Jia Zhenquan, Zhu Hong, Vitto Michael J, Misra Bhaba R, Li Yunbo, Misra Hara P
Division of Biomedical Sciences, Edward Via Virginia College of Osteopathic Medicine, Virginia Tech Corporate Research Center, 2265 Kraft Drive, Blacksburg, VA 24060, USA.
Mol Cell Biochem. 2009 Mar;323(1-2):131-8. doi: 10.1007/s11010-008-9971-6. Epub 2008 Dec 11.
Alpha-lipoic acid (LA) has recently been reported to afford protection against neurodegenerative disorders in humans and experimental animals. However, the mechanisms underlying LA-mediated neuroprotection remain an enigma. Because peroxynitrite has been extensively implicated in the pathogenesis of various forms of neurodegenerative disorders, this study was undertaken to investigate the effects of LA in peroxynitrite-induced DNA strand breaks, a critical event leading to peroxynitrite-elicited cytotoxicity. Incubation of phi X-174 plasmid DNA with the 3-morpholinosydnonimine (SIN-1), a peroxynitrite generator, led to the formation of both single- and double-stranded DNA breaks in a concentration- and time-dependent fashion. The presence of LA at 100-1,600 microM was found to significantly inhibit SIN-1-induced DNA strand breaks in a concentration-dependent manner. The consumption of oxygen induced by 250 microM SIN-1 was found to be decreased in the presence of high concentrations of LA (400-1,600 microM), indicating that LA at these concentrations may affect the generation of peroxynitrite from auto-oxidation of SIN-1. It is observed that incubation of the plasmid DNA with authentic peroxynitrite resulted in a significant formation of DNA strand breaks, which could also be dramatically inhibited by the presence of LA (100-1,600 microM). EPR spectroscopy in combination with spin-trapping experiments, using 5,5-dimethylpyrroline-N-oxide (DMPO) as spin trap, resulted in the formation of DMPO-hydroxyl radical adduct (DMPO-OH) from authentic peroxynitrite and LA at 50-1,600 microM inhibited the adduct signal. Taken together, these studies demonstrate for the first time that LA can potently inhibit peroxynitrite-mediated DNA strand breakage and hydroxyl radical formation. In view of the critical involvement of peroxynitrite in the pathogenesis of various neurodegenerative diseases, the inhibition of peroxynitrite-mediated DNA damage by LA may be responsible, at least partially, for its neuroprotective activities.
最近有报道称，α-硫辛酸（LA）对人类和实验动物的神经退行性疾病具有保护作用。然而，LA介导神经保护作用的潜在机制仍是一个谜。由于过氧亚硝酸盐与多种形式的神经退行性疾病的发病机制密切相关，因此本研究旨在探讨LA对过氧亚硝酸盐诱导的DNA链断裂的影响，这是导致过氧亚硝酸盐引起细胞毒性的关键事件。用3-吗啉代辛二亚胺（SIN-1，一种过氧亚硝酸盐发生器）孵育φX-174质粒DNA，会以浓度和时间依赖性方式导致单链和双链DNA断裂的形成。发现100 - 1600微摩尔的LA以浓度依赖性方式显著抑制SIN-1诱导的DNA链断裂。发现在高浓度LA（400 - 1600微摩尔）存在下，250微摩尔SIN-1诱导的氧气消耗减少，表明这些浓度的LA可能影响SIN-1自氧化产生过氧亚硝酸盐。观察到用纯过氧亚硝酸盐孵育质粒DNA会导致DNA链断裂的显著形成，LA（100 - 1600微摩尔）的存在也能显著抑制这种断裂。使用5,5-二甲基吡咯啉-N-氧化物（DMPO）作为自旋捕获剂的电子顺磁共振光谱结合自旋捕获实验表明，纯过氧亚硝酸盐会形成DMPO-羟基自由基加合物（DMPO-OH），而50 - 1600微摩尔的LA会抑制该加合物信号。综上所述，这些研究首次证明LA可以有效抑制过氧亚硝酸盐介导的DNA链断裂和羟基自由基形成。鉴于过氧亚硝酸盐在各种神经退行性疾病发病机制中的关键作用，LA对过氧亚硝酸盐介导的DNA损伤的抑制作用可能至少部分地解释了其神经保护活性。
