目的 考察1,2,3,4,6-O-五没食子酰基葡萄糖(1,2,3,4,6-penta-O-galloyl-β-D-glucose,β-PGG)对MPP⁺诱导的帕金森病细胞模型中PC12细胞凋亡的保护作用及其机制研究。方法 PC12细胞孵育于高糖DMEM培养基中,在药物处理前1周,将神经生长因子(NGF)加入培养基中,使培养基中NGF的终质量浓度为50 ng/mL。将细胞分为对照组、MPP⁺组以及50 μmol/L β-PGG预处理7、12、20、30 h组,观察预处理不同时间对MPP⁺中PC12细胞存活影响。采用台盼蓝染色法检测细胞死亡情况,MTT法检测细胞活力,免疫印迹法检测Bcl-2、Bax、Fas、FasL、procaspase-3、procaspase-8、procaspase-9蛋白表达情况,并检测caspase-3、caspase-8、caspase-9活力。结果 对照组PC12细胞死亡率最低,MPP⁺组PC12细胞死亡率最高,从β-PGG预处理12 h起PC12细胞死亡率较MPP⁺组均明显降低(P< 0.01)。MPP⁺组PC12细胞活力最低,50 μmol/L β-PGG预处理12 h时PC12细胞活力进一步增高,预处理20 h时细胞活力最高。β-PGG预处理5 h后即可见Bcl-2、procaspase-3、procaspase-8、procaspase-9蛋白含量增加,至15 h时增加达到高峰;与之相反的是,β-PGG预处理5 h后即可见Bax、Fas、FasL蛋白含量减少,至30 h时达最少。50 μmol/L β-PGG预处理PC12细胞15 h后caspase-3、caspase-8、caspase-9的活力分别为MPP⁺组的36.5%、40.2%、42.2%。结论 β-PGG对MPP⁺诱导PC12细胞凋亡具有保护作用,其机制是通过增强Bcl-2的表达、抑制Bax、Fas、FasL的表达以及降低caspase-3、caspase-8、caspase-9的活力实现了抑制MPP⁺引起的PC12细胞凋亡,促进PC12细胞的存活。

Objective To study the protection of 1,2,3,4,6-penta-O-galloyl-β-D-glucose (β-PGG) on apoptosis of PC12 cells from Parkinson's disease models induced by MPP⁺ and its mechanism. Methods PC12 cells were incubated in high glucose DMEM medium. One week before drug treatment, nerve growth factor was added to the cultures at the final concentration of 50 ng/mL. PC12 cells were divided into control group, MPP⁺ group, and 50 μmol/L β-PGG groups pretreated for 7, 12, 20, and 30 h. The survivals of PC12 cells in MPP⁺ were observed after pretreatment with β-PGG for different periods. The death of PC12 cells in MPP⁺ was evaluated with trypan blue staining method, and the activity of the PC12 cells was determined by MTT assay. The expression of Bax,Fas, FasL, procaspase-3, procaspase-8, and procaspase-9 was analyzed by Western blotting method, and then the activity of caspase-3, caspase-8, and caspase-9 was examined. Results The death rates of PC12 cells in control group were the lowest, while those in MPP⁺ group were the highest, and those in β-PGG groups pretreated after 12 h were significantly decreased compared with MPP⁺ group (P < 0.01). The activities of the PC12 cells in MPP⁺ group were the lowest, and those in β-PGG group pretreated for 12 h were further increased, while those in β-PGG group pretreated for 20 h were the highest. The protein contents of Bcl-2, procaspase-3, procaspase-8, and procaspase-9 in β-PGG group pretreated for 5 h were increased, and increased to peak at pretreated for 15 h. On the contrary, protein contents of Bax, Fas, and FasL in β-PGG group pretreated for 5 h were decreased, and decreased to the minimum at pretreated for 30 h. The activities of caspase-3, caspase-8, and caspase-9 of the PC12 cells in β-PGG group pretreated for 15 h were 36.5%, 40.2%, and 42.2% of those in MPP⁺ group, respectively. Conclusion β-PGG has protection against apoptosis of PC12 cells induced by MPP⁺, and its mechanism is related to inhibiting the apoptosis of PC12 cells induced by MPP⁺ and then increase survival rate of PC12 cells by increasing the expression of Bcl-2, inhibiting the expression of Bax, Fas, and FasL, and decreasing activities of caspase-3, caspase-8, and caspase-9.

福建省自然科学基金面上项目(2013J01275);福建省卫生厅中医药研究课题(wzzy201304)