“从肝论治”难治性癫痫对KChIP/Kv4调控A型钾通道的干预研究

It is still unsatisfactory for the clinical treatment of refractory epilepsy. The potassium channel interacting proteins (KChIPs) modulate the structure and function of voltage-gated potassium channels 4 (Kv4) family subunits, control the excitability of neurons and is closely associate with epilepsy. According to TCM "Treatment from Gan" for epilepsy, our previous studies found that "Treatment from Gan" ChaiHuShuGanTang demonstrated antiepileptic activity in a variety of in vivo seizure models and could raised A-type potassium current amplitude. In order to discuss KChIP/Kv4 interaction involved in pathomechanism of epilepsy and the effect mechanism of "Treatment from Gan", We would select li-pilocarpine induced rat refractory epilepsy and culture the hippicampal neurons and use Immunohistochemical staining technology, Western blot technology, RT-PCR technology and Patch-Clamp technology to investigate the effect of TCM "Treatment from Gan" on the molecular mechanism of KChIPs, Kv4, KChIP/Kv4 and the regulative effects on A-type potassium channels. Supply experimental evidence for the target-treatment refractory epilepsy of "Treatment from Gan".

难治性癫痫的治疗仍是临床难点，钾通道相互作用蛋白（KChIPs）调节电压门控性钾通道4（Kv4）结构和功能，调控神经元兴奋性，与癫痫发病密切相关。依据癫痫“从肝论治”中医理论，课题组前期研究发现“从肝论治”复方柴胡疏肝汤有临床抗癫痫和抗实验性癫痫的作用、能够抑制大鼠海马神经元癫痫样放电、上调A型钾通道电流幅度。基于此为了进一步研究“从肝论治”难治性癫痫可能通过干预KChIP/Kv4对A型钾通道结构和功能的调节发挥抗癫痫作用。本课题拟以氯化锂-匹罗卡品诱导的难治性癫痫大鼠及培养的大鼠海马神经元为研究对象，选用荧光免疫组化技术、Western blot、PCR、膜片钳技术等从分子生物学及电生理学角度，观察癫痫大鼠海马神经元KChIPs及Kv4的分子表达特性、KChIP/Kv4对A型钾通道电生理特性的调控作用及“从肝论治”的干预作用。为难治性癫痫“从肝论治”的中医理论提供实验依据。

难治性癫痫的治疗仍是临床难点。钾通道相互作用蛋白（KChIPs）调节电压门控性钾通道4（Kv4）结构和功能，调控神经元兴奋性，与癫痫发病密切相关。前期研究发现“从肝论治”有抗实验性癫痫的作用、能够抑制海马神经元癫痫样放电、上调A型钾通道电流幅度。基于此，本研究首先以氯化锂-匹罗卡品诱导的难治性癫痫大鼠为研究对象，从行为学角度观察了“从肝论治”的抗癫痫作用，接着选用Western-blot技术、膜片钳等技术从分子生物学及电生理学等角度观察了“从肝论治”对难治性癫痫大鼠海马神经元KChIP1/Kv4.2蛋白表达及A型钾通道电生理活性的调节作用。结果提示柴胡疏肝汤及SSa能显著抑制颞叶癫痫大鼠慢性癫痫发作级别、延长癫痫发作潜伏期，能够抑制癫痫大鼠海马脑片海马区CA1区锥体神经元癫痫样放电，能上调癫痫大鼠海马区Kv4.2和KChIP1蛋白的表达，能升高Kv4.2介导的A型钾电流幅度。说明通过增加神经元KChIP1蛋白的表达从而上调Kv4.2蛋白表达，进而提高Kv4.2介导的A型钾电流幅度抑制癫痫发作是“从肝论治”的抗癫痫机制之一。