定量主要吸收成分对柴贝止痫汤抗难治性癫痫效用与机制贡献度的研究

We have found out Chaibei Zhixian Decoction could effectively promote some antiepileptic western drugs, like carbamazepine, passing through the blood brain barrier to treat epilepsy by inhibiting the expression of P-gp and improving the permeability of the blood brain barrier in our previous clinical and basic experiments. Then we detected out eight absorption ingredients in Chaibei Zhixian Decoction through the pharmacokinetic test with the technology of LC-MS. Therefore, we come up with the hypothesis that the antiepileptic effects of Chaibei Zhixian Decoction are related to these eight absorbed bioactive compounds. To test this hypothesis, we will use the eight absorbed compounds alone and Chaibei Zhixian Decoction with carbamazepine respectively to have in vivo and in vitro experiments. After the drugs intervention, we will observe the expression of p-glycoprotein in blood brain barrier, the concentration of carbamazapine in the brain and the seizure frequency and grading. Then we will have a judgment on each absorbed bioactive compound through the simple formula of ‘the effect of absorbed bioactive compound/the effect of Chaibei Zhixian Decoction *100’and find out the exact utility ingredients of Chaibei Zhixian Decoction.

前期临床实验已证实柴贝止痫汤添加治疗难治性癫痫能够显著提高卡马西平等抗痫药的抗痫效用；基础实验进一步佐证了柴贝止痫汤这一功效，并且同时发现其作用机制是抑制血脑屏障中P糖蛋白与多药耐药基因1的表达，从而促进与其联用的抗痫西药入脑发挥抗痫效用，进而降低模型大鼠癫痫发作频次及严重度。前期预实验采用LC-MS技术进行柴贝止痫汤药代动力学测试，揭示了其主要吸收成分有八个。本课题旨在将八个吸收成分(单用)、柴贝止痫汤母方分别联合卡马西平进行体内、外实验，以血脑屏障中多药耐药基因1、P糖蛋白的表达量、大鼠脑内卡马西平浓度以及模型大鼠癫痫发作频次和分级等为效用指标，观察用药前后的变化；通过“成分疗效÷母方疗效×100%”的简单公式，定量主要吸收成分对柴贝止痫汤母方抗难治性癫痫效用与机制的贡献度，在疗效与机制确切的前提下固定出柴贝止痫汤的主要效用成分。

临床上约有20-30%的癫痫为药物难治性，有关药物难治性癫痫发病机制主要存在6种假说，其中研究较为广泛的为多药耐药假说，即癫痫患者血脑屏障和/或致痫灶多药耐药基因1及其编码的P糖蛋白的高表达是药物难治性的主要机制之一。我们前期研究表明复方柴贝止痫汤能够显著抑制血脑屏障中P糖蛋白与多药耐药基因1的高表达，从而促进与其联用的抗痫西药入脑发挥抗痫效用。考虑到方剂的药效物质不清楚，难以获得可重复的疗效，并且还可能存在用药安全隐患等问题。本项目主要的研究内容是通过自发性难治性癫痫大鼠模型与血脑屏障体外模型探究柴贝止痫汤主要吸收成分对自发性难治性癫痫大鼠模型的治疗效用与机制贡献度。在本项目研究中，我们运用LC-MS检测方法建立了复方柴贝止痫汤质量控制标准，明确了复方柴贝止痫汤吸收入血的活性成分，取含量排位前8的活性成分进行下一步实验，建立了自发性难治性癫痫大鼠模型与血脑屏障体外模型，进行了8种活性成分抗癫痫效用及机制的研究。本项目研究发现复方柴贝止痫汤主吸收入血的活性成分中能够下调癫痫大鼠脑组织与大鼠脑微血管内皮细胞中P-gp与多药耐药基因1高表达的为：α细辛醚、β细辛醚、贝母素甲、贝母素乙，以上4种活性成分主要来源于柴贝止痫汤中的化痰药石菖蒲与浙贝母；而来源于疏肝平肝药物天麻、牡蛎、柴胡、地龙的活性成分天麻素、天麻苷元、香草醛、柴胡皂苷则对P-gp高表达以及耐药基因无影响。