大鼠肾脏缺血预处理中P_(38)MAPK、iNOS、COX_2间信号转导关系的研究

The Role of P_(38) Mitogen- activated Protein Kinase( P_(38)MAPK),Nitric Synthase( iNOS)and Cyclooxygenase-2( COX_2) in Rat Renal Ischemic Preconditioning(IPC)

目的:探讨大鼠肾脏缺血预处理中P38MAPK、iNOS与COX2间的信号转导关系。方法:雄性wistar大鼠66只,随机分为6组,分别为:假手术(sham)组,缺血再灌注(IR)组,缺血预处理+缺血再灌注(IPC)组,SB203580药物干预(SB203580)组,AG药物干预(AG)组,NS398药物干预(NS398)组,在再灌注后24 h、48 h两个时间点取材,用苦味酸法测定血清肌酐反映肾功能变化情况;用Western blot法检测再灌注后48 h肾组织P38MAPK、iNOS与COX2蛋白的表达,进行半定量分析。结果:血肌酐在IPC组较IR组低(P<0.05)尤其在IPC后48 h明显(P<0.01);P38MAPK、iNOS与COX2在sham组,IR组,IPC组均有表达,尤在IPC组表达明显(P<0.01);在SB203580组无P38MAPK蛋白的表达,iNOS、COX2的表达均较IPC组低(P<0.05);在AG组无iNOS蛋白的表达,COX2蛋白的表达较IPC组低(P<0.05);在NS398组无COX2蛋白表达,P38MAPK、iNOS蛋白的表达与IPC组相差不明显(P>0.05),在AG与NS398组P38MAPK蛋白表达与IPC组相差不明显(P>0.05)。结论:缺血预处理时P38MAPK、iNOS与COX2蛋白均表达、活化,在其信号转导关系中,P38MAPK是iNOS的上游、并通过iNOS活化介导COX2表达。

Objective： Explore the relations among P38 mitogen- activated protein kinase（ P38MAPK）,nitric synthase（ iNOS） and cyclooxygenase- 2（ COX2） in rat renal ischemic preconditioning（ IPC）. Methods： 66 male Wistar rats were randomly divided into 6 groups： sham group（ n = 6）,ischemic reperfusion（ IR,n = 12） group,ischemic preconditioning ＋ ischemic reperfusion（ IPC,n = 12） group,SB203580（ inhibitor of P38MAPK） group（ n = 12）,AG（ inhibitor of iNOS） group（ n = 12）,NS398（ inhibitor of COX2） group（ n = 12）. Rats were sacrificed,serum and kidney were collected at 24 and 48 h after reperfusion. The renal function was evaluated by testing serum creatinine level. The expression of P38 MAPK,iNOS and COX2 in the kidney was determined by Western blotting. Results： The animals in IPC group showed lower serum creatinine compared with IR group（ P〈0. 05）. The difference was more significant in IPC group at 48 h（ P〈0. 01）. P38 MAPK,iNOS and COX2 were expressed in all groups,in which IPC group showed the highest expression level. There was no P38 MAPK in SB203580 group,and the expression of iNOS and COX2 are lower.（ P〈0. 05） compared with IPC group; No iNOS expressed in AG group,and the expression of COX2 was significantly less than IPC group（ P〈0. 05）; There are no COX2 expression in NS398 group,P38 MAPK and iNOS were not significantly different from IPC group.（ P〉0. 05）; As far as as P38 MAPK expression was concerned,there was no significant deference in AG、NS398 and IPC group（ P〉0. 05）. Conclusion： Ischemic preconditioning showed protective effect in 48 hours post- ischemia reperfusion. The protection was related to P38 MAPK,iNOS and COX2expression、activation. P38 MAPK is the upstream of i NOS,and upregulated COX2 expression by activating iNOS.