Helicobacter pylori promotes inflammatory factor secretion and lung injury through VacA exotoxin-mediated activation of NF-κB signaling

Abstract

Previous reports have shown that Helicobacter pylori (H. pylori) infection is associated with respiratory diseases. However, the pathogenesis remains unclear. Vacuolating cytotoxin A (VacA) is a major H. pylori exotoxin. In this study, we investigated the signaling pathways involved in the inflammatory response to H. pylori infection and VacA. Mice were treated with H. pylori and VacA, and histopathological analysis of lung tissues was performed using hematoxylin-eosin, Masson's trichrome, and periodic acid Schiff staining. The secretion of inflammatory cytokines was evaluated by enzyme-linked immunosorbent assay. The expression of VacA, nuclear factor-kappa B (NF-κB), and p65 NF-κB was analyzed by Western blotting and immunofluorescence. Cell proliferation and apoptosis were assessed using the MTS assay and flow cytometry, respectively. In mice, H. pylori infection and VacA treatment promoted the secretion of the inflammatory factors interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), IL-6, and IL-8, increased p65 NF-κB protein phosphorylation, and induced lung injury. Furthermore, H. pylori infection promoted VacA production. In an in vitro cell model, VacA treatment significantly suppressed the proliferation of WI-38 and BEAS-2B cells, promoted apoptosis, induced TNF-α, IL-1β, IL-6, and IL-8 secretion, and promoted p65 NF-κB protein phosphorylation and NF-κB nuclear transfer. The NF-κB inhibitor BAY11-7082 alleviated VacA-induced inflammation and apoptosis and increased cell viability. In conclusion, VacA promotes the secretion of inflammatory factors and induces lung injury through NF-κB signaling.

Keywords: Helicobacter pylori; NF-κB signaling; VacA; endothelial dysfunction; inflammatory cytokines.

Graphical abstract

Figure 1.

Effect of H. pylori infection and VacA treatment on mouse lung tissue, examined using hematoxylin-eosin (H&E), Masson’s trichrome, and periodic acid Schiff stains. (40 ×).

Figure 2.

Effect of H. pylori infection and VacA treatment on the expression of inflammatory factors and NF-κB signaling in mice. (a) Levels of TNF-α, IL-1β, IL-6, and IL-8 in mouse blood samples after H. pylori infection and VacA treatment were measured by ELISA. (b) VacA, p-p65 NF-κB, and p65 NF-κB protein expression in mouse lung tissue after H. pylori infection and VacA treatment was analyzed by Western blotting. (c) p-p65 NF-κB protein expression in mouse lung tissue after H. pylori infection and VacA treatment was measured by immunofluorescence assay. Red arrows indicate the translocation of p65 NF-κB to the nucleus. (*P < 0.05).

Figure 3.

Effect of VacA treatment on WI-38 and BEAS-2B cell viability and apoptosis. (a and c) Cell viability after VacA treatment was assessed by MTS assay. (b and d) Cell apoptosis after VacA treatment was assessed by flow cytometry. (vs normal, *P < 0.05, vs NC, ^#P < 0.05).

Figure 4.

Effect of VacA treatment on the expression of inflammatory factors and NF-κB signaling in WI-38 and BEAS-2B cells. (a) Levels of TNF-α, IL-1β, IL-6, and IL-8 in cell supernatants after VacA treatment were measured by ELISA. (b) TNF-α, IL-1β, IL-6, and IL-8 mRNA expression in WI-38 and BEAS-2B cells after VacA treatment were measured by RT-qPCR. (c) p-p65, NF-κB, and p65 NF-κB protein expression in WI-38 and BEAS-2B cells after VacA treatment was measured by Western blotting. (d) NF- κB fluorescence intensity after VacA treatment was determined by luciferase activity assay. (e) The expression of p65 NF- κB fluorescence in nucleus and cytoplasm was analyzed by immunofluorescence. Red arrows indicate the translocation of p65 NF-κB to the nucleus. (f) The expression of p65 NF- κB fluorescence in nucleus and cytoplasm was analyzed by Western blotting. (vs normal, *P < 0.05, vs NC, ^#P < 0.05).

Figure 5.

Alleviation of VacA-induced inflammation in WI-38 and BEAS-2B cells by the NF-κB inhibitor BAY11-7082. (a) Protein expression of p-p65, NF-κB, and p65 NF-κB after co-treatment with VacA and NF-κB inhibitor was measured by Western blotting. (b) The expression of p65 NF- κB fluorescence in nucleus and cytoplasm was analyzed by immunofluorescence after after co-treatment with VacA and NF-κB inhibitor. Red arrows indicate the translocation of p65 NF-κB to the nucleus. (c) The expression of p65 NF- κB fluorescence in nucleus and cytoplasm after after co-treatment with VacA and NF-κB inhibitor was analyzed by Western blotting. (d) Levels of TNF-α, IL-1β, IL-6, and IL-8 in cell supernatants after co-treatment with VacA and NF-κB inhibitor were measured by ELISA. (e) TNF-α, IL-1β, IL-6, and IL-8 mRNA expression in cell after after co-treatment with VacA and NF-κB inhibitor were measured by RT-qPCR. (vs VacA group, *P < 0.05, vs VacA + PBS group, ^#P < 0.05).

Figure 6.

Alleviation of VacA-induced apoptosis and suppression of viability by the NF-κB inhibitor BAY11-7082. (a and c) Viability of WI-38 and BEAS-2B cells after VacA treatment was assessed by MTS assay. (b and d) Apoptosis of WI-38 and BEAS-2B cells after VacA treatment was assessed by flow cytometry. (vs VacA group, *P < 0.05, vs VacA + PBS group, ^#P < 0.05).