The inhibitory effect of melatonin on osteoclastogenesis of RAW 264.7 cells in low concentrations of RANKL and MCSF

Abstract

RAW 264.7 cells are one of the most recommended cell lines for investigating the activity and differentiation of osteoclasts.These cells differentiate into osteoclasts in the presence of two critical components: receptor activator of nuclear factor kappa B ligand(RANKL) and macrophage colony stimulating factor (MCSF). Melatonin (MEL) hormone has recently become one of the smallmolecules used in the field of bone regeneration and bone disease treatment, as it has the ability to inhibit the differentiation of osteoclastsdirectly by suppression of the NF-κB signaling pathway. The main aim of the current study is to determine sufficient RANKL/MCSFconcentrations for differentiation of the cells to osteoclasts and to describe the repressive effect of MEL on the osteoclastogenesis of thesecells. In this regard, it was found that 10 ng/mL of RANKL- and MCSF-containing medium is suitable for inducing osteoclastogenesis ofthe cells. In addition, melatonin at doses in the range of 100–1000 µM does not have a cytotoxic effect. Subsequently, results of tartrateresistant acid phosphatase (TRAP) activity, TRAP staining, and relative expressions of cathepsin K, nuclear factor of activated T cellsone (NFATC1), and TRAP genes showed a suppressive effect of MEL —especially 800 µM— on RANKL-induced osteoclastogenesis ofthese cells.