基于吸附胆酸盐和调节肠道菌群作用的荞麦蛋白降低血脂机理研究

The purified protein (BP-1) from buchwheat, a unique crop resource in Chinese region, is studied in this project. The highlight is focused on the activity and characteristics of the BP-1, and its mechanism and leading action pathways of hyperlipidemia improvement.It will be clarified eventually that BP-1 is the important functional factor in buckwheat for hyperlipidemia improvement based on above information. At first, the rapid, effective separation conditions of BP-1 will be set up and optimized through the combined the separation technologies (such as two-dimensional liquid chromatography and other chromatographies) and strategies of keeping the stability of proteins. Then,the relationship between amino acid sequence and the function of cholate adsorption of the BP-1 will be verified from the perspective of adsorption kinetics. The effect of cholesterol reduction and cholate adsorption in rats by BP-1 treatment will be identified. The signal transduction pathways and molecular regulation pathways of cholesterol reduction by BP-1 treatment in HepG2 cell will be elucidated by the analysis of proteins and genes expression. Finally,the effect and regular pattern of BP-1 on the gut flora will be studied in the model animal and microecology by the analysis technologies of proteomics, metabolomics and genomics. Furthermore, it will be confirmed that the effect of hyperlipidemia improvement of BP-1 is mediated by adjustment of gut flora. The project has significant science and social meaning because the results in this project will provide the experimental and theoretical foundation for developing the high quality products from buckwheat with the function of hyperlipidemia improvement.

以中国独特资源荞麦中分离纯化获得的荞麦蛋白（BP-1）为研究对象，重点研究其调节血脂功能所对应的蛋白特征及活性，以及调节血脂的主导作用途径和机理，明晰BP-1是荞麦具有调节血脂功效的重要功能因子。首先将二维色谱、层析等分离纯化技术与目标蛋白质活性稳态化技术相结合，建立并优化BP-1快速高效高活性分离纯化技术条件。以此为基础，从吸附动力学角度，探明BP-1氨基酸序列和吸附胆酸盐功能之间的关系；通过大鼠高脂模型,揭示BP-1在大鼠体内吸附胆酸盐降低胆固醇的作用机制；以蛋白质及基因表达等方法揭示BP-1对HepG2细胞内调节胆固醇的分子调控途径和信号转导途径。利用蛋白质组学、代谢组学和基因组学的技术手段，通过动物和微生态模型研究BP-1对肠道菌群的影响及规律，探明其通过调节肠道菌群调控血脂的作用及机制。为研究开发具有调节血脂功能的高品质荞麦产品提供实验和理论基础，具有重要的科学价值和社会意义。

高脂饮食引发肠道菌群紊乱与血脂代谢异常有着密切关系，调节肠道菌群已成为干预血脂代谢异常的新靶点。本项目不仅建立了荞麦水溶性蛋白（BP-1）的分离纯化及稳态化的关键技术，并初步鉴定其结构是由6对亚基组成的桶状结构。同时，从体内、体外生理活性和细胞内调节胆酸盐的信号转导途径等多水平评价体系，系统揭示了荞麦水溶性蛋白（BP-1）降血脂能力显著，胆酸钠、脱氧胆酸钠和牛磺胆酸钠的吸附量显著提高，且作用后的细胞内胆固醇与甘油三酯含量均有所下降。采用IT-TOF-MSn多级质谱技术和16S高通量测序技术分析高脂膳食小鼠粪便发现，荞麦水溶性蛋白（BP-1）可显著降低厚壁菌门丰度，提高拟杆菌门的丰度，导致Escherichia-shigella增多，同时能够提高Bacteroidales、Blautia等产生短链脂肪酸菌群的丰富度。显著降低Eubacterium coprostanoligenes（一种产粪甾醇真细菌，与降低胆固醇有着密切关系）丰度。同时，荞麦水溶性蛋白（BP-1）组较模型组粪便中乙酸、丙酸等短链脂肪酸显著升高，且肿瘤坏死因子α（TNF-α）、白细胞介素6（IL-6）等炎症因子水平的显著降低。采用代谢组学技术对小鼠血清进行数据采集并结合多元统计分析结果显示高脂膳食小鼠血浆中存在亚油酸、甘油、磷脂酰乙醇胺、半乳糖神经酰胺、脑苷脂、葡糖神经酰胺、磷脂酰胆碱、葡糖苷酸、前列腺素G2、磷脂酸、溶血磷脂酰胆碱、鞘磷脂等12种潜在的生物标志物，荞麦水溶性蛋白（BP-1）可通过调节高脂膳食小鼠体内12 种潜在的生物标志物所对应的6条代谢代谢通路（亚油酸代谢、甘油磷脂代谢、鞘脂类代谢、戊糖-葡萄糖醛酸酯相互转化作用、色氨酸代谢和花生四烯酸代谢），并使其向正常状态转变，从而缓解高脂膳食小鼠代谢紊乱的症状。综合代谢组学、转录组学和肠道菌群结构分析可知，荞麦水溶性蛋白（BP-1）可调节高脂膳食引起肠道菌群失衡，增加短链脂肪酸，抑制炎性反应，从而干预脂类等营养素吸收利用过程，是其有益机体健康作用的重要途径之一。本项目为进一步研究开发高品质、功能独特的荞麦深加工产品提供理论基础和技术指导。