阿曼托双黄酮抗癫痫发生及神经保护作用研究

Epilepsy, one of the most common neurological disorders, is characterized by recurrent, usually unprovoked, epileptic seizures, and followed with the cognitive, psychosocial, and social consequences. The available therapy against epilepsy is only symptomatic and often ineffective. The most important challenge is to prevent the process of epileptogenesis, not merely to treat its symptoms. At present, there is no anti-epileptic drug that performs this function. Thus, there is an urgent need to develop novel therapeutic interventions that are disease modifying-therapies that either completely or partially prevent the emergence of spontaneous recurrent seizures. Epileptogenesis is a process in which various insults ( such as brain insult, stroke, SE, the inflammatin in brain, etc.) induce brain to generate a cascade event, enhance the susceptivity of seizure and generate spontaneous recurrent seizures. In the last decade, Clinical and experimental evidence substantiate that brain inflammation is a common substrate in epilepsies of different etiologies. The use of antiinflammatory drugs in epilepsy has been invoked as a promising therapeutic strategy. In recent years, the research on medicinal plants is a hot point. Amentoflavone, a biflavonoid, widely distributed in plant kingdom.it has been found to possess anti-inflammatory, antioxidative, anti-apoptosis, and neuroprotective effects. Based on the above, it is reasonable to hypothesize that Amentoflavone may play a neuro-protective role in epileptic model and may have the anti-epileptogensis role. Our previous research found that amentoflavone significantly prolonged the latency of seizure attacks and reduced the amplitude and duration of epileptiform burst discharges induced by injection of intraperitoneal Pilocarpine. In addition, Amentoflavone significantly increased the survived neurons of hippocampal CA1, reduced apoptotic of neurons,and inhibited NF-κB p65 expression. On the basis of these considerations, we design the following experiment to investigate antiepileptogenic effects of amentoflavone in the pilocarpine-induced rat model.Spontaneous recurrent seizures (SRSs) were investigated by video-EEG monitoring during the entire procedure. Cognitive function was evaluated by the passive avoidance test and Morris water maze test. Brain tissue damage, oxidative stress parameters, inflammatory cytokines, mossy fiber sprouting, impact to AchE, and GABAA receptors subunit (α1, γ2, and δ) will be assessed by histology and immunohistochemistry. We hope to found new therapies to modify the epileptic process.

癫痫是神经系统常见病，治疗以药物为主。抗痫药物主要抑制症状，难以预防癫痫的发生。近年研究表明脑内炎症可能在癫痫发生中起到决定性作用。阿曼托双黄酮广泛存在于许多药用植物内，具有抗炎、抗氧化、抗凋亡等活性。课题组前期研究显示：阿曼托双黄酮显著缩短致痫小鼠癫痫发作持续时间，减少海马神经元缺失，降低NF-κB p65表达。显示阿曼托双黄酮有可能通过抗炎、抗氧化作用预防癫痫发生。本研究拟建立匹罗卡品致痫模型，应用视频脑电、水迷宫和避暗实验观察阿曼托双黄酮对致痫大鼠自发性癫痫发作及认知功能的影响；应用ELISA、免疫组化、病理学等技术检测海马氧化应激及神经元变性、坏死、凋亡情况，明确阿曼托双黄酮在癫痫发生时的神经保护作用；采用组织学、分子生物学等技术检测炎性相关因子、AchE、GABAA R亚基α1、γ2、δ等，探讨阿曼托双黄酮抗癫痫发生机制，并进一步阐明炎症与癫痫发生的关系，为抗痫新药研发开拓思路。

癫痫是常见的脑部疾病，脑内炎症过程是癫痫发生的一个具有决定性的病理机制，它贯穿于癫痫发生的各个阶段。目前对于癫痫发生的预防和治疗仍存在较大争议，但是癫痫发生与炎症反应的关系及机制是临床与基础研究关注的热点。阿曼托双黄酮（amentoflavone，AF）是具有多种生物活性的天然多酚类化合物，其在抗炎、抗氧化及神经保护等方面可以发挥重要作用。结合前期研究基础，本课题推测AF 能够通过抑制调控癫痫发生时脑内炎症通路中的相关因子，降低神经元损伤预防癫痫的发生。本研究以氯化锂-匹罗卡品致痫大鼠模型为基础，采用 Racine 评分评估癫痫模型的发作等级差异，脑电信号监测各组大鼠电生理变化，Morris 水迷宫和 IntelliCage 智能行为学检测分析系统评价大鼠学习记忆能力，发现AF 的预防性用药可以降低癫痫大鼠发作级别、缩短发作时间、改善空间学习记忆能力的下降，降低癫痫易感性；同时，应用 Nissel 染色及 TUNEL 染色观察癫痫大鼠海马神经元损伤和凋亡情况，免疫组化、Western-Blot 及ELISA方法检测大鼠海马凋亡相关蛋白表达水平及氧化应激水平，发现AF 预处理通过抑制海马氧化应激水平降低癫痫诱发的神经元损伤和凋亡，预防癫痫发生；接着研究 AF 对氯化锂-匹罗卡品诱导的癫痫大鼠神经胶质细胞激活和海马氧化应激反应的影响，发现AF 能够通过抑制匹罗卡品诱导的癫痫大鼠海马 COX-2 和 NF-κBp65 的表达，降低 IL-1β、TNF-α、PGE2水平，抑制小胶质细胞激活和星形胶质细胞的反应性增生。同时，通过进行脑片电生理实验明确了AF对GABA诱发的岛叶神经元GABAR通道电流不产生调节效应，但能够提高海马乙酰胆碱酯酶的活性，从而进一步抑制癫痫发生。这为癫痫预防性药物的筛选提供了新的理论依据。

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