| Objective: Spinal cord injury(SCI)is a serious traumatic disease that results in motor,sensory and autonomic nervous system dysfunction and induces secondary problems including muscle wasting,chronic pain,and urinary infections.These functional deficits are caused by the initial mechanical injury and subsequent secondary injury over a period of weeks or even months.Blood spinal cord barrier(BSCB)is composed of the special cells.The highly developed tight junctions and adhesions can prevent the peripheral circulation substances from entering the spinal cord and provide a unique microenvironment for the spinal cord.After BSCB disruption in the early stage after SCI,blood cells are infiltrated into the injured parenchyma and produce inflammatory mediators such as pro-inflammatory cytokines and chemokines,contributing to more severe injuries.Bone marrow mesenchymal stem cells(BMSCs)have been shown to be effective in treating SCI but also have many limitations.Exosomes are nanoscale extracellular vesicles origin secreted by mesenchymal stem cells,which carry biologically active functional substances,such as DNA,mRNA,miRNA and proteins.However,it is still unclear how exosomes promote the repair of spinal cord injury.This study mainly explores the effects of exosomes on the integrity of the BSCB after SCI and its possible mechanism.Methods: BMSCs were cultured,and exosomes were extracted from cell culture media with Exosome Isolation Kit.Sixty Sprague-Dawley rats were randomly divided into three groups(n=20): Sham,SCI rats treated with PBS(SCI+PBS),SCI rats treated with exosome.The SCI rats were established by modified Allen’s method.On the third day after SCI,Evans blue dye was injected into rats through the caudal vein to evaluate the permeability of BSCB by observing the exudation of the dye.Western blot was used to measure the expression of BSCB-related tight junction proteins,including Claudin-5,Occludin and ZO-1.The expression and activity of MMP-9 were examined by Western blot and gelatin enzyme,respectively.We evaluated the effect of exosome treatment on the blood cells infiltration by immunofluorescence against MPO for neutrophils measured the morphology and tissue loss of the spinal cord using H&E staining.Functional recovery was evaluated for three weeks after injury using BBB rating scale.Results: 1.BMSCs were successfully cultured in vitro,and exosomes were extracted from the p3-p6 cell culture medium with good growth.Exosomes were lipid bilayer vesicles with a diameter of about 100 nm under TEM,and the marker proteins Alix and CD63 were positively expressed by Western Blot(WB).2.The permeability of BSCB was reduced after exosome treatment.We measured the expression of the tight junction(TJ)proteins by Western Blot due to their close relationship to the integrity of BSCB.The results showed that TJ proteins’ expression significantly decreased after spinal cord injury,while exosomes significantly reduced TJ proteins’ degradation.3.Because MMP-9 can degrade tight junctions,we measured the expression and activity of MMP-9 to determine whether MMP-9 mediates the therapeutic effect of exosomes,and the results showed that exosome treatment significantly inhibited the expression and activity of MMP-9.4.The disruption of BSCB leads to neutrophils infiltration,which further aggravates the injury,and immunofluorescence results showed that neutrophils infiltration was significantly reduced after exosome treatment.5.H&E staining results showed that exosomes significantly reduced the area of SCI and the loss of neurons.The BBB score in the exosome treatment group was significantly higher than that in the model group.Conclusion: BMSCs-derived exosome improved functional recovery after SCI in part by inhibiting BSCB disruption and the subsequent infiltration of neutrophils through reducing the degradation of tight junction proteins by inhibiting the expression and activity of MMP-9. |
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