早期反应基因-1(Egr-1)在缺血再灌注肝脏组织中的表达

Expression of early growth response gene-1 in liver following ischemia and reperfusion

目的研究缺血再灌注后肝组织中早期反应基因-1(Egr-1)的表达。方法动脉放血至平均动脉压(MAP)25 mmHg,分别维持1 h或2.5 h,复制小鼠整体肝缺血模型;2.5 h后,回输失血+2倍于失血量的平衡液至MAP 80 mmHg,复制小鼠肝缺血再灌注模型。取肝组织,分别通过Northern印迹法、凝胶电泳迁移分析(EMSA)、蛋白质免疫印迹法,检测肝组织mRNA的表达;肝核提取物中Egr-1蛋白与DNA的结合活性;肝组织、胞浆及核提取物中Egr-1蛋白的表达。结果缺血1 h后,Egr-1mRNA在肝组织中的表达明显增强;缺血2.5 h后肝组织中Egr-1mRNA虽有所下降,但表达水平仍较高。缺血2.5 h+再灌注4 h后,肝组织中Egr-1mRNA消失。缺血2.5 h和缺血2.5 h+再灌注4 h后肝细胞核提取物中Egr-1蛋白的DNA结合活性增加。缺血2.5 h及缺血2.5 h+再灌注4 h肝组织及肝细胞核提取物中Egr-1蛋白表达明显增加;Egr-1蛋白仅在缺血2.5 h肝胞浆提取物中表达。结论本实验结果表明肝缺血再灌注后Egr-1mRNA和蛋白表达均明显增强,其DNA结合活性增加,说明其转录和翻译水平均增加。本研究显示缺血再灌注后肝脏中Egr-1基因被激活,Egr-1可能参与了肝I/R后炎症反应基因的调节,并在肝损伤中起一定的作用。

Objective To investigate the expression of early growth response gene-1 （Egr-1） in the liver following ischemia （I） and reperfusion. Methods C57B16 mice underwent global I by withdrawal of blood to achieve a mean arterial pressure （MAP） of 25 mmHg and the pressure was maintained for 1 h and 2.5 h, respectively. After 2.5 h of I, the mice were resuscitated to an MAP of 80 mmHg by administration of the remaining shed blood plus two times the shed volume in lactated Ringers solution. The expression of EGr-1 mRNA in the liver was detected by Northern blot. DNA binding activity of the Egr-1 protein in liver nuclear extracts was determined by electrophoretic mobility shift assay （EMSA）. Western blot analysis was used to assess the induction of Egr-1 protein in liver tissue, cytoplasm and nuclear extracts. Results Egr-1 mRNA was strongly expressed as early as 1 h after I, the expression was till high but decreased 2.5 h after I as compared with that of 1 h I group. The mRNA disappeared in the liver following 2.5 h I ＋ 4 h R. The Egr-1 DNA binding activity elevated in the nuclear extracts of the livers from 2.5 h I and 2.5 h I ＋ 4 h R mice. Egr-1 protein was increased in the liver following 2.5 h I and 2.5 h I ＋ 4 h R. The protein appeared in the nuclear extracts of the liver following 2.5 h I and 2.5 h I ＋ 4 h R. However, the protein only increased in the cytoplasm of the liver following 2.5 h I, suggesting that the protein translocated from cytoplasm into the nucleus. Conclusions Both Egr-1 mRNA and Egr-1 protein are significantly increased and the binding activity of Egr-1 to its cognate DNA site is enhanced in the liver following I/R, indicating that the Egr-1 transcriptional and translational level is increased. This study provides evidence that Egr-1 gene is activated in the liver during I/R. Egr-1 may be involved in regulating inflammatory response genes and result in liver injury after I/R.