Schwann Cell-derived Exosomes Promote Functional Recovery In Spinal Cord Injury Rats Via Autophage

【Objective】Spinal cord injury causes severe damage to axons and death of neurons,resulting in permanent loss of motor or sensory function.Unfortunately,there are no effective treatments for spinal cord injuries.Exosomes are extracellular vesicles secreted by cells and are involved in intercellular communication.Due to the prominent characteristics of exosomes,they are able to cross the blood-brain barrier,and capable of multiple intravenous（IV）administrations without any side effects,reduce inflammation and enhance neurological and motor functions.In the peripheral nervous system,Schwann cells are the main glial cells,which can promote the dedifferentiation and proliferation of axons after injury,remove myelin sheaths and axon fragments,and their regeneration ability has been applied to the center Damage to the nervous system.Autophage is a metabolic process in which cells are degraded.It plays a key role in the physiological and pathological processes of the body.In addition,autophagosomes have been observed in the nervous system,but their promoting and/or harmful effects in spinal cord injury have not been fully elucidated.Many studies have shown that exosomes are closely related to autophagy.The purpose of this study is to investigate the role of Schwann cell exosomes in axon regeneration after spinal cord injury and its possible mechanisms to provide clues for the treatment of clinical spinal cord injury.【Method】1.Extract primary Schwann cells from the sciatic nerve of Wistar rats,collect the Schwann cell culture supernatant and perform ultracentrifugation for multiple times to obtain exosomes,and identify the exosomes by electron microscopy,CD9,CD63and Alix.2.Observe the rats treated with Schwann cell-derived exosomes（SCDEs）and PBS after spinal cord injury by comparing BBB behavioral scores,HE staining,LFB staining,and immunofluorescence staining.3.To observe the apoptosis of neurons in vivo and to detect the changes of autophagy markers LC3-1/2,Beclin-1,P62 between the control group and the SCDEs treatment group,our experient applied TUNEL staining and Western-blot.Chloroquine was selected to inhibit autophagy expression.The changes of autophagy expression after spinal cord injury were detected under the same concentration of SCDEs.4.Induce PC12 cell line to differentiate into neurons in vitro and make the spinal cord injury model by hydrogen peroxide intervention.Observe the neurons survival between the control group,hydrogen peroxide group and hydrogen peroxide plus SCDEs treatment group by flow cytometry and TUNEL staining.At the same time,the changes of autophagy markers LC3-1/2,Beclin-1,P62 between different groups were detected by Western-blot.5.Western blot was used to detect changes in EGFR,Akt,and m TOR expression levels to explore the signaling pathways of SCDEs on spinal cord injury.【Result】1.This experiment extracted exosomes from rat Schwann cells.And through Western blot,these exosomes were found to express three exosomal markers:CD9,CD63 and Alix.In addition,SCDEs have a size range of 40-100 nm as determined by TEM.2.For in vivo experiments,there was a statistically significant time-dependent change between the BBB scores of SCDEs-treated and control rats（n=6）.Evaluation of myelin content in the spinal cord by HE staining and LFB staining showed that after SCDEs administration,the percentage of myelin content in the myelin area increased significantly,while the cavitation area decreased significantly.At the same time,immunofluorescence results showed that the contents of Neu N and Ch AT at the injury site of rats treated with SCDEs were higher than those in the PBS treatment group,and the difference was statistically significant.3.Apoptosis was measured by TUNEL stanning.Compared with the sham group,the number of apoptotic cells increased in the SCI group and decreased in the SCDEs-treated group significantly.At the same time,the Western-blot expression results of LC3-1/2,Beclin-1 and P62 showed that compared with the untreated SCI group,the amount of autophagy markers expressed in the SCDEs treated group was increased after SCI.In addition,when autophagy was blocked by chloroquine,SCDEs induced an increase in LC3-1/2,Beclin-1 and P62.The difference was statistically significant.4.Assessing apoptosis by in vitro flow cytometry,and the results showed that H₂O₂-induced injury increased apoptosis in neurons,while SCDEs treatment reduced apoptosis.In addition,the expression levels of LC3-1/2,Beclin-1,and P62 in neurons induced by H₂O₂increased after SCDEs treatment and decreased in the presence of chloroquine.The difference was statistically significant.5.In vitro Western-blot results showed that when H₂O₂-induced damage occurs,EGFR is downregulated after SCDEs treatment.Western-blot results of phosphor-Akt and phosphor-m TOR that are involved in autophagy regulation shown that SCDEs treatment decreased the phosphorylation of Akt and m TOR in neurons induced by H2O2.【Conclusion】This experiment showed that SCDEs enhanced autophagy and reduced apoptosis after spinal cord injury in vivo and in vitro experiments,thereby promoting axonal regeneration and motor function recovery.In addition,this experiment found that increased expression of SCDEs led to decreased expression of EGFR,and inhibited the AKT-m TOR signaling pathway,which up-regulated the expression of autophagy and reduced apoptosis.In summary,the research in this experiment shows that SCDEs induce autophagy and reduce apoptosis by inhibiting the EGFR-AKT-m TOR signaling pathway,thereby promoting axon regeneration after spinal cord injury.