基于PPARγ-ApoE的酸枣仁抗阿尔茨海默症协同成分发现与作用机制研究

Nuclear receptor PPARγ increases ApoE expression and thereby clear cerebral Aβ deposition, meanwhile PPARγ inhibits the inflammatory response induced by Aβ, thus becomes a new target for the treatment of Alzheimer’s disease (AD). This project uses a variety of modern analytical techniques to comprehensively analyze the composition of a commonly used Chinese herb Semen Ziziphi Spinosae, as well as their in vivo metabolism and prepare components which enter the brain. We use ultrafiltration-liquid chromatography-mass spectrometry strategy combined with luminescence reporter assay, and through a series of cell based AD pharmacological models, to screen the synergistic components group from Semen Ziziphi Spinosae based on their potential activation of PPARγ-ApoE, as well as their involvement in Aβ42 degradation and clearance processes. By analyzing the association among chemical components-abundance-efficacy, we try to find ingredients which function synergistically; We then use Drosophila and mouse AD model to assess efficacy of the synergistic components group; Furthermore, through real-time monitoring of cerebrospinal fluid metabolites and AD biomarkers such as Aβ42、tau in mouse brain, combined with brain tissue transcriptome data, we are able to describe the functional network by synergistic components group. To investigate the mechanism, we analyze the functional role of synergistic components play in multi-step of AD formation. This study is a useful exploration on how to find out components function synergistically and investigate their mechanisms.

核受体PPARγ可以通过上调ApoE从而清除Aβ在脑内的沉积，并抑制炎症反应，成为抗阿尔茨海默症（AD）重要靶标/通路。本项目拟在前期研究基础上，利用多种现代分析技术，以抗AD常用中药酸枣仁为研究对象，解析并制备其代谢入脑成分，利用超滤-液相色谱-质谱联用结合荧光报告基因法，并通过多种细胞药理模型，筛选发现酸枣仁中围绕PPARγ-ApoE参与的Aβ42降解与清除过程的活性成分（群），并对其进行成分-含量-药效关联，发现具有协同作用的成分组合。利用果蝇及小鼠等模型，验证协同成分组合的抗AD药效。进一步通过脑脊液中内源性代谢产物及Aβ42、tau等生物标记物含量的变化、结合脑组织转录组数据进行关联分析，描绘酸枣仁“协同成分组”的调控网络；并在细胞水平上探讨协同作用成分组中各成分在AD发生过程的多个节点上的多环节协同的抗AD作用机理。以上研究对中药药效物质发现及作用机制进行有益探索。

我们已建立完善的秀丽隐杆线虫平台；构建含有PPARγ结合位点的报告基因载体，并用于高通量筛选活性化合物；结合秀丽隐杆线虫模型及细胞模型发现了一系列具有降低Aβ42毒性蛋白片段累积、增强学习记忆能力的活性小分子天然产物。结合体外细胞模型及哺乳动物体内模型对阳性化合物的药效及机制进行研究：酸枣仁皂苷A可通过激活细胞外调节蛋白激酶Axl，促进HSP90β蛋白水平上升，增强PPARγ蛋白稳定性，恢复PPARγ信号通路活性，促进载脂蛋白E及腺苷三磷酸结合盒转运体1蛋白表达，从而增强小胶质细胞对Aβ蛋白进行清除；金丝桃素可能通过降低核内MKL1蛋白水平抑制oAβ诱导产生的炎性反应，也提示MKL1可能是临床治疗AD的潜在靶点。利用3个表现出与Aβ相关的病理行为的转基因秀丽隐杆线虫（C.elegans）模型，探讨了17种人参皂苷的体内抗Aβ作用。以上研究为AD的治疗提供新的治疗靶点，为AD治疗提供先导化合物，为获得高效、低毒的抗AD治疗药物奠定基础。期间，发表SCI论文3片，其中一篇影响因子>5，培养硕士生2名，博士生1名。