The Relationship Between Cathepsin D Mediated Apoptosis And MLK3after Ischemia-reperfusion Injury

ObjectiveThis experiment aim to establish ischemia reperfusion rats model,and through theintervention of the Cathepsin D inhibitor pepstatin A to detect the change of CathepsinD and pMLK3protein expression, To determine the relationship between Cathepsin Dand MLK3in nerve cells apoptosis after cerebral ischemia-reperfusion injury,offered anew understand about the mechanism of lysosome pathway of apoptosis involved incerebral ischemia-reperfusion injury,and a new targets for the treatment of ischemiccerebrovascular disease.Methods67male Sprague-Dawley rats,(280+20g,10～12weeks old,CLA) wererandomly divided into3groups: sham-operated control group (sham, n=7), modelgroup (M, n=30), Intervention group (I, n=30): inject intraperitoneally lysosomeCathepsin D inhibitor pepstatin A at a dose of2ml/100g before reperfusion andevery12h after reperfusion (whereas the sham-operated control group and modelgroup inject intraperitoneally normal saline at a dose of2ml/100g at the sametime).The model group and intervention group is divided into2hour group (M-2h, n=6; I-2h, n=6),6hours group (M-6h, n=6; I-6h, n=6),24hours group (M-24h, n=6; I-24h, n=6),48hour group (M-48h, n=12; I-48h, n=12). The ratmiddle cerebral artery cerebral ischemia reperfusion model was performed as Longadescribed.restore reperfusion after ischemia2hours.We use Longa ’s of5levelsgrading evaluation to evalueate nerve function defect. We perform TTC staining,TUNEL apoptosis assay and immunohistochemical method to detect cerebralinfarction volume, changes about apoptosis and the expression of Cathepsin D andpMLK3of the cerebral cortex of ischemia side. Results1.The success rate of model:76.14%,the relative cerebral infarction volume ofmodel group48h is37.36±1.44%, and intervention group48h is27.49±2.30%2.Sham-operated control group was not observed infarction, whereas modelgroup and intervention group were observed white infarction in cerebral cortex ofischemia side.but the relative infarction volume of the intervention group48h wasdecrease d compared with the model group (p=0.000), and the neurological defectscore of the intervention group was also significantly improved (p <0.05).3.The cerebral schemia cortex of rats, few apoptotic cells were detected iin thesham-operated control group.In the model group, TUNEL-positive cells can beobserved2h group, and were gradually increased at6,24,48hours of reperfusion(p <0.001).In the intervention group, the TUNEL-positive cells of2h group has nosignificant change compared withthe model group (p=0.127), and the inhibitor cansigificantly reduced the TUNEL-positive cells compared with model group at6,24,48hours.(p=0.000).4.The cerebral schemia cortex of rats, low level expression of Cathepsin D wasdetected in the sham-operated control group. In the model group, the expression ofCathepsin D increased gradually at2,6,24hours of reperfusion,and peak at24h,thendeclined at48h after reperfusion,but higher than thesham-operated control group (p=0.000). The expression of Cathepsin D of intervention group at each time point weresignificantly decreased compared with tne model group24h (p=0.000).5.The cerebral schemia cortex of rats,the sham-operated control group candetected a little expression of pMLK3.The expression of pMLK3increasedgradually at2,6hours of reperfusion,reached peak at6h and declined gradually at24,48h, but higher than thesham-operated control group (p=0.000); InInterventiongroup,the expression of p MLK3at each time point were sigificantly reducedcompared with model group (p <0.05).Conclusions1.Cathepsin D may mediate apoptosis through up-regulate MLK3 phosphorylation after cerebral ischemia-reperfusion injury in rat.2. The inhibition of the Cathepsin D expression by Pepstatin A,maydown-regulate MLK3phosphorylation,reduce cell apoptosis and cerebral infarctionvolume, and act the role of neural protection.