The Effect And Mechanism Of Exosome Derived MiR-124 On The Neuroinflammation And Neurogenesis In Hippocampus After Traumatic Brain Injury

BackgroundTraumatic brain injury(TBI)is an acute neurotrauma disease induced by the sudden external force,one of the major causes of death and long-term disability worldwide.Until recently,no effective treatment was verified to improve neural structural reparation and functional recovery after TBI in clinic trials.Newly studies suggested that neural stem cells(NSCs)reserved in neurogenic regions such as dentate gyrus(DG)of hippocampus in adult mammalian brain played regenerative and reparative roles after TBI,which made the hippocampus neurogenesis to be a research focus.Currently,most of researchers who concentrated on hippocampus neurogenesis after TBI spared their efforts to explore new strategies to promote the NSCs proliferation and differentiation into neurons.The proliferation and differentiation ability of NSCs was affected by various pathological factors,which made the regenerative potential of NSCs was comparatively low and was not sufficient to repair the injuried nerve.Among these factors,neuroinflammation in hippocampus was the major one that could restrict the repairative function of NSCs after TBI.Neuroinflammation played an important role in the pathological and physiological process of TBI.It had protective effects on the injured brain in a way,while the excessive inflammatory response often became a significant driving force leading to reduced hippocampus neurogenesis and worse functional recovery.Microglia(MG)played key roles in activating and regulating neuroinflammation,which was reported to produce double-edged effects on hippocampus neurogenesis.Activated MG had two polarizations,M1 and M2,M1 MG was pro-inflammatory phenotype and M2MG was anti-inflammatory phenotype.Generally,the M1 polarization of MG was adverse for NSCs in hippocampus playing their neurogenesis role,while the M2 polarization of MG was beneficial for NSCs in hippocampus playing their neurogenesis role.Thus,exploring effective strategies to promote the MG to polarize from M1 to M2 phenotype and inhibit the excessive neuroinflammation might has great significance for the improvement of hippocampus neurogenesis and functional recovery after TBI.MicroRNAs(miRNAs)are important molecules enriched in the brain that can play important roles in the modulation of the polarization of MG.Among the known miRNAs,miR-124 is a brain specific miRNA that is highly expressed in MG.In physiological conditions,miR-124 regulates the function of MG and plays a key role in its quiescence.In pathological conditions,the downregulation of miR-124 increased neuroinflammation by polarizing MG into M1 phenotype,while the upregulation of miR-124 reduced neuroinflammation by polarizing MG into M2 phenotype.These studies indicated that miR-124 had the potential to be an effective agent to polarize MG into the M2 phenotype and thus promote hippocampus neurogenesis after TBI.However,the biochemical property of naked miR-124 was very unstable and fragile,it could be degraded and lost its function easily.Therefore,exploring alternative approachs to completely deliver miR-124into the brain and to further play its biology function will have significant value for the basic research and practical application.Latest studies indicate that the exosome might be one promising carrier that can deliver miR-124 into the brain and play its role,which is an endosomal origin membrane-enclosed small vesicle(30–100 nm)containing proteins,lipids,mRNAs and miRNAs.The various advantages of exosome make it have the potential to deliver miR-124 into the brain.First,its ability to cross the(blood brain barrier)BBB makes the delivery of miR-124 into the brain to be a great possibility.Second,its bi-lipid membrane makes the embedded miR-124 will not be disintegrated and destroyed,which will keep the structural integrity and functional activity of miR-124 after delivery.Newly studies have demonstrated that the miR-124 enriched exosome(Exo-miR-124)promoted the M2 polarization of N9 microglia in vitro.However,whether Exo-miR-124 can regulate the polarization of MG and influence the hippocampus neurogenesis after TBI and the underlying mechanisms are still unknown.According to the findings of previous studies,the TLR4 signaling pathway might be involved in the mechanisms of Exo-miR-124 regulating the polarization of MG after TBI.First,the administration of TLR4 activator lipopolysaccharide(LPS)could induce inflammatory responses,which resulted from the activation of MG and inhibited the neurogenesis in hippocampus.Second,LPS stimulation increased the expression of TLR4downstream inflammatory mediator NF-κB and further induced the production of pro-inflammatory cytokines IL-6 and TNF-αultimately,which were widely known to disrupt neurogenesis.And,LPS could activate TLR4 signaling pathway in MG,which further contributed to the depletion of NSCs in DG of hippocampus.Third,newly studies demonstrated that the inhibition of TLR4 induced the M2 polarization of MG and alleviated the development of neuroinflammation,and miR-124 overexpression was shown to inhibit the LPS-induced MG activation by targeting TLR4.Herein,TLR4signaling pathway might play a role in the process of Exo-miR-124 regulating the polarization of MG and affecting hippocampus neurogenesis after TBI.ObjectiveThe purpose of this study was to explore the potential of Exo-miR-124 in regulating the polarization of MG and further affecting the neuroinflammation and neurogenesis in hippocampus after TBI.This study also investigated whether Exo-miR-124 regulated the polarization of MG by targeting TLR4 signaling pathway.The results of this research will have promise to provide an underlying strategy to improve the outcome of TBI through delivering miR-124 into the brain by using the exosome.Methods1.Mesenchymal stem cells(MSCs)were transfected by the plasmid loading with miR-124or Control(Con),and the transfection effect was identified by quantitative real time polymerase chain reaction(qRT-PCR).The exosomes were isolated from the supernatant of cultured MSCs by ExoQuick-TC isolation kit,and the isolated exosomes were identified by electron microscope and Western Blotting(WB),and the expression of miR-124 in exosome-miR-124(Exo-miR-124)and exosome-Con(Exo-Con)was identified by qRT-PCR.2.A total of 168 rats were randomly divided into sham,TBI,TBI+Exo-Con and TBI+Exo-miR-124 groups,with 42 rats in each group.TBI rat model was constructed by controlled cortical injury device,and the Exo-Con or Exo-miR-124 was administrated via tail intravenous injection at 24 hours after TBI.The hippocampus tissue was acquired at3/7/14/28 days after TBI,qRT-PCR was performed to measure the expression of miR-124,reverse transcription polymerase chain reaction(RT-PCR)was performed to measure the expression of M1 MG signature moleculars CD32,IL-1 and M2 MG signature moleculars CD206,Arginase-1,and enzyme-linked immune sorbent assay(ELISA)was performed to measure the expression of pro-inflammatory cytokines including IL-1,IL-6,TNF-αand anti-inflammatory cytokines including IL-4,IL-10,TGF-β.3.In order to analyze the mechanism of Exo-miR-124 regulating MG in hippocampus after TBI and the role of TLR4 signaling pathway in this process,RT-PCR was first performed to measure the expression of TLR4 signaling pathway moleculars TLR4,MyD88,IRAK-1,TRAF6 and NF-κB in hippocampus at 3/7/14/28 days after TBI.Then,BV2 MG was divided into BV2(Con),BV2+LPS,BV2+LPS+Exo-Con and BV2+LPS+Exo-miR-124 groups.qRT-PCR was performed to measure the expression of miR-124 in BV2 MG,WB was performed to measure the expression of TLR4 pathway moleculars indicated above,RT-PCR was performed to measure the expression of M1 MG signature moleculars CD32,IL-1 and M2 MG signature moleculars CD206,Arginase-1,and ELISA was conducted to detect the expression level of pro-inflammatory cytokines including IL-1,IL-6,TNF-αand anti-inflammatory cytokines including IL-4,IL-10,TGF-β.4.In order to explore the effect of Exo-miR-124 on the proliferation and differentiation ability of NSCs in TBI rat hippocampus after regulating the polarization of MG,immunofluorescence(IF)was first performed to detect the number of newly generated NSCs(BrdU~+/SOX2~+cells)and newly generated neuron differentiated from NSCs(BrdU~+/NeuN~+cells)in the hippocampus at 7/28 days after TBI respectively.Then,neurological severity score(NSS)was used to evaluate the neurological function recovery at 7/14/21/28 days after TBI,and morris water maze(MWM)was used to evaluate the recognition and memory function recovery at 24–28 days after TBI.Results1.The exosome with miR-124(Exo-miR-124)high expression and exosome-control(Exo-Con)was successfully constructed.Images of the electron microscope showed that exosomes were round-shaped morphology particles with a size of 30–100 nm.WB indicated that CD9,CD63 and CD81 was highly expressed on these particles.The qRT-PCR indicated that miR-124 was highly expressed in the Exo-miR-124,compared with the Exo-Con.2.The expression of M1 MG signature moleculars CD32,IL-1βand M2 MG signature moleculars Arginase-1,CD206,as well as the pro-inflammatory cytokines including IL-1,IL-6,TNF-αand anti-inflammatory cytokines including IL-4,IL-10,TGF-βwas elevated in hippocampus after TBI.Compared with Exo-Con,the Exo-miR-124 administration increased the expression of miR-124 in hippocampus after TBI,the expression of M2 MG signature moleculars Arginase-1,CD206 and anti-inflammatory cytokines IL-4,IL-10,TGF-βwas increased as well,while the expression of M1 MG signature moleculars CD32,IL-1 and pro-inflammatory cytokines IL-1,IL-6,TNF-αwas decreased.3.The expression of TLR4 pathway moleculars TLR4,MyD88,IRAK-1,TRAF6 and NF-κB in hippocampus was elevated after TBI.Compared with Exo-Con,the administration of Exo-miR-124 decreased the expression of them.After stimulated BV2cells by LPS,the expression of TLR4 pathway moleculars TLR4,MyD88,IRAK-1,TRAF6 and NF-κB was elevated.Compared with Exo-Con,the administration of Exo-miR-124 increased the expression of miR-124 and decreased the expression of TLR4pathway moleculars.The expression of M1 MG signature moleculars CD32,IL-1 and pro-inflammatory cytokines IL-1,IL-6,TNF-αwas also elevated in BV2 cells after LPS stimulation.Compared with Exo-Con,the administration of Exo-miR-124 decreased the expression of M1 MG signature moleculars CD32,IL-1 and pro-inflammatory cytokines IL-1,IL-6,TNF-α,while increased the expression of M2 MG signature moleculars Arginase-1,CD206 and anti-inflammatory cytokines IL-4,IL-10,TGF-β.4.The number of newly generated NSCs(BrdU~+/SOX2~+cells)and newly generated neuron differentiated from NSCs(BrdU~+/NeuN~+cells)in hippocampus was increased after TBI.Compared with Exo-Con,the administration of Exo-miR-124 further increased the number of them.NSS and WMW indicated that the neurological and recognition and memory function of rats was impaired after TBI.Compared with Exo-Con,the administration of Exo-miR-124 promoted the recovery of these neurological functions.4.ConclusionAfter TBI,both M1 and M2 phenotype of MG was activated,the administration of Exo-miR-124 promoted the M1 to M2 polarization of MG.And,both in vivo and in vitro experiments indicated that Exo-miR-124 promoted the M1 to M2 polarization of MG by inhibiting TLR4 signaling pathway.Further,the administration of Exo-miR-124 promoted the M1 to M2 polarization of MG and enhanced the proliferation and differentiation ability of NSCs in hippocampus,and improved neurological function recovery after TBI.