ClC-3氯通道在药物难治性颞叶癫痫中的作用及其机制

Temporal lobe epilepsy(TLE) is the mostly common form of epilepsy, neuronal ectopic discharge in lesions caused by imbalance between excitatory and inhibitory circuit is the greatest reason which result to its onset. It has been reported that voltage-gated chloride channel 3(ClC-3) is broadly expressed in central nervous system and play important role in regulating chloride homeostasis and the release of neurotransmitter, some ClC-3 mutant mice even had spontaneous seizures. In our preliminary experiment, we found that ClC-3 is broadly expressed in human temporal lobe cortex, and the expression of ClC-3 was markedly reduced in the epilepsy lesions from TLE patients at mRNA and protein level, ClC-3 mediated chloride currents also down-regulated significantly. So we speculate that ClC-3 plays an important role in the development and maintenance of TLE. In the present study, with the use of the epileptic lesions from TLE patients and epileptic model in rodents induced by polocarpine, we will utilize molecular biotechnological, electrophysiological and siRNA interfere technology to investigate the pathological relationship between ClC-3 and TLE, and the underlying mechanism of which. This study will clarify the potential role and mechanism of ClC-3 in TLE, and help to understand the etiology of TLE and provide scientific basis for developing new anti-epileptic drug.

颞叶癫痫是最常见的癫痫，病灶神经元兴奋性和抑制性环路失衡导致的神经元异常放电是其发作的重要原因。已有报道发现，氯通道ClC-3在中枢神经系统大量表达，参与调节神经元胞内氯稳态和神经递质的释放，并且部分ClC-3敲除小鼠有自发癫痫发生。我们的预实验结果显示，ClC-3在人颞叶皮层大量表达，其mRNA水平和蛋白水平在颞叶癫痫患者较正常对照皮层显著降低，其介导的氯电流也显著减小。因此我们推测，ClC-3在颞叶癫痫的发生和维持中可能起重要作用。本项目拟利用颞叶癫痫患者的术中切除标本和匹鲁卡品诱导的颞叶癫痫动物模型，采用分子生物学、电生理和siRNA干扰技术等探讨ClC-3与颞叶癫痫中的病理关系及其机制，将有助于进一步了解颞叶癫痫的病因和为开发新的抗癫痫药物提供科学依据。

颞叶癫痫（Temporal lobe epilepsy, TLE）是最常见的癫痫，研究表明，病灶神经元胞内外氯稳态失衡导致其兴奋性异常升高，是癫痫发作的重要诱因，但关于引起氯稳态失衡的具体机制仍需进一步了解。利用临床顽固性TLE患者的术中切除标本，结合海人酸（Kainic acid, KA）诱导的TLE动物模型，我们在本项目中研究发现：与正常对照相比，电压门控性氯通道（Voltgage-gated chloride channels, ClC）介导的氯电流幅度在TLE患者病灶神经元上显著增高，是导致癫痫病灶神经元胞内氯离子蓄积的重要原因之一；氯离子通道阻断剂NPPB, DIDS则可显著抑制TLE病灶神经元上的电压激活性氯电流。膜片钳记录发现，NPPB, DIDS可超极化TLE病灶神经元的静息膜电位，进而降低其兴奋性，且该超极化幅度显著高于其对正常神经元静息膜电位的超极化幅度。高频振荡（High-frequency oscillations, HFOs, 80-500 Hz）表现为突触后电流的高度同步化，为TLE患者癫痫病灶的电生理特点之一，我们在行为学实验中发现：NPPB, DIDS可显著降低KA诱导的急性癫痫发作时海马CA3区HFOs（80-500 Hz）的功率，以及动物癫痫发作等级、强直发作持续时间和动物死亡率。ClC-3是ClC家族的亚型之一，电压激活后可介导一外向整流的电流。我们鉴定发现，ClC-3在人颞叶皮层神经元胞体和轴突上大量表达，而较少表达于星形胶质细胞和小胶质细胞；Western blot结果显示，与正常对照相比，ClC-3在TLE患者病灶的表达量显著升高，极有可能是介导癫痫病灶神经元胞内氯离子蓄积的ClC通道之一。相关性分析结果显示，ClC-3的表达水平与TLE患者癫痫发作时HFOs的发放频率呈显著正相关。因此，我们的结果表明，TLE癫痫病灶神经元上ClC功能异常升高致使神经元胞内氯离子蓄积，是导致TLE病灶神经元兴奋性异常升高和癫痫发作的重要原因之一；以ClC为靶标开发新的抗癫痫药物，极有可能为临床上顽固性癫痫的治疗提供新的思路。