NADPH氧化酶在高脂诱导的人脐静脉血管内皮细胞氧化应激损伤中的作用

摘要

Aim To explore the role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in high fat-induced oxidative stress injury in human umbilical vein endothelial cells (HUVECs).Methods HUVECs were exposed to different concentrations of palmitic acid(0.1,0.2,0.4,0.8 mmol · L-1) for 24 h and different time points of 0.4 mmol · L-1 palmitic acid(0,12,24,48 h).Cell viability was measured by Cell Counting Kit 8,and the protein expression of NADPH oxidase subunits such as p22phox,p47phox,p67phox and gp91phox were determined by Western blot.The expression of reactive oxygen species (ROS) in HUVECs was detected by immunofluorescence.Results Cell proliferation rate of HUVECs stimulated by 0.4 mmol · L-1 palmitic acid for 24 h and 48 h was significantly reduced.In the next experiment,model group was accordingly set as HUVECs stimulated by 0.4 mmol · L-1 palmitic acid for 24 h.The expression of NADPH oxidase subunits such as p22phox,p47phox,p67phox and gp91phox significantly increased at 24 h and 48 h after 0.4 mmol· L-1 palmitic acid stimulation (P ＜ 0.05),and the difference.between the 24 h group and the 48 h group was not significant (P ＞ 0.05).The expression of ROS in HUVECs significantly increased at 24 h and 48 h after O.4 mmol · L-1 palmitic acid stimulation (P ＜ 0.05),and the difference between 24 h group and 48 h group was not significant (P ＜ 0.05).Compared with the model group (0.4 mmol · L-1 palmitic acid stimulation for 24 h),the NADPH oxidase inhibitor diphenyliodonium (DPI,10 μmol · L-1) pretreatment could significantly decrease the expression of ROS in vascular endothelial cells (P ＜ 0.05).Conclusion Activated NADPH oxidase might play an important role in treatment of high fat-induced oxidative stress injury in vascular endothelial cells.%目的 探讨NADPH氧化酶在高脂诱导的人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVECs)氧化应激损伤中的作用.方法 不同浓度(0.1、0.2、0.4、0.8 mmol·L-1)棕榈酸(palmitic acid,PA)刺激HUVECs0、12、24、48 h,CCK-8法检测血管内皮细胞增殖能力;免疫印迹法检测血管内皮细胞NADPH氧化酶亚基p22phox、p47phox、p67phox、gp91phox的表达水平;免疫荧光法检测血管内皮细胞细胞内活性氧簇(reactive oxygen species,ROS)的表达水平.结果 0.4 mmol·L-1 PA刺激HUVECs24、48 h组的细胞增殖率出现明显降低.因此,实验中我们采用0.4 mmol· L-1PA刺激24 h作为模型组;0.4 mmol·L-1PA刺激HUVECs24、48 h时,p22phox、p47phox、p67phox、gp91phox的表达均明显升高(P＜0.05),24h组与48 h组差异不明显(P＞0.05);0.4mmol·L-PA刺激血管内皮细胞24、48 h时,细胞内ROS表达水平均明显增高(P＜0.05),24 h组与48 h组差异不明显(P＞0.05);与模型组(0.4 mmol·L-1 PA刺激24 h)相比,NADPH氧化酶抑制剂diphenyliodonium(DPI,10 μmol·L-1)预处理可以使模型组血管内皮细胞ROS表达水平明显下调(P＜0.05).结论 NADPH氧化酶活性对高脂所致血管内皮细胞氧化应激损伤的防治有重要意义.