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

Figure 3 The effect of osthole and PD98059/wortmannin/dickkopf-1 on the phosphorylation levels of extracellular signal-regulated kinase and protein kinase B and the nuclear translocation of β-catenin. ^aP < 0.05 vs control; ^bP < 0.05 vs osthole alone (one-way analysis of variance with Tukey’s post-hoc test). A: MG-63 cells were treated with various concentrations of osthole (0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM) or control medium for a specified period. Relative mRNA expression levels of osteoprotegerin, receptor activator of nuclear factor-κB ligand, and Osterix were detected by reverse transcription-quantitative polymerase chain reaction, with β-actin as the housekeeping gene for normalization; B: Relative protein expression levels of osteoprotegerin, receptor activator of nuclear factor-κB ligand, and Osterix in MG-63 cells treated with the aforementioned concentrations of osthole, determined by Western blot analysis. β-actin was used as the loading control to ensure equal protein loading. Data are mean ± SD (n = 3 independent experiments); C-E: Detection of phosphorylated extracellular signal-regulated kinase (ERK), ERK, phosphorylated protein kinase B (AKT), AKT and β-catenin proteins in osthole effect by western blot analysis; F and G: Osthole-induced phosphorylation of ERK and AKT in MG-63 cells was partially blocked by PD98059 or wortmannin; H: Osthole-induced nuclear translocation of β-catenin was blocked by dickkopf-1. PD98059 (50 μM), wortmannin (200 nM) or dickkopf-1 (200 ng/mL) was added for 30 minutes before osthole treatment. OPG: Osteoprotegerin; RANKL: Receptor activator of nuclear factor-κB ligand; p-ERK: Phosphorylated extracellular signal-regulated kinase; ERK: Extracellular signal-regulated kinase; PD: 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; p-AKT: Phosphorylated protein kinase B; AKT: Protein kinase B; DKK: Dickkopf-1; PCNA: Proliferating cell nuclear antigen.