Abstract

The effects of tournefolic acid B (TAB) and two ester derivatives, TAB methyl ester (TABM) and TAB ethyl ester (TABE), on N-methyl-d-aspartate (NMDA)-mediated excitotoxicity and the underlying mechanisms were investigated. Treatment with 50 μM NMDA elicited neuronal death by 48.7 ± 5.1%, coinciding with the appearance of injured morphology. TABM (50 μM) attenuated the NMDA-induced cell death by 60.9 ± 19.7%, and to a lesser extent by TABE. The NMDA-mediated activation of calpain was not affected by TABM and TABE, as determined by the cleavage of α-spectrin. NMDA increased the activity of caspases 2, 3, 6, 8, and 9 and reached the maximum after 8-h treatment. TABM and TABE abrogated NMDA-induced activation of caspases 2, 3, 6, and 8 by approximately 80 to 90% and 50 to 60%, respectively, and to a higher extent for caspase 9. TABM and TABE also blocked the NMDA-mediated activation of caspase 12. Furthermore, TABM and TABE eliminated the NMDA-induced accumulation of superoxide anion (). NMDA evoked significant depolarization of mitochondria, whereas TABM elicited a mild decrease of mitochondrial membrane potential as determined by tetramethylrhodamine methyl ester perchlorate. NMDA treatment induced elevation of Ca²⁺ levels in cytosol, endoplasmic reticulum (ER), and mitochondria. TABM (50 μM) significantly diminished the NMDA-induced elevation of Ca²⁺ levels in mitochondria and ER but not cytosol. Therefore, TABM decreased mitochondrial membrane potential and attenuated the NMDA-mediated Ca²⁺-loading in ER and mitochondria. These events subsequently eliminated the accumulation of and blocked the activation of caspase cascade, thereby conferring their neuroprotective effects on NMDA-mediated excitotoxicity.