Objective To investigate the mechanisms of excitotoxic effects of glutamate on human neuroblastoma SH-SY5Y cells. Methods 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. Results 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）. Conclusion Glutamate exerts toxicity in human neuroblastoma SH-SY5Y cells possibly through oxidative damage, not through calcium homeostasis destruction mediated by NMDA receptors.
Objective This report aims to describe the oxidative damage profile in brain ofpresenilinl andpresenilin2 conditional double knockout mice （dKO） at both early and late age stages, and to discuss the correlation between oxidative stress and the Alzheimer-like phenotypes of dKO mice. Methods The protein level of Aβ42 in dKO cortex and free 8-OHdG level in urine were measured by ELISA. Thiobarbituric acid method and spectrophotometric DNPH assay were used to determine the lipid peroxidation and protein oxidation in cortex, respectively. SOD and GSH-PX activities were assessed by SOD Assay Kit-WST and GSH-PX assay kit, separately. Results Significant decrease of Aβ42 was verified in dKO cortex at 6 months as compared to control mice. Although lipid peroxidation （assessed by MDA） was increased only in dKO cortex at 3 months and protein oxidation （assessed by carbonyl groups） was basically unchanged in dKO cortex, ELISA analysis revealed that free 8-OHdG, which was an indicator of DNA lesion, was significantly decreased in urine of dKO mice from 3 months to 1 2 months. Activities of SOD and GSH-PX in dKO and control cortices showed no statistical difference except a significant increase of GSH-PX activity in dKO mice at 9 months. Conclusion Oxidative damage, especially DNA lesion, was correlated with the neurodegenerative symptoms that appeared in dKO mice without the deposition of Aβ42. Triggers of oxidative damage could be the inflammatory mediators released by activated microglia and astrocytes.
