基于网络药理学和实验验证探索荆防合剂治疗甲型H1N1流感的作用机制  

Molecular Mechanism of Jingfang Mixture Against H1N1 Influenza Based on Network Pharmacology and Experimental Verification

作　　者：倪雯婷 马大龙 邵骏菁 尹怡铭 赵方舒 李保宏 孙允红 王晓晴 张晓平[1,2] 田景振 NI Wen-ting;MA Da-long;SHAO Jun-jing;YIN Yi-ming;ZHAO Fang-shu;LI Bao-hong;SUN Yun-hong;WANG Xiao-qing;ZHANG Xiao-ping;TIAN Jing-zhen(Shandong University of Traditional Chinese Medicine,Jinan 250355,China;The Affiliated Hospital of Qingdao University,Qingdao 266005,China)

机构地区：[1]山东中医药大学,济南250355 [2]青岛大学附属医院,山东青岛266005

出　　处：《中国实验方剂学杂志》2022年第12期200-209,共10页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：山东省重大科技创新工程项目(2021CXGC010511);山东省重点研发项目(2020CXGC010505)。

摘　　要：目的:预测荆防合剂治疗甲型H1N1流感的潜在靶点及作用机制,以期为荆防合剂临床应用提供参考依据。方法:通过中药系统药理学分析平台数据库(TCMSP)、SwissTargetPrediction、TargetNet数据库筛选荆防合剂抗甲型H1N1流感的活性成分及作用靶点,通过GeneCards、在线人类孟德尔遗传数据库(OMIM)、DisGeNet数据库获取甲型H1N1流感靶点并统一使用UniProt KB数据库进行标准化,借助Venny 2.1.0在线平台获取两者交集靶点;采用Cytoscape 3.2.1软件建立“药物-成分-靶点”网络图并进行拓扑学属性分析;将交集靶点上传至STRING 11.5数据库获得蛋白质-蛋白质相互作用(PPI)关系网络,并在Metascape平台中进行基因本体(GO)富集分析和京都基因与基因组百科全书(KEGG)分析。最后通过Autodock vina软件将度值靠前的活性成分和关键核心靶点进行对接验证,并用PyMOL软件进行可视化分析。采用BALB/C雌鼠进行实验验证,苏木素-伊红(HE)染色观察肺组织病变情况,酶联免疫吸附测定法(ELISA)检测肿瘤坏死因子-α(TNF-α)、白细胞介素-10(IL-10)、白细胞介素-17(IL-17)因子水平,实时荧光定量聚合酶链式反应(Real-time PCR)和蛋白免疫印迹法(Western blot)检测各组动物肺组织mRNA和蛋白表达水平。结果:荆防合剂中共有活性成分144个,获得靶点基因421个,甲型H1N1流感靶点2 956个,两者交集靶点199个,拓扑学分析显示,荆防合剂治疗甲型流感的核心成分为槲皮素、木犀草素和山柰酚,核心靶点为人前列腺素内过氧化物合酶2(PTGS2)、雌激素受体1(ESR1)、诱导型一氧化氮合酶2(iNOS2)、过氧化物酶体增殖物激活受体γ(PPARG)、环氧合酶-1(PTGS1)等。GO富集分析中生物学过程(BP)得到697个条目(P<0.01),分子功能(MF)得到59个条目(P<0.01),细胞组成(CC)得到21个条目(P<0.01)。KEGG富集分析中共得到132条信号通路(P<0.01)如磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/Akt)信号通�Objective: To predict the potential targets and mechanism of Jingfang mixture in the treatment of H1N1 influenza and provide references for clinical application of Jingfang mixture. Method: The active components and targets of Jingfang mixture against H1N1 influenza were screened out by Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP),SwissTargetPrediction,and TargetNet. The targets of H1N1 influenza were obtained from GeneCards,Online Mendelian Inheritance in Man(OMIM),and DisGeNET and standardized by UniProt KB. The intersection targets were obtained by Venny 2.1.0. The "drug-component-target" network was constructed with Cytoscape 3.2.1 and analyzed for the topological attributes. The intersection targets were uploaded to STRING 11.5 to obtain the protein-protein interaction(PPI)network. Gene Ontology(GO)enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis were carried out by Metascape. Finally,the top active components ranked by degree were docked to the core targets by Autodock vina and visually analyzed by PyMOL. Balb/c female rats were used for experimental verification. Hematoxylin-eosin(HE)staining was used to observe the pathological changes in lung tissues. Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of tumor necrosis factor-α(TNF-α),interleukin-10(IL-10), and interleukin-17(IL-17). Real-time fluorescence-based quantitative polymerase chain reaction(Real-time PCR) and Western blot were used to detect the mRNA and protein expression levels in lung tissues. Result: There were 144 active components in Jingfang mixture. A total of 421target genes of Jingfang mixture and 2 956 targets of H1N1 influenza were identified,including 199 common targets. Topological analysis showed that the core components of Jingfang mixture against H1N1 influenza included quercetin,luteolin,and kaempferol,and the core targets included prostaglandin-endoperoxide synthase 2(PTGS2),estrogen receptor alpha(ESR1),inducible nitric oxide synthas

关 键 词：荆防合剂 甲型H1N1流感 网络药理学 分子对接 实验验证 

分 类 号：R285[医药卫生—中药学] R289[医药卫生—中医学]