桃叶珊瑚苷对谷氨酸诱导的兴奋性神经毒性的抑制作用研究

Study on the Inhibitory Effect of Aucubin on Glutamate-Induced Excitatory Neurotoxicity

关键词:  桃叶珊瑚苷  N-甲基-D-天冬氨酸受体亚基  细胞内Ca²⁺水平  谷氨酸  PC-12

Key words:  aucucbin  N-methyl-D-aspartic acid receptor subunit  intracellular Ca²⁺ level  glutamate  PC-12

摘要:

目的 研究桃叶珊瑚苷对谷氨酸诱导的兴奋性神经毒性的抑制作用。方法 采用20 mmol·L^-1谷氨酸诱导PC-12细胞损伤建立神经毒性模型,加入1,5,10,20,40 μmol·L^-1的桃叶珊瑚苷处理24 h后,通过MTT法检测细胞活力,用流式细胞术检测细胞内Ca²⁺和活性氧(reactive oxygen species,ROS)水平,采用In Cell Western检测谷氨酸受体N-甲基-D-天冬氨酸受体(N-methyl-D-aspartic acid receptor,NMDA)亚基NMDAR1的水平,采用ELISA试剂盒检测细胞上清中超氧化物歧化酶(superoxide dismutase,SOD)、丙二醛(malondialdehyde,MDA)、乳酸脱氢酶(lactate dehydrogenase,LDH)水平。结果 谷氨酸20 mmol·L^-1作用24 h可使体外培养的PC-12细胞活力明显下降(P<0.01),细胞内Ca²⁺、NMDAR1蛋白水平和ROS、MDA、LDH水平明显增加(P<0.01),SOD水平明显降低。10 μmol·L^-1的桃叶珊瑚苷可以明显提高谷氨酸诱导后PC-12细胞的细胞活力(P<0.01),降低细胞内Ca²⁺、NMDAR1蛋白水平和ROS、LDH水平(P<0.01),升高细胞内SOD水平(P<0.01)。结论 桃叶珊瑚苷可能通过抑制NMDAR1的表达,从而降低细胞内Ca²⁺的堆积,并抑制氧化应激水平来改善谷氨酸诱导的PC-12细胞损伤,抑制谷氨酸诱导的兴奋性神经毒性。

Abstract:

OBJECTIVE To investigate the inhibitory effect of aucubin on glutamate-induced excitatory neurotoxicity. METHODS A neurotoxicity model was established using 20 mmol·L^-1glutamate to induce damage in PC-12 cells. After treatment with 1, 5, 10, 20 and 40 μmol·L^-1 aucubin for 24 h, cell viability was measured by MTT, intracellular Ca²⁺ and reactive oxygen species(ROS) levels were measured by flow cytometry, the level of glutamate receptor N-methyl-D-aspartic acid receptor(NMDAR1) were measured by In Cell Western. And superoxide dismutase(SOD), malondialdehyde(MDA), and lactate dehydrogenase(LDH) levels in cell supernatants were measured by using ELISA kits. RESULTS After treated with 20 mmol·L^-1glutamate for 24 h, the viability of PC-12 cells was significantly reduced(P<0.01), the levels of intracellular Ca²⁺, NMDAR1 ROS, MOA, LDH were significantly increased (P<0.01), the level of SOD was significantly reduced. 10 μmol·L^-1 aucubin could significantly increase the cell viability of PC-12 cells injured by glutamate(P<0.01), it could reduce the levels of intracellular Ca²⁺, NMDAR1, ROS and LDH(P<0.01), and elevate intracellular SOD level(P<0.01). CONCLUSION Aucubin may ameliorate glutamate-induced damage in PC-12 cells by inhibiting NMDAR1 expression, thereby reducing the accumulation of intracellular Ca²⁺, and inhibiting oxidative stress levels to suppress glutamate-induced excitatory neurotoxicity.