利拉鲁肽通过 microRNA－33对2型糖尿病小鼠肝损伤的治疗作用

摘要

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T2DM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（P＜0．05）；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（P＜0．05），肝组织AMPK磷酸化水平显著增加（P＜0．01）；且肝组织miR－33水平显著降低（ P＜0．01），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。%AIM:To observe the effects of liraglutide on the level of microRNA-33 (miR-33) and the expres-sion 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 ob-serve 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 .