黄连活性成分药根碱干预T790M突变EGFR-TKIs耐药非小细胞肺癌的机制研究——基于网络药理学及实验验证  

Study on the Mechanism of Jatrorrhizine, the Active Ingredient of Rhizoma Coptidis, Interfering with T790M Mutant EGFR-TKIs Resistant Non-Small Cell Lung Cancer——Based on Network Pharmacology and Experimental Verification

作　　者：姚楷南 李航 李泽丽 周继红 Yao Kainan;Li Hang;Li Zeli;Zhou Jihong(Baoan District Hospital of Traditional Chinese Medicine,Shenzhen 518000,China;Medical College of Acupuncture-Moxibustion and Rehabilitation,Guangzhou University of Chinese Medicine,Guangzhou 510006,China)

机构地区：[1]宝安区中医院,深圳518000 [2]广州中医药大学针灸康复临床医学院,广州510006

出　　处：《世界科学技术-中医药现代化》2022年第9期3491-3503,共13页Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology

基　　金：国家自然科学基金委员会地区科学基金项目(81760802):虎杖提取物白藜芦醇基于miR-506调控SPHK1/S1P/S1PR3通路抑制肺癌侵袭和转移的研究,负责人:周继红。深圳市科技创新委员会科技计划项目(JCYJ20210324131204012):基于PI3K/Akt/mTOR信号通路探讨药根碱干预T790M突变非小细胞肺癌的机制,负责人:周继红;深圳市科技创新委员会科技计划项目(JCYJ20220530141417038):基于超级增强子驱动的PADI2促组蛋白瓜氨酸化探讨黄连碱抗奥希替尼耐药非小细胞肺癌的机制研究,负责人:姚楷南

摘　　要：目的通过网络药理学及分子对接技术预测黄连活性成分药根碱(Jatrorrhizine)干预表皮生长因子受体酪氨酸激酶抑制剂(epidermal growth factor receptor tyrosine kinase inhibitors,EGFR-TKIs)耐药非小细胞肺癌(non-small cell lung cancer,NSCLC)的效果及作用机制,运用体外细胞学实验进行验证。方法通过Swiss Target Prediction数据库、PharmMapper数据库、TCMSP数据库及PubMed数据库获得黄连活性成分药根碱的潜在靶标,使用OMIM、Gene Cards及DisGeNET数据库收集非小细胞肺癌相关基因,经人工检查剔除假阳性基因后通过Venn工具获取药根碱和非小细胞肺癌的交集基因,通过PubMed数据库进一步验证靶标后导入STRING数据库,构建蛋白-蛋白相互作用(protein-protein interaction,PPI)网络,通过Cytoscape软件进行拓扑分析,通过DAVID数据库进行GO富集分析及KEGG通路分析,通过KEGG PATHWAY数据库比对药根碱靶标与EGFR-TKIs耐药通路之间的关系,预测药根碱干预EGFR-TKIs耐药非小细胞肺癌的直接作用靶标及靶通路。借助Auto Dock软件进行分子对接验证,PyMol软件进行可视化。采用EGFR-TKIs耐药非小细胞肺癌细胞模型H1975细胞系(即T790M突变EGFR-TKIs耐药非小细胞肺癌细胞系)进行细胞学实验,通过CCK-8初步考察药根碱对H1975细胞增殖能力的影响,采用蛋白质印迹法(Western blot,WB)验证药根碱对H1975细胞中部分关键靶蛋白的调控作用。结果共获得药根碱靶标328个,非小细胞肺癌疾病基因3965个,药根碱与疾病交集基因69个,有7个核心靶基因为EGFR-TKIs耐药通路相关基因,富集分析显示PI3K-Akt信号通路为重要靶通路,分子对接结果表明药根碱与PI3K及mTOR之间均有较好的结合活性。药根碱可下调靶蛋白PI3K和mTOR的磷酸化,促进H1975细胞凋亡(P<0.01)。结论黄连活性成分药根碱可能通过PI3K-Akt信号通路发挥抗T790M突变EGFR-TKIs耐药非小细胞肺癌的效用。Objective The network pharmacology and molecular docking technology were used to predict the effect and mechanism of Jatrorrhizine,an active ingredient of Rhizoma Coptidis,on the intervention of epidermal growth factor receptor tyrosine kinase inhibitors(EGFR)-resistant non-small cell lung cancer(NSCLC),and the cytological experiment in vitro was used for verification.Methods Swiss Target Prediction database,PharmMapper database,TCMSP database and PubMed database were used to obtain the potential target of rhizobine,the active ingredient of Coptis chindis.The target genes of NSCLC were collected by OMIM,Gene Cards and DisGeNET database.After the false positive genes were removed by manual inspection,the intersection genes of Jatrorrhizine and NSCLC were obtained by Venn tool.The target was further verified by PubMed database and then imported into STRING database to construct PPI network.Topological analysis was conducted by Cytoscape software.GO enrichment analysis and KEGG pathway analysis were conducted by David database.KEGG PATHWAY database was used to compare the relationship between Jatrorrhizine targets and EGFR-TKIs resistance pathway,to predict the direct-action targets and target pathways of Jatrorrhizine intervention in EGFR-TKIs resistant NSCLC.Auto Dock software was used for molecular docking verification,and PyMol software was used for visualization.Cytological experiments were performed on H1975cell line(T790M EGFR-TKIS drug-resistant NSCLC cell line),CCK-8 assay was used to investigate the effect of Jatrorrhizine on the proliferation of H1975 cells.WB was used to verify the regulatory effect of Jatrorrhizine on some key target proteins in H1975 cells.Results There were 328 potential targets of Jatrorrhizine,3965 disease genes,and 69 intersection genes of Jatrorrhizine and disease,of which 7 core target genes were genes related to the EGFR-TKIs resistance pathway,enrichment analysis showed that PI3K-Akt signaling pathway was an important target pathway.The results of molecular docking showed that

关 键 词：网络药理学 分子对接 药根碱 靶向药耐药 非小细胞肺癌 

分 类 号：R285[医药卫生—中药学]