高分子量脂联素在EA.hy926血管内皮细胞中对前炎症因子介导的NF-κB信号通路及其下游因子的影响  

Effects of high molecular weight adiponectin on proinflammatory cytokines mediated NF-κB signaling pathway and its downstream molecules in endothelial EA.hy926 cells

作　　者：刘师伟[1] 王军[2] 王明明[2] 楼晓华 张丽[2] 赵术君 董艳婷[4] 王建华[5] 牛勃[5] 

机构地区：[1]山西医学科学院 山西大医院内分泌科,太原030032 [2]山西医科大学研究生学院 [3]美国休斯敦Methodist代谢研究中心 [4]生物化学与分子生物教研室 [5]首都儿科研究所生物技术研究室

出　　处：《中国药物与临床》2015年第4期445-449,共5页Chinese Remedies & Clinics

基　　金：国家自然科学基金(81200591;81471025);山西省留学回国人员科技活动择优资助项目(2013-251)

摘　　要：目的：本研究通过促炎因子肿瘤坏死因子（TNF）-α和白细胞介素（IL）-1对血管内皮细胞株EA．hy926的作用，来探讨高分子量脂联素对EA．hy926中NF-κB活性及其下游因子的影响，以阐明高分子量脂联素在血管内皮细胞炎症反应中的作用。方法构建NF-κB荧光素酶报告质粒，并稳定转染血管内皮细胞株EA．hy926。在TNF-α和IL-1作用之前，用不同浓度（0-8μg/ml）高分子量脂联素处理转染后的血管内皮细胞EA． hy926。NF-κB的活性通过荧光酶报告分析法测定。采用酶联免疫吸附试验（ELISA）法测定细胞培养上清液中TNF-α、IL-1及IL-6的浓度。分别采用实时荧光定量聚合酶链反应（real time-PCR）法、蛋白印迹法（Western blot）在mRNA和蛋白水平上检测血管内皮细胞的细胞间黏附分子-1（ICAM-1）、血管细胞黏附分子-1（VCAM-1）和单核细胞趋化蛋白-1（MCP-1）的含量。结果研究表明高分子量脂联素抑制促炎因子TNF-α和IL-1介导的NF-κB活性及其下游因子的表达。结论高分子量脂联素通过抑制NF-κB的活性及其下游因子的表达，抑制前炎症因子介导的炎症反应，从而保护血管内皮细胞。Objective To investigate the effects of high molecular weight adiponectin on the activities of nucle-ar factor-kappa B （NF-κB） and its downstream molecules in endothelial EA.hy926 cells induced by proinflammatory cytokines, such as tumor necrosis factor-α （TNF-α） and interleukine-1 （IL-1）, so as to elucidate the role of the high molecular weight adiponectin in the inflammatory response of endothelium. Methods A NF-κB luciferase reporter system was constructed and stably transfected into human endothelial cell line EA.hy926. Following transfection, EA. hy926 cells were pre-treated with various doses of high molecular weight adiponectin （0-8 μg/ml） before TNF-α and IL-1 stimulation. The transcription activity of NF-κB was determined using luciferase reporter assay. Expression levels of NF-κB downstream inflammatory cytokines, TNF-α, IL-1 and IL-6 in the supernatant of cell culture were measured by enzyme-linked immunosorbent assay （ELISA）. Expressions of adhesion molecules and chemokines, intercellular cell adhesion molecule-1 （ICAM-1）, vascular cell adhesion molecule-1 （VCAM-1） and monocyte chemotactic protein-1 （MCP-1） were determined by quantitative real time-PCR and western blot in mRNA and protein levels, respectively. Results The high molecular weight adiponectin was shown to inhibit TNF-α and IL-1 mediated activation of NF-κB and its downstream molecules in a dose-dependent manner （P＆lt;0.05）. Conclusion The high molecular weight adiponectin may protect endothelial cells by suppressing pro-inflammatory cytokines induced inflammation through in-hibition of NF-κB and its downstream molecules.

关 键 词：脂联素 炎症 胰岛素抵抗 血管内皮细胞 

分 类 号：R363[医药卫生—病理学]