神经元外泌体miR-24-3p对小胶质细胞激活及血脑屏障功能的影响OA

Objective To explore the roles of miR-24-3p in neuronal exosomes in the activation of microglia and the function of the blood-brain barrier(BBB).Methods Primary mouse neurons were isolated and cultured,and treated with LPS,then the supernatants were collected to isolate the exosomes.The exosome markers(TSG101,CD63,CD9)were detected by Western blot,and miR-24-3p expression was detected by RT-qPCR.After the neurons were treated with miR inhibitor or the exosome inhibitor GW4869,followed by 10 mg/L LPS,miR-24-3p expression in neurons and exosomes was measured.Exosomes from the above groups were co-cultured with BV2 cells,named as control-Exo group,LPS-Exo group,LPS-miR inhibitor-Exo group and LPS-GW4869-Exo group.BV2 cell viability,migration,polarization(ICAM-1,VCAM-1,iNOS,Arg1,CD206),and secretion of inflammatory cytokines(TNF-α,IL-1β,IL-6,TGF-β,IL-4,IL-10)were assessed by MTT assay,scratch assay,flow cytometry,ELISA,and Western blot.BV2 cells in the above four groups were co-cultured with an in vitro BBB model(neuroendothelial cells,pericytes,astrocytes),named as control-Exo-BV2 group,LPS-Exo-BV2 group,LPS-miR inhibitor-Exo-BV2 group and LPS-GW4869-Exo-BV2 group,then the expressions of tight junction proteins(Occludin,Claudin-5,ZO-1)were detected by Western blot,and BBB permeability and transendothelial electrical resistance(TEER)were evaluated.Results After LPS induction,miR-24-3p expression was significantly increased in neurons and exosomes(P<0.05),the expressions of TSG101,CD63 and CD9 were detected in exosomes but not in supernatants.The miR inhibitor and GW4869 inhibited miR-24-3p expression in exosomes(P<0.05).Compared with control-Exo group,BV2 cell viability and migration capacity were significantly enhanced in LPS-Exo group(P<0.05),the expressions of M1 polarization markers(CD16/32 and iNOS)increased(P<0.05),and the expressions of M2-related markers(Arg1 and CD206)decreased(P<0.05);the levels of pro-inflammatory cytokines(TNF-α,IL-1β,and IL-6)were elevated(P<0.05),while the levels of anti-inflammatory cytokines(TGF-β,IL-4,and IL-10)were reduced(P<0.05).Compared with LPS-Exo group,BV2 cell viability and migration capacity significantly decreased in LPS-miR inhibitor-Exo group and LPS-GW4869-Exo group(P<0.05),the expressions of M2-related markers increased(P<0.05),the levels of pro-inflammatory cytokines decreased(P<0.05),and the levels of anti-inflammatory cytokines were elevated(P<0.05).In the in vitro BBB model,compared with control-Exo-BV2 group,BBB permeability increased in LPS-Exo-BV2 group(P<0.05),TEER decreased(P<0.05),and the expressions of Occludin,Claudin-5,and ZO-1 decreased in BBB endothelial cells(P<0.05).Compared with LPS-Exo-BV2 group,BBB permeability decreased in LPS-miR inhibitor-Exo-BV2 group and LPS-GW4869-Exo-BV2 group(P<0.05),TEER increased(P<0.05),and the expressions of Occludin,Claudin-5,and ZO-1 were elevated(P<0.05).Conclusion The abnormal upregulation of miR-24-3p in neuronal exosomes can promote the activation of microglia,induce the neuroinflammation,and exacerbate BBB dysfunction.