GATA1/AEG-1通路对颞叶癫痫星形胶质细胞炎症免疫的调控及“从肝论治”的干预作用

Epilepsy has become a major public health problem. On aspects of prevention and treatment for Epilepsy, traditional Chinese medicine has unique advantages in both theory and strategy, such as syndrome differentiation of liver. However, the material basis and intervention mechanism syndrome differentiation of liver are still not fully understood. Our previous study discovered the expression of 1 gene (AEG-1) was increased after the activation of the pro-inflammatory factor (IL-1β), and the expression of amino acid transporter (EAAT2) was decreased in astrocytes. Bioinformatics analysis revealed that the transcription factor GATA1 could be combined with AEG-1 and played a role in the acetylation site of AEG-1. Therefore, we put forward our scientific hypothesis: the study on the mechanism of immune regulation of GATA1/AEG-1 in the treatment of temporal lobe epilepsy from prescriptions of syndrome differentiation of liver; Based on CRISPR/cas9 technique and gene knocked-out mice will be used to investigate changes of astrocytes induced by inflammation and immunity in this project and the regulatory mechanisms of GATA1/ AEG-1. We intend to use GATA1 and AEG-1 as the starting point, combined with the technology of CRISPR/cas9 and gene knockout mice platform, analyze the nature of astrocyte inflammatory immune activation from molecular, cell and animal "multi-level and multi-target; and by means of CHSGD and SSa in the intervention," medicine "test" syndrome" reversely analysis of epilepsy from syndrome differentiation of liver connotation, which answer modern molecular biology mechanism and the substance basis of its pharmacology to find new molecular targets for the prevention and treatment of temporal lobe epilepsy.

癫痫严重威胁人类的健康，而中医药具有独特的防治理论及策略，“从肝论治”是其关键之法，但对其治疗的物质基础及干预机制仍尚未明确。基于预实验研究发现IL-1β诱导的星形胶质细胞炎症免疫激活引起星形胶质细胞上调基因1（AEG-1）及其细胞膜上氨基酸转运体2（EAAT2）的表达异常与颞叶癫痫的发病密切相关。经生物信息学分析发现转录因子GATA1与AEG-1启动子序列相结合并通过其乙酰化区域发挥作用。因此，我们提出：“从肝论治”方药通过GATA1/AEG-1途径调控颞叶癫痫星形胶质细胞的炎症免疫。拟以GATA1及AEG-1为切入点，结合CRISPR/cas9技术及敲基因鼠平台，从“分子-细胞-动物”多层次、多靶点分析星形胶质细胞炎症免疫活化的本质；并通过CHSGD及SSa干预，以“药”验“证”反向分析癫痫“从肝论治”的内涵，解答其现代分子生物机制及中药的作用基础，为防治颞叶癫痫寻找新的分子靶点。

癫痫严重威胁人类的健康，而中医药具有独特的防治理论及策略，“从肝论治”是其关键之法，但对其治疗的物质基础及干预机制仍尚未明确。基于预实验研究发现IL-1β诱导的星形胶质细胞炎症免疫激活引起星形胶质细胞上调基因1（AEG-1）及其细胞膜上氨基酸转运体2（EAAT2）的表达异常与颞叶癫痫的发病密切相关。经生物信息学分析发现转录因子GATA1与AEG-1启动子序列相结合并通过其乙酰化区域发挥作用。因此，我们采用“从肝论治”方药探讨GATA1/AEG-1途径调控颞叶癫痫星形胶质细胞的炎症免疫。拟以GATA1及AEG-1为切入点，结合CRISPR/cas9技术及敲基因鼠平台，从“分子-细胞-动物”多层次、多靶点分析星形胶质细胞炎症免疫活化的本质；并通过CHSGD及SSa干预，以“药”验“证”反向分析癫痫“从肝论治”的内涵，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、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IL-1β和AEG-1的表，并增加EAAT2的表达，从而降低神经元兴奋性；为明确癫痫“从肝论治”的作用靶点提供实验依据。