Experimental Study Of HucMSC-Ex Promoting Microglial Polarization To M2 To Relieve Spinal Cord Injury

Objective:Traumatic spinal cord injury?SCI?is a kind of refractory trauma of central nervous system with a high disability and mortality rate.However the existing most of the treatment options are not effective.At present,stem cell therapy has achieved a certain effect in the repair of spinal cord injury,and its promotion of tissue damage repair and regeneration is largely dependent on its paracrine.Exosomes are the main active components secreted by stem cells.Therefore,this article aims to study the role of human umbilical cord mesenchymal stem cell derived exosomes?hucMSC-Ex?in the repair of rat spinal cord injury and to explore its related mechanisms.Methods:In situ injection of hucMSC-Ex to repair the spinal cord injury model of SD rats was constructed.According to continuous 6w BBB score after surgery,the general view of the spinal cord tissue at the injury site,HE staining and TUNEL staining of the spinal cord tissue sections were used to determine the repairing effect of hucMSC-Ex on SCI.Western-blot,immunohistochemistry,immunofluorescence,and qRT-PCR techniques were used in vivo to detect the effects of hucMSC-Ex on the polarization of microglia in spinal cord tissue.Microglial cells were cultured in vitro.A model of microglial inflammation was constructed in vitro using LPS?Lipopolysaccharides?.Western-blot,qRT-PCR,immunofluorescence staining,and ELISA were used to detect the effects of hucMSC-Ex on the polarization state and inflammatory response of microglia.Western-blot,migration experiment,and tubule formation were used to detect the effects of microglial supernatant on PC12,astrocytes,and endothelial cells after treatment with hucMSC-Ex.miRNAs related to the microglial phenotypic polarization were screened from hucMSC-Ex and HFL-Ex miRNA expression differential sequencing profiles.The target gene and downstream signaling pathways were screened by dual luciferase reporter gene experiment,qRT-PCR technology,western-blot and immunofluorescence staining.After knocking down or overexpressing the miRNA,westernblot and immunofluorescence staining were used for further verification.Results:The BBB score,spinal cord macroscopic view,spinal cord tissue HE staining and TUNEL staining results for 6 consecutive weeks suggested that hucMSC-Ex can reduce the degree of hematoma and apoptosis of spinal cord tissue,regulate excessive cell proliferation,reduce tissue structural disorder and promote glial hyperplasia limiting the scope of injury in the early stage.In the long term,hucMSC-Ex can promote the regeneration of spinal cord tissue,enhance the motor function of hind limbs in rats,and promote the recovery of spinal cord function.In vivo experiments showed that hucMSC-Ex can rapidly induce microglial polarization to M2 in the early stage of injury,and can still maintain this effect in the later stage of repair.The results of in vitro LPS-induced microglia inflammation model further supported the result that hucMSC-Ex can promote microglia polarization to M2,and this change had a concentration-dependent relationship with hucMSC-Ex.After treatment,the proinflammatory factors IL-1? and TNF-? decreased in the supernatant of microglia,and the antiinflammatory factor IL-10 increased.The treated microglia supernatant can promote PC12 axon regeneration and synapse formation,can promote astrocytes to A2 polarization,can reduce HUVEC apoptosis,and promote endothelial cell migration and tubule formation.From the sequencing results,hucMSC-Ex highly expressed miRNA NC₀00020.11₁3896?referred to as miR13896?molecules.The results suggested that hucMSC-Ex can carry new miR13896 molecules to play a role.Dual luciferase reporter gene experiments,western-blot,immunofluorescence staining,and qRT-PCR results showed that miR13896 played the role of polarizing microglia by targeting the PDPK1/AKT/TSC2/mTOR axis.Overexpression and knockdown of miR13896 further confirmed the above results.Conclusion : Our findings showed that miR13896 wrapped by hucMSC targeted PDPK1/AKT/TSC2/mTOR axis to promote microglial polarization to M2,thereby promoting axon regeneration and synapse formation,and promoting astrocytes to polarize to A2,promoting endothelial cell migration and tubule formation and reducing endothelial cell apoptosis,which played a key role in repairing spinal cord injury.