目的 通过网络药理学方法和实验验证探讨右归丸治疗卵巢早衰（POF）的潜在作用机制。方法 通过网络药理学分析预测右归丸治疗POF的相关靶点，环磷酰胺（CTX）构建小鼠POF模型进行实验验证，实验分为对照组，模型组，右归丸低、中、高剂量组。通过大鼠血清激素水平、卵巢组织病理学观察等探究药效；通过卵巢组织蛋白激酶B（Akt1）/叉头框蛋白O1（FOXO1）通路相关蛋白荧光强度观察、免疫印迹法对沉寂信息调节因子（SIRT1）/肿瘤蛋白53（TP53）信号通路中的关键蛋白定量分析，研究右归丸改善POF是否与该通路相关。结果 网络药理学分析得出右归丸治疗POF的潜在治疗靶点145个，结合KEGG富集通路分析，其核心靶点可能为TP53、Akt1、SIRT1、FOXO1等。与对照组相比，模型组血清促卵泡激素（FSH）、促性腺激素释放激素（GnRH）水平显著升高（P＜ 0.01），抗穆勒管激素（AMH）水平显著降低（P＜0.01）；卵巢组织卵泡闭锁增加；染色可见模型组大鼠卵巢组织p-丝氨酸和苏氨酸激酶1（p-Akt1）、磷酸化叉头蛋白家族1（p-FOXO1）、B细胞淋巴瘤/白血病-2（Bcl-2）蛋白荧光信号减弱，兔抗人单克隆抗体（Bax）蛋白荧光信号增强； SIRT1表达减少（P＜0.05），Ac-p53表达增加（P＜0.01）。与模型组相比，右归丸各剂量组上述血清激素水平均明显改善（P＜0.05、0.01）；卵巢组织内窦卵泡与闭锁卵泡均可见；免疫荧光染色可见p-Akt1、p-FOXO1、Bcl-2蛋白荧光信号增强，Bax蛋白荧光信号减弱；右归丸高剂量组SIRT1表达较模型组升高（P＜0.05），右归丸各剂量组Ac-p53表达均降低（P＜0.05、0.01）。结论 网络药理学及实验验证发现右归丸能够改善POF大鼠血清激素水平、抑制其病理性卵泡闭锁，其作用可能与影响Akt1/SIRT1/FOXO1/TP53通路相关。

Objective To investigate the potential mechanisms of Yougui Pill (YGP) in treating premature ovarian failure (POF) through network pharmacology and experimental validation. Methods Network pharmacology analysis was employed to predict therapeutic targets of YGP for POF. A cyclophosphamide (CTX)-induced POF mouse model was established and divided into five groups: control, model, low-, medium, and high-doses YGP groups. The therapeutic efficacy of YGP was evaluated through serum hormone levels and ovarian histopathology. The roles of Akt1/FOXO1 and SIRT1/TP53 signaling pathways were investigated via immunofluorescence (IF) staining of pathway-related proteins (p-Akt1, p-FOXO1, Bcl-2, Bax) and quantitative analysis (Western blotting) of SIRT1 and acetylated P53 (Ac-p53). Results Network pharmacology analysis identified 145 potential therapeutic targets of YGP in the treatment of POF. Combined with KEGG pathway enrichment analysis, the core targets may include TP53, Akt1, SIRT1, FOXO1. Compared to the control group, the CTX group exhibited significantly elevated serum FSH and GnRH (P < 0.01), reduced AMH (P < 0.01), increased follicular atresia, weakened IF signals of p-Akt1, p-FOXO1, and Bcl-2, enhanced Bax fluorescence, decreased SIRT1 expression (P < 0.05), and increased Ac-p53 (P < 0.01). Compared to the CTX group, YGP-treated groups showed normalized serum hormones (P < 0.05, 0.01), reduced follicular atresia, enhanced p-Akt1, p-FOXO1, and Bcl-2 signals, attenuated Bax fluorescence, upregulated SIRT1 in YGPH (P < 0.05), and downregulated Ac-p53 across doses (P < 0.05, 0.01). Conclusion YGP ameliorates POF by regulating serum hormones, suppressing follicular atresia, and modulating the Akt1/SIRT1/FOXO1/TP53 pathways, as validated through network pharmacology and experimental studies. Key words: premature ovarian failure; network pharmacology; Yougui Pills; apoptosis; Akt1/FOXO1/SIRT1/TP53 signaling pathway

R285.5

陕西省自然科学基础研究计划项目（2023-JC-YB-733）；陕西中医药大学2021年度研究生质量提升工程专项项目（ZG022）；陕西中医药大学2023年度研究生质量提升工程专项项目（CXSJ202319）