基于网络药理学探讨葛根治疗肝癌的分子机制  

Molecular mechanism of Puerariae Lobatae Radix in treatment of hepatocellular carcinoma based on network pharmacology

作　　者：周玟[1,2] 周元 程晔[2] 许楠 李伟兵[2] 吴承玉[1] 陈妍[2] ZHOU Min;ZHOU Yuan;CHENG Ye;XU Nan;LI Wei-bing;WU Cheng-yu;CHEN Yan(College of Traditional Chinese Medicine•College of Integrated Chinese and Western Medicine,Nanjing University of Chinese Medicine,Nanjing 210046,China;Jiangsu Cancer Hospital&Jiangsu Institute of Cancer Research&the Affiliated Cancer Hospital of Nanjing Medical University,Nanjing 210009,China;Department of General Surgery,the Affiliated Sir Run Run Shaw Hospital of School of Medicine of Zhejiang University,Hangzhou 310016,China)

机构地区：[1]南京中医药大学中医学院•中西医结合学院,江苏南京210046 [2]江苏省肿瘤医院/江苏省肿瘤防治研究所/南京医科大学附属肿瘤医院,江苏南京210009 [3]浙江大学医学院附属邵逸夫医院普外科,浙江杭州310016

出　　处：《中国中药杂志》2020年第17期4089-4098,共10页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金面上项目(81874452);江苏省肿瘤医院院内课题面上项目(ZM201810)。

摘　　要：通过网络药理学和体外细胞实验方法,对葛根治疗肝细胞肝癌(hepatocellular carcinoma,HCC)的主要活性成分及其潜在作用机制进行探讨。运用中药系统药理学数据库与分析平台(TCMSP)获取葛根的主要活性成分及其预测靶点,并通过GeneCards数据库检索获得疾病作用靶点。将疾病及药物预测靶点进行交集,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软件将药物-疾病共同靶点进行关键靶基因GO与KEGG功能富集分析,采用Autodock vina 1.1.2进行分子对接。最后,通过葛根素干预人肝癌细胞SMMC-7402及Huh7细胞增殖的体外实验进行核心靶点和通路的初步验证,采用CCK-8法、EDU酶染色检测细胞的增殖情况,运用Western blot法检测PTEN,PDK1,Akt和GSK3β的表达。该研究筛选出葛根10个活性成分(其中9个活性成分涉及肝癌相关靶点及信号通路),涉及149个肝癌靶点及156个相关信号通路。网络分析结果表明葛根可能通过作用于Akt,IL6,MAPK3,EGFR等关键靶点及PI3K-Akt等关键通路发挥抗肝癌作用。分子对接提示葛根活性成分葛根素、染料木素与大豆苷元与AKT1,MAPK3,MAPK1,CASP3等关键靶点结合性较好,葛根素与AKT1,MAPK3的Vina得分最低且相近。体外细胞实验证实,葛根素对人肝癌细胞的增殖有明显的抑制作用。Western blot结果表明,葛根素可以通过PTEN/Akt/GSK3β信号通路使人肝癌细胞出现PTEN磷酸化表达增加,其下游Akt磷酸化水平下降。该系列研究初步揭示了网络药理学预测并指导实验设计的科学性,证实葛根素可通过阻断PTEN/Akt/GSK3β细胞信号转导通路治疗肝癌,为后续验证葛根治疗肝癌的分子机制研究提供思路。To investigate the potential mechanism of Puerariae Lobatae Radix in the treatment of hepatocellular carcinoma by network pharmacology and in vitro cell experiment.The main active components of Puerariae Lobatae Radix and their predicted targets were obtained from TCMSP,and the disease targets were obtained from GeneCards database.The disease and drug prediction targets were intersected to select the common potential therapeutic targets.The"compound-target-disease"network diagram was constructed in Cytoscape 3.7.1,and the common targets were input into the STRING database to build the PPI network of proteins interaction.GO function and KEGG pathway enrichment analysis on effective targets were performed by using R software.Autodock vina 1.1.2 was used for molecular docking.Finally,the core targets and pathways were preliminarily verified by in vitro experiments.The proliferation of human hepatocellular carcinoma cells was detected by CCK-8 and EDU enzyme staining,and the expressions of PTEN,PDK1,Akt and GSK3 were detected by Western blot.In this study,10 components of Puerariae Lobatae Radix(9 components involved in hepatocellular carcinoma-related targets and signaling pathways),and 149 hepatocellular carcinoma-related targets and 156 signaling pathways were screened out.The results of network analysis indicated that Puerariae Lobatae Radix may play an anti-hepatocellular carcinoma effect on key targets,such as Akt,IL6,MAPK3,EGFR,and key pathways,such as PI3 K-Akt.The results of molecular docking indicated that puerarin,genistein and daidzein had a good binding ability with the key targets such as AKT1,MAPK3,MAPK1 and CASP3,and puerarin had the lowest Vina score with AKT1 and MAPK3 and also similar to them.In vitro cell experiments confirmed that puerarin has a significantly inhibitory effect on the proliferation of human hepatocellular carcinoma cells.Western blot results showed that puerarin could increase the phosphorylation of PTEN in human hepatocellular carcinoma cells through the PTEN/Akt/GSK3βsignaling

关 键 词：网络药理学 葛根 葛根素 肝癌 体外细胞实验 

分 类 号：R285[医药卫生—中药学]