基于网络药理学的逍遥散治疗代谢相关脂肪性肝病作用机制探讨及实验验证

目的 基于网络药理学预测逍遥散治疗代谢相关脂肪性肝病（MAFLD,曾用名非酒精性脂肪性肝炎,NASH）相关机制,并观察逍遥散对MAFLD模型小鼠肝损伤的保护作用及机制。方法 通过TCMSP数据库获取逍遥散各组成味药的活性成分及潜在靶点,利用Gencards、OMIM数据库获取MAFLD疾病靶点,构建蛋白质相互作用（PPI）网络,进行基因本体论（GO）功能及京都基因与基因组百科全书（KEGG）通路富集分析,构建成分-靶点-通路网络,并对核心靶点和主要活性成分进行分子对接验证。通过喂食蛋氨酸及胆碱缺乏（MCD）饲料构建MAFLD小鼠模型,给予多烯磷脂酰胆碱胶囊（178mg·kg^-1·d^-1）或逍遥散低、中、高剂量（1.437、2.874、5.748g·kg^-1·d^-1）进行干预,测定各组小鼠血清天冬氨酸氨基转移酶（AST）、丙氨酸氨基转移酶（ALT）活性及三酰甘油（TG）、总胆固醇（TC）水平;HE染色考察各组小鼠肝组织病理变化;Western blotting法检测PTGS2、ESR1、NOS2及PPARG的蛋白表达情况。结果 经筛选得到逍遥散治疗MAFLD的作用靶点有133个,核心成分主要有槲皮素、木犀草素、山柰酚、猪苓酸C等,关键靶点为PTGS2、ESR1、NOS2及PPARG等,主要的生物学通路为与肿瘤相关信号通路、糖尿病并发症中的AGE-RAGE信号通路、IL^-17信号通路及非酒精性脂肪肝相关信号通路等,分子对接结果显示槲皮素、木犀草素及山柰酚与PTGS2、ESR1、NOS2及PPARG等均有较强的结合。体内动物实验表明,与模型组比较,逍遥散各组小鼠AST、ALT、TG、TC水平均显著降低（P＜0.05）;肝组织病理损伤均得到改善;与模型组比较,逍遥散中、高剂量组小鼠PTGS2、ESR1及NOS2蛋白表达显著降低（P＜0.01）,PPARG蛋白表达显著升高（P＜0.01）。结论 逍遥散可能通过调控PTGS2、ESR1、NOS2及PPARG等靶标,IL^-17信号通路及非酒精性脂肪肝相关信号通路等多种途径治疗MAFLD。

Mechanism of Xiaoyaosan on metabolic associated fatty liver disease based on network pharmacology and experiment verification

Objective To explore the mechanism of Xiaoyaosan in the treatment of metabolic associated fatty liver disease (MAFLD, used to be known as nonalcoholic steatohepatitis, NASH) based on network pharmacology and in vivo experiments.Methods The active components and potential targets of Xiaoyaosan were obtained from TCMSP database, and the disease targets of MAFLD were obtained from Gencards and OMIM database. A protein-protein interaction (PPI) network based on active components and disease common targets was constructed, Metascape platform was used for gene ontology (GO) function and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis, the "component-target-pathway" network was constructed, and autodock software was used for molecular docking verification of key compounds and targets. MAFLD mouse model was established by feeding methionine and choline deficient L-amino acid diet (MCD). Polyene Phosphatidylcholine Capsules (178 mg·kg^-1·d^-1) or Xiaoyaosan (1.437, 2.874, 5.748 g·kg^-1·d^-1) were given to intervene. The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglyceride (TG) and total cholesterol (TC) in serum of mice in each group were measured, pathological changes of liver tissues of mice in each group were investigated by HE staining. The protein expressions of PTGS2, ESR1, NOS2 and PPARG were detected by Western blotting.Results 160 active components of Xiaoyaosan and 619 corresponding action targets were obtained, including 133 action targets for the treatment of MAFLD. The core components of Xiaoyaosan in the treatment of MAFLD were quercetin, luteolin, kaempferol and polyporenic acid C and so on, and the key targets were PTGS2, ESR1, NOS2 and PPARG etc. The main biological pathways are tumor related signaling pathways, AGE-RAGE signaling pathways in diabetic complications, IL-17 signaling pathways, and nonalcoholic fatty liver related signal pathways. Molecular docking results show that quercetin, luteolin and kaempferol are strongly associated with PTGS2, ESR1, NOS2 and PPARG. In vivo animal experiments showed that compared with the model group, the levels of AST, ALT, TG and TC in Xiaoyaosan group were significantly reduced (P <0.05), and the pathological damage of liver tissue was improved. Compared with the model group, the expressions of PTGS2, ESR1 and NOS2 protein in the middle and high dosage groups of Xiaoyaosan were significantly decreased (P <0.01), while the expression of PPARG protein was significantly increased (P <0.01).Conclusion Xiaoyaosan may treat MAFLD by regulating targets such as PTGS2, ESR1, NOS2 and PPARG, IL-17 signal pathway and nonalcoholic fatty liver related signal pathway.