目的 通过网络药理学和体外实验探讨六味地黄丸治疗乳腺癌的作用机制。方法 利用中药系统药理学数据库与分析平台（TCMSP）获取中药有效活性成分；GeneCards、OMIM等数据库筛选六味地黄丸与乳腺癌的相关靶点，并通过Venny2.1.0取二者交集；Cytoscape软件构建“药物-成分-靶点”网络图，DAVID数据库进行基因本体（GO）注释及京都基因与基因组百科全书（KEGG）通路富集分析；将筛选出的活性成分和核心靶点进行分子对接验证。采用CCK-8法及平板克隆检测细胞活力及增殖能力，细胞划痕实验检测迁移能力，流式细胞技术实验检测凋亡；Western blotting法检测蛋白激酶B(AKT1)、p-丝氨酸和苏氨酸激酶1(p-AKT1)、兔抗人单克隆抗体（Bax）、B细胞淋巴瘤/白血病-2(Bcl-2)、cleaved Caspase-3的蛋白表达水平。结果 通过数据分析共筛选出有效成分39个，交集靶点171个，其核心成分是槲皮素、豆甾醇、山柰酚；通过蛋白质-蛋白质相互作用（PPI）网络图得出核心靶点为TP53、JUN及AKT1;GO分析包含生物过程（BP）772条、细胞组成（CC）80条、分子功能（MF）192条富集结果，KEGG分析共包含155条富集通路；分子对接显示豆甾醇与AKT1结合稳定性最好。细胞实验证明，豆甾醇能够抑制MDA-MB-231细胞的生长并诱导其凋亡，并下调p-AKT1/AKT1的蛋白表达，上调Bax/Bcl-2及cleaved Caspase-3的蛋白表达。结论 六味地黄丸的活性成分豆甾醇能抑制MDA-MB-231细胞增殖并诱导其凋亡，作用机制可能是通过调节PI3K/Akt信号通路而发挥作用。

Objective To explore the mechanism of action of Liuwei Dihuang Pills in treating breast cancer through network pharmacology and in vitro experiments. Methods The effective active components of the traditional Chinese medicine were obtained using TCMSP; The relevant targets of Liuwei Dihuang Pills and breast cancer were screened using GeneCards, OMIM, etc., and the intersection of the two was obtained using Venny2.1.0; The “drug-component-target” network diagram was constructed using Cytoscape software, and GO and KEGG enrichment analysis was performed using the DAVID database; The active components and core targets were verified by molecular docking. The cell viability and proliferation ability were detected using the CCK-8 method and plate cloning, the migration ability was detected using the cell scratch assay, and the apoptosis was detected using flow cytometry; The protein expression levels of AKT1, p-AKT1, Bax, Bcl-2, and cleaved Caspase-3 were detected using Western blotting. Results A total of 39 active components were screened, and 171 intersection targets were obtained. The core components were quercetin, stigmasterol, and kaempferol. The core targets were determined to be TP53, JUN, and AKT1 using the protein-protein interaction（PPI） network. GO analysis included 772 biological process（BP）, 80 cellular component（CC）, and 192 molecular function（MF） enrichment results, and KEGG analysis included 155 enriched pathways. Molecular docking showed that stigmasterol had the best binding stability with AKT1. Cell experiments demonstrated that stigmasterol could inhibit the growth of MDA-MB-231 cells and induce their apoptosis, and down-regulate the protein expression of p-AKT1/AKT1, up-regulate the protein expression of Bax/Bcl-2 and cleaved Caspase-3. Conclusion The active component stigmasterol of Liuwei Dihuang Pills can inhibit the proliferation of MDA-MB-231 cells and induce their apoptosis. The mechanism may be through regulating the PI3K/Akt signaling pathways.

R285.5

安徽省高校优秀青年科研项目(2023AH030114); 安徽省重点研究与开发计划项目(202204295107020023)