同型半胱氨酸影响实验性结肠炎大鼠肠黏膜上皮细胞紧密连接蛋白表达的研究

Effect of homocysteine on the expression of tight junction proteins in intestinal mucosal epithelial cell of experimental colitis rats

目的 探讨同型半胱氨酸(Hcy)对实验性结肠炎大鼠肠黏膜上皮细胞紧密连接蛋白表达的影响.方法 把SD大鼠分为正常对照组(生理盐水灌肠+生理盐水皮下注射)、Hcy皮下注射组(生理盐水灌肠+Hcy皮下注射)、2,4,6-三硝基苯磺酸溶液(TNBS)模型对照组(TNBS灌肠+生理盐水皮下注射)和TNBS+Hcy皮下注射组(TNBS灌肠+Hcy皮下注射)共4组.采用TNBS-乙醇(100 mg/kg体质量)灌肠制备大鼠结肠炎模型.采用皮下注射Hcy,0.03μmol/g体质量,2次/d,间隔8h,连续4周,造成高Hcy血症模型.记录疾病活动指数(DAI)评分.实验结束时,检测血浆和结肠标本中Hcy浓度;结肠组织HE染色并进行组织学评分(HI);采用Evans蓝(EB)方法检测小肠黏膜通透性;透射电镜观察小肠上皮细胞紧密连接变化;结肠匀浆检测髓过氧化物酶(MPO)活性;Western blot检测小肠组织中Claudin-1、Claudin-2、Occludin和ZO-1蛋白的表达.结果 与TNBS模型对照组相比,TNBS+Hcy皮下注射组大鼠血浆及结肠Hcy水平增高[(23.44土1.40)μmoYL比(11.40±0.94) μmol/L,t=20.19,P<0.05;(3.06±0.30) μmol/L比(1.69±0.20) μmol/L,t=10.75,P<0.05)];DAI及HI评分增高(2.63±0.78比1.57±0.86,t=2.582,P<0.05;4.98±2.10比3.34±0.43,t=2.16,P<0.05);结肠匀浆MPO活性升高(3.46±0.62比2.31±0.54,t=3.96,P<0.05);小肠黏膜组织Claudin-1、Occludin、ZO-1表达降低,Claudin-2表达升高;小肠组织EB含量明显增高[(829.95±11.42) μg/L比(610.21±11.56) μg/L,t=-38.24,P<0.05);电镜下细胞间隙增宽.结论 Hcy可增加实验性结肠炎大鼠肠黏膜通透性,加重大鼠结肠炎症反应损伤程度,其机制可能与抑制Claudin-1、Occludin、ZO-1表达,促进Claudin-2表达,损伤上皮细胞紧密连接有关.

Objective To investigate the effect of homocysteine(Hcy)on the expressions of tight junction proteins in intestinal epithelial cells of experimental colitis rats.Methods SD rats were divided into 4 groups:normal control group[normal saline(NS)enema and NS subcutaneous injection],Hcy subcutaneous injection group(NS enema and Hcy subcutaneous injection),2,4,6-trinitrobenzene sulfonic acid(TNBS)model control group(TNBS enema and NS subcutaneous injection)and TNBS+Hcy subcutaneous injection group(TNBS enema and Hcy subcutaneous injection).Rat colitis model was established by TNBS-ethanol enema(100 mg/kg body weight).The hyper-homocysteinemia model was induced by subcutaneous injection of Hcy(0.03μmol/g body weight,bid)with an interval of 8 h for 4 weeks.The disease activity index(DAI)score was recorded.At the end of experiment,Hcy levels in plasma and colon specimens were detected;HE staining of colon tissue was used to evaluate histological index(HI)scores;Evans blue(EB)was applied to detect intestinal mucosal permeability;transmission electron microscopy was used to observe the changes of tight junction of intestinal epithelial cells;myeloperoxidase(MPO)activity in colon homogenate was examined;Western blot was applied to detect the expressions of Claudin-1,Claudin-2,Occludin and ZO-1 proteins in the small intestine.Results Compared with TNBS model control group,TNBS+Hcy subcutaneous injection group had higher Hcy level of plasma and colon[(23.44±1.40)μmol/L vs.(11.40±0.94)μmol/L,t=20.19,P<0.05;(3.06±0.30)μmol/L vs.(1.69±0.20)μmol/L,t=10.75,P<0.05];higher DAI and HI scores(2.63±0.78 vs.1.57±0.86,t=2.582,P<0.05;4.98±2.10 vs.3.34±0.43,t=2.16,P<0.05);down-regulated expressions of Claudin-1,Occludin and ZO-1 and up-regulated expression of Claudin-2;higher MPO activity in colon homogenate(3.46±0.62 vs.2.31±0.54,t=3.96,P<0.05);higher EB content in small intestine[(829.95±11.42)μg/L vs.(610.21±11.56)μg/L,t=-38.24,P<0.05];and wider cell gap under electron microscope.Conclusion Hcy can increase the permeability of intestinal mucosa in experimental colitis rats and aggravate the injury degree of colitis in experimental rats,whose mechaoism may be related to down-regulated expressions of Claudin-1,Occludin,ZO-1,up-regulated expression of Claudin-2,and the injury of the tight junction of epithelial cells.