Exosomes containing miR-122-5p secreted by LPS-induced neutrophils regulate the apoptosis and permeability of brain microvascular endothelial cells by targeting OCLN

摘要

Objective: To explore the effect of exosomes containing miR-122-5p secreted by lipopolysaccharide (LPS)-induced neutrophils on the apoptosis and permeability of brain microvascular endothelial cells (BMECs). Methods: Neutrophils in blood were isolated, purified and identified. LPS-induced neutrophils were co-cultured with BMECs. Untreated or LPS-induced neutrophil exosomes were isolated and identified with a transmission electron microscope. miR-122-5p expressions in the exosomes were detected by real-time quantitative polymerase chain reaction, and then the exosomes were co-cultured with BMECs. Bioinformatics analysis was performed to predict the downstream target gene of miR-122-5p, and OCLN was selected as the subject. Dual luciferase reporter assay was carried out to verify the interactive relationship between OCLN and miR-122-5p. LPS and miR-122-5p were used to treat neutrophils, and then exosomes were collected. Exosome or OCLN was embedded in BMECs. The proliferation, colony forming ability and apoptosis of BMECs were detected by cholecystokinin octopeptide, clone formation assay and flow cytometry, respectively. Corresponding kits were used to detect the activities of reactive oxygen species, superoxide dismutase, malondialdehyde and catalase. Vascular endothelial growth factor and tight junction proteins (ZO-1 and Claudin-5) expressions were measured by Western blot for cell permeability evaluation. Results: miR-122-5p had an increased expression in LPS-induced neutrophil exosomes and could promote oxidative stress, apoptosis and permeability increase of BMECs and the inhibition of BMECs proliferation and colony formation (P0.05). miR-122-5p targeted the binding with OCLN and down-regulated OCLN expression. OCLN overexpression partly decreased the malignant effect of miR-122-5p on BMECs (P0.05). Conclusion: LPS can induce neutrophils to secrete exosomes containing miR-122-5p. The down-regulation of OLCN expression can aggravate BMECs injury.