TFP5 protects MPP+ induced PC12 cell apoptosis by specifically inhibiting cyclin-dependent kinase 5/p25activity / 中华神经医学杂志

ABSTRACT

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 ofp35/25,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.