C57BL/6J肥胖小鼠脂肪米色化与自噬调控的中医药干预研究

Obesity induced by energy overload is the cause of many critical chronic diseases. The central mechanism of obesity is the limited activation of beige adipose tissue, a decrease of energy combustion in mitochondria, and the excessive accumulation of white adipose tissue (WAT). Inhibition of autophagy mediated mitochondrial clearance promotes the beigeing of WAT, and the cAMP/PKA signaling pathway activates mTORC1, thus inhibiting autophagy. White adipose accumulation belongs to the concept of “Feiman” in traditional Chinese medicine (TCM), whose core mechanism was found to be "phlegm-heat accumulation in middle-jiao" in TCM syndrome studies. Based on the “phlegm- heat accumulation” mechanism, Dai-Zong-Fang (DZF) is prescribed aiming to clear heat and remove phlegm. Previous studies have confirmed that DZF could ameliorate obesity and improve glucose and lipid metabolism. The mechanism of DZF effects are related to inhibiting adipocyte differentiation, improving the dynamic balance of intracellular lipogenesis and lipolysis, promoting GLUT4 translocation to the plasma membrane in insulin signaling and up-regulating PKA expression. The present study, standing from a perspective of the upstream molecular regulation of energy metabolism in adipocytes, with C57BL/6J obese mice induced by high- fat and high- sugar diets as a model, applies key technology of PET/CT, transmission electron microscopy, immunofluorescence, oxygen bomb calorimeter, RT-PCR and Western blot, aiming to investigate the three aspects of energy metabolism/ WAT beigeing/ autophagy signaling, and to explore the biological basis of DZF effects in obesity treatment, providing a scientific support for clinical application.

能量过剩导致的肥胖是慢性重大疾病始因，核心是米色脂肪激活受限，线粒体能量燃烧降低，白色脂肪组织（WAT）过度堆积。抑制自噬介导的线粒体清除可增加白色脂肪米色化，cAMP/PKA通路可激活mTORC1进而抑制细胞自噬。脂肪堆积属中医“肥满”，中医证候学调查发现“痰热中阻”为基本证候，以“清热涤痰”治法拟定代综方。既往研究证实代综方可减轻患者肥胖，改善糖脂代谢，其机制与抑制脂肪细胞分化、改善胞内脂质合成与分解的动态平衡，促进胰岛素信号通路中GLUT4膜转位，上调PKA表达有关。本研究从脂肪细胞能量代谢上游分子调控的视角，以高脂高糖诱导C57BL/6J肥胖小鼠为模型，采用PET/CT、透射电镜、免疫荧光、氧弹量热计、RT-PCR及Western-blot等关键技术，研究机体能量代谢/脂肪细胞米色化/自噬信号通路三个层次内容，探索代综方治疗肥胖的生物学基础，为临床应用提供科学支撑。

能量过剩是造成肥胖的源头，也是慢性重大疾病始因，核心是米色脂肪细胞激活受限，白色脂肪组织（WAT）过度积聚。脂肪堆积属中医“肥满”，“中满痰浊”为核心病机，“开结涤痰”为基本治法。本课题以开结涤痰中药（代综方）作为干预药物，单纯膳食诱导的肥胖C57BL/6J小鼠模型及3T3-L1脂肪细胞为研究对象，通过对cAMP/PKA-mTORC1信号通路介导的脂肪细胞米色化展开研究。研究证实：（1）代综方减轻C57BL/6J小鼠肥胖程度。降低肥胖C57BL/6J小鼠体重、肥胖Lee's指数及脂肪系数；（2）代综方调节肥胖C57BL/6J小鼠糖脂代谢，改善胰岛素抵抗。DZF给药28d，改善C57BL/6J小鼠OGTT；给药42d，降低小鼠空腹血糖、血清胰岛素、血清抵抗素、胰岛素抵抗指数及血清中TC、TG等的浓度；与模型组比较，代综方上调血清中脂肪细胞因子脂联素、Ghrelin的浓度，下调血清中Leptin、TNF-α的浓度。（3）代综方激活肥胖C57BL/6J小鼠脂肪细胞米色化，促进WAT脂解、抑制胞内脂质积累，调节能量代谢。代综方组WAT细胞体积较模型组减小，细胞数目增多，胞内线粒体数目增多、结构也更为清晰。与模型组比较，脂肪细胞米色化特异性因子（UCP1和PGC-1α）的表达上调，白色脂肪细胞分化基因（C/EBPα、C/EBPβ）的表达减少；能量代谢cAMP/PKA-mTORC1信号通路基因表达增加。（4）代综方通过AMPK/PKA信号通路激活3T3-L1脂肪细胞米色化，增加脂肪细胞线粒体产热，提高能量代谢。给药干预48h，降低成熟脂肪细胞胞内脂质积累、降低胞内TG含量、增加脂滴数目并减少大脂滴体积，下调脂肪生成标记蛋白SREBP-1的表达，增加胞内线粒体数目，上调米色化线粒体产热标记蛋白UCP1、PGC1a的表达，上调能量代谢信号通路AMPK、PKA基因表达。. 本研究阐明代综方通过激活脂肪细胞米色化，促进能量代谢，从而减轻肥胖，改善糖脂代谢的机制。为中医开结涤痰法（代综方）治疗肥胖引起的代谢紊乱提供了现代科学依据，对临床有针对性选择治疗肥胖或代谢综合征的药物具有重要意义。