五子衍宗丸通过调控TGF-β1/PI3K/Akt/mTOR通路抑制颗粒细胞凋亡改善卵巢储备功能减退

doi:10.12122/j.issn.1673-4254.2026.09.17

南方医科大学学报 ›› 2026, Vol. 46 ›› Issue (9): 2185-2195.doi: 10.12122/j.issn.1673-4254.2026.09.17

• • 上一篇

五子衍宗丸通过调控TGF-β1/PI3K/Akt/mTOR通路抑制颗粒细胞凋亡改善卵巢储备功能减退

梁云腾¹(), 高倩², 李悦怡¹, 黄菁宇¹, 刘凯鲁¹, 洪艳丽¹, 周惠芳¹, 聂晓伟¹()

^1.南京中医药大学附属医院生殖医学科，江苏南京 210029
^2.宜兴市中医医院妇产科，江苏宜兴 214200

收稿日期:2025-12-28 出版日期:2026-09-20 发布日期:2026-09-30
通讯作者: 聂晓伟 E-mail:15269546010@163.com;niexiao5715@163.com
作者简介:梁云腾，在读硕士研究生，E-mail: 15269546010@163.com
基金资助:
国家自然科学基金(82474567);国家自然科学基金(82074479);江苏省自然科学基金(BK20251964);宜兴市“陶都之光”科技攻关计划项目(2023SF12);江苏省前沿技术研发计划（卫生健康领域）项目(BF2025618);2025年度江苏省中医院院级课题(Y25004)

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

Yunteng LIANG¹(), Qian GAO², Yueyi LI¹, Jingyu HUANG¹, Kailu LIU¹, Yanli HONG¹, Huifang ZHOU¹, Xiaowei NIE¹()

^1.Department of Reproductive Medicine, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing 210029, China
^2.Department of Obstetrics and Gynecology, Yixing Traditional Chinese Medicine Hospital, Yixing 214200, China

Received:2025-12-28 Online:2026-09-20 Published:2026-09-30
Contact: Xiaowei NIE E-mail:15269546010@163.com;niexiao5715@163.com
Supported by:
National Natural Science Foundation of China(82474567)

摘要/Abstract

摘要：

目的探讨五子衍宗丸对卵巢储备功能减退小鼠的治疗作用及潜在机制。方法利用雷公藤多苷建立卵巢储备功能减退小鼠模型，将雌性C57BL/6J小鼠随机分为对照组、模型组、五子衍宗丸低剂量组（五子衍宗丸-L，1.82 g·kg^-1·d^-1）和五子衍宗丸高剂量组（五子衍宗丸-H，3.64 g·kg^-1·d^-1），8只/组，灌胃给药28 d。计算小鼠卵巢指数，通过ELISA检测激素水平，HE染色观察卵巢组织形态。利用RNA-seq筛选五子衍宗丸治疗卵巢储备功能减退的关键信号通路。结合网络药理学，使用多重数据库分析五子衍宗丸的潜在作用靶点与通路。通过免疫组化和Western blotting检测小鼠卵巢组织中TGF-β1/PI3K/Akt/mTOR通路关键蛋白表达，TUNEL染色及Western blotting评估颗粒细胞（GCs）凋亡水平。此外，收集人GCs样本进行Western blotting，验证该通路在卵巢储备功能减退患者中的表达。结果与模型组相比，五子衍宗丸使卵巢指数恢复（P<0.05），抗缪勒管激素及雌二醇水平升高，卵泡刺激素降低（P<0.05），并改善卵巢组织形态，使GCs功能恢复，闭锁卵泡减少。RNA-seq显示PI3K/Akt通路在五子衍宗丸治疗后显著富集。网络药理学显示，TGF-β、PI3K-Akt及凋亡通路是五子衍宗丸潜在作用的关键通路。免疫组化及Western blotting提示五子衍宗丸上调卵巢组织TGF-β1、PI3K、Akt和mTOR蛋白表达（P<0.05），TUNEL染色及Western blotting表明五子衍宗丸抑制GCs凋亡（P<0.05）。卵巢储备功能减退患者GCs中TGF-β1/PI3K/Akt/mTOR通路蛋白表达下调（P<0.05）。结论五子衍宗丸通过上调TGF-β1/PI3K/Akt/mTOR信号通路，抑制GCs过度凋亡，从而改善卵巢储备功能。

关键词: 卵巢储备功能减退, 五子衍宗丸, 颗粒细胞, 凋亡, TGF-β1/PI3K/Akt/mTOR通路

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

梁云腾, 高倩, 李悦怡, 黄菁宇, 刘凯鲁, 洪艳丽, 周惠芳, 聂晓伟. 五子衍宗丸通过调控TGF-β1/PI3K/Akt/mTOR通路抑制颗粒细胞凋亡改善卵巢储备功能减退[J]. 南方医科大学学报, 2026, 46(9): 2185-2195.

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.

图/表 5

图1 五子衍宗丸减轻TWP诱导的卵巢毒性

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.

图2 RNA-seq检测WZYZP处理前后小鼠卵巢颗粒细胞的转录水平

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).

图3 网络药理学探讨五子衍宗丸(WZYZP)治疗卵巢储备功能减退(DOR)的作用机制

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.

图4 五子衍宗丸上调DOR小鼠中TGF-β1、PI3K及Akt的表达

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.

图7 DOR患者颗粒细胞中TGF-β1/PI3K/Akt/mTOR信号通路下调及凋亡相关蛋白上调

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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五子衍宗丸通过调控TGF-β1/PI3K/Akt/mTOR通路抑制颗粒细胞凋亡改善卵巢储备功能减退

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

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