Schwann Cells Regulate Peripheral Nerve Regeneration Through "Mechanical Tension Sensitive EV-miRNA" By Targeting Nrp1

Peripheral nerve injury is a common complication of trauma,and always a knotty problem in clinical.Injuries have a profound and permanent impact on the patient and their ability to perform activities of daily living,as well as preventing return to work.Microsurgical repair by tensionless epineurial sutures have progressed in recent years,however,the treatment outcome remains unsatisfied.Schwann cell(SC)is the glial cell of peripheral nervous system(PNS),which plays a pivotal role in maintaining the physiology functions of nervous system and supporting nerve regeneration in PNS.After peripheral nerve injury,SCs are induced dedifferentiate to a pro-regenerative phenotype.The regenerative SCs participate in clearing the myelin and axon debris,secreting several neurotrophic factors and form the Biingner bands,which provide essential tracing support for the regenerated axons.SCs,as the key factor in nerve repair,have been extensively investigated by regulating their cellular and molecular biology to promote nerve regeneration.Peripheral nerve is remarkable tissue of tremendous elasticity.Peripheral nerve fibers and SCs subjected to stretches associated with movement of the limbs and mechanical tension stress from daily activities.Accumulating evidence suggested that SCs are sensitive to the stiffness of their environment.Studies have reported several mechanotransduction pathways in SCs,including YAP/TAZ,two transcriptional activators of the Hippo pathway.Nevertheless,it is far from clear that how SCs sense and respond to mechanical signals,as well as their consequent effect on nerve regeneration.The functional and structural integrity of nervous system are critically dependent on proper intercellular communication between neurons and glial cells.In recent years,the important role of extracellular vesicle(EV)mediated intercellular communication in maintaining the functions of nervous system and regulating nerve regeneration process has been gradually recognized.Studies reported that mechanical stimuli can regulate intercellular communication through EV mediation.Studies have shown that osteocytes under mechanical loading regulate bone metabolism by mechanically induced Ca2+oscillations in osteocytes and then controlling osteocyte-specific protein release through EVs.Thus,EV can mediate the response of SCs subjected to mechanical stimuli.Therefore,studies focused on how SCs sense and transduce mechanical stimuli,the role of EV played in the process of SCs mechanotransduction and the effect of EVs derived from mechanically stimulated SCs(MS-SCs-EVs)on nerve regeneration,which will provide insight in understanding the mechanobiology of SC in nerve regeneration.In this study,we established effective SC mechanical stimulation system and applied mechanical tension stimuli on SCs(MS-SCs).Then we obtained EVs from supernatants of SCs and MS-SCs.The effect of EVs derived from SCs(SCs-EVs)and MS-SCs-EVs on neurite outgrowth and nerve regeneration was investigated in vitro and in vivo.Furthermore,we identified the differentially expressed miRNAs in SCs-EVs and MS-SCs-EVs through high throughput miRNA sequencing.Functional examination found that miR-23b-3p showed a predominant role in MS-SCs-EVs.In addition,our results showed that MS-SCs-EVs enhance neurite outgrowth and nerve regeneration in vitro and in vivo via transfer of miR-23b-3p by targeting to neuropilinl(Nrp1).Part Ⅰ:Foundation of Schwann cells mechanical tension stimulation system and purification of EVs from mechanically stimulated SCsBackground:Mechanical tension stimuli are effective in regulating cell biology.Superparamagnetic iron oxide nanoparticles(SPIONs)have excellent superparamagnetic peoperties and are nanoscale particles,which facilitate cell endocytosis.SPIONs are widely used in biomedical applications for transfection of cells,especially for FDA-approved clinical applications as contrast agents for MR imaging.Because of its magnetic properties,combined with external magnetic field(MF),magnetic force can be used to separate cells,guide cell migration and exert mechanical stimulation to cells.Safe and effective mechanical stimuli parameters are the basis of constructing mechanical stimuli system.Objective:Construction of SC effective mechanical tension stimuli system and purification of EVs from cell culture supernatants.Methods:The cytotoxicity of SPIONs on cell viability was evaluated by CCK-8 assay.In addition,the intensity of MF on cell apoptosis was examined by flow cytometry.The intracellular iron was quantified,and the magnetic force acting upon single SC was calculated.The EVs were isolated by gradient centrifugation from cell culture supernatants.The characterization of the obtained EVs was analyzed by NTA,TEM and Western blot analysis.Results:Primary SCs were doubling immuno-stained with S100 and p75NTR.The purity of the primary SCs was 96.3±0.36%.TEM analysis showed that SPIONs had an average diameter of 21.7±2.36 nm.The saturation magnetization information of SPIONs at 3.382 Qe was 61.62 emu/g.The SPIONs showed an average diameter of 121.7±17.43 nm and a positive charge of 18.9±2.94 mV as measured by a ZetaPALS particle analyzer.The results of CCK-8 assay suggested that concentration of SPIONs no more than 2 μg/mL has no potential cytotoxicity for SCs and 2 μg/mL was used as a safe concentration in the remaining experiments.The flow cytometry showed that the intensity of MF no more than 2 mT had no impact on cell apoptosis when the cultures treated with 2 μg/mL SPIONs.In addition,we quantified the intracellular iron of SCs and calculated the magnetic force acting upon a single SC,which further confirmed the effectiveness of mechanical stimulation of SCs.Therefore,the parameters of the mechanical tension stimuli system are 2 μg/mL SPIONs and 2 mT MF.The "cup-shaped" morphology of the obtained EVs was observed by TEM,indicating that these vesicles were consistent with characteristics of EV.Western blot analysis showed that the exosomal markers Alix,CD63 and TSG101 were expressed in MS-SCs-EVs,while calnexin,an integral protein was not detected in MS-SCs-EVs.Interestingly,we found that MS-SCs-EVs also express p75NTR,a marker of dedifferentiated SCs.The NTA results showed that MS-SCs-EVs had a modal peak size of 137.5±1.77 nm.Conclusion:SCs mechanical tension stimuli system is safe and effective and the obtained EVs were consistent with characteristics of EV reported previously.Part Ⅱ:EVs derived from mechanical tension stimulated SCs promote peripheral nerve regeneration after sciatic nerve injury in RatsBackground:The functional and structural integrity of nervous system are critically dependent on proper intercellular communication.EV as a novel mediator for intercellular communication has been gradually recognized.Mechanical tension stimuli are capable of regulating cell biology.SC subjected to stretches associated with movements of the limbs and mechanical tension stress from daily activities.Accumulating evidence suggested that SC is sensitive to the stiffness of surrounding environment.Studies reported that mechanical stimuli can regulate cell biology through EV.Therefore,it is important to explore the role of mechanical tension stimuli on SCs derived EVs and their effect on neurtite outgrowth,which is the basis of understanding the mechanotransduction of SCs.Objective:To explore the effect of MS-SCs-EVs on neurite outgrowth and nerve regeneration in vitro and in vivo.Methods:MS-SCs-EVs were labeled with PKH26 and co-cultured with DRG neurons to examine the internalization of MS-SCs-EVs by neurons.The effect of MS-SCs-EVs on neurite outgrowth was evaluated by DRG neurons and explants in vitro,respectively.In addition,the effect of different concentrations of MS-SCs-EVs on neurite outgrowth was examined.Furthermore,SD rat with 5 mm sciatic nerve defect was used to explore the effect of MS-SCs-EVs on nerve regeneration.Results:PKH26 labeled MS-SCs-EVs co-cultured with DRG neurons.We found that MS-SCs-EVs can be internalized by DRG neurons.MS-SCs-EVs co-cultured with DRG neurons and explants showed stronger ability in enhancing neurite outgrowth compared to SCs-EVs.Furthermore,we found that the enhancement of MS-SCs-EVs on neurite outgrowth is dose-dependent and the facilitating effect maximized when the concentration reached 1×108 per explant.In addition,the enhancement of MS-SCs-EVs on neurite outgrowth is dependent on the integrity of EVs.MS-SCs-EVs also showed further promotion of nerve regeneration compared to SCs-EVs,and are similar with the autologous nerve graft.Conclusion:MS-SCs-EVs showed a stronger ability in enhancing neurite outgrowth and nerve regeneration compared to SCs-EVs.Part Ⅲ:The mechanism of EVs derived from mechanical tension stimulated SCs promote peripheral nerve regeneration after sciatic nerve injury in ratsBackground:EV can be secreted nearly by all type of cells.The diverse function of EV is dependent on its diverse cargoes,including DNA,mRNA,miRNA and proteins.EVs and their miRNA cargo play an important role in intercellular communication in nervous system.MiRNA can be shuttled to axons by microglia and astrocytes derived EV in central nervous system.Similarly,miRNA can be transferred by EV derived from glia cells in peripheral nervous system.Studies reported that SCs,MSCs and macrophage derived EV and their miRNA cargo play an important role in regulating nerve regeneration.MS-SCs-EVs can enhance neurite outgrowth and nerve regeneration in vitro and in vivo,while the mechanism still remained unclear.Therefore,it is important to identify the mechanism that MS-SCs-EVs enhanced neurite outgrowth,and provide a new strategy for peripheral nerve injury repair.Objective:To identify the mechanism of MS-SCs-EVs enhance neurite outgrowth and nerve regeneration.Methods:High throughput miRNA sequencing was used to compare the miRNA profiles in MS-SCs-EVs and SCs-EVs.Differentially expressed miRNAs associated with axon growth were identified with the KEGG pathway enrichment analysis.Further functional analysis showed that miR-23b-3p had a predominant role in these differentially expressed miRNAs in MS-SCs-EVs.The effect of miR-23b-3p on neurite outgrowth was examined in vitro and in vivo.SCs were transfected by lentiviral vectors with miR-23b-3p antisense oligoribonucleotides to knockdown the miR-23b-3p expression in MS-SCs-EVs(miR-23b-3p KD MS-SCs-EVs).The effect of miR-23b-3p in MS-SCs-EVs on neurite outgrowth was further examined in vitro and in vivo.Nrp1 was predicted as the putative target gene of miR-23b-3p and qRT-PCR and Western blot analysis were applied to explore the effect of miR-23b-3p on Nrp1 expression.In addition,the effect of overexpression and knock down expression of Nrp1in neurons on neurite outgrowth was also explored.Results:The miRNAs expression profiles of SCs-EVs and MS-SCs-EVs were compared using high-throughput sequencing.Fold change log2>2 and P<0.05 wer used as the threshold cutoff.We found that 20 miRNAs were significantly altered between SCs-EVs and MS-SCs-EVs.KEGG pathway enrichment analysis suggesting that 5 differentially expressed miRNAs(miR-26a-5p,miR-129-1-3p,miR-186-5p,miR-23b-3p,miR-365-3p)were related to axon guidance and regeneration.MiR-23b-3p significantly decreased Nrp 1-3’-UTR-WT luciferase activity.Therefore,miR-23b-3p played a predominant role in these differentially expressed miRNAs.Our results showed that miR-23b-3p mimics enhanced neurite outgrowth,while miR-23b-3p inhibitors counteracted the enhancement of MS-SCs-EVs on neurite outgrowth.In addition,knock down the expression of miR-23b-3p in MS-SCs-EVs abolished the enhancment of MS-SCs-EVs on neurite outgrowth and nerve regeneration in vitro and in vivo,which suggested that miR-23b-3p in MS-SCs-EVs promote nerve regeneration.The qRT-PCR and Western blot analysis results showed that miR-23b-3p decreased the expression of Nrp1.Knock down the expression of Nrp1 in neurons by Nrp1 siRNA enhanced neurite outgrowth.In addition,the restoration of Nrp1 expression in neurons partially counteracted the enhancement of MS-SCs-EVs on neurite outgrowth.Conclusion:MS-SCs-EVs enhance neurite outgrowth and nerve regeneration via miR-23b-3p transfer and effect on Nrp1.