The changes of Bcl-xL/Bcl-2 associated death promoter in ischemia-reperfusion injury of rabbit spinal cord and its significance

English Keywords:Ischemic-reperfusion injury  Spinal cord  Bcl-xL/Bcl-2 associated death promoter  c-Jun N-terminal kinase

English Abstract:

  【Abstract】 Objective：To observe the changes of Bcl-xL/Bcl-2 associated death promoter（BAD） in reperfusion of ischemic rabbit spinal cord，and discuss its significance.Method：Forty-five white adult New England rabbits were randomly and equally assigned to three groups：sham-operation group（n=5），ischemic reperfusion group（20 rabbits were randomly assigned to four subgroups），and c-Jun N-terminal kinase（JNK） inhibitor group（20 rabbits was randomly assigned to four subgroups）.In sham-operation group and ischemia reperfusion group 25% dimethyl sulfoxide（DMSO） in PBS was intrathecally injected.SP600125（1.0mg/kg） in 25% DMSO in PBS was also intrathecally injected in the JNK inhibitor group 2h prior to the ischemic injury.In sham-operation group，peritoneotomy was performed without abdominal aortic cross-clamping（AACC），however，ischemic reperfusion group and JNK inhibitor group experienced 30min AACC followed by 30min，2h，8h，24h reperfusion respectively.At the end of the experiment，the lumbar spine，from L4 to sacrum，was harvested.Changes in spinal cord were observed through electron microscopy，the level of p-JNK，JNK and BAD，p-BAD and cytochrome C was detected by Westernblot；the expression of p-BAD，14-3-3，BAD，Bcl-xL and Bcl-2 were determined by coimmunoprecipitation analysis.Result：In group A and 0.5h of reperfusion in group C，no apoptotic cell was noted.At 0.5h of reperfusion in group B，2h and 8h of reperfusion in group C，apoptotic cells were visualized occasionally.At 2h，8h and 24h of reperfusion in group B，24h of reperfusion in group C，numerous apoptotic cells could be detected.The percentage of apoptotic cell in the JNK inhibitor group was lower than that of the ischemic reperfusion group among groups at the same time point（P<0.05）.The expression level of BAD，JNK and 14-3-3 were the same in all three groups.The expressions of p-JNK，p-BAD，cytochrome C，Bcl-XL and Bcl-2 did not change significantly in group A，0.5h of reperfusion in group B，0.5h，2h and 8h of reperfusion in group C.The expression level of p-JNK，cytochrome C，Bcl-XL and Bcl-2 increased at 2h，8h and 24h of reperfusion in group B（P<0.05），which increased with the increase of reperfusion time（P<0.05），simultaneously the amount increased at 24h of reperfusion in group C compared with group A（P<0.05）.The level of p-BAD decreased at 2h，8h and 24h of reperfusion in group B（P<0.05），which decreased with the decrease of reperfusion time（P<0.05），simultaneously the amount decreased at 24h of reperfusion in group C compared with group A（P<0.05）.Conclusion：The BAD is activated when induced reperfusion of ischemic spinal cord，which will lead to neurocyte apoptosis.JNK inhibitor can inhibit neurocytes apoptosis by inhibition of the activity of BAD.