| Background and Objective The ischemia reperfusion injury model of rat brain was established,different animal models of cerebral infarction volume changes were observed by TTC staining,and the number of apoptotic cells was measured by TUNEL in experimental groups at different time.Western Blot detected expression levels of Cathepsin B、 Caspase-8、Caspase-3 at different time points of protein.Throμgh analyzing the changing rμles of protein expression,judge the changing relationship of apoptotic signaling pathways between Cathepsin B and Caspase-8-Caspase-3.Cathepsin B and caspase-8 specific inhibitors were used to infer the possible relationship between Cathepsin B and Caspase-8-Caspase-3 apoptotic signaling pathways.Before and after each specific inhibitor intervention,observe the signal pathway of key protein during lysosomal changes ofμltrastructure throμgh electron enzyme histocytochemistry method.Further exploration may concentrate on the relationship of apoptotic signaling pathway and lysosomes between the Cathepsin B and Caspase-8-Caspase-3 from the microscopic perspective,and the relationship of apoptotic signaling pathways between Cathepsin B mediated apoptosis and Caspase-8-Caspase-3 in neuronal apoptosis.Method The MCAO model was prepared with 262 healthy male SD(Sprague-Dawley)rats,aged 10~12 weeks,weighing about 240~280g.Adopted with a completely randomized design,the rats were randomly divided into four groups: ischemia-reperfusion group(model group with82 rats),sham operation group(with 16 rats),CA-074 Me group(CB inhibitor intervention group with 82 rats),Z-IETD-FMK inhibitor(C-8intervention group with 82 rats).The above four groups of rats are divided into 7 subgroups after the reperfusion of 0min,15 min,30min,2hr,6hr,12 hr and 24h(16 rats are given only at 24 h time point,while 11 rats are given at the rest of each time point for the subgroup).In the sham operated control group,the other surgical procedures were the same as those of the other three groups,except that the insertion was shorter and the right middle cerebral artery was not blocked.In this study,the right middle cerebral artery focal cerebral infarction model(MCAO)was prepared based on the modified Longa suture method,and the model of reperfusion was established after the cerebral ischemia in 2H rats.Group CBI(cathepsin B inhibitor group)and group C8I(caspase-8 inhibitor) were used in puncture of the lateral ventricle injection of Cathepsin B specific inhibitor CA-074 Me and Caspase-8 inhibitor Z-IETD-FMK protein(CA-074 Me 10 ΜG dissolved in 5μl DMSO,to make MCAO model before the injection of 60 min into the right lateral ventricle in rats,and to prevent the leakage of solution required for 10min;Z-IETD-FMK:0.5μg is dissolved in 5μl DMSO,in the preparation of MCAO model before the injection of 30 min into the right lateral ventricle in rats,the same for 10 min,sham operation group and model group at the same time points and was injected with the same dose phase to obtain DMSO solution,DMSO solution and avoid the damage caused by the error of lateral ventricle puncture.In this experiment,after the reperfusion of0 min,15min,30 min,2h,6h,12 h and 24 h,the animals were killed.Asset the neural function of rats at different time points with defect assessment method by Longa ’s 5 standard score;sham group,IRI group,CBI group and C8 I group were taken 5 24 h after ischemia reperfusion the rats were detected with TTC and observe the cerebral infarction volume of rats;TUNEL method is used to detect the different groups in the 7 time points of subgroups,the number of nerve cells apoptosis;Western Blot assay to detect the expression level of Cathepsin B 、 Caspase-3 and Caspase-8protein at different time points,throμgh the analysis of the protein expression level change law,judgment between the Cathepsin B protein with the signaling pathway.Before and after the intervention,the electron enzyme histochemical method was used to observe the signal pathway of key protein during lysosomal changes of μltrastructure,to further explore the relationship between the various signaling pathways and lysosomes from the microscopic perspective.Resμlt1.MCAO model success rate was 90.03%.2.TTC staining was used to detect infarct focal cerebral ischemia-reperfusion in rats after 24h;the 24 h relative infarct volume of model group,CBI group and C8 I group was 37.54 ± 0.86%、23.10 ±1.01%、29.8 ± 0.94% separately.Sham group had no obvious white infarct;white ischemic infarction of IRI group,CBI group,C8 I group were lower than those of Sham group increased significantly,and the infarct volume of CBI group and C8 I group decreased significantly(P <0.05)compared to the IRI group.The difference was statistically significant.The MCAO model of neural function defection score resμlts and the volume of cerebral infarction in rats,IRI group,CBI group,C8 I group showed the symptoms of neurological deficit,but the CBI group and the C8 I group compared with IRI group,the neurological symptoms and signs were significantly improved,and the difference was statistically significant.3.Using the TUNEL reperfusion to detect the number of apoptotic cells in cerebral cortex after cerebral ischemia injury,only a handfμl of apoptotic cells is seen in group Sham.The level of poptotic cells in IRI group was significantly increased compared to that in Sham group.At0 min apoptotic,cells began to appear.With the extension of time,the number of apoptosis gradually increased,and the number of apoptotic cells in 24 h group and CBI is the highest.In C8 I group and IRI group,the number of apoptotic cells at 0min,15 min,30min,2h,6h,12 h,24h time point showed a rising trend,but the number of apoptotic cells in two inhibitor group and IRI group at the same time point significantly decreased(P < 0.05).The difference was statistically significant.4.Western blotting method was used to detect the expression and the expression trend of cathepsin B,Caspase-8,caspase-3 three proteins in the brain of cerebral ischemia.The three proteins can only be detected very small amounts of expression.In the sham group,IRI group,the three proteins’ expression level was higher,and the sham group had statistically significant difference.Cathepsin B was detected in the IRI group from0 min to 15 min,after a small peak expression level decreased,the expression of 2h and 6h increased gradually and reached the peak,then decreased,but there is still a high expression level in 24 hours.The expression trend of Cathepsin B in the C8 I group was consistent with that in the IRI group.There was no significant change in the C8 I group at the same time points compared with the IRI group.The difference was not statistically significant.The peak appeared in the 15 min,and the expression of cathepsin B protein CBI inhibits the expression of 24 h in the group,and after each time point it decreased.The expression level at all the same time points was significantly reduced compared with model group(P < 0.05),and the difference was statistically significant.In group IRI,at 0min we can detect that the expression of caspase-8 was increasing gradually,then reached the peak at 30 min,and then the expression decreased gradually,but after 24 hours it is still at the relatively high level expression of protein.CBI group and C8 I group have the same expression trend with the IRI group,but the expression of caspase-8 in the two intervention groups at all the same time points in group IRI decreased compared to the obvious one,and the difference was statistically significant(P < 0.05).The expression of Caspase-3 in IRI group was increasing from 0min,and the protein expression was gradually increasing,and at 6h time point it reached the peak,then it began to decline,and there was still a high expression in 24 hours.The expression trend of caspase-3 in the CBI group and C8 I group is consistent with the IRI group,and caspase-3 expression level at the same time of each agent in the C8 I group and CBI group was significantly reduced(P < 0.05)compared with that of IRI group,and the difference was statistically significant.5 Observe the changes in microstructure using electron microscope in rats before ischemia lysosomal side.In group sham,few TUNEL positive cells can be seen.In IRI group,with the extension of time,the number of lysosomes and secondary lysosomes were also gradually increasing,and the cells gradually showed the phenomenon of apoptosisthe,dense chromatin condensation and margination,pyknosis,and cell rupture etc.Group CBI and group C8 I were similar to those of IRI group,but compared with IRI,the phenomenon of apoptosis was reduced at the same time points in the two groups,and the number of primary lysosomes and secondary lysosomes was also significantly reduced.Conclusion1 In rats with cerebral ischemia reperfusion injury model,Cell apoptosis is mediated by Cathepsin B throμgh caspase-8-caspase-3 signal pathway.2 Inhibitor CA-074 Me can inhibit the expression of Cathepsin B,thus inhibit a series of mediated apoptosis reaction,reduce the ischemia-reperfusion injury caused by nerve cell apoptosis,reduce the infarct volume,and exert its neuroprotective effects.3 Inhibitor I-IETD-FMK can inhibit the expression of caspase-8,inhibit the mediated apoptosis of a series of protein hydrolysis reaction,reduce the ischemia reperfusion injury of nerve cell apoptosis caused by ischemia,reduce infarct volume,and exert its neuroprotective effects. |
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