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 mg/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-a, IL-1b, 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-a, IL-1b, 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. 2013 Surgical Associates Ltd. Published by Elsevier Ltd. All rights reserved.

Study details

Article type

Original Research

Disease / injury model

LPS-stimulated inflammation (in vitro macrophage model)

Dose comparison

The study compared three concentrations of hydrogen (0.15, 0.30, 0.60 mM) in the culture medium to assess dose-dependent effects on cytokine release and HO-1 activity/expression.

Dose-dependent effect

Yes

Outcome

Positive

Findings

Molecular hydrogen dose-dependently attenuates pro-inflammatory cytokines (TNF-a, IL-1b, HMGB1) and increases anti-inflammatory cytokine (IL-10) in LPS-stimulated RAW 264.7 macrophages, an effect mediated at least in part by HO-1 expression and activity.