基于Nrf2/ARE信号通路探讨白藜芦醇治疗非酒精性脂肪性肝炎大鼠的作用机制

The mechanism of resveratrol in the treatment of nonalcoholic steatohepatitis in rats based on Nrf2/ARE signaling pathway

目的基于Nrf2/ARE信号通路探讨白藜芦醇治疗非酒精性脂肪性肝炎大鼠的作用机制。方法利用高脂饮食法进行非酒精性脂肪性肝炎大鼠造模。大鼠分为对照组(CD组)、高脂饮食组(HCD组)、白藜芦醇组(HCD+RSV组)及(白藜芦醇+OAD85)组(HCD+RSV+OAD85组),每组8只。CD组始终喂养普通饲料,其他组自由高脂饮食。(HCD+RSV)组及(HCD+RSV+OAD85)组在喂养第4周开始灌胃给予10 mg/kg RSV或(10 mg/kg RSV+100μg/kg OAD85),连续灌胃给药28 d(OAD85为Nrf2/ARE抑制剂齐墩果酸衍生物)。HE染色观察肝组织病理学改变,肝组织冰冻切片油红O染色观察脂肪量变化,试剂盒检测ALT、AST、TC、TG、HDL、LDL和FFA,Western-blot和qRT-PCR检测Keap1、Nrf2、ARE、NQO1、HO-1蛋白和mRNA表达。结果与CD组相比,HCD组肝脂肪变性、小叶炎症、门静脉炎症、肿胀程度NASH评分、脂肪含量及血清中ALT、AST升高(P<0.05),与HCD组相比,(HCD+RSV)组上述指标均降低(P<0.05),HCD组与(HCD+RSV+OAD85)组差异无统计学意义(P>0.05)。与CD组相比,HCD组NQO1、HO-1、Nrf2、ARE蛋白及mRNA表达均明显升高(P<0.001),Keap1蛋白表达降低(P<0.001),与HCD组对比,(HCD+RSV)组NQO1、HO-1、Nrf2、ARE蛋白及mRNA表达亦明显增加(P<0.001),Keap1蛋白及mRNA表达明显降低(P<0.001),HCD组与(HCD+RSV+OAD85)组差异无统计学意义(P>0.05)。结论白藜芦醇治疗非酒精性脂肪性肝炎大鼠可能是基于Nrf2/ARE信号通路激活机制,从而改善氧化应激水平,可减轻肝病理损伤。

Objective To explore the mechanism of resveratrol in the treatment of nonalcoholic steatohepatitis rats based on Nrf2/ARE signaling pathway.Methods Nonalcoholic steatohepatitis rats were molded by high-fat diet.Rats were divided into control group(CD group),high-fat diet group(HCD group),resveratrol group(HCD+RSV group)and resveratrol+Nrf2/ARE inhibitor oleanolic acid derivative OAD85 group(HCD+RSV+OAD85 group),with 8 rats in each group.The CD group was always fed a normal diet,while other groups were fed a free high-fat diet.The HCD+RSV group and the HCD+RSV+OAD85 group were given 10 mg/kg RSV or 10 mg/kg RSV+100μg/kg OAD85 by intragastric administration for 28 days at the fourth week of feeding.The liver pathological changes were observed by hematoxylin-eosin staining,and the fat changes were observed by oil red O staining of frozen sections of the liver.Kit method was used to detect alanine aminotransferase(ALT),aspartate aminotransferase(AST),cholesterol(TC),triglyceride(TG),high-density lipoprotein(HDL),low density lipoprotein(LDL)and free fatty acid(FFA).Western blot and qRT-PCR were used to detect the protein and mRNA levels of Keap1,Nrf2,ARE,1 NQO1,HO-1.Results Compared with the CD group,hepatic steatosis,lobular inflammation,portal vein inflammation,swelling NASH score,fat content,serum enzyme indicators ALT and AST significantly increased in the HCD group(P<0.05).Compared with the HCD group,all the above indicators in the HCD+RSV group significantly decreased(P<0.05),there were no statistical difference in the above indicators between the HCD group and the HCD+RSV+OAD85 group(P>0.05).Compared with the CD group,the protein expressions of NQO1,HO-1,Nrf2,ARE and mRNA in the HCD group significantly increased(P<0.001),while the expressions of Keap1 protein expression decreased(P<0.001).Compared with the HCD group,the protein expressions of NQO1,HO-1,Nrf2,ARE and mRNA in the HCD+RSV group also increased(P<0.001),while the expressions of Keap1 protein expression and mRNA decreased(P<0.001)There was no statistical difference in the above indicators between HCD group and HCD+RSV+OAD85 group(P>0.05).Conclusions Resveratrol in the treatment of non-alcoholic steatohepatitis rats may be based on the activation mechanism of Nrf2/ARE signaling pathway,thereby improving the level of oxidative stress and alleviating liver pathological damage.