基于环氧合酶-2糖基化探讨雷公藤内酯鼻用纳米粒温敏凝胶改善 “tau蛋白病”的分子机制研究

As a powerful inducer of inflammation, cyclooxygenase-2 (COX-2) is reported to be activated at the early stage of Alzheimer’s disease (AD). Indeed, prior works have revealed the relationship between the dyshomeostasis of COX-2 and the phosphorylation of tau. However, the molecular mechanisms of COX-2 glycosylation in tauopathy remain unknown. To this end, triptolide (TP) was selected as the inhibitor of COX-2, especially its potentially roles in modulating the glycosylation of COX-2. As an insoluble drug, temperature-sensitive nano hydrogel was employed to increase the bioavailability of TP by intranasal administration, which potentially increases the distribution of TP in the brain of mice. In addition, temperature-sensitive nano hydrogel facilitated the TP to bypass the blood brain barrier (BBB) and sustained the release of the drug to access the brain. In the current investigation, acute hypoglycemia and hyperglycemia animal models were established by starvation and hyperglycemic clamp technique; on the other hand, chronic hyperglycemia animal models were established by high-fat diet or leptin mutation in C57BL/6 mice. Using these in vivo experimental models, we demonstrate that the accumulation or uptake of excessive glucose will result in N-glycosylation of COX-2, leading to the instability and degradation of COX-2. In contrast, relative low levels of glucose in tauP301S mice protect COX-2 from N-glycosylation and degradation of COX-2, which cause the increasing the protein levels of COX-2. Apart from these findings, we further aimed to elucidate the involvements of glucose, glucose transporters (GluT), insulin and insulin receptor (IR) in regulating the glycosylation of COX-2. In addition, TP was further used to elucidate the potential roles of COX-2 in regulating the phosphorylation of tau. Finally, the mechanisms of COX-2 glycosylation in regulating the phosphorylation, propagation and deposition of tau will be revealed, which affect the cognitive decline of AD..In light of the above observations in vivo, the neuronal cells derived from either human or mice were employed as an in vitro model system. Taking the advantage of gene intervention, different cell lines were created by knocking down or ectopic expressing of relevant genes. By treating with glucose, insulin or TP, we extended the in vivo results to decipher the mechanisms of COX-2 glycosylation in regulating the phosphorylation, propagation and deposition of tau. Using the multiple and sophisticated biological techniques, we will identify the GluTs that are responsible for transporting glucose to neurons, which induced N-glycosylation and degradation of COX-2. In addition, the mechanisms or signliang pathways that insulin and IR regulate the glycosylation of COX-2 will be deciphered. To verify the pivotal roles of COX-2 in tau phosphorylation, TP were further used for treating neurons. The activity of COX-2 will be confirmed to cause the phosphorylation of tau. On the basis of these observations, we will next explore the mechanisms of COX-2 in regulating the glycosylation of tau, which prevent the phosphorylation of tau during the course of AD development and progression. Especially, the metabolic products of COX-2, prostaglandins (PGs) including PGE2, PGD2, 15d-PGJ2, PGI2, PGF2α and TXA2 will be screened for the glycosylation of tau. Glycosylation of tau will be beneficial for decreasing the phosphorylaiton, propagation and deposition of tau, which improve the cognitive decline of AD. The proposed project will provide a novel insight into the mechanisms of tauopathies and fulfill the gaps between COX-2 glycosylation and cognitive decline of AD. More importantly, these observations will help us developing and improving TP drug delivery system, such as temperature-sensitive nasal nano hydrogels to combat AD.

环氧合酶-2（COX-2）作为重要的炎症诱发因子，其激活是阿尔茨海默病（AD）的致病因素之一，且COX-2代谢紊乱具有上调tau蛋白磷酸化的作用，然而糖基化作为一种重要的蛋白翻译后修饰方式，如何通过影响COX-2代谢及活性影响tau介导的AD发病机制尚无报道。为此，本项目借助COX-2抑制剂雷公藤内酯（TP），采用纳米粒温敏凝胶技术制备高生物利用度的脑靶向药物传递系统，通过鼻饲处理多种外周和脑葡萄糖失稳态的动物模型，结合药理学、分子生物学和行为学手段，以老化进程中的葡萄糖失稳态和胰岛素抵抗为切入点，阐明TP通过抑制不同糖基化修饰的COX-2活性降低脑内tau蛋白磷酸化、“朊病毒样”传播和神经元缠结形成过程中“种子效应”的分子机制，明确TP通过抑制COX-2活性降低tau蛋白活性来改善小鼠学习记忆能力的可能机制，揭示TP通过COX-2调控AD的作用机理，最终为防治AD提供科学依据。

阿尔茨海默病（Alzheimer’s disease, AD）以β-淀粉样蛋白（β-amyloid protein, Aβ）和磷酸化tau蛋白沉积为主要病理特征。该项目以tau蛋白磷酸化这一AD主要病理特征为切入点，结合多种AD小鼠模型，探索环氧合酶-2（cyclooxygenase-2，COX-2）抑制剂，包括：雷公藤甲素，改善AD小鼠学习记忆能力损伤的病理学机制。并将该研究进一步拓展到COX-2通过其代谢产物，前列腺素（prostaglandins, PGs）A1调控tau蛋白磷酸化的分子机制研究，最终绘制COX-2调控tau蛋白磷酸化的分子机制网络。在此基础上，结合COX-2过表达以及与ob/ob小鼠杂交，探索COX-2介导的炎症机制影响tau蛋白糖基化的分子机制，以及其对tau蛋白磷酸化的竞争性抑制作用。最终揭示COX-2调控AD发病过程中tau蛋白磷酸化的分子机制。