基于网络药理学的气滞胃痛颗粒治疗胃炎作用机制探讨和实验验证

Discussion on mechanism of Qizhi Weitong Granules in treating gastritis based on network pharmacology and experimental verification

目的 采用网络药理学的方法预测气滞胃痛颗粒治疗胃炎主要活性成分的作用靶点及其作用机制,并采用细胞实验对部分主要靶点进行实验验证。方法 在中药系统药理学数据库与分析平台(TCMSP)和网络药理学在线数据库(TCMID)中获得气滞胃痛颗粒组方中药柴胡、白芍、枳壳、香附(炙)、延胡索(炙)、甘草(炙)的化学成分,并在TCMSP和PharmMapper数据库中收集活性成分对应的靶点,在药物靶标数据库(TTD)和人类孟德尔遗传数据库(OMIM)数据库中搜索与胃炎相关的蛋白和基因,建立胃炎靶点数据库;通过DIP数据库将成分靶点和疾病靶点进行关联,建立化合物-靶点-疾病网络。将气滞胃痛颗粒治疗胃炎的相关靶点输入STRING数据库中构建靶点之间的蛋白质相互作用(PPI)关系;使用DAVID数据库对靶点进行京都基因与基因组百科全书(KEGG)通路富集分析,使用Cytoscape3.5.1软件自带的Glue GO插件进行基因本体论(GO)生物功能注释;通过人类基因和基因表型综合数据库(OMM)筛选炎症相关细胞相关的基因和蛋白靶点,对气滞胃痛颗粒治疗胃炎的靶细胞进行富集。体外细胞实验以巨噬细胞RAW264.7为对象,采用1μg·mL^(-1)脂多糖(LPS)和0.2μg·mL^(-1)γ干扰素(IFN-γ)制备细胞模型,以含有最大无毒剂量气滞胃痛颗粒内容物的培养基处理RAW264.7模型细胞,采用实时荧光定量PCR (qRT-PCR)法测定各组RAW264.7细胞中环氧合酶2 (COX-2)、诱导型一氧化氮合酶(iNOS)、白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)mRNA表达。结果 网络药理学预测得到气滞胃痛颗粒治疗胃炎的主要潜在的活性成分25个,重要靶点20个,其中关键靶点包括COX-2、iNOS、过氧化物酶体增殖物激活受体γ(PPARγ)、丝裂原活化蛋白激酶14(MAPK-14)、表皮生长因子受体(EGFR)等,作用机制可能与调节TNF信号通路、NOD样受体信号通路、VEGF信号通路等与胃炎密切相关的信号通路有关,其药效作用主要表现为对炎症、血管内稳态、免疫、中枢神经及激素调节等生物过程的影响。气滞胃痛颗粒对RAW264.7模型细胞中COX-2、iNOS、IL-6、TNF-α的mRNA表达具有显著抑制作用。结论 气滞胃痛颗粒可能主要通过修复胃黏膜、减轻炎症损伤、消除感染等实现其治疗胃炎作用。

Objective To predict the target and mechanism of Qizhi Weitong Granule in treating gastritis by network pharmacological method,and to verify some of the main targets by cell experiment.Methods The chemical components of Bupleuri Radix,Paeoniae Radix Alba,Aurantii Fructus,Cyperi Rhizoma,Corydalis Rhizoma and Glycyrrhizae Radix et Rhizoma were obtained from TCMSP and TCMID,and the corresponding target points of active components were collected from TCMSP and PharmaMapper databases,Search the drug target database(TTD) and the human Mendelian genetic database(OMIM) database for proteins and genes related to gastritis,and establish the gastritis target database.The component target and disease target are related through DIP database to establish a compound target disease network.Input the relevant targets of Qizhi Weitong Granule for gastritis into STRING database to build the protein interaction(PPI) relationship between the targets.The DAVID database was used to enrich and analyze the Kyoto encyclopedia of genes and genomes(KEGG) pathway of the target,and the Glue GO plug-in included in the Cytoscape 3.5.1 software was used to annotate the biological function of the gene ontology(GO).The gene and protein targets related to inflammation related cells were screened through the comprehensive database of human genes and gene phenotypes(OMM) to enrich the target cells of Qizhi Weitong Granule in treating gastritis.In vitro cell experiment,macrophage RAW264.7 was used as the object,and the cell model was prepared by 1 μ g · mL^(-1) lipopolysaccharide(LPS) and 0.2 μ g · mL^(-1) γ interferon(IFN-γ).RAW264.7 model cells were treated with the medium containing the largest non-toxic dose of Qizhi Weitong Granules.The mRNA expression of cyclooxygenase-2(COX-2),inducible nitric oxide synthase(iNOS),interleukin-6(IL-6) and tumor necrosis factor α(TNF-α) in RAW264.7 cells in each group were determined by real-time fluorescent quantitative PCR(qRTPCR).Results The network pharmacology predicted that there were 25 main potential active components and 20 important targets of Qizhi Weitong Granule in treating gastritis,and key targets include COX-2,iNOS,peroxisome proliferator activated receptor γ(PPAR-γ) mitogen activated protein kinase 14(MAPK-14),epidermal growth factor receptor(EGFR),etc.The action mechanism of Qizhi Weitong Granule in treating gastritis may be related to the regulation of TNF signal pathway,NOD like receptor signal pathway,VEGF signal pathway and other signal pathways closely related to gastritis.Its pharmacodynamic effect is mainly manifested in its influence on biological processes such as inflammation,vascular homeostasis,immunity,central nervous system and hormone regulation.The mRNA expression of COX-2,iNOS,IL-6,TNF-α in RAW264.7 model cells were significantly inhibited by Qizhi Weitong Granule.Conclusion Qizhi Weitong Granule may achieve its therapeutic effect on gastritis mainly by repairing gastric mucosa,alleviating inflammatory damage and eliminating infection.