Conclusion
Melatonin at pharmacological doses directly inhibited osteoclastogenesis of BMMs by a ROS-mediated but not a SIRT1-independent pathway.
Methods
Primary BMMs were isolated from the femurs and tibias of C57BL/6 mice and were induced toward multinucleated osteoclasts, in the presence of melatonin at either physiological (0.01 to 10 nM) or pharmacological (1 to 100 μM) concentrations. Tartrate-resistant acid phosphatase (TRAP) staining was used to label multinucleated osteoclasts and the levels of osteoclast-specific genes were evaluated. To further explore the underlying mechanisms, the roles of silent information regulator type 1 (SIRT1) and reactive oxygen species (ROS) were evaluated.
Results
We found that melatonin at pharmacological concentrations, rather than at physiological concentrations, significantly inhibited osteoclast formation in a dose-dependent manner. The number of TRAP-positive cells and the gene expression of osteoclast-specific markers were significantly downregulated in melatonin-treated BMMs. The melatonin-mediated repression of osteoclast differentiation involved the inhibition of the nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signaling pathway. The treatment with SIRT1 inhibitors did not affect osteoclast differentiation but, when supplemented with exogenous hydrogen peroxide, a partial rescue of melatonin-suppressed osteoclastogenesis was observed.