【作者】 李丹；

【导师】 杨巍；

【作者基本信息】 华中科技大学 ， 营养与食品卫生学， 2022， 硕士

【摘要】 研究目的:骨骼肌衰老的特征主要表现为其质量和功能随着年龄增长而逐渐下降,如不及时干预或治疗将可能发展为肌肉减少症,并导致一系列不良健康结局发生。已有证据提示亮氨酸摄入与肠道菌群、肌肉健康之间可能存在密切的关联,但其中具体关联作用仍需进一步研究。本研究旨在揭示长期亮氨酸补充是否可以通过AMPKα/SIRT1/PGC-1α通路延缓小鼠骨骼肌衰老,并探究肠道菌群在此过程中发挥的作用。研究方法:本研究选取19月龄雄性C57BL/6j小鼠（n=12/组）,分别给予超纯水、低剂量(500 mg·kg^-1·d^-1)和高剂量亮氨酸(1 250 mg·kg^-1·d^-1)灌胃12周。对干预后小鼠的股四头肌通过超声、组织H&E染色切片、肌纤维横截面积测量和成肌、肌萎缩相关蛋白表达水平（蛋白免疫印迹法）,来评估亮氨酸对骨骼肌再生及萎缩的生物学效应。同时,收集小鼠干预前后的粪便样本,采用16S rDNA扩增子测序方法实现肠道菌群物种构成及相对丰度分析,结合MetaCyc代谢途径数据库预测分析其功能谱,并进一步检测粪便样本中的丙酮酸水平。研究结果:在补充亮氨酸12周后,小鼠肌肉超声和组织学结果较对照组均有改善。高剂量亮氨酸摄入能刺激股四头肌中AMPKα激活,并调节SIRT1、PGC-1α蛋白表达水平,进而调控肌生成、肌萎缩及脂代谢相关蛋白表达水平。同时,亮氨酸补充也改变了肠道菌群的丰富度、多样性及其代谢途径。其中,高剂量亮氨酸使肠道菌群厚壁菌门-拟杆菌门比值显著降低。此外,高剂量亮氨酸补充后的差异性菌种与丙酮酸发酵至丙酸I途径呈负相关,且高剂量亮氨酸组小鼠粪便丙酮酸水平显著升高。结论:长期高剂量亮氨酸干预可调控衰老小鼠肠道菌群及AMPKα/SIRT1/PGC-1α信号通路并改善骨骼肌衰老进程。菌群代谢产物丙酮酸可能在此过程中发挥重要作用。更多还原

【Abstract】 Aims:Muscle aging is characterized by decline in mass and function along with growing age.The aging in skeletal muscle,if without any prevention or treatment,could consequently lead to sarcopenia,accompanied by a series of adverse health outcomes.The previous studies demonstrated that there might be a complex and close relationship among leucine supplement,gut microbiota and muscle health,which required further investigation to affirm.This study aimed to investigate whether chronic leucine supplementation could ameliorate muscle aging through AMPKα/SIRT1/PGC-1αpathway in aging mice and explore the role of gut microbiota in this course.Methods:In this study,19-month-old male C57BL/6j mice（n=12/group）were supplemented respectively with ultrapure water,low dose(500 mg·kg^-1·d^-1)or high dose(1 250 mg·kg^-1·d^-1)of leucine for 12 weeks by oral gavage.The biological effects of leucine supplement on muscle regeneration and atrophy in quadriceps were assessed by ultrasound measurement,H&E staining,myofiber cross-sectional area measurement and expression levels of myogenesis and atrophy related proteins（by Western Blot）at the endpoint.Fecal samples of mice were collected before and after intervention,and the analysis of gut microbiota composition and richness was implemented by 16S r DNA amplicon sequencing.Combined with Meta Cyc metabolic pathways database,the function profiles were predicted and analyzed.Besides,the pyruvate levels were further detected in fecal samples.Results:After 12 weeks of leucine supplementation,the muscle ultrasonography and histology in mice were improved versus the control group.High-dose leucine intake activated the AMPKαand regulated the expression of SIRT1 and PGC-1α,along with the changed expression levels of myogenesis,muscle atrophy and lipids metabolism related proteins in quadriceps.Besides,the richness and diversities of gut microbiota as well as related metabolic pathways were altered after leucine supplementation.The Firmicutes-Bacteroidetes ratio was significantly reduced in high-leucine group.In addition,the differential species in high-leucine group were negatively correlated with pyruvate fermentation to propionate I pathway.Consistently,the fecal pyruvate levels were significantly increased in the high-leucine group compared with control group.Conclusion:Chronic high-dose leucine supplementation changed the gut microbiota,activated AMPKα/SIRT1/PGC-1αpathway and ameliorated skeletal muscle aging in mice.Pyruvate,one of the gut microbial metabolites,might play important roles in this process.更多还原