SARA和TAK1调控TGFβ信号通路参与耐药性颞叶癫痫发病的机制研究

Temporal lobe epilepsy (TLE), as a common type of drug-resistant epilepsy, is associated with neuroinflammation. Studies have shown that transforming growth factor beta (TGFβ) signal pathway involves in epileptogenesis; however, it is still unknown whether TGFβ signal pathway participates in drug resistance in TLE. ..Our previous studies have found that expression of type І transforming growth factor β receptor (TβRІ) increases in the brain of patients with intractable TLE compared to control group, and CSF-TGFβ is also increased in patients with intractable TLE. We thus deduce that TGFβ signal pathway might be involved in the pathogenesis of intractable TLE. The aim of the present application is to test this speculation...The amygdala-kindled model with Wistar rats and astrocytes culture will be established. The important proteins of TGFβ signal pathway will be detected in drug-resistant rats compared with non-resistant rats, including TβRІ, smad anchor for receptor activation (SARA), transforming growth factor beta activated kinase 1 (TAK1), as well as the downstream proteins in different duration of the disease. Then, we will detect the changes of downstream proteins and epilepticform discharges via interference of above three proteins by using reporter gene, RNA interference, patch clamp and other techniques. The mechanisms of these proteins contributed to intractable TLE will be exposed in vivo and in vitro, including neuroinflammation, blood-brain-barrier injury, neuronal apoptosis, alteration of multidrug resistance gene, and changes of neurotransmitters. We hope to understand the roles of TGFβ signal dysfunction in the mechanism of drug-resistant TLE. Finally, we will test above-mentioned proteins in patients with intractable TLE to find biomarkers for early diagnosis of drug resistance...We try to supply new targets of anti-epileptic treatment for intractable epilepsy, and to develop a new therapeutic strategy for intractable epilepsy. Hopefully, we will also diagnose drug-resistant TLE as early as we can via TGFβ-associated inflammatory factors.

颞叶癫痫是一种常见的耐药性癫痫，近年研究发现神经炎症与颞叶癫痫耐药有关，但炎症相关的转化生长因子β(TGFβ)通路是否参与其中尚无报道。我们前期研究发现，耐药性颞叶癫痫患者脑脊液TGFβ1升高，癫痫模型中Ⅰ型TGFβ受体(TβRⅠ)及其调节蛋白表达异常，但内在机制有待研究。本项目以耐药杏仁核点燃模型和星形胶质细胞为研究对象，以TβRⅠ、SARA和TAK1为切入点，评价它们及其下游蛋白在动物模型中表达差异及变化规律；通过报告质粒、基因沉默和过表达、膜片钳等技术从体内外探讨TβRⅠ、SARA和TAK1调控相关信号通路及下游靶基因，影响反复痫样放电和行为学的机制；研究此三种蛋白对炎症信号转导、血脑屏障损伤、多药耐药基因表达、神经元损伤和神经递质变化的调控作用差异；探讨耐药性颞叶癫痫患者脑脊液和外周血中TGFβ通路重要蛋白变化的规律。有望为疾病的新药开发提供精细靶点，为早期诊断提供外周生物标记物。

癫痫是严重危害人类健康的重大神经疾病之一，耐药性癫痫的机制探索仍然是难点，神经炎症与癫痫耐药的关系越来越受到重视。本项目组前期研究表明转化生长因子β（TGFβ）信号通路通过调节炎症反应参与了癫痫的发生发展，提示该通路可能与癫痫耐药有关。本项目以耐药性癫痫患者和动物模型为研究对象，采用多种分子生物学手段、膜片钳技术、病毒干预等技术方法，发现无论是耐药性癫痫患者还是动物模型炎症反应明显，阐明TGFβ信号通路经典途径的Smad 锚定受体激活蛋白（SARA）和非经典途径的转化生长因子激酶-1（TAK1）均参与癫痫耐药和自发性发作，是耐药性癫痫的潜在重要的药物靶点。其中，TGFβ信号通路经典途径的SARA通过Smad3及其磷酸化途径参与耐药性癫痫，非经典途径的TAK1通过TRAF6/TAK1/P-TAK1/NF-κB/IκBα/P-gp通路对癫痫耐药产生影响，减少自发性发作。本研究发现的SARA和TAK1有望为耐药性癫痫的新药研发和干预靶点提供理论证据，具有较好的应用前景和转化价值。此外，我们发现老年人癫痫以部分性发作最常见，尤以复杂部分性发作为多，大多存在明确病因，脑血管疾病最多，颅内肿瘤、中枢神经系统感染、脑外伤常见。提示预防癫痫的策略就是防治脑血管病、预防脑外伤和颅内感染等，将为减少继发性癫痫提供防治策略，惠及老年人群，积极助力健康老龄化。