Effects And Mechanism Of Microglia Exosomes And Its Expressed MiR-124-3p On Neurological Outcome After Traumatic Brain Injury

Objective:Traumatic Brain Injury(TBI)is an important cause of death and disability in the world.Neuroinflammation is a characteristic pathological change in acute neurological deficits and chronic traumatic encephalopathy following TBI.It occurs several minutes after trauma and can last for a long time,running through the entire process of TBI initiation and development.Therefore,finding a way to effectively inhibit excessive neuroinflammatory response is important to improve the prognosis of neural function.Exosomes are 30-100 nm extracellular vesicles that are actively secreted outside the cell after the fusion of the intracellular polycystic bodies with the cell membrane.It contains active substances such as lipids,proteins,miRNAs,etc.,which can achieve cell-to-cell signal transduction by binding to target cell receptors and releasing contents,thereby exerting biological functions.This experiment focused on studying the effects of microglia exosomes miRNAs on damaged neurons.By analyzing the expression of miRNAs in microglia exosomes from the acute phase to the chronic phase of TBI,the most significant changes were found,and The effects and mechanisms of this miRNA on neuroinflammation and neurological function after traumatic brain injury in microglial exosomes were further explored.Methods:1.Isolation of microglia exosomes 3,7,14,28 days after rTBI was performed using gradient centrifugation exosomes isolation technology.Western blot,electron microscope and Nano Sight were used to detect the extracted exosomes.It was detected by microarray technology to find the miRNA with the most significant change expression in exosomes.The expression of this miRNA in microglial and exosomes secreted by microglial after r TBI was verified by q RT-PCR technology.The effect of target miRNA on microglia was detected using flow cytometry.2.In an in vitro neuronal scratch injury model,an enzyme-linked immunosorbent assay(ELISA)was used to detect the secretion of neuronal inflammatory factors.In a mouse r TBI model,q RT-PCR was used to detect the secretion of inflammatory factors in the brain tissue of r TBI mice.Western blot was used to study the pathway of miR-124-3p,and its activator was used for verification;target gene analysis was used to obtain the target gene of miR-124-3p,and verified by Western blot and fluorescein plum report experiment.3.In the in vitro neuronal scratch injury model,immunofluorescence,ion conductivity microscopy,and Western blot were used to detect the improvement of damaged neurites.In the mouse r TBI model,the Morris Water Maze was used to detect the neurological outcome of mice.Results:1.Exosomes isolated by microglia were collected at 3,7,14,and 28 days after rTBI and detected by miRNA microarray.It was found that the expression level of 16 miRNAs increased more than 2-fold,among which miR-124-3p was expressed The most significant level increase.And after up-regulates the expression of miR-124-3p by miR-124-3p mimics in microglia,microglia will polarize to M2 type.2.(1)When miR-124-3p–up-regulated microglial exosomes were added to the neuronal scratch injury model in vitro,tumor necrosis factor ?(TNF-?),interleukin-1?(IL-1?)and interleukin-6(IL-6)decreased,while interleukin-10(IL-10)increased.In mice r TBI model,miR-124-3p–up-regulated microglial exosomes were injected through the rat tail vein,and it was found that miR-124-3p–up-regulated microglial exosomes could reduce TNF-?,The secretion of IL-1? and IL-6 increases the secretion of IL-10.(2)Western blot showed that mammalian rapamycin target protein(m TOR)signaling pathway is involved in the regulation of miR-124-3p on neuroinflammation.miR-124-3p can inhibit the activity of the m TOR signaling pathway.Furthermore,the target gene PDE4 B of miR-124-3p was confirmed by target gene prediction and luciferase reporter assay.3.When miR-124-3p–up-regulated microglial exosomes were added to an in vitro neuronal scratch injury model,it was found that the inhibitors of Rho A,amyloid precursor(APP),and phosphorylated Tau protein(p-Tau)were inhibited.Expression and increase the total length of neurite branches and neurites.In mice TBI model,miR-124-3p-up-regulated microglia exosomes were injected into the tail vein of mice and found that they can reduce the escape latency and increase the target quadrant residence time of mice in the Morris Water Maze.Conclusions:1.After r TBI,the expression of miR-124-3p increased most significantly in microglial exosomes,and miR-124-3p could promote microglial polarization to anti-inflammatory.2.After rTBI,the microglial exosomes and its expressed miR-124-3p can directly target PDE4 B to inhibit m TOR signaling activity,thereby inhibiting neuroinflammation.3.After rTBI,the microglial exosomes and its expressed miR-124-3p can promote the growth of injured neurites and improve the neurological outcome of mice.4.The microglial exosomes and its expressed miR-124-3p can become a therapeutic target for interventional neuroinflammation after TBI.Microglia exosomes that regulate miRNAs may provide a new strategy for the treatment of TBI or other neurological diseases.