基于网络药理学和体外细胞实验探讨芍药甘草汤治疗溃疡性结肠炎的作用机制  

Exploration on mechanism of Shaoyao Gancao Decoction in treating ulcerative colitis based on network pharmacology and in vitro cell experiments

作　　者：何毅豪 王冰 杨骏 张彤[1] HE Yihao;WANG Bing;YANG Jun;ZHANG Tong(School of Pharmacy,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Center for Pharmaceutics Research,Shanghai Institute of Materia Medica,Chinese Academy of Sciences,Shanghai 201203,China;Xiangshan TCM,Huangpu District,Shanghai 200020,China)

机构地区：[1]上海中医药大学中药学院,上海201203 [2]中国科学院上海药物研究所药物制剂研究中心,上海201203 [3]上海市黄浦区香山中医医院,上海200020

出　　处：《上海中医药大学学报》2022年第6期59-69,共11页Academic Journal of Shanghai University of Traditional Chinese Medicine

基　　金：国家科技重大专项(2019ZX09201004-002);上海市领军人才项目(2019100);上海市中医药事业发展三年行动计划项目[(2021-2023)-0502]。

摘　　要：目的:利用网络药理学、差异基因分析、分子对接技术和体外细胞实验,探究芍药甘草汤(SYGCD)治疗溃疡性结肠炎(UC)的初步作用机制。方法:通过中药系统药理学分析平台(TCMSP)和文献检索,获取SYGCD中白芍和甘草的活性成分和作用靶点。从基因表达综合(GEO)数据库中筛选获得GSE47908芯片数据,绘制差异基因热图和火山图。运用Cytoscape软件构建SYGCD治疗UC分子基因网络图,借助Bisogenet和CytoNCA绘制核心靶点拓扑网络,使用R 3.6.3软件对差异基因进行GO功能富集分析、KEGG通路富集分析。采用分子对接解析活性成分与关键靶点的相互作用。采用RAW264.7巨噬细胞,通过MTT实验和RT-qPCR实验初步验证SYGCD的抗炎作用的相关靶点。结果:①获得SYGCD治疗UC的102个活性成分,主要为槲皮素、山柰酚、异鼠李素等,拓扑网络筛选出关键候选基因为过氧化物酶体增殖物激活受体γ(PPARγ)、诱导型一氧化氮合酶(NOS2)等。富集分析显示,与UC治疗相关的信号通路主要为白介素(IL)-17信号通路、肿瘤坏死因子(TNF)信号通路等。②分子对接结果显示,SYGCD中核心成分与关键靶点均能通过氢键结合。③MTT实验结果显示,SYGCD对RAW264.7细胞几乎没有毒性;RT-qPCR结果显示,SYGCD能降低RAW264.7细胞炎症模型中细胞因子TNF-α、IL-1β和IL-6 mRNA表达水平。结论:SYGCD治疗UC具有多成分、多靶点、多途径等特征,作用机制可能是SYGCD中的槲皮素、山柰酚、芒柄花素、异鼠李素等活性成分作用于PPARγ、NOS2、IL-1β等靶点,调控IL-17、TNF、NF-κB等信号通路发挥治疗UC的作用;且SYGCD可通过调节促炎性细胞因子TNF-α、IL-1β和IL-6发挥抗炎效果。Objective:To explore the mechanism of Shaoyao Gancao Decoction(SYGCD)in the treatment of ulcerative colitis(UC)based on network pharmacology,differential gene analysis,molecular docking technology and in vitro cell experiments.Methods:Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)and literature retrieval were used to obtain the active ingredients and targets of SYGCD. GSE47908 microarray data were obtained from GEO database,and differential gene heatmaps and volcano plots were drawn. The molecular gene network map of SYGCD against UC was constructed by Cytoscape software,and the core target topology network was drawn by Bisogenet and CytoNCA. GO functional enrichment analysis and KEGG pathway enrichment analysis of differential genes were performed by R 3.6.3 software. Molecular docking was used to analyze the interaction between active ingredients and key targets. RAW264.7 macrophages were used to preliminarily verify the anti-inflammatory effect of SYGCD by MTT assay and RTqPCR assay in vitro.Results:①102 active ingredients of SYGCD for UC treatment were obtained,mainly including quercetin,kaempferol,isorhamnetin,etc. The key candidate genes such as peroxisome proliferator-activated receptor-γ(PPARγ)and inducible nitric oxide synthase(NOS2) were selected by topological network. Enrichment analysis showed that UC treatment was mainly related to interleukin(IL)-17 signaling pathway and tumor necrosis factor(TNF)signaling pathway,etc.②Molecular docking results showed that the core components of SYGCD could bind to the key targets stably by hydrogen bonding.③MTT assay results showed that SYGCD was almost not toxic to RAW264.7 cells. RT-qPCR results showed that SYGCD could reduce the mRNA expression levels of TNF-α,IL-1β and IL-6 cytokines in RAW264.7 cell inflammation model.Conclusion:SYGCD has the characteristics of multi-component,multi-target and multi-pathway in treating UC,the mechanism of which may be that the active components of SYGCD,such as quercetin,kaem

关 键 词：芍药甘草汤 溃疡性结肠炎 网络药理学 GEO芯片 分子对接 体外细胞实验 

分 类 号：R285[医药卫生—中药学]