摘要
目的采用网络药理学和体外实验探讨小柴胡汤扶正祛邪的核心配伍“柴胡-黄芩-人参”治疗流行性感冒的作用靶点及信号通路。方法筛选“柴胡-黄芩-人参”有效成分并对应靶点基因,并检索流感疾病靶点。应用Cytoscape3.6.1软件构建“柴胡-黄芩-人参”-流感-靶点-通路网络,筛选核心靶点基因,进行GO功能和KEGG富集分析。根据网络药理学结果,基于自噬途径以甲型H1N1流感病毒感染的狗肾(MDCK)细胞为研究对象,以柴胡-黄芩-人参和自噬抑制剂3-MA为干预药物,孵育12 h后,用Western blot法检测自噬标志蛋白LC3-Ⅰ、LC3-Ⅱ及自噬降解底物P62的表达量。结果预测获得“柴胡-黄芩-人参”治疗流感的靶点102个,其主要活性成分有豆甾醇、β-谷甾醇、二甲氧基黄酮、千层纸甲素A、黄芩新素、荠苎黄酮、汉黄芩素等,其核心基因为AKT1、IL6、VEGFA、TNF、TP53、JUN、CXCL8等54个,涉及生物过程及相关代谢通路多与炎症反应、各种代谢、免疫反应、细胞死亡等相关。根据以上网药预测结果及相关文献,筛选自噬途径进行细胞生物学层面的实验验证,结果显示,甲型H1N1病毒感染组与空白对照组相比,LC3-Ⅱ蛋白表达水平显著升高(P<0.01)。“柴胡-黄芩-人参”颗粒剂与病毒感染组相比,有下调LC3-Ⅱ蛋白表达的作用(P<0.01),并上调自噬降解底物P62蛋白的表达(P<0.05)。结论小柴胡汤扶正祛邪的核心配伍“柴胡-黄芩-人参”对流感病毒抑制(如干预病毒吸附)和宿主因素调节(如免疫应答、活性氧调节、细胞死亡、各种代谢等)两方面都发挥着抗流感的作用,尤以调节宿主内环境的稳态(如自噬)为主,并通过体外实验初步验证,“柴胡-黄芩-人参”可能是通过调节AKT抑制甲型H1N1流感病毒诱导的细胞自噬,从而调控甲型H1N1流感病毒的致病作用,这也正与其体现的“扶正祛邪”治法高度吻合。
Objective Pharmacological methods and in vitro experiments were used to screen targets and signaling pathways of"Bupleurum-Scutellaria-Ginseng",which was the core compatibility of Xiaochaihu Decoction.Methods Screen the active ingredients of"Bupleurum-Scutellaria-Ginseng"and correspond to target genes,and search for influenza disease targets.Cytoscape 3.6.1 software was used to construct the"Bupleurum-Scutellaria-Ginseng"-influenza-target-pathway network,screen the core target genes,perform GO function and KEGG enrichment analysis.According to the results of Pharmacological methods,MDCK cells infected by influenza A H1N1 virus were used as the research object based on autophagy pathway,and Bupleurum-Scutellaria-Ginseng and autophagy inhibitor 3-MA were used as intervention drugs.After 12 hours of incubation,the autophagy marker protein immune LC3-Ⅰ,LC3-Ⅱand the expression of autophagy degradation substrate P62 was detected by Western blot.Results A total of 102 predicted chemical ingredients of"Bupleurum-skullcap-ginseng"including Stigmasterol,beta-sitosterol,panicolin,oroxylin a,neobaicalein,moslosooflavone,wogonin,et al..The core genes were AKT1、IL6、VEGFA、TNF、TP53、JUN、CXCL8,etc.The biological processes and related metabolic pathways were mostly related to inflammation,various metabolisms,immune responses,and cell death.According to above results and related research,autophagy pathway was screened for experimental verification from the cellular level.The experimental results showed that compared with the blank control group,the expression level of LC3-Ⅱprotein in the virus-infected group was significantly increased(P<0.01).Compared with the virus-infected control group,"Bupleurum-Scutellaria-Ginseng"granules can downregulate the expression of LC3-Ⅱprotein(P<0.01)and up-regulate the expression of P62 protein,which is a substrate for autophagy degradation(P<0.05).Conclusions Bupleurum-Scutellaria-ginseng,which was the core compatibility of Xiaochaihu Decoction for strengthening the body and eli
作者
王上
杨特达
李家庚
王彦春
蔡羽
王建婷
陈雨
Wang Shang;Yang Teda;Li jiageng;Wang Yanchun;Cai Yu;Wang Jianting;Chen Yu(College of Clinical Chinese Medicine,Hubei University of Chinese Medicine,Wuhan 430065,China;College of pharmacy,Hubei University of Chinese Medicine,Wuhan 430065,China)
出处
《世界科学技术-中医药现代化》
CSCD
北大核心
2022年第9期3419-3431,共13页
Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology
基金
湖北中医药大学“青苗计划”资助项目(2017zzx027):基于自噬途径探讨少阳方核心药对“柴胡-黄芩”防治流感的机制研究
负责人:王上
国家自然科学基金委员会面上项目(82074417):从神经-免疫角度探讨抑郁症LHb与DA、5-HT中心的环路异常及柴-芩抗抑郁的相关机制,负责人:陈雨
关键词
柴胡-黄芩-人参
小柴胡汤
流行性感冒
网络药理学
自噬
Bupleurum-scutellaria-ginseng
Xiaochaihu decoction
Influenza
Network pharmacology
Autophagy
