Cathepsin L Mediated Apoptosis Relationship With Caspase-3 After Cerebral Ischemia And Reperfusion

Background and Objective The model of middle cerebral artery occlusion(referred to as MCAO) of a rat is established in this experiment, and Cathepsin L inhibitor Z-FY-DMK and Caspase-3 inhibitor Z-DEVD-FMK will be use separately, then the volume of cerebral infarction and expression variations of Cathepsin L and Caspase-3 will be tested and analyzed in different time point after CIR. This experiment studies the correlation between Cathepsin L and Caspase-3 in the process of nerve cell apoptosis.Method 162 male Sprague-Dawley Rats(260-300 g, 10-12 weeks old, clean lever animal) were randomly divided into four groups: sham operation group(27), CIRI group(model group, 45), CL intervention group(45), and C-3 intervention group(45). The sham operation group is divided into 4 subgroups according to different reperfusion hours as 6h, 12 h, 24 h,and 48 h, each subgroup has 6 rats while 48 h group has 9 rats. The other three groups are divided into 4 subgroups separately according to different reperfusion hours as 6h, 12 h, 24 h, and 48 h, each subgroup has 10 rats while 48 h groups have 15 rats. Then using Longa suture method to establish focal right brain middle cerebral artery infarction model, the reperfusion will be done when removing suture after 2 hours. The inserted depth of suture for sham operation groups is 10 mm, while remaining steps are the same with other groups. Cathepsin L specific inhibitor Z-FY-DMK CL and Caspase-3 protein inhibitor Z-DEVD-FMK are separately used in CL intervention group and C-3 intervention group by applying the lateral ventricle puncture injection method(5ug, 5ml DMSO MCAO;Z-DEVD-FMK:Preoperative right side of the lateral ventricle of rats injected 30 min 10ul, concentration 0.5ug / 0.5ul, needle retention 10min). Separately reject equal quantity of DMSO solution to sham operation group and model group at the same time and the same part. The TTC staining will be done to 3 rats of sham operation group and 5 rats of model group(with 48 hours reperfusion) and intervention group(with 48 hours reperfusion). Longa’s of 5 levels grading as the evaluation standard. Using TTC staining,TUNEL method and Western blotting method will be applied separately to test the volume of cerebral infarction,the change of nerve cell apoptosis of cerebral cortex in ischemic brain, and the change of expression of protein Cathepsin L andCaspase-3.Result 1.The success rate of model is 88.24%.The relative infarct volume of model group 48 h is 38.25±1.06%.The CL intervention group of 48 h is 38.25±1.06%.The C-3 intervention group of 48 h is 29±1.414%. 2.Infarct is not appeared in sham operation group, while white infarct is appeared at and under cortex of ischemic cell in model group and intervention groups. However, compared to model group, not only the volume of relative cerebral infarction of intervention groups is obviously decreased(P<0.001), but also the symptoms and signs of nerve function defect are obviously improved(P<0.05). 3.In the cortical area of ischemic brain, apoptosis cells of sham operation group are rare, while apoptosis of nerve cells of model group with 6 hours reperfusion are visible, and are gradually increased in the order of 12 hours, 24 hours and 48 hours. At the same time point, the apoptosis cells of CL intervention group and C-3 intervention group(6h, 12 h, 24 h, 48h) are obviously less than model group(p<0.05). 4.In the cortical area of ischemic brain, a small amount of Cathepsin L expression in sham operation group can be detected. For model group, the Cathepsin L expression starts to increase in sub groups with 6 hours reperfusion, reach to a peak in sub groups with 12 hoursand24 hours,remains a high level in sub groups with 48 hours reperfusion.Compared to model group, the Cathepsin L expression of CL intervention group and C-3 intervention group are obviously decreased at all time points(p<0.05). 5.In the cortical area of ischemic brain, a small amount of Caspase-3 expression in sham operation group can be detected. For model group, the Caspase-3 expression starts to increase in sub groups with 6 hours reperfusion, reach to a peak in sub groups with 24 hours reperfusion, and remains a high level in sub groups with 48 hours reperfusion. Compared to model group, the Caspase-3 expression of CL intervention group and C-3 intervention group are obviously decreased at all time points(p<0.05).Conclusion 1.After cerebral Ischemia-reperfusion injury in rat,Cathepsin L mediated cell apoptosis by means of downergulating Caspases pathway, the mutual relationship that may exist with constraints Caspase-3. 2.Z-FY-DMK can inhibit the Cathepsin L signal transduction pathway of apoptosis and reduce cell apoposis,and decrease cerebral infarct volnmu after cerebral ischemia-reperfusion in Rat.