Effect of arctigenin on osteoclast-like cell function.

(A) Effects of arctigenin on the pit-forming activity of osteoclast-like cells cultured on dentin slices. Osteoclast-like cell preparations were cultured on dentin slices in the presence or absence of 1 µM arctigenin. After cultivation for 48 h, cells on the slices were fixed and stained for TRAP. TRAP-positive cells were counted as osteoclast-like cells. Cells were then removed from dentin slices, and slices were stained with Mayer's hematoxylin to visualize resorption pits. Resorption pit areas were measured using ImageJ software. The results were expressed as means +/− SD (n = 6). *, p<0.01. Bar = 50 µm. (B) Effect of arctigenin on actin ring formation by osteoclast-like cells. Osteoclast-like cell preparations were cultured on dentin slices in the presence or absence of 1 µM arctigenin. After cultivation for 24 h, cells on the slices were fixed and stained with Rhodamine-phalloidin. Bar = 50 µm. (C) A hypothetical model for the action of arctigenin on the differentiation and function of osteoclasts. Arctigenin inhibited RANKL-induced osteoclast-like cell formation in BMM cultures, in which the Ca²⁺/calcineurin-dependent NFATc1 pathway was activated. Arctigenin also inhibited osteoclast-like cell formation in co-cultures with osteoblastic cells, in which the osteoblastic cell-dependent NFATc1 pathway was activated. The pit-forming activity of osteoclast-like cells was also inhibited by arctigenin. Arctigenin induced a lower molecular weight species of NFATc1, which may act as the dominant negative inhibitor of NFATc1.