Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, Texas 76107-2699, USA.
J Appl Physiol (1985). 2012 May;112(10):1706-14. doi: 10.1152/japplphysiol.01428.2011. Epub 2012 Mar 8.
Intermittent hypoxia (IH) conditioning minimizes neurocognitive impairment and stabilizes brain mitochondrial integrity during ethanol withdrawal (EW) in rats, but the mitoprotective mechanism is unclear. We investigated whether IH conditioning protects a key mitochondrial enzyme, cytochrome c oxidase (COX), from EW stress by inhibiting mitochondrially directed apoptotic pathways involving cytochrome c, Bax, or phosphor-P38 (pP38). Male rats completed two cycles of a 4-wk ethanol diet (6.5%) and 3 wk of EW. An IH program consisting of 5-10 bouts of 5-8 min of mild hypoxia (9.5-10% inspired O(2)) and 4 min of reoxygenation for 20 consecutive days began 3 days before the first EW period. For some animals, vitamin E replaced IH conditioning to test the contributions of antioxidant mechanisms to IH's mitoprotection. During the second EW, cerebellar-related motor function was evaluated by measuring latency of fall from a rotating rod (Rotarod test). After the second EW, COX activity in cerebellar mitochondria was measured by spectrophotometry, and COX, cytochrome c, Bax, and pP38 content were analyzed by immunoblot. Mitochondrial protein oxidation was detected by measuring carbonyl contents and by immunochemistry. Earlier IH conditioning prevented motor impairment, COX inactivation, depletion of COX subunit 4, protein carbonylation, and P38 phosphorylation during EW. IH did not prevent cytochrome c depletion during EW, and Bax content was unaffected by EW ± IH. Vitamin E treatment recapitulated IH protection of COX, and P38 inhibition attenuated protein oxidation during EW. Thus IH protects COX and improves cerebellar function during EW by limiting P38-dependent oxidative damage.
间歇性低氧（IH）预处理可减轻乙醇戒断（EW）期间大鼠的神经认知障碍并稳定脑线粒体完整性，但其中的保护机制尚不清楚。我们研究了间歇性低氧预处理是否通过抑制涉及细胞色素 c、Bax 或磷酸化-P38（pP38）的线粒体定向凋亡途径来保护关键的线粒体酶细胞色素 c 氧化酶（COX）免受 EW 应激。雄性大鼠完成了为期 4 周的乙醇饮食（6.5%）和 3 周的 EW。一个由 5-10 个 5-8 分钟的轻度低氧（9.5-10%吸入 O2）和 4 分钟复氧组成的 IH 方案，在第一个 EW 期前 3 天开始，连续 20 天。对于一些动物，用维生素 E 替代 IH 预处理以测试抗氧化机制对 IH 保护作用的贡献。在第二次 EW 期间，通过测量从旋转杆上坠落的潜伏期来评估小脑相关的运动功能（旋转棒测试）。第二次 EW 后，通过分光光度法测量小脑线粒体中的 COX 活性，并通过免疫印迹分析 COX、细胞色素 c、Bax 和 pP38 的含量。通过测量羰基含量和免疫化学法检测线粒体蛋白氧化。早期 IH 预处理可防止 EW 期间的运动障碍、COX 失活、COX 亚基 4 的耗竭、蛋白羰基化和 P38 磷酸化。IH 不能防止 EW 期间细胞色素 c 的耗竭，并且 Bax 含量不受 EW ± IH 的影响。维生素 E 处理再现了 IH 对 COX 的保护作用，并且 P38 抑制减轻了 EW 期间的蛋白氧化。因此，IH 通过限制 P38 依赖性氧化损伤来保护 COX 并改善 EW 期间的小脑功能。
