Investigation Of The Relationship Between Cathepsin D-mediated Apoptosis And ERK1/2Signaling Pathway After Cerebral Ischemia-reperfusion In Rat

ObjectiveThe experiment through establish the rat middle cerebral artery occlusionreperfusion model to detect cerebral infarction volume in different time points aftercerebral ischemia reperfusion and the expression of cathepsin D, p-ERK1/2andapoptosis cascade execution protease caspase3. Using pepstatin A, which is theinhibitor of cathepsin D, to interfere the experiment then observe the cerebralinfarction volume in pepstatin A intervention groups and the change of the expressionof three protein above. The aim of the article is to investigate the relationship betweencathepsin D-mediated apoptosis and ERK1/2signaling pathway after cerebralischemia-reperfusion in rat and the neuroprotective effect of pepstatin A.MethodsSeventy-two healthy adult male Sprague-Dawley rats were randomly dividedinto three groups:sham operation group(n=12), saline control group(n=30) andpepstatin A intervention group(n=30). The saline control group and pepstatin Aintervention group were redivided into ischemia-reperfusion6(n=6),12(n=6),24(n=6),48h subgroups(n=12) according to the intervention time points. A model of middlecerebral artery ischemia-reperfusion was induced by the suture method. Pepstatin Awith at a dose of0.2ml/10g or an equal volume of saline were injectedintraperitoneally at ischemia for2h and reperfusion for0,12,24,36h in the pepstatinA intervention group and the saline control group. At ischemia for2h and reperfusionfor0,6,12,24,48h, Longa’s of five standard scoring method were used to evaluatethe neurological deficit. We chose six rats randomly from the sham operation group,saline control group48h subgroup and pepstatin A intervention group48h subgroupfor infarct volume detection respectively. The remaining six rats were used to detect the expression of caspase-3beside cathpsin D, p-ERK1/2like the remaining groupswith western blot.Results1.The modle success rate is81.82%.2.The behavior evaluation showed that the neurological deficit score was0in thesham operation group. In the pepstatin A intervention group, the neurological deficitscores were significantly lower than those in the saline control group at the sameinvervention time points (all P<0.05).3.TTC staining showed no infarction was observed in the sham operation group.There were significant differences (P=0.000) beween the48h subgroups of the salinecontrol group and pepstatin A intervention group with the relative infarction volumes(26.40±1.45)%,(13.14±1.94)%respectively.4.Western blot detection showed that the expression of cathepsin D in the salinecontrol group was increased at6,12,24h after ischemia-reperfusion, reach the peak at24h, and still at the higher level at48h. There were significantly more than the shamoperation group (all P<0.05). The expression of cathepsin D in the pepstatin Aintervention group were significantly less than the saline control group (all P<0.05).5.The expression of p-ERK1/2in the saline control group was increased at6,12hafter ischemia-reperfusion, reach the peak at12h; it was still at the higher level at24hand declined sharply at48h. There were significantly more than the sham operationgroup (all P<0.05). The expression of p-ERK1/2in the pepstatin A intervention groupwere significantly more than the saline control group (all P<0.05).6.The relatively gray value of caspase3in the sham operation group, the48hsubgroups of the saline control group and pepstatin A intervention group were0.2989±0.077,0.9763±0.384,0.5656±0.110, respectively. There were significantdifferences between eath two groups (all P<0.05).Conclusions1.Cathepsin D may mediate apoptosis through up regulating the expression ofcaspase-3, this effect may be associated with ERK1/2signaling pathways. 2. Pepststin A has a neuroprotective effect (reduce neurological deficit and braininfarction volumes) by inhibiting immunoreactivity of cathepsin D and caspase-3.