lncRNA GAS5­mediated miR­23a­3p promotes inflammation and cell apoptosis by targeting TLR4 in a cell model of sepsis.

Sepsis is a syndrome characterized by organ dysfunction and an abnormal immune response to infection. A growing body of research has shown the importance of long non­coding RNAs (lncRNAs) in tumorigenesis, virus replication, inflammatory injury and other pathological processes. The aim of the present study was to explore the role and potential mechanism of the lncRNA growth arrest­specific 5 (GAS5) in the lipopolysaccharide (LPS)­induced inflammation and apoptosis of THP­1 cells. An in vitro sepsis model was established by treating THP­1 cells with LPS. Apoptosis was detected by flow cytometry. The expression levels of IL­6, IL­1ß and TNF­α were detected using reverse transcription­quantitative PCR (RT­qPCR) and ELISA, and those of GAS5, microRNA (miR)­23a­3p and Toll­like receptor 4 (TLR4) were detected by RT­qPCR. The changes in the biological activity of THP­1 cells induced by the silencing of GAS5 and overexpression of miR­23a­3p and TLR4 were investigated. The relationships among GAS5, miR­23a­3p and TLR4 were analyzed using luciferase reporter assays. The results revealed that LPS increased the expression of GAS5 in THP­1 cells, and GAS5 knockdown effectively inhibited inflammation and cell apoptosis in the LPS­induced sepsis model. In addition, the results of the luciferase reporter assays indicated that both GAS5 and TLR4 directly target miR­23a­3p. The expression of miR­23a­3p was downregulated whereas that of TLR4 was upregulated in the septic cells. Further experiments showed that the overexpression of TLR4 attenuated the suppressive effects of miR­23a­3p overexpression and GAS5 knockdown on LPS­induced inflammation and apoptosis. In conclusion, the present study indicates that GAS5 strengthens LPS­induced inflammation and apoptosis via the miR­23a­3p/TLR4 pathway.

Comparative study of trans-oral and trans-tracheal intratracheal instillations in a murine model of acute lung injury.

Animal model is of importance to further elucidate the pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). We envisioned a possibility that there might be the differences in lipopolysaccharide (LPS)-induced acute lung inflammation by the trans-oral and trans-tracheal intratracheal instillations. We compared the LPS-induced early inflammatory responses by these two methods. The evaluative system included bronchoalveolar lavage (BAL) fluid biochemical analysis and differential cell counting, lung wet/dry weight ratio and lung histology. In vitro studies were performed on human bronchial epithelial cell line NCI-H292 and alveolar Type II epithelial cell line A549 stimulated with LPS. Both interleukin (IL)-8 release in the BAL fluid and IL-8 secretions from NCI-H292 and A549 cells were measured. We found that the trans-tracheal intratracheal instillation promoted the LPS-induced cell injury, neutrophil infiltration, and pulmonary edema compared to the trans-oral one. The LPS-induced pathological changes by the trans-oral intratracheal instillation were characterized by pulmonary interstitial edema, but the trans-tracheal intratracheal instillation was exudative pulmonary edema. More IL-8 is produced from A549 cells than from NCI-H292 cells under the treatment of LPS. The increased IL-8 release in the BAL fluid and enhanced inflammatory responses caused by LPS may be due to more LPS delivered into the alveolar spaces by the trans-tracheal intratracheal instillation compared to the trans-oral one. The trans-tracheal intratracheal instillation is proved to be more suitable to establish the murine model of ALI than the trans-oral one and helpful to further elucidate the pathogenesis of ALI/ARDS.

Temporal dynamic changes of connexin 43 expression in C6 cells following lipopolysaccharide stimulation.

Connexin 43, a gap junction protein, is expressed mainly in glia in the central nervous system. Neuroinflammation plays an important role in central nervous system injury. Changes to glial connexin 43 levels and neuroinflammation may trigger brain injury and neurodegenerative diseases. To illustrate the relationship between connexin 43 and neuroinflammation, this study investigated how connexin 43 expression levels change in lipopolysaccharide-stimulated rat C6 glioma cells. C6 cells were treated with 0.05, 0.25, 0.5, 1, 2.5 and 5 µg/mL lipopolysaccharide for 24 hours. The nitrite estimation-detected nitric oxide release level was elevated substantially after lipopolysaccharide stimulation. To test the transcriptional level changes of inducible nitric oxide synthase, tumor necrosis factor-α and connexin 43 mRNA, C6 cells were treated with 5 µg/mL lipopolysaccharide for 3-48 hours. Reverse transcription-PCR showed that the expression of inducible nitric oxide synthase and tumor necrosis factor-α mRNA increased over time, but connexin 43 mRNA levels increased in lipopolysaccharide-stimulated C6 cells at 3 and 6 hours, and then decreased from 12 to 48 hours. Connexin 43 protein expression was detected by immunofluorescence staining, and the protein levels matched the mRNA expression levels. These results suggest that connexin 43 expression is biphasic in lipopolysaccharide-induced neuroinflammation in C6 cells, which may be correlated with the connexin 43 compensatory mechanism.