基于网络药理学的茵陈术附汤抗石胆酸诱导的胆汁淤积性肝损伤作用机制研究

Protective mechanism of Yinchenzhufu decoction against cholestatic liver injury induced by lithic acid based on network pharmacology

茵陈术附汤(Yinchenzhufu decoction,YCZFD)是治疗阴黄证的经典方剂,可改善胆汁淤积性肝损伤,但作用机制尚不完全清楚,本文运用网络药理学、分子对接、动物实验和分子生物学方法,探讨YCZFD抗胆汁淤积性肝损伤机制。采用石胆酸(lithocholic acid,LCA)诱导的急性胆汁淤积小鼠模型,考察YCZFD给药2周对小鼠肝功能指标及肝脏组织形态学的影响。动物实验经上海中医药大学实验动物福利与伦理委员会批准,伦理编号为:PZSHUTCM190823002。结果显示,YCZFD可降低模型小鼠血生化指标水平,改善肝细胞损伤;运用多个数据库预测YCZFD活性成分与胆汁淤积性肝损伤对应靶点。利用String数据库和Cytoscape软件构建交集靶点PPI网络(protein-protein interaction networks),显示YCZFD抗胆汁淤积性肝损伤可能核心靶点与肿瘤坏死因子、白介素-1β、非受体酪氨酸激酶Src、白介素-6等有关,基于Metascape平台进行GO(gene ontology)和KEGG(kyoto encyclopedia of genes and genomes)富集分析,发现YCZFD抗胆汁淤积性肝损伤可能与其调节肿瘤坏死因子信号通路、核因子-κB信号通路、胆汁分泌等相关,并运用了AutoDockTools软件对YCZFD影响的核心靶点和核心成分进行分子对接验证。为验证网络药理学结果,采用UPLC-MS/MS方法测定小鼠肝脏中胆汁酸谱水平,显示YCZFD使模型小鼠肝脏中游离胆汁酸、牛磺酸结合型胆汁酸和总胆汁酸水平明显下降;通过real-time PCR和Western blot方法,发现YCZFD干预可上调胆汁淤积小鼠肝脏法尼醇X受体及代谢酶(尿苷二磷酸葡萄糖醛酸转移酶1a1)、外排转运体(胆盐外排泵、多药耐药相关蛋白2、多药耐药相关蛋白3等)表达,促进胆汁酸代谢和外排,改善胆汁酸稳态,抑制NOD样受体家族3活化介导的细胞焦亡及炎症反应,改善胆汁淤积性肝损伤。

Yinchenzhufu decoction(YCZFD)is a classic formula for treating Yin Huang syndrome,which can improve liver injury caused by cholestasis.However,the mechanism of action of YCZFD still remains unclear.This article used network pharmacology,molecular docking,animal experiments,and molecular biology methods to explore the mechanism of YCZFD in treating liver injury caused by cholestasis.A mouse model of acute cholestasis induced by lithocholic acid was used to investigate the effects of YCZFD on liver injury.The experimental procedures described in this paper were reviewed and approved by the Ethical Committee at the Shanghai University of Traditional Chinese Medicine(approval NO.PZSHUTCM190823002).The results showed that YCZFD could reduce the levels of blood biochemical indicators and improve hepatocyte damage of cholestatic mice.Then,multiple databases were used to predict the corresponding targets of YCZFD active components on cholestatic liver injury.An intersection target protein-protein interaction(PPI)networks based on String database and Cytoscape software was used to demonstrate the possible core targets of YCZFD against cholestatic liver injury.The results indicated that core targets of YCZFD include tumor necrosis factor,interleukin-1β,non-receptor tyrosine kinase Src,interleukin-6,etc.GO(gene ontology)and KEGG(kyoto encyclopedia of genes and genomes)enrichment analysis indicated that YCZFD may regulate the tumor necrosis factor signaling pathway,nuclear factor-κB signaling pathway,bile secretion,and other related factors to ameliorate the cholestatic liver injury.AutoDockTools software was used to perform molecular docking verification on the core targets and components of YCZFD.To verify the results of network pharmacology,UPLC-MS/MS method was used to determine the effect of YCZFD on levels of bile acid profiles in mouse liver tissues.It was found that treatment with YCZFD significantly reduced the content of free bile acids,taurine bound bile acids,and total bile acids in the liver tissues of cholestatic mice.Then,results from real time PCR and Western blot also found that YCZFD can upregulate the expression of hepatic nuclear receptor farnesoid X receptor,metabolizing enzyme(UDP glucuronidase transferase 1a1),and efflux transporters(bile salt export pump,multidrug resistance-associated protein 2,multidrug resistanceassociated protein 3,etc)in cholestasis mice,promote bile acid metabolism and excretion,and improve bile acid homeostasis.Moreover,YCZFD can also inhibit pyroptosis and inflammation by regulating NOD-like receptors 3 pathway,thereby inhibiting cholestatic liver injury.