整肠生对溃疡性结肠炎小鼠肠上皮屏障的保护作用

Effects of Zheng Chang Sheng on intestinal epithelial barrier function in mice with ulcerative colitis

目的探讨整肠生对溃疡性结肠炎小鼠肠道紧密连接蛋白表达以及对氧化应激反应的影响。方法选用雄性8~10周龄C57BL/6小鼠40只,随机分为4组:对照组、模型组(3%DSS)、5-ASA组(3%DSS+5-ASA 200mg/kg灌胃)和整肠生组(3%DSS+联合整肠生及5-ASA灌胃),每组10只,造模7d。观察各组小鼠便血程度、组织学损伤情况,通过投射电镜观察各组肠道上皮间紧密连接改变情况,应用Western blot和RT-PCR的方法,检测小鼠结肠黏膜紧密连接蛋白Occludin、ZO-1、Claudin-2的表达情况。结果 (1)与模型组比较,5-ASA组和整肠生组小鼠便血程度明显减轻,DAI评分显著降低(P<0.05)。整肠生组与5-ASA组比较,便血减轻,DAI评分降低(P<0.05)。(2)电镜显示,对照组肠上皮间紧密连接呈一条致密条带,结构完整,见细胞桥粒,微绒毛光滑、排列整齐,细胞间隙狭窄;模型组肠上皮间紧密连接结构松散、模糊、密度降低,桥粒结构消失,微绒毛稀疏,短缩且长短不一,细胞间隙增宽;各治疗组的紧密连接的破坏情况较模型组有不同程度的改善,整肠生组紧密连接清晰,细胞间隙缩窄,微绒毛排列整齐,出现细胞桥粒。(3)应用Western blot和Real time-PCR法检测,与正常组相比,模型组Occludin、ZO-1蛋白和mRNA表达显著下降,Claudin-2表达显著上调(P<0.05);各治疗组较模型组Occludin、ZO-1蛋白表达上调,Claudin-2蛋白表达下调(P<0.05),整肠生组较单用5-ASA组更明显提高Occludin、ZO-1蛋白和mRNA表达。(4)与正常组相比,模型组MDA含量增高,SOD活性降低,与5-ASA组相比,整肠生组能更显著地降低MDA含量,提高SOD活性(P<0.05)。结论联合应用整肠生通过调节紧密连接蛋白Occludin、ZO-1的表达和降低氧化应激反应,来改善溃疡性结肠炎小鼠肠上皮屏障功能。

Objective To investigate the effects of Zheng Chang Sheng（ZCS）on intestinal expression of tight junction protein and oxidative stress in mice with ulcerative colitis.Methods A total of 40 C57 BL/6 male mice were randomly divided into four groups（n=10）including the control group,DSS group（3%DSS）,5-ASA group（3% DSS and 5-ASA 200 mg/kg）and ZCS group（3% DSS,5-ASA 200 mg/kg and ZCS）.The bloody stools,weight loss and histologic lesions of mice were observed.The changes of intestinal epithelial tight junction were assessed by using transmission electron microscope.The expression levels of tight junction proteins（Occludin,ZO-1 and Claudin-2）were assessed with Western blot and RT-PCR.Results（1）Compared with DSS group,the severity of bloody stool and DAI scores obviously reduced in 5-ASA group and ZCS group（P〈0.05）.Compared with 5-ASA group,the severity of bloody stool and DAI score in ZCS group significantly decreased（P〈0.05）.（2）In control group,the tight junction appeared as an electron-dense belt at the apex of intestinal epithelial cells,with smooth and orderly microvilli,narrow intercellular spaces,and visible desmosomes.In DSS group,the intercellular spaces widened,the tight junction was indistinct with lower density,the desmosomes disappeared,and the microvilli were sparse and short.In treatment groups,the density of tight junctions increased compared with that in the DSS group.In ZCS group,the intercellular spaces were narrow,the microvilli were in an orderly arrangement,and the desmosomes appeared.（3）Compared with control group,the expressions of Occludin and ZO-1 obviously reduced while that of Claudin-2 increased in DSS group（P〈0.05）.Compared with DSS group,the expressions of Occludin and ZO-1 were upregulated while that of Claudin-2 downregulated in treatment groups（P〈0.05）.The expressions of Occluding,ZO-1 and mRNA in ZCS group were significantly higher than in the 5-ASA group.（4）Compared with control group,the MDA content increased and SOD activity decreased in DSS group.Compared with 5-ASA group,ZCS group had lower MDA content and higher SOD activity（P〈0.05）.Conclusion The combined application of ZCS can improve intestinal epithelial barrier function in ulcerative colitis mice by regulating the expression of tight junction proteins（Occludin and ZO-1）and reducing oxidative stress response.