化滞柔肝颗粒减轻非酒精性脂肪性肝炎相关肝纤维化的机制研究  

Revealing the mechanism of Huazhi Rougan Granule in alleviating non-alcoholic steatohepatitis-related fibrosis

作　　者：余思雨 操颖 朱明哲 黄志艳 季光 张贵民 张莉 YU Siyu;CAO Ying;ZHU Mingzhe;HUANG Zhiyan;JI Guang;ZHANG Guimin;ZHANG Li(Institute of Digestive Diseases,Longhua Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai 200032,China;School of Public Health,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;State Key Laboratory of Generic Manufacture Technology of Traditional Chinese Medicine,Linyi,Shandong 276006,China)

机构地区：[1]上海中医药大学附属龙华医院脾胃病研究所,上海200032 [2]上海中医药大学公共健康学院,上海201203 [3]中药制药共性技术国家重点实验室,山东临沂276006

出　　处：《上海中医药杂志》2023年第2期41-50,共10页Shanghai Journal of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(82274448)。

摘　　要：目的结合网络药理学研究方法,探讨化滞柔肝颗粒(HZRG)减轻非酒精性脂肪性肝炎(NASH)相关肝纤维化的作用机制。方法借助网络药理学分析聚焦HZRG治疗NASH的潜在靶点。将雄性C57BL/6小鼠随机分为对照组、模型组和HZRG低、高剂量组,每组8只。除对照组外,其余各组小鼠给予蛋氨酸胆碱缺乏饮食诱导NASH相关纤维化模型,造模的同时给予相应药物干预,连续4周。HE染色观察肝组织病理变化,并行非酒精性脂肪性肝病活动度评分(NAS);天狼猩红和Masson染色法观察肝脏胶原沉积情况;免疫组化法观察肝组织中α-平滑肌肌动蛋白(α-SMA)和Ⅰ型胶原蛋白α1链(Col1a1)的表达情况,并用ImageJ软件分析阳性表达面积。试剂盒检测肝组织总胆固醇(TC)、三酰甘油(TG)水平。采用生化分析仪检测各组小鼠血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、乳酸脱氢酶(LDH)、碱性磷酸酶(ALP)水平,ELISA试剂盒检测血清肿瘤坏死因子-α(TNF-α)水平。Western blot法和免疫荧光法检测α-SMA、Col1a1、p53蛋白表达水平;RT-qPCR法检测p53 mRNA相对表达量。不同浓度转化生长因子-β1(TGF-β1)或Tenovin-6处理人源性肝星状细胞系LX2细胞24 h,检测相关指标。结果①网络药理学研究结果提示,p53是HZRG发挥效应的潜在靶点。②HE染色、Masson染色和天狼猩红染色结果显示,模型组小鼠肝脏脂质累积、炎细胞浸润、纤维化形成,与对照组比较,模型组小鼠肝组织病理α-SMA、Col1a1阳性表达面积增加(P<0.05),NAS评分升高(P<0.05);HZRG低、高剂量组小鼠肝细胞内脂滴减少,肝脏脂肪变和炎细胞浸润程度明显减轻,与模型组比较,HZRG低、高剂量组肝组织病理α-SMA、Col1a1阳性表达面积减少(P<0.05),NAS评分降低(P<0.05)。③与对照组比较,模型组小鼠血清ALT、AST、LDH、ALP水平升高(P<0.05);与模型组比较,HZRG低、高剂量组小鼠血清ALT、AST、LDH、TNF-α水平降低(P<0.Objective To explore the mechanism of Huazhi Rougan Granule(HZRG)in improving non-alcoholic steatohepatitis(NASH)-related fibrosis by network pharmacology methods.Methods Potential targets of HZRG for NASH were investigated through the analysis of network pharmacology.Male C57BL/6 mice were randomly divided into control group,model group and HZRG low dose group and HZRG high dose group,and 8 mice were allocated per group.The mice in each group were given a methionine choline-deficient diet(MCD)to induce NASHrelated fibrosis model except those in the control group,and the corresponding pharmacological interventions were given at the same time of modeling for 4 consecutive weeks.HE staining was used to observe the pathological changes of liver tissues,and the non-alcoholic fatty liver disease activity score(NAS)was used in parallel;Sirius red staining and Masson’s trichrome staining were used to observe the liver collagen deposition;immunohistochemistry was used to detect the expressions ofα-smooth muscle actin(α-SMA)and type I collagenα1 chain(Col1a1)in liver tissues,and the area of positive expression was analyzed by ImageJ.The reagent kit was used to detect TC and TG levels in liver tissues.Biochemical analyzer was used to measure the blood alanine aminotransferase(ALT),aspartate aminotransferase(AST),lactate dehydrogenase(LDH)and alkaline phosphatase(ALP)levels of mice in each group,and ELISA kits was used to measure the level of serum tumor necrosis factor-α(TNF-α).The expressions of fibrosis parametersα-SMA,Col1a1 and p53 were detected by western blot and immunofluorescence,respectively;and the expression of p53 mRNA was detected by RT-qPCR.LX2 cells were treated with different concentrations of TGF-β1 and/or Tenovin-6 for 24 hours,and related indicators were detected.Results①The results of network pharmacology suggested that p53 was a potential target for HZRG to exert its effect on.②The results of HE,Sirius red and Masson’s trichrome staining showed hepatic lipid accumulation,inflammatory cel

关 键 词：非酒精性脂肪性肝炎 肝纤维化 化滞柔肝颗粒 中医药疗法 网络药理学 作用机制 

分 类 号：R285.5[医药卫生—中药学]