cyclin-d1 and Hyperoxia

Bronchopulmonary dysplasia is a serious and lifelong pulmonary disease in premature neonates that influences around one-quarter of premature newborns. The wingless-related integration site /β-catenin signaling pathway, which is abnormally activated in the lungs with pulmonary fibrosis, affects cell differentiation and lung development.. Newborn rats were subjected to hyperoxia exposure. Histopathological changes to the lungs were evaluated through immunohistochemistry, and the activation of disheveled and Wnt /β-catenin signaling pathway components was assessed by Western blotting and real-time PCR. The abilities of proliferation, apoptosis and migration were detected by Cell Counting Kit-8, flow cytometry and scratch wound assay, respectively.. Contrasting with normoxic lungs, hyperoxia-exposed lungs demonstrated larger alveoli, fewer alveoli and thicker alveolar septa. Superoxide dismutase activity was significantly decreased (7th day: P < 0.05; 14th day: P < 0.01) and malondialdehyde significantly increased (7th day: P < 0.05; 14th day: P < 0.01) after hyperoxia exposure. Protein and mRNA expression levels of β-catenin, Dvl-1, CTNNBL1 and cyclin D1 were significantly upregulated by hyperoxia exposure on 7th day (P < 0.01) and 14th day (P < 0.01). In hyperoxic conditions, Dvl-l downregulation and Dvl-l downregulation + MSAB treatment significantly increased the proliferation rates, decreased the apoptosis rates and improved the ability of cell migration. In hyperoxic conditions, Dvl-l downregulation could decrease the mRNA expression levels of GSK3β, β-catenin, CTNNBL1 and cyclin D1 and decrease the protein relative expression levels of GSK3β, p-GSK3β, β-catenin, CTNNBL1 and cyclin D1.. We confirmed the positive role of Dvl-1 and the Wnt/β-catenin signaling pathway in promoting BPD in hyperoxia conditions and provided a promising therapeutic target.

Topics: Animals; Animals, Newborn; beta Catenin; Cyclin D1; Dishevelled Proteins; Glycogen Synthase Kinase 3 beta; Hyperoxia; Lung; Rats; Rats, Sprague-Dawley; RNA, Messenger; Wnt Signaling Pathway

Oxidative stress is regarded as a key regulator in the pathogenesis of prolonged hyperoxia-induced lung injury, which causes injury to alveolar epithelial cells and eventually leads to development of bronchopulmonary dysplasia (BPD). Many studies have shown that hydrogen has a protective effect in a variety of cells. However, the mechanisms by which hydrogen rescues cells from damage due to oxidative stress in BPD remains to be fully elucidated. This study sought to evaluate the effects of hydrogen on hyperoxia-induced lung injury and to investigate the underlying mechanism. Primary type II alveolar epithelial cells (AECIIs) were divided into four groups: control (21% oxygen), hyperoxia (95% oxygen), hyperoxia + hydrogen, and hyperoxia + hydrogen + LY294002 (a PI3K/Akt inhibitor). Proliferation and apoptosis of AECIIs were assessed using MTS assay and flow cytometry (FCM), respectively. Gene and protein expression were detected by quantitative polymerase chain reaction (q-PCR) and western blot analysis. Stimulation with hyperoxia decreased the expression of P-Akt, P- FoxO3a, cyclinD1 and Bcl-2. Hyperoxic conditions increased levels of Bim, Bax, and Foxo3a, which induced proliferation restriction and apoptosis of AECIIs. These effects of hyperoxia were reversed with hydrogen pretreatment. Furthermore, the protective effects of hydrogen were abrogated by PI3K/Akt inhibitor LY294002. The results indicate that hydrogen protects AECIIs from hyperoxia-induced apoptosis by inhibiting apoptosis factors and promoting the expression of anti-apoptosis factors. These effects were associated with activation of the PI3K/Akt/FoxO3a pathway.

Topics: Acute Lung Injury; Alveolar Epithelial Cells; Animals; Apoptosis; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; Cells, Cultured; Cyclin D1; Female; Forkhead Box Protein O3; Gene Expression; Genes, bcl-2; Hydrogen; Hyperoxia; Oxidative Stress; Phosphatidylinositol 3-Kinases; Pregnancy; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; RNA, Messenger; Signal Transduction

Hyperoxia can lead to a myriad of deleterious effects in the lung including epithelial damage and diffuse inflammation. The specific mechanisms by which hyperoxia promotes these pathological changes are not completely understood. Activation of ion channels has been proposed as one of the mechanisms required for cell activation and mediator secretion. The two-pore-domain K(+) channel (K2P) Trek-1 has recently been described in lung epithelial cells, but its function remains elusive. In this study we hypothesized that hyperoxia affects expression of Trek-1 in alveolar epithelial cells and that Trek-1 is involved in regulation of cell proliferation and cytokine secretion. We found gene expression of several K2P channels in mouse alveolar epithelial cells (MLE-12), and expression of Trek-1 was significantly downregulated in cultured cells and lungs of mice exposed to hyperoxia. Similarly, proliferation cell nuclear antigen (PCNA) and Cyclin D1 expression were downregulated by exposure to hyperoxia. We developed an MLE-12 cell line deficient in Trek-1 expression using shRNA and found that Trek-1 deficiency resulted in increased cell proliferation and upregulation of PCNA but not Cyclin D1. Furthermore, IL-6 and regulated on activation normal T-expressed and presumably secreted (RANTES) secretion was decreased in Trek-1-deficient cells, whereas release of monocyte chemoattractant protein-1 was increased. Release of KC/IL-8 was not affected by Trek-1 deficiency. Overall, deficiency of Trek-1 had a more pronounced effect on mediator secretion than exposure to hyperoxia. This is the first report suggesting that the K(+) channel Trek-1 could be involved in regulation of alveolar epithelial cell proliferation and cytokine secretion, but a direct association with hyperoxia-induced changes in Trek-1 levels remains elusive.

Topics: Alveolar Epithelial Cells; Animals; Cell Line; Cell Proliferation; Chemokine CCL2; Chemokine CCL5; Cyclin D1; Hyperoxia; Inflammation Mediators; Interleukin-6; Interleukin-8; Male; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Potassium Channels, Tandem Pore Domain; Proliferating Cell Nuclear Antigen; Pulmonary Alveoli