| BackgroundGlucocorticoids (GC) are the most effective anti-inflammatory and immuno- suppressive drugs and are widely used in various pulmonary diseases such as asthma and chronic obstructive pulmonary disease (COPD). The inflammatory action of GC are mainly mediated through glucocorticoid receptor (GR), which have two major subtypes, GRαand GRβ, of which GRαis the predominant isoform and binds to GC. The expression and state of GRαplays a major role in the anti-imflammatoty action of GC. While much is known about the cellular and molecular mechanisms of GC actions, it is worthwhile to mention that most of studies was carried out under normoxic conditions. While various human cells are exposed to the complicated microenvironment and the changes of microenvironment may lead to the alteration of cellular response to GC. Leonard et al have reported a potentiation of GC effect by hypoxia through induction of mRNA and protein of the GR in human proximal tubular epithelial cells. Some pulmonary diseases such as asthma and COPD are often accompanied with hypoxia. Whether hypoxia could influence the anti-inflammatory action of GC by changing the expression of GR in human alveolar epithelial cells remains unclear.ObjectiveIn the present study, we used A549 cell, a well characteristic representation of human alveolar epithelial cell line as a model to investigate the morphological alteration and cell cycle distribution under hypoxic conditions; moreover we studied the expression of GRαand GRβand dexamethasone mediated inhibition of IL-8 release stimulated by LPS in A549 cells under hypoxic conditions.Method1. Cell culture and Hypoxic treatmentA549 Cells were maintained in DMEM/ ham's F-12 medium supplemented with 10% fetal bovine serum under normoxic (37°C, a humidified atmosphere with 5% CO2) or hypoxic conditions (37°C, 5% CO2 and 95% N2, humidified) for 24h, 48h and 72h respectively.2. Morphological alteration in A549 cellsA549 cells were seeded in 30mm dishes and cultured in normoxic or hypoxic conditions for 24h, 48h and 72h respectively. The morphological alterations of cells were captured by differential interference contrast microscope (DIC).3. Cell cycles analyseA549 cells were cultured in serum free medium for 24h to reach synchronization, then the cells were cultured in complete medium for 48h under normoxic or hypoxic conditions. Cell cycle distribution was estimated by propidium iodide staining in flow cytometry.4. RNA isolation and RT-PCR analysisA549 cells were cultured in normoxic or hypoxic conditions for 24h, 48h and 72h respectively. Total cell RNA was extracted and the expression of GRαand GRβwere measured by RT-PCR.5. Protein extraction and Western blot analysisA549 cells were cultured in normoxic or hypoxic conditions for 24h, 48h and 72h respectively. Total cell protein was extracted and the expression of GRαand GRβwere measured by Western Blot.6. Radioimmune assay for IL-8A549 cells were cultured in 24 -well dishes before cultured in serum free medium and then were stimulated with LPS (10μg/ml) in the absence or presence of 10-8 to 10-6M dexamethasone (DEX) under normoxic or hypoxic conditions for 48h. Supernatants were collected and the levels of IL-8 were analyzed by radioimmune ..7. Statistical methodAll experiments were performed at least four independent times. Data were expressed as the means±standard error and were analyzed for significant differences by independent Student's t test and one-way ANOVA with SPSS 10.0. Differences were considered statistically significant if P value <0.05.Result1. Hypoxia altered the morphology of A549 cellsThere were no visible morphological alterations between the cells exposed to hypoxic and normoxic conditions for 24h. But when cells were exposed to hypoxic conditions for 48h, the cells became bigger and colplanater and the edge of the cells became bleared compared to the cells in normoxic conditions and the differences became more obvious between the cells exposed to hypoxic and normoxic conditions for 72h.2. Hypoxia inhibited cell proliferation of A549 cellsFlow cytometry analysis showed that under hypoxic conditions for 48h, 88.2±0.82% of the cells were in G1 phase compared to 7.2±0.64% of the cells under normoxic conditions for 48h. The results indicated that culture of A549 cells in hypoxia resulted in inhibition of cell proliferation.3. Hypoxia caused a time-dependent down-regulation of GRαin A549 cells.RT-PCR and western blot analysis all indicated that exposure to hypoxia for 24h did not caused significant alteration of GRαin A549 cells compared to that in the control 24h normoxia But when the time of hypoxia extended to 48h and 72h, GRαdecreased significantly compared to the corresponding control normoxia groups respectively and the expression of GRαamong the cells exposed to hypoxia for 24h, 48h and 72h were also significantly different. These results revealed hypoxia caused a time-dependent down-regulation of the GRαin A549 cells.4. Hypoxia caused no significant alteration of GRβin A549 cellsRT-PCR and western blot analysis showed that the expression of GRβin A549 cells exposed to hypoxic conditions for 24h, 48h and 72h had no significant difference compared to that in the corresponding control normoxia groups and the GRβexpression among the three hypoxia groups also had no significant difference .5. Hypoxia caused an attenuation of dexamethasone mediated inhibition of LPS stimulated IL-8 releaseLPS-stimulated IL-8 production in the presence of DEX from 10-7M to 10-6M under hypoxic conditions were all higher than that under normoxic conditions . Furthmore, the concentration of DEX reached the maximum inhibition efficacy under hypoxia was 10-7M. While under normoxic conditions, increase in the concentration of DEX more than 10-7M could further inhibit IL-8 .Conclusion1. Hypoxia time-dependently caused alteration of morphology in A549 cells;2. Hypoxia induced inhibition of cell proliferation in A549 cells;3. Hypoxia down-regulated the expression of GRαboth at mRNA and protein levels in A549 cells time-dependently , while GRβexpression in A549 cells did not change significantly under hypoxia;4. Hypoxia attenuated anti-inflammatory action of dexamethasone. |
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