Glutamate oxidative injury to RGC-5 cells in culture is necrostatin sensitive and blunted by a hydrogen sulfide (H2S)-releasing derivative of aspirin (ACS14)
Glutamate oxidative injury to RGC-5 cells in culture is necrostatin sensitive and blunted by a hydrogen sulfide (H2S)-releasing derivative of aspirin (ACS14)
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DOI：
10.1016/j.neuint.2012.01.015
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发表时间：
2012-03-01
影响因子：
4.2
通讯作者：
Sparatore, Anna
中科院分区：
文献类型：
--
作者：
Osborne, Neville N.;Ji, Dan;Sparatore, Anna
通过暴露于谷氨酸（Glu）和丁硫氨酸-s， r -亚砜胺（BSO）的组合，培养的RGC-5细胞产生氧化应激。通过reazurin -reduction和dead/live测定对细胞存活的影响。此外，DNA的分解，磷脂酰丝氨酸和活性自由基（ROS）的定位及其定量测定。此外，采用Western blot、real - time PCR和免疫细胞化学方法对各种蛋白和mrna进行了研究。我们测试了ACS14及其硫化部分ACS1和阿司匹林对Glu/BSO对RGC-5细胞的负面影响的减弱能力。此外，还进行了试验，以确定这些物质是否影响谷胱甘肽（GSH）。Glu/BSO剂量依赖性杀死RGC-5细胞的机制涉及ROS的升高，伴随着DNA的分解、磷脂酰丝氨酸的表达和p38 MAPK的激活。该过程不受pan caspase抑制剂z-VAD-fmk的影响，不涉及凋亡诱导因子（AIF）的激活，但对活性坏死他汀-1敏感。在细胞活力研究（reazurin还原实验）中，ACS1和ACS14同样抵消了5 mM Glu/BSO对RGC-5细胞的负面影响，但阿司匹林仅在较轻的氧化应激（1 mM Glu/BSO）下有效。在所有其他试验中，ACS14在抵消5 mM Glu/BSO的影响方面比阿司匹林更有效。此外，ACS14和ACS1直接刺激GSH，而阿司匹林无效。此外，ACS14的神经保护作用被非特异性钾通道阻滞剂格列本脲特异性减弱。ACS14 （20 μ M）对5 mM Glu/BSO诱导的Bcl-2、HO-1和XIAP上调的抑制作用也比阿司匹林（20 μ M）更明显。这些数据表明，与阿司匹林相比，ACS14是一种非常有效的神经保护剂。ACS14保持阿司匹林的特性，并具有释放H2S的能力。阿司匹林与H2S以ACS14的形式联合多重作用在青光眼的治疗中值得考虑。(C) 2012 Elsevier Ltd.版权所有。
Oxidative stress to RGC-5 cells in culture was delivered by exposure to a combination of glutamate (Glu) and buthionine-S,R-sulfoximine (BSO). The effect of the insult on cell survival was quantified by the resazurin-reduction and a dead/live assays. Moreover, breakdown of DNA, the localisation of phosphatidylserine and reactive radical species (ROS) and its quantification were determined. In addition, various proteins and mRNAs were studied using Western blot, real time PCR and immunocytochemistry. ACS14, its sulfurated moiety ACS1 and aspirin were tested for their ability to blunt the negative effects of Glu/BSO on RGC-5 cells. In addition assays were carried out to see whether any of these substances influenced glutathione (GSH).Glu/BSO dose-dependently kills RGC-5 cells by a mechanism that involves an elevation of ROS accompanied by a breakdown of DNA, expression of phosphatidylserine and the activation of p38 MAPK. The process is unaffected by the pan caspase inhibitor z-VAD-fmk, does not involve the activation of apoptosis inducing factor (AIF) but is sensitive to active necrostatin-1. In cell viability studies (resazurin-reduction assay), ACS1 and ACS14 equally counteracted the negative effects of 5 mM Glu/BSO to RGC-5 cells but aspirin was only effective with a milder oxidative stress (1 mM Glu/BSO). In all other assays ACS14 was very much more effective than aspirin at counteracting the influence of 5 mM Glu/BSO. Moreover, ACS14 and ACS1 directly stimulated GSH while aspirin was ineffective. In addition the neuroprotecive effect of ACS14 was specifically blunted by the non-specific potassium channel blocker glibenclamide. Also the up-regulation of Bcl-2, HO-1 and XIAP induced by 5 mM Glu/BSO were all attenuated to a greater extent by ACS14 (20 mu M) than aspirin (20 mu M). These data show that ACS14 is a very effective neuroprotectant when compared with aspirin. ACS14 maintains its aspirin characteristics and has the ability to release H2S. The combined multiple actions of aspirin and H2S in the form of ACS14 is worthy to consider for possible use in the treatment of glaucoma. (C) 2012 Elsevier Ltd. All rights reserved.