抑制PERK/CHOP信号通路对降低慢性间歇性低氧海马神经元凋亡的机制研究

Obstructive sleep apnea syndromes (OSAS) is a common respiratory and sleeps disorder which is mainly characterized by intermittent hypoxia (IH) and result in neuronal damage. However, it is lack of effective therapeutic and rehabilitative approach in clinical practice. Previous studies have shown that IH-induced oxidative stress could activate endoplasmic reticulum stress mediated by PERK signaling pathway, leading to neuronal apoptosis and impairment of learning and memory, however, the precise mechanism remains unclear. In this project，chronic IH will be used to induce in vivo animal models of OSAS and in vitro primary cell model while PERK signaling pathway inhibitor through oral gavage and micro RNA interference technique will be applied to suppress ER stress caused by IH and the effect of inhibiting PERK signaling pathway on learning and memory will be detected through multiple methods, including behavior test, electrophysiology and molecular biology. The expression of mitochondrial apoptosis-related proteins via immune electron microscopy, the mitochondrial function and neuronal apoptosis will also be investigated. Finally, the mechanism of PERK/CHOP pathway mediated neuronal apoptosis in hippocampus will be further elucidated and the results will reveal new therapeutic and rehabilitative drug targets for this disease.

阻塞性睡眠呼吸暂停综合征(obstructive sleep apnea syndrome, OSAS)是一种常见呼吸和睡眠障碍性疾病。该疾病以间歇性低氧 (intermittent hypoxia, IH)为主要特征，引起神经元的损伤，目前缺乏有效的治疗与康复手段。本课题组以往研究表明IH能够通过氧化应激诱导内质网应激中PERK信号通路的激活，引起神经元的凋亡以及学习记忆能力的下降，但具体调控机制未明。本项目拟通过慢性IH来诱导OSAS动物模型和原代细胞模型，用PERK通路抑制剂以及小RNA干扰技术来抑制PERK信号通路激活，利用行为学，电生理学，分子生物学等方法来观察抑制PERK通路后对其学习记忆能力的影响。同时，免疫电镜观察线粒体凋亡相关蛋白表达情况，并检测线粒体功能以及神经元凋亡，明确PERK/CHOP信号通路介导海马神经元凋亡的分子机制，并为该疾病治疗与康复提供相应的药物靶点。

在本课题中，我们观察到内质网应激中的双链RNA依赖的蛋白激酶样激酶(protein kinase RNA-like ER kinase, PERK) 通路对间歇性低氧(intermittent hypoxia, IH)所导致细胞凋亡起到重要的作用。通过水迷宫和新物体识别实验，我们发现到PERK抑制剂-GSK2606414能够明显改善海马依赖的认知功能降低。同时，GSK2606414能够提高海马突触可塑性，使得CHOP表达下降，并减少细胞凋亡的发生。而IH模型中CHOP诱导的细胞凋亡与线粒体存在的一定的关系。研究表明IH处理后BAX/BAK 表达水平在线粒体中表达升高，而在胞浆中降低，同时细胞色素C从线粒体中溢出，从而诱导caspase-3激活。而GSK2606414能够减少线粒体中BAX/BAK和胞浆中细胞色素C的表达。另外，在细胞模型中，GSK2606414抑制PERK通路后使得细胞凋亡明显减少，细胞形态结构改善。结果说明PERK/CHOP信号通路在IH所诱导细胞凋亡中起到重要的作用。