基于AMPK-TSC2/TFEB和mTORC1-Ulk1通路介导的巨噬细胞自噬障碍研究肥胖虚实体质差异及调体干预作用机制

Traditional Chinese Medicine believes that obesity can be categorized into excess and deficiency types, and the modern medicine treating method of reducing excess is not suitable for all. It was previously confirmed that qi deficiency and phlegm-dampness constitutions are the main types of obesity. Wang Qi’s two prescription – YQJY for Qi deficiency constitution and HTQX for Phlegm-dampness constitution - are effective in clinical application. Autophagy is an important mechanism for cleaning up metabolic waste and producing energy, and its obstacle is closely related to obesity. It was found that macrophage autophagy disorder of the people with excess and deficiency obesity constitution occurred in two different stages: the initial stage and the degradation stage. A hypothesis is proposed in combination with herbal molecular target prediction: The underlying mechanism of body for intervention in obesity is different, HTXS acts on mTORC1-Ulk1 pathway to promote that formation of autophagy double-layer membrane and recover the garbage recovery in the autophagy initial stage; YQJY acts on AMPK-TFEB/TSC2 pathway to improve lysosomal function and mitochondrial autophagy, and to restore waste incineration and productivity during degradation. Clinical intervention of the two groups – excess and deficiency - of obesity and dyslipidemia to confirm that the two prescriptions’ effectiveness in improving metabolism by regulating the different stages of autophagy; Establishing macrophage hyperlipidemia model, knocking down and overexpressing pathway core molecules, and applying drug-containing serum to intervene to clarify that both parties improve autophagy by regulating the above two pathways respectively. This study will provide an objective basis for intervention of obesity from the perspective of deficiency and excess constitution, and provide a reference target for drug research and development from different stages of autophagy.

中医认为肥胖有虚实之分，不可一味泻实。前期证实气虚质和痰湿质是肥胖主要虚实体质分型，王琦国医大师拟益气健运方和化痰祛湿方调体临床有效。自噬是清理代谢垃圾并产能的重要机制，其障碍与肥胖密切相关。前期发现实/虚体质肥胖巨噬细胞自噬障碍分别发生在起始和降解两个不同阶段。结合方药分子靶标预测提出假说：调体干预实/虚体质肥胖的内在机制不同，化痰祛湿方作用于mTORC1-Ulk1通路促进自噬小体双层膜形成，恢复自噬起始阶段垃圾回收；益气健运方作用于AMPK-TFEB/TSC2通路改善溶酶体功能及线粒体自噬，恢复降解阶段垃圾焚化及产能。首先通过两方干预实/虚肥胖兼血脂紊乱人群，确证二者通过调控自噬不同阶段改善肥胖及代谢；进一步以高脂环境培养巨噬细胞，敲低或过表达通路核心分子，进行含药血清干预，阐明两方分别通过调控上述两条通路改善自噬。本研究将为从虚实角度干预肥胖、从自噬不同阶段进行药物研发提供客观依据。

肥胖是代谢紊乱及心脑血管并发症的高风险人群，但临床发现单纯减重并没有带来降低代谢紊乱和心脑血管疾病发病率的预期。中医认为肥胖有虚实之分，不可一味泻实减重。气虚质和痰湿质是肥胖主要虚实体质分型，王琦国医大师拟益气健运方和化痰祛湿方调体，在临床中根据肥胖虚实偏重的不同，灵活配伍加减，取得显著效果。.自噬是清理代谢垃圾并产能的重要机制，其障碍与肥胖及相关代谢紊乱密切相关，明确自噬作用靶点和环节对有效干预肥胖和药物研发至关重要。本项目首先聚焦肥胖与动脉硬化，基于气虚/痰湿/平和体质肥胖临床数据及自噬差异基因分析，联合高脂诱导RAW264.7巨噬细胞炎症模型（模拟肥胖）、ox-LDL诱导RAW264.7巨噬细胞泡沫化模型（模拟动脉硬化）方药干预实验及自噬蛋白芯片检测，证实二者自噬机制存在差异，而相应调体方药自噬作用环节不同，益气健运更侧重于上调自噬ATGs基因，而化痰祛湿侧重于上调溶酶体LAMP1的表达。.进一步聚焦肥胖与MAFLD，建立MAFLD金黄地鼠模型和HepG2脂质沉积模型，深入探讨化痰祛湿与益气健运配伍对自噬及后续游离脂肪酸β氧化的作用机制，发现二者配伍可有效减重、降脂、改善肝脏脂质沉积。结合Compound C抑制AMPK后，双荧光自噬流观察及下游蛋白WB检测，认为其作用可能与激活AMPK/SIRT1/TFEB通路靶向Beclin1/ATG3和P62/LAMP1作用于自噬起始和降解环节恢复自噬流有关，同时益气健运配伍靶向线粒体β氧化相关转录因子PPARα、RXRα以及下游蛋白CPT-1A、ACOX、ACADL、ACADM等，更加有利于后续游离脂肪酸的氧化产能。本研究转变了肥胖认知理念，注重肥胖虚实程度之不同，将为从虚实角度干预肥胖及相关代谢紊乱提供客观依据。