血管紧张素Ⅱ诱导Toll样受体4在腹膜间皮细胞的表达及其对脂多糖诱导CD40表达的影响  

Role of TLR4 expression induced by angiotensin Ⅱ in LPS-induced CD40 expression in rat peritoneal mesothelial cells

作　　者：伍军[1] 阳晓[1] 张云芳[1] 张锐[1] 董秀清[1] 范瑾瑾[1] 刘眉[1] 余学清[1] 

机构地区：[1]中山大学附属第一医院肾内科,广州510080

出　　处：《中华肾脏病杂志》2008年第10期711-717,共7页Chinese Journal of Nephrology

摘　　要：目的观察血管紧张素Ⅱ（AngⅡ）对原代培养大鼠腹膜间皮细胞（RPMC）Toll样受体4（TLR4）表达的影响及其在脂多糖（LPS）诱导的核转录因子κB（NF－κB）活化及CD40表达中的作用。方法分离及培养RPMC。用不同浓度AngⅡ（10。、10、10、10。mt，l／l一）刺激细胞及用10’mol／L AngⅡ刺激细胞不川时问（mltNA为1、2、4、8、12、24、48h和蚩r1为6、12、24、36、4811），观察血管紧张素l刑受怵（ATIR）阻滞剂洛沙m（10。mol／L）和血竹紧张素2剐受休【AT2It）阻滞制PDl23177（10_ll（11／l。）对AngⅡ诱岢TLt／4表达的影响？将细胞随机分为下列4组：埘照组、AngⅡ（10mol／I。）组、LPSllmg／L）组、AngⅡ（10。mo]／L）＋LPS（1mg／L）组，观察AngⅡ对I。l，s诱导的NF-κB激活和CD40表达的影响。RT—PCR检测TLR4、CD40ml＇lNA表达；Weslern印迹愉测rFLtl4、IκBc~、磷酸化IκBα（p-IκBα）、NF-κBp65、磷酸化NF—κB（p—p65）蛋白表达；免疫荧光检测细胞NF－κB p65形单位的表达及分布。结果（1）10^-9、10^-8、10^-7、10^-6mol／L AngⅡ作用RPMC 12h，TLR4 mRNA表达分别增加70．5％、89．5％、102．9％和121．9％；作用24h TLR4蛋白表达分别增加12．1％、27．7％、51．2％和41．6％。AngⅡ（10^-7mol／L）作用RPMC不同时间，TLR4 mRNA表达高峰为8h和12l·（P〈0．01），蛋白表达高峰为12h和24h（P〈0．01）。（2）洛沙坦阻断后，AngⅡ诱导的TLR4表达与未阻断组比较，下调33．5％（P〈0．05）。PD123177对AngⅡ诱导的TLR4表达无显著影响（P〉0．05）。（3）与正常对照组比较，LPS作用60min p-IκBα／IκBα、p-p65／p65表达分别上调362．6％（P〈0．01）和67．4％（P〈0．05）；作用4h CD40 mRNA表达上调299．9％（P〈0．01）；与LPS组比较，AngⅡ预刺激24h加LPS作用60min，p-IκBα／IκBα、p-p65／p65表达分别上调49．1％（P〈0．01）和29．3％（P〈0．05）�Objective To investigate the effects of angiotensin Ⅱ（Ang Ⅱ ） on the expression of TLR4 and its role in lipopolysaccharide （LPS）-induced NF-κB activation and CD40expression in rat peritoneal mesothelial cells （RPMCs）. Methods RPMCs were harvested from Sprague-Dawley rat peritoneal cavity and maintained under defined in vitro condition. The cells were treated with Ang Ⅱ at different concentrations （10^-9, 10^-8, 10^-7, 10^-6 tool/L） and exposed to AngⅡ （10^-7 tool/L） for different times （1, 2, 4, 8, 12, 24, 48 h for mRNA and 6, 12, 24, 36, 48 h for protein, respectively）. Meanwhile, the influence of AT1 receptor antagonist （AT1R, losartan, 10.5 mol/L） and AT2 receptor blocker （AT2R, PD123177, 10 5 mol/L） on the TLR4 induced by Ang Ⅱ was observed. After synchronization for 24 hours, the ceils were randomly assigned to four groups： the control group, the Ang Ⅱ （10^-7 mol/L） group, the LPS （1 mg/L） group, the Ang Ⅱ （10^-7 mol/L） plus LPS （1 mg/L） group, which were used to investigate the effects of Ang Ⅱ on the NF-κB activation and CD40 expression induced by LPS. The mRNA expression of TLR4 and CD40 was measured by RT-PCR and the protein abundance of TLR4, NF-κB p65, phospho-p65, IκBα and phospho-IκBα were analyzed by Western blot. hnmunofluoreseence was performed to determine the subcellular localization of p65 subunit of NF-κB. Results （1） Treatment of RPMCs with Ang IκBα resulted in a concentration-dependent increase in the expression of TLR4. Ang Ⅱ at 10^-9, 10^-8, 10^-7 and 10^-6 mol/L increased TLR4 mRNA expression by 70.5%, 89.5%, 102.9%, and 121.9%, respectively and protein expression by 12.1%, 27.7%, 51.2%, and 41.6%, respeetively （P〈0.01）. Treatment of RPMCs with 10-7 mol/L Ang Ⅱ resulted in a time-dependent increase in the expression of TLR4, with the peak of mRNA expression at 8 and 12 h （P〈0.01） and the protein expression at 12 and 24 h （P〈0.01）. （2） Losartan antagonized Ang Ⅱ-stimulated expression

关 键 词：血管紧张素Ⅱ 核转录因子ΚB 炎症 脂多糖类 Toll样受体 4 CD40 腹膜间皮细胞 

分 类 号：R572.2[医药卫生—消化系统] R459.5[医药卫生—内科学]