Ceccon M, Giusti P, Facci L, Borin G, Imbesi M, Floreani M, Skaper S D
Department of Pharmacology, University of Padova, 35131, Padova, Italy.
Brain Res. 2000 Apr 17;862(1-2):83-9. doi: 10.1016/s0006-8993(00)02074-6.
Glutathione (GSH) is a key component of the cellular defence cascade against injury caused by reactive oxygen species. Kainic acid (KA) is a potent central nervous system excitotoxin. KA-elicited neuronal death may result from the generation of ROS. The present study was undertaken to characterize the role of GSH in KA-induced neurotoxicity. Cultures of cerebellar granule neurons were prepared from 8-day-old rats, and used at 8, 14 and 20 days in vitro (DIV). Granule neurons displayed a developmental increase in their sensitivity to KA injury, as quantified by an ELISA-based assay with the tetrazolium salt MTT. At DIV 14 and 20, a 30-min challenge with KA (500 microM) reduced cell viability by 45% after 24 h, significantly greater (P<0.01) than the 22% cell loss with DIV 8 cultures. Moreover acute (30 min) KA exposure concentration-dependently reduced intracellular GSH and enhanced reactive oxygen species generation (evaluated by 2', 7'-dichlorofluorescein diacetate). In comparison to control, KA (500 microM) lowered GSH levels in DIV 8 granule neurons by 16% (P=0. 0388), and by 36% (P=0.0001) in both DIV 14 and DIV 20 neurons, after 30 min. Preincubation of granule neurons with the membrane permeant GSH delivery agent, GSH ethyl ester (5 mM), for 30 min significantly increased intracellular GSH content. Importantly, GSH ethyl ester reduced the toxic effects of KA, becoming significant at 1 mM (P=0.007 vs. KA-treated group), and was maximal at >/=2.5 mM (P<0.0001). GSH ethyl ester displayed a similar dose-dependence in its ability to counteract KA-induced depletion of cellular GSH. The data strengthen the notion that cellular GSH levels have a fundamental role in KA-induced neurotoxicity.
谷胱甘肽（GSH）是细胞防御由活性氧引起的损伤的级联反应中的关键成分。海人酸（KA）是一种强效的中枢神经系统兴奋性毒素。KA引发的神经元死亡可能源于活性氧的产生。本研究旨在阐明GSH在KA诱导的神经毒性中的作用。从小鼠制备小脑颗粒神经元培养物，于体外培养8、14和20天（DIV）使用。通过基于四唑盐MTT的ELISA测定法量化，颗粒神经元对KA损伤的敏感性呈现发育性增加。在DIV 14和20时，用KA（500 microM）刺激30分钟后，24小时细胞活力降低45%，显著高于DIV 8培养物中22%的细胞损失（P<0.01）。此外，急性（30分钟）KA暴露浓度依赖性地降低细胞内GSH水平并增强活性氧的产生（通过2'，7'-二氯荧光素二乙酸酯评估）。与对照相比，KA（500 microM）在30分钟后使DIV 8颗粒神经元中的GSH水平降低16%（P=0.0388），在DIV 14和DIV 20神经元中降低36%（P=0.0001）。用膜渗透性GSH递送剂谷胱甘肽乙酯（5 mM）预孵育颗粒神经元30分钟可显著增加细胞内GSH含量。重要的是，谷胱甘肽乙酯降低了KA的毒性作用，在1 mM时变得显著（与KA处理组相比，P=0.007），在≥2.5 mM时达到最大效果（P<0.0001）。谷胱甘肽乙酯在抵消KA诱导的细胞内GSH消耗的能力方面表现出类似的剂量依赖性。这些数据强化了细胞内GSH水平在KA诱导的神经毒性中起基本作用的观点。
