TFP5通过特异性抑制CDK5／p25活性抑制MPP＋诱导的PCI2细胞凋亡

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

目的探讨TFP5（p35的一个裂解片段）特异性抑制细胞周期蛋白依赖蛋白激酶5（CDK5）/p25活力后在1-甲基-4-苯基吡啶（MPP＋）诱导的PC12细胞凋亡过程中的保护作用及其机制。方法使用100 μg/L β神经生长因子将体外常规培养的PCI2细胞诱导分化为多巴胺能神经元，加入不同浓度MPP＋（0、100、200、300、400、600、800、1000 μmol/L）处理，应用CCK8法检测细胞活力，以明确MPP＋诱导PC12细胞凋亡的合适浓度。将诱导分化后的PC12细胞分为4组：PBS＋PBS组、MPP＋＋PBS组、MPP＋＋TFP5组和MPP＋＋CDK5抑制剂Roscovitine组；TFP5与Roscovitne提前12 h加入培养基中预处理（终浓度分别为100 nmol/L、10 nmol/L）。分别应用流式细胞仪及Hoechst33258染色检测细胞凋亡情况，应用Western blotting检测细胞中p35/p25、半胱氨酸蛋白酶-3、裂解半胱氨酸蛋白酶-3蛋白表达水平。结果CCK8法检测结果显示：MPP＋浓度为300 μmol/L时，PC12细胞的存活率为（64.84±1.58）%。流式细胞仪检测结果显示：MPP＋＋PBS组细胞凋亡率[（25.61±2.74）%]最高，MPP＋＋TFP5组细胞凋亡率[（13.33±1.24）%]较低，比较差异有统计学意义（P〈0.05）。Hoechst33258染色结果显示：MPP＋＋PBS组细胞核固缩及碎裂最明显，较多凋亡小体形成，而MPP＋＋TFP5组与MPP＋＋Roscovitine组仅有少量凋亡小体形成。Western blotting检测结果显示：与PBS＋PBS组比较，MPP＋＋PBS组、MPP＋＋TFP5组、MPP＋＋Roscovitine组p25蛋白表达水平均明显增高，差异均有统计学意义（P〈0.05）。MPP＋＋PBS组裂解半胱氨酸蛋白酶-3蛋白表达水平较PBS＋PBS组明显升高，差异有统计学意义（P〈0.05）；MPP＋＋TFP5组裂解半胱氨酸蛋白酶-3蛋白表达水平较MPP＋＋PBS组明显降低，差异有统计学意义（P〈0.05）。结论TFP5通过特异性抑制CDK5/p25表达，减少裂解半胱氨酸蛋白酶-3蛋白的产生，对MPP＋诱导的PC12细胞凋亡起抑制作用，从而对细胞产生保护作用。

Objective To determine whether the apoptosis of PC12 cells induced by MPP＋ can be protected when the activity ofcyclin-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 1000μmol/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）. Coneluslon TFP5 has protective effect against the apoptosis of PC 12 cells induced by MPP＋ through inhibiting the CDK5/p25 expression and reducing the cleaved caspase-3 protein production.