荔枝果肉酚类成分群基于miRNA的调节脂质代谢作用机制

Changing the expression of target genes by regulating miRNA has become a novel research strategy to investigate the action mechanism of botanical polyphenols. Our previous results showed that phenolic extracts from litchi pulp improved the lipid metabolism in high fat loaded mice significantly. However, the exact bioactive components and the specific action mechanism are not clear up till now. In this research, we will use the iterative screening strategy of bioactive equivalent combinatorial components of the Chinese traditional medicine pharmacodynamics as a reference to screen the bioactive combinatorial phenolics from litchi pulp. The phenolics which do not contribute significant roles in maintaining the lipid homeostasis in oleic acid loaded HepG2 cells will be knocked out through preparative HPLC by analyzing the cellular contents of triglyceride and total cholesterol and then the bioactive combinatorial phenolics will be selected. The chemical structure of each component in the selected bioactive combinatorial phenolics will be characterized. Transient transfection technique will be used to get lipid metabolism miRNA overexpressed or inhibited HepG2 cells including miR-33 and miR-122. The effects of bioactive combinatorial phenolics on the expression of cholesterol and fatty acid metabolism related genes including ABCA1, FAS and CPT1a etc. will be investigated using miRNA overexpressed or inhibited HepG2 cells. The substance basis and action mechnism of litchi pulp regulating lipid metabolism will be elucidated focusing on the controll of miRNA. These results ill be able to enrich the theoretical basis of phenolic bioactivity, and to supplement scientific foundation for the fine processing and functional foods development of lichi.

通过调节miRNA影响其靶基因表达成为近年来探讨植物多酚生物活性机制的新方向，本项目针对前期研究发现荔枝果肉酚类提取物明显改善高脂负荷小鼠脂质代谢，而其具体活性成分及作用机制不明确等问题，拟借鉴中药药效学“等效成分群迭代反馈筛选策略”，采用油酸负荷的HepG2细胞模型，以细胞内甘油三酯和胆固醇含量等为评价指标，分级和逐一剔除对活性无显著贡献的多酚类群和单体，迭代筛选确定荔枝果肉调节脂质代谢作用的有效酚类成分群，并表征各化合物的化学结构；采用瞬时转染技术，建立脂代谢相关miRNA miR-33或miR-122过表达或抑制的肝细胞模型，探讨荔枝果肉有效酚类成分群对细胞内ABCA1、FAS和CPT1a等胆固醇和脂肪酸代谢相关基因表达的影响，围绕miRNA的调节阐释荔枝果肉调节脂代谢作用的主要物质基础及分子机制。研究结果将丰富酚类生物活性的理论基础，为荔枝精深加工和功能食品研发提供科学依据。

本项目围绕荔枝果肉酚类物质调节脂质代谢的有效酚类成分及其作用机制开展研究。通过比较F1-F4四个荔枝果肉酚类成分群对油酸诱导的肝细胞脂质聚集的影响，发现F2、F3和F4三个成分群均具有改善脂代谢作用，进而通过多级柱层析分离结合波谱分析，表征出酚类成分不明确的F2和F4成分群中的主要酚类化合物，从荔枝果肉中首次分离到异鼠李素3-O-鼠李糖基(1→4)-鼠李糖基(1→6)-葡萄糖苷、山奈酚3-O-鼠李糖基(1→4)-鼠李糖基(1→6)-葡萄糖苷等9个黄酮类化合物，通过成分“敲出”前后活性比较分析，明确了槲皮素-3-O-芸香糖-7-O-α-L-鼠李糖苷（QRR）等5个黄酮类化合物为荔枝果肉调节脂代谢主效酚类成分。分别通过油酸诱导的肝细胞模型和高脂膳食诱导的NAFLD模型从体外和体内水平揭示了荔枝果肉酚类成分调节肝脏脂质代谢的分子机制，发现荔枝果肉酚类物质通过促进肝细胞内AMPK的磷酸化，进而调节PPARα等核受体的活化，促进脂肪酸分解代谢，通过抑制SREBP1活性或直接磷酸化修饰ACC抑制脂肪酸合成是其抑制肝细胞脂质过量储集的主要机制。. 本项目全面完成合同内容，部分超出合同指标。发表学术论文14篇，其中SCI论文9篇，申请并获得授权国家发明专利1件。以本项目研究发现为重要内容的科技成果获得广东省科技进步一等奖和广东省农业技术推广奖一等奖各1项。培养硕士研究生3名。项目主持人被评为全国农产品加工业十佳青年科技人才，广东省“三八红旗手”，于2017年入选广东省农业科学院第一层次的青年人才培养项目—金颖之光；项目核心成员入选国家“万人计划”科技创新领军人才。