基于巨噬细胞自噬启动和降解障碍研究实性和虚性体质肥胖的差异及表观遗传调控机制

Despite of identifying obesity by the appearance, Traditional Chinese Medicine found a new way of analyzing it from the viewpoint of access and deficiency. Previous epidemiological survey showed that Qi-deficiency constitution-which belongs to deficiency constitution and Phlegm-dampness constitution-which belongs to access constitution mainly constituent the obesity. Metabolomics study found that although both of them are obesity but energy metabolism showed the opposite signs. Recent studies showed that macrophage autophagy can achieve metabolic regulation by the initiation step-garbage collection and storage and degradation step-garbage incineration and energy supply and disorders of either step can cause obesity, so to clarify where the inhibition occurs is significant in clinical obesity prevention and treatment. Based on the results of Metabolomics and early gene expression profile, this project hypothesizes that "the macrophage autophagy inhibition of both access constitutional obesity and deficiency constitutional obesity occurs at the initiation and degradation step respectively ", establishes balanced constitution group as control, selects both access and deficiency obesity people, uses vitro single macrophage autophagy and introduces the autophagy flow detection techniques to observe the difference between the two in autophagy’s intiation and degradation abilities dynamically. Further, this project clarifies the regulatory mechanisms underlying the existence of difference by detecting the transcription levels, protein phosphorylation levels and promoter DNA methylation of autophagy’s key regulatory molecules such as mTORC1，AMPK，Beclin1 and Rab7，etc. This not only provides scientific basis for differentiating obesity by access and deficiency constitution, changes the conception of obesity, but also provides a basis for the research and development of obesity drugs from different aspects of autophagy.

中医除从外形认识肥胖外，尚从虚和实角度认识肥胖。前期流调发现气虚质(虚性体质)和痰湿质（实性体质)是肥胖两大主要体质。代谢组研究显示，两者虽同为肥胖，能量代谢却呈现相反趋势。新近研究表明巨噬细胞自噬可通过启动（垃圾回收及储能）和降解（垃圾焚化及供能）实现代谢调节，任何环节障碍均可造成肥胖，明确障碍所在环节对肥胖防治意义重大。基于代谢组及前期基因表达谱结果，提出“实性和虚性体质肥胖自噬障碍分别发生在启动和降解两个不同环节”的假说，设立平和质对照，选取虚实体质肥胖人，体外诱导单核巨噬细胞自噬，引入自噬流检测技术，动态观察两种肥胖自噬启动和降解能力差异；进一步检测mTORC1、AMPK、Rab7、Beclin1等自噬关键调控分子转录水平和蛋白磷酸化水平及启动子区DNA甲基化，明确差异存在的调控机制。为肥胖虚实体质分型提供科学依据，转变肥胖认知理念，也为从自噬不同环节进行肥胖新药研发奠定基础。

项目背景：中医除从外形认识肥胖外，尚从虚和实角度认识肥胖。前期流调发现气虚质(虚性体质)和痰湿质（实性体质)是肥胖两大主要体质。代谢组研究显示，两者虽同为肥胖，能量代谢却呈现相反趋势。新近研究表明巨噬细胞自噬可通过启动（垃圾回收及储能）和降解（垃圾焚化及供能）实现代谢调节，任何环节障碍均可造成肥胖，明确障碍所在环节对肥胖防治意义重大。基于代谢组及前期基因表达谱结果，提出“实性和虚性体质肥胖自噬障碍分别发生在起始和降解两个不同环节”的假说。.研究内容：设立平和质对照，选取虚实体质肥胖人，体外诱导单核巨噬细胞自噬，引入自噬流检测技术，动态观察两种肥胖自噬启动和降解能力差异；并进一步开展两者自噬差异背后的表观遗传调控机制研究。.关键数据：①通过基础数据比较发现，痰湿肥胖代谢危险性最高，气虚肥胖代谢危险性介于正常与痰湿肥胖之间；②通过自噬比较发现，痰湿肥胖和气虚肥胖基础自噬水平均低于平和质，痰湿肥胖者在自噬起始阶段呈现明显障碍、降解阶段接近正常水平；气虚肥胖在起始阶段接近正常水平，降解阶段呈现明显障碍；③痰湿肥胖差异基因有7条富集GO与肌动蛋白（actin）支架组装有关，而肌动蛋白支架组装是自噬小体双层膜形成的必要条件，说明痰湿肥胖可能在自噬起始阶段自噬小体双层膜结构形成障碍，垃圾无法回收；气虚质肥胖差异基因富集到溶酶体信号通路、线粒体自噬调节，气虚肥胖很有可能存在自噬溶酶体功能障碍和线粒体自噬障碍，导致产能不足，耗能下降，脂肪囤积；④气虚肥胖和痰湿肥胖自噬差异背后的表观遗传调控机制探索，发现LOC101930316、RPL29P2等lncRNA在调控两者自噬早期自噬体形成（Rab1a）方面发挥重要作用；DNA甲基化修饰在调控两者自噬降解阶段发挥重要作用（差异位点cg22198907被注释到SQSTM1，编码P62蛋白）。.科学意义：从自噬障碍发生不同环节揭示虚性和实性体质肥胖的差异及可能的表观遗传调控机制，为从中医角度建立肥胖虚实分型提供科学依据，也为从自噬不同环节进行肥胖药物研发奠定基础。