应用蛋白质组学策略研究难治性癫痫患儿丙戊酸耐药的发生机制

The unresponse to valproic acid (VPA), a broad-spectrum antiepileptic drug, is a thorny problem in treating patients with refractory epilepsy, however the mechanism of the development of VPA unresponse remains unclear. Although the overexpression of P-glycoprotein and the changes of drug-targets were considered the primary factors contributing to refractory epilepsy, a number of studies have shown that VPA is not the substrate of P-glycoprotein and does not cause P-glycoprotein overexpression. In addition, currently known VPA targets are unlikely attributed to its effective discrepancy. Our preliminary study has shown that CREB, a nuclear transcription factor closely associated with epilepsy, was significantly upregulated in the hippocampus of VPA-unresponsive rats, suggesting that CREB overexpression may be a key factor in the insensitivity of VPA. The present project intends to use a LC-MS/MS-based label-free quantitative proteomics approach to identify differentially expressed proteins between VPA-responsive and VPA-unresponsive subjects, to analyze the target proteins related to CREB and their signaling pathways, to observe the efficacy of VPA with the knockdown and overexpression of CREB by in situ transduction to verify the valid target proteins and signal pathways of CREB involved in effective discrepancy of VPA using lentiviral vector. This project will reveal the mechanisms of differential efficacy of VPA and the role of CREB in the insensitivity, which may lead to the development of an improved strategy for treating refractory epilepsy.

广谱抗癫痫药物丙戊酸不敏感是临床治疗的棘手问题，但机制仍不明确。虽然传统认为P-糖蛋白的过表达和药物靶点结构的变化是药物难治性癫痫产生的首要因素，但现有文献表明丙戊酸并不是P-糖蛋白的底物且与P-糖蛋白的过表达不相关。此外，目前已知丙戊酸靶点无法解释丙戊酸疗效差异的发生。申请者前期转录组研究结果显示，与癫痫发生密切相关的核转录因子CREB在丙戊酸不敏感大鼠海马中显著提高，提示CREB的过表达可能是丙戊酸不敏感的关键因素。本课题拟基于质谱技术采用Label-free的定量蛋白质组学研究策略，寻找丙戊酸敏感和不敏感组中差异表达靶蛋白；分析其中与CREB相关的靶蛋白及其信号通路；采用慢病毒载体干预CREB的表达观察丙戊酸的疗效，验证CREB参与丙戊酸疗效差异发生的效应靶蛋白及信号通路。本项目旨在深入探讨丙戊酸疗效差异发生的机制及其与CREB的相关性，为丙戊酸不敏感的难治性癫痫治疗提供新思路。

围绕阐明耐药癫痫发生的分子机制的研究目标，本项目综合运用了分子生物学、电生理学、药理学等多学科交叉方法，从有效血药浓度、靶蛋白和疾病分子状态，耐药癫痫相关非编码RNA的筛选与临床验证，以及小胶质细胞BK通道在颞叶癫痫发生过程中的功能定位等三方面研究内容，探讨了耐药癫痫的发生机制。.本项目初步研究成果揭示了：1）通过临床调查和癫痫大鼠中发现丙戊酸的血药浓度与其疗效差异不相关；部分丙戊酸作用靶点的基因多态性与其疗效差异不相关；过度自噬参与丙戊酸耐药的发生；Homer1b/c过表达通过诱导自噬、mGluR1和mGluR5重分布介导丙戊酸耐药的发生。2）miRNA-34c-5p通过调控HMGB1表达促进耐药性癫痫的神经炎症；miRNA-221-3p调控HIF-1α表达介导耐药癫痫神经炎症的变化和小胶质细胞活化。3）发现了一个全新的KCNMA1基因(编码BK通道α亚基)突变位点，BK 通道的缺失，除了影响神经元动作电位的复极化，也可能调控小胶质细胞溶酶体 TRPML1的钙释放功能，抑制细胞自噬，介导神经炎症与癫痫的发生。.本项目结果丰富了介导耐药癫痫发生的分子病因，为临床应对难治性癫痫的药物干预提供可能的分子靶标。

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