基于AMPK/SIRT1-FoxO1信号通路探讨葛根芩连汤对db/db糖尿病小鼠肝脏氧化应激的影响

Effects of Gegen Qinlian Decoction on Oxidative Stress in Liver of db/db Diabetes Mellitus Mice Based on AMPK/SIRT1-FoxO1 Signaling Pathway

目的观察葛根芩连汤对db/db小鼠肝脏氧化应激的影响,探讨其改善2型糖尿病的作用机制。方法将75只自发性2型糖尿病db/db小鼠随机分为模型组、二甲双胍组和葛根芩连汤高、中、低剂量组,每组15只,另取15只db/m小鼠作为空白组,分别予相应药物灌胃12周。检测小鼠体质量、空腹血糖(FPG)及糖化血红蛋白(HbA1c)含量,HE染色观察肝脏组织病理改变,化学发光免疫分析法检测肝组织丙二醛(MDA)含量和谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)活性,Western blot检测肝组织腺苷酸活化蛋白激酶(AMPK)、p-AMPK、沉默信息调节因子1(SIRT1)、叉头框蛋白1(FoxO1)、p-FoxO1蛋白表达,实时荧光定量PCR检测肝组织AMPK、SIRT1、FoxO1 mRNA表达。结果与空白组比较,模型组小鼠体质量、FPG及HbA1c含量显著增加(P<0.01);肝细胞脂肪变性、点状坏死,肝窦淤血;肝组织MDA含量显著增加,GSH-Px、CAT活性显著降低(P<0.01),AMPK、p-AMPK和SIRT1蛋白表达显著降低,FoxO1和p-FoxO1蛋白表达显著升高(P<0.01),AMPK和SIRT1 mRNA表达显著降低,FoxO1 mRNA表达显著升高(P<0.01)。与模型组比较,各给药组小鼠体质量、FPG及HbA1c含量显著减少(P<0.05,P<0.01);肝组织病理改变有所减轻;肝组织MDA含量显著减少,GSH-Px、CAT活性显著升高(P<0.05,P<0.01),p-AMPK、AMPK和SIRT1蛋白表达显著升高,FoxO1和p-FoxO1蛋白表达显著降低(P<0.05,P<0.01),AMPK和SIRT1 mRNA表达显著升高,FoxO1 mRNA表达显著降低(P<0.05)。与二甲双胍组比较,葛根芩连汤高剂量组各指标变化更显著(P<0.05)。结论葛根芩连汤可能通过调节AMPK/SIRT1-FoxO1信号通路降低db/db小鼠肝脏氧化应激水平,改善2型糖尿病。

Objective To observe the effects of Gegen Qinlian Decoction on oxidative stress in liver of db/db mice;To explore its mechanism of improving type 2 diabetes mellitus.Methods Totally 75 db/db mice with spontaneous type 2 diabetes mellitus were randomly divided into model group,metformin group and Gegen Qinlian Decoction high-,medium-and low-dosage groups,with 15 mice in each group,and another 15 db/m mice were taken as blank group.The corresponding drugs were given by gavage for 12 weeks.The body weight,fasting plasma glucose(FPG)and HbA1c of mice were detected,HE staining was used to observe the pathological changes of liver tissue,chemiluminescence immunoassay was used to detect malondialdehyde(MDA)content,glutathione peroxidase(GSH-Px)and catalase(CAT)activity in liver tissue,Western blot was used to detect adenylate-activated protein kinase(AMPK),p-AMPK,silent information regulator 1(SIRT1),forkhead box 1(FoxO1),and p-FoxO1 protein expression in liver tissue,real-time fluorescence quantitative PCR was used to detect the mRNA expressions of AMPK,SIRT1 and FoxO1 in liver tissue.Results Compared with the blank group,the body weight,FPG,and HbA1c content in the model group increased significantly(P<0.01);hepatocyte steatosis,punctate necrosis,hepatic sinus congestion;MDA content significantly increased in liver tissue,GSH-Px and CAT activities significantly decreased(P<0.01),the protein expressions of AMPK,p-AMPK and SIRT1 significantly decreased,and the protein expressions of FoxO1 and p-FoxO1 significantly increased(P<0.01),the mRNA expressions of AMPK and SIRT1significantly decreased,and the expression of FoxO1 mRNA significantly increased(P<0.01).Compared with the model group,the body weight,FPG,and HbA1c content were significantly reduced in each administration group(P<0.05,P<0.01);the pathological changes of liver tissue alleviated;the content of MDA in liver tissue significantly reduced,the activities of GSH-Px and CAT significantly increased(P<0.05,P<0.01),AMPK,p-AMPK and SIRT1protein expressions significantly increased,FoxO1 and p-FoxO1 protein expressions significantly decreased(P<0.05,P<0.01),the mRNA expressions of AMPK and SIRT1 significantly increased,and the expression of FoxO1 mRNA significantly decreased(P<0.05).Compared with the metformin group,the changes of each index in the Gegen Qinlian Decoction high-dosage group were more significant(P<0.05).Conclusion Gegen Qinlian Decoction may reduce the level of liver oxidative stress in db/db mice and improve type 2 diabetes mellitus by regulating the AMPK/SIRT1-FoxO1 signaling pathway.