基于网络药理学和实验验证分析鸡血藤治疗卵巢癌的分子机制

Molecular mechanism of Spatholobi Caulis in treatment of ovarian cancer based on network pharmacology and experimental verification

通过网络药理学和分子对接及体外细胞实验,对鸡血藤Spatholobi Caulis治疗卵巢癌(ovarian cancer,OC)的主要活性成分及其潜在的作用机制进行探讨。运用中药药理学数据库与分析平台(TCMSP)获取鸡血藤的主要活性成分及其预测靶点,通过GeneCards、OMIM、PharmGkb、TTD、DrugBank数据库检索获得疾病基因。将疾病及药物预测靶点进行交集处理,筛选出共同的潜在治疗靶点。运用Cytoscape 3.7.1软件构建"药物-成分-疾病-靶点"相互作用网络图;运用STRING数据库构建蛋白相互作用的PPI网络。运用R 4.0.5进行GO与KEGG功能富集分析。分析对接虚拟筛选的处理和优化由Schr9dinger Maestro软件完成。该研究筛选出鸡血藤23个活性成分,涉及75个卵巢癌靶点及178个相关信号通路;网络分析结果表明鸡血藤可能通过作用于GSK-3β、Bcl-2、Bax等关键靶点,发挥抗卵巢癌作用;分子对接结果显示GSK-3β与活性成分樱黄素结合活性较好。最后体外实验将筛选出的鸡血藤活性成分樱黄素对人卵巢癌细胞SKOV3进行核心靶点和通路的初步验证。采用CCK-8法检测细胞增殖情况,使用流式细胞仪检测樱黄素对人卵巢癌细胞SKOV3凋亡的影响。运用Western blot法检测樱黄素靶点及相关调控蛋白的表达。体外细胞实验证明,樱黄素对卵巢癌细胞的增殖有明显的抑制作用,并且樱黄素对SKOV3有诱导凋亡的作用。Western blot实验结果表明,樱黄素可以通过抑制GSK-3β磷酸化的发生,调控下游Bcl-2、Bax蛋白的表达,诱导SKOV3细胞凋亡的发生。该研究揭示了网络药理学预测并指导实验设计的科学性,证实樱黄素可通过阻断GSK-3β/Bcl-2/Bax细胞信号转导通路治疗卵巢癌,为鸡血藤治疗卵巢癌的机制研究提供依据。

The present study explored the main active ingredients and the underlying mechanism of Spatholobi Caulisin the treatment of ovarian cancer(OC) by network pharmacology,molecular docking,and in vitro cell experiments.The active ingredients and their predicted targets(AITs) were first acquired online with the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP).Theoretical disease targets(DTs) were obtained through professional databases including GeneCards,OMIM,PharmGkb,TTD,and DrugBank.The common targets in the intersection of AITs and DTs were used for the construction of a "drug-ingredient-disease-target" network by Cytoscape 3.7.1.STRING database was used to construct a protein-protein interaction(PPI) network.R 4.0.5 was used for GO and KEGG functional enrichment analyses.Schr9 dinger Maestro was used to perform and optimize the molecular docking and virtual screening.Twenty-three active ingredients of Spatholobi Caulis were screened out,involving 75 OC targets and 178 signaling pathways.Network analysis revealed that Spatholobi Caulis presumedly exerted an anti-OC effect by acting on key protein targets such as GSK-3β,Bcl-2,and Bax.Molecular docking showed that GSK-3β possessed goodbinding activity to prunetin.In vitro cell experiments preliminarily verified the core targets and pathways of prunetin,the active ingredient of Spatholobi Caulis against human OC SKOV3 cells.CCK-8 assay was used to detect the cell proliferation,and flow cytometry was used to detect the effect of prunetin on apoptosis of human OC SKOV3 cells.The expression of prunetin targets and related regulatory proteins was detected by Western blot.In vitro cell experiments demonstrated that prunetindisplayed significant inhibitory effects on the proliferation of OC cells and could induce apoptosis of SKOV3 cells.Western blot showed that prunetin could induce SKOV3 cell apoptosis by inhibiting GSK-3β phosphorylation and regulating the expression of downstream Bcl-2 and Bax proteins.This study reveals the scientific nature of network pharmacology in the prediction and guidance of experimental design,confirming that prunetin can treat OC by blocking the GSK-3β/Bcl-2/Bax cell signal transduction pathway.The findings are expected to provide a basis for the investigation of the mechanism of Spatholobi Caulis in the treatment of OC.