The Relationship Between Cathepsin B-mediated Apoptosis And ROCK-JNK Signaling Pathway After Cerebral Ischemia And Reperfusion Injury In Rat

Background and ObjectiveIschemia-reperfusion induced neuronal damage including necrosis,apoptosis andautophagy,which the apoptosis is an important way of brain cell death and it occursmainly in the marginal zone of Ischemic infarction in tne central area around,alsoknown as ischemic penumbra zone,while reducing neuronal apoptosis in this regionis the key.The broader molecular mechanisms involved in nerve cells apoptosis,lysosomes and ROCK-JNK signaling Pathway also involved,but the exact mechanismremains to be into a research and clarify.The aim of the present study attemps tointervene by cathepsinB specific inhibitor of CA-074in rats,and through detectionand analysis of the expression changes of ROCK Ⅱ and p-MKK7,exploring therelationship cathepsinB with ROCK-JNK signaling pathway in neuronalapoptosis,which provides a new direction of thinking for the prevention and treatmentof ischemic strokeMethodsSixty-seven male Sprague-Dawley(280±20g,10-12weeks old,CLA) wereRandomly divided into ten groups:sham-operated group(Sham,n=7),model group1/2h（M-1/2h,n=6）,model group6h（M-6h,n=6）,model group12h（M-12h,n=6）,modelgroup24h（M-24h,n=12）,Intervention group1/2h（I-1/2h,n=6）,Intervention group6h（I-6h,n=6）,Intervention group12h（I-12h,n=6）and Intervention group24h（I-24h,n=12）,which the sham-operated group take a rat,the model group24h andintervention group24h were taken six rats for TTC staining.Transient middle cerebralartery occlusion was performed as Longa described,and recovery reperfusion after therat cerebral ischemia2h,the sampling time point after reperfusion is divided into 1/2h,6h,12h,24h.The Intervention group was injected with Cathepsin B specificinhibitor CA-074through the lateral ventricle puncture(20ug CA-074dissolved in1ulDMSO at30minutes before MCAO injected into the right lateral ventricle[1]).Therats of other groups were injected with the same volume of DMSO solution at thesame point in time and location.The neurologic deficit scores were evaluated withlonga’s five-point scale standard scoring method.TTC Staining using infarctvolume,HE staining pathological changes,TUNEL method and immunohistochemistrywere used to detect neuronal apoptosis and expressions of ROCKⅡ a nd p-MKK7inthe cortex of cerebral ischemic.Results1.78.82%of the model success rate, Cortical and subcortical visible whiteinfarction were detected in model group and intervention group, the relative infarctvolume of model group24h is27.72±3.54%,but the intervention group24h is12.70±1.37%. The relative infarct volume of the intervention group were significantlyreduced compared with the model group(p<0.001),and the symptions and signs ofneurologic deficit has also been significantly improved(p<0.05).Sham-operated groupwas not observed infarction, and normal neurological function.2.HE staining showed cerebral pathology of24h after reperfusion in eachgroup:the brain tissue slices of sham-operated group of24h is visible for glialcells,nerve cells and capillaries with normal morphology,integrated and clearstructure,the nucleus of pyramidal cell is largr and round,and nucleolus is obvious.Themodel group of24h showed ischemic changes,the structure of normal tissuedisappeared in the infarct area,and the delineation of the nucleus and the cytoplasm isunclear,the volume of pyramidal cell is reduced,nucleus pyknosis, cytoplasm iseosinophilic,partial cells is necrosis,and the necrosis were cribriform,interstitialedema and inflammatory cell infiltration.The ischemic changes compared with themodel group was significantly reduced in intervention group of24h,and the extent ofnecrosis is decreased,complete cell structure,less nucleus pyknosis,and interstitialedema of cells is light. 3.In the ischemia side of cortex region, a few scattered TUNEL-positive cellscan be observed in the model1/2h group,and were gradually increased at6,12,24hours of reperfusion(p<0.001);Cathepsin B specific inhibitor CA-074significantlyreduced apoptotic cells was observed compared with the model group induced byischemia-reperfusion at6,12,24hours(p<0.05),no significant chang at1/2hours(p>0.05); the sham-operated group apoptotic cells were rarely seen in thecorresponding region.4. In the model group,there was a little growth in ROCK Ⅱpositive expression inthe ischemia side of cortex region at the1/2hours of reperfusion,and a significantincrease during the6-hours12-hours and24-hours reperfusion,and a peak at24-hoursafter reperfusion.(p<0.001);Except the1/2h group no significant decrease,theintervention group by CA-074were significantly decreased in the expression ofROCKⅡ compared with the model group at the various time points(p<0.05).In thesham-operated group,no ROCK Ⅱp ositive expression was detected in thecorresponding region.5. In the model group,there was a large numble of growth in p-MKK7positiveexpression in the ischemia side of cortex region at the1/2hours of reperfusion,downto the bottom at6hours,and then gradually increased at12hours,again reached thepeak at24hours after reperfusion,showing two peaks (p<0.05);The interventiongroup p-MKK7positive cells were significantly decreased compared with the modelgroup at24hours (p<0.001),other each time point p-MKK7positive cells weresignificantly reduced compared with the model group(p<0.05). In the sham-operatedgroup,no p-MKK7staining was detected in the corresponding region.ConclusionsCathepsin B may through its specific inhibition of CA-074down ROCK-JNKsignal pathway mediated apoptosis signal transduction, reduce apoptosis and infarctvolume.