Mesenchymal Stem Cell Derived EVs Mediate Neuroprotection After Spinal Cord Injury In Rats Via The MiRNA-21-5p/FasL Gene Axis

Part I Neuroprotective effect of EVs derived from mesenchymal stem cells on spinal cord injury[Objective]To investigate the effect of MSCs-EVs on attenuating neuronal apoptosis and improving neurological dysfunction after spinal cord injury.[Methods]1.BMSCs were purified and cultured from rat bone marrow in vitro.The supernatant of BMSCs was collected and purified.The extracellular vesicles were extracted by ultracentrifugation and purified by qEV kit.Their microstructures were observed by transmission electron microscopy,and the expression levels of their surface markers CD9 and TSG101 were detected by Western blot.2.PKH67 labeled MSCs-EVs was used to detect its localization in the injured spinal cord tissue.3.MSCs-EVs were injected into rats with spinal cord injury through tail vein,and posterior limb motor function(basso,beattie and Bresnahan,BBB)was evaluated at each time point after spinal cord injury.4.Rats were sacrificed 3 days after spinal cord injury,and Nissl staining was used to observe the morphological changes of spinal cord tissues in each group;the expression of apoptosis-related proteins Bcl-2,Bax,caspase-3,and cleaved-caspase-3 were detected by Western blot.TMT quantitative proteomics was used to detect the differential expression of protein before and after MSCs-EVs treatment.[Results]1.This study successfully isolated and labeled EVS from the culture supernatant of bone marrow mesenchymal stem cells.The morphology of extracellular vesicles was observed by transmission electron microscopy,and the extracellular surface markers CD9 and TSG101 were detected by Western blot.The results were the same as those reported in domestic and foreign literatures.2.Fluorescence microscopy was used to observe the localization of PKH67 labeled MSCs-EVs in the spinal cord,and the results showed that MSCs-EVs targeted at the site of spinal cord inj ury and mainly combined with neurons in the injured area.3.MSCs-EVs significantly inhibited the ratio of Bax/Bcl-2 and cleaved-caspase-3/caspase-3 and finally exerted an anti-apoptotic effect.Meanwhile MSCs-EVs significantly improved the motor function score(BBB score)after spinal cord inj ury in rats.4.Nissl staining showed that the number of Nissl bodies was increased,the staining was enhanced,and the cells were arranged in an orderly manner after treatment with MSCs-EVs compared with the SCI group.5.Our proteomics analysis identified 883 proteins,of which 72 showed opposite trends between the SCI and MSCs-EVs groups.Databases such as GO,KEGG and COG were used to functionally annotate the identified proteins and thus understand the functional properties of these differential proteins.We found that some of these proteins were associated with notable effects of SCI,such as apoptosis(Programmed cell death 6-interacting protein,Bcl2-associated athanogene-4)and inflammation(Annexin Al,Neutrophil antibiotic peptide NP-4).[Conclusions]1.MSCs-EVs can improve the neurological dysfunction of rats after spinal cord injury.2.The mechanism may be related to the targeting of MSCs-EVs on neuronal cells and the inhibition of neuronal apoptosis.Part ? Mesenchymal stem cell derived EVs mediate neuroprotection after spinal cord injury in rats via the miRNA-21-5p/FasL gene axis[Objective]Whether MSCs-EVs translocate miR-21-5p within neurons to inhibit the expression of the pro-apoptotic target gene FasL to promote cell survival.[Methods]1.Luciferase reporter plasmid was constructed to verify the targeting relationship between miR-21-5p and FasL gene.2.MSCs transfection:MSCs were cultured and then transfected with either the miR-21-5p inhibitor or its negative controls using Lipofectamine(?)2000 reagent according to the manufacturers' instructions and MSCs-EVs were extracted separately.3.Rats were randomly allocated into 3 groups:(1)SCI group:treated with an equal volume of PBS(0.5 mL),(2)SCI + EVs-miR-21-5p inhibitor-NC group:treated with MSCs-EVs which were transfected with the miR-21-5p inhibitor-NC and(3)SCI+ EVs-miR-21-5p inhibitor group:treated with MSCs-EVs which were transfected with the miR-21-5p inhibitor.All administrations of MSCs-EVs or PBS were injected via tail vein at 1 h following SCI insult.4.Tunel staining and BBB score confirmed that the neuroprotective effects of extracellular vesicles could be partially reversed by miR-21-5p inhibitor transfection.[Results]1.There is abundant expression of miR-21-5p in MSCs-EVs.2.miR-21-5p in MSCs-EVs can be combined with FasL gene to inhibit its expression,promote the recovery of motor function and reduce apoptosis after spinal cord inj ury in rats.3.The neurotropic protective effect of MSCs-EVs can be partially reversed by miR-21-5p inhibitor transfection.[Conclusions]The neuroprotective effect of MSCs-EVs is partially mediated by miR-21-5p targeted inhibition of FasL gene.