全蝎提取物BmKIT2调控海马神经元电压门控钠通道Nav1.6抗癫痫发生的机制研究

The mechanism of temporal lobe epilepsy (TLE) is still unclear and the effect of clinical treatment is limited. It is reported that the gene mutation as well as abnormal expression of voltage-gated sodium channel Nav1.6 could induce the imbalance of neuronal calcium homeostasis, mediate neuron pyroptosis and cause status epilepticus. In clinical application of TCM, scorpion was found to have strong antiepileptic effects. In preliminary studies of the project, 13 scorpion components were extracted, and it was found that BmK IT2 could significantly inhibit Nav1.6-mediated persistent sodium currents, protect neurons and alleviate the status of epileptogenesis in mice. Therefore, it is speculated that BmK IT2 can regulate intracellular calcium signal of neurons by inhibiting Nav1.6 activity, prevent neuron pyroptosis pathway, and intervent epileptogenesis. This project aims to investigate the following subjects by using the TLE mouse model, Nav1.6 mutant V929F transgenic mice and Nav1.6 CKO conditional knockout mice combined with patch-clamp, SPR, calcium imaging, scanning electron microscopy, computational biology and so on: investigating the electrophysiological and behavioral effects of BmK IT2 on the epileptogenesis of TLE in mice; identifying the molecular structure information of Nav1.6 which been recognized by BmK IT2; regulating the mechanism of which BmK IT2 regulates intracellular free calcium concentration and the molecular integration mechanism of calcium signal and pyroptosis pathway. It provides a theoretical basis for analyzing the effect of scorpion "wind extinguishing and antispasmodic", and a possible molecular template for the design of antiepileptic drugs.

颞叶癫痫的发生机制尚不明晰，临床治疗效果有限。资料表明电压门控钠通道Nav1.6的基因突变与表达异常会诱发神经元钙稳态失衡，介导神经元焦亡造成癫痫持续状态。中医临床实践发现全蝎具备较强的抗癫痫作用。本项目前期提取了13种活性成分，其中组分BmK IT2可显著抑制Nav1.6介导的持续性钠电流，具有神经元保护、缓解小鼠癫痫发生的作用。故推测BmK IT2可通过下调Nav1.6活性，异化神经元胞内钙信号，阻断神经元焦亡途径，干预癫痫病状。为此，本项目拟利用颞叶癫痫小鼠、Nav1.6 V929F转基因癫痫小鼠及Nav1.6敲除小鼠模型结合膜片钳、脑电、钙成像、扫描电镜和计算生物学等手段，探讨BmK IT2干预小鼠颞叶癫痫发生的电生理与行为学效应；识别Nav1.6的分子结构信息；调控神经元钙信号与焦亡途径的分子整合机理。为解析全蝎“熄风止痉”的功效提供理论依据，为抗癫痫药物设计贡献可能的分子模板。

颞叶癫痫（TLE）发病机制复杂。中药全蝎作为我国治疗癫痫的经典药物，具有“熄风止痉、攻毒散结、通络止痛”的功效。本项目负责人所在团队率先发现了BmK IT2等多个全蝎抗癫痫活性提取物，为此我们利用TLE小鼠模型和Nav1.6 KO条件下的基因敲除小鼠，结合动物行为学、电生理学和计算生物学等技术，揭示：1）BmK IT2可有效延长小鼠癫痫发作的潜伏期，减少发作的总时程，抑制癫痫大发作的次数、降低癫痫的死亡率，并可显著抑制癫痫小鼠海马脑区各频率场电位的功率谱密度；2）BmK IT2能显著减少海马兴奋性标志物c-Fos的表达；同时尼氏染色结果发现给药组小鼠海马区神经元死亡情况显著优于模型组，提示BmK IT2在癫痫发生过程中对神经元具有保护作用；3）BmK IT2能够改善癫痫引起的小鼠学习记忆能力下降，自主探索能力下降及焦虑抑郁情绪；4）BmK IT2能够剂量依赖性的抑制Nav1.6介导的持久性钠电流，在分子水平解析了BmK IT2与Nav1.6的结构和相互作用，识别了BmK IT2可能作用于Nav1.6的关键结合位点Domain II 588D、598F与Domain III 1187E；5）BmK IT2通过选择性地抑制Nav1.6的活性来调节神经元的细胞内钙信号，减少小鼠海马区焦亡和炎症相关因子NLRP1、caspase-1、GSDMD和IL-18、IL-1β等表达，提示颞叶癫痫的发生于与神经元焦亡发生密切相关，BmK IT2可通过下调Nav1.6活性，降低神经元胞内钙信号，阻断神经元经典焦亡途径，干预癫痫发生。本项目为解析全蝎“熄风止痉”的功效提供了理论依据，为设计抗癫痫发生药物提供了一个可能的分子模板。