核桃壳黄酮调节脂代谢的活性成分及其作用机制研究

The increased prevalence of dyslipidemia in Chinese population has become one of the main causes for the rapid rise of sub-health population. It was found that some of dietary flavonoids were able to regulate dyslipidemia. Our early research found that walnut shells were rich in flavonoids with good anti-hyperlipidenmia activity on mice and inhibitory effects of differentiation on preadipocyte 3T3-L1 and several digestive enzymes. Yunnan has the largest area under walnut cultivation and production of walnut in China. However, a lot of walnut shells have been used only as fuel or just discarded as trash, which cause a tremendous waste of nature resources as well as the environmental pollution. This project will further systematically screen the effective parts and active components on the cellular level by applying to 3T3-L1 preadipocyte, L-02 humam hepatocytes, and fatty-changed L-02 human hepatocytes. And then,high fat diet rat, hyperlipemia rat and obese rat will be used to test the regulation of lipid metabolism of active components from walnut shell flavonoids. At last, the mechanism on the molecular level will be exposed by molbiological methods and the active components of walnut shell flavonoids will be identified structurally by LC-MS, NMR ect. It will allow multi-level revealing the active components and the molecular mechanism of walnut shell flavonoids on dyslipidemia regulation at vivo animals, cells, and molbiological aspects, which will lay the scientific foundation for the comprehensive development and utilization of walnut waste and the exploration of dyslipidemia transcriptomics.

近年我国人群脂代谢异常明显增加，是亚健康人群快速上升的主因之一。有些黄酮类化合物对脂代谢异常有良好的调节作用。我们的前期研究发现核桃壳不仅富含黄酮类化合物，而且核桃壳黄酮对3T3-L1前脂肪细胞的增殖分化和部分消化酶都有良好的抑制作用，对实验小鼠的降脂效果与化学降脂药非诺贝特相当。云南核桃栽培面积及产量均居全国第一，取仁后的大量核桃壳多作燃料或丢弃,造成资源的极大浪费和环境污染。本项目将进一步采用3T3-L1前脂肪细胞、人肝L-02细胞及脂肪变性的人肝L-02细胞模型和动物模型，从细胞水平和整体动物水平系统研究核桃壳黄酮降脂的活性成分或成分群，采用光谱、质谱、核磁等技术鉴定其化学结构；采用生化试剂盒和高通量测序等技术和方法，从酶水平和分子水平研究其降脂机制；从整体-细胞-分子多个层次，揭示核桃壳黄酮调节脂代谢异常的活性成分及作用机制，为核桃壳资源的高效开发利用和探索脂代谢转录组学奠定基础。

脂代谢异常是我国心脑血管疾病及亚健康人群快速上升的主因之一，植物多酚黄酮降脂是研究热点。云南核桃面积产量均居全国第一，取仁后的大量核桃壳富含多酚黄酮。本项目利用反复柱层析分级分离技术从核桃壳中分离纯化得到并鉴定了15个单体化合物；利用细胞模型和柱层析分离技术探明了核桃壳黄酮降脂的活性成分群（WS）及其单体化合物，分析鉴定了WS中的14个化合物；利用细胞模型、高脂血症动物模型探明了WS具有显著预防和调节脂代谢异常的作用，可预防高脂饮食引起的大鼠高脂血症；可显著降低脂肪变性L-02人肝细胞和3T3-L1成熟细胞的TG和TC；显著降低高脂血症模型鼠血清胰岛素及血清和肝脏的TC、TG、LDL-C、MDA、NO、瘦素、ALT、AST水平（P<0.001）；显著升高血清CAT（P<0.01）、肝脏HDL-C、脂联素、内脂素水平及CAT、GSH（P<0.001）和脂蛋白脂肪酶（P<0.01）活力；显著降低高脂血症鼠体重、Lee's指数、肝脏指数、附睾脂肪指数和肾周脂肪指数（P<0.05）；探明了WS对高脂血症脂蛋白脂肪酶、激素敏感性脂肪酶、脂肪酸合成酶、谷胱甘肽过氧化物酶、谷草转氨酶和谷丙转氨酶等脂代谢关键酶的影响，对瘦素、脂联素、内脂素、PPAR-γ等脂代谢因子的影响；从细胞水平和动物水平研究了WS对脂代谢关键酶及脂代谢相关因子在转录组水平上表达量的影响，获得了WS显著调节脂代谢异常在细胞水平上的相关酶基因74个，脂代谢相关因子基因80个，在动物水平上的主要相关酶基因79个，丰富了脂代谢异常及调节的相关基因转录水平表达内容，为脂代谢机理的研究提出了新的方向；阐明了WS对高脂血症模型大鼠肠道菌群的影响，揭示了核桃壳黄酮调节脂代谢异常的物质基础和作用机制，为核桃壳资源的开发利用和探索脂代谢转录组学奠定了基础。项目实施期间发表相关论文14篇，其中SCI收录5篇（含一区SCI刊物两篇），SCI他引13篇次；申请发明专利3件，其中1件已获授权；参与申报并获云南省科技进步特等奖1项。