S-亚硝基谷胱甘肽对小鼠肠急性缺血再灌注损伤后肠屏障功能的影响

摘要

目的：探讨 S-亚硝基谷胱甘肽（GSNO）预处理对小鼠肠急性缺血再灌注（I／R）所致的肠黏膜屏障损伤的影响。方法6～8周龄雄性 C57BL／6小鼠24只，分为 Sham 组、I／R 组、I／R＋GSNO 组，每组8只。采用夹闭肠系膜上动脉30 min，再灌注6 h作为小鼠肠 I／R 模型。苏木素-伊红（HE）染色观察小肠组织形态学的变化；免疫组织化学观察肠上皮细胞间紧密连接蛋白 clau-din-1的表达情况；Western blot 检测 claudin-1蛋白水平变化。结果 HE 染色显示，与 Sham 组比较，I／R 组肠黏膜明显肿胀，绒毛部分脱落、变粗，绒毛倒伏、断裂，而 I／R＋GSNO 组肠黏膜无明显肿胀，绒毛完整，形态接近正常；免疫组织化学观察显示 I／R刺激可造成肠上皮表面 claudin-1连续性明显破坏，阳性表达率明显降低，I／R＋GSNO 组肠上皮 claudin-1连续性明显恢复，阳性染色显著增加；肠上皮 claudin-1蛋白定量分析显示：与 Sham 组比较，I／R 组及 I／R＋GSNO 小肠上皮紧密连接蛋白 claudin-1表达分别下降32．5％，13．8％（P ＜0．05）；与 I／R 组比较，I／R＋GSNO 组小鼠肠上皮紧密连接蛋白 claudin-1表达上调27．8％（P ＜0．05）。结论小肠急性 I／R 刺激可造成明显的肠黏膜损伤及肠上皮间紧密连接的显著破坏；GSNO 预处理可通过上调 claudin-1表达，有效缓解 I／R 对肠黏膜结构与肠上皮屏障功能的损伤。%Objective To investigate the effect of S-Nitrosoglutathione (GSNO)on acute ischemia reperfusion (I/R)induced in-testinal barrier function lesion in a mouse model.Methods Twenty-four 6-8-year-old C57BL/6 mice were divided into 3 groups,8 for each:(1)the sham group;(2)the I/R group;(3)the I/R+GSNO group.The mouse intestine I/R model was established by the occlusion of the superior mesenteric artery temporarily followed by reperfusion for 6 h.Histological changes in the small intestine were observed after HE staining;the expression of claudin-1 protein in the intestine epithelium was assessed by immunohistochem-istry staining as well as western blot analysis.Results Both HE staining and immunohistochemistry results showed the integrate intestinal villi with the continuous Claudin-1 expression alone the villi in the sham group;the intestinal villi of the I/R group partial-ly detached,thickened,crooked and fractured,with the obvious disconnection of Claudin-1 staining alone the top of the villi;while the intestinal villi of the I/R+GSNO group were neatly arranged and damage to intestinal mucosa was much alleviated,accompanied with the marked restoration of the continuity of claudin-1 staining.Compared to the sham group,claudin-1 protein level of for the I/R group and the I/R+GSNO group decreased by 32.5% and 13.8% respectively (P <0.05);and compared to the I/R group,clau-din-1 protein level of the I/R+GSNO group increased by 27.8% (P <0.05).Conclusion Protein level of claudin-1 would decrea-ses after I/R,and pretreatment with GSNO can effectively relieve the damage of intestinal mucosal structure as well as intestinal tight junction barrier through upregulating the expression of claudin-1 protein.