Gorina Roser, Sanfeliu Coral, Galitó Aida, Messeguer Angel, Planas Anna M
Department of Brain Ischemia and Neurodegeneration, Institut d'Investigacions Biomèdiques de Barcelona (IIBB)-Consejo Superior de Investigaciones Científicas (CSIC), Barcelona, Spain.
Glia. 2007 Oct;55(13):1313-24. doi: 10.1002/glia.20542.
The JAK/STAT pathway is activated in response to cytokines and growth factors. In addition, oxidative stress can activate this pathway, but the causative pro-oxidant forms are not well identified. We exposed cultures of rat glia to H2O2, FeSO4, nitroprussiate, or paraquat. We assessed oxidative stress by measuring reactive oxygen species (ROS) and oxidated proteins, we determined phosphorylated Stat1 (pStat1), and we evaluated the effect of antioxidants (trolox, propyl gallate, and N-acetylcysteine) and of Jak2 (Janus tyrosine kinases) inhibitors (AG490 and Jak2-Inhibitor-II). Pro-oxidant agents induced ROS and protein oxidation, excluding nitroprussiate that induced protein nitrosylation. H2O2, and to a lesser extent FeSO4, increased the level of pStat1, whereas nitroprussiate and paraquat did not. Trolox and propyl gallate strongly prevented ROS formation but they did not abolish H2O2-induced pStat1. In contrast, NAC did not reduce the level of ROS but it prevented the increase of pStat1 induced by H2O2, evidencing a differential effect on ROS formation and on Stat1 phosphorylation. H2O2 induced pStat1 in mixed glia cultures and, to a lesser extent, in purified astroglia, but not in microglia. Jak2 inhibitors reduced H2O2-induced pStat1, suggesting the involvement of this kinase in the increased phosphorylation of Stat1 by peroxide. Unexpectedly, AG490, but not Jak2-Inhibitor-II, reduced ROS formation, and it abrogated lipid peroxidation in microsomal preparations. Furthermore, AG490 reduced ROS in glial cells that were transfected with siRNA to silence Jak2 expression. These findings reveal previously unrecognized Jak2-independent antioxidant properties of AG490, and show that Jak2-dependent Stat1 activation by peroxide is dissociated from ROS generation.
JAK/STAT信号通路会因细胞因子和生长因子而被激活。此外，氧化应激也能激活该通路，但引发这一过程的促氧化剂形式尚未完全明确。我们将大鼠神经胶质细胞培养物分别暴露于过氧化氢（H2O2）、硫酸亚铁（FeSO4）、硝普盐或百草枯中。我们通过测量活性氧（ROS）和氧化蛋白来评估氧化应激，测定磷酸化Stat1（pStat1），并评估抗氧化剂（生育三烯酚、没食子酸丙酯和N - 乙酰半胱氨酸）以及Jak2（Janus酪氨酸激酶）抑制剂（AG490和Jak2 - 抑制剂 - II）的作用。促氧化剂会诱导ROS和蛋白质氧化，但硝普盐除外，它会诱导蛋白质亚硝化。H2O2以及在较小程度上的FeSO4会增加pStat1的水平，而硝普盐和百草枯则不会。生育三烯酚和没食子酸丙酯能强烈抑制ROS的形成，但它们并不能消除H2O2诱导的pStat1。相比之下，N - 乙酰半胱氨酸（NAC）不会降低ROS水平，但它能阻止H2O2诱导的pStat1增加，这表明其对ROS形成和Stat1磷酸化有不同的影响。H2O2在混合神经胶质细胞培养物中诱导pStat1，在纯化的星形胶质细胞中诱导程度较小，但在小胶质细胞中则不会。Jak2抑制剂能降低H2O2诱导的pStat1，这表明该激酶参与了过氧化物导致的Stat1磷酸化增加。出乎意料的是，AG490而非Jak2 - 抑制剂 - II能降低ROS的形成，并消除微粒体制剂中的脂质过氧化。此外，AG490能降低用小干扰RNA（siRNA）转染以沉默Jak2表达的神经胶质细胞中的ROS。这些发现揭示了AG490此前未被认识到的不依赖Jak2的抗氧化特性，并表明过氧化物通过Jak2依赖的方式激活Stat1与ROS生成是分离的。
