Objective: To observe the influence of ascorbic acid (AA) on osteoclastogenesis induced by receptor activated nuclear factor kappaB ligand (RANKL) in RAW264.7 cells and to study the mechanism of AA in osteoclast differentiation.
Methods: Mouse mononuclear cells of the line RAW264.7 were cultured. AA and/or RANKL were added. Cellular respiration of RAW264.7 cells was assayed by MTT cell proliferation method. RAW264.7 cells were inoculated and RANKL and AA of different concentrations were added. TRAP staining was used to observe the formation of osteoclasts. RAW264.7 cells were inoculated on OAAS, RANKL and AA were added. The number of bone absorption pits was counted. RT-PCR was used to measure the expression of RANK, matrix metalloproteinase 9, TRAF6, TRAP, integrin alpha v, integrin beta 3, cathepsin K, and carbonic anhydrase II (CA II).
Results: AA of the concentration of 500 microg/ml significantly inhibited the proliferation of RAW264.7 cells (0.13 +/- 0.05 vs 1.11 +/- 0.22, P < 0.05). RANKL significantly inhibited the proliferation of RAW264.7 cells as well (1.58 +/- 0.22 vs 1.26 +/- 0.17). AA showed no synergistic action to RANKL. AA alone could not induce the formation of TRAP-positive osteoclasts from RAW264.7 cells and 0 approximately 100 microg/ml AA inhibited the RANKL-induced formation of osteoclast-like multinuclear cells from RAW264.7 cells. AA alone up-regulated the mRNA expression of CA II, cathepsin K, and TRAP by 10, 1.5, and 1.5 times respectively. RANKL plus AA significantly down-regulated the mRNA expression of CAII and RANK by 60% and 20% respectively. No bone absorption pit was seen after addition of AA. The number of pits on OAAS after addition of AA + RANKL was significantly lower than that after addition of RANKL alone (P < 0.05). Western blotting showed that no difference was seen in the protein expression of CA I between the RANKL group and the control group, however, the protein expression of CA II in the low concentration AA + RANKL group was significantly lower than that in the RANKL alone group (P < 0.05).
Conclusion: AA directly inhibits RANKL induced osteoclast formation and its function in vitro.