护肝解毒方调控NAFLD大鼠机制的网络药理学及实验研究

Network pharmacology mechanism and experimental verification of Hugan Jiedu Fang regulating nonalcoholic fatty liver disease in rats

目的运用网络药理学等生物信息学技术预测以及非酒精性脂肪肝病(NAFLD)大鼠模型实验验证研究,揭示护肝解毒方改善NAFLD的主要作用机制。方法通过网络药理学方法筛选出护肝解毒方主要作用成分及其NAFLD主要作用靶点,并进行基因通路分析和GO富集分析,定位护肝解毒方作用的关键通路和生物学功能。动物实验采用Wistar雄性大鼠30只随机分为正常组、模型组、护肝解毒方组,每组10只。检测实验前后各组大鼠血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、γ-谷氨酰转肽酶(γ-GT)、总胆固醇(TC)、甘油三酯(TG)、空腹血糖(FBG)、肝脏组织活性氧(ROS)等生化指标、肝组织病理以及NAFLD关键靶点蛋白mRNA表达水平变化。结果网络药理学方法筛选出了护肝解毒方9个主要作用成分以及12个NAFLD相关作用靶点,预测出沉默信息调节因子1(SIRT1)、固醇调节元件结合蛋白1(SREBF1)等是护肝解毒方主要成分-NAFLD相关作用靶点中的核心蛋白;KEGG通路分析和GO富集分析表明护肝解毒方主要作用靶点与NAFLD的相关信号通路、病理和生物过程密切相关。动物实验生化指标检测显示模型组大鼠的ALT、AST、γ-GT、TC、TG、FBG、ROS等指标均较正常组显著升高,护肝解毒方组大鼠的ALT、AST、γ-GT、TC、TG、FBG、ROS等指标较模型组显著降低,差异均有显著统计学意义(P<0.01);HE染色可见护肝解毒方对NAFLD大鼠肝脏组织脂肪变性具有明显的减轻作用;Real-TimePCR实验显示,模型组大鼠肝脏Sirt1 mRNA表达水平显著低于正常组,Srebp1 mRNA表达水平显著高于正常组,护肝解毒方组大鼠肝脏Sirt1 mRNA表达显著高于模型组,Srebp1 mRNA表达显著低于模型组,差异均有显著统计学意义(P<0.01)。结论护肝解毒方能有效调控NAFLD大鼠SIRT1和SREBF1等肝脂代谢相关基因表达、改善糖脂代谢、减少肝脏氧化应激,减轻肝细胞脂肪变性。

Objective To explore the main mechanism of Hugan Jiedu Fang in improving nonalcoholic fatty liver disease(NAFLD)by using network pharmacology and other bioinformatics techniques,as well as experimental ver-ification.Methods The main active components and targets of Hugan Jiedu Fang and NAFLD related targets were screened by network pharmacology method,and the gene pathway analysis and GO enrichment analysis were carried out to locate the key pathways and biological functions of Hugan Jiedu Fang.In animal experiment,30 male Wistar rats were randomly divided into normal group,model group,and Hugan Jiedu Fang group,with 10 rats in each group.Serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),γ-Glutamyl transpeptidase(γ-GT),total cholesterol(TC),tri-glyceride(TG),fasting blood glucose(FBG),reactive oxygen species(ROS)of liver tissue,liver histopathology,and mRNA expression levels of key NAFLD target proteins were detected before and after the experiment.Results Nine ma-jor components and 12 NAFLD-related action targets were screened out by network pharmacological method.Silent infor-mation regulator 1(SIRT1)and sterol regulatory element-binding protein 1(SREBF1)are the core proteins in NAFLD-re-lated action targets and the main components of liver protection and detoxification.KEGG pathway analysis and GO en-richment analysis indicated that the main action targets of Hugan Jiedu Fang were closely related to the signal pathways,pathology and biological processes of NAFLD.Biochemical index tests showed that ALT,AST,γ-GT,TC,TG,FBG,ROS and other indexes in model group were significantly higher than those in normal group,while ALT,AST,γ-GT,TC,TG,FBG,ROS and other indexes in Hugan Jiedu Fang group were significantly lower than those in model group,with statistically significant differences(P<0.01).HE staining showed that Hugan Jiedu Fang alleviated steatosis of NAFLD rats.Real-time PCR showed that the expression level of Sirt1 mRNA in the liver of rats in the model group was significantly lower than that in the normal group,and the expression level of Srebp1 mRNA was significantly higher than that in the normal group.The expression level of Sirt1 mRNA in the liver of rats in the Hugan Jiedu Formula group was significantly higher than that in the model group.Meanwhile,the mRNA expression of Srebp1 was significantly low-er than that of model group,with statistically significant differences(P<0.01).Conclusion Hugan Jiedu Fang can effec-tively regulate the expression of SIRT1,SREBF1 and other genes related to liver lipid metabolism in NAFLD rats,im-prove glucose and lipid metabolism,reduce oxidative stress in the liver,and thereby alleviate hepatocyte steatosis.