基于网络药理学和体外验证实验探索斑蝥素治疗肝癌的作用机制

Mechanism of Cantharidin in Treating Liver Cancer Based on Network Pharmacology and Its in vitro Experimental Verification

目的:利用网络药理学分析斑蝥素抗肝癌的作用机制,并通过分子对接技术和体外实验进行验证。方法:采用TCMSP数据库和Swiss Target Prediction平台预测斑蝥素的潜在靶点集。通过检索GeneCards和TTD数据库获取肝癌的靶点,取肝癌和斑蝥素靶点的交集,构建疾病药物共同靶点网络图,并依据拓扑网络分析得到斑蝥素抗肝癌的核心靶蛋白。通过DAVID数据库对作用靶点进行GO注释和KEGG通路富集分析,利用AutoDock将核心靶点与斑蝥素进行分子对接,并通过Real-time PCR和Western blot实验对相关的核心靶蛋白及通路进行验证。结果:共筛选出31个共同靶点和12个核心靶点。共同靶基因主要参与调控细胞凋亡、增殖等生物学过程,在蛋白质磷酸化和去磷酸化过程中发挥作用,主要涉及PI3K/Akt、MAPK、p53等信号通路。分子对接结果显示斑蝥素和核心靶点具有较稳定的结合活性。细胞实验表明,斑蝥素能够抑制HepG2细胞的增殖,显著提高TP53、PTEN、ABCB1和PTGS2的mRNA表达,降低PI3K/Akt信号通路中PI3K、Akt的蛋白表达。结论:斑蝥素主要通过上调TP53、PTEN、ABCB1、PTGS2等靶点的mRNA表达,抑制PI3K/Akt信号通路的激活及HepG2细胞的增殖实现抗肝癌的作用。

Objective:To analyze the anti-hepatoma mechanism of cantharidin based on network pharmacology and to verify it by molecular docking technique and in vitro experiments.Methods:The potential targets of cantharidin was predicted by TCMSP database and Swiss Target Prediction platform.The target of liver cancer was obtained by searching GeneCards and TTD databases,and the common target network map of disease drugs was constructed by taking the intersection of liver cancer and cantharidin targets.According to the topological network analysis,the core target protein of cantharidin against liver cancer was obtained.The target was analyzed by GO annotation and KEGG pathway enrichment analysis through DAVID database.The core target was docked with cantharidin by AutoDock,and the related core proteins and pathways were verified by Real-time PCR and Western blot.Results:A total of 31 common targets and 12 core targets were selected.Common target genes were mainly involved in the regulation of biological processes,such as apoptosis and proliferation,and played a role in the process of protein phosphorylation and dephosphorylation,which mainly involved PI3K/Akt,MAPK and p53 signaling pathways.The results of molecular docking showed that cantharidin had stable binding activity to the core target.Cell experiments showed that cantharidin could inhibit the proliferation of HepG2 cells,increase the mRNA expressions of TP53,PTEN,ABCB1 and PTGS2,and decrease the expressions of PI3K and Akt in PI3K/Akt signaling pathway.Conclusion:Cantharidin has an anti-hepatoma effect mainly by up-regulating the mRNA expressions of TP53,PTEN,ABCB1 and PTGS2,inhibiting the activation of PI3K/Akt signaling pathway and the proliferation of HepG2 cells.