神经元限制性沉默因子REST/NRSF参与调控癫痫作用及机制研究

Role and mechanism of neuronal restriction silencing factor REST/NRSF in regulation of epilepsy

目的探究神经元限制性沉默因子(REST/NRSF)参与调控癫痫作用及分子机制。方法采用免疫组织化学染色、免疫荧光、Western blot和qPCR检测癫痫患者病灶组织和海人藻酸(kainic acid,KA)癫痫小鼠海马脑区REST/NRSF表达水平的变化;采用病毒注射,脑电图记录和行为学方法检测在海马CA1区分别敲低或过表达REST/NRSF后对小鼠癫痫发作的影响。结果癫痫患者病灶REST/NRSF表达水平相对于脑外伤患者脑组织明显升高;KA模型组小鼠海马CA1区REST/NRSF蛋白和mRNA水平均明显升高,Kv7.2、Kv7.3钾通道mRNA表达水平明显下调;脑内注射NMDA兴奋海马脑区小鼠REST/NRSF表达水平明显上调;海马CA1区敲低REST/NRSF明显升高Kv7.2、Kv7.3钾通道mRNA表达水平,明显降低小鼠脑电图棘波、尖波发放频率以及癫痫发作等级;海马CA1区过表达REST/NRSF明显降低Kv7.2、Kv7.3钾通道mRNA表达水平,明显升高小鼠棘波、尖波发放频率,癫痫症状明显加重。结论小鼠海马脑区REST/NRSF通过转录调控Kv7.2、Kv7.3钾通道参与癫痫疾病发生发展。

Aim To investigate the effect and role of neuronal restriction silencing factor(REST/NRSF)in epilepsy disorder.Methods Immunohistochemistry,immunofluorescence,Western blot and qPCR techniques were used to detect REST/NRSF expression levels in hippocampal tissues of mice induced by kainic acid and human brain tissue.Viral injections,EEG recordings and behavioral methods were used to test the effects on epileptic mice after knockdown and overexpression of REST/NRSF in the hippocampal CA1 region,respectively.Results The positive rate of REST/NRSF in the lesions of epileptic patients was significantly higher compared with that in the control group.The levels of REST/NRSF protein and mRNA in the CA1 region of the hippocampus of mice in the KA model group were significantly higher.Kv7.2 and Kv7.3 potassium channel mRNA expression levels were significantly down-regulated.Significant up-regulation of REST/NRSF expression levels was observed in mouse hippocampus after NMDA injection.Knockdown of REST/NRSF in the CA1 region of hippocampus significantly elevated the expression levels of Kv7.2 and Kv7.3 potassium channel mRNAs.The frequency of EEG spiking and sharp-wave issuance and epileptic seizure grade were significantly lower.Overexpression of REST/NRSF in the CA1 region of hippocampus significantly reduced the mRNA expression levels of Kv7.2 and Kv7.3 potassium channels.The frequency of EEG spiking and sharp-wave issuance was significantly higher and epileptic symptoms were exacerbated.Conclusion REST/NRSF in mouse hippocampal brain regions is involved in epileptic disease development through transcriptional regulation of Kv7.2 and Kv7.3 potassium channels.