Heme oxygenase-1 mediates the anti-inflammatory effect of molecular hydrogen in LPS-stimulated RAW 264.7 macrophages

Abstract

Background: Molecular hydrogen (H2) as a new medical gas has an anti-inflammatory effect. In the present study, we investigated whether heme oxygenase-1 (HO-1) contributes to the anti-inflammatory effect of H2 in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages.

Methods: RAW 264.7 macrophages were stimulated by LPS (1 μg/mL) with presence or absence of different concentrations of H2. Cell viability and injury were tested by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay and lactate dehydrogenase (LDH) release, respectively. The cell culture supernatants were collected to measure inflammatory cytokines [TNF-α, IL-1β, HMGB1 (high mobility group box-1) and IL-10] at different time points. Moreover, HO-1 protein expression and activity were tested at different time points. In addition, to further identify the role of HO-1 in this process, zinc protoporphyrin (ZnPP)-IX, an HO-1 inhibitor, was used.

Results: H2 treatment had no significant influence on cell viability and injury in normally cultured RAW 264.7 macrophages. Moreover, H₂ treatment dose-dependently attenuated the increased levels of pro-inflammatory cytokines (TNF-α, IL-1β, HMGB1), but further increased the level of anti-inflammatory cytokine IL-10 at 3 h, 6 h, 12 h and 24 h after LPS stimulation. Furthermore, H₂ treatment could also dose-dependently increase the HO-1 protein expression and activity at 3 h, 6 h, 12 h and 24 h in LPS-activated macrophages. In addition, blockade of HO-1 activity with ZnPP-IX partly reversed the anti-inflammatory effect of H₂ in LPS-stimulated macrophages.

Conclusions: Molecular hydrogen exerts a regulating role in the release of pro- and anti-inflammatory cytokines in LPS-stimulated macrophages, and this effect is at least partly mediated by HO-1 expression and activation.

Keywords: 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide; CO; DMEM; Dulbecco's modified eagle medium; ECL; EDTA; EGTA; ELISA; FBS; H(2); HMGB1; HO-1; Heme oxygenase-1; I/R; IL-10; IL-1β; IL-6; Inflammatory cytokines; LDH; LPS; MCP; MIP; MTT; Macrophage; Molecular hydrogen; NADPH; Nrf2; P/S; PMSF; PVDF; RIPA; TBST; TNF-α; Tris-buffered saline with Tween; ZnPP; carbon monoxide; enhanced chemiluminescence; enzyme-linked immunosorbent assay; ethylene glycol tetraacetic acid; ethylenediaminetetraacetic acid; fetal bovine serum; heme oxygenase-1; high mobility group box-1; interleukin-1 beta; interleukin-10; interleukin-6; ischemia–reperfusion; lactate dehydrogenase; lipopolysaccharide; macrophage inflammatory protein; molecular hydrogen; monocyte chemoattractant protein; nicotinamide adenine dinucleotide phosphate; nuclear factor erythroid 2-related factor 2; penicillin/streptomycin solutions; phenylmethanesulfonyl fluoride; polyvinylidene fluoride; radioimmunoprecipitation assay; tumor necrosis factor-alpha; zinc protoporphyrin.