Effect of tacolimus postconditioning on serine-threonine protein kinase signaling pathway via regulating generation of reactive oxygen species after spinal cord ischemia-reperfusion injury

Objective To explore the influence of tacrolimus postconditioning (TP) on serine-threonine protein kinase (Akt) signaling pathway afier spinal cord ischemia-reperfusion injury (SCIRI)and the role of reactive oxygen species (ROS) in the mechanism.Methods The rat model of SCIRI was prepared on the basis of Taira' s method.Eighty male SD rats were randomly divided into 4 groups:sham operation (SO) group,ischemia-reperfusion (IR) group,TP group,and TP plus N-Acetylcysteine (TP +N) group.IR group underwent reperfusion for 20 min after spinal cord ischemia.TP group experienced a single injection of tacrolimus (0.5 mg/kg) through the left common carotid artery right at the onset of reperfusion,with remaining procedures the same as IR group.TP + N group was injected through the left common carotid artery with N-Acetylcysteine (150 mg/kg) 5 min before spinal cord ischemia,with remai-ning procedures the same as TP group.SO group received exclusively femoral artery catheterization.Fluo-rospectrophotometry was employed to detect the level of ROS in injured spinal cord segments,and immuno-blotting was used to determine the expression of phosphorylated Akt (p-Akt) at 15 min after reperfusion.Flow cytometry was applied to assess apoptosis of neural cells at 24 h after reperfusion.Nissl's staining was utilized to observe pathological changes of injured spinal cord segments at 7th day after reperfusion.Re-sults The ROS levels in spinal cord tissue were 49.0 ± 3.5,145.3±11.7,79.0±6.3 and12.8 ± 0.9in SO group,IR group,TP group,and TP + N group at 15 rain after reperfusion,respectively.The ROSlevel in TP group was significantly lower than that in IR group at 15 min after reperfusion (P ＜0.01),and that in TP + N group was significantly lower than the other three groups (P ＜ 0.01).The p-Akt expression in spinal cord tissue was 0.54 ± 0.06,0.34 ± 0.03,0.42 ± 0.07,and 0.28 ± 0.05 in SO group,IR group,TP group,and TP + N group at 15 min after reperfusion respectively.The p-Akt expression was sig-nificantly lower in IR group than in SO group (P ＜ 0.01),higher in TP group than in IR group (P ＜0.05),and lower in TP + N group than in SO group (P ＜0.01) or in TP group (P ＜0.01).The percentages of apoptotic cells in spinal cord tissue were (7.5 ± 1.8) ％,(31.4 ± 5.5) ％,(22.3-± 4.0) ％,and (33.3 ± 3.1) ％ in SO group,IR group,TP group,and TP + N group at 15 min after reperfusion respectively.The percentage of apoptotic cells was significantly higher in IR or TP + N group than in TP group (P ＜0.01),while there was no significant difference between IR group and TP + N group (P ＞0.05).The spinal cord sections revealed that pathological changes could hardly be observed in SO group,and mild in TP group but serious in IR and TP + N groups.Conclusion TP produces protective effect on ischemic spinal cord via positive regulation of Akt signaling pathway,and ROS,acting as a signaling molecule,plays an indispensable role in initiating the mechanism. Key words: Spinal cord ischemia;  Tacrolimus ;  Reactive oxygen species ;  Serine-threonine protein kinase