硫氧还蛋白通过下调Nox4活性抑制血管内皮细胞黏附蛋白的表达

Thioredoxin inhibits the expression of human vascular endothelial cell adhesion molecules by down- regulating Nox4 activity

目的探讨动脉粥样硬化始动环节中硫氧还蛋白（thioredoxin，Trx）对还原型辅酶Ⅱ（NADPH）氧化酶Nox4亚型的作用，及其参与内皮细胞黏附蛋白表达的分子机制研究。方法应用腺病毒感染的方法在原代人脐静脉内皮细胞（HUVECs）中建立高表达硫氧还蛋白（Ad—Trx）及其对照（Ad—GFP）的细胞模型。以致动脉粥样硬化重要危险因子氧化型低密度脂蛋白（ox-LDL）为刺激剂。用免疫印迹法检测细胞间黏附蛋白-1（ICAM-1）、Nox4及P22phox的表达；用间接免疫荧光的方法检验Trx在细胞内的表达；提取细胞膜组织，用酶学方法测定内皮细胞NADPH氧化酶的活性；应用荧光探针DCFH—DA进行细胞内活性氧检测。结果Trx过表达及NADPH氧化酶特异性的抑制剂Apocynin可以显著下调ox-LDL刺激下内皮细胞ICAM-1的表达和活性氧产生。与对照组相比过表达Trx抑制了内皮细胞膜组分中ox-LDL刺激下Nox4的表达、NADPH氧化酶活性增加以及P22phox的表达增加。结论Trx通过下调内皮细胞Nox4的活性，抑制内皮细胞ICAM-1的表达，减轻炎性损伤，保护内皮细胞功能。

Objective To investigate the effect of Thioredoxin （Trx）on dihydronicotinamide adenine dinucleotide phosphate （NADPH）oxidase subtype Nox4, and the molecular mechanisms of Nox4 regulating intracellular adhesion molecular-1 （ICAM-1）expression in human vascular endothelial cells during the initiation of atherosclerosis. Methods Adenovirus vector gene transfer technology was used in human umbilical vein endothelial cells（HUVECs）to establish the cell models of Trx- overexpression cells（Ad-Trx）and their control cells（Ad-GFP）. Oxidized low density lipoprotein,a risk factor for atherosclerosis, was used as a stimulator. Western blot was conducted to detect protein expression levels of intracellular adhesion molecule-1 （ICAM-1）, Nox4 and P22 phox. Indirect immunofluorescence was used to detect Trx expression in endothelial cells. Cell membrane tissues were extracted,and the activity of NADPH oxidase in endothelial cells was determined by an enzymatic method. Production of cellular reactive oxygen species （ROS）was detected by the fluorescent probe dichloro-dihydro-fluorescein diacetate（DCFH-DA）. Results Both overexpression of Trx and the specific inhibitor of NADPH oxidase（apocynin）significantly reduced ICAM=I expression and ROS generation in ox-LDL stimulated endothelial cells. Compare with the control group, Trx overexpression inhibited NOX4 expression of cell membrane fractions, the increase of NADPH oxidase activity and P22 phox expression in ox-LDL stimulated endothelial cells. Conclusions Trx inhibits ox-LDL- induced ICAM-1 expression by down-regulating endothelial Nox4 activity, thus reduces inflammatory damage and protects endothelial cell function.