Effect Of Butylphthalide On The Expression Of XIAP And BNIP3 In Model Of Focal Cerebral Ischemia Referfusion Rats

Objectives According to observe the effect of dl-3N-butylphthalide(NBP) post processing of different dozes on the expression of XIAP,BNIP3 in the neuron cells of rats of surrounding infarct locus cortex area and hippocampus CA1 in focal cerebral IR(ischemia reperfusion),and then to explore the brain protection of NBP,and to observe whether expression of XIAP, BNIP3 and NBP have dose-dependent relationship.Methods 160 clean male SD rats were split to five groups:Sham group(Sham,n=16),middle cerebral artery occlusion group(MCAO, n=36),NBP group of doses of 2mg/kg(n=36) randomly,NBP group of doses of 4mg/kg(n=36),NBP group of doses of6mg/kg(n=36).The later four groups were further divided into five time points according to reperfusion 6h,12 h,24h,48 h and 72 h after ischemia 2h.Line plug method as the right middle cerebral artery ischemia reperfusion rats model,and NBP groups with the method of the intraperitoneal injection of drugs immediately after ischemia 2 hour.In each group we select five rats to TTC staining after reperfusion 24 hour.In MCAO group and NBP groups at each time point we take five rats to HE staining, TUNEL staining and immunohistochemical staining.Compare with groups of rats nerve function defect scale,cerebral infarction volume,structural change in the neurons and XIAP and BNIP3 positive cells.According to the immunohistochemical results,it is concluded that XIAP and BNIP3 positive cells expression peak time,and further compare XIAP,BNIP3 m RNA expression in peak time of NBP different doses.Results 1 Nerve function defect scale result Rats in Sham group moved freely.With the extension of reperfusion time,nerve function defect scale showed a trend of first drop later and then reduced in MCAO group and NBP groups;NBP group of adjacent time point defects of rats score comparison was less than MCAO group,the difference was significant(P<0.05).With NBP dose increased,scale reduced,and the difference was significant(P<0.05).2 TTC staining result Sham group didnot see ischemia brain tissue.MCAO group had the most evident infarcts compared with NBP groups;compared low group with NBP middle and high dose group,smaller infarcts in turn, and the difference was statistically significant(P<0.05).3 HE staining result Sham group didnot see the pathological histology change.Then in the cortex area surrounding infarct locus and hippocampus CA1 we observed the changes of MCAO group to the disorder of fuzzy cellular structure,morphological abnormalities,dissolved of pyramidal cells,the nucleus pycnosis,structure is not clear,to late to vacuolate,nuclear fragmentation,deep dyeing and interstitial cells obviously loose,NBP groups over time showed similar trends with the MCAO group.But in the same point,neurons structure damage less in NBP groups than MCAO group,with neuron structure damage the slightest in NBP high dose group.4TUNEL staining result Sham group accidentally see apoptosis positive neurons expression. Compared with Sham group,MCAO group and NBP groups of apoptosis positive cells increased obviously,the difference was significant(P<0.05).Positive cells expression in MCAO group showed rose later lower,then the largest number in reperfusion 24 h,NBP groups have the expression of similar trend.The same point in time,apoptotic cells number of NBP groups decreased than MCAO group,the difference was significant(P<0.05);the difference was existed compared with NBP groups(P<0.05).5 Immunohistochemical result Sham group accidentally see XIAP and BNIP3 positive neurons expression.Compared with Sham group,in MCAO group and NBP groups expression of XIAP and BNIP3 positive cells increased,the difference was significant(P<0.05).The extension of reperfusion time,in MCAO group XIAP positive cells first increased and then decreased, at reperfusion 12 h XIAP positive cells were the most,NBP groups have similar trends; in the same point in time,XIAP positive cells in NBP groups increased compared with MCAO group,the difference was existed(P<0.05),with positive cells to increase the most significant in NBP high dose group.With the extension of reperfusion time,in MCAO group BNIP3 positive cells first decreased and then increased,at reperfusion 24 h BNIP3 positive cells were the most,NBP groups have similar trends;the same point in time,BNIP3 positive cells in NBP groups decreased compared with MCAO group,with positive cells to increase the most significant in NBP high dose group,the difference was existed(P<0.05).6 PCR result According to immunohistochemical result,XIAP positive cells expressing peak time is at 12 h, in this time XIAPm RNA express result comparison:Compared with Sham group, XIAPm RNA expression of the focal ischemic cortex and hippocampal area in MCAO group and NBP groups increased(P<0.05);compared with MCAO group, XIAP m RNA expression increased in NBP groups.With the increase of NBP dose,XIAPm RNA expression has a tendency to increase,the difference was existed(P<0.05).BNIP3 positive cells expressing peak time is at 24 h,in this time BNIP3 m RNA express result comparison:Compared with Sham group, BNIP3 m RNA expression of the focal ischemic cortex and hippocampal area in MCAO group and NBP groups increased(P<0.05);compared with MCAO group,BNIP3 m RNA expression quantity in NBP each dose group reduced.With the increase of NBP dose,BNIP3 m RNA expression has a tendency to reduce,there is still a statistically difference(P<0.05).Conclusions 1 NBP post processing have certain brain protection in focal cerebral ischemia reperfusion rat, the changes were that nerve function defect scale reduced,cerebral infarction volume decreased, and then the pathological tissue damage reduced,the apoptosis of cells reduced.2 NBP in a certain dose range may be by increasing expression of XIAP positive cells and m RNA,decreasing expression of BNIP3 positive cells and m RNA in the cortex area surrounding infarct locus and hippocampus CA1 area of focal cerebral ischemia,to regulate apoptosis to play a role of brain protection.