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目的探讨茶多酚(tea polypheonols,TP)对甲基汞(methylmercury,MeHg)所致大鼠大脑皮质神经元氧化损伤的防护作用及机制。方法进行大鼠大脑皮质神经元原代培养,细胞成熟后给予0.01、0.1、1、2μmol/L MeHg分别处理0.5、1、3、6、12 h,通过测定培养液中乳酸脱氢酶(lactate dehydrogenase,LDH)活力来进行MeHg细胞毒性分析;根据测定结果选择最具代表性的1μmol/L MeHg暴露6 h作为MeHg染毒组。应用同样方法进行TP预处理组选定,向培养液中分别加入终浓度为5、10、20及40μmol/L TP,分别预处理0.5、1、3及6 h后,再加入终浓度为1μmol/L MeHg,继续培养6 h后测定培养液LDH漏出,根据实验结果选定5、10、20μmol/L预处理3 h作为TP预处理剂量及时间;细胞经各剂量TP预处理后,再暴露于1μmol/L MeHg 6 h,测定神经元细胞凋亡率、非蛋白巯基(non-protein sulfhydryl,NPSH)含量、活性氧簇(reactive oxygen species,ROS)水平及Na+-K+-ATPase和Ca2+-ATPase活力。结果与对照组比较,随着染MeHg剂量的升高,培养液中LDH活力逐渐升高,呈现剂量和时间依赖性的效应关系。TP预处理后,LDH活力逐渐降低,在10、20μmol/L TP预处理组显著降低(P<0.05或P<0.01);1μmol/L MeHg导致神经元凋亡率显著升高,NPSH含量显著降低,ROS水平显著升高,Na+-K+-ATPase和Ca2+-ATPase活力显著降低,差异均有统计学意义(P<0.01),TP预处理对上述指标的拮抗作用呈现剂量-效应关系,差异均有统计学意义(P<0.05或P<0.01)。结论 TP对MeHg所致大鼠大脑皮质神经元氧化损伤具有一定的防护作用。
Objective To investigate the protective effect and mechanism of tea polypheonols (TP) on oxidative damage of cerebral cortex neurons induced by methylmercury (MeHg) in rats. Methods Primary cultured rat cerebral cortical neurons were cultured and treated with 0.01, 0.1, 1 and 2 μmol / L MeHg for 0.5, 1, 3, 6 and 12 h after maturation. The lactate dehydrogenase dehydrogenase (LDH) activity was used to analyze the cytotoxicity of MeHg. According to the results, the most representative 1 μmol / L MeHg was exposed for 6 h as MeHg exposure group. The same method was used to select the TP pretreatment group. The final concentrations of 5, 10, 20 and 40 μmol / L TP were added to the culture medium, respectively. After 0.5, 1, 3 and 6 h pretreatment, / L MeHg, continue to culture 6 h after the determination of LDH leakage of culture medium, according to the experimental results selected 5,10,20 μmol / L pretreatment 3 h as TP pretreatment dose and time; cells pretreated with each dose of TP before exposure The apoptosis rate, the content of non-protein sulfhydryl (NPSH), reactive oxygen species (ROS) and the levels of Na + -K + -ATPase and Ca2 + -ATPase in neurons were measured at 1μmol / L MeHg for 6 hours. vitality. Results Compared with the control group, the LDH activity in the culture medium increased gradually with the increase of the dose of MeHg, showing a dose-dependent and time-dependent effect. After pretreatment with TP, the activity of LDH decreased gradually, decreased in 10 and 20μmol / L TP preconditioning groups (P <0.05 or P <0.01). The apoptosis rate of neurons increased significantly and the content of NPSH decreased significantly (P <0.01). The antagonism of TP pretreatment on these indexes showed a dose-effect relationship, the difference was statistically significant (P <0.01), the level of ROS was significantly increased, the activities of Na + -K + -ATPase and Ca2 + -ATPase were significantly decreased Statistical significance (P <0.05 or P <0.01). Conclusion TP can protect MeHg-induced neuronal oxidative damage in the cortex of rats.