Abstract

Abstract:

Objective To investigate the efficacy of Wuzi Yanzong Pill (WZYZP) for improving diminished ovarian reserve (DOR) in mice and explore the underlying mechanism. Methods Female C57BL/6J mice were randomly divided into control group, Tripterygium wilfordii polyglycoside-induced DOR model group, and two WZYZP groups treated with WZYZP at daily doses of 1.82 or 3.64 g/kg for 28 days (n=8). Ovarian index of the mice was calculated, hormone levels were detected with ELISA, and ovarian histopathological changes were observed using HE staining. RNA sequencing was performed to identify the key signaling pathways involved in WZYZP treatment for DOR. Network pharmacology analysis was used to predict the potential targets and pathways of WZYZP. Immunohistochemistry and Western blotting were used to detect ovarian expressions of the key proteins in the TGF‑β1/PI3K/Akt/mTOR pathway. Granulosa cell (GC) apoptosis levels were assessed with TUNEL staining and Western blotting. In addition, human GCs samples were collected from DOR patients for validating the expressions of the TGF‑β1/PI3K/Akt/mTOR pathway using Western blotting. Results In the mouse models of DOR, WZYZP treatment significantly improved the ovarian index, increased anti-Müllerian hormone (AMH) and estradiol (E₂) levels, decreased follicle stimulating hormone (FSH) level, alleviated ovarian pathologies, enhanced GC function, and reduced atretic follicles. RNA-seq revealed significant enrichment of the PI3K/Akt pathway after WZYZP treatment. Network pharmacology analysis suggested that the TGF‑β, PI3K‑Akt, and apoptosis pathways were the key pathways potentially targeted by WZYZP. WZYZP treatment significantly upregulated the expressions of TGF‑β1, PI3K, Akt, and mTOR in mouse ovarian tissues and inhibited GC apoptosis. Western blotting of human GCs further verified the down‑regulation of TGF‑β1/PI3K/Akt/mTOR pathway in DOR patients. Conclusion WZYZP improves ovarian reserve by upregulating the TGF‑β1/PI3K/Akt/mTOR signaling pathway and inhibiting excessive apoptosis of GCs.

Key words: diminished ovarian reserve, Wuzi Yanzong Pill, granulosa cells, apoptosis, TGF-β1/PI3K/Akt/mTOR pathway

Yunteng LIANG, Qian GAO, Yueyi LI, Jingyu HUANG, Kailu LIU, Yanli HONG, Huifang ZHOU, Xiaowei NIE. Wuzi Yanzong Pill ameliorates diminished ovarian reserve in mice by inhibiting granulosa cell apoptosis via the TGF-β1/PI3K/Akt/mTOR pathway[J]. Journal of Southern Medical University, 2026, 46(9): 2185-2195.

Figures/Tables 5

Fig.1 Wuzi Yanzong Pill (WZYZP) alleviates Tripterygium wilfordii polyglycoside-induced ovarian toxicity. A: Animal experimental protocol. B: Comparison of ovarian index among the groups. C-E: ELISA detection of AMH, FSH, and E2 levels in each group. F: HE staining of the ovarian tissues in each group (Scale bar=100 μm or 50 μm). Data are presented as Mean±SD (n=8). DOR: Diminished ovarian reserve; AMH: Anti-Müllerian hormone; E2: Estradiol; FSH:Follicle stimulating hormone. *P<0.05, ***P<0.001 vs CON group, #P<0.05, ##P<0.01, ###P<0.001 vs DOR group.

Fig.2 RNA-seq of transcriptional levels in mice granulosa cells (GCs) before and after WZYZP treatment. A: Volcano plot of differentially expressed genes in mice GCs before and after WZYZP treatment (n=3). B: Heatmap of differentially expressed genes in mice GCs before and after WZYZP treatment (n=3). C: GO analysis of differentially expressed genes (n=3). D: Dotplot of KEGG pathway enrichment analysis (n=3).

Fig.3 Network pharmacology analysis of the mechanisms of WZYZP for treating diminished ovarian reserve (DOR). A: Venn diagram of shared targets between WZYZP and DOR. B: Medicine-component-target-disease map. C: PPI network diagram. D: GO analysis of the shared targets, categorized into Biological Processes (BP), Cellular Components (CC), and Molecular Functions (MF). E: Dotplot of KEGG pathway enrichment analysis.

Fig.4 WZYZP upregulates ovarian expressions of TGF-β1, PI3K, and Akt in DOR mice. A: Immunohistochemistry for TGF-β1, PI3K and Akt in mouse ovaries (Scale bar=50 μm or 20 μm). B-D: Expression levels of TGF-β1, PI3K and Akt in mouse ovaries (Mean±SD, n=5). *P<0.05, **P<0.01, ***P<0.001 vs CON group; #P<0.05, ##P<0.01, ###P<0.001 vs DOR group.

Fig.7 Down-regulation of the TGF-β1/PI3K/Akt/mTOR signaling pathway and up-regulation of apoptosis-related proteins in GCs of DOR patients. A: Protein bands of TGF-β1, PI3K, Akt, and mTOR detected by Western blotting. B: Quantitative analysis of TGF-β1, PI3K, Akt, and mTOR protein expression levels. C: Protein bands of caspase-3, BAX, and Bcl-2 detected by Western blotting. D: Quantitative analysis of caspase3, BAX, and Bcl-2 protein expression levels. Data are presented as Mean±SD (n=3). **P<0.01, ***P<0.001 vs CON group.

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