【摘要】 目的 运用网络药理学、体外实验验证结合分子对接技术探讨17-羟-岩大戟内酯B(HJB)治疗乳腺癌的作用机制。方法 通过利用化合物靶点预测工具PharmMapper数据库和Swiss Target Prediction数据库获取HJB的潜在作用靶点，利用GeneCards、OMIM收集乳腺癌作用相关靶点；筛取HJB作用靶点和疾病靶点的交集靶点，即作为HJB治疗乳腺癌的潜在靶点；利用STRING蛋白互作数据库构建靶点的蛋白质之间相互作用网络，将结果导入生物网络绘图软件Cytoscape进行拓扑分析并提取核心靶点；运用Metascape基因富集在线分析平台对交集靶点进行基因本体(GO)和京都基因与基因组百科全书(KEGG)通路富集分析，构建“成分—靶点—疾病—通路”网络。采用流式细胞术检测HJB对乳腺癌MCF-7细胞的诱导凋亡作用、检测对细胞内线粒体膜电位和活性氧水平的影响；采用Western blot免疫印迹实验检测HJB对相关蛋白表达的影响。最后通过AutoDockTools进行分子对接，验证HJB和核心通路蛋白的相互作用。结果 共获得疾病靶点19 143个、交集靶点368个、核心靶点99个；KEGG和GO富集分析取前20个；流式细胞术结果显示，采用10、20、40μmol/L HJB作用MCF-7细胞48 h后，细胞的总凋亡率显著升高；线粒体膜电位(MMP)水平显著降低；活性氧ROS水平显著升高。Western blot结果显示，经EP处理后的MCF-7细胞内Bax、Cyt C、p-JNK、p-p38、p-PI3K、p-AKT蛋白表达显著升高，而Bcl-2蛋白显著下调。进一步分子对接结果表明HJB与PI3K、AKT、JNK、p38均具有良好的结合活性。结论 HJB可能通过激活JNK/p38 MAPK信号通路和PI3K-AKT信号通路诱导乳腺癌MCF-7细胞凋亡。更多还原

【Abstract】 Objective To investigate the mechanism of 17-hydroxyjolkinolide B(HJB) in the treatment of breast cancer using network pharmacology, in vitro experiments, and molecular docking technology.Methods The potential action targets of HJB were obtained by using the compound target prediction tools PharmMapper database and Swiss Target Prediction database, and the targets related to the action of breast cancer were collected by using GeneCards and OMIM.The intersection targets of HJB’s action targets and disease targets were screened out, as potential targets for HJB in the treatment of breast cancer.The protein-protein interaction network of the target was constructed by using the STRING protein-protein interaction database, and the results were imported into the biological network mapping software Cytoscape for topological analysis and extraction of the core target.The Metascape gene enrichment online analysis platform was used to conduct gene ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analyses on the intersection targets, and a "component-target-disease-pathway" network was constructed.The apoptotic induction effect of HJB on breast cancer MCF-7 cells and its influence on mitochondrial membrane potential and reactive oxygen species levels within the cells were detected by flow cytometry.The effect of HJB on the expression of related proteins was detected by Western blot immunoblotting experiment.Finally, molecular docking was performed through AutoDockTools to verify the interaction between HJB and core pathway proteins.Results A total of 19 143 disease targets, 368 overlapping targets, and 99 core targets were obtained.The top 20 items from KEGG and GO enrichment analyses were selected.Flow cytometry results showed that after treatment with 10,20,and 40 μmol/L HJB for 48 h, the total apoptosis rate of MCF-7 cells was significantly increased, mitochondrial membrane potential was significantly decreased, and ROS levels were significantly elevated.Western blot results showed that after HJB treatment, the protein expression levels of Bax, Cyt C,p-JNK,p-p38,p-PI3K,and p-AKT in MCF-7 cells were significantly increased, while Bcl-2 expression was significantly downregulated.Furthermore, molecular docking results indicated that HJB exhibited good binding affinity with PI3K,AKT,JNK,and p38.Conclusions HJB may induce apoptosis in breast cancer MCF-7 cells by activating the JNK/p38 MAPK signaling pathway and the PI3K-AKT signaling pathway.更多还原