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 SHSY5Y 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).
MTT法检测SH-SY5Y细胞存活率; 测定乳酸脱氢酶释放量观察细胞损伤程度; DAPI染色法观察细胞凋亡形态学特点; 钙流法检测胞浆钙离子浓度变化; 以胞内谷胱甘肽、 超氧化物歧化酶活性和胞外丙二醛含量检测谷氨酸引发SH-SY5Y细胞的氧化应激状态。
谷氨酸导致SH-SY5Y细胞受损, 包括存活率下降、 乳酸脱氢酶释放量增多及形态结构发生改变; 谷氨酸处理20 min 后, 胞浆钙离子浓度无显著改变, 而处理24 h 后, 胞浆钙离子大量增加, 且MK801 (NMDA受体拮抗剂)及LY341495 (代谢型谷氨酸受体拮抗剂)均不能抑制钙离子内流的增多; 谷氨酸可导致SH-SY5Y氧化损伤, 包括胞内谷胱甘肽含量减少、 超氧化物歧化酶活性降低、 胞外脂质过氧化产物丙二醛水平升高等, 而丹参酮IIA (一种抗氧化剂)可减轻这些氧化损伤。