应激水平糖皮质激素对NLRP-1炎症小体的调节作用及在海马神经元损伤中的作用研究

Alzheimer's disease (AD) is a chronic neurodegenerative disorder marked by a progressive loss of memory and cognitive function. Plasma stress level glucocorticoids (GCs) are correlated with dementia progression in patients with AD. It is reported that GCs can enhance NMDA neurotoxicity through facilitating [Ca2+](i) increment and attenuating the NR2A-ERK1/2-mediated neuroprotective signaling. GCs can reduce hippocampal dendritic complexity and can even cause hippocampal cells death. Our previous studies showed that twenty one days exposure of dexamethasone (DEX) induced impairment of memory and learning, accompanied by severe histological damage in the hippocampus in senescent mice. Furthermore, DEX treatment increased ROS production and the activity of caspase-3, facilitated Aβ25-35 neuronal cytotoxicity and induced cortex and hippocampus neurons apoptosis. Our preliminary experiment showed that DEX significantly activated BK ion channel and enhanced K+ efflux.While the ROS accumulation and K+ efflux can activate the NLRP-1 inflammsome. So we hypothesize that chronic stress level of GCs may upregulate and activate NLRP-1 inflammsome in hippocampal neurons, which may be related with the GCs induced hippocampal injury. In this study, patch clamp, immunofluorescence, immunoblotting,co-immunoprecipitation, Real time PCR, confocal microscopy and flow cytometry will be used to tudy the effects and mechanisms of stress level of GCs on neurodegeneration. ① The effects and mechanisms of stress level of GCs on the regulation of NLRP-1 inflummsome in hippocampal neurons in wild and NLRP-1 knock-out mice. ② The effects and mechanisms of stress level of GCs on regulation of BK ion channel and activation of NLRP-1inflammsome in hippocampal neurons. ③ The effects and mechanisms of NLRP-1inflammsome on GCs mediated hippocampus neuronal injury.This study will provide new idea and target point for prevention and therapy of large dose GCs and chronic stress induced neurodegeneration.

应激水平的糖皮质激素（GC）对海马神经元具有损伤作用，与老年性痴呆的发病密切相关。GC可损伤动物的学习记忆功能，促进钙离子内流而增加NMDA的神经毒性，增加神经元ROS生成，增加Caspase3活性，促进Aβ对神经元的毒性和损伤。我们预实验发现，GC能明显激活BK通道，促进钾离子外流，而ROS增加和细胞内低钾可以激活炎症小体。据此我们提出慢性应激水平GC可以上调和激活NLRP-1炎症小体导致海马神经元损伤的假说。本研究利用膜片钳、免疫荧光、蛋白印迹、免疫共沉淀、定量PCR、激光共聚焦等技术：①用野生和NLRP-1敲除小鼠研究GC对NLRP-1的调节作用及对海马神经元的损伤作用及机制；②研究GC对海马神经元BK通道的调节及对NLRP-1激活的影响；③研究NLRP-1炎症小体在慢性应激水平GC致海马神经元损伤中的作用及其机制。期望为防治大剂量GC应用和慢性应激对神经元的损伤提供新思路和新靶点。

阿尔茨海默病(AD)又称“老年性痴呆”，是当今世界上老年人最常见的中枢神经系统退行性疾病。应激水平的糖皮质激素（GCs）对海马神经元具有损伤作用，与老年性痴呆的发病密切相关。本课题主要研究了不同水平GCs作用不同时间对海马神经元糖皮质激素受体（GR）和NLRP-1炎症小体激活的影响，研究了高糖皮质激素对海马神经元NLRP-1炎症小体调节的机制研究，同时研究了GCs环境下海马神经元NLRP-1炎症小体在海马神经元损伤中的作用及其机制。我们的研究结果显示，应激水平的GCs长期作用可明显降低小鼠海马区脑组织GR的表达，增加NLRP-1炎症小体的表达和炎症因子的生成，导致小鼠学习记忆下降，皮层和海马神经元的损伤；体外实验也显示，应激水平糖皮质激素作用3d能明显降低原代培养海马神经元GR表达，增加NLRP1炎症小体的表达和炎症因子的生成，导致海马神经元老化损伤，其发生机制可能与海马神经元BK通道的激活导致细胞内低钾有关。我们进一步研究了NLRP1炎症小体在慢性GCs诱导神经元损伤中的作用。研究结果显示，GR受体阻断剂（RU486）、人参皂苷Rg1、caspase1抑制剂、BK受体阻断剂及NLRP1-siRNA干扰均可下调NLRP1炎症小体的表达，对慢性GCs诱导的海马神经元损伤有一定的保护作用，表明NLRP1炎症小体激活在慢性GCs诱导海马神经元损伤中起着重要作用。本研究结果表明，慢性应激水平的GCs长期作用能明显降低培养海马神经元GR的表达，增加BK通道表达和钾离子外流，增加NLRP-1炎症小体及其相关炎症因子的表达，导致海马神经元的损伤，可能与神经退行性疾病的发生发展密切相关。同时RU486和人参皂苷Rg1对皮层和海马区神经元GR都具有上调作用，对NLRP-1炎症小体的激活都具有抑制作用，能明显改善小鼠的学习记忆功能，减轻脑组织病理损伤，进而为防治慢性应激和糖皮质激素诱导的神经退行性疾病提供了理论依据。