植物食品miRNA在胃肠环境中的稳定性和吸收机理的研究

Recent reports show that some plant-derived miRNAs can regulate nutrient metabolism by binding the complementary transcriptional sequences in vivo. The findings immediately raised numerous follow-up studies, however, the stability and the absorption mechanism of the plant-derived miRNAs in gastrointestinal environment, which serve as the basis for their regulation effects, are still unclear. This has become the scientific bottleneck problem hindering the research on nutritional functions of plant-derived miRNAs. For this reason, the present study is designed to investigate the stability and the absorption mechanism of the plant miRNAs in gastrointestinal environment from the perspective of food digestion and absorption, in order to clarify the cause of molecular tolerance against nuclease degradation and the structural characteristics of the stable miRNAs, and to illuminate the influence of food ingredients on the stability of the plant miRNAs. By using Caco-2 model, absorption mechanism and uptake efficiency of the plant-derived miRNAs are expected to be revealed. Animal experiments on rats are also designed to explore the in vivo stability characteristics and absorption process of the miRNAs. This study is devoted to clarify the stability and the absorption mechanism of plant-derived miRNAs in gastrointestinal environment, which will provide the experimental evidence for the plant-derived miRNAs to play regulatory functions in vivo. The results can be used to re-evaluate the relationship between nucleic acids intake and human nutrition, and to provide new perspective and theory basis for development of active ingredients from food and for establishment of nucleic acid-related balanced diet theory.

新近报道，植物食品中的miRNA可以靶向结合转录序列进而调节机体营养物质代谢。此发现立即引发了大量跟进研究，但目前对于植物miRNA发挥营养调节作用的基础，即植物miRNA在胃肠环境下的稳定性和吸收机理尚不清楚，已成为阻碍其营养功能研究的瓶颈科学问题。为此，本课题拟从食品消化吸收的角度出发，研究植物miRNA在胃肠液中稳定性，揭示耐降解miRNA的序列结构特征，明确食品共存组分对miRNA稳定性的影响；利用Caco-2模型研究细胞对miRNA的摄取效率和吸收机制。同时结合大鼠实验，考察植物miRNA的生物利用度、稳定性miRNA的特征结构域和吸收过程。本课题的研究将阐明植物miRNA稳定性的成因和吸收机理，为其营养调节作用的进一步探索提供实验证据。相关成果会使人们重新审视食品核酸摄入与人体营养的关系，为食品有效成分的发掘和饮食核酸均衡膳食理论的建立提供全新的视角和理论依据。

新近研究表明，植物来源的微小RNA（Plant-derived miRNA）能够以跨界调节（Cross-kingdom regulation）的方式影响哺乳动物的营养代谢。这一新发现打破了RNA在体外不能稳定存在的原有认识，预示着植物miRNA同六大营养素一样，可对人体产生营养调节作用。然而后续的相关研究过度关注植物miRNA的生物活性，而忽略了“植物miRNA在消化道的稳定性”和“细胞对耐受性miRNA吸收过程”这两个植物miRNA发挥跨界调节作用的基础性问题。为此，本项目以植物食品高丰度的六种miRNA为对象，研究其分子结构在口腔、胃、肠道环境中的稳定性；深入探讨植物食品miRNA在相应条件下的存在状态和吸收机制；在此基础上，进一步考察了高稳定性miRNA同肠道微生物的相互作用。研究结果表明：植物miRNA对消化环境的耐受性具有显著差异，osa-miR168a在胃液中的半衰期可长达42.5 min，而sly-miR157a不到其1/4（9.2 min）。经肠液消化后，gma-miR160的终浓度为2.1 pM，远高于sly-miR157a的8.4 fM。从口腔到小肠的整个消化过程对植物miRNA的降解存在瓶颈，miRNA浓度在降低到一定阈值后随降解时间的延长不再减小，终浓度和初始剂量之间不存在依赖关系。分子模拟揭示，基于miRNA的碱基组成的高级结构决定了其在消化环境中的稳定性，miRNA分子内自配对（Self-paring）形成茎-环（Stem-loop）基序能有效提高其耐受性。植物miRNA中的2′-O-Methyl修饰基团可提高其在消化道中的稳定性。此外，同植物miRNA共摄入（co-ingested）的多糖、蛋白质和脂质等食品基质可保护miRNA免于唾液中核糖核酸酶（RNase）的降解。肠上皮Caco-2细胞对植物miRNA的吸收具有序列依赖性（Sequence-dependent），网格蛋白（Clathrin）和小窝蛋白（Caveolin）介导的内吞是Caco-2摄入植物miRNA的主要途径。bol-miR159（一种源自西蓝花的植物miRNA）可调节小鼠肠道菌群（Gut Microbiota，GM）的组成，具体体现为Firmicutes和Desulfobacterota的降低，以及Bacteroidota、Proteobacteria和Actinob