Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198-5215, USA.
Free Radic Biol Med. 2011 Oct 15;51(8):1601-9. doi: 10.1016/j.freeradbiomed.2011.06.020. Epub 2011 Jun 28.
Oxidative damage of the endothelium disrupts the integrity of the blood-brain barrier (BBB). We have shown before that alcohol exposure increases the levels of reactive oxygen species (ROS; superoxide and hydroxyl radical) and nitric oxide (NO) in brain endothelial cells by activating NADPH oxidase and inducible nitric oxide synthase. We hypothesize that impairment of antioxidant systems, such as a reduction in catalase and superoxide dismutase (SOD) activity, by ethanol exposure may elevate the levels of ROS/NO in endothelium, resulting in BBB damage. This study examines whether stabilization of antioxidant enzyme activity results in suppression of ROS levels by anti-inflammatory agents. To address this idea, we determined the effects of ethanol on the kinetic profile of SOD and catalase activity and ROS/NO generation in primary human brain endothelial cells (hBECs). We observed an enhanced production of ROS and NO levels due to the metabolism of ethanol in hBECs. Similar increases were found after exposure of hBECs to acetaldehyde, the major metabolite of ethanol. Ethanol simultaneously augmented ROS generation and the activity of antioxidative enzymes. SOD activity was increased for a much longer period of time than catalase activity. A decline in SOD activity and protein levels preceded elevation of oxidant levels. SOD stabilization by the antioxidant and mitochondria-protecting agent acetyl-L-carnitine (ALC) and the anti-inflammatory agent rosiglitazone suppressed ROS levels, with a marginal increase in NO levels. Mitochondrial membrane protein damage and decreased membrane potential after ethanol exposure indicated mitochondrial injury. These changes were prevented by ALC. Our findings suggest the counteracting mechanisms of oxidants and antioxidants during alcohol-induced oxidative stress at the BBB. The presence of enzymatic stabilizers favors the ROS-neutralizing antioxidant redox of the BBB, suggesting an underlying protective mechanism of NO for brain vascular tone and vasodilation.
内皮细胞的氧化损伤破坏了血脑屏障（BBB）的完整性。我们之前已经表明，酒精暴露通过激活 NADPH 氧化酶和诱导型一氧化氮合酶增加脑内皮细胞中活性氧（ROS；超氧自由基和羟自由基）和一氧化氮（NO）的水平。我们假设，乙醇暴露对抗氧化系统的损害，如过氧化氢酶和超氧化物歧化酶（SOD）活性的降低，可能会使内皮细胞中的 ROS/NO 水平升高，导致 BBB 损伤。本研究探讨了抗氧化酶活性的稳定是否通过抗炎剂抑制 ROS 水平。为了验证这一观点，我们确定了乙醇对原代人脑内皮细胞（hBEC）中 SOD 和过氧化氢酶活性以及 ROS/NO 生成的动力学特征的影响。我们观察到 hBEC 中乙醇代谢导致 ROS 和 NO 水平的产生增加。hBEC 暴露于乙醇的主要代谢物乙醛后，也发现了类似的增加。乙醇同时增加了 ROS 的产生和抗氧化酶的活性。SOD 活性增加的时间比过氧化氢酶活性增加的时间长得多。SOD 活性的下降和蛋白水平的下降先于氧化剂水平的升高。抗氧化剂和线粒体保护剂乙酰-L-肉碱（ALC）和抗炎剂罗格列酮稳定 SOD 抑制了 ROS 水平，NO 水平略有升高。乙醇暴露后线粒体膜蛋白损伤和膜电位下降表明线粒体损伤。ALC 可防止这些变化。我们的研究结果表明，在 BBB 处酒精诱导的氧化应激过程中，氧化剂和抗氧化剂存在相互拮抗的机制。酶稳定剂的存在有利于 BBB 的 ROS 中和抗氧化剂的氧化还原，这表明 NO 对脑血管张力和血管扩张具有潜在的保护机制。
