Hydrogen gas protects against ovariectomy-induced osteoporosis by inhibiting NF-κB activation.

Osteoporosis is a prevalent condition among postmenopausal women, and lacks satisfactory therapeutic options. Hydrogen (H2) has been shown to be effective in alleviating many diseases. This study aimed to investigate the effects of H2 on inhibiting osteoclastogenesis and bone loss in ovariectomized mice. Osteoclast differentiation from Raw264.7 cells was induced with receptor activator NF-κB ligand (RANKL) with or without 60% H2. The number and resorption activity of osteocalsts were assessed by tartrate-resistant acid phosphatase staining and pit formation assay, respectively. The expression of osteoclast markers and NF-κB phosphorylation were detected by western blot. NF-κB nuclear translocation was assessed by immunofluorescence. NF-κB transcriptional activity was analyzed by luciferase assay. Bone loss in mice was induced by ovariectomy (OVX). OVX mice were given either regular air or 60% H2. Bone structure was analyzed by micro-computed tomography and hematoxylin and eosin staining. Cytokine levels were measured by enzyme-linked immunosorbent assay. The data were analyzed with one-way or two-way ANOVA followed by Bonferroni post hoc tests. H2 did not have any measurable effect on the proliferation of Raw264.7 cells. The number of osteoclasts and size of resorption pits of RANKL+H2-treated cells were 3 to 4 times less than RANKL treated cells. The expression of osteoclast marker genes of RANKL+H2-treated cells was 30% to 60% lower than RANKL-treated cells (P < 0.05). H2 markedly inhibited RANKL-induced activation, nuclear translocation, and transcriptional activity of NF-κB (P < 0.05, RANKL+H2 vs RANKL). The amount and density of trabecular bone and bone mineral density of ovariectomized mice were significantly less than sham-operated mice (P < 0.05 OVX vs sham). The amount of trabecular bone and bone mineral density of OVX mice that inhaled H2 were more than 40% higher, whereas the levels of serum proinflammatory cytokine interleukin 1β, IL-6, and tumor necrosis factor-α were more than 50% lower than those of OVX mice (P < 0.05). These results demonstrated that H2 could be an effective therapeutic agent of postmenopausal osteoporosis.