在患有急性肺损伤的小猪的肺泡巨噬细胞中一氧化氮,表面活性剂和糖皮质激素对核因子-κB和激活蛋白-1的活性的调控作用

AIM: To investigate whether acute lung injury (ALI) in ventilated piglets with bacterial infection affects NF-κB and AP-1 expression in alveolar macrophages (AM) and whether nitric oxide (NO), surfactant (Surf), glucocorticoids (GC) affect NF-κB and AP-1 activation in AM in vivo and in vitro. METHODS: The animals were intraperitoneally injected Escherichia coli, which caused ALI. Nuclear extracts of AM were analyzed by electrophoretic mobility shift assay (EMSA) for the nuclear factor-kappa B (NF-κB) and activation protein-1 (AP-1) expression. Detection of IκB-α protein was from cytoplasmic extract by Western blotting. Immunocytochemistry staining was used for intracellular location of p65 subunits of NF-κB. RESULTS: In ex vivo experiments, strikingly higher expression of NF-κB and AP-1 by EMSA was found 6 h after bacterial injection in contrast to the Normal group. In the NO, SNO, and GC groups, markedly attenuated NF-κB and AP-1 activation was observed. The NF-κB and AP-1 activation in Surf group showed lower levels of the expression. Immunoblotting of AM cytoplasmic extract showed low expression of IκB-α protein in the Control and Surf groups. The stronger expression was observed in the NO, GC, and SNO groups. AM of the Control and Surf groups showed intense nuclear staining, with decreased nuclear staining in the NO, GC and SNO groups. In in vitro experiment, it caused a significant increase in NF-κB and AP l activity in AM 1 h after exposure to lipopolysaccharides (LPS). In AM treated by LPS+SNP and LPS+GC, all showed decrease of DNA binding activity of NF-κB and AP-1 compared to those exposed to LPS+Surf. Immunoblotting of AM cytoplasmic extract showed that LPS stimulation of AM resulted in the low expression of Iκ B-α protein, which was not observed in the presence of SNP and methylprednisolone. However, the surfactant did not show such effect. LPS+Surf-exposed AM had intense nuclear staining, whereas decreased nuclear staining in the LPS+NO and LPS+GC-treated cultures was found, confirming a decrease in NF-κB activity. CONCLUSION:Activation of NF-κB was found in AM of ventilated piglets with bacterial ALI. NO and GS could prevent NF-κB and AP-1 activation in vivo and in vitro. Surfactant has limited effects on NF-kB and AP-1 activity.