[Microvesicles derived from LPS-induced microglia aggravate the injury of tight junction in rat brain microvascular endothelial cells under oxygen-glucose deprivation].

Objective To study the effect of the administration of the circulating microvesicles (MV) obtained from lipopolysaccharide (LPS)-stimulated microglia supernatant into rat brain endothelial cells (RBECs) on the injury of tight junction in RBECs under the condition of oxygen-glucose deprivation (OGD) as well as the underlying mechanism. Methods The circulating MV was isolated from the supernatant of microglia stimulated with LPS 1 mg/L for 24 hours and subjected to morphological identification. The expression of miR-27a in MV was detected by real-time PCR. RBECs were randomly divided into control group, MV-RBECs control group, OGD 6-hour group and OGD-MV group. The expressions of occludin and claudin-5 were detected by immunofluorescence staining in the four groups. Western blot analysis was used to investigate the expressions of occludin, claudin-5, Toll-like receptor 4 (TLR4), NF-κBp65 and p38 proteins in RBECs, and ELISA was applied to detect the levels of interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) in RBECs. Results The shape of MV was approximately circular double membrane vesicles, with an average diameter of 150 nm, in accordance with the morphological characteristics of MV. Under LPS stimulation, the level of miR-27a in the circulating MV was abnormally elevated. Compared with the control group, RBECs were not obviously influenced by the incubation of MV; under OGD condition, tight junction of RBECs was damaged with the decreasing expressions of occluding and claudin-5. The degree of injury was further damaged after the treatment with MV. Fluorescence intensity of occludin and claudin-5 were further reduced. Meanwhile, Western blot analysis showed the levels of occludin and claudin-5 proteins decreased in the OGD group after MV treatment, which was consistent with immunofluorescence staining. Compared with the control group, the expression of TLR4 protein and the phosphorylation of NF-κBp65 and p38 proteins increased in OGD group; after MV treatment, the level of TLR4 protein and the phosphorylation of NF-κBp65 and p38 proteins further increased, and the release of IL-1β and TNF-α increased as well. Conclusion Treatment with the circulating MV containing miR-27a obtained from LPS-stimulated microglia supernatant damages the tight junction of RBECs under the OGD condition. The mechanism may be related to up-regulation TLR4 and phosphorylation of NF-κBp65 and p38.