基于网络药理学的复方鱼腥草合剂清热解毒药效物质基础及作用机制研究

Pharmacodynamic material basis and mechanism of Fufang Yuxingcao Mixture for the treatment of heat-clearing and detoxification based on network pharmacology

探索复方鱼腥草合剂清热解毒功效的药效物质基础及网络调控机制。首先利用网络药理学的方法,选取复方鱼腥草合剂中25个化合物为研究对象,依据反向药效团匹配方法和TCMSP、UniProt等数据库预测化合物作用靶点,借助Omicsbean软件及KEGG数据库对获得靶点进行基因本体(gene ontology,GO)功能富集分析、信号通路分析,利用Cytoscape软件构建"化合物-靶点-通路-药理作用-功效"网络药理图。最后采用脂多糖(lipopolysaccharide,LPS)诱导的RAW264.7细胞炎症模型,验证复方鱼腥草合剂及方中10个重要单体成分的抗炎作用。网络药理预测实验发现,25个化合物可通过211个靶点干预97条信号通路,其中RAC-α丝氨酸/苏氨酸蛋白激酶(RAC-alpha serine/threonine-protein kinase,AKT1)、胰岛素(insulin,INS)、血管内皮生长因子α(vascular endothelial growth factor A,VEGFA)、白介素-6(interleukin-6,IL-6)、细胞肿瘤抗原p53(cellular tumor antigen p53,TP53)、肿瘤坏死因子(tumor necrosis factor,TNF)、转录因子AP-1(transcription factor AP-1,JUN)、半胱氨酸蛋白酶-3(caspase-3,CASP3)、基质金属蛋白酶-9(matrix metalloproteinase-9,MMP9)、白介素-8(interleukin-8,IL-8)、前列腺素G/H合酶2(prostaglandin G/H synthase 2,PTGS2)、原癌基因c-fos(proto-oncogene c-Fos,FOS)、酪氨酸蛋白激酶SRC(tyrosine-protein kinase SRC,SRC)、丝裂原活化蛋白激酶-8(mitogen-activated protein kinase 8,MAPK8)和雌激素受体1(estrogen receptor 1,ESR1)等15个核心靶点和核转录因子κB信号通路(NF-kappa B signaling pathway)、Toll样受体信号通路(Toll-like receptor signaling pathway)、丝裂原活化蛋白激酶信号通路(MAPK signaling pathway)、白介素-17信号通路(IL-17 signaling pathway)、花生四烯酸代谢(arachidonic acid metabolism)、环磷酸腺苷信号通路(cAMP signaling pathway)、T细胞受体信号通路(T cell receptor signaling pathway)、钙离子信号通路(calcium signaling pathway)、TRP通道炎性介质调节(inflammatory mediator regulation of TRP channels)、趋化因子信号通路(chemokine signaling pathway)、Th1和Th2细胞分化(Th1 and Th2 cell differentiation)、自然杀伤细胞介导的细胞毒性(natural killer cell mediated cytotoxicity)等46条信号通路与抗炎、解热、免疫调节、镇痛等过程相关,推测复方鱼腥草合剂通过干预以上过程发挥清热解毒功效,体现了其多成分、多靶点、多途径的治疗作用。体外细胞实验表明,复方鱼腥草合剂及10个单体成分(异槲皮苷、木犀草苷、黄芩素、汉黄芩素、黄芩苷、连翘苷、连翘酯苷A、绿原酸、异绿原酸A、獐牙菜苷)均能显著降低LPS诱导的RAW264.7炎性细胞上清中一氧化氮(nitric oxide,NO)、TNF-α和IL-6的表达(P<0.05),推测以上10个成分可能为复方鱼腥草合剂的关键药效物质基础。

We explored the pharmacodynamic material basis and network regulatory mechanism of Fufang Yuxingcao Mixture(FYM)for the treatment of fever and inflammation.Targets of the 25 compounds in FYM were predicted according to the reverse pharmacophore method and TCMSP,UniProt database.Gene ontology(GO)function enrichment and pathway analysis of the targets was analyzed by Omicsbean software and the Kyoto Gene and Genome Encyclopedia(KEGG)database.A"compound-target-pathway-pharmacological action-effect"network was established with Cytoscape 3.6.1 software.The lipopolysaccharide(LPS)-induced RAW264.7 cell inflammation model was used to verify the anti-inflammatory effects of FYM and its 10 important components.The network pharmacology experiment showed that 25 compounds affected 97 pathways through 211 targets,of which 15 key targets[including RAC-alpha serine/threonine-protein kinase(AKT1),insulin(INS),vascular endothelial growth factor A(VEGFA),interleukin-6(IL-6),cellular tumor antigen p53(TP53),tumor necrosis factor(TNF),transcription factor AP-1(JUN),caspase-3(CASP3),matrix metalloproteinase-9(MMP9),interleukin-8(IL-8),prostaglandin G/H synthase 2(PTGS2),proto-oncogene c-Fos(FOS),tyrosine-protein kinase SRC(SRC),c-Jun N-terminal kinase1(MAPK8),estrogen receptor 1(ESR1)]and 46 pathways(including NF-kappa B signaling pathway,Toll-like receptor signaling pathway,MAPK signaling pathway,IL-17 signaling pathway,arachidonic acid metabolism,cAMP signaling pathway,T cell receptor signaling pathway,calcium signaling pathway,inflammatory mediator regulation of TRP channels,chemokine signaling pathway,Th1 and Th2 cell differentiation,natural killer cell mediated cytotoxicity,etc.)were related to anti-inflammatory,antipyretic,immune regulation,and analgesia.In vitro cell experiments showed that FYM and the 10 components(including isoquercitrin,luteoloside,baicalein,wogonin,wogonoside,phillyrin,forsythoside A,chlorogenic acid,isochlorogenic acid A,and sweroside)could significantly reduce the expression of nitric oxide(NO),TNF-αand IL-6 in cell supernatants,indicating that the above 10 components may be the key pharmacodynamic material basis of FYM.