Osthole exerts osteoprotective effect by activating ER, PI3K/AKT, MEK/ERK, and Wnt/β-catenin signaling pathways

Figure 6 Effects of conditioned medium from osthole-treated MC3T3-E1 cells (with or without signaling pathway inhibitor pretreatment) on RAW264. 7 cell functions and osteoclastogenic markers.^aP < 0.05 vs control; ^bP < 0.05 vs osthole alone; ^cP < 0.05 vs blank conditioned medium (CM); ^dP < 0.05 vs CM (1 nM Ost) (one-way analysis of variance with Tukey’s post-hoc test). Data are mean ± SD from three independent experiments (n = 3). A: Viability of RAW264.7 cells cultured in CMs derived from MC3T3-E1 cells under different treatments (including control, CM alone, CM + dickkopf (200 ng/mL), CM + fulvestrant (100 μM), CM + 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (50 μM), CM + wortmannin (200 nM), CM + Ost, and CM + Ost combined with each inhibitor), assessed using a cell viability assay; B: Cytotoxicity of RAW264.7 cells cultured in the aforementioned CMs, determined via a cellular cytotoxicity assay; C and D: Receptor activator of nuclear factor-κB ligand secretion in MC3T3-E1 cells subjected to treatments consistent with CM generation conditions, quantified by the corresponding detection method; E and F: Relative mRNA expression levels of osteoclast-specific genes (TRAP, CTR, RANK, MMP-9, CatK) in RAW264.7 cells cultured in the specified CMs, analyzed by reverse transcription-quantitative polymerase chain reaction; expression was normalized to an appropriate housekeeping gene; G: Tartrate-resistant acid phosphatase (TRAP) staining of RAW264.7 cells cultured under different CM conditions (original magnification, × 100). Experimental groups include: Control, CM from MC3T3-E1 cells treated with 1 nM Ost [CM (1 nM Ost)], CM alone, CMs from MC3T3-E1 cells treated with individual inhibitors [dickkopf 200 ng/mL, fulvestrant 100 μM, 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one 50 μM, wortmannin 200 nM], and CMs from MC3T3-E1 cells treated with Ost combined with each respective inhibitor, blue arrows indicate TRAP-positive cells; H and I: Osteoclast viability (per field) of RAW264.7 cells cultured in the indicated CMs, counted under light microscopy following TRAP staining; J and K: TRAP activity of RAW264.7 cells cultured in the specified CMs, measured using a TRAP activity assay; activity levels were normalized relative to the control group as indicated. Ost: Osthole; CM: Conditioned medium; Wort: Wortmannin; PD: 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; ICI: Fulvestrant; DKK: Dickkopf.