基于DYRK1A信号通路多途径调控AD作用的苦木总生物碱体内药效物质及作用机制研究

Multi-pathway intervention of the onset and development of Alzheimer’s disease (AD) is considered as a novel and effective strategy for the treatment of this serious disease. The overactivation of DYRK1A plays important roles on the activation of many pathogenic pathways including the formations of NFTs and Aβ, which makes it be a new target for treating AD. Previous work in our lab suggest that the total alkaloide from Picrasma quassioides (TAPQ) has the capability to treat AD, the possible mechanisims are that β-carboline alkaloides or their metabolites can reduce the formations of NFTs and Aβ through suppressing the activities of DYRK1A and its downstream AD-related signal pathways, and thereby alleviating some symptoms of AD. In this project, we plan to test our hypothesis using a new research pattern called “Chemical constituents analysis - In vivo metabolites identification - Bioactive compounds screening- In-vitro mechanism deciphering”. The components in feces, urine and bile after oral administration of TAPQ will be analyzed by LC-Q-TOF-MS/MS and LC-DAD-MS/MS combine with chemical compositions and chemical fingerprinting of TAPQ to clarify its metabolites in vivo. Then, the metabolites will be further screened by pharmacokinetic in rat brain and DYRK1A kinase assay in vitro to comfire the internal active components. Finally, the influence of bioactive metabolites to DYRK1A-induced tau protein phosphorylation and the formation of Aβ can be tested by using several cell lines. Thus, systematically investigate and decipher the internal active components and action mechanisms of TAPQ for AD.

多途径干预复杂性疾病AD的发生和发展为治疗AD的新的有效策略，DYRK1A在诱发AD的多个重要途径中起着核心作用，为抗AD的新靶点。本课题组前期研究表明苦木总生物碱(TAPQ)具有抗AD作用，可能的作用机制为：β-咔巴啉生物碱的体内代谢物质（原型或代谢产物）通过抑制DYRK1A活性及其下游AD相关信号通路，减少NFTs和Aβ生成，从而改善AD。因此，本项目拟在TAPQ化学成分和指纹图谱研究的基础上解析体内代谢物质，再以“体内—脑内”分析关联、分离/合成和DYRK1A抑制活性测试相结合的方法确认并获得靶器官—脑内药效物质；用多个细胞模型研究脑内药效物质对DYRK1A介导的tau蛋白磷酸化和Aβ生成的影响。以这种“有效部位化学成分研究—体内代谢物质解析—脑内药效物质确证—作用机制研究”层间递进的研究模式，系统阐明TAPQ抗AD的体内效应物质及作用机制。

本项目基于多途径干预AD的策略，以现代化学、分析学和生物学手段研究了苦木抗AD的药效物质基础和作用机制。以L-谷氨酸诱导的 PC12细胞损伤模型 和 Aβ25-35 诱导的 SH-SY5Y 细胞损伤模型初步筛选出苦木抗AD的有效部位为总生物碱部位，再以AD小鼠模型验证了其显著的抗AD作用，并初步阐明了其作用机制与抑制DYRK1A活性、神经炎症以及减少Aβ1-42 聚集有关；采用现代分离技术和波谱学手段从有效部位中鉴定出35个化合物，包括β-卡巴林类生物碱18个，铁屎米酮类生物碱8个，苦味素类化合物5个，其它类化合物4个。通过多个细胞模型筛选发现了10个活性显著的神经保护活性，且能够显著抑制DYRK1A和丁酰胆碱酯酶活性的生物碱类化合物，并总结了它们的构型关系，研究了它们与蛋白的结合方式，阐明了部分活性成分的体内代谢规律；建立了有效部位的指纹图谱，并对图谱中主要色谱峰和活性色谱峰进行了指认。此外，以现代合成方法全合成了两个活性化合物，并基于靶点设计合成了21个β-卡巴林生物碱衍生物，通过L-谷氨酸诱导的 PC12细胞损伤模型、Aβ25-35诱导的SH-SY5Y细胞损伤模型以及冈田酸诱导的 SH-SY5Y 细胞损伤模型筛选发现了4个具有显著神经保护活性的衍生物。以上研究为苦木抗AD开发提供了参考，同时为开发抗AD药物提供新的模板分子。