金银花多糖抑制Aβ42聚集的构效关系及相关机制研究

Amyloid-beta protein (Aβ) is a key factor of causing Alzheimer's diease (AD). Our previous report had found that the polysaccharide named as LJW0F2 from flowers of Lonicera japonica, Thunb could significantly inhibit Aβ42 aggregation in a dose-dependent-manner in vitro, by the sulfur element fluorescence and atomic force microscope detection. Besides, it could attenuate the cytotoxicity induced by Aβ42 aggregation in SH-SY5Y neuroblastoma cells. To the best of our knowledge, this was the first report that the exogenous plant-derived polysaccharide might block Aβ42 aggregation directly and reduce its toxicity in SH-SY5Y cells. However, its structure-activity relationship and molecular mechanism are still vague. On this basis, using circular dichroism spectra, computer simulation, site-specific mutagenesis and QCM technologies, the molecular mechanism of Aβ42 aggregation inhibition of LJW0F2 and the specific amino acid sequence are going to be elucidated. Inaddition, the structure-activity relationship of LJW0F2 and the active structral domain will be also elucidated through the investigation of the inhibitory activity influence by different molecular weights and substituents of LJW0F2. The pharmacological activities of polysccahraides are going to be futher evaluated in SH-SY5Y cells and in the APP/PS1 double transgenic mouse model. The study will help to further extend the medicinal fields of the traditional Chinese flowers of Lonicera japonica, Thunb and provide theoretical foundation for the further development of its value of medicinal resources. Besides, these reslults would provide important theoretical basis and research paradigm for the potential AD therapy by the carbohydrate-based new drug development.

本实验室以AD发病的关键因素β淀粉样蛋白为切入点，采用硫磺素荧光和原子力显微镜检测技术，首次发现中药金银花多糖可在体外抑制Aβ42聚集，并可抑制Aβ42诱导神经细胞SH-SY5Y毒性，但其构效关系及分子作用机制不详。本课题在体外活性筛选基础上，拟利用圆二色谱、计算机模拟、氨基酸定位突变及石英微晶天平技术阐明多糖抑制Aβ42聚集的分子作用机制和特定氨基酸结合序列。进而，采用酶法及化学法对多糖LJW0F2进行降解和衍生化，通过考察不同分子量及多种取代基对其抑制Aβ42聚集活性的影响，阐明多糖抑制Aβ42聚集的构效关系和活性多糖结构域。在阐明其构效关系的基础上，进一步在人神经母细胞瘤株SH-SY5Y细胞水平上和APP/PS1双转基因小鼠动物水平上，对活性多糖进行初步药效评价。该研究结果将为传统中草药金银花的活性物质基础研究及其药物靶向性研究奠定理论基础。

本实验室以AD发病的关键因素β淀粉样蛋白为切入点，采用硫磺素荧光和原子力显微镜检测技术，首次发现中药金银花多糖可在体外抑制Aβ42聚集，并可抑制Aβ42诱导神经细胞SH-SY5Y毒性，但其构效关系及分子作用机制不详。在此项目资助基础上，我们进一步阐明：1）均一多糖LJW0F2的精细结构为α1,4链接葡聚糖；2）通过部分酸水解制备系列不同分子量片段的多糖、此外制备不同取代基取代的多糖衍生物及不同链接方式的葡聚糖，通过硫磺素荧光检测，考察其抑制Aβ42聚集的构效关系，考察其产生活性所需多糖结构域，初步明确了该多糖抑制Aβ42聚集的活性与其分子量大小、取代基及链接方式密切相关；3）通过SH-SY5Y细胞模型和APP/PS1转基因雄性早老痴呆小鼠模型，对系列多糖进行药理活性评价，发现β1.3链接的葡聚糖可减轻阿尔茨海默症造成的认知障碍，减少脑剖的β淀粉样蛋白沉积，并且促进海马体的神经前驱细胞新生。该研究结果为传统中草药金银花的活性物质及其药物靶向性研究奠定了理论基础，也为新型抗阿尔兹海默糖类药物研发提供新的物质基础和研究思路。.项目完成情况：1）发表受此项目资助的SCI收录期刊论文4篇。2）获得授权国家发明专利1项并申请1项国家专利；3）培养博士后1名，硕士研究生1名。