The role of superoxide and nuclear factor-κB signaling in N-methyl-D-aspartate-induced necrosis and apoptosis
The role of superoxide and nuclear factor-κB signaling in N-methyl-D-aspartate-induced necrosis and apoptosis
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DOI：
10.1124/jpet.301.2.478
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发表时间：
2002-05-01
影响因子：
3.5
通讯作者：
Johnson, KM
中科院分区：
文献类型：
--
作者：
McInnis, J;Wang, C;Johnson, KM
N-甲基-D-天冬氨酸（NMDA）受体介导的细胞死亡是复杂的，可能涉及坏死和凋亡的元素。这种现象背后的机制尚未完全理解，但已被认为涉及活性氧物质，如一氧化氮和超氧阴离子（O-2（自由基阴离子））和核因子-kappaB（NF-κ B）信号传导。在这项研究中，我们使用了选择性非肽超氧化物歧化酶模拟物（M40403）和SN 50，NF-κ B易位的肽抑制剂，研究的作用，O-2（自由基阴离子）和潜在的下游信号分子在细胞死亡诱导的NMDA受体激活。应用NMDA混合神经元/神经胶质前脑培养导致细胞质乳酸脱氢酶（LDH）的释放早期增加，在4小时达到峰值。随后，染料MTT [3-（4，5-二甲基噻唑-2-基）-2，5-二苯基四唑溴盐]的线粒体代谢减少，在整个20小时暴露期间持续减少。在7和20 h时，通过对DNA相关组蛋白（核小体）特异性的酶联免疫吸附测定（ELISA）测量，观察到DNA片段化显著增加。M40403和SN 50可阻断NMDA诱导的2、4和20 h LDH释放变化、4和20 h MTT代谢变化以及20 h DNA片段化变化（通过ELISA和末端dUTP缺口末端标记增加测定）。M40403还阻止NMDA诱导的NF-κ B的核转运，并增加Bax相对于Bcl-X-L的表达。SN 50还能够阻断NMDA诱导的细胞死亡以及增加的Bax/Bcl-X-L比率。时间进程的研究和SN 50和M40403的实验表明，O-2（自由基阴离子）的生产和NF-κ B易位可能参与坏死和凋亡，但后者也需要增加Bax的表达。M40403在该级联反应中相对早期预防NMDA诱导的细胞死亡的能力表明其在与NMDA受体介导的O-2（自由基阴离子）产生增加相关的中枢神经系统疾病（如中风）中的潜在治疗效用。
N-Methyl-D-aspartate (NMDA) receptor-mediated cell death is complex, probably involving elements of necrosis and apoptosis. The mechanisms underlying this phenomenon are incompletely understood but have been suggested to involve reactive oxygen species such as nitric oxide and superoxide anion (O-2(radical anion)) and nuclear factor-kappaB (NF-kappaB) signaling. In this study, we used a selective nonpeptidyl superoxide dismutase mimetic (M40403) and SN50, a peptide inhibitor of NF-kappaB translocation, to investigate the role of O-2(radical anion) and the potential downstream signaling molecules in cell death induced by activation of the NMDA receptor. Application of NMDA to a mixed neuronal/glial forebrain culture resulted in an early increase in the release of cytoplasmic lactate dehydrogenase (LDH), which peaked at 4 h. This was followed by a reduction in mitochondrial metabolism of the dye MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide] that continued to decrease throughout the 20-h exposure. A substantial increase in DNA fragmentation as measured by an enzyme-linked immunosorbent assay (ELISA) specific for DNA-associated histone proteins (nucleosomes) was observed at 7 and 20 h. M40403 and SN50 blocked NMDA-induced changes in LDH release at 2, 4, and 20 h, MTT metabolism at 4 and 20 h, and DNA fragmentation at 20 h as measured by the ELISA and by an increase in terminal dUTP-nick end labeling. M40403 also prevented NMDA-induced nuclear transport of NF-kappaB and increased expression of Bax relative to Bcl- X-L. SN50 was also able to block NMDA-induced cell death as well as the increased Bax/Bcl-X-L ratio. Time course studies and experiments with SN50 and M40403 suggest that O-2(radical anion) production and NF-kappaB translocation may be involved in necrosis and apoptosis, but the latter also requires an increased expression of Bax. The ability of M40403 to prevent NMDA-induced cell death relatively early in this cascade suggests its potential therapeutic utility in central nervous systems diseases such as stroke that are associated with increased NMDA receptor-mediated production of O-2(radical anion).