Abstract
Pulmonary inflammation strongly promotes alveolar hypercoagulation and fibrinolytic inhibition. NF-κB signaling regulates the expression of molecules associated with coagulation and fibrinolytic inhibition in type-II alveolar epithelial cells (AECII) stimulated by lipopolysaccharide. However, whether TNF-α-induced alveolar hypercoagulation and fibrinolysis inhibition is also associated with the NF-κB pathway remains to be determined. The aim of the present study was to determine whether BAY11-7082, an inhibitor of the NF-κB pathway, inhibits the expressions of tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) in AECⅡ in response to TNF-α. Rat AECII were treated with BAY11-7082 for 24 h and stimulated with TNF-α for 1 h. The expression of TF and PAI-1 were determined using western blotting and reverse transcription-quantitative PCR. The concentrations of TF and PAI-1 in culture supernatant were also measured by ELISA. Moreover, levels of NF-κB p65 (p65), phosphorylated (p)-p65 (p-p65), inhibitor of NF-κB α (IκBα) and p-IκBα were also evaluated. Immunofluorescence was used to detect p65 levels in cell nuclei. TNF-α significantly promoted TF and PAI-1 expression either at the mRNA or protein level in AECII cells. Concentrations of TF and PAI-1 in supernatant also significantly increased upon TNF-α stimulation. Furthermore, TNF-α upregulated the levels of p-IκBα, p65, and p-p65 in the cytoplasm. Immunofluorescence analysis indicated that TNF-α increased p65 translocation from the cytoplasm to the nucleus. However, AECII pre-treated with BAY11-7082 expressed lower levels of TF and PAI-1 following TNF-α treatment. Levels of p-IκBα, p65 and p-p65 in the cytoplasm also decreased, and translocation of p65 from cytoplasm into the nucleus was inhibited by BAY11-7082 pretreatment. These findings suggest that BAY11-7082 improves the hypercoagulation and fibrinolytic inhibition induced by TNF-α in alveolar epithelial cells via the NF-κB signaling pathway. BAY11-7082 might represent a therapeutic option for alveolar hypercoagulation and fibrinolytic inhibition in acute respiratory distress syndrome.