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目的研究ATP敏感性钾离子通道开放剂二氮嗪、环孢菌素A及两者协同对β-淀粉样蛋白(Aβ1-42)致原代培养基底前脑胆碱能神经元凋亡的影响。方法采用2μmol/L的Aβ1-42对原代培养大鼠胆碱能神经元进行干预,建立阿尔茨海默病(AD)细胞凋亡模型;采用500μmol/L二氮嗪(预处理1 h)、20μmol/L环孢菌素A以及两者协同对其干预,作用不同时间(24、72 h)后置于倒置显微镜下观察细胞形态;采用噻唑蓝(MTT)比色法检测细胞存活率,Wes-tern blot法检测细胞内凋亡相关蛋白表达水平的变化。结果①Aβ1-42作用胆碱能神经元72 h后,Bcl-2表达量降低(P<0.01),细胞色素C、Caspase-3、Cleaved caspase-3的表达量增加(P<0.01,P<0.001,P<0.01),Bax未见明显变化,Bcl-2/Bax比值降低(P<0.01),细胞形态发生明显损伤性变化,细胞存活率明显降低(P<0.001);②二氮嗪、环孢菌素A以及两者协同作用24 h后Bcl-2的表达增加(P<0.05,P<0.01,P<0.01),作用72h能明显增加Bcl-2的表达(P<0.05,P<0.01,P<0.01),并提升Bc-2/Bax比率而抑制细胞色素C、Caspase-3、Cleaved caspase-3表达量(P<0.05,P<0.01,P<0.001),细胞存活率均增高(P<0.01,P<0.001)。结论Aβ1-42作用胆碱能神经元72 h能促使细胞凋亡,二氮嗪与环孢菌素A及两者协同可以明显拮抗Aβ1-42致细胞凋亡作用。
Objective To investigate the effects of diazoxide, cyclosporine A and their combination on the apoptosis of cholinergic neurons in the basal forebrain of β-amyloid (Aβ1-42) -induced ATP-sensitive potassium channel openers . Methods Apoptosis model of Alzheimer’s disease (AD) was established by intervention of choline (2 μmol / L) Aβ1-42 on primary cultured rat cholinergic neurons. 500 μmol / L diazoxide (pretreatment for 1 h) , 20μmol / L cyclosporin A and their synergistic intervention. The cells were observed under inverted microscope at different time (24 and 72 h), and cell viability was measured by MTT assay. Wes-blot assay was used to detect the expression of apoptosis-related proteins. Results ① The expression of Bcl-2 was decreased (P <0.01) and the expression of cytochrome C, Caspase-3 and Cleaved caspase-3 was increased in Aβ1-42-treated cholinergic neurons after 72 h , P <0.01). There was no significant change in Bax and Bcl-2 / Bax ratio (P <0.01). The morphology of cells was obviously damaged and the cell survival rate was significantly decreased (P <0.001) (P <0.05, P <0.01, P <0.01), and the expression of Bcl-2 increased significantly at 72h (P <0.05, P <0.01) (P <0.01, P <0.01), and increased the Bc-2 / Bax ratio and inhibited the expression of cytochrome C, Caspase-3 and Cleaved caspase-3 P <0.01, P <0.001). Conclusion Aβ1-42-induced cholinergic neurons induce apoptosis in 72 h. Synergistic effect of diazoxide and cyclosporin A can significantly antagonize Aβ1-42-induced apoptosis.