TFP5 protects MPP+ induced PC12 cell apoptosis by specifically inhibiting cyclin-dependent kinase 5/p25activity

Objective To determine whether the apoptosis of PC12 cells induced by MPP+ can be protected when the activity of cyclin-dependent kinase 5(CDK5)/p25 is inhibited specifically by TFP5. Methods The 100 μg/L of beta nerve growth factor (β-NGF) was used to induce PCI2 cells differentiating into dopaminergic neurons in vitro. Different concentrations of MPP+ (0, 100, 200, 300, 400, 600, 800 and 1000 μmol/L) were added to the cells; CCK8 assay was used to determine the cell activities and adequate concentration of MPP+. After induction, four groups were designed: PBS and PBS group, MPP+ and PBS group, MPP+ and TFP5 group, and MPP+ and Roscovitine group. Pretreatment of TFP5 and Roscovitne for 12 h was given to the MPP+ and TFP5 group and MPP+ and Roscovitine group, respectively. Hochest33258 staining and flow cytometry were used to detect the cell apoptosis. Western blotting was used to detect the protein expressions of p35/25, caspase3, cleaved caspase3. Results CCK8 assay showed that the survival rate of PC12 cells was (64.84±1.58)% when the MPP+ concentration was 300 μmol/L. Flow cytometry indicated significant differences in the apoptosis rate between different groups, which was the highest in MPP+ and PBS group ([25.61±2.74]%), following by MPP+ and TFP5 group ([13.33±1.24]%), MPP+ and Roscovitine group ([9.94±1.70]%), and PBS and PBS group ([8.68±0.21]%); significant difference was noted between MPP+ and TFP5 group and MPP+ and Roscovitine group (P<0.05). Hochest33258 staining indicated the most obvious nucleus condensation and fragmentation and more apoptotic bodies in MPP+ and PBS group, While few apoptotic bodies were found in MPP+ and TFP5 group and MPP+ and Roscovitine group. Western blotting showed that as compared with that in the PBS and PBS group, the p25 protein level in the MPP+ and PBS group, MPP+ and TFP5 group, and MPP+ and Roscovitine group was significantly increased (P<0.05). The cleaved caspase-3 protein expression in the MPP+ and PBS group was significantly higher than that in the PBS and PBS group (P<0.05); the cleaved caspase-3 protein expression in the MPP+ and PBS group was significantly higher than that in the MPP+ and TFP5 group (P<0.05). Conclusion TFP5 has protective effect against the apoptosis of PC12 cells induced by MPP+ through inhibiting the CDK5/p25 expression and reducing the cleaved caspase-3 protein production. Key words: Parkinson's disease; TFP5; Cyclin-dependent kinase 5; Caspase-3