Fig 2. Effects of sudachitin on osteoclastogenesis in a co-culture of isolated osteoblasts and osteoclast precursors and the mRNA expression levels of RANKL and OPG in isolated osteoblasts.

Isolated osteoblasts and osteoclast precursors were co-cultured in the presence of IL-1β (10 ng/ml) and PGE₂ (10 μM) with various concentrations of sudachitin (Sud.) for 5 days. Then, the cells were stained for the detection of the TRAP activity. The photographs represent the TRAP-stained co-cultures (A). The TRAP-positive MNCs in the co-cultures were counted (B). The presented values represent the mean ± SD (n = 4). *P < 0.05 vs. co-culture in the absence of IL-1β and PGE₂. **P < 0.05 vs. co-culture with IL-1β and PGE₂ in the absence of sudachitin. In addition, the isolated osteoblasts were treated with various concentrations of sudachitin in the absence or presence of IL-1β (10 ng/ml) and PGE₂ (10 μM) for 6 h. Then, the total RNA was extracted; the mRNA levels of RANKL (rankl, C) and OPG (opg, D) were measured by quantitative real-time RT-PCR, and the ratio of rankl mRNA/opg mRNA was calculated (E). The presented values represent the mean ± SD (n = 3). In C and E, *P < 0.05 vs. culture with IL-1β and PGE₂ in the absence of sudachitin; **P < 0.05 vs. culture with IL-1β and PGE₂ alone. In D, **P < 0.05 vs. culture without IL-1β, PGE₂ and sudachitin. NS indicates that the difference is not significant.