海参高硫酸化寡糖的制备、结构及其对Aβ诱导的神经元毒性的保护机制研究

Food-derived acid polysaccharides have been widely reported to have neuron-protective activity. However, as the complexity of the polysaccharides structure and limitation in structure analytical skills, the structure-activity relationship was still unclear. This study was based on three sulfated polysaccharides with well-repeated units, which were identified from sea cucumber in our previously research. The oligosaccharides of these polysaccharides were prepared either by oxidative degradation or acid degradation, and then purified by geltration followed with SAX-HPLC. The structures of these oligosaccharides were identified by combination of mass spectrometry and high-resolution nuclear magnetic resonance technology. The neuron-protection activities of the oligosaccharides were firstly tested by an oxidative stress damage cell model, which was to investigate the effect of the sulfation pattern, molecular size, and branched chain on the neuron-protective activity.Then, the exact neuron-protection mechanism of the oligosaccharides was further tested by an Aβ-induced nerve cell damage model, the cell viability as well as the changes in Aβ aggregation state and BACE1 activity were tested to evaluate the activities; finally, to clarify the mechanism of the interaction mechanisms and structure-activity relationship, the binding of the Aβ with the sulfated oligosaccharides was examed by a combination of SPR, and circular dichroism and atomic force microscopy, which was to investigated the binding sites and the change of the secondary structure of Aβ during the process. The research results of this project will not only proposed a common structure-activity relationship for the the food derived sulfated oligosaccharides on anti-Alzheimer, but also provide a theoretical basis for molecular size controlling of the polysaccharide during food processing.

食源性酸性多糖的神经保护活性近年来已广有报道，然而受限于多糖结构的复杂性和结构分析技术的滞后，其构效机理目前仍不清楚。 本研究以课题组前期从海参中纯化鉴定的三种具重复序列结构的硫酸多糖为对象，利用氧化降解、酸降解以及色谱纯化技术制备系列寡糖，并结合质谱和高分辨核磁共振技术鉴定其结构；应用氧化应激损伤神经细胞模型，研究寡糖硫酸基取代方式、分子量、支链取代基对其神经保护活性的影响；在此基础上，以Aβ诱导的海马神经细胞损伤模型，以Aβ蛋白聚集状态和BACE1酶活变化为切入点，阐明海参寡糖对神经细胞保护机制；并以计算机模拟、SPR技术研究Aβ分子上的硫酸寡糖结合区域，圆二色谱和原子力显微镜技术明确寡糖结构对Aβ处理过程中二级结构的影响，阐明寡糖靶向Aβ机制和构效机理。 本课题的研究成果不仅将为食源性硫酸寡糖抗老年痴呆活性的构效机理研究探索共性规律，也为食品加工过程多糖分子量的控制等提供理论依据。

为深入阐明食源性海洋硫酸多糖的神经保护及构效机理，本研究以课题组前期从海参纯化鉴定的三种具有重复序列结构的硫酸多糖为对象，采用氧化降解，酸降解，色谱纯化技术制备系列寡糖，并结合液质联用，质谱和高分辨核磁共振等技术鉴定了硫酸寡糖结构；研究了不同硫酸基取代方式，不同分子量的硫酸寡糖的抗凝血抗血栓和降血糖血脂活性，完善硫酸寡糖发挥预防老年痴呆症的作用途径；运用氧化应激损伤神经细胞模型和和Aβ诱导的神经细胞损伤模型，研究了硫酸寡糖对神经细胞的保护机制；以计算机模拟和SPR技术研究了Aβ分子上的硫酸寡糖结合区域，并用SPR技术和核磁研究硫酸寡糖与Tau蛋白的相互作用。本项目解决或部分解决了快速制备食源性海洋硫酸寡糖问题，寡糖分离纯化问题，硫酸寡糖结构鉴定问题，硫酸寡糖抗老年痴呆构效关系问题，为食源性硫酸寡糖应用于抗老年痴呆症提供了充分的理论依据。