文献类型：期刊文献

中文题名：基于网络药理学和体外实验探讨通关藤口服液抑制胃癌细胞增殖及联用厄洛替尼和阿帕替尼的增敏效应

英文题名：The Inhibitory Action and Mechanisms of Marsdenia Tenacissima Oral Liquid on the Proliferation of Gastric Cancer Cells Based on Network Pharmacology and in vitro Experiments and the Sensitization Effect of Combining Erlotinib and Apatinib

作者：王蒙[1,2,3];李海龙[1,2,4];宁月[1,2,3];邵利华[1,2,3];高夏青[1,2,3];杨春婷[1,2,3];张志明[2];陈凤琴[4]

第一作者：王蒙

机构：[1]甘肃中医药大学第一临床医学院,兰州730000;[2]甘肃省中西医结合肿瘤临床医学研究中心,兰州730000;[3]甘肃省中药新产品创制工程实验室/甘肃省中医方药挖掘与创新转化重点实验室,兰州730000;[4]甘肃中医药大学附属医院,兰州730000

第一机构：甘肃中医药大学临床医学院

年份：2024

卷号：30

期号：7

起止页码：1178

中文期刊名：中国中医基础医学杂志

外文期刊名：JOURNAL OF BASIC CHINESE MEDICINE

收录：CSTPCD;;CSCD:【CSCD_E2023_2024】；

基金：甘肃省科技计划资助项目(18JR2FA001);甘肃省中西医结合肿瘤临床医学研究中心2021年开放基金项目(zlzx2021-3);甘肃省自然科学基金项目(22JR5RA614)。

语种：中文

中文关键词：网络药理学;通关藤口服液;胃癌;分子对接;EGFR/PI3K/AKT/m TOR信号通路

外文关键词：Network pharmacology;Marsdenia Tenacissima oral liquid;Gastric cancer;Molecular docking;EGFR/PI3K/AKT/mTOR signaling pathway

摘要：目的利用网络药理学方法、分子对接及体外细胞实验探究通关藤(marsdenia tenacissima,MT)口服液抑制胃癌(gastric cancer,GC)增殖的潜在作用机制。方法通过搜索文献、检索TCMSP、Swiss Target Prediction数据库完成MT化学成分、靶点基因的收集;采用Gene Cards数据库获取疾病相关靶点;利用STRING平台和Cytoscape软件构建交集靶点的蛋白互作网络(protein-protein interaction,PPI);采用Metascape网络平台进行GO和KEGG信号通路分析;运用Auto Dock对部分活性成分与潜在靶点进行分子对接验证;采用四甲基偶氮唑盐(methyl thiazolyl tetrazolium,MTT)法检测MT口服液对GC细胞增殖的抑制作用,分别与厄洛替尼和阿帕替尼联用的协同作用、流式细胞术检测MT口服液诱导GC细胞凋亡及对胃癌细胞周期的影响;采用RT-qPCR法检测MT口服液对GC细胞周期、凋亡相关基因表达的影响;采用Western blot法验证网络药理学和分子对接筛选的靶点。结果共得到MT主要活性成分17个,胃癌关键靶点1880个,主要信号通路167条。体外实验结果表明,40~200 mg/m L的MT口服液可有效抑制GC细胞增殖(P<0.01),与厄洛替尼和与阿帕替尼联用时,具有协同效应。40~160 mg/m L的MT口服液可诱导胃癌细胞凋亡(P<0.01),阻滞细胞周期G0/G1期(P<0.01)。RT-qPCR结果显示,通关藤口服液可以上调B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)相关X蛋白(Bcl-2-associated X protein,BAX)的表达量,下调Bcl-2、细胞周期蛋白依赖性激酶(cyclin dependent kinase,CDK)4、CDK6(P<0.05)的表达量。Western blot结果显示MT口服液,抑制磷酸化-表皮生长因子受体(phosphorylated-epidermal growth factor receptor,p-EGFR)、磷酸化-磷脂酰肌醇-3激酶(phosphorylated-phosphatidylinositol 3-kinase,p-PI3K)、磷酸化AKT丝氨酸/苏氨酸激酶(p-AKT)、磷酸化-雷帕霉素激酶的机制靶点(phosphorylated-mechanistic target of rapamycin kinase,p-mTOR)的表达水平,降低p-EGFR/EGFR、p-PI3K/PI3K、p-AKT/AKT1、p-mTOR/m TOR比值;同时上调BAX、肿瘤蛋白p53(tumor protein p53,P53)、细胞周期蛋白依赖性激酶抑制剂(cyclin dependent kinase inhibitor 1A,CDKN1A/P21),下调Bcl-2、CDK4、CDK6(P<0.05)水平。结论MT口服液可有效抑制胃癌细胞的增殖,阻滞细胞周期并诱导胃癌细胞凋亡,并提高小分子靶向药物厄洛替尼和阿帕替尼治疗胃癌的敏感性。其机制与经调控PI3K/AKT/m TOR信号通路上调BAX、P53、P21,下调Bcl-2、CDK4、CDK6的表达,并抑制EGFR信号通路有关,实验结果验证了网络药理学和分子对接的结果。
Objective To investigate the potential mechanism of Marsdenia Tenacissima(MT)oral liquid in inhibiting gastric cancer(GC)by network pharmacology,molecular docking and in vitro cell experiments.Methods The chemical components and target genes of MT were collected by literature searching,TCMSP and Swiss Target Prediction databases.Disease-related targets were obtained by GeneCard database.The protein interaction(PPI)network of intersection targets was constructed by STRING platform and Cytoscape software.GO functional enrichment and KEGG signaling pathways were obtained by Metascape network platform,molecular docking validation of some active components and potential targets was performed by AutoDock Vina and other software.Methyl thiazolyl tetrazolium(MTT)method was used to detect the inhibiting effect of MT oral liquid on the proliferation of GC cells and the synergistic effects of MT oral solution combined with Erlotinib and Apatinib on GC cells were also observed.Flow cytometry was used to detect the apoptosis induced by MT oral liquid and its effect on the cell cycle of GC cells.RT-qPCR was used to detect the effects of MT oral liquid on the cell cycle and apoptosis-related gene expression in GC cells.Western blot was used to verify the targets screened by network pharmacology method and molecular docking.Results A total of 17 main active components,1880 key targets of GC and 167 main KEGG pathways were obtained.The MTT tests showed that MT oral liquid with the concentration of 40~200 mg/mL had an inhibitory effect on the proliferation of GC cells in a concentration-dependent and time-dependent manner(P<0.01).When combined with Erlotinib and Apatinib,it had a synergistic effect(P<0.01).The results of flow cytometry showed that MT oral liquid with the concentration of 40~160 mg/mL could change the cell cycle distribution of GC block it in G0/G1 phase and induce its apoptosis(P<0.01).RT-qPCR results showed that MT oral liquid could up-regulate the expression of Bcl-2 associated X protein(BAX)and down-regulate the expressions of Bcl-2 apoptosis regulator(Bcl-2),cyclin dependent kinase(CDK)4 and CDK6(P<0.05).The results of WB showed that MT oral liquid could downregulate the phosphorylation expression levels of p-EGFR,p-PI3K,p-AKT,and p-mTOR,thereby reducing the ratio of p-EGFR/EGFR,p-PI3K/PI3K,p-AKT/AKT1,and p-mTOR/mTOR,up-regulate the expressions of Bax,P53 and P21,and down-regulate the expressions of Bcl-2,CDK4 and CDK6 significantly(P<0.05).Conclusion MT oral liquid could inhibit the proliferation of GC cells effectively,arrest cell cycle,induce apoptosis of GC cells,and improve the sensitivity of small molecule target drugs including Erlotinib and Apatinib in the treatment of GC.The mechanism is related to the up-regulation of Bax,P53 and P21 proteins,and down-regulation of Bcl-2,CDK4 and CDK6 proteins and inhibition of EGFR signaling pathway via regulating PI3K/AKT/mTOR signaling pathway.The experimental results had verified the results of network pharmacology and molecular docking.