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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/m L。将细胞分为对照组、MPP+组以及50μmol/Lβ-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、Fas L、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,50μmol/Lβ-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、Fas L蛋白含量减少,至30 h时达最少。50μmol/Lβ-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、Fas L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 investigate the effect of 1,2,3,4,6-O-galloyl-β-D-glucose (β-PGG) on MPP + Protective effect and mechanism of PC12 cell apoptosis in a model of Parkinson ’s disease. Methods PC12 cells were incubated in high glucose DMEM medium. NGF was added into the medium one week before drug treatment, so that the final concentration of NGF in culture medium was 50 ng / m L. The cells were divided into control group, MPP + group and 50μmol / Lβ-PGG preconditioning for 7, 12, 20 and 30 h groups. The effect of pretreatment on the survival of PC12 cells in MPP + was observed. The cell viability was detected by trypan blue staining, the cell viability was detected by MTT assay, the protein expressions of Bcl-2, Bax, Fas, Fas L, procaspase-3, procaspase-8 and procaspase-9 were detected by Western blotting and caspase -3, caspase-8, caspase-9 activity. Results The PC12 cell death rate in the control group was the lowest. The mortality rate of PC12 cells in MPP + group was the highest. Compared with MPP + group, the PC12 cell death rate was significantly decreased after β-PGG pretreatment for 12 h (P <0.01). The viability of PC12 cells was the lowest in MPP + group. The viability of PC12 cells was further increased after pretreatment with 50μmol / Lβ-PGG for 12 hours, and the cell viability was highest at 20 h after pretreatment. The content of Bcl-2, procaspase-3, procaspase-8 and procaspase-9 increased after pretreatment with β-PGG for 5 h, and reached a peak at 15 h. In contrast, pretreatment with β-PGG for 5 h The content of Bax, Fas and Fas L decreased after 30 h, reaching the least at 30 h. The viability of caspase-3, caspase-8 and caspase-9 in PC12 cells pretreated with 50μmol / Lβ-PGG for 15 h were 36.5%, 40.2% and 42.2% of those in MPP + group, respectively. Conclusions β-PGG can protect PC12 cells from MPP + -induced apoptosis by increasing the expression of Bcl-2, inhibiting the expression of Bax, Fas and Fas L and decreasing the activities of caspase-3, caspase-8 and caspase-9 It can inhibit the apoptosis of PC12 cells induced by MPP + and promote the survival of PC12 cells.