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Abstract：
OBJECTIVE: To investigate the role of the Wuwei Zishen formula (WWZSF) in treating and preventing perimenopausal syndrome (PMS) and to understand its mechanism.
METHODS: Network pharmacology and molecular docking was used to predict active compounds, potential targets, and pathways for PMS treatment using WWZSF. Female Sprague-Dawley (SD) rats were induced with D-galactose (D-gal) to establish a PMS model and treated with Kunbao pill (KBP) and WWZSF. Estrus cycles were observed using vaginal smears. Serum sex hormones were measured using the enzyme-linked immunosorbent assay (ELISA). Histological changes in the uterus and ovaries were evaluated using hematoxylin-eosin staining (HE). Western blot was used to assess the protein expression levels of Cleaved Caspase-3, p62, BAX/Bcl-2, p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR in the uterus and ovaries.
RESULTS: A total of 70 active compounds and 440 potential targets were screened out. Important targets and pathways, including AKT1, Bcl-2, Caspase-3, mTOR, and the PI3K/AKT/mTOR pathways, and molecular docking verified their high affinities to key WWZSF components. In vivo experiments showed that WWZSF can ameliorate the morphological abnormalities of the uterus and ovaries, increase sex hormone levels and organ index, and restore the estrus cycles in PMS rats. Moreover, the western blot results showed decreased Cleaved Caspase-3 and BAX/Bcl-2 protein levels in the ovarian and uterine tissues after WWZSF therapy. Concurrently, there was an increase in the expression of p62 and the ratios of p-AKT/AKT, p-mTOR/mTOR, and p-PI3K/PI3K.
CONCLUSIONS: The PI3K/AKT/mTOR signaling pathway-mediated apoptosis and autophagy pathways may be how WWZSF efficiently reduces PMS.
METHODS: Network pharmacology and molecular docking was used to predict active compounds, potential targets, and pathways for PMS treatment using WWZSF. Female Sprague-Dawley (SD) rats were induced with D-galactose (D-gal) to establish a PMS model and treated with Kunbao pill (KBP) and WWZSF. Estrus cycles were observed using vaginal smears. Serum sex hormones were measured using the enzyme-linked immunosorbent assay (ELISA). Histological changes in the uterus and ovaries were evaluated using hematoxylin-eosin staining (HE). Western blot was used to assess the protein expression levels of Cleaved Caspase-3, p62, BAX/Bcl-2, p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR in the uterus and ovaries.
RESULTS: A total of 70 active compounds and 440 potential targets were screened out. Important targets and pathways, including AKT1, Bcl-2, Caspase-3, mTOR, and the PI3K/AKT/mTOR pathways, and molecular docking verified their high affinities to key WWZSF components. In vivo experiments showed that WWZSF can ameliorate the morphological abnormalities of the uterus and ovaries, increase sex hormone levels and organ index, and restore the estrus cycles in PMS rats. Moreover, the western blot results showed decreased Cleaved Caspase-3 and BAX/Bcl-2 protein levels in the ovarian and uterine tissues after WWZSF therapy. Concurrently, there was an increase in the expression of p62 and the ratios of p-AKT/AKT, p-mTOR/mTOR, and p-PI3K/PI3K.
CONCLUSIONS: The PI3K/AKT/mTOR signaling pathway-mediated apoptosis and autophagy pathways may be how WWZSF efficiently reduces PMS.
摘要：
目的:探讨五味滋肾方(WWZSF)治疗和预防围绝经期综合征(PMS)的作用及机制。
方法：网络药理学和分子对接用于预测活性化合物，潜在目标,以及使用WWZSF治疗PMS的途径。用D-半乳糖（D-gal）诱导雌性Sprague-Dawley（SD）大鼠建立PMS模型，并用昆宝丸（KBP）和WWZSF治疗。使用阴道涂片观察发情周期。使用酶联免疫吸附测定（ELISA）测量血清性激素。使用苏木精-伊红染色（HE）评估子宫和卵巢的组织学变化。Westernblot用于评估裂解的Caspase-3，p62，BAX/Bcl-2，p-PI3K/PI3K的蛋白表达水平，p-AKT/AKT,子宫和卵巢中的p-mTOR/mTOR。
结果：共筛选出70种活性化合物和440种潜在靶标。重要的目标和途径，包括AKT1、Bcl-2、Caspase-3、mTOR、和PI3K/AKT/mTOR通路，和分子对接验证了它们对关键WWZSF组件的高度亲和力。体内实验表明，WWZSF可以改善子宫和卵巢的形态异常，增加性激素水平和器官指数，恢复经前综合症大鼠的发情周期。此外，Westernblot结果显示WWZSF治疗后,卵巢和子宫组织中的cleavedCaspase-3和BAX/Bcl-2蛋白水平降低.同时,p62的表达和p-AKT/AKT的比例增加，p-mTOR/mTOR,和p-PI3K/PI3K。
结论：PI3K/AKT/mTOR信号通路介导的细胞凋亡和自噬通路可能是WWZSF有效降低PMS的途径。
方法：网络药理学和分子对接用于预测活性化合物，潜在目标,以及使用WWZSF治疗PMS的途径。用D-半乳糖（D-gal）诱导雌性Sprague-Dawley（SD）大鼠建立PMS模型，并用昆宝丸（KBP）和WWZSF治疗。使用阴道涂片观察发情周期。使用酶联免疫吸附测定（ELISA）测量血清性激素。使用苏木精-伊红染色（HE）评估子宫和卵巢的组织学变化。Westernblot用于评估裂解的Caspase-3，p62，BAX/Bcl-2，p-PI3K/PI3K的蛋白表达水平，p-AKT/AKT,子宫和卵巢中的p-mTOR/mTOR。
结果：共筛选出70种活性化合物和440种潜在靶标。重要的目标和途径，包括AKT1、Bcl-2、Caspase-3、mTOR、和PI3K/AKT/mTOR通路，和分子对接验证了它们对关键WWZSF组件的高度亲和力。体内实验表明，WWZSF可以改善子宫和卵巢的形态异常，增加性激素水平和器官指数，恢复经前综合症大鼠的发情周期。此外，Westernblot结果显示WWZSF治疗后,卵巢和子宫组织中的cleavedCaspase-3和BAX/Bcl-2蛋白水平降低.同时,p62的表达和p-AKT/AKT的比例增加，p-mTOR/mTOR,和p-PI3K/PI3K。
结论：PI3K/AKT/mTOR信号通路介导的细胞凋亡和自噬通路可能是WWZSF有效降低PMS的途径。
