文献类型：期刊文献

中文题名：基于网络药理学及体外实验探讨黄芪甲苷治疗胃癌的机制

英文题名：Study on the Mechanism of AstragalosideⅣin the Treatment of Gastric Cancer Based on Network Pharmacology and In Vitro Experiments

作者：陈凤琴[1];樊涛[2,3];宁月[2,3];王蒙[2,3];王宏伟[1];李海龙[1,2,3]

第一作者：陈凤琴

机构：[1]甘肃中医药大学附属医院,甘肃兰州730000;[2]甘肃中医药大学第一临床医学院内科学教研室,甘肃兰州730000;[3]甘肃中医方药挖掘与创新转化重点实验室/甘肃省中药新产品创制工程实验室,甘肃兰州730000

第一机构：甘肃中医药大学第二附属医院

年份：2023

卷号：34

期号：9

起止页码：1236

中文期刊名：中药新药与临床药理

外文期刊名：Traditional Chinese Drug Research and Clinical Pharmacology

收录：CSTPCD;;北大核心:【北大核心2020】；CSCD:【CSCD2023_2024】；

基金：甘肃省自然科学基金项目(20JR5RA189,21JR7RA579);甘肃省中医药研究中心2019-2020年度开放课题(zyzx-2020-10)。

语种：中文

中文关键词：黄芪甲苷;胃癌;网络药理学;分子对接;PI3K/AKT信号通路;MAPK信号通路;体外实验

外文关键词：astragalosideⅣ;gastric cancer;network pharmacology;molecular docking;PI3k-AKT signaling pathway;MAPK signaling pathway;in vitro experiments

摘要：目的基于网络药理学方法及体外实验探讨黄芪甲苷治疗胃癌的分子机制。方法(1)通过SwissTarget、TargetNet、Super-PRED、pharmMapper数据库检索黄芪甲苷作用靶点;利用OMIM、GeneCards、TTD数据库筛选胃癌疾病相关靶点;取上述二者的交集靶点(共同靶点),即为黄芪甲苷治疗胃癌的潜在作用靶点。将交集靶点通过STRING平台建立蛋白互作(PPI)网络,并筛选出黄芪甲苷治疗胃癌的核心靶点。利用R语言包clusterProfiler软件进行潜在作用靶点的GO功能及KEGG通路富集分析,利用Cytoscape 3.8.2软件绘制“疾病-药物-通路-靶点”网络图。使用AutoDock 1.5.6软件进行核心靶点与黄芪甲苷的分子对接验证,并对关键靶点进行体外实验验证。(2)使用不同浓度(20、40、80、120、160、200μg·mL^(-1))黄芪甲苷分别干预MKN-45、HGC-27胃癌细胞24、48、72、96 h后,采用MTT法检测细胞增殖情况。采用80、120、160μg·mL^(-1)黄芪甲苷分别干预MKN-45、HGC-27胃癌细胞48 h后,采用Western Blot法检测细胞PIK3R1、PIK3CA、HSP90AA1、MAPK1、HRAS蛋白表达水平。结果(1)共得到87个黄芪甲苷治疗胃癌的潜在作用靶点;筛选出20个黄芪甲苷治疗胃癌的核心靶点;其中PIK3R1、PIK3CA、HSP90AA1、MAPK1、HRAS等核心靶点与黄芪甲苷的结合能<-5.0 kcal·mol-1,有较好的结合活性;潜在作用靶点主要涉及蛋白激酶信号通路、氧化、化学应激等生物学过程,以及PI3K/AKT信号通路、MAPK信号通路、Ras信号通路、Rap1信号通路等多条通路。(2)与对照组比较,黄芪甲苷在20~200μg·mL^(-1)浓度范围,分别干预24、48、72、96 h后,MKN-45、HGC-27细胞相对活力均明显降低(P<0.05,P<0.01),且呈现明显的时间、浓度依赖性。与对照组比较,120、160μg·mL^(-1)黄芪甲苷分别干预MKN-45、HGC-27胃癌细胞48 h后,PIK3R1、PIK3CA、HSP90AA1、MAPK1、HRAS蛋白表达水平均明显降低(P<0.05,P<0.01),且呈现明显的浓度依赖性。结论黄芪甲苷通过PIK3R1、PIK3CA、HSP90AA1、MAPK1、HRAS等多靶点,调控PI3K/AKT信号通路、MAPK信号通路等多通路,发挥治疗胃癌的作用。
Objective To explore the molecular mechanism of astragalosideⅣ(AS-Ⅳ)in the treatment of gastric cancer based on network pharmacological methods and in vitro experiments.Methods(1)Retrieve targets of ASⅣthrough SwissTarget,TargetNet,Super-PRED and pharmMapper databases;use OMIM,GeneCards and TTD databases to screen targets related to gastric cancer.The intersection target(common target)was taken as the potential target of AS-Ⅳfor gastric cancer treatment.The intersecting targets were used to establish a proteinprotein interaction(PPI)network through the STRING plaform,and the core targets of AS-Ⅳfor gastric cancer were screened out.The GO function and KECG pathway enrichment of potential targets were analyzed using R package clusterProfiler software,and the network diagram of"disease-drug-pathway-target"was drawn using Cytoscape 3.8.2 software.AutoDock 1.5.6 software was used to validate the molecular docking between the core target and AS-Ⅳ,and in vitro experiments were carried out to validate the key targets.(2)After intervening in MKN-45 and HGC-27 gastric cancer cells at different concentrations(20,40,80,120,160,200μg·mL^(-1))of AS-Ⅳfor 24,48,72 and 96 hours,respectively,the cell proliferation was detected by MTT assay.The protein expression levels of PIK3R1,PIK3CA,HSP90AA1,MAPK1 and HRAS were detected by Western Blot assay after 48 hours of intervention of MKN-45 and HGC-27 gastric cancer cells with 80,120,160μg·mL^(-1)AS-Ⅳ,respectively.Results(1)A total of 87 potential targets of AS-Ⅳfor the treatment of gastric cancer have been obtained;20 core targets of AS-Ⅳfor the treatment of gastric cancer have been screened;among them,core targets such as PIK3R1,PIK3CA,HSP90AA1,MAPK1,HRAS,have better binding activities with AS-Ⅳwith binding energies<-5.0 kcal·mol^(-1).The potential targets of action mainly involve protein kinase signaling pathway,oxidation,chemical stress and other biological processes,as well as multiple pathways such as PI3K/AKT signaling pathway,MAPK signaling pathway,Ras signaling pathway,Rapl signaling pathway and so on.(2)Compared with the control group,the relative viability of MKN-45 and HGC-27 cells was significantly reduced by AS-Ⅳin the concentration range of 20-200μg·mL^(-1),and after 24,48,72,and 96 hours of separate interventions(P<0.05,P<0.01),and showed obvious time-and concentration-dependence.Compared with the control group,after 120 and 160μg·mL^(-1)AS-Ⅳintervened in MKN-45 and HCC-27 gastric cancer cells for 48 hours,respectively,the protein expression levels of PIK3R1,PIK3CA,HSP90AA1,MAPK1,and HRAS were significantly reduced(P<0.05,P<0.01),and showed obvious concentration-dependent.Conclusion AS-Ⅳexerts its therapeutic effect on gastric cancer by regulating multiple pathways such as PI3K/AKT signaling pathway and MAPK signaling pathway through multi-targets such as PIK3R1,PIK3CA,HSP90AA1,MAPK1,HRAS and so on.