基于神经炎症研究短柄枹栎中三萜类抗阿尔茨海默病活性成分及其作用机制

Alzheimer's disease (AD) serious harm to human health and its pathogenesis is very complex. The neuroinflammation caused by activated microglia, which is activated by amyloid β (Aβ) pledges, is one of the core mechanisms. Searching for the neuroinflammatory inhibitors from natural resources is an important strategy for R&D of the new anti-AD drug... Our previous study found that the total triterpenoids in the seeds of Quercus serrata var. brevipetiolata exhibited obvious anti-neuroinflammatory and neuroprotective activity, and could significantly improve the spatial learning and memory ability of AD transgenic mice, reduce the Aβ deposition in the areas of mouse cerebral cortex and hippocampus, and inhibit neuroinflammation in the brain. .. This project will be focused on isolation and purification of the triterpenoids with anti-AD activity from the seeds of Quercus serrata var. brevipetiolata by a variety of chemical and chromatographic methods. The chemical structure of triterpenoids will be identified through the integrated spectroscopic and chemical methods. LC-MS/MS (MRM) will be applied to establish the characteristic profile of the triterpenoids to track and isolate the triterpenoids. Anti-neuroinflammation bioactivity and mechanism of the isolated triterpenoids would be predicted by the network models of systems biology, and then verified by molecular pharmacological methods to screen out the lead compounds which have clear anti-AD mechanism. .. Subsequently, preliminary experiments in pharmacokinetics (ADME/T) of the lead compounds were also carried out to the candidates with potent anti-AD activity through Pipeline Pilot software. For the candidates with good druggability and prospects, the in vivo anti-AD activity would be conducted in APP/PS1 transgenic AD mice to clarify their anti-AD mechanisms. Thus, these data may consider together to screen out anti-AD candidate in the new drug development.

阿尔茨海默病(AD)严重危害人类健康，发病机理十分复杂，脑内β-淀粉样蛋白(Aβ)沉积激活小胶质细胞引起的神经炎症是AD的核心病理机制之一，寻找抑制神经炎症天然先导化合物是研发防治AD新药的重要策略。我们前期研究发现短柄枹栎种子中总三萜具有显著的抗神经炎症及神经保护活性，同时能有效改善AD小鼠空间学习记忆能力，减少大脑皮层和海马区域Aβ沉积及明显抑制脑内神经炎症。本项目拟对短柄枹栎中三萜类进行分离鉴定，通过LC-MS/MS(MRM)建立三萜类化合物的特征轮廓谱，追踪分离三萜成分，利用抗神经炎症及神经细胞保护活性模型筛选出活性化合物。利用生物信息学网络模型及分子药理学手段预测和验证活性化合物抗AD的作用机制，筛选出抗AD作用机制明确的先导化合物。采用Pipeline Pilot软件探讨先导化合物的成药性，对成药性良好的先导化合物进行转基因AD小鼠体内活性及作用机制研究，优选出抗AD候选药物。

阿尔茨海默病（AD）严重危害人类健康，脑内β-淀粉样蛋白沉积激活小胶质细胞引起的神经炎症是AD的核心病理机制之一。短柄枹栎（Quercus serrata var. brevipetiolata）是壳斗科（Fagaceae）栎属植物枹栎（Quercus serrata）的一个变种，其种子中药名为橡实，其总三萜能通过抑制胶质细胞神经炎发挥体内外抗AD作用。本项目通过多种色谱学分离手段对枹栎种子中的三萜类成分进行分离，并利用理化性质、现代波谱学以及化学等手段鉴定了它们的结构，共分离鉴定103个三萜类化合物，其中新化合物39个。利用抗神经炎症细胞模型，系统筛选了三萜类化合物的抗AD活性，部分三萜类化合物活性显著。进一步，对活性显著的三萜类单体化合物Q43开展了抗AD作用机制研究。结果表明，Q43可剂量依赖性抑制LPS诱导的BV2细胞释放NO及抑制PGE2的生成。采用qRT-PCR和Western blot检测，发现Q43在mRNA水平和蛋白水平上对iNOS和COX-2的表达均有抑制作用，推测Q43对BV2细胞中NO和PGE2释放的影响可能与抑制iNOS和COX-2表达有关。qRT-PCR实验结果表明，Q43可抑制LPS诱导的IL-1β、IL-6和TNF-α mRNA表达，其中对IL-1β mRNA的抑制作用最明显。ELISA实验发现，Q43可剂量依赖性地抑制IL-6和TNF-α在细胞上清中的释放。Western blot结果表明，Q43与抑制剂SP600125联合作用可增强Q43对p-JNK蛋白表达的抑制作用，进一步证明了Q43对JNK通路的抑制作用。同时，Q43可抑制LPS诱导的BV2细胞IκBα蛋白磷酸化和泛素化降解，抑制NF-κB蛋白磷酸化，阻碍NF-κB核转位。免疫荧光的结果与Western blot结果一致，证明Q43的抗炎作用可能与调节NF-κB通路有关。综上所述，Q43可抑制LPS诱导的BV2细胞产生炎症反应，包括抑制NO、PGE2、ROS、IL-6和TNF-α的释放以及IL-1β、iNOS和COX-2的表达，其抗炎的作用机制为抑制MAPK和NF-κB通路的激活，可作为抗AD候选药物做进一步研发。.通过本项研究，阐明了枹栎抗AD药效物质和作用机制，为枹栎三萜是进一步开发奠定了基础。本项目已发表SCI论文7篇，获国家发明专利3项。