Although exosomes, as byproducts of human umbilical cord mesenchymal stem cells (hUC-MSCs), have been demonstrated to be an effective therapy for traumatic spinal cord injury (SCI), their mechanism of action remains unclear. We designed and performed this study to determine whether exosomes attenuate the lesion size of SCI by ameliorating neuronal injury induced by a secondary inflammatory storm and promoting neurite outgrowth. We determined the absolute levels of all exosomal miRNAs and investigated the potential mechanisms of action of miR-199a-3p/145-5p in inducing neurite outgrowth in vivo and in vitro. miR-199a-3p/145-5p, which are relatively highly expressed miRNAs in exosomes, promoted PC12 cell differentiation suppressed by lipopolysaccharide (LPS) in vitro through modulation of the NGF/TrkA pathway. We also demonstrated that Cblb was a direct target of miR-199a-3p and that Cbl was a direct target of miR-145-5p. Cblb and Cbl gene knockdown resulted in significantly decreased TrkA ubiquitination levels, subsequently activating the NGF/TrkA downstream pathways Akt and Erk. Conversely, overexpression of Cblb and Cbl was associated with significantly increased TrkA ubiquitination level, subsequently inactivating the NGF/TrkA downstream pathways Akt and Erk. Western blot and coimmunoprecipitation assays confirmed the direct interaction between TrkA and Cblb and TrkA and Cbl. In an in vivo experiment, exosomal miR-199a-3p/145-5p was found to upregulate TrkA expression at the lesion site and also promote locomotor function in SCI rats. In summary, our study showed that exosomes transferring miR-199a-3p/145-5p into neurons in SCI rats affected TrkA ubiquitination and promoted the NGF/TrkA signaling pathway, indicating that hUC-MSC-derived exosomes may be a promising treatment strategy for SCI.

中文翻译：

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脐带间充质干细胞来源的外泌体通过miR-199a-3p / 145-5p介导的NGF / TrkA信号通路促进大鼠脊髓功能恢复

尽管已证明外泌体作为人脐带间充质干细胞（hUC-MSC）的副产物是一种有效的创伤性脊髓损伤（SCI）疗法，但其作用机理仍不清楚。我们设计并进行了这项研究，以确定外泌体是否通过减轻继发性炎性风暴引起的神经元损伤并促进神经突向外生长来减轻SCI的病变大小。我们确定了所有外泌体miRNA的绝对水平，并研究了miR-199a-3p / 145-5p在体内和体外诱导神经突生长的潜在作用机制。miR-199a-3p / 145-5p是外泌体中表达相对较高的miRNA，通过调节NGF / TrkA途径在体外促进了脂多糖（LPS）抑制的PC12细胞分化。我们还证明了Cblb是miR-199a-3p的直接靶标，而Cbl是miR-145-5p的直接靶标。Cblb和Cbl基因敲低导致TrkA泛素化水平显着降低，随后激活NGF / TrkA下游途径Akt和Erk。相反，Cblb和Cbl的过度表达与TrkA泛素化水平的显着增加有关，随后使NGF / TrkA下游途径Akt和Erk失活。Western印迹和免疫共沉淀试验证实了TrkA和Cblb与TrkA和Cbl之间的直接相互作用。在体内实验中，发现外泌体miR-199a-3p / 145-5p上调损伤部位的TrkA表达并促进SCI大鼠的运动功能。总之，