兔脊髓缺血再灌注损伤时Bcl-xL/Bcl-2相关死亡启动因子的变化及其意义

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

中文关键词:  缺血再灌注损伤  脊髓  Bcl-xL/Bcl-2相关死亡启动因子  c-Jun氨基末端激酶

英文关键词:Ischemic-reperfusion injury  Spinal cord  Bcl-xL/Bcl-2 associated death promoter  c-Jun N-terminal kinase

中文摘要:

  【摘要】 目的：观察兔脊髓缺血再灌注损伤时Bcl-xL/Bcl-2相关死亡启动因子（Bcl-xL/Bcl-2 associated death promoter，BAD）的变化情况，探讨BAD变化的意义。方法：45只新西兰白兔随机分为假手术组（A组，5只）、缺血再灌注组（B组，20只）和c-Jun氨基末端激酶（c-Jun N-terminal kinase，JNK）抑制组（C组，20只）。手术前2h，A组及B组动物L4硬膜外注射25%二甲基亚砜（DMSO），C组动物注射溶于25%DMSO的JNK抑制剂SP600125。A组动物麻醉后仅腹腔切开，B组及C组采用腹主动脉阻断法（夹闭腹主动脉30min后松开，再灌注至0.5h、2h、8h、24h）建立脊髓缺血再灌注模型。取L4节段以下脊髓组织，电镜观察神经细胞形态学改变，Western blot检测磷酸化JNK（p-JNK）、JNK、磷酸化BAD（p-BAD）、BAD及细胞色素C的表达，免疫共沉淀检测p-BAD、14-3-3、BAD、Bcl-xL及Bcl-2的表达。结果：电镜检查A组及C组再灌注0.5h未见脊髓神经细胞凋亡，B组再灌注0.5h及C组再灌注2h、8h时偶见脊髓神经细胞凋亡，B组再灌注2h、8h、24h及C组再灌注24h时可见部分脊髓神经细胞凋亡，C组神经细胞凋亡率与同时间点B组比较明显降低（P<0．05）。各组JNK、BAD、14-3-3的表达无明显差异。p-JNK、p-BAD、细胞色素C、Bcl-xL和Bcl-2的表达在A组、B组再灌注0.5h和C组再灌注0.5h、2h、8h无明显差异。B组再灌注2h、8h、24h p-JNK、细胞色素C、Bcl-xL、Bcl-2的表达较A组明显增加（P<0．05），且表达强度随着再灌注时间的延长而增强（P<0．05）；C组再灌注24h较A组增加（P<0．05），C组再灌注2h、8h、24h表达量与同时间点B组比较明显降低（P<0．05）。B组再灌注2h、8h、24h时p-BAD较A组明显减少（P<0．05），且表达强度随着再灌注时间的延长而减弱（P<0．05）；C组再灌注24h较A组减少（P<0．05），C组再灌注2h、8h、24h表达量与同时间点B组比较明显增加（P<0．05）。结论：兔脊髓缺血再灌注时，BAD被激活，诱发了脊髓神经细胞的凋亡。JNK抑制剂可抑制BAD的活性，减少缺血再灌注损伤过程中脊髓神经细胞的凋亡。

英文摘要:

  【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.