二氮嗪抑制Aβ_(1～42)对U251细胞存活率、线粒体膜电位和细胞内活性氧水平的影响

Influence of diazoxide inhibition of Aβ_(1-42) on cellular survival rate,mitochondrial membrane potential and intracellular ROS level

目的探讨线粒体膜上ATP敏感的钾离子通道开放药物二氮嗪防治Aβ1～42细胞毒性作用及其分子学机制。方法采用不同浓度Aβ1～42和二氮嗪同时处理神经胶质细胞瘤U251细胞24h,以四唑盐比色法测定细胞存活率;四氯四乙基苯并咪唑基羰花青碘化物(JC-1)检测线粒体膜电位;2',7'-二氯荧光黄双乙酸盐(DCFH-DA)检测细胞内活性氧水平;膜联蛋白荧光素荧光染色检测U251细胞凋亡。结果(1)二氮嗪组神经胶质细胞瘤U251细胞在不同浓度Aβ1～42作用下,细胞存活率明显高于单纯Aβ1～42组(P<0.05)。(2)半定量分析显示,单纯Aβ1～42组(5μmol/L)U251细胞线粒体膜红/绿荧光强度比值(590/527nm)明显低于正常对照组(P<0.01);而二氮嗪组U251细胞红/绿荧光强度比值(590/527nm)明显高于单纯Aβ1～42组(P<0.01)。(3)流式细胞术检测显示,单纯Aβ1～42组(5μmol/L)U251细胞内的活性氧水平明显增高,与对照组相比差异有统计学意义(P<0.05);经二氮嗪(1mmol/L)预处理后U251细胞内的活性氧水平下降,与单纯Aβ1～42组相比差异有统计学意义(P<0.05)。(4)流式细胞荧光强度分析显示,无论Aβ1～42或Aβ42～1均不能引起U251细胞发生早期或晚期凋亡或坏死。结论Aβ1～42的细胞毒性作用明显早于其致细胞凋亡作用。提示尽早应用二氮嗪保护细胞线粒体功能状态,可预防长时间暴露于Aβ1～42导致细胞凋亡所引起的神经细胞数量的减少。

Objective To investigate whether K^＋ channel opener diazoxide could protect cells from Aβ1～42 induced cytotoxicity and its molecular mechanism. Methods Neuroglioma U251 cells were exposed to different concentrations of Aβ1～42 and diazoxide for 24 h in vitro. Cellular viabilities were assessed with MTT. The mitochondria membrane potential and intracellular reactive oxygen species （ROS） levels were detected by 5, 5＇, 6, 6＇-tetrachloro-1, 1＇, 3, 3＇-tetraethyl-benzamidazolocarbocyanin iodide （JC-1） and 2＇, 7＇-dichlorodihydrofluorescin diacetate （DCFH-DA） respectively, and cell apoptosis by Annexin-V-FITC/PI fluorescence flow cytometry. Results 1） In different concentration of Aβ1～42 the survival rate of neuroglioma U251 cell （U251 cell） in diazoxide group was significantly higher than that in simple Aβ1～42 group （P〈 0.05）. 2） In semi-quantitative analysis, red/green fluorescence light intensity ratio of mitochondrial membrane （590/527 nm） in simple Aβ1～42 group （5 μmol/L） was significantly lower than that in normal control group （P〈 0.01）; U251 cell red/ green fluorescence light intensity ratio in diazoxide group （590/527 nm） was significantly higher than that in simple Aβ1～42 group （P〈 0.01）. 3） Flow cytometry showed that U251 intracellular ROS level in simple Aβ1～42 group （5 μmol/L） was significantly higher than that in control group （P〈 0.05）; after pretreatment with diazoxide （1 mmol/L）, U251 intracellular ROS level decreased significantly comparing with simple Aβ1～42 group （P〈 0.05）. 4） Whether Aβ1～42 or Aβ42～1 did not cause early or later cell apoptosis or necrosis as compared to control in fluorescence flow cytometry. Conclusion The cellular toxic effect of Aβ1～42 occurs earlier than its induction of cell apoptosis. It suggested that earlier diazoxide could protect mitochondrial function and prevent nerve cells decreasing cell induced by cell apoptosis in long-term Aβ1～42 exposure.