NF-κB p65 Knock-down inhibits TF, PAI-1 and promotes activated protein C production in lipopolysaccharide-stimulated alveolar epithelial cells type II

Abstract

Purpose/aim: Activated coagulation and reduced fibrinolysis in alveolar compartment are an important characteristics in acute respiratory distress syndrome (ARDS). Alveolar epithelial cell type II (AECII) participates in regulating the intra-alveolar abnormalities of coagulation and fibrinolysis mainly through adjusting the productions of tissue factor (TF), plasminogen activator inhibitor (PAI)-1 and activated protein C (APC) in ARDS. NF-κB signal pathway may be involved in coagulation regulation in sepsis-induced ALI. The purpose of this study was to testify the hypothesis that NF-κB p65 (p65) knock-down would improve the abnormalities of coagulation and fibrinolysis mediated by lipopolysaccharide (LPS) stimulation in AECII. Materials and Methods: p65 gene knock-down in AECII was achieved by small interfering RNA (siRNA) transfection. Rat AECII (RLE-6TN) with or without p65 gene knock-down were stimulated by LPS for 24 hours. And then cytolysate was used for TF, PAI-1 expression examination, and supernatant was collected for TF, PAI-1 and PC concentrations determination. Activation of NF-κB canonical pathway was simultaneously checked by western-blotting, RT-PCR and immunofluorescence respectively. Results: TF, PAI-1 expressions in normal cells obviously increased under LPS stimulation with NF-κB canonical pathway activation represented by high levels of p65, p-p65, p-IκB with increased nuclear translocation of p-p65. Cells with NF-κB p65 knock-down, however, showed significant decreases in TF, PAI-1, p65, p-p65, p-IκB expressions following LPS stimulation with significant reduction in p-p65 nuclear translocation as compared to normal and siRNA control cells. The high concentrations of TF, PAI-1 and low level of APC in supernatant induced by LPS in normal cells were significantly reversed through p65 knock-down. Conclusions: The experimental findings demonstrate that NF-kB signaling pathway is involved in regulating the expressions of coagulation and fibrinolysis factors in LPS-stimulated AECII, which suggest that NF-kB signaling pathway may be a new target to correct intra-alveolar coagulation and fibrinolytic abnormalities in ARDS.

Keywords:

• Alveolar epithelial cell type II (AECII)
• NF- kappa B p65
• siRNA
• cell transfection
• coagulation
• fibrinolysis
• lipopolysaccharide (LPS)

Disclosure statement

The authors declare no conflict of interest.

Additional information

Funding

This study was supported by the grants from Major Research Project of Innovation Group in Education Department of Guizhou Province ([2016] 034), Science and technology supportive plan project of Guizhou Province ([2017]2876), Science and Technology Innovation Project of Overseas Students in Guizhou ([2016] No. 19).