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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 investigate the effect of tea polypheonols (TP) on calcium overload and N-methyl-D-aspartate (N-methyl-D-aspartate) in rat cerebral cortical neurons induced by methylmercury (Me Hg) Antagonism and Mechanism of Abnormal Expression of Aspartic Acid and NMDA Receptors. Methods Primary cultured rat cerebral cortical neurons were cultured. After maturation, cells were treated with 0.01, 0.1, 1 and 2 μmol / L Me Hg Cl for 0.5, 1, 3, 6 and 12 h, respectively. Cell viability was measured by 1 μmol / Me Hg Cl exposure 6 h as Me Hg Cl exposure group TP pretreatment and other indicators determination. The cells were pretreated with TP for 5, 10 and 20 μmol / L for 3 h as the TP pretreatment group. The levels of ROS and free Ca2 +, the activities of calpain and the expressions of NR1, NR2A, NR2B m RNA and protein in neurons were measured. Results Compared with the control group, the viability of neurons decreased gradually with the dose of Me Hg Cl increasing, showing a dose-response relationship. The viability of neurons at 6 h exposure to 1 μmol / L Me Hg Cl was 53.15 %, Close to IC50. TP pretreatment, compared with 6 h exposure to 1 μmol / L Me Hg Cl, the viability of cells pretreated with 10 and 20 μmol / L TP significantly increased (P <0.05 or P <0.01). Me Hg Cl resulted in the increase of ROS, the intracellular free Ca2 + level and the increase of calpain activity, and the decrease of NR1, NR2A m RNA and protein expression (P <0.05 or P <0.01) Compared with 1 μmol / L Me Hg Cl group, the antagonism of these indexes showed a dose-effect relationship. Compared with the 1 μmol / L Me Hg Cl group, the level of ROS, intracellular free Ca 2+ and the activity of calpain decreased and the expression of NR 1, NR 2 A m RNA and protein increased There was statistical significance (P <0.05 or P <0.01). Conclusion TP can antagonize the neuronal toxicity, intracellular calcium overload and NMDA receptor abnormalities induced by Me Hg in rats.