Role And Mechanism Of HucMSC-Ex Promoting Astrocyte Polarization To Relieve Spinal Cord Injury

Objective:Spinal cord injury(SCI)is a neurological disorder that severely damages the central nervous system.The existing clinical treatment cannot cure SCI.Previous studies of our group have found that human umbilical cord mesenchymal stem cell derived exosomes(hucMSC-Ex)can alleviate SCI in rats,but the mechanism remains unclear.Astrocytes are one of the most abundant heterogenous glial cells in the central nervous system,which influence the neural microenvironment.In this study,our aim is to explore the role and mechanism of hucMSC-Ex in phenotypic polarization of astrocytes in order to provide a novel therapeutic strategy for SCI.Methods:(1)huc MSCs were cultured,and hucMSC-Ex was isolated and identified.(2)Rats were randomly divided into three groups: sham operation group,SCI treated with PBS group and SCI treated with hucMSC-Ex group.The healing effect of huc MSCEx on SCI was evaluated in 2 weeks after surgery by combining motor function of rat hind limbs and HE staining and immunohistofluorescence staining of spinal cord tissues.The phenotypic changes of astrocytes were detected by immunohistochemistry,immunohistofluorescence staining,western blot and real-time fluorescence quantitative polymerase chain reaction(q RT-PCR).(3)Astrocytes were isolated,cultured and identified,and then treated with three ways:PBS,lipopolysaccharide(LPS)and LPS merged with hucMSC-Ex.After 6 hours,immunofluorescence,western blot and q RT-PCR were used to detect cell phenotypes and the expression of inflammatory factors.Transwell migration assay and scratch assay were applied to detect the ability of cell migration.(4)The effects of astrocyte conditioned medium on microglia and nerve cells PC12 and BV2 were detected by western blot and immunofluorescence staining.(5)The protein AP1B1 related to the polarization of A2 type astrocytes in hucMSC-Ex was screened by mass spectrometry.Western blot,immunohistochemistry and immunohistofluorescence staining were used to verify the results.The hucMSC-Ex with AP1B1 knockdown was used to treat astrocytes,and the expression level of S100a10,migration ability and supporting effect on PC12 were detected,so as to explore the biological function of AP1B1.Results:(1)huc MSCs had the ability of adipogenesis and osteogenesis,and hucMSC-Ex exhibited the typical characterization of exosomes.(2)hucMSC-Ex could alleviate SCI in rats,reduce the structure disorder of spinal cord tissue and promote nerve regeneration.hucMSC-Ex suppressed the polarization of astrocytes from resting state to inflammatory(A1)phenotype and promoted the activation of protective(A2)phenotype in vivo.(3)hucMSC-Ex reversed the LPS-induced A1 type polarization of astrocytes by inhibiting the phosphorylation of nuclear factor-κ gene binding(NF-κB)signaling pathway.At the same time,the polarization of A2 type,the expression of anti-inflammatory factors and migration ability of astrocytes were promoted in vitro.(4)The conditioned medium of A2 astrocytes could support microenvironment cells,promote axon regeneration,synaptic outgrowth and polarization of protective type(M2)microglia.(5)AP1B1 protein was enriched in hucMSC-Ex,AP1B1 and S100a10 were highly expressed in the tissues and target cells after hucMSC-Ex intervention.The expression of AP1B1 and S100a10 in astrocytes after the intervention of hucMSC-Ex with AP1B1 knockdown were downregulated,the astrocyte migration ability and support function to PC12 of astrocytes were weakened.Conclusions:Our findings indicate that under the inflammatory state,hucMSC-Ex reduces the polarization of A1 astrocytes and inhibits inflammation by inhibiting the phosphorylation of NF-κB signaling pathway.hucMSC-Ex transfers AP1B1 protein to promote the polarization of A2 astrocytes,thereby supporting neurons and alleviating SCI.hucMSC-Ex will provide a novel theraptic strategy for SCI.