Experimental Study On Apoptosis-regulatory Genes Expression In Rats Following Focal Cerebral Ischemic Reperfusion And The Effect Of EGb761 On Them

Objective To observe the dynamic changes of XIAP , Smac and Caspase-3P17 protein expression in rats following focal cerebral ischemic reperfusion and the effect of Egb761 action on them.To further explore the pathogenesis of cerebral ischemic reperfusion and the possible mechamism of EGb761 for cerebral protection. Methods Male Wistar rats weighing 250-300g were prepared .MCAO for 1.5h reperfusion models were performed according to Longa's method,which were divided into sham-operation group(groupA) ,MCAO reperfusion group(groupB ),MCAO reperfusion group treated with 50mg/kg of EGb761 (groupC) and MCAO reperfusion group treated with 100mg/kg of EGb761 (groupD). EGb761 were given to rats in the latter two groups intraperitoneally 1h before operation respectively,qd . Each group were divided into 6h,12h,24h,48h and 72h subgroups respectively . Except ten rats were prepared at 24h after cerebral ischemic reperfusion(five rats were used to measure infarct volumes), the other subgroups each had five rats .we observed tissue pathological changes by Hematoxylin-Eosin (H.E) staining and the neuronal survial by Nissel body staining. Infarct volumes were assessed by TTC staining 24h post-reperfusion and the expression of XIAP , Smac and Caspase-3P17 protein was performed by immunohistochemical procedures. Results There were minimal XIAP and Smac immune positive cells in the rat brains of sham-operation group and the controlateral non-ischemic hemisphere in MCAO reperfusion group,but no Caspase-3P17 appearred.The three genes expression elevated after focal cerebral ischemic reperfusion, located in ischemic penumba. Compared with sham-operation group, XIAP expression increased at 6h post-reperfusion(P=0.000),peaking at 12h timepoint. Then XIAP expression declined and became normalized at 48 h post-reperfusion(p=0.649). There was an increase in Smac expression at 6h post-reperfusion(p=0.002), and peaked at 24h after ischmic reperfusion.Smac expression decreased afterwards and return to normal at 72h after reperfusion(P=0.505). Caspase-3P17 protein incresed slightly at 6h post-reperfusion and reached its peak at 24h timepoint. There was much expression till 72h timepoint of reperfusion,compared with that of 6h post reperfusion, the difference was statistically significant(P=0.000). When different doses of EGb761 were injected intraperitoneally to rats, the expression of XIAP protein increased but that of Smac(except 72h timepoint) and Caspase-3P17 declined.The ration between XIAP and Smac increased too.The infarct volumes was reduced and neurological function injury was ameliorated as well. Compared with MCAO group,the differences were statistically significant. Conclusions 1. Focal cerebral ischmia could induce the expression of XIAP and Smac genes, the disturbance between them probably leaded to the activation of Caspase-3, suggesting the apoptotic regulatory genes Smac and XIAP were implicated in the pathophysiological mechanisms of ischemic reperfusion injury. 2. EGb761 conferred neuroprotection against ischemic reperfusion injury by regulating XIAP,Smac signaling pathway and inhibiting Caspase-3 activation, indicating a relationship between dose and effect .EGb761 might be a avaluable neuroprotective agent to ameliorate cerebral dysfunction associated with ischemic reperfusion injury,presumedly reacting upstream of Caspase-3 cascade.