Mechanism of Gan Dou Ling in improving liver fibrosis in Wilson disease based on network pharmacology and experimental verification

Objective:To explore and verify the mechanism of Gan Dou Ling in improving liver fibrosis in Wilson disease(WD)by network pharmacology and copper loaded mice experiments.Methods:The main chemical components and corresponding gene targets of each drug in Gan Dou Ling were obtained by using TCMSP database.The database of gene mutation and disease related gene was searched through the GeneCards database,DrugBank database,PharmGKB database and the DisGeNET database.After the intersection of drug and disease target genes.The STRING website was used to analyze the protein-protein interaction degree of target genes,and import the data to Cytoscape software 38.2 to analyze protein interaction network.The GO databases and KEGG databases were obtained in R language for enrichment analysis.On this basis,Masson staining were used to observe the degree of liver fibrosis in copper loaded mouse model,and the results of network pharmacological analysis were verified by Western Blot(WB).Results:A total of 108 drug disease intersection genes were analyzed by network pharmacology.Through PPI network analysis,JUN was found to be the key genes.The enrichment analysis of KEGG pathway showed that MAPK signal pathway was the important potential target pathways.Animal experiments showed that Gan Dou Ling could reduce liver fibrosis and inhibit the phosphorylation of P38,JNK and C-JUN in copper loaded mice.Conclusion:Gan Dou Ling may achieve the effect of treating WD liver fibrosis by inhibiting P38/JNK signaling pathway.

基于网络药理学与实验验证探讨肝豆灵改善Wilson病肝纤维化机制

目的:通过网络药理学和铜负荷小鼠实验验证探讨肝豆灵改善Wilson病(Wilson disease,WD)肝纤维化作用机制。方法:使用TCMSP平台获取肝豆灵中各药的主要化学成分及对应的基因靶点。通过基因卡片数据库(GeneCards)、药物总数据库(DrugBank)、遗传药理学与药物基因组学数据库(PharmGKB)、疾病相关的基因与突变位点数据库(DisGeNET)搜索筛选疾病主要靶点。取药物-疾病靶基因交集后,使用STRING网站分析靶基因的蛋白质间相互作用联系度,将分析出的数据导入到Cytoscape3.8.2软件中分析蛋白质相互作用网络,并用R语言获取GO与KEGG数据库进行富集分析。在此基础上,通过MASSON染色观察铜负荷小鼠模型肝组织纤维化程度,使用蛋白免疫印迹法(western blot,WB)验证网络药理学分析结果。结果:网络药理学分析出药物-疾病交集基因108个,通过PPI网络分析发现JUN是关键基因,KEGG通路富集分析结果显示MAPK信号通路是重要潜在通路。动物实验表明肝豆灵可减轻铜负荷小鼠肝纤维化,抑制P38、JNK、C-JUN磷酸化。结论:肝豆灵治疗WD肝纤维化可能与其抑制P38/JNK信号通路有关。