钠氢交换体-1通过对钙信号调控参与难治性癫痫神经元凋亡机制的研究

Neuronal apoptosis is very important pathological basis of intractable epilepsy. However, the mechanism of these pathological changes caused by recurrent seizures is not clear. Our previous study found that the increasing expression of μ-calpain protein were closely related to neuronal apoptosis in brain tissue. We further found that the expression of sodium hydrogen exchanger 1 (NHE1) which was closely related to the calcium overload in brain specimen with intractable epilepsy increased. Peers have reported calcium overload was an important reason for the activation of calpain protein. Our hypothesis is that recurrent seizures lead to brain hypoxia and neuron acidosis. Then, it promotes the expression of NHE1 protein which result in excessive Na+ into the neurons. NHE1 will enhanced calcium concentratin through removing Na+ in neuron. Overloaded calcium can activate Calpain which cleave cytoskeletal protein or promote neuronal apoptosis through the Calpain signaling pathway. In this study, we explore the expression of NHE1 protein and gene on epileptic rats brain and epileptic cell model. The regulation mechanism of NHE1 by calcium signal pathway was further investigated. Then, the effect of analysis on the regulation of calcium signal pathway was evaluated by the mutation on two specific functional sites of NHE1. Finally, the NHE1 regulatory mechanisms on calcium signal pathwa in vivo will be explored. In all, our aim is to elucidate the pathogenesis of NHE1 in the course of intractable epilepsy. It will contribute to the development of antiepileptic drugs.

神经元凋亡是难治性癫痫重要病理基础，产生机制尚不清楚。本课题组发现反复痫性发作后与脑组织中神经元凋亡密切相关的μ-Calpain蛋白表达增高。进一步研究发现与神经元内钙超载密切相关的钠氢交换体1（NHE1）表达增高。而细胞内钙超载是μ-Calpain蛋白表达增高的重要原因。故我们推测：反复痫性发作后神经元缺氧酸中毒导致NHE1蛋白表达增高，通过排H+导致Na+过度进入神经元，钠钙交换体激活在排Na+过程中导致Ca2+过度进入胞内，钙超载诱导Calpain信号途径促进神经元凋亡。我们拟通过难治性癫痫大鼠及细胞癫痫模型分析NHE1蛋白及基因表达特点；进一步探讨上/下调NHE1表达对钙信号途径调控机制；再针对NHE1两个特定功能位点进行突变，分析对钙信号途径调控影响；最后在体试验验证NHE1对钙信号通路的调控机制。以期阐明难治性癫痫病程中NHE1致病机制，为抗癫痫药物研发提供依据。

神经元凋亡是难治性癫痫重要病理基础，产生机制尚不清楚。本课题组前期发现反复痫性发作后与难治性癫痫患者术后脑组织中神经元凋亡密切相关的μ-Calpain蛋白表达增高。进一步研究发现与神经元内钙超载密切相关的钠氢交换体1（NHE1）表达增高。从而建立NHE1参与神经元凋亡的研究假设。基于该假设课题组开展相关研究工作，并按要求完成相关任务。课题组成功制作癫痫细胞模型和动物模型，验证了NHE1表达特征，以及神经元凋亡的情况，建立了NHE1和神经元凋亡的关联。进一步通过上调 NHE1 表达和下调NHE1表达，证实NHE1通过Ca2+/ Calpain信号通路调节海马神经元凋亡的发生。进一步通过构建 NHE1 两个功能区突变体，转染神经元，探讨转染细胞信号通路变化，证实两个突变体对于Ca2+/ Calpain信号通路的影响，并通过在体实验进行了细胞学相关实验结果的验证。基于本课题研究结果，课题组丰富和发展反复癫痫发作后神经元凋亡的病理生理机制，从干预NHE1/Ca2+/ Calpain信号的视角为寻求难治性癫痫及相关疾病防治提供崭新的策略，为开发 NHE1 相关药物治疗癫痫提供新的实验证据。对于难治性癫痫患者，临床可以考虑加入NHE1抑制剂来达到干预癫痫病程进展的目的。另外通过本课题培养硕士研究生 3 名；博士研究生2名。目前发表中文期刊8篇，发表SCI论文4篇，贵州省医学科技奖奖项1项（贵州省医学会），贵州省自然科学奖1项（贵州省科技厅），在中华医学会神经病学分会大会做专题汇报1次，实用新型专利1项。