Carvacrol inhibits osteoclast differentiation induced by RANKL in RAW264.7 cells

Abstract

RAW264.7, a murine macrophage cell line, an osteoclast model used for the differentiation of Osteoclast by inducing RANKL. Carvacrol, a monoterpenoid phenol that possess various medicinal property, was used for the treatment of osteoclast cells in this study. We investigate the effect of carvacrol in osteoclast cells induced by RANKL in RAW264.7 cells. RAW264.7 cells, cultured along with 40ng/ml of RANKL, on day 5, the cells were treated with TRAP staining to assess the formation of osteoclast cells, the presence of multinucleated TRAP positive cells were visualised using an inverted light microscope. The osteoclast cells were treated with varying concentrations of carvacrol (0-200µm) respectively for 48 hours. By MTT assay, it was found that there was no cytotoxic effect induced by carvacrol in RAW264.7 cells, whereas in RANKL induced osteoclast cells, there was a significant change in a dose dependent manner for 48 hours. By western blot and agarose gel electrophoresis, the levels of osteoclastogenic marker genes such as TRAP and cathepsin k were assessed and the developed osteoclast cells were treated with 150µm of carvacrol and found a significant change on treatment with carvacrol when compared with control. The present study reveals the anti-osteoclastogenic effect of carvacrol on osteoclast cells induced by RANKL in RAW264.7 cells.