Humanin抑制NR1亚基表达和NR2B亚基磷酸化的神经保护作用

Neuroprotective effects of Humanin by inhibiting expression of NR1 subunit and NR2B subunit phosphorylation

目的:探讨Humanin(HN)通过抑制N-甲基-D-天门冬氨酸(NMDA)受体NR1亚基的表达和NR2B亚基的磷酸化在兴奋性神经毒中发挥的保护作用,并阐明其可能的作用机制。方法:利用原代培养的Wistar大鼠皮层神经元建立NMDA诱导的兴奋性神经毒模型,将神经元分为对照组、NMDA组、NMDA+MK-801(MK-801)组和NMDA+HN(HN)组。流式细胞术荧光定量检测各组神经元膜NMDA受体NR1亚基蛋白的表达,Western blotting法检测各组神经元膜NMDA受体NR2B亚基Tyr1472位点的磷酸化水平。结果:倒置相差显微镜观察,NMDA组中的神经元胞体肿胀,个别细胞变成圆形,胞体周围光晕模糊,神经元突起大部分消失,表现出明显的神经毒性作用。流式细胞术免疫荧光定量检测,与对照组比较,NMDA组NR1亚基蛋白的平均荧光强度值明显增加(P<0.05);与NMDA组比较,MK-801组和HN组的NR1亚基蛋白的平均荧光强度值明显降低(P<0.05)。Western blotting法检测,各组神经元的NR2B亚基总蛋白的表达水平无改变;与对照组比较,NMDA组NR2B亚基蛋白Tyr1472位点的磷酸化水平明显升高(P<0.05);与NMDA组比较,MK-801组和HN组均能降低NR2B亚基蛋白Tyr1472位点的磷酸化水平(P<0.05)。结论:NMDA受体NR1亚基蛋白表达水平的增加和NR2亚基磷酸化可能参与NMDA诱导的兴奋性神经毒作用;HN可能通过抑制NR1亚基的表达和NR2B亚基的磷酸化而发挥对神经元的保护作用。

Objective To study the neuroprotective effects of Humanin（HN） on exciatory neurotoxicity based on inhibiting the expressions of NMDA receptor NR1 subunits and phosphorylation of NR2B subunits,and to explore the possible mechanisms.Methods Primary cultured cerebral cortical neurons of Wistar rats were used to set up excitatory neuotoxicity model and were divided into control group,NMDA group,NMDA＋MK-801（MK-801） group and NMDA＋HN group.The expressions of NR1 subunit proteins of NMDA receptors were detected by flow cytometry（FCM）.The levels of NR2B subunits of NMDA receptors phosphorylation at Tyr1472 in various groups were detected by Western blotting.Results The results of inverted phase contrast microscope showed that in NMDA group the cerebral cortical neurons were obvious neuronal loss,few cells became round and there was halofuzzy around the cells and the neurices were mostly gone,and the cells showed obvirous excitatory neurotoxicity.The mean fluorescence intensity value of NR1 subuint protein of NMDA receptor dsignificantly was increased compared with control group（P0.05）,the mean fluorescence intesity values of NR1 subunit protein were reduced in MK-801 group and HN group compared with NMDA group（P0.05）.The Western blotting results showed that there were no significant changes of total NR2B subunit proteins in various groups.The level of NR2B phosphorylation at Tyr1472 induced by NMDA was increased significantly compared with control group（P0.05）,while the pretreatment of HN（1 μmol·L-1） attenuated the level of phosphorylated of NR2B subunit in primary cultured rat cerebral cortical neurons compared with control group（P0.05）.Conclusion The up-regulation of the expression level of NR1 subuint protein of NMDA receptor and the increaing of the level of the phosphorylated of NR2B subunit might be responsible for the excitatory neurotoxicity induced by NNDA.HN might contribute to the neuroprotection against excitatory neurotoxicity by inhibiting the expression of NR1 subunits and reduction of phosphorylated of NR2B.