The Role Of Bad In Ischemia-reperfusion Injury Of Spinal Cord

Objective:To elucidate the role and mechanism of BAD in reperfusion of ischemic spinal cord, and investigate the the neuroprotective effect of JNK inhibitor by inhibition of the activity of BAD.Methods:Forty-five white adult New England rabbits were randomly assigned to one of the three groups: Group I: sham-operation group(n = 5), Group II: Ischemic reperfusion group: 25% DMSO in PBS was intrathecally injected 2 h prior to the ischemic injury, (ischemia 30 minutes/reperfusion 30 minutes group( group A, n=5) , ischemia 30 minutes/reperfusion 2 hour group( group B, n=5) and ischemia 30 minutes/reperfusion 8 hours group ( group C, n=5), ischemia 30 minutes/reperfusion 24 hours group ( group D, n=5)),and Group III :JNK inhibitor group:SP600125(1.0 mg/kg) in 25% DMSO in PBS was intrathecally injected in the JNK inhibitor group 2 h prior to the ischemic injury,Specific groups are exactly the same as Group II. Changes in spinal cord morphology were observed by Hematoxylinand eosin (HE) stain and Electron Microscopy; mitochondrial morphology were observed by Electron Microscopy; the activation of p- JNK, JNK and BAD, p-BAD,cytochrome C,Bcl-xL,Bcl-2,14-3-3 was detected by Westernblot; the interactions between 14-3-3 and BAD, BAD and Bcl-XL or Bcl-2 were performed by coimmunoprecipitation analysis; The localization of 14-3-3 and p-BAD were analyzed by immunohistochemistry.Results:ⅰ)For HE stain, hemorrhagic focuses were found and the neuronal cells were swollen in group B, C, D of ischemic reperfusion group and in group D of JNK inhibitor group.ⅱ) Electron microscopic examination of spinal cord revealed demyelination, coarse chromatin condensation, and breakdown of the nucleus into discrete fragments in group B, C, D of ischemic reperfusion group and in group D of JNK inhibitor group.ⅲ) Electron microscopic examination of mitochondria shows that the cristae appeared dense, and the boundaries of mitochondria were irregular in group B, C, D of Ischemic reperfusion group and in group D of JNK inhibitor group.ⅳ) In ischemic reperfusion group,the expressions of p-JNK and cytochrome C were increased, and enhanced With the extension of time was gradually increasing in group B, C, D of ischemic reperfusion group and in group D of JNK inhibitor group. While the expression of p-BAD was decreased.ⅴ) The dissociation of ASK1 from 14-3-3 and the Bcl-XL/BAD and Bcl-2/BAD dimerization were increased, and subsequently increased in group B, C, D of ischemic reperfusion group. While the decrease was observed in group D of JNK inhibitor group.ⅵ) p-BAD and 14-3-3 were mainly observed in neuronal cells in the sham-operation group. The amount of cytoplasmic staining of p-BAD and 14-3-3 was decreased in group B of ischemic reperfusion group, and subsequently decreased in group C and group D of ischemic reperfusion group. While the decrease was observed in group D of JNK inhibitor group. Conclusion:(1) The activity of BAD is induced in the reperfusion of ischemic spinal cord, which lead to the decrease in BAD/14-3-3 dimerization, the increase in dimerization of BAD with Bcl-XL and Bcl-2, the release of cytochrome c, and the neurocyte apoptosis via the mitochondrion-dependent mechanism.(2) JNK inhibitor could inhibit the apoptosis of neurocytes by inhibition of the activity of BAD.