Fig. 7

EC-18 inhibited osteoclastogenesis in mouse and human cells. A The vehicle or EC-18 (125 mg/kg) was administered to mice once daily from day 21 after the first immunization. At 70 days after the first immunization with CII, sections of joint tissues were stained with antibodies against TRAP (original magnification ×40 or ×400) (n = 5/group). Representative histological features are shown. The graphs present the number of TRAP-positive osteoclasts (left) and quantitative analysis of osteoclast surface/bone surface (right) in the talus endosteal surface of joint section. B, C Bone marrow-derived monocytes/macrophages (BMMs) (1 × 10⁵ cells/500 μl) from normal C57BL/6 mice were cultured with macrophage colony-stimulating factor (M-CSF) for 3 days. Then nonadherent cells were washed out and adherent cells were cultured in the presence of M-CSF and receptor activator of nuclear factor kappa-Β ligand (RANKL) with EC-18 (10 or 100 μg/ml) or vehicle for 4 days. Cells were stained to assess tartrate-resistant acid phosphatase (TRAP) activity, and the number of multinucleated TRAP⁺ cells was determined (upper). Osteoclasts were generated in OAAS plates in the presence of EC-18 for 6 days to assess their functional activity (lower, original magnification, ×100). Resorbed areas were examined using the Tomoro analySIS TS Lite program (B). The mRNA levels of TRAP, carbonic anhydrase II, and calcitonin receptor were determined using real-time PCR (C). D Human PBMCs from healthy controls (5 × 10⁵ cells/500 μl) were incubated for 2 h and then the adherent cells were cultured with M-CSF for 3 days. Then the cells were stimulated with EC-18 (10 or 100 μg/ml) or vehicle in the presence of M-CSF and RANKL for 9 days. Cells were stained to assess TRAP activity, and the number of multinucleated TRAP⁺ cells was determined. Data are presented as the means ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle-treated control