阿托伐他汀对C反应蛋白诱导的CD14＋单核细胞Toll样受体4表达影响的实验研究  

The effects of atorvastatin on C-reactive protein induced Toll-like receptor 4 expression on CD14^＋monocyte

作　　者：彭隆[1] 罗艳婷[1] 刘金来[1] 

机构地区：[1]中山大学附属第三医院心内科,广州510630

出　　处：《中华心血管病杂志》2011年第7期664-669,共6页Chinese Journal of Cardiology

基　　金：广东省自然科学基金（07001556）

摘　　要：目的 观察阿托伐他汀对C反应蛋白（CRP）诱导的外周血CD14＋单核细胞Toll样受体4（TLR4）表达,以及TLR4信号传导下游炎症因子肿瘤坏死因子α（TNFα）、白细胞介素-6（IL-6）和基质金属蛋白酶-9（MMP-9）的影响,探讨阿托伐他汀的抗炎机制.方法 不同浓度（0、5、25、50、100 μg/ml）和不同作用时间（0、6、12、24、48 h）的C反应蛋白（CRP）刺激正常人外周血CD14＋单核细胞.给予CRP 50 μg/ml预先干预CD14＋单核细胞2 h后,再用不同浓度的阿托伐他汀（1.0、2.5、5.0、7.5、10.0 μmol/L）进行干预.应用流式细胞仪检测细胞表面TLR4蛋白的表达,并应用定量PCR方法检测TLR4 mRNA和髓样分化蛋白2（myeloid differentiation protein,MD2）mRNA的表达,ELISA法检测刺激前后细胞上清液TNFα、IL-6和MMP-9的蛋白水平.结果 （1）CRP 0 μg/ml组TLR4蛋白表达量为19.59%,CRP 5 μg/ml组的TLR4蛋白表达为29.33%,差异有统计学意义（P〈0.01）,并随着CRP浓度的增加,TLR4蛋白表达量逐渐增加.以CRP 0 h组为空白对照,CRP 6 h组的TLR4蛋白表达量为25.75%,差异有统计学意义（P〈0.01）,并随着时间的增加,TLR4表达量逐渐增加.（2）以CRP 50 μg/ml组为对照组,CRP 50 μg/ml和阿托伐他汀1.0 μmol/L组、2.5 μmol/L组、5.0 μmol/L组、7.5 μmol/L组、10.0 μmol/L组,TLR4蛋白表达分别为（68.17±1.71）%、（52.43±1.38）%、（27.72±4.55）%、（17.46±3.20）%、（9.99±2.81）%.（3）以CRP 50 μg/ml组为标准,CRP 50 μg/ml〖JP〗和阿托伐他汀1.0 μmol/L组、2.5 μmol/L组、5.0 μmol/L组、7.5 μmol/L组、10.0 μmol/L组,TLR4 mRNA分别为对照组的82.72%、67.34%、48.16%、30.88%、13.85%.MD2 mRNA分别为对照组的81.78%、71.04%、47.85%、27.06%、18.30%.随着阿托伐他汀浓度增加,TLR4和MD2的mRNA含量减少.（4）当单核细胞未受CRP和阿托伐他汀刺激时,分泌TNFα、IL-6和MMP-9的基础值分别为（16.3±5.8）pg/ml、（31.1±9.5）pg/ml 和（155.Objective To investigate the effects of atorvastatin on C-reactive protein （CRP）induced Toll-Like receptor 4（TLR4）expression on CD14＋ monocyte, and the production of proinflammatory cytokines tumor necrosis factor α （TNFα）, interleukin-6 （IL-6）, matrix metalloproteinases-9 （MMP-9）, and to study the anti-inflammatory mechanisms of statins. Methods The monocytes were isolated from blood of healthy volunteers by the Ficoll density gradient and stimulated by CRP with different doses （5, 25, 50, 100 μg/ml）and different exposure time （6, 12, 24, 48 h）. Cells were also incubated with atorvastatin of different doses （1.0, 2.5, 5.0, 7.5, 10.0 μmol/L）in the presence of CRP 50 μg/ml. The protein expression of TLR4 was measured by flow cytometry, mRNA expression of TLR4 and of myeloid differentiation protein （MD2）was detected by quantitative PCR. TNFα, IL-6, MMP-9 concentrations in supernatants of cultured medium were measured by ELISA.Results （1）Compared with the un-stimulated control group, enhanced TLR4 protein expression was already detected at a concentration of 5 μg/ml of CRP and increased in a dose-dependent manner （32.22±2.80）%, （49.94±5.58）%, （74.82±3.24）% and （90.82±2.88）% at 5, 25, 50 and 100 μg/ml CRP. （2）TLR4 protein expression on 50 μg/ml CRP stimulated cells also increased in a time-dependent manner （29.80±2.70）%, （47.44±4.41）%, （81.71±2.92）% and （50.57±3.34）% after 6 h, 12 h, 24 h, 48 h.（3）When monocytes were incubated with CRP 50 μg/ml and atorvastatin （1.0, 2.5, 5.0, 7.5, 10.0 μmol/L）, protein expression [（68.17±1.71）%, （52.43±1.38）%, （27.72±4.55）%, （17.46±3.20）%, （9.99±2.81）%]and mRNA expression （82.72%, 67.34%, 48.16%, 30.88%, 13.85%）of TLR4 as well as mRNA expression of MD2 （81.78%, 71.04%, 47.85%, 27.06%, 18.30%）were reduced in a dose-dependent manner. （4）Level of TNFα, IL-6 and MMP-9 in supernatants was significantly reduced by atorvastatin （2.5 μmol/L）comp

关 键 词：炎症 C反应蛋白 TOLL样受体4 单核细胞 阿托伐他汀 

分 类 号：R9[医药卫生—药学]