Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal.
Neurobiol Dis. 2012 Jan;45(1):206-18. doi: 10.1016/j.nbd.2011.08.005. Epub 2011 Aug 10.
The current study was undertaken to address the role of mitochondrial reactive oxygen species (ROS), and hypoxia inducible factor-1 alpha (HIF-1α) signaling pathway in the protection against high glucose levels in brain endothelial and NT2 neuron-like cells. Rat brain endothelial cells (RBE4) treated with non-toxic concentrations of cyanide (≤1 μM; 1h) exhibited an increase in ROS levels, particularly hydrogen peroxide (H(2)O(2)). Cyanide also induced a modest mitochondrial depolarization, an increase in oxygen consumption and a structural (smaller mitochondria) and spatial (perinuclear region) reorganization of mitochondrial network. The stabilization and nuclear activation of HIF-1α in the presence of cyanide were also observed, which resulted in an increase in vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS) and erythropoietin (EPO) protein levels reflecting an adaptive response. Importantly, preconditioning induced by cyanide protected brain endothelial cells against high glucose-mediated damage by the prevention of apoptotic cell death. In mitochondrial DNA-depleted NT2 (NT2 ρ0) cells, cyanide (0.1 μM) was unable to stimulate ROS production and, consequently, protect against glucotoxicity. Conversely, in NT2 cells, the parental cells with functional mitochondria, cyanide significantly increased ROS levels protecting against high glucose-induced neuronal cell loss and activation of caspase-3. The free radical scavenger N-acetyl-L-cysteine and the specific HIF-1α inhibitor 2-methoxyestradiol completely abolished the protective effects of cyanide preconditioning. Altogether our results demonstrate that mitochondrial preconditioning induced by cyanide triggers a protective response mediated by mitochondrial ROS and HIF-1α activation and signaling, which render brain endothelial and neuronal cells resistant against glucotoxicity.
本研究旨在探讨线粒体活性氧（ROS）和缺氧诱导因子-1α（HIF-1α）信号通路在保护脑内皮细胞和 NT2 神经元样细胞免受高糖水平损伤中的作用。用无毒浓度的氰化物（≤1 μM；1 小时）处理大鼠脑内皮细胞（RBE4）可增加 ROS 水平，特别是过氧化氢（H2O2）。氰化物还可诱导轻微的线粒体去极化、耗氧量增加以及线粒体网络的结构（较小的线粒体）和空间（核周区）重排。氰化物还观察到 HIF-1α 的稳定和核激活，导致血管内皮生长因子（VEGF）、内皮型一氧化氮合酶（eNOS）和促红细胞生成素（EPO）蛋白水平增加，反映出适应性反应。重要的是，氰化物诱导的预处理通过防止细胞凋亡来保护脑内皮细胞免受高糖介导的损伤。在缺少线粒体 DNA 的 NT2（NT2 ρ0）细胞中，氰化物（0.1 μM）不能刺激 ROS 产生，因此不能保护细胞免受糖毒性。相反，在具有功能线粒体的亲本 NT2 细胞中，氰化物可显著增加 ROS 水平，从而保护细胞免受高糖诱导的神经元细胞死亡和 caspase-3 的激活。自由基清除剂 N-乙酰-L-半胱氨酸和特异性 HIF-1α 抑制剂 2-甲氧基雌二醇完全消除了氰化物预处理的保护作用。总之，我们的研究结果表明，氰化物诱导的线粒体预处理通过线粒体 ROS 和 HIF-1α 激活和信号转导触发保护性反应，使脑内皮细胞和神经元细胞对糖毒性具有抗性。
