基于环氧合酶-2乙酰化，探讨乙酰水杨酸抑制β-淀粉样蛋白聚集和沉积的分子机制

Cyclooxygenase-2 (COX-2) is activated at the early stage of Alzheimer’s disease, which is associated with the abnormal production of β-amyloid protein (Aβ). Our prior works have demonstrated that acetylsalicylic acid (ASC), a COX-2 inhibitor has ability to acetylate COX-2, which result in disrupting its binding on Aβ. As slightly soluble in water, the bioavailability of ASC is relative low. If we want to achieve high brain bioavailability, the toxicity will be highly induced by increasing the dosage of ASC. In view of these problems, acetylsalicylic acid-loaded albumin nanoparticles (ASC-BSA-NPs) were employed to increase the solubility of ASC by intranasal administration, which potentially contribute to increase the distribution of ASC in the brains of mice. To extend the residence time of ASC in the location of absorption, temperature-sensitive hydrogels were further prepared to encapsulate ASC-BSA-NPs, which maintain the drug at the nasal mucosa. Using this preparation, we will continue to elucidate the mechanisms of ASC in regulating the aggregation and accumulation of Aβ during the course of AD development and progression.. Using bioinformatics techniques, we firstly forecasted the possible acetylation sites of COX-2 including Ser530, Val523, Arg513, Val434, Tyr385, Tyr348 and Arg120. By mutating possible acetylation sites of COX-2, we identified that Tyr385 is the key acetylation site of the enzyme for ASC treatment. Once the acetylation sites of COX-2 were determined, we will continue to elucidate the roles acetylation in the activity of COX-2 by determining the production of its metabolic products including PGH2, PGE2, PGD2, 15d-PGJ2, PGI2, PGF2α and TXA2. Given their potential contribution to the production of Aβ, the activity of acetylated COX-2 on the expression of β-secretase, such as BACE-1 and γ-secretases including presenilin 1/2 (PS1/2), APH-1α/1β, nicastrin (NCT), presenilin enhancer 2 (PEN2) as well as Aβ will be further determined. More importantly, the formation of hetero-oligomers between COX-2 and Aβ will be determined in COX-2Y385F cells. Moreover, the propagation and clearance of Aβ will be studied in ASC-treated or COX-2Y385F cells by bimolecular fluorescence complementation (BIFC) system. . Based on the above in vitro observations, we will further established COX-2Y385F mice, which abolished the acetylation of the enzyme by ASC treatment. After crossed with APP/PS1 or COX-2/APP/PS1 mice, we will continue to reveal the effects of COX-2 acetylation on the production, aggregation and accumulation of Aβ in ASC-treated mice. In detail, the expression of β- or γ-secretases as well as Aβ will be determined in COX-2Y385F/APP/PS1 mice. Using these different groups of mice, the formation of hetero-oligomers between COX-2 and Aβ will be measured by immunoprecipitation and western blots. In addition, the aggregation and deposition of Aβ will be further determined by immunofluorescence (IF). The degrading mechanisms of Aβ will be also monitored in these mice. Finally, the learning ability of the mice will be measured by morris maze test or nest construction assay. The proposed project will provide a novel insight into the mechanisms of AD and fulfill the gaps between COX-2 acetylation and cognitive decline of AD. More importantly, these observations will help us developing and improving ASC drug delivery system, such as ASC-BSA-NPs to combat AD.

环氧合酶-2（COX-2）激活在阿尔茨海默病（AD）中扮演了重要角色，与β-淀粉样蛋白（Aβ）生成密切相关。我们在前期研究中发现COX-2抑制剂类药物乙酰水杨酸（ASC）具有通过乙酰化COX-2抑制其与Aβ形成异二聚体的作用。但由于ASC微溶于水，极大的降低了其脑生物利用度及增加了高剂量给药的毒副作用。鉴于此，本项目拟采用白蛋白纳米粒包裹ASC（ASC-BSA-NPs），结合温敏水凝胶技术制备高生物利用度的ASC脑靶向给药传递系统。通过鼻饲处理Aβ脑失稳态的AD动物模型，以COX-2乙酰化为切入点，结合行为学评估、神经病理学和分子生物学研究手段，探讨ASC通过乙酰化COX-2抑制其与Aβ形成异二聚体，继而影响其聚集和沉积的分子机制。明确ASC通过抑制COX-2与Aβ形成异二聚体降低老年斑（APs）沉积，进而促进Aβ清除，最终改善小鼠学习记忆能力的可能机制，为防治AD提供理论依据和实践基础。

阿尔茨海默病（Alzheimer’s disease，AD）的主要病理特征之一是脑内出现（β-amyloid protein，Aβ）形成的老年斑（β-amyloid plaques, APs）沉积。基于环氧合酶2（cyclooxygenase 2，COX-2）在调控Aβ产生、传播及沉积中的关键作用，进一步阐明非甾体抗炎药，乙酰水杨酸（Acetylsalicylic acid，ASA）通过乙酰化COX-2，调控Aβ的产生、传播和沉积的分子机制。在AD病理学研究方面，ASA还可以通过调控神经元的细胞周期再进入抑制神经元凋亡的发生，从而改善AD小鼠的学习记忆损伤。在此基础上，进一步将研究拓展到COX-2通过其代谢产物，前列腺素（prostaglandin，PG）A1经过氧化物酶体增殖物激活受体（peroxisome proliferator-activated receptor（PPAR）γ/ ATP结合盒转运体A1（ATP-binding cassette transporter A1，ABCA1）途径调控胆固醇转运，进而抑制早老素增强子2（presenilin enhancer 2，PEN2）表达减少Aβ单体、寡聚体和纤维体的形成；另外PGA1还可以与蛋白磷酸酶（protein phosphotase，PP）2A亚基PPP2A1的Cys377位点结合，激活PP2A来抑制tau蛋白的磷酸化，最终改善了APP/PS1和tauP301S转基因小鼠的认知功能下降。

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