Functions of transcription factors NF-κB and Nrf2 in the inhibition of LPS-stimulated cytokine release by the resin monomer HEMA.

ABSTRACT

OBJECTIVE: Resin monomers like 2-hydroxyethyl methacrylate (HEMA) interfere with effects induced by stressors such as lipopolysaccharide (LPS) released from cariogenic microorganisms. In this study, mechanisms underlying monomer-induced inhibition of the LPS-stimulated secretion of inflammatory cytokines from immunocompetent cells were investigated. METHODS: Secretion of pro-inflammatory cytokines TNF-α, IL-6 and the anti-inflammatory IL-10 from RAW264.7 mouse macrophages exposed to LPS and HEMA (0-6-8mM) was determined by ELISA. The formation of reactive oxygen (ROS) and nitrogen species (RNS) was determined by flow cytometry (FACS) after staining of cells with specific fluorescent dyes. Cell viability was analyzed by FACS, and protein expression was detected by Western blotting. RESULTS: Secretion of TNF-α, IL-6 and IL-10 from LPS-stimulated cells increased after a 24h exposure. A HEMA-induced decrease in cytokine secretion resulted from the inhibition of LPS-stimulated NF-κB activation. Nuclear translocation of NF-κB was inhibited possibly as a result of enhanced levels of hydrogen peroxide (H2O2) and nitric oxide (NO) in HEMA-exposed cells. Oxidative stress caused by HEMA-induced formation of H2O2 and LPS-stimulated peroxynitrite (ONOO) also enhanced nuclear expression of Nrf2 as the major regulator of redox homeostasis, as well as Nrf2-controlled stress protein HO-1 to inhibit NF-κB activity. HEMA inhibited the LPS-stimulated expression of NOS (nitric oxide synthase) to produce NO but counteracted the expression of Nox2, which forms superoxide anions that combine with NO to peroxynitrite. CONCLUSIONS: Resin monomers like HEMA inhibit LPS-stimulated NF-κB activation essential for cytokine release as a crucial response of immunocompetent cells of the dental pulp to invading cariogenic pathogens.