Relationship Between Cathepsin L And P-JNK In The Regulation Of Apoptosis After Cerebral Ischemia-reperfusion Injury In Rats

Objective:The mechanism of cerebral ischemia-reperfusion injury is closely related to apoptosis.Many researchers have confirmed that Cathepsin L and p-JNK plays an important role on apoptosis. However, whether there are some connection and some correlation between the cathepsin L and p-JNK,there was no reported.The purpose of this study was to investigate the correlation between Cathepsin L and p-JNK in the rat after cerebral ischemia-reperfusion,in order to provide the potential molecular mechanism for the treatment of ischemic stroke.Methods:In this experiment,the model of middle cerebral artery occl-usion was established Longa method.Clean and healthy male Sprague-D aeley(SD) rats 216(260~300g,10~12weeks old),as the random number table method,are divided into five groups:sham-operated group(n=54), ischemia-reperfusion group(IRI group, n=54), Cathepsin L intervention group(CLI group,n=54),p-JNK intervention group(n=54).These groups a-re segmented into 4 subgroups 2h,6h,12 h,and 24 h,based on different rep- erfusion hours.Sham operated group was inserted into the vascular depth of 8~10mm,whille other steps were consistent with the model group.Inter-vention groups of cathepsin L and p-JNK were injected Z-FY-DMK(20 ug/1ul*5ul) and SP600125(30ug/10ul*10ul) in 30 minutes before ischem-ia into lateral intracerebroventricular.In the sham operation group and m-odel group injected into an equivalent amount of DMSO solution.Tunel method was used to count the apoptotic cell death and western blotting was used to carry out detection of cathepsin L and p-JNK in each of the subgroups(6 rats), respectively.TTC staining method measuring infarct volume in 24 h subgroups(6 rats).Results:1.The success rate of model of the cerebral ischemia-reperf-usion is 83.72%. 2.The neurological deficit score of SP600125 group an-d CLI group less than the model group(P<0.01). 3.The normal group and sham operation group showed no obvious infarction.The relative infarct v-olume of model group 24 h is 28.05±1.04%,SP600125 group is 15.27±1. 28%and CLI group is 14.80±1.64.The model group and intervention grou-p were seen pale infarcts in ischemic cortex(P<0.01). 4.TUNEL staining was used to check apoptotic cells.In model group,the number of apoptotic cells in 2h, 6h, 12 h was increased, 24 h reached the peak, the apoptotic cells in each time point was statistically significant difference compared with the sham operation group(P<0.01).The expression of apoptosis cell in SP600125 group and Z-FY-DMK group at each time point was reduced compared with the model group in same time point of apoptosis cell expression(P<0.01). 5.Blotting Western can detect a small amount of Cathepsin L protein expression in the sham operation group in the cerebral cortex. In model group, the expression of Cathepsin L protein upward at 2h, 6h, 12 h reached the peak, slightly decline at 24 h.There was statistically significant difference in each time point between the model group and the sham group(P<0.01). 6.Blotting Western can detect a small amount of p-JNK protein expression in the sham operation group in the cerebral cortex. In model group, the expression of p-JNK protein upward at 2h, 6h, 12 h, 24 h reached the peak. There was statistically significant difference in each time point between the model group and the sham group(P<0.01).7.In CLI group, the expression of p-JNK and Cathepsin L protein at 2h, 6h, 12 h, 24 h were reduced compared with the model group in same time(P<0.01). 8. In SP600125 group, the expression of p-JNK protein at 2h, 6h, 12 h, 24 h was reduced compared with the model group in same time point(P<0.01).Cathepsin L protein was no statistically difference compared with the model group at 2h, 6h, 12 h, 24h(P>0.05). 9.Spearman’s rank correlation coefficient methodreveals that cathepsin L and p-JNK have a positive correlation in model group(r=0.813,P=0.001).Conclusions:1.Cathepsin L may be located upstream of the p-JNK to regulate jointly apoptosis. 2.The Cathepsin L inhibitors can inhibited Cathepsin L, p-JNK protein expression, reduced cerebral infarction volume and apoptotic cells, improved nerve function score, proved nerve protective effect.