目的:基于超高效液相色谱-四级杆飞行时间质谱(UPLC-Q-TOF-MS/MS)技术和网络药理学探讨柔肝方治疗肝纤维化的作用机制.方法:将8 只C57BL/J小鼠随机分成空白血清组和柔肝方含药血清组,每组4 只.利用UPLC-Q-TOF-MS/MS技术结合Xcalibur软件分析空白血清和柔肝方含药血清数据,结合二级谱图鉴定柔肝方的入血成分.通过Swiss Tar-get Prediction数据库和中药系统药理学数据库与分析平台(Traditional Chinese Medicine Systems Pharmacology Database and A-nalysis Platform,TCMSP)检索柔肝方入血成分的靶点.利用GeneCards、OMIM、PharmGKB、DrugBank Online和Therapeutic Tar-get Database数据库检索肝纤维化的靶点.采用Cytoscape软件构建"疾病-化合物-靶点"网络,并筛选柔肝方抗肝纤维化的关键成分.借助STRING数据库构建蛋白质-蛋白质相互作用(protein-protein interaction,PPI)网络,通过拓扑分析筛选柔肝方抗肝纤维化的核心靶点.采用DAVID数据库进行基因本体(gene ontology,GO)和京都基因与基因组百科全书(kyoto ency-clopedia of genes and genomes,KEGG)分析.结果:柔肝方入血成分主要包括氧化槐果碱、槐果碱、高黄芩素、新补骨脂异黄酮等.通过检索获取柔肝方入血成分相关靶点303 个,肝纤维化相关靶点6 331 个,两者的交集靶点250 个.柔肝方抗肝纤维化的关键成分包括槲皮素、芹菜素、山柰酚、芒柄花黄素等.柔肝方抗肝纤维化的核心靶点包括原癌基因酪氨酸蛋白激酶Src(SRC)、信号转导和转录激活因子3(signal transducer and activator of transcription 3,STAT3)、丝裂原活化蛋白激酶1(mitogen-activated protein kinase 1,MAPK1)、丝氨酸/苏氨酸蛋白激酶(protein kinase B,PKB,又称AKT1)等.GO分析主要涉及细胞因子介导的信号通路、凋亡过程的负调控、炎症反应、蛋白激酶活性等.KEGG分析主要涉及PI3K-Akt信号通路、MAPK信号通路、白细胞介素(interleukin,IL)-17 信号通路、HIF-1 信号通路等.结论:柔肝方可能通过槲皮素、芹菜素、山柰酚等化合物调控PI3K-Akt、MAPK、IL-17 等信号通路,作用于SRC、STAT3、MAPK1 等靶点,从而发挥抗肝纤维化的作用,体现了多成分、多途径、多靶点的药理特征.
Study on the Anti-Fibrotic Mechanism of Liver-Softening Formula Based on UPLC-Q-TOF-MS/MS Technology and Network Pharmacology
Objective:To study the therapeutic mechanism of Liver-Softening Formula in treating liver fibrosis based on ultra-high-per-formance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS/MS)technology and network pharma-cology.Methods:A total of 80 C57BL/J mice were randomly divided into the blank serum group and the group of drug-containing serum from Liver-Softening Formula,with 4 mice in each group.The data of blank serum and drug-containing serum from Liver-Softening For-mula were analyzed by UPLC-Q-TOF-MS/MS technology combined with Xcalibur software,and the blood-entering components of Liver-Softening Formula were identified combined with secondary mass spectrum.By using Swiss Target Prediction database and Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP),the targets of blood-entering components in Liver-Softening Formula were searched.The targets of liver fibrosis were searched using GeneCards,OMIM,PharmGKB,DrugBank Online,and Therapeutic Target Database databases.Using Cytoscape software,a"disease-compound-target"network was constructed,and the key components of Liver-Softening Formula in its anti-liver fibrosis effect were screened.The protein-protein interaction(PPI)network was constructed with the help of STRING database,and the core targets of Liver-Softening Formula in its anti-liver fibrosis were screened by topological analysis.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)were analyzed using the DAVID database.Results:The main components of Ruogan prescription into blood included Oxysophocarpine,Sophocarpine,Scutellarein,Neobavaisoflavone,etc.A total of 303 related targets and 6 331 related targets of liver fibrosis,and 250 intersection targets of the two were obtained.The key components of Liver-Softening Formula in its anti-liver fibrosis effect include Quercetin,Apigenin,Kaempferol,Formononetin and so on.The core targets of Liver-Softening Formula in its anti-liver fibrosis effect included proto-oncogene tyrosine pro-tein kinase Src(SRC),signal transducer and activator of transcription 3(STAT3),mitogen activated protein kinase 1(MAPK1),ser-ine/threonine protein kinase B(PKB,also known as AKT1),etc.The GO analysis mainly involved the Cytokine-mediated signaling pathway and the negative regulation of apoptotic process process,inflammatory response,protein kinase activity,etc.KEGG analysis mainly involved PI3K-Akt signaling pathway,MAPK signaling pathway,IL-17 signaling pathway,HIF-1 signaling pathway,etc.Conclu-sion:Liver-Softening Formula may regulate PI3K-Akt,MAPK,IL-17 and other signaling pathways through quercetin,apigenin,kaemphiol and other compounds,and act on SRC,STAT3,MAPK1 and other targets,thus playing an anti-fibrosis role,reflecting the pharmacologi-cal characteristics of multi-components,multi-pathways and multi-targets.
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