miR-17调控自噬保护高糖环境下内皮细胞

The Impact of miR-17 Regulated Autophagy on Endothelial Cells under High Glucose Condition

目的:研究miR-17调控的自噬对高糖环境下内皮细胞氧化应激反应和凋亡的影响。方法:体外培养人脐静脉内皮细胞(HUVECs),并加入5.5mmol/L(对照组)和16.7mmol/L(高糖组)的葡萄糖进行干预,通过检测超氧化物歧化酶(SOD)活性,丙二醛(MDA)含量及自噬相关基因LC3、Beclin-1和SQSTM1的表达,比较各组HUVECs氧化应激和自噬水平的改变。再将HUVECs转染miR-17 mimics,并以自噬抑制剂3-甲基腺嘌呤(3-MA)干预,非转染miR-17mimics作为对照,观察miR-17对HUVECs自噬、氧化应激和凋亡的影响,并评价3-MA抑制HUVECs自噬后上述指标的改变。结果:与对照组比较,高糖组HUVECs的SOD活性下降,MDA水平增加,同时LC3II/I比例和Beclin-1上调,SQSTM1表达减少(P<0.01)。在高糖环境下,转染miR-17 mimics后的HUVECs的LC3II/I比例和Beclin-1表达上调,SQSTM1水平降低,SOD活性部分恢复,MDA水平下降,细胞凋亡减少(P<0.01)。经3-MA干预后,HUVECs上述效应明显减弱(P<0.01)。结论:miR-17可上调内皮细胞的自噬水平,减轻高糖诱导的内皮细胞氧化应激反应,减少内皮细胞凋亡。

Objective:To study the impact of miR-17 regulated autophagy on endothelial cell oxidative stress and apoptosis under high glucose condition.Method:In vitro cultured human umbilical vein endothelial cells were treated with different concentrations of glucose(5.5 mmol/L and 16.7 mmol/L).Superoxide dismutase activity,malonaldehyde and autophagy related gene expression,including LC3,Beclin-1 and sequestosome 1 were measured by using superoxide dismutase activity detection kit,malonaldehyde detection kit and western blotting methods.Human umbilical vein endothelial cells were transfected with miR-17 mimics and treated with 3-Methyladenine under different concentrations of glucose.After transfection,the autophagy,oxidative stress and apoptosis of human umbilical vein endothelial cells were evaluated.Results:Under high glucose condition,superoxide dismutase activity declined and malonaldehyde level increased when compared with normal glucose group.Meanwhile,the LC3II/I ratio and expression of Beclin-1 elevated,accompanied with sequestosome 1 decrease(P<0.01).Forced expression of miR-17 in human umbilical vein endothelial cells increased the LC3II/I ratio and Beclin-1 expression,and decreased the levels of sequestosome 1,malonaldehyde and cell apoptosis.Over expression of miR-17 also partially restored superoxide dismutase activity(P<0.01).After inhibiting autophagy with 3-Methyladenine,the effect generated by miR-17 mimics transfection was diminished.Conclusion:Forced expression of miR-17 enhances autophagy and therefore decreases oxidative stress and apoptosis in endothelial cells.