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目的:本研究应用原代培养星形胶质细胞,探讨炎症因子白细胞介素-1β(IL-1β)对星形胶质细胞抗氧化应激能力的影响。方法:在原代星形胶质细胞培养中分别加入1 ng/ml IL-1β,流式细胞仪检测不同作用时间(0、24、48、72 h)细胞线粒体膜电位改变,激光共聚焦显微镜观察活性氧(ROS)生成,分光光度法检测细胞内谷胱甘肽(GSH)含量,RT-PCR、Western Blot检测星形胶质细胞谷胱甘肽过氧化物酶(GSH-Px)的表达变化。结果:1 ng/ml IL-1β处理原代培养星形胶质细胞24~72 h,细胞线粒体膜电位无明显改变,细胞未出现明显损伤。IL-1β孵育24 h,细胞内ROS生成减少,GSH生成增加;48 h细胞内ROS生成增加,GSH较24 h组减低(P<0.05);72 h,原代培养星形胶质细胞内ROS生成明显增加,GSH较0 h组降低(P<0.05)。1 ng/ml IL-1β处理原代培养星形胶质细胞24 h,细胞内GSH-Px mRNA及蛋白水平较0 h组升高(P<0.05);但仅是一过性反应,IL-1β孵育72 h后,原代培养星形胶质细胞内GSH-Px mRNA及蛋白水平均较0 h组降低(P<0.05)。结论:持续炎性因子刺激下,星形胶质细胞抗氧化应激能力呈现动态改变,并且细胞内抗氧化物参与此变化过程。
Objective: In this study, primary cultured astrocytes were used to investigate the effect of interleukin-1β (IL-1β) on the antioxidant capacity of astrocytes. Methods: The primary astrocytes were cultured with 1 ng / ml IL-1β respectively. The changes of mitochondrial membrane potential (MIP) were observed by flow cytometry (FCM) at different time points (0, 24, 48 and 72 h) The content of glutathione (GSH) was determined by reactive oxygen species (ROS) generation and spectrophotometry. The expression of glutathione peroxidase (GSH-Px) in astrocytes was detected by RT-PCR and Western Blot . Results: Primary cultured astrocytes treated with IL-1β at 1 ng / ml for 24-72 h had no significant changes in mitochondrial membrane potential and no obvious cell damage. After incubated with IL-1β for 24 h, ROS generation decreased and GSH production increased; ROS production increased at 48 h, GSH decreased compared with that at 24 h (P <0.05); at 72 h, ROS production in primary cultured astrocytes The production of GSH was significantly higher than that of 0 h group (P <0.05). The level of GSH-Px mRNA and protein in primary cultured astrocytes treated with 1 ng / ml IL-1β for 24 h was higher than that in 0 h group (P <0.05), but only a transient response. IL- After 72 h incubation, the level of GSH-Px mRNA and protein in primary cultured astrocytes was lower than that in 0 h group (P <0.05). CONCLUSIONS: With the stimulation of persistent inflammatory cytokines, the antioxidant capacity of astrocytes changes dynamically, and intracellular antioxidants participate in this process.