银杏酮酯抑制β-淀粉样蛋白诱导海马神经元细胞凋亡的研究

Ginkgo biloba Extract 50 inhibit beta-amyloid-induced apoptosis in hippocampal neurons

目的:研究银杏酮酯(GBE50)抑制由β-淀粉样蛋白(Aβ)诱导的海马神经元凋亡现象及机制,来探讨GBE50防治阿尔茨海默病(AD)的机制。方法:通过体外原代培养海马神经细胞,经20μmol/L Aβ25-35诱导细胞出现凋亡现象,采用MTT法和流式细胞仪,观察不同浓度的GBE50对细胞活力和细胞凋亡的影响。然后,运用Western blot法分别检测凋亡相关基因Bcl-2、Bax和caspas-3的蛋白表达,来探讨机制。结果:与正常对照组比较,20μmol/L的Aβ25-35可以使细胞活力明显降低(P<0.05);GBE50各浓度组均能够明显改善细胞活力,呈现浓度依赖性。其中,除了浓度10μg/mL之外的其他各浓度组与Aβ组之间也存在显著性差异(P<0.05)。20μmol/L的Aβ25-35可以使细胞凋亡率和死亡率分别提高14.5%和8.6%,且与正常对照组之间呈显著性差异(P<0.01);而50μg/mL的GBE50可使凋亡率和死亡率分别降低约47.7%和59.5%,且与Aβ组之间也呈显著性差异(P<0.05)。蛋白检测的结果显示,20μmoL/L的Aβ25-35能够使Bcl-2/Bax表达量比值减小,而使活化的caspase-3表达量升高,且与正常对照组之间呈显著性差异(P<0.05);而GBE50能够使Bcl-2/Bax表达量比值增大,使活化的caspase-3表达量降低,且呈现浓度依赖性。而且,50和100μg/mL GBE50组与正常对照组之间均呈现显著性差异(P<0.05)。结论:20μmol/L的Aβ25-35能够诱导海马神经元出现凋亡现象,而GBE50通过促进抗凋亡蛋白Bcl-2的表达,抑制促凋亡蛋白Bax的表达和caspase-3的活化,从而抑制Aβ诱导的细胞凋亡,提高细胞活力,从而保护神经元。

Objective： To study the mechanism of Ginkgo biloba Extract 50 （GBE50） inhibit beta-amyloid （Aβ）- induced apoptosis in hippocampal neurons in vitro. Methods： The primary hippocampal neuron was cultured in vitro and induced to apoptosis by 20μmol/L Aβ25-35. The MTT and flow cytometry were used to observe the effect of GBES0 with different concentrations on cell viability and apoptosis in neurons. Furthermore, the Western blot was used to detect expressions of apoptosis-related protein Bcl-2, Bax and caspase-3. Results： Compared with control group, 20μmol/L Aβ25-35 could reduce the cell vitality significantly （P〈0.05）. There were statistical differences between GBE50 group with different concentrations （10μg/ mL except） and Aβ group （P〈0.05）. GBE50 could improve cell vitality with certain concentration dependence. The cell apoptosis rate and necrosis rate were obviously increased by 20μmol/L Aβ25-35 （P〈0.01）. However, 50μg/mL GBE50 could significantly reduce the cell apoptosis and necrosis rate （P〈0.05）. The result of protein detection showed that, compared with control group, 20μmol/L Aβ25-35 could reduce the ratio of Bcl-2/Bax and enhance the activated caspase-3 expression （P〈0.05）. But GBE50 could make this ratio of Bcl-2/Bax increase and weaken the expression of activated caspase-3 with certain concentration dependence. What＇s more, the statistical difference was significant between GBE50 group （50 and 100pg/mL） and the control group （P〈0.05）. Conclusion： 20μmol/L Aβ25-35 could induce the apoptosis of hippocampal neuron. However, GBE50 could enhance the anti- apoptosis protein Bcl-2 expression and reducing expressions of two pro-apoptosis proteins Bax and caspase-3, thus it could inhibit the apoptosis of neurons and improve cell vitality. These effects consequently contributed to protect hippocampal neurons.