硫氧还蛋白通过Smad3／AP-1通路抑制人血管内皮细胞黏附蛋白的表达  

Thioredoxin inhibits human vascular endothelial cell adhesion molecules expression via Smad3/AP-1 pathway

作　　者：陈北冬[1] 王文东[2] 赵革新[1] 马丽娜[3] 刘雪青[1] 齐若梅[1] 

机构地区：[1]卫生部北京医院卫生部北京老年医学研究所卫生部老年医学重点实验室,100730 [2]北京市海淀区卫生局卫生监督所 [3]北京中医药大学

出　　处：《中华老年医学杂志》2013年第5期469-472,共4页Chinese Journal of Geriatrics

基　　金：国家自然科学基金青年基金项目（30900627）

摘　　要：目的 研究硫氧还蛋白（Trx）在动脉粥样硬化中对血管内皮细胞保护作用的分子机制. 方法 应用腺病毒感染的方法在原代人脐静脉内皮细胞（HUVECs）中建立过表达硫氧还蛋白及其对照的细胞模型.以致动脉粥样硬化重要危险因子氧化型低密度脂蛋白（oxLDL）为刺激剂.应用免疫印迹及间接免疫荧光法检测Trx,黏附分子（ICAM-1,VCAM-1）及其上游信号分子（Smad3,AP-1）的蛋白表达及细胞定位.应用胰岛素还原法检测Trx的活性,应用荧光探针DCFHDA进行细胞内活性氧检测. 结果 和对照组相比过表达Trx组Trx表达量明显提高,活性检测显示Ad-Trx的活性上调率为（26.2±3.3）％,细胞内活性氧（ROS）检测提示过表达Trx显著抑制细胞内ROS的产生.和对照组相比在基础及氧化型低密度脂蛋白（ox-LDL）刺激下过表达Trx组明显下调了内皮细胞黏附分子的表达（P＜0.05）,显著提高了内皮细胞中Smad3的磷酸化（P＜0.05）.而应用Smad3磷酸化特异性的抑制剂SIS3预处理细胞反转了Trx对黏附蛋白的抑制作用.SIS3预处理细胞进一步上调了oxLDL刺激下AP-1亚基c-Fos的核蛋白表达. 结论 Trx抑制内皮细胞黏附分子表达的作用可能是通过上调Smad3蛋白的磷酸化及抑制核转录因子AP-1亚基c-Fos的核表达来调节的.Objective To investigate the molecular mechanisms of protective effects of thioredoxin （Trx） on human vascular endothelial cells in atherosclerosis.Methods The cell models of Trx-overexpressing cells （Ad Trx） and the control cells （Ad-con） were established by adenovirus vector gene transfer technology in human umbilical vein endothelial cells （HUVECs）.The oxidized low density lipoprotein,a risk factor of atherosclerosis,was used as a stimulator.Western blot and indirect immunofluorescence were used to detect the protein expression levels and the cellular localization of Trx,adhesion molecules （ICAM-1,VCAM-1） and the upstream signal pathways.Trx activity was detected by insulin disulfide reduction assay,and cellular reactive oxygen species （ROS）production was detected by fluorescent probe DCFH-DA.Results As compared with control group,Trx protein expression level was enhanced in Ad-trx group and the Trx activity in Ad-Trx group was upregulated by （26.2 ±3.3）%.The result of ROS detection showed that overexpression of Trx significantly inhibited the cellular ROS generation.As compared with control group,overexpression of Trx obviously inhibited the adhesion molecules expression but markedly promoted the phosphorylation of Smad3 in endothelial cells with or without oxLDL stimulation （P〈0.05）.Pretreatment of cells with SIS3,a specific inhibitor of Smad3 phosphorylation,reversed Trx-induced inhibition of adhesion molecules expression.Further studies showed that pretreatment of cells with SIS3 enhanced oxLDL-induced AP-1 subunit c-fos nuclear expression.Conclusions The enhancement of Smad3 phosphorylation and c-Fos nuclear expression are mainly responsible for the Trx-induced downregulation of adhesion molecules.

关 键 词：硫氧还蛋白质类 血管细胞黏附分子-1 Smad3蛋白质 转录因子AP-1 

分 类 号：R54[医药卫生—心血管疾病]