电磁辐射对原代培养的皮层神经元中tau蛋白磷酸化的影响

Effect of electromagnetic radiation on tau protein phosphorylation in primarily cultured cortical neurons

目的研究电磁辐射对原代培养皮层神经元tau蛋白磷酸化的影响。方法原代培养大鼠皮层神经元分为假辐照组和辐照组,辐照组采用平均功率密度为90 mW/cm2的电磁波辐照10 min,分别于辐照后0、1、3、6、12 h取材。采用CCK-8检测电磁辐射辐照后神经元活性,Western blot检测tau蛋白磷酸化位点ser199/202、ser396、ser404磷酸化变化,同时观察细胞周期素依赖性蛋白激酶5(cyclin dependent kinase 5,CDK5)抑制剂Roscovitine、糖原合成激酶-3β(glycogen synthase kinase-3β,GSK-3β)抑制剂氯化锂(LiCl)和钙激活蛋白酶(calpain)抑制剂PD150606对电磁辐射暴露后tau蛋白磷酸化水平的影响。结果电磁辐射辐照后6 h和12 h,原代培养大鼠皮层神经元细胞活性明显降低[与假辐照组比较,分别下降19%(P<0.05)和30%(P<0.01)]。电磁辐射辐照后1 h和3 h,原代培养大鼠皮层神经元tau蛋白ser404位点磷酸化程度一过性增强[与假辐照组比较,分别升高89%(P<0.01)和46%(P<0.05)],而ser199/202、ser396位点磷酸化程度变化不明显(P>0.05)。Roscovitine、LiCl和PD150606对电磁辐射引起的tau蛋白ser404位点过度磷酸化有显著抑制作用(P<0.01)。结论电磁辐射可引起原代培养的皮层神经元tau蛋白ser404位点过度磷酸化,CDK5、GSK-3β和calpain参与其中。

Objective To determine the effect of electromagnetic radiation on tau protein phosphorylation in primarily cultured cortical neurons.Methods Primarily cultured rat cortical neurons were divided into sham radiation group and radiation group.Rats in radiation group were radiated with electromagnetic wave at the average power density of 90 mW/cm^2 for 10 min.Tissue samples were taken at 0,1,3,6 and 12 h after radiation.Viability of neurons was assayed with CCK-8,tau protein phosphorylation in primarily cultured neurons at ser199/202,ser396 and ser404 was detected by Western blotting,and effect of cyclin-dependent kinase5（CDK5） inhibitor Roscovitine,glycogen synthase kinase-3β（GSK-3β）inhibitor lithium chloride（LiCl）,and calpain inhibitor PD150606 on tau protein phosphorylation was detected after radiation.Results The viability of primarily cultured neurons was 19% and 30% lower respectively at 6 and 12 h after radiation in radiation group than in sham radiation group（P〈0.05,P〈0.01）.The tau protein phosphorylation level in primarily cultured neurons at ser404 was 89% and 46% higher respectively at 1 and 3 h after radiation in radiation group than in sham radiation group（P〈0.05）.No significant difference was found in tau protein phosphorylation level in primarily cultured neurons at ser199/202 and ser396 after radiation（P 0.05）.The inhibitory effect of Roscovitine,LiCl and PD150606 on tau protein phosphorylation in primarily cultured neurons at ser404 was lower in radiation group than in sham radiation group after radiation（P〈0.01）.Conclusion Electromagnetic radiation induces over tau protein phosphorylation in primarily cultured neurons at ser404 with involvement of CDK5,GSK-3β and calpain.