食源性TMAO靶向肝细胞Nrf2-Keap1/ARE通路的氧化应激及苹果黄烷醇调节的生化机制

Recent findings show that choline and phosphatidylcholine as traditional nutritional methyl-donor foods can be metabolized by the gut microbiota and liver to trimethylamimine N-oxide (TMAO), leading to the vascular damage. Furthermore, we have preliminarily found that foodborne TMAO plays a strong harmful role in the liver, and dietary flavonols exhibit antagonistic effects against the damage. However, the specific mechanism of hepatoxic damage of TMAO and its regulation remains fully unclear, and therefore, the investigation based on metabolism and molecular levels is an urgent need. For this reason, the present study is designed to further explore the metabolism and transport, liver-targeted distribution and hepatomicrosome enzyme-based regulation of TMAO in choline- or egg yold-feeding/administrating rats. This study will also investigate the intervention effect and molecular mechanism of TMAO targeting the genes of hepatocellular Nrf2-Keapl/ARE antioxidative pathway in rats, which is further linked to hepatoxic molecular mechanism via injurying upstream gen of MAPKs, and downstreat molecules of antioxidant enzymes including HO-1 and phase II detoxifying enzymes including NQO1. In addition, we will evaluate the detoxifying molecular mechanism, interaction characteristics and structure-activity relationship of the tested apple flavonols with different molecular structures against TMAO-induced hepatocellular metabolism, distribution and oxidative stress hapatoxicity in rats. Finally, the present study will discover the harmful new pathway of foodborne TMAO, and also find the molecular intervention target for liver oxidative stress. This study will also provide the theoretical basis for the scientific knowledge on nutrition and safety of methyl-donor foods.

新近报道，胆碱、卵磷脂等甲基供体食物可被肠道菌群代谢而最终在肝脏中生成氧化三甲胺（TMAO），并介导血管损伤。同时，我们初步发现该食源性TMAO对肝脏有强效危害作用，而食物成分黄烷醇有拮抗效应，但该具体损害及其调控机制尚不清楚，急需从代谢与分子水平深入探究。为此，本项目采用大鼠摄入胆碱或富含卵磷脂的鸡蛋黄原始食物的方式，研究体内代谢物TMAO在肝细胞中的代谢转化、肝靶向分布及其对肝微粒体酶的调节作用；探究TMAO对大鼠肝细胞Nrf2-Keapl/ARE抗氧化通路及其上游MAPKs、下游HO-1等抗氧化酶和NQO1等Ⅱ相解毒酶基因转录与表达的干预作用及机制；考察不同结构特性的苹果黄烷醇对食源性TMAO在大鼠肝细胞代谢、分布及氧化应激损伤中的减毒机制、互作特性和构-效关系。揭示食源性TMAO的体内危害新途径，发现TMAO肝氧化应激的干预靶点，为科学认知甲基供体食物的营养与安全提供理论依据。

美国SL Hazen研究组相继在Nature (2011)、Nature Medicine (2013)和Cell (2015)报道，卵磷脂、胆碱、L-左旋肉碱等甲基供体食物可被肠道菌群代谢成三甲胺（TMA）而最终在肝脏中生成氧化三甲胺（TMAO）进而诱发血管损伤。近年来，团队验证了TMAO的微弱血管毒性，发现并率先报导了TMAO摄入明显触发小鼠肝细胞氧化应激损伤与肝脂代谢异常的食品毒理特性，并报导了几种饮食类黄酮和茯砖茶汤可显著拮抗TMAO的血管与肝损伤。. 在此基础上深入研究了食源性代谢物TMAO肝损伤的分子毒理机制。为此，我们选择小鼠等动物喜好的高剂量（3%）L-左旋肉碱饮用水为肝损伤的食品造模剂（即采用3%左旋肉碱水溶液喂养12周的雄性昆明小鼠为动物模型），以“肠-肝轴”损伤途径为研究突破口，综合利用液-质联用技术（UPLC-MS/MS）、16S rRNA测序、肠道AKK菌体外厌氧发酵技术、代谢组和转录组学等生化与分子生物学新技术，研究高左旋肉碱诱导小鼠的肝毒性、分子机理与类黄酮的调控机制。. 首先，我们成功地构建了一种更快、更简便的血清及尿液中左旋肉碱代谢产物TMAO、TMA和DMA的UPLC-MS/MS同时分析检测方法；首次发现小鼠结肠Akkermansia_ muciniphila（AKK）在TMAO诱导的小鼠肝损伤中的强效“肠-肝轴”调控作用，并第一次从AKK菌体外发酵及菌群回补两方面验证了肠道AKK的肠菌群代谢与肝毒性调控作用；同时，利用转录组学和代谢组学技术，率先揭示了TMAO调控小鼠肝脏胆汁酸代谢通路、AMPK信号通路及PPAR信号通路中相关基因差异表达，从基因水平阐释了TMAO诱导的小鼠肝毒性分子机制；此外，证实槲皮素可剂量依赖性拮抗高左旋肉碱诱导的小鼠肝脏氧化应激毒性效应。. 相关研究成果已在农业食品领域Molecular Nutrition & Food Research、Journal of Agricultural and Food Chemistry、Food & Function等国际高水平SCI论文43篇，且受到国内外学术界高度关注，被 SCI 期刊大量引用，当前已完成项目合同的全部任务；此外，部分研究成果正在整理中，拟发表在更高水平的杂志上。