The Effects And Mechanisms Of Melatonin-modified Astrocyte-derived Exosomes On Optic Nerve Injury

All Retinal Ganglion Cells(RGCs)axons pass through the wall of the eye and converge to form the optic nerve.As a part of the Central Nervous System(CNS),it performs an important function of transmitting visual information from the retina to the brain.Glaucoma,traumatic disease and genetic disease and other diseases can lead to optic nerve injury,the main pathological changes are a large number of RGCs loss and the difficulty of axon regeneration.These can lead to serious consequences such as reduced vision and even loss of vision.Saving RGCs and promoting axon regeneration are the basis of improving visual function.However,it is difficult for neurons to regenerate after injury in CNS.Clinically,neurotrophic factor,ganglioside,hormone and other therapeutic measures are mainly symptomatic treatment,but the therapeutic effect is poor.The research of transplantation therapy provides a new way to find safe and effective treatment.Astrocytes(ASTs)play a key role in maintaining the normal structure and function of the CNS.ASTs are involved in important processes under physiological and pathological conditions including the regulation of blood flow,extracellular homeostasis,ions and transmitters,energy supply,axonal transmission,and myelination.Recent studies have highlighted that ASTs and neurons communicate information through delivery of Extracellular vesicles(EVs).There are three types of EVs,of which exosomes are the smallest vesicles.The contents and functions of exosomes depend on the cell type from which they are derived,and can be involved in pathophysiological processes such as immune response,cell migration,cell differentiation,regeneration and repair.The development of exosomes related drugs is a new strategy for the treatment of major diseases of nervous system.Drug preconditioning,mechanical stimulation,cytokines and biological scaffold support can change the key molecules carried by exosomes,enhance the biological function of exosomes,and improve the therapeutic effect of exosomes.The neuroendocrine hormone Melatonin(MT)is secreted by the pineal gland,It plays a role in regulating biological rhythms,immune regulation,metabolism,and cell protection.Current evidence suggests that melatonin acts as a protective agent in glaucoma,diabetic retinopathy,and retinopathy with ischemia/reperfusion injury.Melatonin pretreated mesenchymal stem cell-derived exosomes have been shown to alleviate interferon gamma and tumor necrosis factor-α induced inflammatory responses by delivering microRNAs(miRNAs)including miR-34a,miR-124,and miR135b.Based on the above research background,we propose the following hypothesis:Melatonin-modified astrocyte-derived exosomes(MT-EVs)have therapeutic effects on mice with Optic Nerve Crush(ONC).Treatment with MT-EVs can improve the survival rate of RGCs after ONC,promote axon regeneration and long-term visual function recovery.The specific action mechanism of MT-EVs was explored by screening key miRNA in MT-EVs.In order to explore the role and mechanism of MT-EVs,the following studies were carried out:We selected C57BL/6J adult female mice as subjects and used tweezers(Dumont#5;Roboz)clamped the optic nerve at 1.5 mm behind the eyeball for 5s to establish the ONC model.After astrocyte-derived exosomes not treated with melatonin(ADEVs)and MTEVs treatment,the recovery of visual function and RGCs survival,retinal ASTs activation and optic nerve regeneration were evaluated by electrophysiological,behavioral,immunofluorescence staining and transmission electron microscopy.Next,Illumina HiSeq 2500 high throughput sequencing(miRNA-seq),Dual-luciferase reporter gene assay,qRT-PCR,Western blot and other molecular detection methods screen and verify the differentially expressed miRNAs and their downstream targets in exosomes of ASTs that are not treated with melatonin(ADEVs)and MT-EVs,and explore the specific mechanism of the action of the key miRNA.The results are as follows:1.CCK-8,GFAP immunofluorescence staining,nanoparticle tracking analysis,transmission electron microscopy,and Western blot showed that the characteristics of two types of exosomes were similar,and melatonin had no effect on the size and morphology of exosomes.2.Electrophysiological and visual behavior tests showed that MT-EVs treatment could improve the visual dysfunction of mice after ONC,and promote the recovery of the visual level of mice.3.The results of GAP43 immunofluorescence staining and transmission electron microscopy showed that the increased fluorescence expression level of GAP43 after MT-EVs treatment suggested that the difficult regeneration of optic nerve axons was improved,and the results of transmission electron microscopy showed that the myelin structure of optic nerve was recovered.4.The results of immunofluorescence staining of RBPMS and TUJ1 showed that the number of RGCs increased after MT-EVs treatment,suggesting that MT-EVs promoted the survival of RGCs.The increased fluorescence density of TUJ1 suggested that MT-EVs promoted axon recovery in RGCs.5.The results of GFAP immunofluorescence staining showed that the fluorescence density of GFAP decreased after MT-EVs treatment,suggesting that MT-EVs inhibited the abnormal activation of retinal ASTs.PKH26 staining showed that both retinal RGCs and ASTs can absorb MT-EVs.The results in vitro were consistent with those in vivo.6.miRNA expression profiles of ADEVs and MT-EVs were identified by Illumina HiSeq 2500 high-throughput sequencing(miRNA-Seq).The results of heat map and scatter map showed that there were significantly differentially expressed miRNAs in the two types of exosomes.KEGG results showed that the enrichment of miRNAs target gene-related pathways were related to axon growth and regeneration,including axon guidance,mTOR signaling pathway and ErbB signaling pathway.GO enrichment analysis showed that the biological functions of target genes were related to dendrite development,axon regeneration,and vesicle localization and transport.Differential miRNAs expression was verified by qRT-PCR,and Dual-Luciferase Reporter was further used to determine their targeting relationships to candidate genes.The result of qRT-PCR showed that miRNAs differential expression was consistent with sequencing results.The results of Dual-Luciferase Reporter showed that there was a targeting relationship between miR-223-3p and Mapk10.7.After transfection of miR-223-3p mimic and miR-223-3p inhibitor into ASTs,qRT-PCR detected the expression changes of miR-223-3p in ASTs and derived exosomes.The result of qRT-PCR showed that miR-223-3p levels in cells and exosomes were increased after transfection with miR-223-3p mimic.miR-223-3p expression was decreased in cells and exosomes after transfection with miR-223-3p inhibitor.The expression of Mapk10 in RGCs after Oxygen Glucose Deprivation(OGD)was detected by qRT-PCR.The result of qRT-PCR showed that compared with OGD group,the expression of Mapk10 in ADEVs group,MT-EVs group and miR-223-3p mimics group was significantly decreased.However,Mapk10 expression was decreased in MT-EVs group with miR-223-3p inhibitor pretreatment,suggesting that miR-223-3p was negatively correlated with Mapk10 expression.Research conclusionThis study confirmed that MT-EVs can regulate the expression of Mapk10 in RGCs by delivering miR-223-3p to RGCs,thus improving the survival and axon regeneration of RGCs after injury.At the same time,MT-EVs can inhibit the activation of ASTs,improve the environment around injury,and promote the recovery of visual function in mice.This study suggests that ADEVs play an important role in the repair of nerve damage,and MT as a preconditioning drug can change exosomes contents and greatly improve its therapeutic effect.The development of ADEVs resources and its key miRNA is expected to become a new important strategy in the treatment of optic nerve injury and other nervous system diseases,providing theoretical basis and research basis for subsequent studies.It has important clinical value.