Ezrin蛋白在肿瘤坏死因子致大鼠肺微血管内皮细胞炎性损伤中表达及Rac 1信号通路的影响  

Role of Ezrin in the injury of rat pulmonary microvascular endothelial cells induced by tumor necrosis factor-αand the impact of Rac 1

作　　者：唐思慧[1] 岳扬[1] 孙耕耘[1] 

机构地区：[1]安徽医科大学第一附属医院呼吸内科,合肥230022

出　　处：《中华危重病急救医学》2014年第11期785-788,共4页Chinese Critical Care Medicine

基　　金：国家自然科学基金（81370170）

摘　　要：目的：探讨肿瘤坏死因子-α（TNF-α）诱导大鼠肺微血管内皮细胞（PMVEC）中Ezrin蛋白及其磷酸化（p-Ezrin）表达的变化，以及小G蛋白家族成员Rac 1对其的影响。方法将体外培养的SD大鼠PMVEC分别进行TNF-α刺激时效实验和Rac 1通路干预实验。①时效实验：以10μg/L TNF-α分别刺激PMVEC 0、0.25、0.5、1、3、6、12、24 h后，采用蛋白质免疫印迹试验（Western Blot）检测Ezrin及p-Ezrin的表达。②Rac 1通路干预实验：以200μmol/L Rac 1特异性抑制剂NSC 23766与PMVEC预孵育0.5 h再加入10μg/L TNF-α继续孵育，3 h后检测p-Ezrin表达，同时设空白对照组（1%胎牛血清）及TNF-α或NSC 23766单独刺激组。结果①PMVEC中仅低量表达Ezrin，且TNF-α刺激对Ezrin无明显影响。TNF-α刺激PMVEC 0 h时p-Ezrin蛋白表达〔即p-Ezrin/Ezrin（灰度值）〕为0.21±0.03，刺激0.25 h时p-Ezrin蛋白表达即开始升高（0.53±0.19），3 h达高峰（1.68±0.30），然后逐渐下降，24 h时仍高于基础水平（0.87±0.18），组间比较差异有统计学意义（F＝62.200，P＝0.000），说明TNF-α可呈时间依赖性增加Ezrin磷酸化。②与空白对照组比较，TNF-α和NSC 23766单独刺激组均能诱导p-Ezrin表达增加（TNF-α组：0.92±0.12比0.68±0.16， t＝-2.864，P＝0.020；NSC 23766组：1.33±0.24比0.68±0.16，t＝-5.429，P＝0.000）；NSC 23766与TNF-α共同孵育可使p-Ezrin表达较TNF-α单独刺激组进一步增加（2.14±0.18比0.92±0.12，t＝-14.670，P＝0.000），各组间差异有统计学意义（F＝73.810，P＝0.000）。结论 Ezrin磷酸化表达增加参与了TNF-α诱导的PMVEC损伤，抑制Rac 1信号通路可通过上调Ezrin磷酸化表达参与TNF-α对PMVEC的致伤效应。Objective To investigate the role of Ezrin and its phosphorylation（p-Ezrin）in the modulation of rat pulmonary microvascular endothelial cell（PMVEC）injury induced by tumor necrosis factor-α（TNF-α）and the impact of Rac 1. Methods Cultured PMVECs of Sprague-Dawley（SD）rats were randomly divided into time-dependent injury group induced by TNF-αand intervention group in which cells were pretreated with Rac 1 inhibitor （NSC 23766）.①In the time-dependent injury group, Western Blot was used to detect the expression of Ezrin and p-Ezrin after 10μg/L TNF-αstimulation for 0,0.25,0.5,1,3,6,12,24 hours.②In the intervention group,after pre-treatment with 200μmol/L NSC 23766 for 0.5 h,PMVECs were treated with 10μg/L TNF-α,and the expression of p-Ezrin was detected by Western Blot after 3 hours. Besides these groups,there were control（1% fetal bovine serum simulation）,single NSC 23766 or TNF-α simulation groups. Results ① Few Ezrin expression was found in PMVEC,and TNF-α could not affect Ezrin expression. p-Ezrin protein expression（p-Ezrin/Ezrin,gray scale） of PMVECs at 0 hour after TNF-αstimulation was 0.21±0.03,and elevated at 0.25 hour（0.53±0.19）,peaked at 3 hours（1.68±0.30）,then it was gradually lowered,but it remained at higher level at 24 hours（0.87±0.18）with significant difference（F=62.200,P=0.000）. It demonstrated that TNF-αcould increase Ezrin phosphorylation in a time-dependent manner.②Compared with blank control group,in single NSC 23766 or TNF-αsimulation group, p-Ezrin expression was induced（TNF-αgroup：0.92±0.12 vs. 0.68±0.16,t=-2.864,P=0.020;NSC 23766 group：1.33±0.24 vs. 0.68±0.16,t=-5.429,P=0.000. When NSC 23766 was pre-treated with PMVECs,the expression of p-Ezrin was significantly increased compared with that in single TNF-αsimulation group（2.14±0.18 vs. 0.92±0.12,t=-14.670,P=0.000）with significant difference（F=73.810,P=0.000）. Conclusion Ezrin proteins are phosphorylated by TNF-α. Rac 1 signaling pathway inhibition plays

关 键 词：EZRIN蛋白 肿瘤坏死因子-α 肺微血管内皮细胞 RAC 1信号通路 

分 类 号：R563.8[医药卫生—呼吸系统]