Objective: This study aimed to determine the combined effect of SBM and CAPE on the initial differentiation of osteoclasts.
Methods: Pre-cursor osteoclast cells (RAW 264.7) were allowed to proliferate, and frozen until needed. RANKL was used to obtain initial differentiation. The optimal dose for RANKL and the best treatment time were established. The effects of different doses of CAPE or SBM were tested. Subsequently, RAW 264.7 cells seeded in phenol red-free c-DMEM supplemented with 10% charcoal/dextran treated FBS for 24h, were treated with CAPE (1 and 2.5μM) or SBM (50 and 150 μg/ml) with 150ng/ml RANKL for 24h and incubated at 37°C under 5% CO2 for 30min. Gene expression of differentiation biomarkers (c-Fun, c-Jos and NF-κB) were obtained using qRT-PCR. The Mouse Osteogenesis RT² Profiler PCR Array was used to analyze the lowest dose for SBM, CAPE and (SBM+CAPE).
Results: CAPE significantly up-regulated while SBM down-regulated the expression of all three genes (c-Fos, c-Jun, and NF-KB). The effects of CAPE+SBM combinations mostly followed the trend of CAPE alone. The gene array scatter plots showed that the effect of the SBM+CAPE combination was different from the individual effects.
Conclusions: SBM down-regulated while CAPE and (CAPE+SBM) up-regulated the expression of all three genes. However, since it is known that protein levels might not always follow gene expression, our results on the SBM+CAPE effect need confirmation at the protein level. [Supported by NIAMS/NIH grant AR056208].