Increases In MiR-124-3p In Microglial Exosomes Confer Neuroprotective Effects By Regulating Neuronal Autophagy Following Traumatic Brain Injury

Objective:Traumatic brain injury(TBI)is one of the leading causes of death and disability worldwide.Exploring the pathogenesis of neurological impairment after TBI has always been the hot point of TBI research.Autophagy is an evolutionarily conserved intracellular lysosome degradation pathway.Autophagy can degrade misfolded harmful proteins,remove damaged and redundant organelles,and maintain the homeostasis of intracellular environment.More and more studies have shown that there is an up-regulation of autophagy level in neurons after TBI.This activated autophagy may play an important role in regulating neurological damage after TBI.Exosome is a kind of membrane vesicles with biological activity which can be secreted by various cells in vivo and in vitro.Exosomes can be used as carriers to transfer functional substances between cells,thus playing a role in physiological processes and pathological processes of different diseases.In our previous research,we found that the level of microRNA-124-3p in microglial exosomes increased after TBI by using microarray technology.Moreover,miR-124-3p has the potential to regulate autophagy.Therefore,this study will mainly elucidate the mechanism of microglial exosomes with high expression of miR-124-3p in regulating neuronal autophagy after TBI and its effect on neurological damage after TBI in vitro and in vivo.This study provides a theoretical basis for exploring new strategies for the treatment of TBI with exosomes and microRNAs.Methods:1.Establish rTBI mice model and stimulate BV2 microglia cell line in vitro by homogenate of brain tissue of rTBI mice at different stages to simulate the surrounding environment of microglia in acute and chronic stage after injury.Microglial exosomes were isolated and extracted by ultracentrifugation.The exosomes were identified by transmission electron microscopy,exosome marker protein detection and nanosights.The expression of miR-124-3p in microglia and microglia exosomes at different time points after TBI was detected by qRT-PCR.2.HT-22 neuronal cell line was cultured and the TBI model in vitro was established by scratch test.Western Blot technique was used to detect the expression of autophagy marker protein and apoptosis-related protein.Immunofluorescence and confocal imaging were used to detect the expression of autophagy marker protein LC3 in order to evaluate the activation of neuron autophagy after TBI and the effect of neuron autophagy on neuron apoptosis after TBI.3.Liposome transfection technique was used to exogenously regulate the expression of related genes;Western Blot technique was used to detect the changes of autophagy marker protein and apoptosis-related protein expression levels;in order to evaluate the regulatory effect of microglial exosomes with high expression of miR-124-3p on autophagy of neurons after TBI and apoptosis of neurons after injury.Bioinformatics analysis was used to predict the possible target genes of miR-124-3p regulating autophagy.Luciferase reporter assay and gene silencing/replenishment assay were used to verify that miR-124-3p regulates neuronal autophagy by acting on related target genes.The effects of microglial exosomes with high expression of miR-124-3p on neurological and cognitive impairment after TBI were further investigated in vivo by tail vein injection technique and neurological function evaluation methods(mNSS,rod rotation test and water maze test).Results:1.(1)The microglia-derived exosomes were extracted by ultracentrifugation and identified by transmission electron microscopy,marker protein detection and nanosights.(2)RT-PCR was used to detect microglia stimulated by rTBI mice brain homogenate and total RNA in their exosomes.The results showed that the expression of miR-124-3p in microglia was significantly higher than that in control group at different time points(3d,7d,14 d,21d)after rTBI injury.The day reached its peak and then declined slightly in 21 days.In microglial exosomes,the expression of miR-124-3p showed the same trend.2.(1)The autophagy and apoptosis-related proteins in HT22 after scratch injury were detected.The results showed that scratch injury could induce the activation of autophagy and aggravate the apoptotic damage of HT22 neurons.(2)After inhibiting scratch-induced neuronal autophagy by chloroquine,the autophagy and apoptotic proteins in HT22 neurons were detected again.The results showed that the degree of neuronal apoptosis decreased with the decrease of autophagy level after scratch injury,indicating that inhibiting scratch-induced neuronal autophagy could reduce neuronal apoptosis.3.(1)Microglia with high expression of miR-124-3p were co-cultured with scratched neurons.It was found that microglia with high expression of miR-124-3p could inhibit neuronal autophagy and reduce apoptotic damage.GW4869 was used to inhibit exosome secretion,which confirmed that microglia exosome was the key to play the above role.(2)By exogenous regulation of the level of microglial exosome miR-124-3p,it is confirmed that microglial exosome mainly inhibits neuronal autophagy through its transport of microglial miR-124-3p.(3)Through target gene prediction and Luciferase Report experiments,FIP200 was confirmed to be an autophagy-related target gene of miR-124-3p,and miR-124-3p could regulate neuronal autophagy after TBI by targeting FIP200 inhibition.(4)Through gene silencing/gene replenishment experiments,it was further confirmed that miR-124-3p could directly inhibit FIP200-mediated neuronal autophagy and play a protective role in TBI model neurons.(5)Microglial exosomes with high expression of miR-124-3p were injected into tail vein of rTBI mice at last.It was found that microglial exosomes with high expression of miR-124-3p could improve neurological impairment,learning ability and memory recovery of rTBI mice.Conclusions:1.The expression of miR-124-3p in microglia-derived exosomes increased after TBI,and this increase lasted from acute stage to chronic stage(21 days after TBI).2.TBI can lead to over-activation of neuron autophagy;inhibition of over-activation of neuron autophagy after TBI can reduce the neuron damage.3.Microglia with high expression of miR-124-3p could inhibit the autophagy of neurons after TBI by secreting exosomes and then alleviate neuronal damage.4.Microglial exosomes with high expression of miR-124-3p mainly inhibit TBI-induced neuronal autophagy through their transport of miR-124-3p.5.miR-124-3p regulates neuronal autophagy after TBI by targeting FIP200.6.Intravenous injection of microglial exosomes with high expression of miR-124-3p by tail vein injection can improve neurological impairment and cognitive recovery in rTBI mice,providing a promising therapeutic strategy for the treatment of TBI.