基于orexin-A信号转导特性探讨睡眠剥夺损害学习记忆的分子与细胞机制

Sleep deprivation, as a state of sleep loss induced by a variety of reasons, seriously affects the function of learning and memory, which is closely associated with the hippocampus. However, there are a limited number of therapeutics that are able to improve learning and memory in the face of more and more serious insufficient sleep. The fundamental reason is that the mechanisms about the impairment of sleep deprivation in learning and memory and intervention targets have not been identified. Clinical results from our studies suggested that the level of orexin-A, produced from the lateral hypothalamus with being important effects on the regulation of sleep/wakefulness, was significantly increased in the patients of primary insomnia with memory deficit. Therefore, we speculate that the over-expression of orexin-A induced by chronic sleep loss in the patients with primary insomnia disrupts learning and memory function. In order to verify the hypothesis from the cellular and molecular mechanisms, we will use the measures of a dominant-negative mutant, siRNA gene silencing, Western blot, confocal, water maze etc. in the rat model of REM sleep deprivation to investigate the critical effects of orexin-A signal transduction on hippocampus-dependent learning and memory. It further shed light on the molecular and cellular mechanisms that sleep deprivation impairs learning and memory via orexin-A signal pathways. The project will yield new targets for the development of more effective drugs and therapeutics in treating insomnia and reversing the impairment caused by sleep deprivation.

睡眠剥夺是各种原因导致的睡眠缺乏状态，严重影响了人类的学习记忆功能，与海马功能受损密切相关。然而，面对越来越重的睡眠缺乏，能够有效改善学习记忆的治疗方法和措施却极为有限，其根本原因是睡眠剥夺损害记忆的机制和干预靶点不明确。研究显示，在伴有记忆减退的原发性失眠患者以及睡眠剥夺动物模型中，下丘脑产生的调节睡眠觉醒神经肽orexin-A水平明显升高。为此，我们推测睡眠剥夺诱导的orexin-A过度表达对学习记忆功能有损害作用。为了探索这一假说，我们将利用睡眠剥夺大鼠模型，采用显性失活突变、siRNA基因沉默、共聚焦、水迷宫等手段，从分子、神经细胞再生与迁移以及学习记忆等方面，探讨orexin-A信号转导在海马依赖性学习记忆中的作用，进一步揭示睡眠剥夺通过诱导orexin-A信号过度激活，进而影响学习记忆的分子与细胞机制。这一研究将为开发药物有效治疗失眠和逆转睡眠剥夺所致的学习记忆损害提供新靶点。

当今社会的发展使得睡眠缺乏成为常见的问题之一，严重影响了人类的学习记忆功能，与海马功能受损密切相关。但能解决因睡眠缺乏引起的学习记忆的方法和措施极为有限，根本原因是因为睡眠剥夺损害记忆的机制和干预靶点不明确。资料及前期研究显示伴有记忆损害的原发性失眠患者及睡眠剥夺动物模型中，下丘脑产生的调节睡眠觉醒神经肽orexin-A水平明显增高，基于此，我们针对 orexin-A的信号转导通路开展研究，通过建立动物实验模型，并利用慢病毒载体介导siRNA的方法沉默海马orexin-1受体(OX1R)和orexin-2受体(OX2R)，给予睡眠剥夺后检测海马内P-ERK1/2表达、海马齿状回区BrdU+、BrdU+/DCX+及BrdU+/NeuN+细胞表达，从分子、神经细胞再生与迁移以及行为学实验等方面，探索下调orexin受体后睡眠剥夺对于学习记忆的是否有改善作用。研究结果发现，阻断OXR能改善睡眠剥夺大鼠的空间学习记忆能力，海马齿状回神经细胞增殖能力强，这说明睡眠剥夺诱导的Orexin-A过度表达可能通过激活ERK1/2信号通路，抑制海马齿状回细胞增殖，损害空间学习记忆。本研究的意义在于进一步说明了orexin系统中orexin-A的信号转导通路在睡眠剥夺引发认知障碍机制中的作用，通过特异性阻断和调控OXR，既能改善睡眠，又能改变睡眠剥夺所致的学习记忆损害，可为开发推进OXR拮抗剂治疗失眠并有效预防和逆转失眠造成的学习记忆损害提供新靶点。