基于“治痰为先”探讨柴胡加龙骨牡蛎汤调控颞叶癫痫海马神经元焦亡的机制

The pathogen of the Temporal lobe epilepsy (TLE) is related to the phlegm mainly, and it will exist through the entire course of TLE. The treatment to TLE should be focused on the phlegm firstly. Our previous study found that, the expression of NLRP3, caspase-1 and IL-1 increased in the hippocampus of TLE patients, and Chaihu-longgu-muli decoction (CLMD) could reduce the epileptiform discharge, thus relieve the symptoms of epilepsy. It has been reported that the activation of NLRP3 inflammasome is the key to regulate the pyroptosis. The bioinformatics analysis showed miR-146a may regulate pyroptosis by adjusting TLR signal pathway. Therefore, we suppose that the mechanism of TLE on the state of phlegm is associated with the hippocampal pyroptosis induced by inflammation, and CLMD may adjust the TLR4 signal pathway induced by miR-146a, inhibit the activation of NLRP3 inflammasome, down-regulate expression of different inflammatory factor, affect the hippocampal pyroptosis, thus improve the TLE symptoms. In the present study, the hippocampal neurons will be the research target. Detection methods include syndrome differentiation, macroscopic assessment of Chinese Medicine, EEG, RT-PCR, Western-blot, patch clamp, confocal microscopy, electron microscope, etc. This study will be carried out with the aspects of clinical study, overall level and experiment in vitro. The result will offer novel treatment methods and targets for TLE with TCM.

颞叶癫痫（TLE）病因主要责之于痰，痰邪贯穿疾病始终，治疗当以“治痰为先”。我们前期研究发现：TLE患者海马NLRP3、caspase-1和IL-1表达升高，柴胡加龙骨牡蛎汤（CLMD）通过减少痫样放电控制癫痫发作。研究证实NLRP3炎症小体的活化是调控神经元细胞焦亡的核心环节。生物信息学分析显示，miR-146a可通过介导TLR受体通路调控神经元焦亡。我们推测：“痰”状态下TLE 的病理机制与炎症反应导致海马神经元焦亡有关，CLMD可能通过miR-146a介导TLR4信号通路，抑制NLRP3炎症小体活化，下调炎症因子表达，最终调控海马神经元焦亡，缓解TLE痫性发作。本项目拟重点选择海马神经元作为研究靶点，采用证素辨证、中医宏观表征观察、脑电图、RT-PCR、蛋白质印迹、膜片钳、激光共聚焦、电镜等方法，从临床、整体和离体实验三个方面来验证该假说，为TLE的治疗提供新思路和新靶点。

颞叶癫痫（TLE）病因主要责之于痰，痰邪贯穿疾病始终，治疗当以“治痰为先”。大量研究证明柴胡加龙骨牡蛎汤（CLMD）能有效缓解癫痫患者相关症状，但其起效的机制尚不明确。本项目选择海马神经元作为研究靶点，采用证素辨证、中医宏观表征观察、脑电图、qRT-PCR、蛋白质印迹、激光共聚焦、电镜等方法，从临床、整体和离体实验三个方面进行研究，尝试阐释CLMD有效治疗TLE的机制是否跟改善海马神经元的焦亡及自噬有关。结果发现：柴胡加龙骨牡蛎汤（CLMD）可减少TLE痫样放电，降低癫痫发作频率，减少海马齿状回自噬小体的数量；TLE大鼠海马齿状回miR-146a-5p和miR-146a-3p mRNA表达显著升高，TLR4、IRAK1、TRAF6、TAB、NF-κB和IL-1β蛋白表达亦明显上升，经CLMD干预后，以上指标均显著下降；TLE大鼠血清及海马L-1β、TNF-α表达显著上升，海马NLRP3、Caspase-1表达显著上升，经CLMD干预后均显著下降；海马齿状回Beclin-1和 LC3表达显著上升，mTOR显著下降，经CLMD干预后均显著回调。结果证实：“痰”状态下TLE的病理机制与炎症反应可导致海马神经元焦亡及自噬，CLMD可能通过下调miR-146a-3p和miR-146a-5p的表达，抑制TLR4通路，下调炎症因子表达，最终改善海马神经元焦亡及自噬，缓解TLE痫性发作。该研究可为TLE的中医药治疗提供新思路和新靶点。