陈皮显效物质的新发现及其治疗非酒精性脂肪肝作用机制

Pharmacodynamic material basis is the difficulty and the emphasis of Chinese medicine research. With breakthroughs in gene sequencing technology over the past years, plant miRNAs as new natural active substances are verified to regulate human genes through daily diet. However, studies on its applications in the field of traditional Chinese medicine are still lacking. In previous studies, we found that besides flavonoids, miRNAs sequenced from Citri Reticulatae Pericarpium can be absorbed orally into the blood, and possesses bioactivities, thus proving that miRNA could be one of the pharmacodynamic materials in C. Reticulatae Pericarpium for non-alcoholic fatty liver disease (NAFLD) treatment. Considering the fact that therapeutic effect of the herbal medicine is based on the synergic action of its mass constituents, this study is aimed to investigate the correlation between blood concentration of flavonoids and miRNAs from C. Reticulatae Pericarpium and their pharmacodynamic actions on rodent and cell models,which is analyzed by multiple qPCR, LC/MS, protein chip and microdialysis technology.In addition, to investigate the effectiveness, safety and development value, synergistic effects of flavonoids and miRNAs are also illustrated as well as their network regulation mechanism by the chemical composition, distribution, metabolism, pharmacodynamics mechanism and transcriptomics analysis. This study will explore synergistic effects between the miRNA and plant secondarymetabolites of traditional Chinese medicine pharmacodynamic substance, expand the existing research model on secondary metabolites as the main effective components, and providing evidence for development of new herbal miRNA drugs.

药效物质基础是中药研究的重点和难点。随着近年来基因测序技术的突破，最新研究表明植物微小核糖核酸(miRNA)是一类新的天然活性物质，但在中药研究领域鲜有报道。项目组前期发现，除黄酮外，陈皮miRNA可通过饮食方式吸收入血，具有多种生物活性，可能是陈皮治疗非酒精性脂肪肝(NAFLD)的显效物质之一。鉴于中药药效的发挥是多组分联合作用的结果。本项目采用多重qPCR、LC/MS、膜蛋白芯片及微透析技术，通过构建多种NAFLD体内外模型，从化学组分、代谢分布、药效机制和转录组学4个层次，实时、同步分析陈皮黄酮和miRNA血药浓度与其生物活性效应的关联性，探讨陈皮miRNA的有效性、安全性及研发价值，阐明陈皮治疗NAFLD的药效物质基础，揭示中药miRNA类药效物质与植物次生代谢产物之间的协同作用机制，拓展现有以次生代谢成分为主体的药效物质研究模式，为中药的质量控制及药效评价提供一种全新的研究方法。

药效物质基础是中药研究的重点和难点。随着近年来基因测序技术的突破，最新研究表明植物微小核糖核酸(miRNA)是一类新的天然活性物质，但在中药研究领域鲜有报道。项目组前期发现，除黄酮外，陈皮miRNA可通过饮食方式吸收入血，具有多种生物活性，可能是陈皮治疗非酒精性脂肪肝(NAFLD)的显效物质之一。因此，本项目分别以非酒精性脂肪肝的纤维化和脂质沉积等病理特征为依据，1）通过TGF-β1诱导HSC细胞建立肝纤维化细胞模型，以索拉菲尼为阳性对照，对陈皮中14种miRNAs进行活性筛选。结果发现，陈皮miRNA-159a可能是陈皮抗肝纤维化作用的药效物质之一，其能抑制TGF-β1诱导的HSC细胞增殖，促进HSC细胞凋亡，还能显著改善CCl4诱导的小鼠肝纤维化，其机制可能是miRNA-159a靶向作用于GSK-3β，调控GSK-3β通路关键蛋白的表达，抑制HSC细胞的活化与增殖从而抑制肝纤维化。2）通过棕榈酸诱导LO2细胞建立脂质沉积肝细胞模型，以辛伐他丁为阳性对照，对陈皮中9种主要酚酸类成分进行活性筛选。结果发现，迷迭香酸对棕榈酸诱导LO2细胞脂质沉积的抑制作用最佳。并且能降低高脂饲料诱导非酒精性脂肪肝小鼠的血脂及血糖水平，减少肝组织脂质沉积和肝细胞脂质空泡，其机制可能是通过靶向FYN/ERK通路而发挥疗效。通过本项目的研究，不仅阐明了陈皮治疗NAFLD的药效物质基础，还为中药的质量控制及药效评价提供一种以miRNA为切入点的全新的研究方法。