基于网络药理学与分子对接和体外试验探讨车前草治疗肝癌的作用机制  

Study on the Mechanism of Plantago asiatica L.in the treatment of liver cancer based on network pharmacology,molecular docking and in vitro experiments

作　　者：聂小雅 吴晓欢 许达 朱金容 NIE Xiaoya;WU Xiaohuan;XU Da;ZHU Jinrong(School of Life Sciences and Biopharmaceutics,Guangdong Pharmaceutical University,Guangzhou 510006,China)

机构地区：[1]广东药科大学生命科学与生物制药学院,广东广州510006

出　　处：《广东药科大学学报》2024年第4期18-26,共9页Journal of Guangdong Pharmaceutical University

基　　金：2023年省级大学生创新创业训练计划项目(202310573018)。

摘　　要：目的采用网络药理学结合分子对接及体外试验验证,探究车前草治疗肝癌的活性成分、关键作用靶点及潜在的分子机制。方法通过TCMSP和SwissTarget Prediction数据库获取车前草主要活性成分及潜在靶点,构建“成分-靶点”网络。检索DisGeNET和GeneCards数据库获取肝癌相关的疾病靶点,运用String数据库结合Cytoscape软件对交集靶点进行PPI网络分析,选取车前草主要活性成分与核心靶点进行分子对接验证。进一步通过体外肝癌Hep3B细胞试验验证车前草活性成分抗肿瘤活性,采用CCK-8试验、平板克隆形成试验及软琼脂克隆形成试验检测增殖抑制作用,采用K-LISAAKT活性试剂盒检测AKT活性水平,采用qRT-PCR检测mRNA的表达。结果车前草的活性成分有山萝花苷、6羟基木犀草素、豆甾醇、黄芩苷、豆甾醇棕榈酸酯、谷甾醇、木犀草素、黄芩素、高车前素和β-谷醇棕榈酸酯;获得车前草和肝癌交集基因168个,PPI网络图表明交集基因之间关系密切,其中潜在靶标为AKT1、TNF、ESR1、EGFR等;富集分析得到GO功能316个,以及相关通路94条,相关的通路主要有PI3K-AKT信号通路等;分子对接结果显示,主要活性成分木犀草素与核心靶点(AKT1、EGFR、TNF、ESR1、SRC、MAPK3)结合稳定。体外试验结果显示,木犀草素对肝癌细胞具有抑制增殖作用,干预肝癌细胞克隆形成以及降低AKT信号通路的活性,且BCL2、FASL等凋亡相关基因mRNA表达升高。结论车前草能够通过多靶点、多通路治疗肝癌,其主要活性成分为木犀草素。木犀草素可能通过抑制AKT1等关键靶点从而抑制PI3K-AKT信号通路,进而达到抑制肝癌细胞增殖、促进凋亡的作用,具有潜在的抗肝癌活性。Objective To explore the active components,key targets and potential molecular mechanisms of Plantago asiatica L.in the treatment of liver cancer by network pharmacology combined with molecular docking and in vitro experiment verification.Methods The main active components and potential targets of P.asiatica L.were obtained from TCMSP and Swiss Target Prediction databases,and a"component-target"network was constructed.The liver cancer-related disease targets were obtained from DisGeNET and GeneCards databases,and the PPI network analysis of the intersection targets was performed by String database combined with Cytoscape software.The GO and KEGG enrichment analysis was performed by DAVID database,and the main active components of P.asiatica L.were selected for molecular docking verification with the core targets.Furthermore,the anti-tumor activity of the active components of P.asiatica L.was verified through in vitro Hep3B cell experiments.The proliferation inhibition effect was detected by CCK-8 experiment,and the tumor formation ability was detected by plate colony formation experiment and soft agar colony formation experiment.The AKT activity level was detected by K-LISA AKT activity kit,and the mRNA expression was detected by qRT-PCR.Results The core active components of P.asiatica L.included melampyroside,6-OH-luteolin,stigmasterol,baicalin,stigmasteryl palmitate,sitosterol,luteolin,baicalein,dinatin,β-sitosteryl palmitate.168 intersection genes between P.asiatica L.and liver cancer were obtained,and the PPI network showed that the intersection genes were closely related,and the potential targets were AKT1,TNF,ESR1,EGFR,etc.316 GO functions and 94 related pathways were obtained by enrichment analysis,and the related pathways mainly included PI3K-AKT signaling pathway,etc..Molecular docking results showed that the main active component luteolin bound stalely with the core target(AKT1,EGFR,TNF,ESR1,SRC,MAPK3).In vitro experiment results showed that luteolin had inhibitory effects on the proliferation of liver

关 键 词：车前草 肝癌 网络药理学 木犀草素 体外试验 

分 类 号：R282[医药卫生—中药学] R735.7[医药卫生—中医学]