The Relationship Between Cathepsin L Mediated Apoptosis And P38 Protein After Cerebral Ischemia And Reperfusion Injury In Rats

Objective:Apoptosis occurs after cerebral ischemia and reperfusion. The lysosomal cathepsin L and p38 protein are both involved in apoptosis, but their relationship is unclear. In this study,we established ischemia-reperfusion injury model in rat and used cathepsin L inhibitor Z-FY-DMK and p38 inhibitor SB203580 intervened, to detect the dynamic expression of cathepsin L and p-p38 protein in ischemic penumbra in respective time points.It was designed to investigate the possible relationship between cathepsin L and p38 protein to provide new idea for the treatment of ischemic stroke.Methods:216 male Sprague-Dawley Rats(260~300g, 10~12 weeks old, clean lever animal) were randomly divided into four groups: sham-operated control group(sham group,n=54), ischemia-reperfusion group(IRI group, n=54), cathepsin L inhibitor intervention group(CLI group, n=54) and p38 inhibitor intervention group(SB group,n=54). Using Longa method to establish brain middle artery ischemia-reperfusion model in rats.Sham group, IRI group, CLI group and SB group were randomly divided into four subgroups. CLI group and SB group were injected intracerebroventricularly Z-FY-DMK(20μg/1μl×5μl)and SB203580(0.1nmol/μl×5μl)respectively preoperative 30 min prior to surgery. Sham group and IRI group were injected intracerebroventricularly the same amount of 10ml/L DMSO at the same time and same position. Using Longa’s five levels grading to mark the neurological impairment.Using TTC staining method to detect the infarct volume at 24 h subgroup. Using TUNEL method to observe the apoptotic cells in the ischemic cortex of rats.Using western blot method to detect the cathepsin L and p-p38 protein expression.Results:1.The successful rate of modeling was 87%.2.TTC staining detected, sham group had no significant brain infarction. IRI group, CLI group and SB group brain infarct volume were 22.89%, 17.09%, 18.37% respectively. Compared with IRI group, CLI group and SB group brain infarct volume decreased(p<0.05).3.TUNEL method observed, sham group had no significant apoptic cells, IRI group at 2h, 6h, 12 h, 24 h can be seen apoptotic cells(nuclei were stained yellow or brown), and with the extension of the time point of apoptotic cells positive rate increased gradually. CLI group, SB group at 2h, 6h, 12 h, 24 h can also be seen apoptotic cells and significantly reduced compared with IRI group(p <0.05).4. Western blot detected the expression of cathepsin L protein. In sham group,there was little visible cathepsin L protein expression.In IRI group, the expression of cathepsin L protein upward at 2h, 6h, reached peak at 12 h, decreased at 24 h. Compared with sham group,the expression of cathepsin L protein was significantly increased(p<0.05).The expressin of cathepsin L protein in CLI group was significantly reduced compared with IRI group(p<0.05). The expressin of cathepsin L protein in SB group and IRI group had no significantly difference( p >0.05).5. Western blot detected the expression of p38 protein.In IRI group, the expression of p38 protein at 2h, 6h, 12 h, 24 h remains a continuous upward trend. Compared with sham group, the expression of p-p38 protein was significantly increased(p<0.05). The expressin of p-p38 protein in CLI group was significantly reduced compared with IRI group(p<0.05). The expressin of p38 protein in SB group and IRI group had no significantly difference(p<0.05).6. Cathepsin L and p-p38 expression was positively correlated respectively in the IRI group and CLI group.In IRI group, r=0.789, p <0.01.In CLI group, r =0.889, p <0.01.Conclusions:1.After cerebral ischemia-reperfusion injury in rats, the expression of cathepsin L and p38 protein may be relevant.2.After cerebral ischemia-reperfusion injury in rats, cathepsin L inhibitor Z-FY-DMK can reduce infarct size and cell apoptosis, which may play an anti-apoptotic role by down regulating the expression of p38 protein.