网络药理学结合GEO芯片技术探讨黄芎方治疗缺血性脑卒中的作用机制及实验验证  

Network pharmacology combined with GEO chip to investigate mechanism of action and experimental validation of Huangxiong Formula in treatment of ischemic stroke and experimental verification

作　　者：赵赛红 王磊 张平平 黄莹莹 汪宁 ZHAO Saihong;WANG Lei;ZHANG Pingping;HUANG Yingying;WANG Ning(College of Pharmacy,Anhui University of Chinese Medicine,Hefei 230012,China;Anhui Province Key Laboratory of Research&Development of Chinese Medicine,Hefei 230012,China;Anhui Province Key Laboratory of Chinese Medicinal Formula,Hefei 230012,China;Institute for Evaluation of Efficacy and Safety of Chinese Medicines,Anhui Academy of Chinese Medicine,Hefei 230012,China)

机构地区：[1]安徽中医药大学药学院,安徽合肥230012 [2]安徽省中药研究与开发重点实验室,安徽合肥230012 [3]安徽省中药配方重点实验室,安徽合肥230012 [4]安徽省中医药研究院中药疗效与安全评价研究所,安徽合肥230012

出　　处：《药物评价研究》2023年第5期983-996,共14页Drug Evaluation Research

基　　金：安徽省高校优秀科研创新团队(2022AH010034);安徽省高等学校科学研究项目(2022AH050478);安徽中医药大学自然重点项目(2021zrzd10);安徽中医药大学人才支持计划项目(2020rcyb005)。

摘　　要：目的基于GEO芯片联合网络药理学方法探讨黄芎方治疗缺血性脑卒中的作用机制,并构建脑缺血/再灌注(I/R)大鼠模型验证黄芎方药效和核心靶点。方法利用中药系统药理学数据库与分析平台(TCMSP)数据库及文献检索,获得黄芎方的主要活性成分,通过Swiss Target Prediction数据库检索其相应的靶点。通过R软件limma包对GEO平台缺血性脑卒中患者数据集GSE16561进行差异基因分析。利用Venny 2.1.0进行黄芎方成分靶点和缺血性脑卒中差异基因靶点交集靶点分析,采用DAVID数据库对交集靶点进行基因本体(GO)功能富集分析和京都基因与基因组百科全书(KEGG)通路富集分析。采用Cytoscape 3.9.0软件构建成分-靶点-通路网络并筛选核心成分,通过STRING数据库与Cytoscape 3.9.0软件构建蛋白质相互作用(PPI)网络并筛选核心靶点基因。利用PyMOL软件对度值排名靠前的成分和靶点基因进行分子对接。通过single sample Gene Set Enrichment Analysis(ssGSEA)进行免疫浸润分析。采用线栓法制备I/R大鼠模型,除假手术组外(n=21),将69只大鼠随机分为模型组、黄芎方组(3.6 g·kg^(-1))组、阿司匹林(9 mg·kg^(-1))组。黄芎方组和阿司匹林组每日ig给药1次,假手术组和模型组ig给予等量生理盐水,持续7 d。采用Longa分级评分法对大鼠进行神经行为评分,TTC染色测定大鼠脑梗死体积,免疫组织化学染色法检测离子钙接头蛋白分子1(Iba1)阳性表达,ELISA法检测脑组织中肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、白细胞介素-6(IL-6)的表达,实时荧光定量PCR(qRT-PCR)检测大鼠脑组织Toll样受体4(TLR4)、信号转导和转录激活因子3(STAT3)、缺氧诱导因子1A(HIF1A)、髓过氧化物酶(MPO)、基质金属蛋白酶9(MMP9)的mRNA表达。结果通过TCMSP数据库和文献检索共筛选得到黄芎方的有效活性成分35个,成分靶点754个,缺血性脑卒中基因芯片差异表达基因677个,交集�Objective To explore the mechanism of Huangxiong Formula(HXF)in treatment of ischemic stroke(IS)based on GEO chip combined with network pharmacology,and to verify its pharmacodynamics and core targets by using the rat ischemiareperfusion(I/R)model.Methods The active ingredients of HXF were searched by using the TCMSP database and the literature,and their corresponding targets were searched by using the Swiss Target Prediction database.GEO platform IS dataset GSE16561 was used for variance analysis by R software limma package.The Venny 2.1.0 was used to perform intersection target analysis,and the DAVID database GO and KEGG pathway enrichment analysis on intersection target.Cytoscape 3.9.0 software was used to construct the components-target-pathway network and screen the core ingredients,and the STRING database and Cytoscape 3.9.0 software was used to construct the protein-protein interaction(PPI)network and screen the core target genes.The top ranked components and target genes based on the degree values were molecularly docked using PyMOL software.Immuno-infiltration analysis was performed by single sample Gene Set Enrichment Analysis(ssGSEA).A rat model of cerebral I/R was prepared by the wire bolus method.Totally 69 rats were randomly divided into the model group,the Huanxiong Formula(3.6 g·kg^(-1))group and the aspirin(9 mg·kg^(-1))group,except for the sham-operated group(n=21).Huanxiong Formula and Aspirin groups were given ig once a day,and equal amount of saline was given to the sham-operated and model groups ig for 7 d.The rats were scored for neurobehaviour using the Longa grading scale.TTC staining was used to determine the volume of brain infarction in the rats.Immunohistochemical staining was used to detect positive expression of ion calcium junction protein molecule 1(Iba1).The levels of tumour necrosis factorα(TNF-α),interleukin 1β(IL-1β)and interleukin 6(IL-6)in brain tissue was measured by ELISA.The expression of Toll-like receptor 4(TLR4),signal transducer and activator of transcription 3(

关 键 词：黄芎方 缺血性脑卒中 网络药理学 GEO数据库 分子对接 Α-细辛醚 3 4 5-三甲氧基肉桂酸 Β-细辛醚 异阿魏酸 阿魏酸 Toll样受体4 信号转导和转录激活因子3 缺氧诱导因子1A 髓过氧化物酶 基质金属蛋白酶9 

分 类 号：R285.5[医药卫生—中药学]