Plasma extracellular vesicle-derived miR-296-5p is a maturation-dependent rejuvenation factor that downregulates inflammation and improves survival after sepsis

There is a progressive decline in physiological function with age, and aging is associated with increased susceptibility to injury and infection. However, several reports have indicated that the agility of youth is characterized by transferable rejuvenating molecular factors, as was observed previously in heterochronic parabiosis experiments. These experiments demonstrated a rejuvenating effect of young blood in old animals. There have been several efforts to characterize these youthful or maturation-associated factors in the young blood. In this report, we demonstrate the resilience of young mice, at or before puberty, to polymicrobial sepsis and show an age-dependent effect of plasma extracellular vesicles on the outcome following sepsis. The EVs from the young mice were cytoprotective, anti-inflammatory, and reduced cellular senescence markers. MicroRNA sequencing of the extracellular vesicles showed an age-associated signature and identified miR-296-5p and miR-541-5p to progressively reduce their levels in the blood plasma with increasing age. We further show that the levels of these miRNAs decline with age in multiple organs. The miRNAs miR-296-5p and miR-541-5p showed a reparatory effect in an in vitro wound healing model and the miR-296-5p, when given intraperitoneally, reduced mortality in the mouse model of sepsis. In summary, our studies demonstrate that EVs from very young mice have a reparative effect on sepsis, and the reparative factors are likely maturation-dependent. Our observation that miR-296-5p and miR-541-5p are plasma EV constituents that significantly reduce with age and can reduce inflammation suggests a therapeutic potential for these miRNAs in inflammation and age-associated diseases. Overall design: Total RNA from young, mature, 1year and 2 year old mice EVs was extracted using the Total Exosome RNA Isolation Kit (Invitrogen, 4478545). miRNA Library was prepared by using Illumina TruSeq stranded mRNA kit and sequencing was carried out at Novogene

机体生理功能随年龄增长呈渐进性衰退，衰老与损伤和感染的易感性升高密切相关。既往多项异时共生（heterochronic parabiosis）实验已证实，年轻血液可使老年动物产生返老还童效应，提示年轻个体存在可传递的分子返老还童因子。目前已有多项研究尝试对年轻血液中的年轻相关或成熟相关因子进行系统表征。本研究证实，青春期前后的年轻小鼠对多重微生物败血症（polymicrobial sepsis）具有较强抵抗力，并揭示了血浆细胞外囊泡（extracellular vesicles, EVs）对败血症预后的年龄依赖性调控作用。年轻小鼠来源的EVs可发挥细胞保护、抗炎效果，并降低细胞衰老标志物的表达水平。通过对EVs进行microRNA测序，我们鉴定出与年龄相关的表达谱特征，并发现miR-296-5p与miR-541-5p的血浆水平随年龄增长逐渐下调。我们进一步证实，在多个小鼠器官中，这两种miRNA的表达水平同样随年龄增长而降低。体外实验显示，miR-296-5p与miR-541-5p可促进伤口愈合；而通过腹腔注射给予miR-296-5p，能够降低败血症小鼠模型的死亡率。综上，本研究表明，极年轻小鼠来源的EVs对败血症具有显著修复作用，其核心修复因子大概率具有成熟依赖特性。我们的研究发现，miR-296-5p与miR-541-5p作为血浆EV的组成成分，其水平随年龄显著降低且可有效减轻炎症，提示这两种miRNA在炎症性疾病及年龄相关疾病中具备治疗应用潜力。实验整体设计：采用Total Exosome RNA Isolation Kit（Invitrogen, 4478545）提取年轻、成熟、1年龄及2年龄小鼠EVs的总RNA；使用Illumina TruSeq stranded mRNA试剂盒构建miRNA文库，测序工作由诺禾致源（Novogene）完成。