塞来昔布脂质体鼻凝胶抑制tau蛋白磷酸化及其“类朊蛋白样”传播的分子机制研究

Alzheimer’s disease (AD) is (patho)physiologically characterized by the formation of amyloid plaques (AP) and tau hyperphosphorylation. Epidemiological evidence has demonstrated that long-term treatment of osteoarthritis (OA) patients with nonsteroidal anti-inflammatory drugs (NSAIDs), such as Celecoxib (CB) markedly reduces the risk of AD. Most CB act upon local inflammatory events by inhibiting the expression of cyclooxygenase-2 (COX-2). Although COX-2 and its metabolic product, prostaglandins (PGs) are elevated in the brain of AD patients, the mechanisms remain unknown. As an insoluble drugs, liposomes incorporated nasal gel was employed to increase the distribution of CB in the brain. Using glial and neuron cells derived from either human or mouse as an in vitro model system, we demonstrate that COX-2 played pivotal roles in the pathogenesis of AD, which was reversed by the incubation of CB. Indeed, COX-2 mediated the reciprocal regulation of proinflammatory cytokines and tau phosphorylation between glial and neuron cells. In addition, we will elucidate the mechanisms or signaling pathways that COX-2 regulates the production of proinflammatory cytokines in glial cells, which in turn modulates the phosphorylation of tau in neuron cells. On the basis of these observations, we will next explore the mechanisms of tau propagation between cells by transfecting cells with GFP- or mCherry-tau plasmids. Once phosphorylated tau obtained the ability of transmission between cells, it is possibly able to accelerate the formation of neurofibrillary tangles (NFTs). To this end, bimolecular fluorescence complementation (BiFC) system will be used to determine the aggregation of phosphorylated tau in cultured cell level.. In light of the above observations in vitro, the biological functions of CB containing liposomes will be further administered via nasal feeding in the AD model of C57BL/6J mice. Specifically, 1-month old COX-2, tau or COX-2/tau transgenic mice were adminitrated with CB liposomes incorporated nasal gel for 8 months. The activities of signaling molecules and the phosphorylation of tau will be determined in CB liposomes-treated transgenic mice. As compared with non- or CB suspension-treated mice, the pivotal roles of COX-2 and its metabolic products, PGs in tau phosphorylation will be addressed. In addition, BiFC will be used to determine the cell transmission and synapse cross by cerebral cortex, hippocampus or cerebral ventricles injection in transgenic mice. Finally, the pivotal roles of phosphorylated tau in NFTs formation will be eluciated by tau injection to mesentery of mice. The proposed project will provide completely novel insights into the mechanisms of taupathies and fullfill the gaps between COX-2 metabolic disturbance and cognitive decline in AD. More importantly, these observations will help us developing and improving CB drug delivery system, such as lipososmes incorporated nasal gel to combat AD.

流行病学调查发现长期服用环氧合酶-2（COX-2）特异性抑制剂塞来昔布（CB）的病人AD的发病率明显降低，并且COX-2代谢紊乱具有上调tau蛋白磷酸化的作用，然而CB作为一种难溶性药物如何透过血脑屏障及如何通过稳定COX-2代谢抑制tau介导的阿尔茨海默病（AD）发病机制尚不清楚。为此，本项目拟借助COX-2特异性抑制剂，采用脂质体鼻凝胶技术制备脑靶向CB药物传递系统，通过鼻饲处理Tau和Tau/COX-2转基因小鼠，借助CB共培养的新鲜脑片和原代培养细胞，结合药剂学、药理学、分子生物学和行为学等手段，阐明CB通过抑制COX-2活性降低脑内tau蛋白磷酸化、“类朊蛋白样”传播和NFTs形成过程中“种子效应”的分子机制，明确CB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CB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) 是一种神经退行性疾病，病理进程伴随着神经元生成的缺失，并诱发大量的神经元凋亡。塞来昔布是环氧合酶-2（Cyclooxygenas，COX-2）的特异性抑制剂，对神经元具有保护作用，但其脑部生物利用度很低，加之心脑血管副作用，使其临床应用受到严重限制。因此，本项目构建具有改善脑部生物分布、生物相容性良好的载药系统，对于AD的治疗具有重要意义。 .项目构建了塞来昔布-脂质体和红细胞膜载药系统，粒径均匀，性质稳定。以MTT法考察载药系统对细胞的毒性，显示载体本身无细胞毒性，而载药系统可明显改善塞来昔布的细胞毒性。体外释放结果表明红细胞膜载药系统具有更优越的缓释特征。组织分布、脑部分布及细胞摄取实验结果显示，红细胞膜载药系统具有较高的细胞摄取能力，能在脑部保持较长时间的有效浓度，且在非靶器官没有明显滞留。应用APP/PS1转基因小鼠为AD动物模型，并采用鼻饲给药处理，发现塞来昔布载药系统能明显抑制β-淀粉样蛋白 (β-amyloid，Aβ)的产生和聚集。水迷宫及絮窝实验证明，塞来昔布载药系统能改善APP/PS1转基因小鼠的认知障碍。进一步对塞来昔布载药系统调控神经元生成和凋亡的分子机制进行研究。采用Western Blot和Real-Time PCR技术检测塞来昔布-载药系统对相关基因表达水平的影响，结果表明塞来昔布载药系统可使SOD2、14-3-3ζ和ARRB1的表达上调，而BIK的表达量下调，确证了载药系统可以促进神经元生成。微管实验和Transwell实验表明，塞来昔布载药系统可显著提高神经元细胞的迁移能力。流式细胞仪实验证实，塞来昔布载药系统能够明显改善Aβ寡聚体引起的神经元凋亡。分析COX-2的代谢产物前列腺素E2（PGE2）和前列腺素D2（PGD2）的作用， 发现塞来昔布载药系统能够通过调控PGE2和PGD2的平衡，增强神经元生成，并且抑制神经元凋亡来缓解AD病理进程。塞来昔布-红细胞膜载药系统比传统塞来昔布-脂质体载药系统具有更优越的治疗效果。