海马神经元癫痫样放电后ERK1/2信号通路与c-fos的相关性

目的 研究海马神经元癫痫样放电后细胞外信号调节激酶(ERK1/2)与c-fos之间的相关性.方法 用无镁细胞外液建立海马神经元癫痫样放电模型,分为模型组、抑制剂组(加入10μmol/L U0126)和对照组(不作任何处理,只取0 min).运用免疫荧光双标标记,在激光共聚焦扫描显微镜下观察建模后30 min,磷酸化ERK1/2(p-ERK1/2)和c-fos在神经元内的分布;采用Western印迹检测p-ERK1/2和c-fos在相应处理后0、30 min,2、6、12和24 h的表达.结果 免疫荧光双标显示:癫痫样放电后30 min p-ERK1/2在神经元胞核与胞质内都有表达,而c-fos只在核内表达.Western印迹表明:模型组中各时间点p-ERK1/2都有表达,c-fos与P-ERK1/2变化趋势相似,30 min达峰值(1.849±0.059).抑制剂组中,P-ERK1/2表达完全被抑制,c-fos表达明显减少,且各时间点表达强度相近(30min时0.127±0.029),模型组与抑制剂组及对照组(0.241±0.030)在相应时间点之间比较差异有统计学意义(均P＜0.01).结论 海马神经元癫痫样放电后ERK1/2信号通路被持久激活,阻滞ERK1/2磷酸化,可以下调c-fos基因的表达.

Correlative research between ERK1/2 cascade and c-fos expression after hippocampal neuron epileptiform discharge

Objective To investigate the correlation between ERK1/2 cascade and c-fos after hippocampal neuron epileptiform discharge. Methods Hippocampal neurons were collected from Wistar rats and cultured in magnesium-free extra-cellular fluid to establish hippocampal neuron epileptiform discharge model. Then the hippocampal neurons were divided into 2 groups: inhibitor group, added with 10 μmol/L U0126, inhibitor of ERK 1/2 pathway, and control group without any treatment, got at 0 min). Using double-label immunofluorescence, laser scanning confocal microscopy was performed to detect the disposition of phosphorylated-ERKl/2(p-ERKl/2) and c-fos in the neurons. Western blotting was used to detect the expression of p-ERKl/2 and c-fos 0 min, 30 min, 2 h, 6 h, 12 h, and 24 h following corresponding treatment Results Double-label immunofluorescence examination displayed that there was p-ERKl/2 in both the cytoplasm and nucleus, but c-fos in the nucleus only. Western blotting manifested that p-ERKl/2 was observed at each time points in the model group, and the expression pattern of c-fos was the same as p-ERK1/2, which peaked 30 min after treatment In the inhibitor group, p-ERKl/2 was inhibited completely, and c-fos was decreased obviously. The c-fos expression levels at different time points of the inhibitor group were all significantly lower than those of the model group (all P <0. 01). Conclusion After hippocampal neuron epileptiform discharge, ERK1/2 is activated for a long time, and the expression of c-fos is down-regulated by blocking the phosphorylation of ERK1/2.