大鼠体外循环后小肠黏膜基质金属蛋白酶9及其组织型抑制剂1表达与肠屏障功能的关系

Expression of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in gut mucosa and its relationship to intestinal barrier function in rats after cardiopulmonary bypass

目的探讨大鼠体外循环(CPB)后小肠黏膜基质金属蛋白酶9(MMP-9)及其组织型抑制剂(TIMP-1)表达与肠屏障功能的关系,为CPB后肠屏障功能保护提供新靶点。方法健康成年雄性清洁级SD大鼠40只随机分为CPB组(n=20)和假手术组(SH组,n=20)。另取10只作为正常对照组(N组)。分光光度法测定CPB后血浆二胺氧化酶(DAO)活性,转录酶-逆转录聚合酶链反应及蛋白质斑迹法检测肠黏膜MMP-9,TIMP-1的mRNA和蛋白表达变化,明胶酶谱法测定肠黏膜MMP-9活性变化,并进行统计学分析。结果 CPB组血浆中DAO活性比SH组和N组显著增高(P<0.05);RT-PCR显示CPB组小肠黏膜MMP-9 mRNA表达水平较N组和SH组明显增加(P<0.01);CPB组TIMP-1 mRNA表达水平与N组和SH组无显著差异(P>0.05);Western blot显示CPB组MMP-9蛋白表达显著高于SH组和N组(P<0.01);三组之间TIMP-1蛋白表达无明显差异(P>0.05)。凝胶成像系统分析结果显示,CPB组小肠黏膜组织中MMP-9、MMP-2活性与SH和N组比较,均显著增强(P<0.01);线性回归及相关分析结果显示:CPB后肠黏膜组织MMP-9活性和血浆DAO活性呈显著正相关(r=0.821,P<0.01)。结论 CPB后小肠黏膜MMP-9表达上调及其酶活性增加导致MMP-9/TIMP-1失衡在肠屏障功能障碍发生发展中起重要作用。调控MMP-9活性有可能成为CPB过程中肠屏障功能保护新靶点。

Objective To explore the expression of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1（MMP-9/TIMP-1） in gut mucosa and its relationship to intestinal barrier function in rats after cardiopulmonary bypass（CPB）. Methods CPB model in rats was established. 40 SD rats were randomly divided into CPB group（n = 20） and sham-operation（SH） group（n = 20）,another 10 SD rats were taken as normal control（N） group. The serum diamine oxidase（DAO） activity was measured with spectrophotometry. The changes of mRNA and protein expression of MMP-9,TIMP-1 in the intestinal mucosa were detected by RT-PCR and Western blot respectively. The changes of MMP-9 activity were detected by Gelatin Zymography. Statistical analysis was performed by using software SPSS 16. 0. Results The plasma DAO activity in CPB group（1. 817±0. 359 U/ml） was significantly higher than those in SH group and N group（P = 0. 018）,and the expression of MMP-9 mRNA in intestinal mucosa detected by RT-PCR was enhanced more significantly than those in the other two groups. Meanwhile no significant difference was observed in expression of TIMP-1 mRNA in three groups. Expression of MMP-9 protein in intestinal mucosa analysed by Western blot was increased more significantly in CPB group than those in the other two groups,and no significant difference was observed in expression of TIMP-1 protein in three groups. The gelatinlytic activity of MMP-9 and MMP-2 enhanced more significantly in CPB group than those in SH group and N group. The activity of MMP-9 protein in gut mucosa in CPB group was significantly positively correlated with the activity of plasma DAO by linear correlative analysis. Conclusion Unbalance of MMP-9/TIMP-1 caused by the upregulated expression and enhanced activity of MMP-9 in intestinal mucosa after CPB play important role in the pathogenesis of intestinal barrier dysfunction. Regulating of the activity of MMP-9 may be a new target for the pretection of intestinal barrier function during CPB.