丙酮酸作用下大鼠移植小肠缺血再灌注损伤中核转录因子的变化

Changes in nuclear factor during ischemia/reperfusion injury in rat small intestine transplantation affected by pyruvate

目的:研究证实丙酮酸对移植小肠缺血再灌注损伤具有保护作用,并且与其影响细胞因子水平有关。核转录因子在各种细胞外刺激介导的细胞信号转导调控中起核心作用,实验拟观察丙酮酸作用下移植小肠缺血再灌注损伤中核转录因子κB的变化并分析其意义。方法:①实验于2003-10/2004-04在解放军第四军医大学西京医院胃肠外科实验室完成。选用成年雄性SD大鼠78只,动物处置方法符合动物伦理学标准。②按随机数字表法分为3组:假手术组(n=6)行剖腹、左侧肾切除与移植组对照;移植小肠缺血再灌注组和丙酮酸处理组(n=36)均建立移植小肠缺血再灌注动物模型,丙酮酸处理组于供体小肠阻断血流、灌洗前10min向肠腔内注入含有分析纯丙酮酸的营养液。③分别于缺血45,90min和再灌注30,180min留取移植小肠组织标本(n=6),光镜下观察组织学改变;另外分离、提取核蛋白并定量,通过电泳迁移改变分析实验检测肠组织中的核转录因子κB的活性。结果:78只大鼠全部进入结果分析。①缺血再灌注不同时相移植小肠缺血再灌注组小肠黏膜组织损伤程度均重于假手术组(P﹤0.01);而丙酮酸处理组损伤程度轻于移植小肠缺血再灌注组(P﹤0.01),与假手术组差异无显著性意义(P﹥0.05)。②再灌注30min时移植小肠缺血再灌注组核转录因子κB活性高于其他两组(P﹤0.01);丙酮酸处理组核转录因子κB活性高于假手术组(P﹤0.05)。再灌注180min时移植小肠缺血再灌注组核转录因子κB活性略降低,但仍高于其他两组(P﹤0.01),丙酮酸处理组核转录因子κB活性与假手术组差异无显著性意义(P﹥0.05)。结论:丙酮酸作用下大鼠移植小肠缺血再灌注损伤过程中核转录因子κB的活性降低,该结果可能是丙酮酸保护移植小肠缺血再灌注损伤的重要作用途径之一。

AIM： Pyruvate possesses protective effect on ischemia/reperfusion injury of small intestine transplantation, which is correlated with cytokine levels. Nuclear factor （NF） has a crucial effect in signal transduction mediated by various extracellular stimulations. This study aimed to investigate the changes and significances of NF- κB on ischemia/reperfusion injury in rat small bowel transplantation afforded by pyruvate. METHODS： Experiments were performed at the Laboratory of Department of Gastrointestinal Surgery, Xijing Hospital, Fourth Military Medical University of Chinese PLA from October 2003 to April 2004. Totally 78 adult male SD rats were selected. Animal disposal in the experiment was accorded with the animal ethical standard. All rats were divided into three groups at random. Rats in the sham operation group （n=6） received belly open and nephrectomy on left kidney. Rat models of ischemia/reperfusion were made in the ischemia/reperfusion group and pyruvate treated group （n=36）. Blood flow was blocked in donor small intestine and nutrient fluid containing analytical pure pyruvate was injected into the enteric cavity 10 minutes before lavation. Samples of intestinal tissues （n=6） were obtained 45 and 90 minutes after ischemia and 30 and 180 minutes after reperfusion. Changes in histology were observed under light microscope. Nucleoprotein was isolated, extracted and quantitated. Activity of NF- κB in intestinal tissues was analyzed by electrophoretic mobility shift assay （EMSA）. RESULTS： Totally 78 rats were included in the final analysis. Intestinal tissues injury was severe in the ischemia/reperfusion group compared to the sham operation group at different time points （P〈 0.01）. Injury degree was mild in the pyruvate treated group compared to the ischemia/reperfusion group （P 〈 0.01）. There was no significant difference compared to the sham operation group （P 〉 0.05）. The activity of NF- κB was higher in the ischemia/reperfusion group compared to the other two groups 30 minutes after reperfusion （P 〈 0.01）. The activity of NF- κB was higher in the pyruvate treated group than in the sham operation group （P 〈 0.05）. The activity of NF-κB was low in the ischemia/reperfusion group 180 minutes after reperfusion, but still higher than in the other two groups （P 〈 0.01）. There was no significant difference in activity of NF- κB in the pyruvate treated group （P 〉 0.05）. CONCLUSION： The activity of NF- κB decreases during ischemia/reperfusion injury in grafted small intestine in rat small bowel transplantation, which may be one of important protective mechanisms of pyruvate.