利拉鲁肽通过microRNA-33对2型糖尿病小鼠肝损伤的治疗作用

目的:观察利拉鲁肽对2型糖尿病小鼠肝组织微小RNA-33(microRNA-33,miR-33)、腺苷酸活化蛋白激酶(AMPK)及肝细胞凋亡通路相关蛋白的影响,探讨其可能作用机制,为临床应用提供药效学依据。方法:采用高脂饮食联合链脲佐菌素(STZ)腹腔注射复制C57BL/6小鼠2型糖尿病模型。小鼠随机分为4组,每组15只:对照组为正常小鼠皮下注射等量的生理盐水;模型组为T2DM小鼠皮下注射等量的生理盐水;利拉鲁肽低剂量组为了2DM小鼠皮下注射100μg·kg~(-1)·d~(-1)利拉鲁肽;利拉鲁肽高剂量组为T2DM小鼠皮下注射200μg·kg~(-1)·d~(-1)利拉鲁肽。给药4周后进行口服葡萄糖耐量试验(OGTT),检测各组小鼠空腹血糖(FBG)、甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、丙氨酸氨基转移酶(ALT)及天冬氨酸氨基转移酶(AST)的含量;HE染色检测肝组织病理学变化;免疫荧光检测肝组织的cleaved caspase-3;Western blot法检测p-AMPK/AMPK及凋亡相关蛋白Bcl-2、caspase-3的水平;实时定量PCR检测肝组织miR-33的水平。结果:与模型组相比,利拉鲁肽高、低剂量组小鼠FBG、TG、TC、LDL-C、ALT及AST含量明显降低,HDL-C含量明显升高(P0.05);肝组织病理结构的紊乱得到明显改善;免疫荧光结果可见,利拉鲁肽高、低剂量给药组小鼠的cleaved caspase-3明显降低;Western blot实验结果可见,利拉鲁肽高、低剂量给药组小鼠肝组织的Bcl-2表达明显增高,caspase-3表达显著下降(P0.05)肝组织AMPK磷酸化水平显著增加(P0.01);且肝组织miR-33水平显著降低(P0.01),且呈一定的剂量依赖性。结论:利拉鲁肽可以缓解2型糖尿病小鼠肝损伤,其机制可能与降低肝组织miR-33表达,从而增加AMPK磷酸化水平,进而抑制肝细胞凋亡有关。

Liraglutide ameliorates liver injury of type 2 diabetic mice via micro-RNA-33-AMPK pathway

AIM: To observe the effects of liraglutide on the level of microRNA-33 (miR-33) and the expression of AMP-activated protein kinase (AMPK) and apoptosis-related proteins in mice with type 2 diabetes mellitus (T2DM), and to explore its possible mechanism. METHODS: High-fat diet and intraperitoneal injection of streptozocin were used to establish the type 2 diabetic model in C57BL/6 mice. The mice were randomly divided into 4 groups (n=15):in control group, the normal mice were subcutaneously injected with equivalent volume of saline; in model group, the T2DM mice were subcutaneously injected with equivalent volume of saline; in low-and high-dose liraglutide treatment groups, the T2DM mice were subcutaneously injected with 100 and 200 μg·kg^-1·d^-1, respectively. After 4 weeks of administration, the levels of FBG, TG, TC, HDL-C, LDL-C, ALT and AST were determined. HE staining was used to observe the pathological changes of the liver tissues. The protein level of cleaved caspase-3 in the liver tissue was detected by the technique of immunofluorescence. The protein levels of p-AMPK/AMPK and apoptosis-related proteins were detected by Western blot. The expression of miR-33 in the liver tissues was detected by real-time PCR. RESULTS: Compared with model group, the contents of FBG, TG, TC, LDL-C, ALT and AST were decreased significantly, while the content of HDL-C was increased significantly in low-dose liraglutide group and high-dose liraglutide group (P<0.05). The protein levels of phosphorylated AMPK and Bcl-2 were up-regulated significantly, and the expression of cleaved caspase-3 was down-regulated significantly (P<0.05). The level of miR-33 was decreased significantly (P<0.01). CONCLUSION: Liraglutide alleviates liver injury in type 2 diabetic mice, and the mechanism may be associated with reducing the level of miR-33 and increasing the phosphorylation of AMPK in the liver tissues, thereby inhibiting hepatocyte apoptosis.