Wei Wei, Han Bing-she, Guan Li-ying, Huang Fang, Feng Lei, Yang Yang, Xu Cai-min
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, CAMS and PUMC, Beijing 100005, China.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2007 Jun;29(3):324-8.
To investigate the role of reactive oxygen species (ROS) and ROS-caused mitochondrial transmembrane potential loss in sodium selenite-induced apoptosis in NB4 cells.
ROS production was measured by ROS-specific probe DCFH-DA. Sodium selenite mitochondrial transmembrane potential loss was evaluated by flow cytometry with Rh123 staining. Protein levels of cytochrome C, Bid, Bcl-xl, and Bax were measured by Western blot using protein-specific antibodies. NB4 cells were pre-incubated by MnTmPy or BSO before selenite treatment to further confirm the effects of ROS on NB4 cells.
20 micromol/L sodium selenite induced ROS production and mitochondrial transmembrane potential loss in NB4 cells time-dependently. Cytochrome C accumulated in cytoplasm after selenite treatment. Sodium selenite also downregulated Bcl-xl and activated Bax and Bid at protein level. Pretreatment with antioxidant MnTmPy almost fully abrogated the proapoptotic effect of sodium selenite prevented the cleavage of Bid protein and in turn the mitochondrail transmembrane potential loss. On the contrary, pretreatment with BSO intensified the mitochondrail transmembrane potential loss induced by sodium selenite.
Sodium selenite may induce apoptosis by inducing ROS production in NB4 cells, which leads to the downregulation of Bcl-xl, upregulation of Bax, and cleavage and activation of Bid. Bax and tBid then agregate on mitochondrial membrane, which in turn causes a decrease of mitochondrial transmembrane potential and release of cytochrome C into cytoplasm.
探讨活性氧(ROS)及ROS导致的线粒体跨膜电位丧失在亚硒酸钠诱导NB4细胞凋亡中的作用。
采用ROS特异性探针DCFH-DA检测ROS生成。用Rh123染色通过流式细胞术评估亚硒酸钠导致的线粒体跨膜电位丧失。使用蛋白特异性抗体通过蛋白质免疫印迹法检测细胞色素C、Bid、Bcl-xl和Bax的蛋白水平。在亚硒酸钠处理前用MnTmPy或BSO对NB4细胞进行预孵育,以进一步证实ROS对NB4细胞的影响。
20 μmol/L亚硒酸钠可时间依赖性地诱导NB4细胞产生ROS并导致线粒体跨膜电位丧失。亚硒酸钠处理后细胞色素C在细胞质中积聚。亚硒酸钠还在蛋白水平下调Bcl-xl并激活Bax和Bid。用抗氧化剂MnTmPy预处理几乎完全消除了亚硒酸钠的促凋亡作用,阻止了Bid蛋白的裂解,进而防止了线粒体跨膜电位丧失。相反,用BSO预处理增强了亚硒酸钠诱导的线粒体跨膜电位丧失。
亚硒酸钠可能通过诱导NB4细胞产生ROS而诱导凋亡,这导致Bcl-xl下调、Bax上调以及Bid的裂解和激活。Bax和tBid随后聚集在线粒体膜上,进而导致线粒体跨膜电位降低以及细胞色素C释放到细胞质中。