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硫氧还蛋白对血管内皮细胞Nox2通路调控的研究 被引量:2

Thioredoxin regulating Nox2 pathway in vascular endothelial cells
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摘要 目的探讨动脉粥样硬化发生发展中硫氧还蛋白(Trx)对NADPH氧化酶Nox2亚型的调控作用及机制。方法应用腺病毒感染的方法,在原代人脐静脉内皮细胞(HUVECs)建立高表达硫氧还蛋白(Ad-Trx)及其对照(Ad-GFP)的细胞模型,将致动脉粥样硬化重要危险因子氧化型低密度脂蛋白(ox-LDL)作为刺激剂,应用免疫印迹法检测Nox2、Rac1的表达及Akt的磷酸化;用免疫印记及免疫荧光的方法检验Trx在内皮细胞中的蛋白表达水平;DCFH-DA法检测细胞内活性氧(ROS)水平;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。结果免疫印记结果显示腺病毒感染在内皮细胞中成功上调了Trx的表达,与对照组相比上调了3.3倍(P<0.01)。与对照组相比,过表达Trx明显下调了ox-LDL刺激下内皮细胞Nox2、Rac1及p-Akt的表达(分别下调了31.5%、23.8%和20.8%,均为P<0.05),抑制了ox-LDL诱导的ROS增加(25.3%,P=0.0247)及NADPH氧化酶活性增加(32.6%,P=0.004)。结论过表达Trx通过减少ox-LDL刺激下内皮细胞Nox2、Nox2结合蛋白Rac1的表达及Nox2下游信号分子Akt的磷酸化,抑制了内皮细胞Nox2通路的过度活化及氧化应激,减轻炎症损伤,保护内皮细胞功能。 Objective To investigate the effect and molecular mechanism of Thioredoxin (Trx) on NADPH oxidase isoform Nox2 in human vascular endothelial cells in Atherosclerosis. Methods Adenovirus vector gene transfer technology was used in human umbilical vein endothelial cells (HUVECs) to establish the cell model of Trx-overexpression cells ( Ad-Trx) and its control cells (Ad-GFP). Oxidized low density lipoprotein ( ox-LDL) ,a risk factor of atherosclerosis, was used as stimulator. Western Blot was used to detect the protein expression level of Nox2, Racl and Akt phosphorylation. Western Blot and immunofluorescence were used to detect Trx protein level in endothelial cells. DCFH-DA was used to detect intracellular reactive oxygen species ( ROS) production. Extracting the cell membrane fractions, NADPH oxidase activity was measured by enzymatic method. Results Western blot analysis showed adenoviral infection in endothelial cells successfully raised the expression of Trx, (by 3. 3-fold,P <0. 01) compare with the Ad-GFP cells. Trx overexpression significantly reduced ox-LDL-induced Nox2, Racl and p-Akt expression ( down regulated 31. 5% , 23. 8% and 20. 8% , respectively, P < 0. 05 ) , suppressed the enhancements of ROS production (25. 3% ,P =0. 0247) and NADPH oxidase activity (32. 6% ,P =0. 004) in ox-LDL stimulated endothelial cells. Conclusions Trx overexpression inhibited ox-LDL-induced Nox2 pathway excessive activation and oxidative stress by down regulating endothelial Nox2, Nox2 binding protein Racl expression and Nox2 downstream signaling molecule Akt phosphorylation, reduced inflammation damage and protected endothelial cell function.
作者 陈北冬 赵革新 赵艳阳 陈坤 鲍利 齐若梅 Chen Beidong;Zhao Gexing;Zhao Yanyang;Chen Kun;Bao Li;Qi Ruomei(The Key Laboratory of Geriatrics, Beijing Institute of Geriatrics, Beijing Hospital, Beijing 100730, China)
出处 《中国心血管杂志》 2016年第3期236-240,共5页 Chinese Journal of Cardiovascular Medicine
基金 国家自然科学基金面上项目(81270854) 国家自然科学基金青年基金项目(30900627)~~
关键词 动脉粥样硬化 硫氧还蛋白 Nox2 RAC1 血管内皮细胞 Atherosclerosis Thioredoxin Nox2 Racl p-Akt Vascular endothelial cells
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