二甲双胍激活AMPK对高糖诱发内皮细胞氧化应激的影响

Effects of AMP-activated protein kinase activation on oxidative stress in endothelial cells

目的探讨二甲双胍激活AMPK对高糖诱发的血管内皮细胞氧化应激过程的影响。方法高糖培养诱导人主动脉内皮细胞HAEC氧化应激,用AMPK激动剂5-氨基咪唑-4-甲酰胺-1-B-呋喃核糖苷(AICAR,2 mmol/L)和二甲双胍(metformin,2mmol/L)进行干预,分别用Western blot和免疫荧光检测内皮细胞AMPK/T-172磷酸化蛋白表达水平及细胞内氧化应激相关蛋白p47phox和p67phox的表达。雄性C57BL/6小鼠随机分为正常对照组(NC,n=10)、链脲菌素诱导糖尿病模型组(DM,n=10)和二甲双胍干预糖尿病模型组(MT,n=10),造模成功后MT组经饮水给予二甲双胍50mg/(kg/d),NC组、DM组给予正常饮水。期间定期测定小鼠体重,并检测各阶段血糖。二甲双胍干预一周后处死小鼠,取主动脉做免疫组化观察血管内皮细胞的氧化应激状态。结果在内皮细胞中二甲双胍可通过激活AMPK来抑制NADPH氧化酶胞质内亚基p47phox和p67phox的表达。动物实验显示,与糖尿病模型组小鼠相比,二甲双胍干预组小鼠血糖降低,体重增加,差异均有统计学意义(P<0.05)。免疫组化分析显示,小鼠主动脉血管内皮细胞的氧化应激标志性产物3-NT(3-nitrotyrosine),MDA(malondialdehyde)和HNE(4-hydroxy-2-nonenal)的水平均明显降低。结论二甲双胍可通过激活AMPK来缓解内皮细胞中出现的氧化应激,可能对糖尿病血管并发症具有预防和治疗作用。

Objective To observe the effects of AMPK activated by metformin on intracellular oxidative stress induced by high glucose in endothelial cells.Methods Our study was divided into a cell experiment and an animal experiment.Firstly,oxidative stress was induced by high glucose in endothelial cells,and AMPK was activated by AICAR and metformin respectively.Then Western blot and immunofluorescent assay were used to detect the AMPK activation and expression of NADPH subunits p47 phox and p67 phox.In the animal experiment,30 male C57BL/6mice were divided into 3groups:normal control group(NC,n=10),diabetic model group induced by STZ(DM,n=10),and diabetic model group treated with metformin(MT,n=10).The MT group was given a dose of 50mg/(kg/d)of metformin in drinking water,while NC and DM groups were given normal drinking water.After measuring body weight and blood glucose for a week,mice were sacrificed and aortas were examined by immunohistochemical analysis.Results Our in vitro results showed that metformin increased AMPK activation and inhibied the expression of NADPH subunits p47 phox and p67 phox.The animal experiment showed that metformin decreased blood glucose and increased body weight in diabetic model mice,and importantly,decreased the level of iconic products of oxidative stress 3-NT(3-nitrotyrosine),MDA(malondialdehyde)and HNE(4-hydroxy-2-nonenal).Conclusion Metformin can relieve intracellular oxidative stress through AMPK activation,and these may be used for the prevention and treatment of vascular complications of diabetis.