基于糖脂代谢微环境探讨脂肪性肝病“痰浊”的物质基础及化痰方药干预机制

Fatty liver disease (FLD) has become a major public health problem. On aspects of prevention and treatment for FLD, traditional Chinese medicine has unique advantages in both theory and strategy, on basis of syndrome differentiation of phlegm. However, the material basis and intervention mechanism of phlegm are still not fully understood. In the multi-center clinical trial, our previous study discovered a close correlation between the levels of glucolipid metabolism related genes, Caveolin-1 and advanced glycation end products (AGEs), and the pathogenesis of FLD. Therefore, we hypothesize that glucolipid metabolism dysfunction plays a key role in micro-syndrome differentiation and treatment of turbid phlegm in FLD. Based on multiple levels and targets of Caveolin-1 and receptors of advanced glycation end products (RAGE) in FLD, CRISPR/cas9 technique, metabonomics, zebrafish high-throughput analysis and gene knocked-out mice will be used to investigate the regulatory mechanisms and changes of microenviroment of glucolipid metabolism in this project. Moreover, the material basis of turbid phlegm can be reversely analyzed by the effects of formula for resolving phlegm on FLD. By elucidating the mechanisms of the pathogenesis and targeted regulation of phlegm syndrome in FLD and explaining the molecular biological mechanisms and functions of principle of treatment of resolving phlegm, this study will find a novel target for effective prevention and regulation of FLD and provide a new idea for drug discoveries.

脂肪性肝病严重威胁人类的健康，传统中医药具有独特的防治脂肪肝的理论及治疗策略，从痰论治是其关键之法，然而有关痰邪的物质基础及干预机制仍不完全清楚。基于前期研究成果以及课题组多中心临床研究发现糖脂代谢关键基因Caveolin-1及终末糖基化产物（AGEs）与脂肪肝发病密切相关，我们提出：糖脂代谢紊乱是脂肪肝痰浊微观辨证及治疗的关键，并拟以膜蛋白Caveolin-1、终末糖基化产物受体（RAGE）为切入点，结合CRISPR/cas9技术、代谢组学、斑马鱼高通量分析以及敲基因小鼠平台，多层次、多靶点分析脂肪肝糖脂代谢微环境改变本质以及Caveolin-1、RAGE调控机制；同时通过化痰方药干预治疗，以“药”验“证”反向分析痰浊内涵。阐明脂肪性肝病痰证病机发病基础及靶标调控机制，同时解答化痰治疗原则的现代分子生物机制及作用基础，为脂肪性肝病有效预防及调控寻找新的分子靶点，并为药物开发提供新的思路。

脂肪性肝病作为我国发病率首位的肝脏疾病，已严重威胁人类的健康。传统中医药具有独特的防治脂肪肝的理论及治疗策略，从痰论治是其关键之法，然而有关痰邪的物质基础及干预机制仍不完全清楚。本项目结合CRISPR/cas9技术、斑马鱼高通量分析以及敲基因小鼠平台，成功构建多种脂肪肝模型，明确了化痰方药二陈汤及化橘红改善脂肪肝发生发展的治疗作用，并筛选出多种有效活性成分：柚皮素、柚皮苷、橙皮苷、柠檬苦素等，初步解析了化痰方药防治脂肪性肝病的分子机制及物质基础。我们首次提出铁过载是脂肪肝“痰浊”组成部分，与糖脂代谢紊乱、炎症浸润及氧化应激等疾病病理共同构成脂肪肝痰浊微观辨证及治疗的关键，并以细胞微环境及糖脂代谢调控关键蛋白（Cav-1和RAGE）作为研究切入点，分别从体外实验、体内斑马鱼和基因敲除小鼠三种模型上进行研究，多层次、多途径证实了脂肪肝“痰浊”发生发展的生物学基础以及分子靶标（Cav-1和RAGE）的调控作用。本项目明确阐释了Cav-1在NAFLD过程中的保护作用，其作用机理涉及抑制脂质过氧化和抑制肝脏铁过载，从而减轻肝细胞损伤及脂肪性变。同时阐释了RAGE在AFLD过程中的损伤作用，其作用机理涉及促进炎性浸润和肝脏铁过载，从而加重肝细胞损伤及脂肪性变。最后，我们进一步通过化痰方药干预小鼠脂肪肝模型，明确了二陈汤和化橘红对脂肪肝组织中Cav-1和RAGE表达的靶向调控，并证实了化痰方药降低血清甘油三酯和游离脂肪酸、抑制促炎因子、铁过载和自由基过剩的作用效果。.本项目进一步解答了脂肪性肝病“从痰论治”的现代分子生物机制及作用基础，同时证实膜蛋白Cav-1 和RAGE 可作为脂肪肝细胞微环境以及痰证研究的重要切入点，以其靶向调控糖脂代谢稳态、铁稳态、自由基及免疫紊乱进而干预脂肪性肝病转归，为脂肪性肝病有效预防及调控寻找新的分子靶点，并为药物开发提供新的思路。