The Protective Effects Of MiR-126-3p/-5p And M2 Microglia Exosome On The Blood-brain Barrier In Cerebral Ischemic Mice

Background:Blood-brain barrier(BBB)disruption is a critical pathological feature after stroke.micro RNA-126(miR-126)maintains BBB integrity by regulating endothelial cell function during development.However,the role of miR-126-3p and-5p in BBB integrity after stroke is unclear.Here,we investigated whether miR-126-3p and-5p overexpression regulates BBB integrity after cerebral ischemia.M2 microglia derived exosome attenuates brain injury of ischemic mice,and reduces nuronal apopotosis.However,the role of M2 microglia derived exosome in BBB integrity after stroke is unclear.Here,we investigated whether M2 microglia derived exosome regulates BBB integrity after cerebral ischemia.Methods:A lentivirus carrying genes encoding miR-126-3p or-5p was stereotactically injected into adult male Institute of Cancer Research mouse brains.Permanent middle cerebral artery occlusion(MCAO)was performed 2 weeks after virus injection.Brain infarct volume,edema volume,and modified neurological severity score were assessed at 1 and 3 days after ischemia.Immunostaining of zonula occludens-1(ZO-1)and occludin was used to evaluate BBB integrity.Interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),vascular cell adhesion molecule-1(VCAM-1),and E-selectin expression levels were determined by real-time polymerase chain reaction(Real-time PCR)and Western blot analysis.M2 microglia derived exosomes were isolated and purified by ultracentrifugation.Nanoparticle tracking analysis,transmission electron microscopy and western blot were used to identify the exosomes.M2 microglia derived exosomes were injected into the MCAO mice by tail vein injection.Brain infarct volume,edema volume,and modified neurological severity score were assessed at 3 days after ischemia.Immunostaining of ZO-1,Occludin and Claudin-5 was used to assess BBB integrity.The downstream target genes regulated by M2 derived exosomes were identified by RNA sequencing and database prediction.Results:The expression of miR-126-3p and-5p decreased at 1 and 3 days after ischemia(p<0.05).Injection of lentiviral miR-126-3p or-5p reduced brain infarct volume and edema volume(p<0.05)and attenuated the decrease in ZO-1/occludin protein levels and Ig G leakage at 3 days after stroke(p<0.05).Injection of lentiviral miR-126-5p improved behavioral outcomes at 3 days after stroke(p<0.05).miR-126-3p and-5p overexpression downregulated the expression of proinflammatory cytokines IL-1βand TNF-αand adhesion molecules VCAM-1 and E-selectin,as well as decreased myeloperoxidase positive(MPO~+)cell numbers at 3days after ischemia(p<0.05).M2 microglia derived exosomes reduced brain infarct volume and edema volume(p<0.01)and attenuated the decrease in ZO-1/Occluding/Claudin-5 protein levels and Ig G leakage at 3 days after stroke(p<0.001,p<0.01),improved behavioral outcomes at 3 days after stroke(p<0.05).Conclusions:miR-126-3p and-5p overexpression reduced the expression of proinflammatory cytokines and adhesion molecules,as well as M2 microglia derived exosomes regulated NFκB pathway,thereby attenuating BBB disruption after ischemic stroke,suggesting that miR-126-3p/-5p and M2 microglia derived exosomes are new therapeutic targets in the acute stage of stroke.