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

Objectives: Osteoporosis is a prevalent condition among postmenopausal women, and lacks satisfactory therapeutic options. Hydrogen (H-2) has been shown to be effective in alleviating many diseases. This study aimed to investigate the effects of H-2 on inhibiting osteoclastogenesis and bone loss in ovariectomized mice. Methods: Osteoclast differentiation from Raw264.7 cells was induced with receptor activator NF-kappa B ligand (RANKL) with or without 60% H-2. 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-kappa B phosphorylation were detected by western blot. NF-kappa B nuclear translocation was assessed by immunofluorescence. NF-kappa B 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% H-2. 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. Results: H-2 did not have any measurable effect on the proliferation of Raw264.7 cells. The number of osteoclasts and size of resorption pits of RANKL+H-2-treated cells were 3 to 4 times less than RANKL treated cells. The expression of osteoclast marker genes of RANKL+H-2-treated cells was 30% to 60% lower than RANKL-treated cells (P < 0.05). H-2 markedly inhibited RANKL-induced activation, nuclear translocation, and transcriptional activity of NF-kappa B (P < 0.05, RANKL+H-2 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 H-2 were more than 40% higher, whereas the levels of serum proinflammatory cytokine interleukin 1 beta, IL-6, and tumor necrosis factor-alpha were more than 50% lower than those of OVX mice (P < 0.05). Conclusions: These results demonstrated that H-2 could be an effective therapeutic agent of postmenopausal osteoporosis.