RhoA在富氢液减轻脂多糖诱发人肠屏障功能障碍中的作用：离体实验  

Role of RhoA in amelioration of lipopolysaccharide-induced human intestinal barrier dysfunction by hydrogen-rich saline： in vitro experiment

作　　者：杨涛[1] 谢克亮[1] 陈红光[1] 于洋[1] 刘玲玲[1] 李媛[1] 王国林[1] 于泳浩[1] 

机构地区：[1]天津医科大学总医院麻醉科天津市麻醉学研究所,300052

出　　处：《中华麻醉学杂志》2016年第1期92-96,共5页Chinese Journal of Anesthesiology

基　　金：国家自然科学基金（81071533,81101409,81372033）;天津市应用基础及前沿技术研究计划（11JCYBJC12900）;天津市医药卫生资助项目（2011KZ108）

摘　　要：目的 评价RhoA在富氢液减轻脂多糖（LPS）诱发人肠屏障功能障碍中的作用.方法 将Caco-2细胞以2×10^5个/ml的密度接种于transwell小室,每孔400μl,采用随机数字表法,将其分为5组（n=50）：对照组（C组）、富氢液组（H2组）、LPS组、LPS＋富氢液组（LPS＋HRS）组和LPS＋ RhoA抑制剂C3胞外酶组（LPS＋C3组）.H2组和LPS＋HRS组用含0.6 mmol/L富氢液的培养基培养,LPS组、LPS＋HRS组和LPS＋C3组加入LPS 100 μg/ml;于加入LPS前1h时,LPS＋C3组加入C3胞外酶2.5 μg/ml.每组分别于LPS孵育前即刻、3、6、12、24、36、48 h时取5孔,测定上皮细胞电阻（TEER）和渗透系数（PE）,每组于LPS孵育24 h时取5孔,采用Western blot法和免疫荧光法测定occludin与E-cadherin的表达,采用GST pull-down法测定RhoA活性.结果 与C组比较,LPS组、LPS＋HRS组和LPS＋C3组LPS孵育6-48 h时TEER降低,PE和RhoA活性升高,occludin和E-cadherin的表达下调（P＜0.05）;H2组上述指标差异无统计学意义（P＞0.05）;与LPS组比较,LPS＋ HRS组和LPS＋C3组LPS孵育6-48 h时TEER升高,PE和RhoA活性降低,occludin和E-cadherin的表达上调（P＜0.05）;与LPS组比较,LPS＋HRS组和LPS＋C3组occludin和E-cadherin在细胞膜处分布部分增多,在细胞质中分布减少,“铁丝网”样形态的连续性与完整性部分恢复.结论 富氢液可减轻LPS诱发人肠屏障功能障碍,其机制与抑制RhoA激活,从而上调occludin和E-cadherin的表达及抑制其分布紊乱有关.Objective To evaluate the role of RhoA in amelioration of lipopolysaccharide （LPS）-induced human intestinal barrier dysfunction by hydrogen-rich saline.Methods Caco-2 cells were plated on transwells at a density of 2× 10^5/ml （400 μl/well）,and randomly divided into 5 groups （n =50 each） using a random number table：control group （group C）,hydrogen-rich saline group （HRS group）,LPS group,LPS ＋ hydrogen-rich saline group （group LPS＋HRS）,and LPS ＋ RhoA inhibitor C3 exoenzyme group （group LPS＋C3）.Cells were cultured in the medium containing 0.6 mmol/L hydrogen-rich saline in HRS and LPS＋HRS groups,or in the medium containing 100 μg/ml LPS in LPS,LPS＋HRS and LPS＋C3 groups.In group LPS＋C3,C3 exoenzyme 2.5 μg/ml was added at 1 h before addition of LPS.Five wells were selected at 0,3,6,12,24,36 and 48 h of incubation with LPS,and the transepithelial electrical resistance （TEER） and permeability coefficient （PE） were measured.Five wells were selected at 24 h of incubation with LPS for determination of occludin and E-cadherin expression （by Western blot and immunofluorescence） and RhoA activity （using GST pull-down assay）.Results Compared with group C,TEER was significantly decreased,PE and RhoA activity were increased,and the expression of occludin and Ecadherin was down-regulated at 6-48 h of incubation with LPS in LPS,LPS＋ HRS and LPS＋C3 groups （P〈 0.05）,and no significant change was found in the variables mentioned above in group HRS （P〉0.05）.Compared with group LPS,TEER was significantly increased,PE and RhoA activity were decreased,and the expression of occludin and E-cadherin was up-regulated at 6-48 h of incubation with LPS in LPS＋HRS and LPS＋C3 groups （P〈0.05）.Compared with group LPS,the distribution of occluding and E-cadherin in the cell membrane was significantly increased,the distribution in the cytoplasm was decreased,and the structures of occludin and E-cadherin partly recovered in LPS＋HRS and LPS＋C3 groups.Conclus

关 键 词：RHOA GTP结合蛋白质 氢 内毒素血症 肠黏膜 

分 类 号：R614[医药卫生—麻醉学]