腺苷酸A2A受体对小鼠小肠急性缺血再灌注损伤中肠屏障功能的影响

Effect of adenosine 2a receptor on intestinal barrier function in mice with acute reperfusion injury

目的探讨腺苷酸A2A受体(A2AR)在小鼠小肠急性缺血再灌注(I/R)刺激所致肠黏膜屏障损伤机制中的作用。方法 6~8周龄雄性C57BL/6小鼠12只,分为假手术组和I/R组;A2AR基因敲除雄性C57BL/6(A2AR^(-/-))小鼠12只,分为A2AR^(-/-)+假手术组和A2AR^(-/-)+I/R组(n=6)。采用夹闭肠系膜上动脉30 min,再灌注6 h建立小鼠肠I/R模型。HE染色观察小肠黏膜的形态学变化;免疫荧光及蛋白质印迹检测肠上皮细胞紧密连接蛋白ZO-1蛋白变化,尤斯灌流仪检测肠上皮通透性变化。结果 HE染色显示假手术组和A2AR^(-/-)+假手术组小肠黏膜完整、无水肿,两组无明显差异;而I/R组和A2AR^(-/-)+I/R组小肠黏膜明显水肿、绒毛断裂、部分脱落,且A2AR^(-/-)+I/R组损伤程度明显重于I/R组。小肠黏膜上皮通透性检测发现,假手术组和A2AR^(-/-)+假手术组没有显著性差异;与假手术组相比,I/R组和A2AR^(-/-)+I/R组小肠黏膜跨上皮电阻(trans-epithelium electrical resistant,TER)值均显著下降(P<0.01),且A2AR^(-/-)+I/R组相比I/R组TER值下降更为明显(P<0.05)。免疫荧光及蛋白定量分析显示假手术组与A2AR^(-/-)+假手术组肠上皮高表达ZO-1,而在I/R组和A2AR^(-/-)+I/R组中ZO-1表达明显减少,同时A2AR^(-/-)+I/R组中ZO-1的表达显著低于I/R组(P<0.05)。结论小肠I/R刺激可诱导肠上皮细胞紧密连接蛋白ZO-1表达与分布异常,造成肠黏膜屏障损伤;而A2AR可通过调控肠上皮细胞ZO-1表达加强肠黏膜屏障功能,在肠I/R损伤中发挥潜在的保护作用。

Objective To investigate the effect of adenosine 2a receptor （A2AR） on intestinal barrier function in a mouse model of acute intestinal ischemia reperfusion （I/R） injury. Methods Twelve 6 to 8-week-old male C57BL/6 mice and another 12 ones with background A2AR gene knockout （A2AR-/- ） were separately randomized into 2 groups for sham operation or occlusion of the superior mesenteric artery for 30 min followed by reperfusion for 6 h to induce acute intestinal I/R group injury. HE staining was used to observe the histological changes of the small intestinal mucosa, and the expression of the tight junction protein ZO-1 was detected with immunofluorescence assay and Western blotting. Results HE staining showed intact intestinal mucosal structure in both the wild-type and A2AR-/- mice receiving the sham operation. Acute intestinal I/R injury induced obvious changes in the intestinal mucosal structure, and the pathology was more prominent in A2AR-/- mice （P 〈 0.05）. In Ussing chamber experiments, the trans-epithelium electrical resistance （TER） was similar in the wild-type and A2AR -/- mice receiving the sham operation, but decreased significantly in the 2 I/R injury groups, especially in A2AR-/- mice （P 〈0. 01 ）. Immunofluorescence assay and Western blotting showed high expression of ZO-1 protein in the intestinal epithelial cells in the 2 sham groups, but its expression level was decreased significantly following I/R injury, and the reduction was significantly greater in A2AR -/- mice （P 〈 0. 05）. Conclusion Acute intestinal I/R injury induces obvious damages of the intestinal mucosal structure and increases the mucosal permeability. A2AR has potential protective effect on the intestinal mucosa by modulating the expression of ZO-I in the epithelial cells.