【摘要】：目的探討腺苷酸A2A受體(A2AR)在小鼠小腸急性缺血再灌注(I/R)刺激所致腸黏膜屏障損傷機(jī)制中的作用。方法 6~8周齡雄性C57BL/6小鼠12只,分為假手術(shù)組和I/R組;A2AR基因敲除雄性C57BL/6(A2AR~(-/-))小鼠12只,分為A2AR~(-/-)+假手術(shù)組和A2AR~(-/-)+I/R組(n=6)。采用夾閉腸系膜上動脈30 min,再灌注6 h建立小鼠腸I/R模型。HE染色觀察小腸黏膜的形態(tài)學(xué)變化;免疫熒光及蛋白質(zhì)印跡檢測腸上皮細(xì)胞緊密連接蛋白ZO-1蛋白變化,尤斯灌流儀檢測腸上皮通透性變化。結(jié)果 HE染色顯示假手術(shù)組和A2AR~(-/-)+假手術(shù)組小腸黏膜完整、無水腫,兩組無明顯差異;而I/R組和A2AR~(-/-)+I/R組小腸黏膜明顯水腫、絨毛斷裂、部分脫落,且A2AR~(-/-)+I/R組損傷程度明顯重于I/R組。小腸黏膜上皮通透性檢測發(fā)現(xiàn),假手術(shù)組和A2AR~(-/-)+假手術(shù)組沒有顯著性差異;與假手術(shù)組相比,I/R組和A2AR~(-/-)+I/R組小腸黏膜跨上皮電阻(trans-epithelium electrical resistant,TER)值均顯著下降(P0.01),且A2AR~(-/-)+I/R組相比I/R組TER值下降更為明顯(P0.05)。免疫熒光及蛋白定量分析顯示假手術(shù)組與A2AR~(-/-)+假手術(shù)組腸上皮高表達(dá)ZO-1,而在I/R組和A2AR~(-/-)+I/R組中ZO-1表達(dá)明顯減少,同時A2AR~(-/-)+I/R組中ZO-1的表達(dá)顯著低于I/R組(P0.05)。結(jié)論小腸I/R刺激可誘導(dǎo)腸上皮細(xì)胞緊密連接蛋白ZO-1表達(dá)與分布異常,造成腸黏膜屏障損傷;而A2AR可通過調(diào)控腸上皮細(xì)胞ZO-1表達(dá)加強(qiáng)腸黏膜屏障功能,在腸I/R損傷中發(fā)揮潛在的保護(hù)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.
【作者單位】： 第三軍醫(yī)大學(xué)新橋醫(yī)院普通外科;
【基金】：國家自然科學(xué)基金面上項(xiàng)目(81470803,81270451)~~
【分類號】：R574

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