Shanghai Institute of Brain Functional Genomics, East China Normal University, Shanghai 200062, China.
Neurosci Bull. 2010 Feb;26(1):8-16. doi: 10.1007/s12264-010-0813-7.
To investigate the mechanisms of excitotoxic effects of glutamate on human neuroblastoma SH-SY5Y cells.
SH-SY5Y cell viability was measured by MTT assay. Other damaged profile was detected by lactate dehydrogenase (LDH) release and by 4', 6-diamidino-2-phenylindole (DAPI) staining. The cytosolic calcium concentration was tested by calcium influx assay. The glutamate-induced oxidative stress was analyzed by cytosolic glutathione assay, superoxide dismutase (SOD) assay and extracellular malondialdehyde (MDA) assay.
Glutamate treatment caused damage in SH-SY5Y cells, including the decrease of cell viability, the increase of LDH release and the alterations of morphological structures. Furthermore, the concentration of cytoplasmic calcium in SH-SY5Y cells was not changed within 20 min following glutamate treatment, while cytosolic calcium concentration significantly increased within 24 h after glutamate treatment, which could not be inhibited by MK801, an antagonist of NMDA receptors, or by LY341495, an antagonist of metabotropic glutamate receptors. On the other hand, oxidative damage was observed in SH-SY5Y cells treated with glutamate, including decreases in glutathione content and SOD activity, and elevation of MDA level, all of which could be alleviated by an antioxidant Tanshinone IIA (Tan IIA, a major active ingredient from a Chinese plant Salvia Miltiorrhiza Bge).
Glutamate exerts toxicity in human neuroblastoma SH-SY5Y cells possibly through oxidative damage, not through calcium homeostasis destruction mediated by NMDA receptors.
研究谷氨酸对人神经母细胞瘤 SH-SY5Y 细胞兴奋毒性作用的机制。
通过 MTT 测定法检测 SH-SY5Y 细胞活力。通过乳酸脱氢酶（LDH）释放和 4'，6-二脒基-2-苯基吲哚（DAPI）染色检测其他损伤特征。通过钙内流测定法检测细胞质钙离子浓度。通过细胞质谷胱甘肽测定法、超氧化物歧化酶（SOD）测定法和细胞外丙二醛（MDA）测定法分析谷氨酸诱导的氧化应激。
谷氨酸处理导致 SH-SY5Y 细胞损伤，包括细胞活力下降、LDH 释放增加和形态结构改变。此外，谷氨酸处理后 20 分钟内 SH-SY5Y 细胞细胞质钙离子浓度没有变化，而谷氨酸处理后 24 小时内细胞质钙离子浓度显著增加，这种增加不能被 NMDA 受体拮抗剂 MK801 或代谢型谷氨酸受体拮抗剂 LY341495 抑制。另一方面，谷氨酸处理的 SH-SY5Y 细胞观察到氧化损伤，包括谷胱甘肽含量和 SOD 活性下降，MDA 水平升高，所有这些都可以被抗氧化剂丹参酮 IIA（丹参酮 IIA，一种来自中国植物丹参的主要活性成分）缓解。
谷氨酸对人神经母细胞瘤 SH-SY5Y 细胞产生毒性作用可能是通过氧化损伤，而不是通过 NMDA 受体介导的钙离子内稳态破坏。
