BACKGROUND Spinal cord injury (SCI) can lead to severe motor and sensory dysfunction with high disability and mortality. In recent years, mesenchymal stem cell (MSC)-secreted nano-sized exosomes have shown great potential for promoting functional behavioral recovery following SCI. However, MSCs are usually exposed to normoxia in vitro, which differs greatly from the hypoxic micro-environment in vivo. Thus, the main purpose of this study was to determine whether exosomes derived from MSCs under hypoxia (HExos) exhibit greater effects on functional behavioral recovery than those under normoxia (Exos) following SCI in mice and to seek the underlying mechanism. METHODS Electron microscope, nanoparticle tracking analysis (NTA), and western blot were applied to characterize differences between Exos and HExos group. A SCI model in vivo and a series of in vitro experiments were performed to compare the therapeutic effects between the two groups. Next, a miRNA microarray analysis was performed and a series of rescue experiments were conducted to verify the role of hypoxic exosomal miRNA in SCI. Western blot, luciferase activity, and RNA-ChIP were used to investigate the underlying mechanisms. RESULTS Our results indicate that HExos promote functional behavioral recovery by shifting microglial polarization from M1 to M2 phenotype in vivo and in vitro. A miRNA array showed miR-216a-5p to be the most enriched in HExos and potentially involved in HExos-mediated microglial polarization. TLR4 was identified as the target downstream gene of miR-216a-5p and the miR-216a-5p/TLR4 axis was confirmed by a series of gain- and loss-of-function experiments. Finally, we found that TLR4/NF-κB/PI3K/AKT signaling cascades may be involved in the modulation of microglial polarization by hypoxic exosomal miR-216a-5p. CONCLUSION Hypoxia preconditioning represents a promising and effective approach to optimize the therapeutic actions of MSC-derived exosomes and a combination of MSC-derived exosomes and miRNAs may present a minimally invasive method for treating SCI.

中文翻译：

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低氧预适应的间充质干细胞穿梭穿梭的miR-216a-5p通过转移小胶质细胞M1 / M2极化来修复脊髓损伤。

背景技术脊髓损伤（SCI）可导致严重的运动和感觉功能障碍，并具有很高的残疾和死亡率。近年来，间充质干细胞（MSC）分泌的纳米大小的外泌体已显示出促进SCI后功能行为恢复的巨大潜力。然而，MSC通常在体外暴露于常氧，这与体内低氧微环境有很大的不同。因此，本研究的主要目的是确定在SCI后小鼠体内缺氧（HExos）下衍生自MSC的外泌体是否对正常行为表现出更大的作用，并寻找其潜在机制。方法采用电子显微镜，纳米粒子跟踪分析（NTA）和蛋白质印迹法来鉴定Exos和HExos组之间的差异。进行了体内SCI模型和一系列体外实验，以比较两组之间的治疗效果。接下来，进行了miRNA微阵列分析，并进行了一系列抢救实验，以验证缺氧性外泌体miRNA在SCI中的作用。蛋白质印迹，荧光素酶活性和RNA芯片用于研究潜在的机制。结果我们的结果表明，HExos通过在体内和体外将小胶质细胞极化从M1型转变为M2型来促进功能行为恢复。miRNA阵列显示miR-216a-5p最富含HExos，并可能参与HExos介导的小胶质细胞极化。TLR4被确定为miR-216a-5p的目标下游基因，并且miR-216a-5p / TLR4轴已通过一系列功能获得和丧失的实验得以证实。最后，我们发现TLR4 /NF-κB/ PI3K / AKT信号级联可能参与了缺氧性外泌体miR-216a-5p对小胶质细胞极化的调节。结论缺氧预处理是一种有前途且有效的方法，可优化MSC来源的外泌体的治疗作用，并且MSC来源的外泌体和miRNA的组合可提供一种微创方法来治疗SCI。