利拉鲁肽通过AMPK/mTOR信号通路诱导自噬改善肝脂肪变性

目的探讨腺苷酸活化蛋白激酶(AMPK)/雷帕霉素靶蛋白(mTOR)通路在利拉鲁肽(LG)改善肝细胞脂肪变性中的作用及机制。方法动物实验:将小鼠分为对照组、模型组和LG组，各10只，对照组普通饲料喂养, 另2组高脂饲料喂养建立肝脂肪变性模型。LG组应用LG腹腔注射0.6 mg·kg-1·d-1，另2组应用等量0.9%氯化钠溶液，连续4周。检测小鼠血清丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、低密度脂蛋白(LDL)、胆固醇(TC)、三酰甘油(TG)；应用HE染色和油红O染色观察肝细胞形态、脂肪变性和脂滴情况；应用蛋白质印迹(Western blotting)法检测p-AMPK、p-mTOR及自噬蛋白LC3B、Beclin1表达情况。细胞实验:应用棕榈酸(PA)诱导建立HepG2肝细胞脂肪变性模型，分为模型组(PA)、LG组(PA+LG)和AMPK抑制剂组(PA+LG+Compound C)。应用免疫荧光检测自噬相关蛋白LC3B的表达；应用Western blotting法检测p-AMPK、p-mTOR、LC3B、Beclin1的蛋白表达情况。结果动物实验:模型组ALT、AST、LDL、TC、TG升高，应用LG后下降，差异均有统计学意义(P < 0.05~P < 0.01)；HE染色、油红O染色发现，模型组肝细胞可见大片脂肪变性、大量融合脂滴，LG组脂变减轻、脂滴含量减少。Western blotting结果显示，模型组p-AMPK、LC3B、Beclin1的蛋白表达下降，而LG组中升高，模型组p-mTOR升高，而LG组中降低(P < 0.05~P < 0.01)。细胞实验:免疫荧光染色LC3B发现，LG组较模型组增加，而AMPK抑制剂组，LC3B的表达受到抑制；Western blotting结果示:和模型组相比，LG组p-AMPK、LC3B、Beclin1的蛋白表达升高，而应用AMPK抑制剂后表达受抑制，而p-mTOR相反, 差异均有统计学意义(P < 0.05~P < 0.01)。结论LG通过AMPK/mTOR通路来改善肝细胞脂肪变性，自噬可能参与其中。

Liraglutide induces autophagy through AMPK / mTOR signaling pathway and improves hepatocyte steatosis

ObjectiveTo investigate the role and possible mechanism of adenylate activated protein kinase(AMPK)/rapamycin target protein(mTOR) pathway in the improvement of liraglutide(LG) on hepatocyte steatosis.MethodsIn animal experiment, the mice were randomly divided into control group, model group and LG group, with 10 mice in each group.The mice in the control group were fed with ordinary diet, and the mice in other groups were fed with high-fat diet to construct hepatic steatosis model.In the LG group, the mice were injected with LG at 0.6 mg·kg-1·d-1 intraperitoneally, and in the other two groups, the mice were treated with the same amount of 0.9% sodium chloride solution for 4 weeks.The levels of serum alanine aminotransferase(ALT), aspartate aminotransferase(AST), low density lipoprotein(LDL), cholesterol(TC) and triglyceride(TG) were detected; the changes of morphology, steatosis and lipid droplets of hepatocytes were observed through HE and oil red O staining methods.The expressions of p-AMPK, p-mTOR, autophagy-related proteins LC3B and Beclin1 were detected by Western blotting.In cell experiment, the palmitic acid(PA) induced steatosis model in HepG2 hepatocytes was established, the cells were equally divided into model group(PA), LG Group(PA+LG) and AMPK inhibitor group(PA+LG+compound C).The expression of autophagy-related protein LC3B was detected by immunofluorescence; the protein expressions of p- AMPK, p-mTOR, LC3B and Beclin1 were detected by Western blotting.ResultsIn contrast to control group, the levels of ALT, AST, LDL, TC and TG in model group were increased significantly, and decreased significantly after LG treatment (P < 0.05 to P < 0.01);HE and oil red O staining showed that in the model group, there had large areas of steatosis and a large number of fused lipid droplets.The steatosis and lipid droplets content was decreased significantly in the LG group.Western blotting results showed that the protein expressions of p-AMPK, LC3B and Beclin1 were decreased significantly in model group, but increased in LG group, p-mTOR was increased in model group, and decreased in LG group(P < 0.05 to P < 0.01).Cell experiment: immunofluorescence staining showed that LC3B expression in LG group was significantly higher than that in model group, while LC3B expression was inhibited in AMPK inhibitor group.Western blotting results showed that compared with the model group, the protein expressions of p-AMPK, LC3B and Beclin1 in LG group were significantly increased, but the expressions were inhibited after the application of AMPK inhibitor, while p-mTOR expression was on the contrary, which had statistically significance(P < 0.05 to P < 0.01).ConclusionsLiraglutide improved hepatocyte steatosis through AMPK/mTOR pathway, and autophagy may be involved.