银杏叶聚戊烯醇抗Aβ_(25-35)介导dPC12细胞损伤的保护机制

Study on Protective Mechanism of the Polyprenols from the Ginkgo biloba Leaves Against Aβ_(25-35)-Induced Neurotoxicity in dPC12 Cells

目的:研究银杏叶聚戊烯醇(GP)抗Aβ_(25-35)诱导dPC12细胞损伤的保护机制。方法:以鼠肾上腺嗜铬细胞瘤单克隆细胞系(PC12细胞)为研究对象,通过LDH比色法检测GP对Aβ_(25-35)处理后dPC12细胞存活率的影响;流式细胞术检测不同浓度GP对dPC12细胞凋亡的影响;应用活性氧(ROS)、丙二醛(MDA)试剂盒检测Aβ_(25-35)对dPC12细胞的氧化损伤,并研究GP的抗氧化作用及其机制;RT-PCR法检测Bax、Bcl-2和Caspase-3在mRNA水平的表达,并采用Western blot法检测Bax、Bcl-2、Caspase-3及Cleaved Caspase-3等凋亡相关因子在蛋白水平的表达。结果:GP可显著降低LDH漏出率以及细胞内ROS水平和MDA含量,对dPC12细胞有显著的抗氧化作用,具有显著的量效关系;GP对Aβ_(25-35)引起的dPC12细胞凋亡有一定的抑制作用,以高剂量组作用显著;GP能显著下调Aβ_(25-35)诱导损伤dPC12细胞内Caspase-3、Bax mRNA水平,并上调Bcl-2 mRNA水平,Bax/Bcl-2 mRNA比值降低,抑制细胞凋亡;GP能降低Aβ_(25-35)诱导损伤dPC12细胞内Caspase-3、Bax蛋白表达,升高Bcl-2蛋白表达,降低Bax/Bcl-2比值,抑制凋亡执行因子Caspase-3活化,从而抑制细胞凋亡。结论:GP可减少Aβ_(25-35)致损伤dPC12细胞的凋亡,其作用可能与其抗氧化作用和抑制细胞凋亡途径激活有关。

Objective： To study protective mechanism of Polyprenols from Ginkgo biloba leaves（GP） against Aβ（25-35）-induced neurotoxicity in dPC12 cells.Methods： The mice adrenal pheochromocytoma monoclonal cells（PC12） was used to study protective mechanisms of the GP.Lactate dehydrogenase（LDH） assay was used to detect the protective effect by GP on Aβ（25-35）-injured dPC12 cells; Flow cytometric was used to analyzed apoptosis ratio of dPC12 cells; and ROS assay and MDA assay were used to measured the oxidative damage of dPC12 cells by Aβ（25-35） respectively,and the antioxidation and the mechanism of GP were studied.RT-PCR method was used to detect the protein expression levels of Bax,Bcl-2 and Caspase-3 mRNA.Western blot assay was used to detect the expression level of protein regulating apoptosis as Bax,Bcl-2,Caspase-3 and Cleaved Caspase-3.Results： The results showed that GP could lower the LDH leakage and decreased the level of ROS and MDA in dPC12 cells,which indicated that GP have a significant antioxidant effect on Aβ（25-35）-injured dPC12 cells,and had significant dose effect relationship.GP had a inhibitory effect on the cells apoptosis which induced by β25-35 and especially obvious effect in high dose group.GP caused an downregulation of Caspase-3 and Bax mRNA level of dPC12 cells damage which induced by Aβ（25-35）,and upregulation of Bcl-2 mRNA level,the ratio of Bax/Bcl-2 mRNA was decreased on dPC12 cells,which indicated that GP could inhibit cell apoptosis at the level transcription of apoptosis-associated protein.GP could downregulate the protein expression level of Caspase-3 and Bax and upregulate the level of Bcl-2,and the lower value of Bax/Bcl-2,and the protein level of Cleaved Caspase-3 was decreased,inhibited the cells apoptosis.Conclusion： The resluts indicate that GP can inhibit the apoptosis of dPC12 cells damage which induced by Aβ（25-35）,the protective effect might be through antioxidant pathways and regulation of the level of cells apoptosis.