牛磺酸预处理抑制IRAK-4信号通路对大鼠移植肝脏再灌注损伤的保护机制

Protective mechanisms of IRAK-4 inhibited by taurine preconditioning in liver graft ischemia/reperfusion injury in rats

目的观察牛磺酸预处理后SD大鼠移植肝脏的白细胞介素-1受体相关激酶-4(interleukin-1 receptor associated kinase-4,IRAK-4)表达水平的变化,探讨牛磺酸预处理抑制肝脏缺血再灌注损害的相关机制。方法雄性SD大鼠128只,完全随机化分为生理盐水对照组(NS组)和牛磺酸预处理组(TA组),每组64只,用Kamada’s袖套法经行肝移植。NS组切取供肝前10 min经腰静脉注射生理盐水1.5 ml,TA组切取供肝前10 min经腰静脉注射牛磺酸(300 mmol/L)1.5 ml。于再灌注后0、60 min及180 min,蛋白免疫印记法及实时荧光定量PCR测定肝组织中IRAK-4 mRNA和蛋白表达水平;凝胶电泳迁移率分析法(EMSA)检测肝组织NF-κB活性及血清TNF-α含量;全自动血清自动生物化学仪检测血清转氨酶;光镜切片检测肝脏组织学。结果牛磺酸预处理能明显提高大鼠1周生存率(P<0.01),改善肝功能,减轻肝组织病理学改变。再灌注后0 min,血清转氨酶、IRAK-4 mRNA与蛋白表达水平、NF-κB活性以及TNF-α含量2组间比较差异无统计学意义(P>0.05);NS组各项指标水平于再灌注60 min出现峰值,但TA组各项指标水平明显低于NS组(P<0.01);再灌注后180 min,2组IRAK-4 mRNA与蛋白表达水平、NF-κB活性以及TNF-α含量均较60 min时有所下降,且TA组下降水平较NS组更为明显(P<0.01)。结论牛磺酸对大鼠移植肝脏的缺血再灌注损伤有明显抑制作用,抑制IRAK-4及其下游的NF-κB、TNF-α表达是牛磺酸预处理减轻肝脏缺血再灌注损害的重要机制之一。

Objective To observe the changes in interleukin-1 receptor associated kinase-4 （IRAK-4） in ischemia/reperfusion （I/R） liver preconditioned with taurine and to explore the protective mechanisms of taurine preconditioning against hepatic I/R injury after liver transplantation. Methods Male Sprague-Dawley rats, weighing 180 -220 g, were randomized into two groups in a blind fashion： control, normal saline group （ NS group）, and taurine preconditioning group （ TA group）. After organ harvest, standardized liver transplantation was performed. At 0, 60 min and 180 min after reperfusion, and the liver tissue IRAK-4 gene and protein level were determined by real-time quantitative fluorogenic PCR （FQ-PCR） and Western blotting. The activity of NF-κB in liver tissue was determined by electrophoretic mobility shift assay （EMSA）. The serum tumor necrosis factor-α （TNF-α） level was detected by ELISA. Serum transaminases, liver histology, graft and animal survivals were also investigated. Results Taurine preconditioning greatly enhanced the one-week survival rate （P 〈 0.01 ）, improved graft function, and ameliorated the histopathologic signs of injury. The liver tissue IRAK-4 mRNA expression level, NF-κB binding activity, and TNF-α production in TA group were signifi-cantly lower than those in NS group （P 〈 0.01 ）. Conclusion The findings from our study suggest that taurine can protect hepatic I/R injury after liver transplantation and the protective effects may be via down-regulation of IRAK4 and its downstream NF-κB and TNF-α expression.