Xiong Yiqin, Rabchevsky Alexander G, Hall Edward D
Spinal Cord and Brain Injury Research Center, University of Kentucky Chandler Medical Center, Lexington, KY 40536, USA.
J Neurochem. 2007 Feb;100(3):639-49. doi: 10.1111/j.1471-4159.2006.04312.x. Epub 2006 Dec 1.
Peroxynitrite (PON, ONOO(-)), formed by nitric oxide synthase-generated nitric oxide radical ( NO) and superoxide radical (O(2) (-)), is a crucial player in post-traumatic oxidative damage. In the present study, we determined the spatial and temporal characteristics of PON-derived oxidative damage after a moderate contusion injury in rats. Our results showed that 3-nitrotyrosine (3-NT), a specific marker for PON, rapidly accumulated at early time points (1 and 3 h) and a significant increase compared with sham rats was sustained to 1 week after injury. Additionally, there was a coincident and maintained increase in the levels of protein oxidation-related protein carbonyl and lipid peroxidation-derived 4-hydroxynonenal (4-HNE). The peak increases of 3-NT and 4-HNE were observed at 24 h post-injury. In our immunohistochemical results, the co-localization of 3-NT and 4-HNE results indicates that PON is involved in lipid peroxidative as well as protein nitrative damage. One of the consequences of oxidative damage is an exacerbation of intracellular calcium overload, which activates the cysteine protease calpain leading to the degradation of several cellular targets including cytoskeletal protein (alpha-spectrin). Western blot analysis of alpha-spectrin breakdown products showed that the 145-kDa fragments of alpha-spectrin, which are specifically generated by calpain, were significantly increased as soon as 1 h following injury although the peak increase did not occur until 72 h post-injury. The later activation of calpain is most likely linked to PON-mediated secondary oxidative impairment of calcium homeostasis. Scavengers of PON, or its derived free radical species, may provide an improved antioxidant neuroprotective approach for the treatment of post-traumatic oxidative damage in the injured spinal cord.
过氧亚硝酸盐（PON，ONOO⁻）由一氧化氮合酶产生的一氧化氮自由基（NO）和超氧阴离子自由基（O₂⁻）反应生成，是创伤后氧化损伤的关键因素。在本研究中，我们测定了大鼠中度挫伤后PON所致氧化损伤的时空特征。我们的结果显示，PON的特异性标志物3-硝基酪氨酸（3-NT）在早期时间点（1小时和3小时）迅速积累，与假手术组大鼠相比显著增加，并持续至损伤后1周。此外，蛋白质氧化相关的蛋白质羰基水平和脂质过氧化衍生的4-羟基壬烯醛（4-HNE）水平同时持续升高。3-NT和4-HNE的峰值增加出现在损伤后24小时。在我们的免疫组织化学结果中，3-NT和4-HNE的共定位表明PON参与了脂质过氧化以及蛋白质硝化损伤。氧化损伤的后果之一是细胞内钙超载加剧，这会激活半胱氨酸蛋白酶钙蛋白酶，导致包括细胞骨架蛋白（α-血影蛋白）在内的多个细胞靶点降解。对α-血影蛋白降解产物的蛋白质印迹分析表明，由钙蛋白酶特异性产生的α-血影蛋白145-kDa片段在损伤后1小时即显著增加，尽管峰值增加直到损伤后72小时才出现。钙蛋白酶的后期激活很可能与PON介导的钙稳态继发性氧化损伤有关。PON或其衍生自由基的清除剂可能为治疗脊髓损伤后的创伤后氧化损伤提供一种改进的抗氧化神经保护方法。
