ENT1介导的p38MAPK信号通路参与神经胶质互动在癫痫形成中的机制研究

Epilepsy is closely related to the imbalance of the neurotransmitter. Equilibrative nucleoside transporter-1 (ENT1) regulates the level of intracellular and extracellular inhibitory neurotransmitter adenosine due to changes of the micro environment. A lot of literature reveals the adenosine level is regulated by neuroglia interaction， and to adjust the adenosine level is effective for epilepsy treatment, but its specific molecular signaling mechanism is not clear. Recent studies have shown that p38 mitogen activated protein kinase（p38MAPK） is an upstream intervention factor of ENT1, and the applicant's preliminary work found an upregulation of p38MAPK phosphorylation after epilepsy. There is no literature about ENT1 mediated by p38MAPK signaling pathway involves in neuroglia interaction leading to epilepsy formation. The project intends to build the ENT1 lentiviral plasmid to conduct function research by observing the rat electroencephalogram (EEG)，behavior, using patch clamp to monitor electrophysiological changes in the brain slice, and detecting related molecular expression in the neurons and astrocytes by molecular biology technology. The ENT1 change will be observed by using p38MAPK specific inhibitor, and the brain ENT1, p38MAPK and related protein expression will be compared between epilepsy patient and non epilepsy patient. The applicant expect to reveal epilepsy mechanisms from the molecular signaling perspective, and provide a new target for the prevention and treatment of epilepsy.

癫痫形成与神经递质的平衡失调密切相关。1型平衡型核苷转运体（ENT1）可因微环境的变化而调节细胞内外抑制性神经递质腺苷的水平。大量文献揭示腺苷水平是通过神经元和星形胶质细胞互动调节完成，且调节腺苷水平对治疗癫痫有效，但其发生的具体分子信号机制尚不清楚。近期研究显示p38丝裂原激活蛋白激酶是ENT1的上游干预因子，申请人的前期工作发现癫痫发作后p38MAPK磷酸化水平升高。ENT1是否介导p38MAPK信号通路参与神经胶质互动导致癫痫形成尚无文献报道。本项目拟构建ENT1慢病毒质粒对其进行功能研究，观察动物脑电行为以及膜片钳监测脑片电生理的变化，分子生物学技术检测神经元和星形胶质细胞相关分子的表达；观察p38MAPK特异性抑制剂使用前后ENT1变化情况；最终比较癫痫和非癫痫患者脑组织中ENT1、p38MAPK及相关蛋白的表达，从分子信号机制的角度揭示癫痫形成机制，期望为癫痫的防治提供新靶点。

研究背景：本课题以CREB信号转导通路为切入点，探讨ENT1参与癫痫发作的功能与机制。.主要研究内容：1.检测临床难治性癫痫患者及大鼠癫痫模型中p38MAPK 及ENT1表达情况；2.在癫痫大鼠中通过ENT1特异性抑制剂NBTI干预其表达并观察急性期大鼠癫痫发作潜伏期及发作程度，检测干预后鼠海马组织p-CREB表达情况；3.离体海马脑片中通过干预ENT1后，膜片钳检测海马CA1区锥体神经元兴奋性及兴奋性突触后电流情况，测定ENT1是否通过腺苷A1受体调控神经递质在突触间的传递，影响神经元兴奋性及突触后电流情况。.结果：1. p38MAPK 及ENT1在难治性颞叶癫痫患者脑标本及癫痫大鼠海马中明显升高；ENT1主要表达于癫痫大鼠海马神经元与星形胶质细的胞膜和胞浆；p-p38 MAPK广泛表达于海马神经元中。2. NBTI海马立体定位注射及外周注射干预后可明显延长大鼠匹罗卡品诱发的首次癫痫发作潜伏期，降低大鼠癫痫发作程度，并且可抑制癫痫大鼠海马内CREB的磷酸化；而定位注射 p38MAPK特异性抑制剂SB203580可明显抑制癫痫大鼠海马 ENT1蛋白表达。3.在离体癫痫大鼠海马脑片膜片钳试验中，NBTI可模拟腺苷降低eEPSCs幅值，这效应能被腺苷A1受体抑制剂DPCPX所逆转，同时可降低癫痫大鼠海马脑片CA1区锥体神经元mEPSC频率，并且能抑制癫痫大鼠AMPAR和NMDAR两种受体介导的eEPSC幅值。.结论：ENT1通过腺苷A1受体调控神经递质在突触间的传递，从而介导CREB的磷酸化参与癫痫的形成。.成果：已有SCI 文章2篇，中文期刊文章6篇，其他数据已整理成文投递SCI杂志，有望在2017年发表。.意义：本项目通过干预p38 MAPK及ENT1表达，结合大鼠行为学、形态学、分子生物学及电生理学等技术，在癫痫患者脑组织标本、癫痫动物模型及离体癫痫脑片模型中探讨ENT1与癫痫的关系，进而从分子角度揭示癫痫的发病机制。本研究成果有望为癫痫的临床治疗提供新的方案和药物靶点。