基于网络药理学方法探析汉黄芩素治疗肝细胞癌作用机制和体外实验研究  

Mechanism and in Vitro Experiment of Wogonin in Treatment of Hepatocellular Carcinoma Based on Network Pharmacology

作　　者：杨安银 刘红丽 陈妙洋 郑玉凤 徐志远 杨永峰 YANG Anyin;LIU Hongli;CHEN Miaoyang;ZHENG Yufeng;XU Zhiyuan;YANG Yongfeng(Department of Liver Disease,the Second Hospital of Nanjing/Nanjing Hospital Affiliated to Nanjing University of Chinese Medicine,Nanjing 210000,China;School of Medicine,Southeast University,Nanjing 210000,China;School of Public Health,Nanjing Medical University,Nanjing 210000,China)

机构地区：[1]南京中医药大学附属南京医院南京市第二医院肝病科,江苏省南京市210000 [2]东南大学医学院,江苏省南京市210000 [3]南京医科大学公共卫生学院,江苏省南京市210000

出　　处：《中国全科医学》2024年第32期4040-4049,共10页Chinese General Practice

基　　金：国家自然科学基金面上项目(81970454)。

摘　　要：背景肝细胞癌是癌症相关死亡的主要原因,目前的防治形势依然严峻,对新的肝细胞癌治疗药物进行探索研究具有科学意义。目的通过网络药理学方法分析汉黄芩素干预肝细胞癌的作用机制,并进行体外实验验证。方法在TCMSP数据库中检索汉黄芩素的药物靶点,从TTD、Gen Card、OMIM、Dis Gent数据库中收集肝细胞癌的疾病靶点。将收集的药物靶点和疾病靶点取交集,作为药物干预疾病的潜在靶点。对交集靶点运用R软件进行富集分析,使用STRING数据库和Cytoscape软件对交集靶点构建蛋白互作网络和筛选核心靶点。在GIEPA数据库对核心靶点行进一步分析。最后通过体外实验对前期分析结果进行验证:采用CCK-8试剂盒测定细胞活性;采用平板克隆形成实验测定细胞增殖;采用划痕实验测定细胞迁移;采用Western-blotting(WB)实验测定蛋白质表达水平。结果分析结果发现汉黄芩素的吸收、分布、代谢、排泄特性符合小分子药物成药规则并且毒性分析结果表明无毒性。收集到汉黄芩素靶点135个,肝细胞癌靶点8238个,两者交集靶点113个。通过对构建的蛋白互作网络筛选出的前10位的核心基因进行分析,发现细胞周期蛋白依赖性激酶1(CDK1)、原癌基因酪氨酸蛋白激酶Src(SRC)在肝细胞癌组织中m RNA水平较正常肝组织上调(P<0.05),并且在肝细胞癌患者中高表达与不良预后相关(P<0.05)。KEGG富集分析发现交集基因富集在PI3K/AKT信号通路上最多,分子对接结果显示汉黄芩素与CDK1、SRC结合构型活力较强。CCK-8试剂盒检测结果显示,加入汉黄芩素75.0、150.0、300.0μmol/L组Hep G2细胞活性均低于对照组(P<0.05);平板克隆形成实验结果显示,加入汉黄芩素37.5、75.0、150.0μmol/L组Hep G2细胞克隆形成数均低于对照组(P<0.05);划痕实验结果表明,加入汉黄芩素37.5、75.0、150.0μmol/L组Hep G2细胞迁移率均低于对照组(P<0.05);WB实Background Hepatocellular carcinoma(HCC)is the leading cause of cancer-related deaths.The current prevention and treatment situation remains critical.It is of scientific significance to explore new therapeutic agents for HCC.Objective To analyze the mechanism of wogonin on HCC by network pharmacology and to verify it in vitro.Methods The drug targets of wogonin were searched in TCMSP database,and the disease targets of HCC were collected from TTD,GenCard,OMIM,DisGent databases.The collected drug targets and disease targets were intersected as potential targets for drug intervention in diseases.R software was used for enrichment analysis of intersection targets,STRING database and Cytoscape software were used to construct protein interaction network and screen core targets.The core targets were further analyzed in GIEPA database.Finally,the preliminary analysis results were verified by in vitro experiments,including cell activity determination using CCK-8 kit,cell proliferation determination using plate clone formation experiment,cell migration determination using scoring test,protein expression level determination using Western-blotting(WB)assay.Results The AMDE characteristics of wogonin were found to be in accordance with the rules for small molecule drug formation and the toxicity analysis showed no toxicity.A total of 135 wogonin targets and 8238 HCC targets were collected,and 113 targets were intersected.Through the analysis of the core genes of TOP10 screened by the constructed protein interaction network,it was found that the mRNA levels of CDK1 and SRC in liver cancer tissues were higher than those in normal liver tissues(P<0.05),and the high expression levels in liver cancer patients were related to poor prognosis(P<0.05).KEGG enrichment analysis showed that the intersection genes were enriched in the PI3K/AKT signaling pathway,and the molecular docking results showed that wogonin had strong binding configuration activity with CDK1 and SRC.The results of CCK-8 kit showed that the activity of HepG2 cells

关 键 词：癌 肝细胞 汉黄芩素 网络药理学 体外实验 

分 类 号：R730.261[医药卫生—肿瘤]