重症急性胰腺炎大鼠紧密连接蛋白-1的表达与肠黏膜屏障损伤

Expression of Zonula Occludens-1 and Mucosal Barrier Injury in Rats with Severe Acute Pancreatitis

目的研究紧密连接蛋白-1(ZO-1)在重症急性胰腺炎(SAP)大鼠回肠组织中的表达情况,初步探讨SAP时肠黏膜屏障损伤的可能机理。方法 SD雄性大鼠50只,按完全随机法分为假手术组和SAP组,SAP组按取材时间不同又分为3、6、12及24 h 4个亚组,每组10只。SAP组采用Aho法逆行穿刺胆胰管注射5%牛磺脱氧胆酸钠建立SAP模型,分别于造模后第3、6、12及24 h处死大鼠,假手术组直接处死。建模成功后检测肿瘤坏死因子-α(TNF-α)水平及二胺氧化酶(DAO)活性,同时观察胰腺和回肠组织的病理改变,分别用免疫组织化学蛋白定位及逆转录-聚合酶链反应(RT-PCR)检测回肠组织中ZO-1蛋白和mRNA表达水平。结果与假手术组〔TNF-:α(10.83±0.96)ng/L;DAO:(354.79±3.67)U/L;ZO-1蛋白:(10.40±0.45)分;ZO-1 mRNA:0.878±0.014 8〕相比较,SAP组制模后不同时相大鼠血清中TNF-α水平〔3 h:(125.30±0.94)ng/L;6 h:(181.89±4.93)ng/L;12 h:(230.58±1.28)ng/L;24 h:(198.89±4.83)ng/L〕明显升高(P<0.05),血浆中DAO活性〔3 h:(235.77±0.67)U/L;6 h:(117.22±5.58)U/L;12 h:(106.69±1.39)U/L;24 h:(91.18±1.09)U/L〕明显降低(P<0.05);回肠组织中ZO-1蛋白〔3 h:(8.70±0.22)分;6 h:(3.73±0.19)分;12 h:(3.92±0.22)分;24 h:(4.29±0.30)分〕和mRNA(3 h:0.806±0.020 7;6 h:0.370±0.015 8;12 h:0.502±0.019 2;24 h:0.562±0.030 3)表达明显下调(P<0.05);同时胰腺组织及回肠黏膜绒毛上皮坏死、血管破裂出血、炎性细胞浸润。结论大鼠回肠组织中ZO-1表达下调是SAP肠黏膜屏障损伤的重要原因之一,可能与炎性细胞因子TNF-α的过度释放及DAO活性降低有关。

Objective To study the expression of zonula occludens-1（ZO-1） in ileum tissues and the possible mechanism of intestinal mucosal barrier injury in rats with severe acute pancreatitis（SAP）.Methods Fifty SD male rats were randomly divided into sham operation group and SAP group,then SAP group was divided into four subgroups with 10 rats in each subgroup according to the sampling time of 3,6,12,and 24 h.The SAP model was made by injecting 5% bovine sodium deoxycholate into biliary-pancreatic duct with Aho＇s method.The rats were killed at 3,6,12,and 24 h after making model.The rats in the sham operation group were killed directly.Tumor necrosis factor-α（TNF-α）,diamine oxidase（DAO）,and histological changes in pancreatic and intestinal pathologies were observed.At the same time,the ZO-1 protein and mRNA expressions of ileum tissues were detected by immunohistochemistry and RT-PCR,respectively.Results Compared with the sham operation group（TNF-α：（10.83±0.96） ng/L;DAO：（354.79±3.67） U/L;ZO-1 protein：（10.40±0.45） score;ZO-1 mRNA： 0.878±0.014 8）,the levels of TNF-α at different time（3 h：（125.30±0.94） ng/L;6 h：（181.89±4.93） ng/L;12 h：（230.58±1.28） ng/L;24 h：（198.89±4.83） ng/L） were significantly higher（P0.05）,the activities of DAO（3 h：（235.77±0.67） U/L;6 h：（117.22±5.58） U/L;12 h：（106.69±1.39） U/L;24 h：（91.18±1.09） U/L） were significantly lower（P0.05）,ZO-1 protein（3 h：（8.70±0.22） score;6 h：（3.73±0.19） score;12 h：（3.92±0.22） score;24 h：（4.29±0.30） score） and mRNA（3 h： 0.806±0.020 7;6 h： 0.370±0.015 8;12 h： 0.502±0.019 2;24 h： 0.562±0.030 3） expressions of the ileum tissues were significantly lower（P0.05） in the SAP group;Meanwhile,the necrosis of ileum mucous membrane chorioepithelium,angiorrhexis and hemorrhage,and inflammatory cell infiltration in the pancreatic and ileum tissues were also observed.Conclusion The decrease of expression of ZO-1 in ileum tissues is one of the vital causes for mucosal barrier injury in SAP,probably through acts the excessive release of inflammatory cytokines TNF-α and the decrease of DAO activity.