Kinetics of nuclear factor κB in LPS stimulated rat alveolar macrophages and its regulative effect on tumor necrosis factor α expression

Objective To investigate the activation of nuclear factor κB (NF-κB) induced by lipopolysaccharides (LPS) in rat alveolar macrophages (AM) and its regulative role in tumor necrosis factor (TNF-α) expression. Methods The dynamic activity changes of NF-κB DNA induced by LPS (E.coli 026:B6) were determined with electrophoretic mobility shift assay (EMSA). The phosphorothioate oligodeoxynucleotides (S-ODN) decoy was transfected into AM 12 hours prior to LPS stimulation. The effect of NF-κB S-ODN decoy on expression of TNF-α in AM stimulated by LPS were measured with enzyme-linked immunosorbent assay (ELISA) kit. Results NF-κB could be activated remarkably after 0.5 hour of LPS stimulation at concentration of 100 ng/ml, reached the highest level 1 hour after LPS stimulation and gradually decreased. But the activation of NF-κB could last at least 8 hours. The dose for LPS stimulation was related to activation of NF-κB in a dose-dependent fashion, ie, the activation of NF-κB gradually strengthened with dose increase of LPS. Supershift assays proved that p50 and p65 were involved in the activation of NF-κB. NF-κB S-ODN decoy could markedly (not completely) inhibit LPS-induced TNF-α production. Conclusions NF-κB plays an important role in LPS induced inflammatory response. However, entire inhibition of the activity of NF-κB can not completely prevent TNF-α expression induced by LPS in rat AM, which implies that other nuclear factors may participate in TNF-α expression.