Neuropeptide P5 Plays A Protective Role In Neonatal Rats By Inhibiting Cdk5 Activity After Hypoxia/Ischemia Injury

Objective: To explore the neuroprotective mechanisms of p5, a mimetic peptide of p35, on hypoxic/ischemic(HI) injury in neonatal rats.Methods: The 7-day-old Sprague-Dawle(SD) rats were divided into sham group, and hypoxic/ischemic(HI) injury group. The rats were anesthetized and sacrificed at different time points after HI injury. The expression levels of Cdk5, p35, p39, p25 were detected by Western blot analysis. Seven-days old rats were randomly assigned into four groups:sham+NS group, sham + p5-TAT group, HI+NS group and HI+ p5-TAT group for setting up the hypoxic/ischemic(HI) injury neonatal model. Pre-treatment with p5(p5-TAT) 50μg/kg or same volume of normal saline(intraperitoneal injection) 1 h before HI injury.Cerebral infarction volume was measured. The expression levels of Cdk5, p35, p25, p39,p-Tau, and p-GR 8 h after HI injury were detected by Western blot analysis.Immunoprecipitation was used to investigate the interaction of biotinylated p5 with Cdk5,p35, p39, p25 in brain tissues.Results: Infarct volumes of the rats were increased after HI injury. At different time points(0, 4, 8, 12, and 24 h) after HI injury, Cdk5 expression levels were not changed, but the levels of p-GR and p-Tau, two markers of Cdk5 activity, were significantly increased at8 h after HI injury. Meanwhile p35 and p39 level decreased at 0 h and continued to decrease at 4 h and 8 h, but recovered at 12 h and 24 h compared with sham group after HI injury. Contrary to p35, p25 was hardly detected in sham group, but significantly increased at 0 h and reached a peak at 8 h, then reduced at 12 h and restored to a low level at 24 h after HI injury. Our findings provided the evidences that p5 pre-treatment significantly reduced infarct volume. Contrasted with sham group, p35 was significantly reduced and p25 was increased obviously after HI injury, whereas p5 treatment did not affect the expression of p35 and p25. Similarly, p5 had no influence on p39 level after HI injury. We explored the role of p5-TAT, which is facilitated to cross the cellular membrane by a TAT peptide, on the levels of p35, p25, and p39, the results were consistent with those of p5. HIinjury caused robust phosphorylation of Tau and GR compared with sham group; however,p5-TAT treatment decreased the levels of p-tau and p-GR. After immunoprecipitation with biotinylated p5, the proteins p35, p25, p39, and Cdk5 were detected; however, only Cdk5 was found to interact with the peptide p5. At the same time, all these proteins had no interactions with the negative control peptide p0.Conclusion: The results indicated that Cdk5 activity, as indicated by p-tau and p-GR,was significantly increased Cdk5 activators p35, p39, and p25 were altered after HI injury.P5, a mimetic peptide of p35, was proved to interact with Cdk5. Although p5 did not affect the expressing of p35 and p39, it inhibited Cdk5 activity. Our findings indicated that targeting Cdk5 activity could be a novel therapeutic strategy after HI injury.