大鼠肾脏缺血预处理中COX-2和iNOS间信号转导关系的研究

Association of COX-2 and iNOS in ischemic preconditioning in rat kidney

目的探讨大鼠肾脏缺血预处理中COX-2和iNOS的表达及其两者之间的信号转导关系,从而进一步探明肾脏缺血预处理的保护机制。方法60只大鼠随机分为五组:假手术组(Sham组)、缺血再灌注组(I/R组)、缺血预处理+缺血再灌注组(IPC组)、NS398干预组(NS398组)和AG干预组(AG组)。在再灌注后24 h、48 h两个时间点取材,测定血清Scr;光镜观察形态学改变;采用Western blot法测定COX-2和iNOS的蛋白表达量,进行半定量分析。结果与I/R组相比较,无论24 h、48 h,IPC组和两给药组的Scr值均明显降低,具有统计学差异(P<0.05)。HE染色形态学也表明IPC组损伤明显减轻,与I/R组相比差异显著。NS398组和AG组两个给药组的损伤程度介于I/R组和IPC组之间。IPC组中COX-2和iNOS均活化、表达,其表达量与sham组和I/R组相比明显增多,差异显著(P<0.05),而且48 h的表达量多于24 h;AG组COX-2表达量明显少于IPC组(P<0.05);NS398组和IPC组间iNOS表达量无显著差异(P>0.05)。结论本实验证实了肾脏IPC对I/R损伤的保护效应;肾脏IPC时COX-2和iNOS均活化、表达,参与了其保护效应,而且主要参与其晚期保护作用;在其信号转导中,iNOS是COX-2的上游,iNOS介导COX-2活化表达。

Objective To investigate the role and asseciation of COX-2 and iNOS in the rat renal ischemic preconditioning; and thereby to explore the mechanism of renal ischemic preconditioning, Methods Sixty Wistar rats were randomly divided into five groups： sham operation group（sham）, ischemic reperfusion group （I/R）, ischemic preconditioning group （IPC）, NS398 （ inhibitor of COX-2） treated group （NS398） ;and AG （inhibitor of iNOS）treated group （AG）, Renal I/R injury model was induced by clamping bilateral renal arteries for 45 rain followed by reperfusion. Rats in IPC group underwent four cycles of IPC （8-min ischemia followed by 5-rain reperfusion）. Rats in AG treated group or NS398 treated group were pretreated with AG or NS398 respectively before ischemic preconditioning. Rats were killed at 24 and 48 h after reperfusion. Renal function was determined by measuring serum creatinine. Changes of renal morphology were measured by hematoxylin- eosine（HE） staining. Western blot analysis was used to detect protein expressions of COX-2 and iNOS. Results Serum creatinine significantly increased after renal I/R injury and the difference was significant in comparison with the IPC group and the other two treated groups（P 〈 0.05）. Kidneys of I/R injury modal rats displayed most significant histology changes including a loss of brush-border membranes, tubular dilatation, flattened tubular epithdium, cast formation, luminal debris and interstitial infiltration. The morphology damage in the IPC group became slighter compared to the I/R group. The degree of damages in AG group and NS398 group were between those of degress of IPC group and I/R group. COX- 2 and iNOS were all activated and expressed in IPC group, and the expression amount was much plentiful than sham group and I/R group. Additionally the expression amount at 48 h was higher than 24 h. The expression of COX-2 in AG group was significantly decreased in comparison with IPC groups（P 〈 0.05） ; and the difference in the amount of iNOS was not significant between NS398 group and IPC group（P〈0.05）. Conclusion Ischemic preconditioning has protective effect on renal I/R injury, which may be at least partly by activating COX-2 and iNOS; moreover iNOS may be an up-stream of COX-2, and may mediate COX-2 expression.