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目的探讨腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)在重症急性胰腺炎SD大鼠肠道紧密连接损伤中的作用。方法给予SD大鼠腹腔注射不同剂量的20%L-精氨酸溶液,分别制备大鼠重症急性胰腺炎(severe acute pancreatitis,SAP)及轻症急性胰腺炎(mild acute pancreatitis,MAP)模型,通过检测血清淀粉酶水平、胰腺病理改变以证实胰腺炎模型构建成功。对SAP大鼠分别给予AMPK抑制剂Compound C和谷氨酰胺灌胃处理,小肠组织切片染色观察大鼠小肠组织损伤情况;FITC-Dextran通透实验检测肠道通透性;免疫组化和Western blot方法分别检测肠黏膜细胞αSNAP、AMPK和紧密连接蛋白occludin的表达情况。结果病理切片证实胰腺炎模型构建成功,并且抑制AMPK后明显降低重症急性胰腺炎的病理损伤;SAP大鼠肠黏膜损伤明显,而加入AMPK抑制剂Compound C或肠黏膜保护剂谷氨酰胺可明显抑制SAP大鼠肠黏膜损伤;SAP组建模48 h(410.25±7.80)的肠道通透性相对于对照组(0.51±0.61)显著增强(P<0.05),而加入AMPK抑制剂Compound C 48 h的Com组(372.95±9.33)或肠黏膜保护剂谷氨酰胺的Gln组(367.93±18.90)(P<0.05),可明显降低SAP大鼠肠道通透性;重症急性胰腺炎时,αSNAP表达下调(P<0.05),而AMPK表达明显增强(P<0.05),加入AMPK抑制剂可以明显上调occludin的表达(P<0.01),提示AMPK对occludin蛋白存在负性调控。结论 AMPK信号在大鼠重症急性胰腺炎肠黏膜损伤中过度活化,可通过负性调控occludin蛋白的表达导致肠黏膜损伤,从而促进肠黏膜通透性增强。
Objective To investigate the role of AMPK (Activated Protein Kinase) in the intestinal tight junction injury in SD rats with severe acute pancreatitis. Methods SD rats were intraperitoneally injected with different doses of 20% L-arginine solution to prepare rat models of severe acute pancreatitis (SAP) and mild acute pancreatitis (MAP) Serum amylase levels were measured, pathological changes in the pancreas to confirm the successful construction of pancreatitis model. The SAP rats were treated with AMPK inhibitor Compound C and glutamine gavage respectively, and the small intestine tissue sections were stained to observe the damage of small intestine. The permeability of intestinal tract was detected by FITC-Dextran permeation test. Immunohistochemistry and Western blot Methods The expressions of αSNAP, AMPK and tight junction protein occludin in intestinal mucosa were detected respectively. Results Pathological sections confirmed that the model of pancreatitis was successfully constructed and the pathological damage of severe acute pancreatitis was inhibited significantly after AMPK was inhibited. The intestinal mucosal injury was obvious in SAP rats, while the addition of AMPK inhibitor Compound C or intestinal mucosal protective agent glutamine significantly inhibited Intestinal mucosal injury was induced in SAP rats. The intestinal permeability of SAP group at 48 h (410.25 ± 7.80) was significantly increased compared with control (0.51 ± 0.61) (P <0.05), while AMPK inhibitor Compound C 48 h (372.95 ± 9.33) or Gln (367.93 ± 18.90) (P <0.05), a protective agent of intestinal mucosa, significantly decreased the intestinal permeability of SAP rats. In severe acute pancreatitis, αSNAP expression (P <0.05), while the expression of AMPK was significantly increased (P <0.05). The addition of AMPK inhibitor significantly up-regulated the expression of occludin (P <0.01), suggesting that AMPK negatively regulated the occludin protein. Conclusion AMPK signal is overexpressed in intestinal mucosal lesion of severe acute pancreatitis in rats, which may lead to intestinal mucosal damage through the negative regulation of occludin protein expression, thereby enhancing intestinal mucosal permeability.