miR-146a-5p-modified hUCMSC-derived exosomes facilitate spinal cord function recovery by targeting neurotoxic astrocytes

Abstract

Background: Acute spinal cord injury (SCI) is a devastating result of neurological trauma with subsequent microenvironment dyshomeostasis that induces neurotoxic phenotype acquisition by astrocytes, exacerbating neurological function impairment. Exosomes derived from human umbilical cord mesenchymal stem cells (hUCMSCs) have demonstrated essential therapeutic effects after central nervous system trauma. However, whether hUCMSC-derived exosomes exert therapeutic effects on neurotoxic astrocytes to facilitate SCI function recovery remains unclear. Additionally, the limited efficiency of single exosomes may restrict the optimization of exosomal biological functions. Methods: We first determined that exosomes reduce the deleterious effects of neurotoxic astrocytes in vitro and in vivo. Then, we identified critical functional microRNAs (miRNAs). miR-146a-5p was overexpressed in exosomes, and then, miR-146a-5p-modified exosomes were used to investigate the ability of exosomes to reduce neurotoxic astrocyte effects, preserve neurons and promote neurological function recovery in rats with SCI. Results: Cell counting kit-8 and neurite length analyses revealed that exosomes partially reduced the negative effects of neurotoxic astrocytes on PC12 cell viability and neurites in vitro. The exosomes also attenuated inflammatory responses, reduced the number of neurotoxic astrocytes and preserved neural tissue in rats with SCI. Immunofluorescence assays suggested that the number of neurotoxic astrocytes was rapidly increased by injury, reaching a peak 5 days post-injury (dpi) and returning to the normal level 14dpi. Exosomal miR-146a-5p was identified as the critical functional miRNA. Overexpression of miR-146a-5p in exosomes strengthened the biological function of the exosomes. Therefore, the modified exosomes exerted more powerful therapeutic effects than the unmodified exosomes, reducing the deleterious effects of neurotoxic astrocytes both in vitro and in vivo and promoting locomotor function of the hindlimbs in the rats with SCI. Through a series of gain- and loss-of-function experiments, Traf6 and Irak1 were identified as targets of exosomal miR-146a-5p. Ultimately, we found that miR-146a-5p-modified exosomes exerted their function by targeting Traf6/Irak1/NFκB pathway in neurotoxic astrocytes. Conclusions: In summary, miR-146a-5p-modified exosomes exerted a more powerful effect than unmodified exosomes to promote neurological function recovery in rats with SCI by targeting neurotoxic astrocytes. Therefore, miR-146a-5p-modified exosomes are promising therapeutics for SCI.