目的 考察1,2,3,4,6-O-五沒食子酰基葡萄糖(1,2,3,4,6-penta-O-galloyl-β-D-glucose,β-PGG)對(duì)MPP⁺誘導(dǎo)的帕金森病細(xì)胞模型中PC12細(xì)胞凋亡的保護(hù)作用及其機(jī)制研究。方法 PC12細(xì)胞孵育于高糖DMEM培養(yǎng)基中,在藥物處理前1周,將神經(jīng)生長(zhǎng)因子(NGF)加入培養(yǎng)基中,使培養(yǎng)基中NGF的終質(zhì)量濃度為50 ng/mL。將細(xì)胞分為對(duì)照組、MPP⁺組以及50 μmol/L β-PGG預(yù)處理7、12、20、30 h組,觀察預(yù)處理不同時(shí)間對(duì)MPP⁺中PC12細(xì)胞存活影響。采用臺(tái)盼藍(lán)染色法檢測(cè)細(xì)胞死亡情況,MTT法檢測(cè)細(xì)胞活力,免疫印跡法檢測(cè)Bcl-2、Bax、Fas、FasL、procaspase-3、procaspase-8、procaspase-9蛋白表達(dá)情況,并檢測(cè)caspase-3、caspase-8、caspase-9活力。結(jié)果 對(duì)照組PC12細(xì)胞死亡率最低,MPP⁺組PC12細(xì)胞死亡率最高,從β-PGG預(yù)處理12 h起PC12細(xì)胞死亡率較MPP⁺組均明顯降低(P< 0.01)。MPP⁺組PC12細(xì)胞活力最低,50 μmol/L β-PGG預(yù)處理12 h時(shí)PC12細(xì)胞活力進(jìn)一步增高,預(yù)處理20 h時(shí)細(xì)胞活力最高。β-PGG預(yù)處理5 h后即可見Bcl-2、procaspase-3、procaspase-8、procaspase-9蛋白含量增加,至15 h時(shí)增加達(dá)到高峰;與之相反的是,β-PGG預(yù)處理5 h后即可見Bax、Fas、FasL蛋白含量減少,至30 h時(shí)達(dá)最少。50 μmol/L β-PGG預(yù)處理PC12細(xì)胞15 h后caspase-3、caspase-8、caspase-9的活力分別為MPP⁺組的36.5%、40.2%、42.2%。結(jié)論 β-PGG對(duì)MPP⁺誘導(dǎo)PC12細(xì)胞凋亡具有保護(hù)作用,其機(jī)制是通過增強(qiáng)Bcl-2的表達(dá)、抑制Bax、Fas、FasL的表達(dá)以及降低caspase-3、caspase-8、caspase-9的活力實(shí)現(xiàn)了抑制MPP⁺引起的PC12細(xì)胞凋亡,促進(jìn)PC12細(xì)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.

福建省自然科學(xué)基金面上項(xiàng)目(2013J01275);福建省衛(wèi)生廳中醫(yī)藥研究課題(wzzy201304)