阿托伐他汀通过抑制PKC的激活对抗高糖诱导的人脐静脉内皮细胞氧化应激反应

目的:探讨阿托伐他汀对高糖诱导的人脐静脉血管内皮细胞(HUVECs)产生氧化应激的影响及其作用机制。方法:体外培养HUVECs,以25 mmol/L葡萄糖干预,模拟糖尿病患者体内环境,通过流式细胞术和共聚焦显微镜检测细胞内的活性氧(ROS)水平,采用Lucigenin分析方法测定还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶活性,分别应用实时荧光定量PCR和免疫印迹杂交的方法检测 NADPH氧化酶亚基Nox4和Nox2/gp91^phox的表达水平,用免疫印迹杂交方法检测蛋白激酶C(PKC)蛋白的磷酸化水平。结果:(1)在高糖环境(终浓度为25 mmol/L)下,HUVECs内ROS生成显著增加,NADPH氧化酶的活性显著增强,NADPH 氧化酶Nox4和Nox2/gp91^phox亚基的mRNA和蛋白表达水平显著上调;(2)阿托伐他汀可显著抑制高糖诱导的ROS 生成、NADPH氧化酶活性的增强及NADPH 氧化酶Nox4和Nox2/gp91^phox亚基表达水平的增加幅度,且具有浓度依赖性;(3)PKC抑制剂(PKC inhibitor peptide, 20 μmol/L)可显著抑制高糖环境下ROS的生成、NADPH氧化酶活性的增强及NADPH 氧化酶Nox4和Nox2/gp91^phox亚基表达水平的增加幅度;(4)阿托伐他汀可抑制高糖诱导的PKC蛋白的磷酸化。结论:PKC的活化参与了高糖诱导的HUVECs产生的氧化应激反应。阿托伐他汀通过抑制PKC蛋白的活化对抗高糖诱导的内皮细胞产生的氧化应激反应。

Atorvastatin inhibits high glucose-induced oxidative stress by depressing PKC activity in human umbilical vein endothelial cells

AIM:To explore the effect of atorvastatin on high glucose-induced oxidative stress and underlying mechanisms in human endothelial cells. METHODS:Human umbilical vein endothelial cells(HUVECs) were cultured in medium 199 containing normal concentration of glucose(5.5 mmol/L). For high glucose treatment, glucose solution was added to the final concentration of 25 mmol/L. Reactive oxygen species(ROS) were detected by flow cytometry and confocal microscopy. The activity of nicotinamide adenine dinucleotide phosphate(NADPH) oxidase was measured by lucigenin assay. Phosphorylated protein kinase C(PKC) and the expression levels of NADPH oxidase subunits Nox4 and Nox2/gp91^phox were determined by quantitative real-time PCR and immunoblotting. RESULTS:High glucose increased ROS production, NADPH oxidase activity and the expression of Nox4 and Nox2/gp91^phox subunits. Treatment of endothelial cells with atorvastatin resulted in significant inhibition(in a concentration-dependent manner) of high glucose-induced ROS production, NADPH oxidase activation and the expression of Nox4 and Nox2/gp91^phox subunits. PKC inhibitor showed a similar effect to that of atorvastatin on high glucose-induced oxidative stress. Furthermore, atorvastatin rapidly inhibited high glucose-induced activation of protein kinase C, an upstream activator of NADPH oxidase. CONCLUSION:PKC is involved in high glucose-induced oxidative stress in HUVECs. Atorvastatin inhibits high glucose-induced oxidative stress by depressing PKC activity in human endothelial cells.