星形胶质细胞条件培养基通过抑制 TNF-α 表达保护前额皮质神经元免受谷氨酸诱导的细胞死亡。

Astrocyte-Conditioned Medium Protects Prefrontal Cortical Neurons from Glutamate-Induced Cell Death by Inhibiting TNF-α Expression.

机构信息

Research Institute for Marine Drugs and Nutrition, Food Science and Technology, Guangdong Ocean University, Zhanjiang, China,

Graduate Institute of Biomedical Sciences, College of Medicine, China Medical University, and Departments of Medical Research, China Medical University Hospital, Taichung, Taiwan,

出版信息

Neuroimmunomodulation. 2019;26(1):33-42. doi: 10.1159/000495211. Epub 2019 Jan 30.

DOI:10.1159/000495211

PMID:30699428

Abstract

OBJECTIVE

Both excitotoxicity and neurotrophin deficiency may contribute to the etiology of depression and neurodegeneration. Astrocytes not only regulate glutamate metabolism and clearance, they also produce neurotrophins in the brain. However, the direct interaction between neurons and astrocytes remains unknown.

METHODS

This study evaluated the cellular mechanisms by which astrocyte-conditioned medium (ACM) protects prefrontal cortical neurons from glutamate-induced death by measuring cell viability and morphology as well as mRNA and protein expression of brain-derived neurotrophic factor (BDNF), BDNF receptors, glial cell line-derived neurotrophic factor (GDNF), and the proinflammatory cytokine, tumor necrosis factor (TNF)-α. Neurons and astrocytes were purified from the brains of neonatal 1-day-old Sprague-Dawley rats. ACM was harvested after exposing astrocytes to culture medium containing 100 μM glutamate for 48 h.

RESULTS

Glutamate insult (100 μM for 6 h) significantly reduced neuronal cell viability and increased the mRNA expression of BDNF. Glutamate (24 h) decreased neuronal viability and the expression of BDNF, but increased mRNA expression of GFAP, p75 neurotrophin receptor (p75NTR), and TNF-α. ACM pretreatment (2 h) reversed glutamate-decreased cell viability and increased BDNF, but reduced the expression of GDNF, P75NTR, and TNF-α at the mRNA level. Western blotting generally confirmed the mRNA expression following 24 glutamate insults. Furthermore, the glutamate-induced decrease in the protein expression of BDNF and full-length TrkB receptor and increase in pro-BDNF, truncated TrkB isoform 1 receptor, p75NTR, GDNF, and TNF-α were significantly attenuated by ACM pretreatment.

CONCLUSIONS

The study demonstrates that ACM exerts neuroprotective effects on cell viability, and this effect is most likely mediated through the modulation of neurotrophin and TNF-α expression.

摘要

目的

兴奋性毒性和神经营养因子缺乏都可能导致抑郁和神经退行性变的发生。星形胶质细胞不仅调节谷氨酸代谢和清除，而且在大脑中产生神经营养因子。然而，神经元和星形胶质细胞之间的直接相互作用仍然未知。

方法

本研究通过测量细胞活力和形态以及脑源性神经营养因子（BDNF）、BDNF 受体、胶质细胞源性神经营养因子（GDNF）和促炎细胞因子肿瘤坏死因子（TNF）-α的 mRNA 和蛋白表达，评估星形胶质细胞条件培养基（ACM）保护前额皮质神经元免受谷氨酸诱导死亡的细胞机制。神经元和星形胶质细胞从新生 1 天大的 Sprague-Dawley 大鼠脑中分离。星形胶质细胞暴露于含 100μM 谷氨酸的培养基中 48 小时后收获 ACM。

结果

谷氨酸刺激（6 小时 100μM）显著降低神经元细胞活力并增加 BDNF 的 mRNA 表达。谷氨酸（24 小时）降低神经元活力和 BDNF 的表达，但增加 GFAP、p75 神经营养因子受体（p75NTR）和 TNF-α的 mRNA 表达。ACM 预处理（2 小时）逆转了谷氨酸降低的细胞活力和增加的 BDNF，但降低了 GDNF、p75NTR 和 TNF-α的 mRNA 表达。Western blot 通常证实了 24 小时谷氨酸刺激后的 mRNA 表达。此外，ACM 预处理显著减弱了谷氨酸诱导的 BDNF 和全长 TrkB 受体蛋白表达的降低以及 pro-BDNF、截断的 TrkB 同工型 1 受体、p75NTR、GDNF 和 TNF-α的增加。