[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-1ß (IL-1ß) 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-1ß 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.

[Mechanism of heart and lung injury induced by cerebral ischemia/reperfusion in both young and old mice].

Objective To study the mechanism of heart and lung injury after cerebral ischemia/reperfusion in mice. Methods C57BL/6J mice were divided into young and old groups according to their ages, the former being 5-6 months old and the latter being 20-21 months old. Each group was divided into five subgroups subjected to sham operation, middle cerebral artery occlusion for 1-hour ischemia followed by 1-, 12-, 24-, 48-hour reperfusion. At different reperfusion time, HE and TUNEL staining were used to observe the morphological changes of heart and lung tissues; meanwhile, chemical colorimetry was performed to determine the changes of cardiac Na+-K+-ATPase and Ca2+-ATPase; the lung indexes were evaluated; the levels of nuclear factor (NF)-κBp65, p-NF-κBp65, IκBα, p-IκBα were detected by Western blotting; the levels of interleukin 1ß (IL-1ß), tumor necrosis factor α (TNF-α) were determined by ELISA; and the release of NO was examined by colorimetry. Results We observed inflammatory responses in the lung tissues of young and old mice at 24-hour reperfusion and 1-hour reperfusion, respectively, and hemorrhage in the heart tissues of young and old mice at 24-hour reperfusion and 12-hour reperfusion, respectively.Lung tissues showed earlier response to the stimulation of cerebral ischemia/reperfusion than heart tissues did. Meanwhile, the results of Na+-K+-ATPase, Ca2+-ATPase, lung index, NF-κB signaling pathway and inflammatory cytokines in young and old mice were consistent with histological changes of heart and lung tissues. Conclusion Cerebral ischemia/reperfusion can cause heart and lung tissue injury in the old mice, and energy metabolism and inflammation cascade are the main mechanisms of the injury.

[Prediction of regulating network of innate immune signaling molecule hsa-miR-181a in stroke development based on bioinformatics analysis].

OBJECTIVE: To predict the regulating network of innate immunity signaling molecule hsa-miR-181a in stroke based on the methods of bioinformatics. METHODS: The UCSC genome browser, the human miRNA disease database (HMDD), the transcription factor-miRNA regulation database (TransmiR), the database on predicted and validated miRNA targets (miRwalk), the Genecards, the long non-coding RNA (LncRNA) disease database, the DIANA LAB-LncBase and the ConSite were employed to study the upstream transcription factor, downstream target genes and the interactive LncRNA of hsa-miR-181a and to draw the core regulating network of hsa-miR-181a. To verify the hsa-miR-181a regulating network, we used lipopolysaccharide (LPS) to stimulate the BV2 cells transfected by lentivirus and real-time quantitative PCR to detect the changes of Toll-like receptor 4 (TLR4), tumor protein 63 (p63), miR-181a and nuclear factor κB (NF-κB) p65. RESULTS: The UCSC genome browser showed that hsa-miR-181a had two subtypes, which were demonstrated with high conservatism in several species. Diseases analysis and literatures investigation revealed that the hsa-miR-181a was related with many diseases, especially ischemia diseases. Bioinformatics analysis indicated that hsa-miR-181a was regulated by the transcription factors p63, and at the same time, it could regulate 58 target genes such as brain-derived neurotrophic factor (BDNF), TLR4 etc. IncRNA CDKN2B-AS1 and its transcription factors Snail and n-MYC might also interact with hsa-miR-181a. All the relative genes composed a regulatory network with hsa-miR-181a as a core and played important roles in the process of stroke. In LPS-stimulated BV2 cells, the expression levels of TLR4, p63, miR-181a were up-regulated; while the levels of p63, miR-181a and NF-κB p65 decreased in the lentivirus-infected BV2 cells, indicating that p63 was the key signaling molecule in the process of TLR4 regulating miR-181a. CONCLUSION: The bioinformatics analysis and preliminary experimental verification predicted and demonstrated the regulating network of hsa-miR-181a in stroke.