NMDAR“偶联”Kv4.2通道参与癫痫病理机制及“从肝论治”的干预作用

Despite the fact that there are a large number of available antiepileptic drugs (AEDs) in the market, the treatment of epilepsy remains unsatisfactory in terms of adverse effects.Thus, there is a growing demand for developing newer medications to treat epilepsy.Traditional Chinese Medicine (TCM) has been used for treatment epilepsy for a long time,and TCM that "Treatment from Gan"has well effects for epilepsy.Our studies found that TCM "Treatment from Gan" demonstrated antiepileptic activity in a variety of in vivo seizure models and could reduce NMDAR current amplitude, raised A-type potassium current amplitude.It was reported NMDAR and Kv4.2 was be closely related to the pathomechanism of epilepsy and can be used for clinical epilepsy treatment targets.In order to discuss NMDAR "couple" Kv4.2 channel 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 Immune living imaging 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 NMDAR, NR2B, Kv4.2 signal pathway such as CaMKII and KChIP1. To supply new evidence for the target-treatment epilepsy of "Treatment from Gan".

临床常用抗癫痫药有多种毒副作用，明确癫痫发病机制与高效、毒副作用小的药物的研发直接相关，NMDAR与钾通道与癫痫发病密切相关，可作为临床抗癫痫治疗靶点，文献报道NMDAR与钾通道可能存在相互调节作用，参与癫痫病理机制。中医药"从肝论治"癫痫临床疗效确切，课题组前期研究发现"从肝论治"有抗实验性癫痫作用、能降低NMDAR电流幅度、上调A-型钾电流幅度。为了探讨NMDAR与Kv4.2通道"偶联"参与癫痫病理机制及"从肝论治"的干预机制，课题组以建立大鼠锂-匹罗卡品癫痫模型及培养大鼠海马神经元为研究对象，选用免疫-活体成像技术、Western-blot技术、RT-PCR技术、膜片钳技术等，分别从行为学、分子生物学、电生理学角度观察"从肝论治"对NMDAR、NR2B亚单位、Kv4.2通道及所涉及的细胞内CaMKII、KChIP 1等信号通路的干预作用。为明确癫痫"从肝论治"的作用靶点提供实验依据。

癫痫是神经科常见疾病，临床常用抗癫痫药表现多种毒副作用，进一步明确癫痫发病机制，指导高效、毒副作用小的靶向药物的研制任重道远，中医药“从肝论治”癫痫临床疗效确切，课题组前期研究发现“从肝论治”有抗实验性癫痫作用，且能降低NMDAR电流幅度，上调A-型钾电流幅度。据报道NMDAR与钾通道与癫痫发病密切相关，可作为临床癫痫治疗靶点。为了探讨NMDAR与Kv4.2通道“偶联”机制参与癫痫病理机制及“从肝论治”的干预作用，课题组以建立大鼠锂-匹罗卡品癫痫模型及培养大鼠海马神经元为研究对象，选用免疫-活体成像技术、Western-blot技术、RT-PCR技术、膜片钳技术等，分别从行为学、形态学、分子生物学、电生理学角度显示了柴胡疏肝汤及SSa明显减少了癫痫发作次数、发作时间、降低了大鼠发作级别，并且改善了模型大鼠的一般状态；尼氏染色技术结果显示了柴胡疏肝汤及SSa增加了尼氏小体的表达，从而起到保护神经元的作用；免疫组化及WB、RT-PCR技术结果显示了柴胡疏肝汤及SSa能够下调NR2B和CamkⅡ的表达，降低神经元兴奋性；同时上调Kv4.2和KCHIP1蛋白的表达，上调A型钾电流抑制癫痫放电。为明确癫痫“从肝论治”的作用靶点提供实验依据。