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

发布时间：2018-10-26 12:55

【摘要】：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(P0.01),且A2AR~(-/-)+I/R组相比I/R组TER值下降更为明显(P0.05)。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(P0.05)。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。
[Abstract]:Objective to investigate the role of adenylate A 2A receptor (A2AR) in intestinal mucosal barrier injury induced by acute small intestinal ischemia reperfusion (I / R) in mice. Methods Twelve male C57BL/6 mice aged 6 to 8 weeks were divided into sham operation group and I / R group. Twelve A2AR gene knockout male C57BL/6 (A2AR- / -) mice were divided into A 2AR- (- / -) sham-operated group and A _ 2AR- /-Irr group (n = 6). The intestinal I / R model of mice was established by clipping superior mesenteric artery for 30 min, and reperfusion for 6 h. The morphological changes of intestinal mucosa were observed by HE staining. The changes of ZO-1 protein in intestinal epithelial cells were detected by immunofluorescence and Western blotting. Results HE staining showed that the intestinal mucosa of sham operation group and A _ 2AR- ~ (- / -) sham-operation group were intact without edema, and there was no significant difference between the two groups. In the I / R and A _ 2AR- / _ (-) I / R groups, the intestinal mucosal edema, villi rupture and partial shedding were observed, and the degree of injury in the A _ 2AR- /-I _ (-) R group was significantly more severe than that in the I / R group. There was no significant difference between the sham-operated group and the A _ 2AR- ~ (- / -) sham-operated group. Compared with the sham-operated group, the intestinal mucosal transdermal resistance (trans-epithelium electrical resistant,TER) in the I / R and A _ 2AR- ~ (- / -) I / R groups decreased significantly (P0.01). The TER of A _ 2AR- /-I / R group was significantly lower than that of I / R group (P0.05). Immunofluorescence and protein quantitative analysis showed that ZO-1, was highly expressed in intestinal epithelium of sham-operated group and A2AR- /-group, but the expression of ZO-1 in I / R group and A2AR- /-I / R group was significantly decreased. At the same time, the expression of ZO-1 in A2AR- /-I / R group was significantly lower than that in I / R group (P0.05). Conclusion intestinal I / R stimulation can induce abnormal expression and distribution of tight junction protein (ZO-1) in intestinal epithelial cells, resulting in intestinal mucosal barrier damage. A2AR can enhance the intestinal mucosal barrier function by regulating the expression of ZO-1 in intestinal epithelial cells and play a potential protective role in intestinal I / R injury.
【作者单位】：第三军医大学新桥医院普通外科;
【基金】：国家自然科学基金面上项目(81470803,81270451)~~
【分类号】：R574

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