基于网络药理学及实验验证探析雷公藤治疗三阴性乳腺癌的分子机制  

Mechanism of Tripterygium wilfordii in Treatment of Triple Negative Breast Cancer Based on Network Pharmacology and Experimental Validation

作　　者：吴沁航[1] 朱丽文 陈子慧 陶宇洋 万朋 潘扬[1] 冷雪娇[1] WU Qin-hang;ZHU Li-wen;CHEN Zi-hui;TAO Yu-yang;WAN Peng;PAN Yang;LENG Xue-jiao(School of Pharmacy,Nanjing University of Chinese Medicine,Nanjing 210023,China)

机构地区：[1]南京中医药大学药学院,南京210023

出　　处：《中国实验方剂学杂志》2022年第6期131-141,共11页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金青年基金项目(82104451);高等学校博士学科点专项(20133237120011);南京中医药大学国自然青年基金经费配套项目(XPT82104451)。

摘　　要：目的:利用网络药理学预测雷公藤治疗三阴性乳腺癌(TNBC)的有效成分、靶点及相关信号通路,体外细胞模型验证其分子机制。方法:通过中药系统药理数据库(TCMSP)筛选雷公藤活性成分;疾病相关基因与突变位点数据库(DisGeNET)和基因数据库(GeneCards)获取TNBC疾病靶点;Venny平台整合雷公藤治疗TNBC的潜在靶点;String数据库构建蛋白质-蛋白质相互作用(PPI)网络;DAVID数据库进行基因本体(GO)功能注释和京都基因与基因组百科全书(KEGG)富集分析;AutoDock Vina对雷公藤甲素与核心靶点进行分子对接;噻唑蓝(MTT)比色法检测雷公藤甲素(0、5、10、20、30、40、50、60、80 nmol·L^(-1))对MDA-MB-231细胞的增殖抑制活性;Hoechst 33342染色法分析雷公藤甲素(0、12.5、25、50 nmol·L^(-1))诱导MDA-MB-231细胞凋亡;蛋白免疫印迹法(Western blot)检测雷公藤甲素(0、25、50 nmol·L^(-1))对关键靶点的表达调控。结果:预测结果显示雷公藤23个活性成分对应55个TNBC作用靶点,靶点共涉及生物过程103种,细胞组成15种,分子功能35种,参与脂质与动脉粥样硬化、细胞凋亡等细胞信号通路140条;雷公藤甲素与核心靶点蛋白激酶B1(Akt1)、血管内皮生长因子A(VEGFA)、细胞肿瘤抗原p53(p53)、转录因子AP-1(JUN)、信号转导及转录激活因子3(STAT3)、肿瘤坏死因子(TNF)、丝裂原活化蛋白激酶8(MAPK8)、前列腺素G/H合成酶2(PTGS2)、半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)的对接结合活性较高;MTT比色法结果表明,与空白组比较,雷公藤甲素(20、30、40、50、60、80 nmol·L^(-1))能明显抑制三阴性乳腺癌细胞MDA-MB-231的增殖(P<0.05,P<0.01);Hoechst 33342染色结果显示,与空白组比较,雷公藤甲素(12.5、25、50 nmol·L^(-1))MDA-MB-231细胞凋亡率升高(P<0.05,P<0.01);Western blot实验表明,与空白组比较,雷公藤甲素(50 nmol·L^(-1))作用后细胞中p-Akt、VEGFA和TNF-α相对表达水平降低(P<0.05,P<0.01),雷�Objective:To explore the active ingredients,therapeutic targets,and relative signaling pathways of Tripterygium wilfordii in the treatment of triple negative breast cancer(TNBC)based on network pharmacology,and to verify the mechanism through in vitro cell model.Method:The active ingredients of T.wilfordii were screened from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP).The targets of TNBC were obtained from DisGeNET and GeneCards.Venny was used to identify the potential therapeutic targets of T.wilfordii against TNBC.Protein-protein interaction(PPI)network was constructed with String database.Gene ontology(GO)annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment were carried out with DAVID to predict the mechanisms of potential targets.The molecular docking between triptolide and key targets were performed with AutoDock Vina.The effect of triptolide(0,5,10,20,30,40,50,60,80 nmol·L^(-1))on the proliferation of MDA-MB-231 cells was determined through methyl thiazolyl tetrazolium(MTT)assay.The effect of triptolide(0,12.5,25,50 nmol·L^(-1))on the apoptosis of MDA-MB-231 cells was detected with Hoechst 33342 staining.Western blot was performed to detect the effect of triptolide(0,25,50 nmol·L^(-1))on the expression levels of key targets.Result:T.wilfordii had 23 active ingredients related to 55 potential targets of TNBC.GO and KEGG enrichment revealed that the potential targets were associated with 103 biological processes,15 cellular components,and 35 molecular functions,and were involved in 140 signaling pathways including atherosclerosis and apoptosis.The results of molecular docking demonstrated that triptolide could bind with the targets including threonine kinase 1(Akt1),vascular endothelial growth factor A(VEGFA),cellular tumor antigen p53(p53),transcription factor AP-1(JUN),signal transducer and activator of transcription 3(STAT3),tumor necrosis factor(TNF),mitogen-activated protein kinase 8(MAPK8),prostaglandin G/H synthase 2(PTGS2),an

关 键 词：雷公藤 三阴性乳腺癌 网络药理学 分子机制 实验验证 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学]