晚期糖基化终产物促进血管外膜成纤维细胞炎性反应机制及坎地沙坦对其干预作用  

Advanced glycation end products enhance inflammatory response of vascular adventitial fibroblasts and effect of candesartan

作　　者：刘亚洋[1] 梁春[1] 刘星[1] 潘晓明[1] 任雨笙[1] 樊民[1] 吴宗贵[1] 

机构地区：[1]第二军医大学附属长征医院心内科,上海200003

出　　处：《上海医学》2009年第4期276-282,共7页Shanghai Medical Journal

基　　金：国家重点基础研究发展计划(973计划)资助项目(2005CB523309)

摘　　要：目的观察晚期糖基化终产物(AGEs)对血管外膜成纤维细胞(AF)炎性反应的影响及其机制,以及坎地沙坦对该影响的干预作用。方法用组织贴块法培养Sprague-Dawley大鼠的AF,应用逆转录-聚合酶链反应(RT-PCR)及Western印迹法检测AGEs受体(RAGE)的表达。应用RT-PCR检测单核细胞趋化因子(MCP)-1、白细胞介素(IL)-6、血管内皮细胞黏附分子(VCAM)-1mRNA表达,应用酶联免疫吸附试验检测培养上清液中MCP-1、IL-6、VCAM-1蛋白表达。应用Western印迹法及免疫电泳迁移率分析检测核因子(NF)-κB和NF-κB抑制蛋白α(I-κB-α)。结果不同浓度糖基化人血清白蛋白(AGE-HSA,50、100、150、200、300mg/L)可呈浓度依赖性地上调RAGEmRNA和蛋白的表达,与对照组和人血清白蛋白(HSA)组的差异均有统计学意义(P值均<0.05),200mg/L时达到峰值。AGE-HSA200mg/L作用AF后0.5、1.0、2.0h,细胞核内NF-κB均激活,作用后0.5、1.0、2.0h活性均显著高于作用后0h(P值均<0.05)。AGE-HSA200mg/L作用后细胞质内I-κB-α磷酸化激活,1.0h达峰值,0.5和1.0h均显著高于0h(P值均<0.05)。3、30nmol/L坎地沙坦均能抑制AGE-HSA200mg/L作用后I-κB-α的磷酸化激活及NF-κB核内移,以30nmol/L更显著。不同质量浓度AGE-HSA(50、100、200、300mg/L)均可使AFIL-6、VCAM-1、MCP-1mRNA表达较对照组显著上调(P值分别<0.05、0.01),且呈剂量依赖性。用RAGE中和抗体、NF-κB抑制剂MG-132、坎地沙坦预处理可减少由AGE-HSA所致的IL-6、VCAM-1、MCP-1mRNA表达及上清液中蛋白表达量(P值分别<0.01、0.05)。结论AGE-HSA通过RAGE、NF-κB途径促使炎性因子表达上调。坎地沙坦可有效抑制I-κB-α磷酸化、NF-κB核内移及炎性因子的表达,提示坎地沙坦可通过抑制AGEs引起的AF炎性反应起到延缓糖尿病血管并发症的作用。Objective To investigate the effects and mechanism of advanced glycation end products （AGEs） on the inflammatory responses of rat vascular adventitial fibroblasts and the interventional effects of candesartan. Methods Isolated vascular adventitial fibroblasts of Sprague-Dawley （SD） rats were cultured. Expression of receptor for advanced glycation end products （RAGE） was examined by reverse transcriptase polymerase chain reaction （RT-PCR） and Western blotting assay. Expression of monoeyte chemoattractant protein （MOP）-1, interleukin （IL）-6, and vascuolar cell adhesion molecule （VCAM）-I mRNA was also analyzed by RTPCR. Levels of MOP-1, IL-6, and VCAM-1 in the supernatants were determined by enzyme-linked immunosorbent assay （ELISA）. Nuclear factor （NF）-κB and inhibitory protein of NF-κB α（I-κB-α） was analyzed by electrophoretic mobility shift assay （EMSA） and Western blotting. Results AGE-human serum albumin （HSA） （50, 100, 150, 200 and 300 mg/L） up-regulated the expression of RAGE at mRNA and protein levels in a concentration-dependent manner;the expression was significantly higher than those of the control group and HSA group （both P〈0.05）. The expression of RAGE peaked at the concentration of 200 mg/L, which was significantly higher than that of the other concentration groups （P〈0.05）. NF-κB transcriptional activity was increased after treatment with AGE-HSA （200 mg/L） for 0.5 h, 1.0 h and 2.0 h. Incubation of AFs with AGE-HSA （200 mg/L） for 0.5 h and 1.0 h induced phosphorylation of I-KB-a. AGE-HSA （200 mg/L） treatment increased nuclear NF-κB-α candesartan co-treatment inhibited this translocation. I-κB-α phosphorylation in response to AGE-HSA （200 mg/L） was also suppressed by candesartan. AGEs （50, 100, 200, and 300 mg/L） elevated the mRNA expression of MOP-l, IL-6, and VCAM-1. Compared with control, the expression of IL-6, VCAM-1, and MOP-1 mRNA increased significantly in AGE-HSA 50, 100 mg/L groups （P〈0.05） an

关 键 词：晚期糖基化终产物 血管外膜成纤维细胞 炎性反应 核因子-ΚB 丝裂原活化蛋白激酶 坎地沙坦 

分 类 号：R587.2[医药卫生—内分泌]