Carbon Monoxide Releasing Molecule 3 Inhibits Osteoclastogenic Differentiation of RAW264.7 Cells by Heme Oxygenase-1

Abstract

Background/Aims: Increased osteoclastogenic differentiation may disrupt the balance of bone resorption and formation, giving rise to bone defective disease. The study aimed to investigate the influence of carbon monoxide releasing molecule 3 on osteoclastogenic differentiation of RAW264.7 cells, and explore the possible mechanism underlying the regulatory effect. Methods: Influence of CORM-3 on the proliferation of RAW264.7 cells was determined by CCK-8 assay. RAW264.7 cells were divided into four groups: Control group; Osteoclastogenic differentiation group, in which cells were induced osteoclastogenic differentiation in medium supplemented with 100μg/L RANKL and 50μg/L M-CSF; Degassed CORM-3-osteoclastogenic differentiation group, in which cells were pretreated with 200μmol/L degassed CORM-3 for 6hrs, and then induced osteoclastogenic differentiation; CORM-3-osteoclastogenic differentiation group, in which cells were pretreated with 200μmol/L CORM-3, and then induced osteoclastogenic differentiation. The mRNA and protein expression of RANK, TRAP, MMP-9, Cts-K and HO-1 of the cells during the osteoclastogenic differentiation was checked by RT-qPCR and Western blot. The induced osteoclasts were identified by TRAP staining. The HO-1 expression of the RAW264.7 cells was silenced by lentivirus transfection, and the expression of RANK, TRAP, MMP-9 and Cts-K was examined by RT-qPCR and Western blot. Results: CORM-3 promoted the proliferation of RAW264.7 cells at the concentration of 200μmol/L. Pretreatment with CORM-3, but not degassed CORM-3, significantly decreased the mRNA and protein expression of osteoclast-specific marker TRAP, RANK, MMP-9 and Cts-K induced by RANKL and M-CSF on day 5, 7 and 9 during the osteoclastogenic differentiation (P< 0.05). After HO-1 was silenced by lentivirus transfection, the mRNA and protein expression of TRAP, RANK, MMP-9 and Cts-K in group with CORM-3 pretreatment maintained the same level as in osteoclastogenic differentiation group. Conclusion: CORM-3 inhibits osteoclastogenic differentiation of RAW264.7 cells via releasing CO. The inhibitory effect is mediated partially by HO-1 pathway. The results suggest the potential application of CORM-3 on some bone defective diseases.