茶多酚对甲基汞致大鼠脑皮质神经元钙超载及N-甲基-D-天冬氨酸受体异常表达的拮抗作用

Protective effects of tea polyphenols against methylmercury-induced intracellular calcium overloading and alterations of N-methyl-D-aspartic acid receptor expression in rat primary cultured cortical neurons

目的探讨茶多酚(tea polypheonols,TP)对甲基汞(methylmercury,Me Hg)所致大鼠大脑皮质神经元钙超载及N-甲基-D-天冬氨酸(N-methyl-D-aspartic acid,NMDA)受体异常表达的拮抗作用及机制。方法进行大鼠大脑皮质原代神经元培养,细胞成熟后给予0.01、0.1、1、2μmol/L Me Hg Cl分别染毒0.5、1、3、6、12 h,通过测定细胞活力选择1μmol/L Me Hg Cl暴露6 h作为Me Hg Cl染毒组进行TP预处理及其他指标测定。通过测定细胞活力选择5、10、20μmol/L TP预处理3 h作为TP预处理组,并测定神经元内ROS和游离Ca2+水平、钙蛋白酶活力及NR1、NR2A、NR2B m RNA和蛋白表达水平。结果与对照组比较,随着Me Hg Cl染毒剂量的升高,神经元细胞活力逐渐降低,呈剂量-效应关系,其中1μmol/L Me Hg Cl暴露6 h组的细胞活力为对照组的53.15%,接近IC50。TP预处理后,与1μmol/L Me Hg Cl暴露6 h组比较,10、20μmol/L TP预处理组细胞活力明显升高(P<0.05或P<0.01)。Me Hg Cl导致神经元ROS、细胞内游离Ca2+水平及钙蛋白酶活力升高,NR1、NR2A m RNA及蛋白表达水平降低,差异均有统计学意义(P<0.05或P<0.01);TP预处理对上述指标的拮抗呈剂量-效应关系,与1μmol/L Me Hg Cl组比较,神经元ROS、细胞内游离Ca2+水平及钙蛋白酶活力降低,NR1、NR2A m RNA及蛋白表达水平升高,差异均有统计学意义(P<0.05或P<0.01)。结论 TP对Me Hg所致大鼠脑皮质神经元毒性、细胞内钙超载及NMDA受体异常表达均有一定的拮抗作用。

Objective To explore the protective effects of tea polyphenols（TP） against methylmercury（Me Hg）-induced neuronal Ca2 ＋overloading and alterations of N-methyl-D-aspartic acid（NMDA） receptor expression in rat primary cultured cortical neurons. Methods The primary cultured cortical neurons were exposed to 0.01, 0.1, 1 and 2 μmol/L Me Hg Cl for 0.5, 1, 3, 6and 12 h, respectively. After cell viability quantification, we selected 1 μmol/L Me Hg Cl treated for 6 h as the Me Hg treatment group, for TP pre-treatment and other indicators evaluation. In addition, we selected 5, 10, 20 μmol/L TP pre-treatment for 3 h as TP pre-treatment groups for investigation of neuronal ROS formation, intracellular free Ca^2＋levels,calpain activity, and NR1,NR2 A, NR2 B m RNA and protein expressions. Results The cell viability decreased in a dose- and time-dependent manner after Me Hg Cl exposure with different concentration and time course, when comparing with those in control. The cell viability was 53.15% of control in the 1 μmol/L Me Hg Cl treatment for 6 h. TP pre-treatment resulted in increased cell viability with a dose- and time-dependent manner, which was significant in 10 and 20 μmol/L TP pre-treatment for 3 h（P〈0.05 or P〈0.01）,relative to those in 1 μmol/L Me Hg Cl group. In addition, Me Hg Cl caused an obvious elevation of neuronal ROS formation,intracellular free Ca^2＋levels, and calpain activity, as well as a decrease in NR1, NR2 A m RNA and protein expressions（P〈0.05 or P〈0.01）. TP pre-treatment could partially antagonize these toxic effects in a dose-dependent manner（P〈0.05 or P〈0.01）.Conclusion TP has the abilities to prevent Me Hg-induced neuronal toxicity and intracellular Ca^2＋overloading, as well as the alterations of NMDA receptors expression.