基于PPARγ通路的A型原花青素肠道菌群代谢物抗动脉粥样硬化作用与机制研究

A-type procyanidins of litchi fruit pericarp (LFP) with strikingly low absorptivity can prevent atherosclerosis (AS). We found that the function might be attributed in part to the metabolites of A-type procyanidins by gut microflora. Therefore, the active metabolites of procyanidin A2 and A-type procyanidin trimer from LFP by the gut microflora will be found according to the inhibitory activity on macrophage-to-foam cell transformation in vitro. Then, the active metabolites will be indentified with HPLC/MS and NMR. The possible effects and mechanism of the active metabolites will be explored in macrophage foam cells based on the signal pathway of PPARγ and the mRNA and protein expression of key genes in the reverse cholesterol transport and inflammation. In order to understand the anti-atherogenic effect of the metabolites of A-type procyanidins by gut microflora, the normal and germ-free ApoE-/- mice will be fed with A-type procyanidins from LFP. The atherosclerotic plaques and PPARγ-signaling pathway will be investigated. The microbial metabolites in plasma and urine will also be compared by HPLC analysis. The anti-atherogenic effect of microbial metabolites of A-type procyanidins and the molecular mechanism of activating PPARγ-signaling pathway will be clarified. The research will enrich the theoretical basis of AS-preventing effects of procyanidins, and provide the scientific basis for the development and application of natural plant products.

荔枝果皮A型原花青素具有极低的吸收率和显著的抗动脉粥样硬化（AS）活性。项目前期的研究表明，荔枝果皮A型原花青素抗AS活性可能与其肠道菌群代谢物有关。为明确肠道菌群代谢物对A型原花青素抗AS活性的作用与机制，本项目拟采用ox-LDL 刺激的巨噬细胞泡沫化模型，筛选确定其代谢产物的主活性组分，采用HPLC/MS、NMR等方法表征其化学结构；分析主活性产物对ox-LDL刺激的巨噬细胞PPARγ活化的影响，探讨细胞内与胆固醇逆向转运及炎症相关的关键基因表达和蛋白水平的变化；结合比较分析A型原花青素对正常与缺失肠道菌群ApoE-/-小鼠的动脉粥样硬化斑块形成、血浆和尿液中其代谢物HPLC谱的差异，以及PPARγ与其下游关键基因表达的变化，揭示以荔枝果皮为代表来源的A型原花青素肠道菌群代谢物抗AS的作用及分子机制。研究结果将丰富原花青素抗AS的理论基础，为特色植物天然产物的开发应用提供科学依据。

采用UPLC-Q-TOF-MS/MS对原花青 A2（procyanidin A2, PCA2）体外肠道菌群代谢物进行了分析，转化生成的代谢产物主要为3-(4-羟苯基)丙酸（HPPA）、对羟基苯乙酸（4-HPAA）、表儿茶素、B型原花青素、5.7.2＇-三羟基黄烷等。利用ox-LDL刺激的巨噬细胞模型，研究主要活性代谢物抗AS机制，发现PCA2肠道菌群主要代谢物HPPA和4-HPAA能显著抑制巨噬细胞泡沫化。HPPA显著上调ABCA1、SR-B1 mRNA 的表达水平，4-HPAA显著上调PPARγ、ABCG1 mRNA 的表达水平；HPPA及4-HPAA 均能显著下调CD36的表达水平。HPPA抑制炎症性细胞因子IL-6和IL-1β的分泌，显著降低NF-kB蛋白表达水平。动物实验表明，PCA2能明显减轻高脂诱导ApoE-/-动脉粥样硬化模型小鼠主动脉窦处斑块形成及巨噬细胞浸润，降低氧化应激和炎症因子分泌，转录组测序显示PCA2主要通过调节PPARγ、NF-κB信号通路促进胆固醇转运、抑制炎症因子生成。采用抗生素剔除肠道菌群后，可显著削弱PCA2对高脂饮食诱导的AS的改善作用。在PCA2干预后，高脂饮食小鼠的肠道菌群结构得到调整，硬壁菌门与拟杆菌门的比例明显下降，阿克曼菌属的丰度显著升高，抗生素饮水的伪无菌小鼠肠道菌群多样性显著降低，并且大量定植了变形菌门的细菌。对照组小鼠血浆PCA2肠道菌群代谢物主要为对HPPA、4-HPAA、对羟基苯乙酸、苯甲酸、阿魏酸、3，4-二羟基苯乙酸、3，4-二羟基苯丙酸等，其主要代谢物HPPA、4-HPAA的血浆浓度显著高于伪无菌小鼠。研究结果表明PCA2与肠道菌群相互作用在PCA2改善高脂诱导ApoE-/-小鼠动脉粥样硬化活性中发挥了重要作用。