Effect of arctigenin on osteoclast-like cell formation in the co-culture with osteoblastic cells.

(A) Effects of arctigenin and CsA on osteoclast-like cell formation in the co-culture. Osteoblastic cells and bone marrow cells were co-cultured in the presence of 1α,25(OH)₂D₃ together with or without 1 µM arctigenin or 1 µg/mL CsA. After cultivation for 6 days, cells were fixed and stained for TRAP. TRAP-positive multinucleated cells containing more than three nuclei were counted as osteoclast-like cells. Bar = 50 µm. (B) Effects of arctigenin on RANKL and OPG expression in osteoblastic cells. Osteoblastic cells were cultured for 24 h in the presence or absence of 10 nM 1α,25(OH)₂D₃ together with or without 1 µM arctigenin. The expression of Rankl and Opg mRNAs was analyzed by quantitative RT-PCR. The expression level was normalized to Gapdh and expressed relative to the vehicle control. (C) Alkaline phosphatase staining of osteoblastic cell cultures. Osteoblastic cells were cultured for 6 days in the presence or absence of 1 µM arctigenin. Cells were then fixed and stained for alkaline phosphatase. Alkaline phosphatase-positive cells appeared as blue cells. Bar = 50 µm. (D) Effects of arctigenin on the proliferation of osteoblastic cells. Osteoblastic cells were cultured with increasing concentrations of arctigenin. Cell proliferation was evaluated on days 0, 1, and 2 using an Alamar Blue assay. The results were expressed as means +/− SD (n = 3). p<0.05.