MiR-214-Enriched Bone Mesenchymal Stem Cell Exosomes Induce Cerebral Protection Against Prolonged Deep Hypothermic Circulatory Arrest

OBJECTIVE: Deep hypothermia circulatory arrest(DHCA)remains an indispensable technique in the treatment of congenital heart in complex neonates,adult congenital heart and aortic arch lesions,but postoperative neurological complications remain the primary Complications.Although a variety of factors may be involved in postoperative neurological complications,it is currently believed that its core mechanism is global cerebral ischemia-reperfusion injury caused by DHCA.Bone marrow mesenchymal stem cell exosomes are currently used in the treatment of cerebral infarction,brain trauma and neurodegenerative diseases.However,there is no relevant research report on the role of specific neurological complications caused by DHCA.At present,another research hotspot increases the therapeutic effect by changing the level of miRNA carried in the exosomes of bone marrow mesenchymal stem cells.Based on the background of this study,the authors speculate that bone marrow mesenchymal stem cell exosomes can alleviate neurological complications after DHCA.And by overexpressing the level of miR-214 in bone marrow mesenchymal stem cells,the expression level of miR-214 in exosomes is further increased.To investigate whether miR-214-enriched mesenchymal stem cell exosomes,comparing to natural bone marrow mesenchymal stem cell exosomes,can enhance the brain protection after DHCA,and further study its mechanism.METHODS: Rat bone marrow mesenchymal stem cells were cultured in vitro and transfected into bone marrow mesenchymal stem cells by transfection with miR-214 lentivirus or blank lentivirus.Exosomes were extracted from their culture medium by ultracentrifugation,and the expression level of miR-214 in exosomes was detected by qRT-PCR.A cardiopulmonary bypass(CPB)model was constructed by rat arteriovenous cannulation and cooled to 18 ° C and DHCA for 60 min.SD rats were randomly divided into Sham group,Control group,Exo group,Vector-Exo group and miR-214-Exo group.One day before surgery,15 μl of PBS,bone marrow mesenchymal stem cell exosomes,transfected blank virus bone marrow mesenchymal stem cell exosomes,and transfected miR-214 bone marrow mesenchymal stem cell exosomes were pretreated through the lateral ventricle.At 6 hours after operation,the expression levels of miR-214,inflammatory factors IL-1β and TNF-α and the expression levels of PTEN,p-AKT/AKT,Bim,Bax,Bcl-2 and cleaved Caspase-3 were detected in hippocampus.The recovery period after operation within 14 days,and the recovery of neurological function was observed by water maze and balance beam.Histopathological changes were observed by HE and Nissl staining.Neonatal cells were labeled by EdU,and neuroangiogensis was observed and vascular immunofluorescence staining were used to observe the microcirculation state.RESULTS: Bone marrow mesenchymal stem cells transfected with miR-214 significantly increased miR-214 levels in secreted exosomes,P < 0.001 versus bone marrow mesenchymal stem cell exosomes or bone marrow Stem cell exosomes transfected with blank virus.Compared with Control,all exosome pretreatment groups effectively inhibited the production of inflammatory factors IL-1β and TNF-α in the early postoperative period.In the long-term,they promoted neuroangiogensis and increased hippocampal tissue vascular density and the number of normal nerves.Exosome-pretreatment markedly improved the spatial learning and memory function and vestibulomotor function.Compared with the Exo group and the Vector-Exo group,miR-214-enriched exosomes remarkably enhanced the expression of miR-214 in hippocampus,increased the expression of p-Akt and Bcl-2,and inhibited the expression of PTEN,Bim,Bax and cleaved Caspase-3,and further increased the number of hippocampal neuron survival and neurological recovery.Conclusion: Exosomes derived Bone marrow mesenchymal stem cell conduct obvious therapeutic effects in brain injury after DHCA.The main mechanism may be related to inhibition of inflammatory reaction and promotion of neuroangiogensis.The genemodified exosomes overexpressing miR-214 can further enhance the therapeutic effect of exosomes from mesenchymal stem cell.The mechanism may be that miR-214 binds to its target proteins PTEN,Bim and Bax,and inhibits acute neuronal cell apoptosis.miR-214-enriched exosomes have no significant effect on inhibiting inflammatory response and promoting neuroangiogensis.