Ravati A, Ahlemeyer B, Becker A, Krieglstein J
Institut für Pharmakologie und Toxikologie, Philipps-Universität Marburg, Ketzerbach 63, D-35032, Marburg, Germany.
Brain Res. 2000 Jun 2;866(1-2):23-32. doi: 10.1016/s0006-8993(00)02210-1.
The current study was performed to determine the role of reactive oxygen species (ROS) in preconditioning against different forms of neuronal damage. Primary cultures of chick embryonic neurons were treated with either FeSO(4) (100 microM; 15 min) to generate hydroxyl radicals or xanthine/xanthinoxidase (10 microM/0.5 mU ml(-1); 15 min; =X/XO (pre)) to produce superoxide radicals. Both stimuli moderately enhanced ROS formation as measured by fluorescence microscopy. This preconditioning significantly protected the neurons against subsequent glutamate (1 mM)-induced excitotoxic damage, staurosporine (200 nM)-induced neuronal apoptosis and oxidative damage caused by exposure to xanthine/xanthinoxidase (500 microM/5 mU ml(-1); 1 h; =X/XO (dam)). The antioxidants vitamin E (10 microM) and 2-OH-estradiol (1 microM), present during the 15-min preconditioning period, completely abolished the protective effect of X/XO (pre). Furthermore, glutamate, staurosporine or X/XO (dam) markedly enhanced oxygen radical formation. Preceding preconditioning by mild ROS stimulation with X/XO (pre) or Fe(2+) reduced this oxygen radical burst. Again, the effect of X/XO (pre) could be blocked by coadministration of vitamin E or 2-OH-estradiol. However, the FeSO(4)-mediated preconditioning was not abolished by the radical scavengers. To address this phenomenon, the effect of vitamin E and 2-OH-estradiol on Fe(2+)- and X/XO (pre)-induced ROS formation kinetics within the 15 min of preconditioning was monitored. The moderate rise of intracellular ROS content during preconditioning was only reduced permanently by the antioxidants, when the neurons were treated with X/XO (pre), but not when Fe(2+) was used. Thus, an immediate and constant radical scavenging seems to be indispensable to abolish the ROS-induced neuronal preconditioning. The current results indicate that preconditioning by moderate ROS-stimulation protects cultured neurons against different damaging agents and prevents against the subsequent massive oxygen radical formation.
本研究旨在确定活性氧（ROS）在针对不同形式神经元损伤的预处理中的作用。鸡胚神经元原代培养物分别用硫酸亚铁（FeSO₄，100微摩尔；15分钟）处理以产生羟自由基，或用黄嘌呤/黄嘌呤氧化酶（10微摩尔/0.5毫单位/毫升；15分钟；=X/XO（预处理））处理以产生超氧阴离子自由基。通过荧光显微镜测量，两种刺激均适度增强了ROS的形成。这种预处理显著保护神经元免受随后的谷氨酸（1毫摩尔）诱导的兴奋性毒性损伤、星形孢菌素（200纳摩尔）诱导的神经元凋亡以及由暴露于黄嘌呤/黄嘌呤氧化酶（500微摩尔/5毫单位/毫升；1小时；=X/XO（损伤））引起的氧化损伤。在15分钟预处理期间存在的抗氧化剂维生素E（10微摩尔）和2-羟基雌二醇（1微摩尔）完全消除了X/XO（预处理）的保护作用。此外，谷氨酸、星形孢菌素或X/XO（损伤）显著增强了氧自由基的形成。用X/XO（预处理）或Fe²⁺进行轻度ROS刺激先于预处理可减少这种氧自由基爆发。同样，维生素E或2-羟基雌二醇的共同给药可阻断X/XO（预处理）的作用。然而，自由基清除剂并未消除硫酸亚铁介导的预处理。为了解决这一现象，监测了维生素E和2-羟基雌二醇在预处理的15分钟内对Fe²⁺和X/XO（预处理）诱导的ROS形成动力学的影响。仅当神经元用X/XO（预处理）处理时，抗氧化剂才会永久降低预处理期间细胞内ROS含量的适度升高，而使用Fe²⁺时则不会。因此，立即且持续的自由基清除似乎是消除ROS诱导的神经元预处理所必需的。目前的结果表明，适度ROS刺激的预处理可保护培养的神经元免受不同损伤剂的影响，并防止随后大量氧自由基的形成。
