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目的研究低氧条件下人肺泡上皮细胞糖皮质激素受体(GR)的两种亚型GRα和GRβ水平的表达及在地塞米松抗炎效应的变化。方法以人A549肺泡上皮细胞系为模型,细胞分别在常氧(37℃,950 mL/L空气、50 mL/L CO2)和低氧(37℃,950 mL/L N2、250 mL/L CO2)条件下培养24、48、72 h,Western blot法检测各组细胞GRα和GRβ的蛋白表达水平;脂多糖(LPS 10μg/mL)和不同浓度的地塞米松(DEX 10-8mol/L～10-6mol/L)一起加入A549细胞培养液中,分别常氧和低氧培养48 h,放射免疫法检测细胞培养上清中的IL-8水平。结果 24 h低氧组的GRα水平与24 h常氧组相比无明显差别(P>0.05),48 h和72 h低氧组的GRα水平与相应时间点的常氧组相比明显下降(P<0.05,P<0.01),24、48、72 h低氧组GRα水平呈进行性下降(P<0.05),而各相应时间点的常氧组GRα水平无明显差别(P>0.05);低氧组和常氧组各时间点GRβ的表达无明显差别。在10-7mol/L和10-6mol/L DEX浓度时,低氧培养48 h的A549细胞上清的IL-8均高于相应的常氧组的IL-8水平(P<0.05)。结论低氧可以时间依赖性的导致人肺泡上皮细胞A549 GRα表达水平下降并减弱地塞米松的抗炎效应。
Objective To investigate the expression of GRα and GRβ in two subtypes of glucocorticoid receptor (GR) in human alveolar epithelial cells under hypoxic conditions and their anti-inflammatory effects on dexamethasone. Methods Human A549 alveolar epithelial cell line was used as a model. The cells were cultured under normoxia (37 ℃, 950 mL / L air, 50 mL / L CO2) and hypoxia (37 ℃, 950 mL / L N2, 250 mL / ) For 24 h, 48 h and 72 h, Western blot was used to detect the expression of GRα and GRβ in each group. Lipopolysaccharide (LPS 10μg / mL) and different concentrations of dexamethasone (DEX 10-8mol / L ~ 10 -6mol / L) were added to A549 cell culture medium, respectively, under normal oxygen and hypoxia for 48 h, radioimmunoassay detection of cell culture supernatant IL-8 levels. Results The levels of GRα in hypoxia group at 24 h were not significantly different from those at 24 h (P> 0.05). The levels of GRα in hypoxia group at 48 h and 72 h were significantly lower than those at normoxia group (P <0.05, P <0.01). The levels of GRα in hypoxia group decreased progressively at 24, 48 and 72 h (P <0.05), while there was no significant difference in the level of GRα at each time point between normoxia group and control group (P> 0.05) There was no significant difference in GRβ expression between hypoxia group and normoxia group at each time point. At DEX concentrations of 10-7 mol / L and 10-6 mol / L, IL-8 in supernatants of A549 cells cultured in hypoxia for 48 h was higher than that of corresponding normoxic groups (P <0.05). Conclusion Hypoxia can decrease the expression of GRα in human alveolar epithelial cells and attenuate the anti-inflammatory effect of dexamethasone in a time-dependent manner.