Lycopus lucidus Turcz Inhibits the Osteoclastogenesis in RAW 264.7 Cells and Bone Loss in Ovariectomized Rat Model

Figure 2

wLL inhibits RANKL-induced osteoclast differentiation and bone resorption in RAW 264.7 cells. RAW 264.7 cells were seeded at 5 × 10³/well on a 96-well plate (for osteoclast differentiation) or osteo assay surface plate (for bone resorption). After 24 hours, RAW 264.7 cells were incubated with RANKL (100ng/ml) for 4 days with or without the presence of LL (1, 10, and 100 μg/mL). For osteoclast staining, cells were fixed with 4% formalin and stained using TRAP kit. To measure the bone resorption activity, the RAW 264.7 cells on osteo assay plate were removed with 4% sodium hypochlorite. Pit area was subjected to visualization with inverted microscope. ((a) left) TRAP-positive cells detected by TRAP staining, and ((a) right) pit area on the osteo assay plate observed under the inverted microscope (original magnification ×100). (b) TRAP-positive cells with more than three nuclei, and (c) TRAP activity of media measured by an ELISA reader. (d) Pit areas measured using Image J software. Data represent the mean ± SEM of the three independent experiments.