Treatment with hydrogen molecules prevents RANKL-induced osteoclast differentiation associated with inhibition of ROS formation and inactivation of MAPK, AKT and NF-kappa B pathways in murine RAW264.7 cells

The bone protective effects of the hydrogen molecule (H₂) have been demonstrated in several osteoporosis models while the underlying molecular mechanism has remained unclear. Osteoclast differentiation is an important factor related to the pathogenesis of bone-loss related diseases. In this work, we evaluated the effects of incubation with H₂ on receptor activator of NFκB ligand (RANKL)-induced osteoclast differentiation. We found that treatment with H₂ prevented RANKL-induced osteoclast differentiation in RAW264.7 cells and BMMs. Treatment with H₂ inhibits the ability to form resorption pits of BMMs stimulated by RANKL. Treatment with H₂ reduced mRNA levels of osteoclast-specific markers including tartrate resistant acid phosphatase, calcitonin receptor, cathepsin K, metalloproteinase-9, carbonic anhydrase typeII, and vacuolar-type H⁺-ATPase. Treatment with H₂ decreased intracellular reactive oxygen species (ROS) formation, suppressed NADPH oxidase activity, down-regulated Rac1 activity and Nox1 expression, reduced mitochondrial ROS formation, and enhanced nuclear factor E2-related factor 2 nuclear translocation and heme oxygenase-1 activity. In addition, treatment with H₂ suppressed RANKL-induced expression of nuclear factor of activated T cells c1 and c-Fos. Furthermore, treatment with H₂ suppressed NF-κB activation and reduced phosphorylation of p38, extracellular signal-regulated kinase, c-Jun-N-terminal kinase, and protein kinases B (AKT) stimulated with RANKL. In conclusion, hydrogen molecules prevented RANKL-induced osteoclast differentiation associated with inhibition of reactive oxygen species formation and inactivation of NF-κB, mitogen-activated protein kinase and AKT pathways.