Study The Effect Of Ionizing Radiation On Primary Hippocampal Neurons NFATc4/3 Signaling Pathway

Part one:Investigate the effects of ionizing radiation on primary hippocampal neurons in the role of NFATc4/3 signaling pathway in vitroObjective: Preliminary experiments show that ionizing radiation inhibited neurite growth, and NFATc4/3 signaling pathway is important for neuronal growth, synapse formation and neuronal survival.This study is to investigate the effect of radiation on primary hippocampal neurons in the role of NFATc4/3 signaling pathway in vitro.Methods: To explore effects of radiation on primary hippocampal neuronal death by MTT method; the use of Q-PCR(Quantitative Real-time PCR) analyses the change of BDNF in gene level in neurons; Western-Blot was used to measure the changes in protein level of NFATc4/3, phosphorylation of NFATc4/3(P-NFATc4/3), glycogen synthase kinase-3β(GSK-3β) and calcineurin.Result: MTT experimental results showed that in the first day and third day after irradiation, as the dose increased, primary hippocampal neuronal death increased, the difference was statistically significant(one-way ANOVA, first day and third day, P<0.0001), but the difference was not statistically significant between 4 Gy, 8 Gy, 10 Gy and 12 Gy groups(one-way ANOVA, first day, P=0.5188; third day, P=0.3428); namely, more than a certain dose after radiation, the primary hippocampal neuronal death has no dose-dependent. Q-PCR results showed that hippocampal neurons after radiation in first day and third day, with the dose increasing, the expression of BDNF in m RNA level gradually decreased, the difference was statistically significant(one-way ANOVA, first day, P<0.0001; third day, P=0.001). Western Blot results showed hippocampal neurons after irradiation in the first day and third day, with dose increases, dephosphorylation of NFATc4/3 decreased, the difference was statistically significant(one-way ANOVA, first days, P=0.033; third day, P=0.015). Phosphorylated NFATc4/3(P-NFATc4/3), with dose increases, P-NFATc4/3 increased, the difference was statistically significant(one-way ANOVA, first day, P=0.035; third day, P=0.022). But GSK-3β and calcineurin, whether it is in the first day or third day after irradiation, the difference was not statistically significant(one-way ANOVA, first day, GSK-3β, P=0.997; calcineurin, P=0.913; third day, GSK-3β, P=0.996; calcineurin, P=0.923).Conclusion: Radiation can inhibit the NFATc4/3 signaling pathway on primary hippocampal neurons.Part two: BDNF and cyclosporin A regulation the effect of ionizing radiation onprimary hippocampal neurons NFATc4/3 signaling pathwayObjective: To use of brain-derived neurotrophic factor(BDNF) and cyclosporine A(Cs A) regulation of ionizing radiation on primary hippocampal neural in the role of NFATc4/3 signaling pathway in vitro.Methods: Q-PCR(Quantitative real-time PCR) analysis the changes of BDNF in m RNA level on primary hippocampal neurons which co-culture with BDNF and Cs A after radiation.Western-Blot was used to measure the changes in protein level of NFATc4/3, phosphorylation of NFATc4/3(P-NFATc4/3), glycogen synthase kinase 3β(GSK-3β) and calcineurin on primary hippocampal neurons which co-culture with BDNF and Cs A after radiation.Results: Q-PCR results showed that, when co-culteur with BDNF and Cs A in the first day after irradiation, in 0 Gy, 2 Gy, 8 Gy group, only 8 Gy group difference was not statistically significant(one-way ANOVA, 0 Gy, P<0.0001; 2 Gy, P<0.0001; 8 Gy, P=0.087). When co-culture with BDNF and Cs A in the third day after irradiation, in 0 Gy, 2 Gy, 8 Gy group, the difference was not statistically significant(one-way ANOVA, 0 Gy, P=0.130; 2 Gy, P=0.058; 8 Gy, P=0.140). Western Blot results showed that in the first day and third day on primary hippocampal neurons which co-culture BDNF and Cs A after radiation, the changs in protein level of dephosphorylation of NFATc4/3, the differences between all the groups were statistically significant(one-way ANOVA, first day, 0 Gy, P=0.002; 2 Gy, P=0.001; 8 Gy, P= 0.001; third day, 0 Gy, P<0.0001; 2 Gy, P=0.001; 8 Gy, P<0.0001). Within each group, after adding BDNF, the expression of dephosphorylation of NFATc4/3 in protein levels were increased; after adding Cs A, expresson of dephosphorylation of NFATc4/3 in protein levels were decreased, the differences were statistically significant. The expression of P-NFATc4/3 at protein level is opposite with NFATc4/3. Both in the first day or the third day after irradiation, the difference between all the groups were statistically significant(one-way ANOVA, first day, 0 Gy, P<0.0001; 2 Gy, P=0.001; 8 Gy, P<0.0001; third day, 0 Gy, P=0.002; 2 Gy, P=0.002; 8 Gy, P=0.001). After joining BDNF, the expression of P-NFATc4/3 in protein levels were decrease; after joining Cs A, expresson of P-NFATc4/3 in protein levels were increased, the differences were statistically significant. For GSK-3β and calcineurin, whether it is in the first day or the third day after irradiation which co-cultured with BDNF and Cs A, the differences between the groups were not statistically significant(one-way ANOVA, first day, GSK-3β, 0 Gy, P=0.378; 2 Gy, P=0.751; 8 Gy, P=0.333; calcineurin, 0 Gy, P=0.864; 2 Gy, P=0.660; 8 Gy, P=0.121; 3d, GSK-3β, 0 Gy, P=0.691; 2 Gy, P=0.263; 8 Gy, P=0.818; calcineurin, 0 Gy, P=0.895; 2 Gy, P=0.082; 8 Gy, P=0.526).Conclusion: By the adding of promoter BDNF and inhibitors Cs A can regulation the effect of ionizing radiation on primary hippocampal neurons in the role of NFATc4/3 signaling pathway,the adding of BDNF can relieve the radiation suppression effect on NFATc4/3 signal pathway, which may provide a potential therapeutic approach for clinical.