The Relationship Between Cathepsin B Mediated Apoptosis And Caspase-9-Caspase-3 Signaling Pathway After Cerebral Ischemia And Reperfusion Injury In Rats

Background and ObjectiveThe death mode of ischemic penumbra after cerebral ischemia reperfusion injury is apoptosis.Cathepsin B(Cathepsin B),Caspase9 and Caspase3 proteins in the Caspase family proteins play an important role,but there are a few researches about them.In this study,we established a rat model of middle cerebral artery ischemia reperfusion injury.Used cathepsin B and caspase-8 specific inhibitors to intervene,and to further detect the three kinds of proteins expression levels in different time points after cerebral ischemia reperfusion,in order to analyze the possible correlation between Cathepsin B and Caspase-9-Caspase-3apoptotic signaling pathway in rats with cerebral ischemia-reperfusion injury.Using the electron microscopy to oberve the ultrastructural changes of lysosomes in each phase before and after the intervention of CA-074 ME and Z-LEHD-FMK,to further explore the relationship between the signal pathway and lysosome from microscopic aspect.MethodsThe MCAO model was prepared by 262 healthy male SD(Sprague-Dawley)rats,aged 10~12 weeks,weighing about 240~280g.Using a completely randomized design,the rats were randomly divided into four groups: the rats were divided into ischemia-reperfusion group(model group 82)and Sham operation group(Sham group 16),CA-074 Me inhibitor intervention group(CBI group 82),Z-LEHD-FMK inhibitor intervention group(C9I group 82).Except the Sham group,the other three groups of rats after reperfusion 0min,15 min,30min,2hr,6hr,12 hr and 24 hr are divided into 7 subgroups(except 24 hr time point of 16 rats,the rest of each time point for the subgroup of 11).The Sham operation group did not need to insert the thread.The rat model of middle cerebral artery ischemia reperfusion injury(middle cerebral artery,MCAO)was made by modified longa suture method,CBI group was injected CA-074ME(2ug/ul × 5ul)into the right lateral ventricle 60 minprior to surgery,C9 I group was injected Z-LEHD-FMK(0.96ug/ul ×5ul)into the right lateral ventricle 30 min prior to surgery,Sham operation group and model group at the same time points and was injected with the same dose phase to obtain DMSO solution.In this experiment,animals were sacrificed after reperfusion 0min,15 min,30min,2h,6h,12 h and 24 h,neural function defect assessment method in rats at different time points by Longa ’s 5standard score;Taking 5 rats of 24 h after ischemia reperfusion to detected with TTC method and observe the cerebral infarction volume in rats;Using TUNEL method to detect the nerve cells apoptosis numbers ofdifferent groupsin the 7 timepoints subgroups;Western Blotting assay to detect the expression level of Cathepsin B 、 Caspase-3 and Caspase-9 protein at different time pointsin the penumbra zone.Using electron microscopy to observe the ultrastructural changes of lysosome in each phase before and after the intervention of specific inhibitors in each pathway.Results1.The success rate of modeling was 88.5%.There were no neurological deficits symptoms in Sham group.IRI group,CBI group,C9 I group rats showed neurological deficit symptoms.Compared with IRI group,the neurological deficit score in CBI group and C9 I group was lower.(P < 0.05).2.TTC staining method found there was no obvious infarction in Sham group.IRI group,CBI group and C9 I group visible white infarcts.but the CBI group and the C9 I group compared with IRI group,the neurological symptoms and signs were significantly improved,the difference was statistically significant.3.TUNEL method detected there were no obvious apoptotic cells in Sham group.IRI group at 0min、15min、30min、2h,6h,12 h,24h can be seen apoptotic cells,and the positive rate of apoptotic cells increased with the extension of the time point of ischemia reperfusion.CBI group,C9 I group at the corresponding time points can also be seen apoptotic cells and the positive rate of apoptotic cells in two groups was significantly lower than that in IRI group at the same time point.(P<0.05).4.Western blotting methodobserved the expression ofcathepsin B,caspase9,caspase3 proteins.Sham group showed a small amount of the three kinds of proteins.The expressionlevel of the threekinds of proteinsin IRI group was higher,and compared with the Sham group IRI group had statistically significant difference.Detected cathepsin B proteins expression in the IRI group,it was upward at 0min,reached peak at 15 min,it was decreased at 30 min,2h,at 6h there is another expression peak,then began to declined at 12 h,decreased significantly at 24 h.but there is still a high expression in 24 hours.The expression trend of Cathepsin B in the C9 I group was consistent with that in the IRI group.There was no significant change in the C9 I group at the same time points compared with the IRI group,the difference was not statistically significant(P>0.05).The peak of cathepsinB protein expression in CBI group is at 15 min,and then showed a downward trendat 30 min,2h,6h,12 h,24h,the expression level in all the same time point was significantly reduced compared with model group(P < 0.05),the difference was statistically significant.IRI group,caspase 9 protein expression was upward at 0min,reached peak at 15 min,showed a downward trend at 30 min,2h,6h,12 h,24h.The expression trend of CBI group and C9 I group was consistent with the IRI group,but the expression of two intervention groups in all the same time point in C-9 group was obvious reduced,compared with the IRI group,and the difference was statistically significant(P<0.05).The expression of caspase3 protein in IRI group upward at 0min,increased at 30 min,2h,the expression peak was at 6h,then showed a downward trend at 12 h,24h.Caspase 3proteinexpression trend in CBI group and C9 I group were consistent with IRIgroup,and the protein expression of both group were significantly declined compared with IRI group(P<0.05).5.Electron microscopic observation showed that the volume of lysosomes increased in the ischemic penumbra,and the number of secondary lysosomes increased.Neuronal apoptosis were appeared in the late stage of cerebral ischemia reperfusion injury.Compared with IRI group,the cell structure is clearer in CBI group and C9 I group at the same time.Conclusions1.After the cerebral ischemia-reperfusion injuryin rats,Cathepsin B,Caspase-9 and Caspase-3 protein expression may be interrelated in cerebral ischemic penumbra.Cathepsin B may mediate apoptosis via Caspase-9-Caspase-3endogenous signaling pathway.2.In the cerebral ischemia-reperfusion injury of rats,CA-074 ME may reduces apoptosis and infarct volume by inhibiting Cathepsin B;Z-LEHD-FMK may inhibit the apoptosis cascade of Caspase-9-Caspase-3 by inhibiting Caspase-9,exerting neuroprotective effect and reducing apoptosis.