Wang Chuen-Neu, Pan Hsien-Chia, Lin Yun-Lian, Chi Chih-Wen, Shiao Young-Ji
National Research Institute of Chinese Medicine, No. 155-1. Sec. 2, LiNung St., Peitou, Taipei, Taiwan, Republic of China.
Mol Pharmacol. 2006 Mar;69(3):950-9. doi: 10.1124/mol.105.018770. Epub 2005 Dec 19.
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 microM NMDA elicited neuronal death by 48.7 +/- 5.1%, coinciding with the appearance of injured morphology. TABM (50 microM) 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 alpha-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 (O2-). 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 microM) 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 O2- and blocked the activation of caspase cascade, thereby conferring their neuroprotective effects on NMDA-mediated excitotoxicity.
研究了表叶酸B（TAB）及其两种酯衍生物，即TAB甲酯（TABM）和TAB乙酯（TABE）对N-甲基-D-天冬氨酸（NMDA）介导的兴奋性毒性作用及其潜在机制。用50微摩尔/升的NMDA处理可导致48.7±5.1%的神经元死亡，同时伴有损伤形态的出现。TABM（50微摩尔/升）使NMDA诱导的细胞死亡减少了60.9±19.7%，TABE的作用程度较小。通过α-血影蛋白的裂解测定，NMDA介导的钙蛋白酶激活不受TABM和TABE的影响。NMDA增加了半胱天冬酶2、3、6、8和9的活性，并在处理8小时后达到最大值。TABM和TABE分别使NMDA诱导的半胱天冬酶2、3、6和8的激活减少了约80%至90%和50%至60%，对半胱天冬酶9的作用程度更高。TABM和TABE还阻断了NMDA介导的半胱天冬酶12的激活。此外，TABM和TABE消除了NMDA诱导的超氧阴离子（O2-*）积累。NMDA引起线粒体显著去极化，而用四甲基罗丹明甲酯高氯酸盐测定，TABM引起线粒体膜电位轻度下降。NMDA处理导致细胞质、内质网（ER）和线粒体中Ca2+水平升高。TABM（50微摩尔/升）显著降低了NMDA诱导的线粒体和ER中Ca2+水平的升高，但对细胞质中Ca2+水平无影响。因此，TABM降低了线粒体膜电位，减弱了NMDA介导的ER和线粒体中的Ca2+负载。这些事件随后消除了O2-*的积累并阻断了半胱天冬酶级联反应的激活，从而赋予它们对NMDA介导的兴奋性毒性的神经保护作用。
