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星形胶质细胞通过谷胱甘肽保护MN9D细胞抵抗鱼藤酮所致氧化应激

曹倩, 魏灵荣, 鲁玲玲, 赵春礼, 赵焕英, 杨慧

首都医科大学北京神经科学研究所,北京神经再生修复研究重点实验室,教育部神经变性病学重点实验室.北京 100069

摘要

星形胶质细胞维持神经元微环境，给予营养和代谢支持，并调节其对损伤的反应。鱼藤酮特异阻断线粒体复合物Ⅰ，长期暴露于鱼藤酮可能增加患帕金森病的几率，并引起帕金森综合征。然而，星形胶质细胞在鱼藤酮所致多巴胺能神经元损伤过程中的作用尚无报道。该研究采用多巴胺能神经元细胞系MN9D细胞模型，将经过或未经过星形胶质细胞条件培养基处理的MN9D细胞暴露于不同浓度的鱼藤酮中，用计数法测生长曲线，MTT法测细胞活性，DCFH染色流式细胞仪测氧化应激水平，比色法测还原型谷胱甘肽含量。结果显示，MN9D细胞在条件和普通培养基培养条件下生长曲线无明显差别；鱼藤酮浓度依赖性地降低细胞活性；不同浓度鱼藤酮作用24、48h后，经条件培养基处理的细胞其活性显著高于普通培养基培养的细胞：不同浓度的条件培养基都有保护作用，纯的条件培养基保护作用稍弱：预先24h条件培养基处理或同时给予鱼藤酮和条件培养基处理都有保护作用，鱼藤酮作用12h后再给予条件培养基则无保护作用；经条件培养基处理的细胞氧化应激水平降低：另外，条件培养基提高了细胞内还原型谷胱甘肽含量，缓解了鱼藤酮所致的谷胱甘肽耗竭。结果提示，星形胶质细胞可保护MN9D细胞抵抗鱼藤酮所致的氧化应激，还原型谷胱甘肽可能参与了该保护过程。

关键词： 星形胶质细胞; 氧化应激; 鱼藤酮; 谷胱甘肽

Astrocytes protect MN9D neuronal cells against rotenone--induced oxidative stress by a glutathione--dependent mechanism

Cao Qian, Wei Longrong, Lu Lingling, Zhao Chunli, Zhao Huanying, Yang Hui

Beijing Institute for Neuroscience, Capital Medical University,Beijing Center for Neural Regeneration and Repair,Key Laboratory for Neurodegenerative Disease of the Ministry of Education.Beijing 100069

Abstract

Astrocytes maintain homeostasis of neuronal microenvironment, provide metabolic and trophic support to neurons and modulate neuronal responses to injury. Rotenone specifically inhibits mitochondrial complex Ⅰ, and long exposure to rotenone may increase the risk for Parkinson's disease （PD） and cause Parkinsonism, However, little is known about the role of astrocytes in the process of rotenone-induced dopaminergic neuron injury. In order to investigate this issue, we used MN9D cells as a cell model of dopaminergic neurons and rotenone as a toxin to initiate mitochondrial deficiency. MN9D cells treated with the normal medium or astrocyte-conditioned medium （ACM） were exposed to different concentrations of rotenone for different time followed by cell viability measurement by MTT assay. Besides, various concentrations of ACM and temporally different treatments were devised to evaluate protective efficiency of ACM. Growth curve of cells in the normal medium or ACM was continuously assessed by cell counting for 8 d. The influence of rotenone and ACM on cellular oxidative stress was determined by DCFH-DA staining followed by flow cytometric analysis. Glutathione （GSH） content after treatment of ACM or rotenone was measured by GSH assay kit. Our results showed that rotenone decreased viability of MN9D cells in a dose-dependent manner and ACM treatment significantly attenuated rotenone toxicity at each concentration. No significant difference in growth rate was observed between the normal medium and ACM treatment. Four concentrations of ACM, namely 1/3ACM, 1/2ACM, 2/3ACM and pure ACM, all displayed protection, increasing cell viability to （124.15±0.79）%, （126.59±0.82）%, （125.84±0.61）% and （117.15±1.63）% of the cells exposed directly to rotenone, respectively. Treatment with ACM through the whole experiment except the initial 24 h, 24 h before or at the same time of rotenone addition all exerted protective effects, with cell viability being （110.11±2.52）%, （113.30±2.36）%, （114.42±2.00）% of the cells exposed directly to rotenone, respectively. Conversely, ACM treatment 12 h after rotenone addition had no protective effect, with cell viability being （102.54±1.36）% of the cells exposed directly to rotenone. Moreover, ACM treatment up-regulated GSH level in MN9D cells nearly twofold. Incubation with 100 nmol/L rotenone for 24 h depleted GSH level by nearly two thirds of the control, but ACM treatment mitigated the drop of GSH level, maintaining its content at （147.83±0.63）% of the control. Consistent with GSH change, rotenone administration resulted in a positive rate of 96.24% of DCF staining, implying a great extent of oxidative stress, whereas treatment with ACM reduced the extent of oxidative stress to a positive rate of 78.31%. Taken together, these findings suggest that astrocytes protect MN9D cells from oxidative stress caused by rotenone, and GSH partially accounts for the protection. Therefore, astrocytes may play a protective role in the process of PD.

Key words: astrocytes;oxidative stress;rotenone;glutathione

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引用本文：

曹倩, 魏灵荣, 鲁玲玲, 赵春礼, 赵焕英, 杨慧. 星形胶质细胞通过谷胱甘肽保护MN9D细胞抵抗鱼藤酮所致氧化应激[J]. 生理学报 2007; 59 (3): 253-259.

Cao Qian, Wei Longrong, Lu Lingling, Zhao Chunli, Zhao Huanying, Yang Hui. Astrocytes protect MN9D neuronal cells against rotenone--induced oxidative stress by a glutathione--dependent mechanism. Acta Physiol Sin 2007; 59 (3): 253-259 (in Chinese with English abstract).