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

Figure 1 Molecular docking analysis of osthole with target proteins. A: Visualised molecular docking models of osthole with target proteins at the minimum binding energy. The colour blue is used to represent the overall structure of target proteins, cyan to indicate osthole molecules, green to mark the protein structural regions interacting with osthole, and yellow dashed lines to denote the formed hydrogen bonds; B: Minimum binding energy values between osthole and each target protein (estrogen receptor α, osteoprotegerin, receptor activator of nuclear factor-κB ligand, extracellular signal-regulated kinase 1, extracellular signal-regulated kinase 2, β-catenin, protein kinase B 1, 2, and 3). The binding energy, which ranges from -5.0 kcal/mol to -7.0 kcal/mol indicates high binding affinity between osthole and the target proteins. OPG: Osteoprotegerin; ESR: Estrogen receptor; RANKL: Receptor activator of nuclear factor-κB ligand ; AKT: Protein kinase B; ERK: Extracellular signal-regulated kinase.

Figure 2 Osthole modulates osteoblastic proliferation, differentiation, mineralization, and apoptosis. ^aP < 0.05 vs control (one-way analysis of variance followed by Dunnett’s post-hoc test); ^bP < 0.05 vs cisplatin alone (one-way analysis of variance with Tukey’s post-hoc test). A: MG-63 cells were treated with different concentrations of osthole (0.01-10000 nM) or vehicle dimethyl sulfoxide in medium containing 1% fetal bovine serum for 2 days, 4 days, and 6 days, and cell proliferation was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Data are mean ± SD (n = 5 independent experiments); B: Alkaline phosphatase(ALP) activity of MG-63 cells treated with the above concentrations of osthole was measured by ALP activity assay. Cell lysates were collected after treatment, and ALP activity was normalized to the absorbance value of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide at 490 nm. Data are mean ± SD (n = 5); C: After rat calvarial osteoblasts were cultured with different concentrations (0.01-10000 nM) of osthole for 18 days, the number of mineralized bone nodules was quantified by Alizarin Red S staining (original magnification × 100). The absorbance of the released Alizarin Red S stain was measured at 570 nm. Data are mean ± SD (n = 3); D: The mean percentage of apoptotic MG-63 cells was measured by flow cytometry. Data are mean ± SD (n = 3). ALP: Alkaline phosphatase; ROBs: Rat calvarial osteoblasts; FL1-H: FL1-height; FITC: Fluorescein isothiocyanate.

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.

Figure 4 Estrogen receptor, mitogen-activated protein kinase kinase/extracellular signal-regulated kinase, phosphoinositide 3-kinase/protein kinase B, and Wnt/β-catenin pathways mediate osthole-induced modulation of MG-63 proliferation, alkaline phosphatase activity, and osteoprotegerin/receptor activator of nuclear factor-κB ligand/Osterix expression. ^aP < 0.05 vs control; ^bP < 0.05 vs osthole alone (one-way analysis of variance with Tukey’s post-hoc test). MG-63 cells were pretreated with 100 μM estrogen receptor inhibitor fulvestrant (ICI) 182780, 50 μM PD98059, 200 nM wortmannin, 200 ng/mL dickkopf-1 (DKK-1), or vehicle dimethyl sulfoxide in medium containing 1% fetal bovine serum for 30 minutes, followed by treatment with 1 nM osthole for 2 days. Data are mean ± SD from three independent experiments (n = 3). A: Cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay; B: Alkaline phosphatase activity was detected using an alkaline phosphatase assay kit. Experiments were independently repeated three times, and data are presented as mean ± SD; C: MG-63 cells were divided into six treatment groups: Control, DKK-1 (200 ng/mL, Wnt/β-catenin pathway inhibitor), ICI 182780 (100 μM, estrogen receptor inhibitor), osthole (1 nM), osthole + DKK-1, and osthole + ICI 182780. After respective treatments, relative mRNA expression levels of osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand (RANKL), and Osterix were analyzed by reverse transcription-quantitative polymerase chain reaction; D: Relative protein expression levels of OPG, RANKL, and Osterix in MG-63 cells subjected to the treatment conditions described in panel C, detected by western blot. β-actin served as the loading control; E: MG-63 cells were assigned to six treatment groups: Control, PD98059 (50 μM, mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway inhibitor), wortmannin (200 nM, phosphoinositide 3-kinase/protein kinase B pathway inhibitor), osthole (1 nM), osthole + PD98059, and osthole + wortmannin. Following treatment, relative mRNA expression levels of OPG, RANKL, and Osterix were measured by reverse transcription-quantitative polymerase chain reaction; F: Relative protein expression levels of OPG, RANKL, and Osterix in MG-63 cells treated as specified in panel E, determined by western blot analysis with β-actin as the loading control. OPG: Osteoprotegerin; RANKL: Receptor activator of nuclear factor-κB ligand; ICI: Fulvestrant; DKK: Dickkopf; PD: 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one.

Figure 5 Effects of osthole at different concentrations on MC3T3-E1 cell viability, and impacts of conditioned medium from osthole-treated MC3T3-E1 cells on RAW264. 7 cell functions and osteoclast-related indicators.^aP < 0.05 vs control; ^bP < 0.05 vs osthole alone; ^cP < 0.05 vs blank conditioned medium (CM) (one-way analysis of variance with Tukey‘s post-hoc test). Data are mean ± SD from three independent experiments (n = 3). A: Cell viability of MC3T3-E1 cells treated with various concentrations of osthole (Ost) (0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 25 μM, 50 μM), assessed via a cell viability assay; results are presented as a percentage relative to the control group; B: Cell viability of RAW264.7 cells treated with various concentrations of Ost (0.01 nM to 50 μM), determined using a cell viability assay; results are presented as a percentage relative to the control group; C: Cell viability of RAW264.7 cells cultured in control medium, blank CM from untreated MC3T3-E1 cells, CM from MC3T3-E1 cells treated with 1 nM Ost [CM (1 nM Ost)], or CM from MC3T3-E1 cells treated with 10 nM Ost [CM (10 nM Ost)], measured via a cell viability assay; results are presented as a percentage relative to the control group; D: RANKL protein expression in MC3T3-E1 cells treated with 1 nM or 10 nM Ost, quantified by the corresponding detection method; data are expressed in pg/mL; E: Relative mRNA expression levels of osteoclast-specific genes (TRAP, CTR, RANK, MMP-9, CatK) in RAW264.7 cells cultured in the aforementioned CMs, analyzed by reverse transcription-quantitative polymerase chain reaction; expression levels were normalized to an appropriate housekeeping gene; F: Tartrate-resistant acid phosphatase (TRAP) activity of RAW264.7 cells cultured in different CMs, measured using a TRAP activity assay; results are presented as a percentage relative to the control group; G: Osteoclast viability per field of RAW264.7 cells cultured in different CMs, counted under a light microscope following TRAP staining; H: TRAP staining of RAW264.7 cells subjected to different treatments [control, CM, CM (1n M Ost), CM (10 nM Ost)]; blue arrows indicate TRAP-positive cells. Original magnification: × 100; scale bar = 100 μm; I and J: MC3T3-E1 cells were pretreated with 100 μM estrogen receptor inhibitor fulvestrant 182780, 50 μM PD98059, 200 nM wortmannin, 200 ng/mL dickkopf-1, or vehicle dimethyl sulfoxide in medium containing 1% fetal bovine serum for 30 minutes, followed by treatment with 1 nM Ost for 2 days. Cell proliferation was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Ost: Osthole; CM: Conditioned medium; RANKL: Receptor activator of nuclear factor-κB ligand; ICI: Fulvestrant; DKK: Dickkopf.

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.

Figure 7 Schematic diagram of the osteoprotective mechanism of osthole. Created with BioGDP.com. OPG: Osteoprotegerin; RANKL: Receptor activator of nuclear factor-κB ligand; ER: Estrogen receptor; ERK: Extracellular signal-regulated kinase; AKT: Protein kinase B.