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

Abstract

BACKGROUND

Osteoporosis is the result of an imbalance between bone resorption and bone formation. Osthole is a coumarin compound derived from medicinal plants that exerts a significant osteoprotective effect, however, its precise molecular mechanism has not yet been fully elucidated.

AIM

To investigate whether osthole activates the estrogen receptor (ER), phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT), mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK), and Wnt/β-catenin pathways in osteoblasts, thereby indirectly suppressing osteoclastogenesis via osteoblast-derived signals.

METHODS

Molecular docking was used to predict osthole-target binding. The proliferation, differentiation, and mineralisation of osteoblasts (MG-63, primary rat osteoblasts, and MC3T3-E1) were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and alkaline phosphatase activity, and Alizarin Red staining. Flow cytometry was used to measure apoptosis. Reverse transcription-quantitative polymerase chain reaction and western blot were performed to analyse the expression of osteoprotegerin, receptor activator of nuclear factor-κB ligand (RANKL), Osterix, and pathway molecules (phosphorylated ERK, phosphorylated AKT, and β-catenin). enzyme-linked immunosorbent assay was used to quantify RANKL. Conditioned medium from osthole-treated MC3T3-E1 cells was applied to RAW264.7 cells. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, tartrate-resistant acid phosphatase staining/activity, and reverse transcription-quantitative polymerase chain reaction were used to evaluate osteoclast viability, differentiation, and gene expression. Specific inhibitors (fulvestrant 182780, wortmannin, PD98059 and dickkopf-1) were used to verify pathway involvement.

RESULTS

Docking results predicted that osthole binds to the active pockets of ERα, AKT, and ERK with high binding affinity, suggesting potential interactions. Osthole promoted the proliferation, differentiation, and mineralisation of MG-63, rat osteoblast, and MC3T3-E1 cells in a concentration-dependent manner, and inhibited cisplatin-induced apoptosis of MG-63 cells. Additionally, osthole was found to upregulate osteoprotegerin and Osterix expression, downregulate RANKL expression, and activate the ER, PI3K/AKT, MEK/ERK, and Wnt/β-catenin signaling pathways in osteoblasts. Conditioned medium from osthole-treated MC3T3-E1 cells inhibited the viability, differentiation, and expression of osteoclast-specific genes (TRAP, CTR, RANK, CatK and MMP-9) in RAW264.7 cells. The osteoprotective effects of osthole were reversed by specific pathway inhibitors.

CONCLUSION

Osthole exerts osteoprotective effects by promoting the function of osteoblasts and indirectly suppressing osteoclastogenesis in an osteoblast-dependent manner. This process primarily occurs through the activation of the ER, PI3K/AKT, MEK/ERK, and Wnt/β-catenin signalling pathways in osteoblasts.

Key Words: Osthole; Bone; Estrogen receptor; Phosphoinositide 3-kinase/protein kinase B; Mitogen-activated protein kinase kinase/extracellular signal-regulated kinase; Wnt/β-catenin

Core Tip: This study elucidates that osthole, a natural coumarin compound, exerts osteoprotective effects through a novel multi-target mechanism involving simultaneous activation of estrogen receptor, phosphoinositide 3-kinase/protein kinase B, mitogen-activated protein kinase kinase/extracellular signal-regulated kinase, and Wnt/β-catenin signaling pathways in osteoblasts. By directly promoting osteoblast function and indirectly inhibiting osteoclast differentiation via osteoprotegerin/receptor activator of nuclear factor-κB ligand modulation, osthole emerges as a promising natural therapeutic candidate for osteoporosis.

INTRODUCTION

Osteoporosis is a long-term metabolic bone disease characterised by disruption of bone homeostasis and consequent bone loss. A recent epidemiological study shows that the incidence of osteoporosis is 3.3% in people aged 45-49 years, and the incidence of osteoporosis is as high as 50.3% by the age of 85 years[1]. The fundamental pathological feature of osteoporosis is the imbalance between bone formation (mediated by osteoblasts) and bone resorption (mediated by osteoclasts), resulting in bone mass loss and microarchitectural deterioration. Current clinical drugs for osteoporosis, such as bisphosphonates and receptor activator of nuclear factor-κB (RANK) ligand (RANKL) inhibitors, have limitations including adverse effects[2] (e.g., osteonecrosis of the jaw, atypical femoral fractures) and long-term safety concerns. This highlights the need for novel anti-osteoporotic agents that are both highly efficacious and low in toxicity.

Traditional Chinese medicine has emerged as a research hotspot for anti-osteoporotic drug development due to its multi-component, multi-target, and low-toxicity characteristics[3]. Osthole is a coumarin compound that is derived from several medicinal plants. It has a variety of pharmacological effects, including anti-inflammatory, anti-tumour, bone protection, and cardiovascular protection[4]. Research has demonstrated that osthole reduces the occurrence of fragility fractures in osteoporotic conditions[5]. However, it remains unclear whether this effect is mediated by promoting bone formation, inhibiting bone resorption, or both. However, the molecular mechanism of osthole in the treatment of osteoporosis remains to be elucidated, necessitating further investigation. The hormone oestrogen has been demonstrated to both promote the formation of new bone tissue and inhibit the process of bone resorption. Consequently, the loss of bone mass that occurs in postmenopausal women is principally associated with oestrogen deficiency[6]. Research has demonstrated that osthole exhibits oestrogenic activity and promotes osteoblast differentiation by activating the estrogen receptor (ER) signaling pathway[7]. The phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT), mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK), and Wnt/β-catenin signaling pathways have been demonstrated to regulate cell survival, proliferation, and differentiation[8-10]. In addition, the PI3K/AKT, MEK/ERK, and Wnt/β-catenin signaling pathways have been demonstrated to stimulate the proliferation, differentiation, and mineralization of osteoblasts, while concomitantly inhibiting the differentiation and viability of osteoclasts[11,12]. It is therefore vital to understand the role of these pathways in bone metabolism. Yan et al[13] reported that osthole inhibited damage to bone marrow-derived neural stem cells by activating the PI3K/AKT signaling pathway. Kuo et al[14] discovered that osthole promoted osteoblast differentiation by activating the ERK1/2 signaling pathway. In addition, Jin et al[15] found that osthole promoted bone formation by activating the Wnt/β-catenin signaling pathway. However, the potential of osthole to affect bone formation by regulating the PI3K/AKT signaling pathway remains to be elucidated. Of particular significance is the observation that the process of bone remodelling is characterised by a close interaction between osteoblasts and osteoclasts. Osteoblasts are responsible not only for the formation of bone tissue but also for the secretion of key regulators such as RANKL and osteoprotegerin (OPG) which are essential for the control of osteoclast differentiation. While osthole has been shown to activate several individual signaling pathways in osteoblasts, the precise mechanism by which it regulates the cross-talk between osteoblasts and osteoclasts - particularly via paracrine signals - remains to be elucidated[16,17].

The MG-63 cell is an immature human osteoblast and is widely used in the study of bone metabolism. The advantages of this approach include the absence of interspecific differences, unlimited proliferation, and a hormone administration response similar to that observed in human osteoblasts. However, the disadvantages of this method include inconsistent cell mineralization[18,19]. Primary rat osteoblast (ROB) is derived from rat skull and is able to mimic bone remodelling and healing in vitro. Mouse osteoblastic MC3T3-E1 cells and osteoclast precursor RAW264.7 cells are also well-established models for studying osteoblast-osteoclast interaction[20,21]. In this study, MG-63 cells were employed to evaluate the capacity of osthole to stimulate osteoblast proliferation, differentiation, and anti-apoptosis, while ROB was utilised to assess osthole’s impact on osteoblast mineralization[22]. In the present study, the effect of osthole on the expression of key osteogenesis-related molecules in the MC3T3-E1 cell line was first determined. An indirect co-culture model of MC3T3-E1 and RAW264.7 was then established to investigate whether osthole regulates osteoclast differentiation via a paracrine mechanism. In this study, an indirect co-culture system was established using conditioned medium (CM) collected from osthole-treated MC3T3-E1 cells. This CM was then applied to RAW264.7 cells to investigate whether osthole indirectly regulates osteoclast differentiation via osteoblast-derived paracrine signals. The present study employed four cell models to investigate the osteoprotective effects of osthole, specifically examining its capacity to regulate the ER, PI3K/AKT, MEK/ERK, and Wnt/β-catenin signaling pathways in osteoblasts. The study also investigated the modulation of osteoblast-osteoclast communication, and the subsequent regulation of osteoclastic differentiation in a paracrine manner.

MATERIALS AND METHODS

Reagents

Osthole (purity > 98%) powder was obtained from the National Institute for Food and Drug Control (Beijing, China) and dissolved in dimethyl sulfoxide (DMSO) (final DMSO concentration < 0.1%). Fulvestrant (ICI) 182780 (ER antagonist), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide, and Annexin V were procured from Sigma (St. Louis, MO, United States). Wortmannin (AKT inhibitor), PD98059 (ERK inhibitor), and dickkopf-1 (DKK-1) (Wnt inhibitor) were obtained from Calbiochem (San Diego, CA, United States). Trizol was supplied by Invitrogen (San Diego, CA, United States). The reverse transcription-quantitative polymerase chain reaction (RT-qPCR) kit was procured from Yeasen Biotechnology (Shanghai, China). Antibodies against OPG, RANKL, β-catenin, and β-actin were obtained from Santa Cruz (CA, United States); anti-Osterix from Abcam; anti-ERK and anti-phosphorylated (p)-ERK from BD Biosciences (San Diego, CA, United States); anti-AKT and anti-p-AKT from Cell Signaling Technology (Beverly, MA, United States). The chemiluminescent substrate kit was procured from Pierce Biochemicals. The tartrate-resistant acid phosphatase (TRAP) staining kit and Triton X-100 were procured from Solarbio (Beijing, China). The TRAP activity assay kit was procured from Beyotime (Shanghai, China). The mouse RANKL enzyme-linked immunosorbent assay (ELISA) kit was procured from Yite Life (Tianjin, China). The remaining chemicals were obtained from local commercial sources.

Cell culture

The MG-63 cells utilised in this study were procured from (Rockville, MD, United States). Primary ROBs were obtained from rat skulls as previously described[23]. Mouse osteoblastic MC3T3-E1 cells and mouse osteoclast precursor RAW264.7 cells (American Type Culture Collection) were maintained in our laboratory. The MG-63, ROB and MC3T3-E1 cells were cultivated in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) medium containing 10% fetal bovine serum (FBS) (supplemented with 100 IU/mL penicillin and 100 μg/mL streptomycin). The Raw264.7 cells were cultivated in RAW 264.7 Cell Complete Medium from Procell (Wuhan, China).

Molecular docking

The PubChem database (https://pubchem.ncbi.nlm.nih.gov/) was utilised to obtain osthole. The Protein Data Bank (PDB) database (https://www.rcsb.org/) was utilised to procure protein receptors: ERα (PDB ID: 1SJ0), OPG (PDB ID: 3URF), RANKL (PDB ID: 3URF), ERK1 (PDB ID: 4QTB), ERK2 (PDB ID: 2OJG), β-catenin (PDB ID: 1JDH), AKT1 (PDB ID: 1UNQ), AKT2 (PDB ID: 1O6 L), AKT3 (PDB ID: 2X18). AutoDockTools-1.5.6 and pymol.exe were used to run and process the molecular docking.

MTT assay

MG-63 cells: MG-63 cells were seeded in 96-well plates at a density of 6 × 10³ cells/well and cultured for 24 hours. The medium was then replaced with 1% FBS DMEM and the cells were cultured for a further 24 hours. The cells were then treated with osthole (0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1000 nM, 10000 nM) or with an equivalent volume of DMSO to act as a control for 2 days, 4 days, or 6 days. Following the administration of treatment, 0.5 mg/mL MTT solution was added to each well and incubated at 37 °C for 4 hours. Thereafter, the MTT solution was removed, and DMSO was added to dissolve the formazan crystals. Absorbance at 490 nm was measured using a microplate reader, and cell viability was calculated relative to the control group.

MC3T3-E1 cells: MC3T3-E1 cells were seeded in 96-well plates at a density of 8 × 10³ cells/well and cultured for 24 hours. The medium was then replaced with 1% FBS DMEM for synchronization for a further 24 hours, then treated with osthole (0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1000 nM, 10000 nM, 20000 nM, 50000 nM, 80000 nM) for 48 hours. Cell viability was detected by MTT assay as described above.

RAW264.7 cells: RAW264.7 cells were seeded in 96-well plates at a density of 4 × 10³ cells/well and cultured for 24 hours. The medium was then replaced with 1% FBS DMEM for 24 hours, then treated with osthole (0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1000 nM, 10000 nM) or CM for 48 hours. Cell viability was detected by MTT assay.

Alkaline phosphatase activity assay

MG-63 cells were seeded at the amount of a density of 4 × 10⁴ cells/well in 12-well plates and cultured for 24 hours. The medium was substituted with a new medium containing 1% FBS and cultured for 24 hours. Osthole was then administered as previously outlined. Following the osthole treatment, MG-63 cells were rinsed with phosphate-buffered saline. MG-63 cells were sonicated three times for 10 seconds in 50 mmol/L Tris hydrochloride, pH 7.5, with 0.1% Triton X-100. The cell lysates were then subjected to centrifugation, after which 1 mL of a mixed solution (5 mmol/L disodium phenyl phosphate in 0.05 M bicarbonate buffer, pH 10.15, containing 2.25 mmol/L 4-amino antipyrine) was added. Following a 15-minute incubation at 37 °C, the reaction was terminated by the addition of 1.5 mL of the stop solution (5.8 mmol/L potassium ferricyanide containing 0.21 M boric acid). The measurement of the absorbances was conducted at a wavelength of 520 nm. The activity of alkaline phosphatase (ALP) was evaluated by dividing the value of ALP activity (U/100 mL) by the A490 value of the MTT assay.

Alizarin red staining assay

ROBs were seeded at a density of 4 × 10⁵ cells/mL in 6-well plates and cultured for 24 hours. The medium was substituted with a new one containing 2% FBS and cultured for 24 hours. Subsequently, the administration of osthole (0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1000 nM, or 10000 nM) or vehicle DMSO was conducted. When the confluence reached approximately 50%, ascorbic acid (100 μg/mL) and beta-glycerol phosphate (10 mmol/L) were added. The medium was changed every 2 days. Following an 18-day period, the ROBs were subjected to fixation with 95% ethanol for 30 minutes. This was followed by staining with 0.1% alizarin red S for 30 minutes at a temperature of 37 °C. The stain was subsequently dissolved using a solution of 10% cetylpyridinium chloride (pH 7.0), following which the solution was shaken for 15 minutes. Alizarin Red S was measured at a wavelength of 570 nm.

Flow cytometry

The MG-63 cells were seeded at a density of 2 × 10⁵ cells/well in 6-well plates and subsequently cultured for 24 hours. The medium was substituted with 1% FBS and the cells were cultured for 24 hours. Osthole (0.1, 1 nM) or vehicle DMSO was added and cultured for 24 hours. Then 10 μmol/L cisplatin was administered for 24 hours to induce apoptosis in MG-63 cells. The MG-63 cells were stained using the fluorescein isothiocyanate Annexin V Apoptosis Detection Kit. After 2 hours of dark incubation at 4 °C, the sample was analysed by flow cytometry.

RT-qPCR assay

The trizol reagent was utilised for the isolation of total RNA. The design and synthesis of RT-qPCR primers was accomplished through the utilisation of Vector NTI 8 software and TaKaRa Biotechnology Co.Ltd. The RT-qPCR Kit was utilised for the execution of the RT-qPCR assay. The results were obtained using a real-time fluorescence quantitative PCR instrument. The expression levels of the target protein were then normalised using β-actin as the reference standard. The primer sequences are presented in Table 1.

Table 1 Primer sequences used for quantitative real-time polymerase chain reaction.

Gene	Primer sequences
OPG	Forward 5’-GCTAACCTCACCTTCGAG-3’
	Reverse 5’-TGATTGGACCTGGTTACC-3’
RANKL	Forward 5’-AACAGGCCTTTCAAGGAGCTGTGC-3’
	Reverse 5’-AAGAGGACAGACTCACTTTATGGG-3’
Osterix	Forward 5’-ATGCAGGCATTTCACCAGGCCC-3’
	Reverse 5’-TGCTCAGGTGGTCACTTCGGGT-3’
TRAP	Forward 5’-TAACCGTCACCTGGCTGAAC-3’
	Reverse 5’-CACGGGACCCTAGTGCATTT-3’
CTR	Forward 5’-TCACTGCTACATGTCACGCC-3’
	Reverse 5’-GTCACTGTCAAGGGTCCAGT-3’
RANK	Forward 5’-GCATCCCTTGCAGCTCAACA-3’
	Reverse 5’-ATGGAAGAGCTGCAGACCAC-3’
CatK	Forward 5’-CCGAAAAGAGCCTAGCGAAC-3’
	Reverse 5’-GGTCATGTCTCCCAAGTGGT-3’
MMP-9	Forward 5’-CCAGCCGACTTTTGTGGTCT-3’
	Reverse 5’-TGGCCTTTAGTGTCTGGCTG-3’
β-actin	Forward 5’-TGGAATCCTGTGGCATCCATGAAAC-3’
	Reverse 5’-TAAAACGCAGCTCAGTAACAGTCC-3’

Western blot assay

Radio-immunoprecipitation assay lysis buffer was administrated to obtain the cell lysate. The bicinchoninic acid kit was utilised to ascertain the protein concentration. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a range of 10%-12%, and transferred to polyvinylidene difluoride membranes. After blocking, these membranes were then subjected to an overnight incubation with the following primary antibodies: Anti-OPG, anti-RANKL, anti-β-actin, anti-β-catenin, anti-Osterix, anti-p-ERK, and anti-p-AKT. The membranes were then subjected to an incubation with secondary antibodies. The membranes were stripped to allow the reprobing with anti-β-actin, anti-ERK, or anti-AKT antibodies once more. ERK, p-ERK, AKT, and p-AKT were then analysed. The target proteins were then subjected to visualisation using a chemiluminescent substrate kit.

Preparation of CM and co-culture

MC3T3-E1 cells were treated with 1 nM or 10 nM osthole (concentrations selected based on preliminary dose-response experiments) or vehicle (DMSO) for 48 hours.The CM was collected, subjected to a centrifugation process in order to remove cell debris, and stored at a temperature of -80 °C. RAW264.7 cells were seeded in 96-well plates (for the purpose of assessing viability and TRAP activity) or 12-well plates (for gene expression analysis). Following adhesion, the culture medium was replaced with a mixture of fresh medium and CM from different groups at a 1:3 ratio (supernatant: Minimum essential medium α). For differentiation assays, the medium was refreshed every 2 days. Specifically, RAW264.7 cells were cultured for 2 days for gene expression analysis, and for 6 days for TRAP staining and activity assays. The composition of the experimental groups is outlined below: (1) Control: RAW264.7 cells were cultured in normal medium; (2) CM-control: RAW264.7 cells were cultured with CM from untreated MC3T3-E1 cells; (3) CM-osthole (1 nM): RAW264.7 cells were cultured with CM from MC3T3-E1 cells treated with 1 nM osthole; and (4) CM-osthole (10 nM): RAW264.7 cells were cultured with CM from MC3T3-E1 cells that had been treated with 10 nM osthole. Based on preliminary dose-response assays, 1 nM and 10 nM were selected as the optimal concentrations, as they significantly enhanced cell viability and ALP activity relative to the control (P < 0.05), achieving maximal or near-maximal effects, while concentrations ≥ 100 nM showed no further improvement and tended to suppress viability.

Inhibition of signaling pathways in the co-culture system

In order to investigate the involvement of specific pathways, MC3T3-E1 cells were pretreated for 30 minutes with ICI 182780 (ER inhibitor, 100 μM), PD98059 (MEK/ERK inhibitor, 50 μM), wortmannin (PI3K/AKT inhibitor, 200 nM), DKK-1 (Wnt inhibitor, 200 ng/mL), or an equal volume of vehicle (DMSO) before being stimulated with the optimal concentration of osthole for 48 hours. CM was then collected and applied to RAW264.7 cells as described above. Groups included: Control (DMSO), inhibitor alone, osthole alone, osthole + inhibitor. to exclude cytotoxicity, MC3T3-E1 cells were treated with each inhibitor alone (ICI 182780 100 μM, PD98059 50 μM, wortmannin 200 nM, DKK-1 200 ng/mL) for 48 hours, and cell viability was assessed by MTT assay. None of the inhibitors significantly reduced cell viability compared to the control group (P > 0.05 for all comparisons).

Assays for osteoclast function

Viability of RAW264.7 cells: Raw264.7 cells were seeded at a density of 4 × 10³ cells/well in 96-well plates for 24 hours. The medium was then substituted with 1% FBS and cultured for 24 hours. RAW264.7 cells were treated for 2 days with osthole at 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1000 nM or 10000 nM, or with CM. After 48 hours of culture with CM, cell viability was assessed by MTT assay.

RANKL secretion: Conditioned media from MC3T3-E1 cells treated with the two effective concentrations of osthole were harvested. Centrifuge at 800 rpm and 4 °C for 10 minutes was then employed to remove cellular debris. The sample was subjected to a process of aliquoting, followed by storage at a temperature of -80 °C, with the intention of facilitating subsequent analysis. RANKL levels were determined in duplicate using a commercial mouse RANKL ELISA kit according to the manufacturer’s protocol. Absorbance was measured at 450 nm and used to calculate the RANKL concentration in each sample.

Osteoclast gene expression: Total RNA was extracted from RAW264.7 cells after 2 days of culture with CM. RT-qPCR was performed to measure the mRNA expression levels of osteoclast-specific genes, including TRAP, CTR, RANK, CatK, and MMP-9. Primer sequences are listed in Table 1.

Osteoclast differentiation: Following a six-day culture period, the cells were fixed and stained for TRAP using a TRAP staining kit. TRAP-positive multinucleated cells (nuclei ≥ 3) were enumerated as mature osteoclasts. TRAP activity was also quantified using a TRAP activity assay kit.

Statistical analysis

The data are presented as the mean ± SD. The statistical analysis was conducted using SPSS version 13.0 software. One-way analysis of variance was used to assess the differences, followed by the Bonferroni post hoc test. Results were considered statistically significant at a P value of less than 0.05.

The statistical methods of this study were reviewed by Associate Professor Huai-En Bu, Head of the Department of Public Health, School of Public Health and Health Sciences, Tianjin University of Traditional Chinese Medicine.

RESULTS

Molecular docking

The binding affinity between small molecule ligands and protein receptors can be evaluated by the minimum binding energy. The hypothesis is that the binding affinity is high when the minimum binding energy level which is between -5.0 kcal/mol and -7.0 kcal/mol[24]. AKT kinase contains AKT1, AKT2, and AKT3, and molecular docking results indicated that osthole exhibited high binding affinity with AKT1, AKT2 and AKT3. Mitogen-activated protein kinase (MAPK) 1/ERK2 and MAPK3/ERK1 have been identified as playing key roles in the MAPK/ERK signaling cascade. As anticipated, osthole demonstrated a high binding affinity for ERK1 and ERK2. Furthermore, the present study investigated the binding capacity of osthole and OPG, RANKL, Osterix, and ERα. Osthole demonstrated a high binding affinity with OPG, RANKL, Osterix and ERα (Figure 1).

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.

Effect of osthole on cell viability, differentiation, mineralization and apoptosis of osteogenic precursor cells. The MTT assay was utilised to assess the impact of varying concentrations of osthole on the viability of MG-63 cells. The MTT assay results indicated that low concentrations of osthole (0.01 nM, 0.1 nM, 1 nM, 10 nM) stimulated cell proliferation, whereas high concentrations of osthole (1000 nM, 10000 nM) inhibited cell proliferation. On days 2, days 4, and days 6, osthole at 1 nM exhibited the most significant effect on cell viability (Figure 2A). The up-regulation of ALP activity is a hallmark of early osteoblast differentiation. The formation of mineralized nodules occurs during the terminal stage of osteoblast differentiation[25]. The ALP activity assay demonstrated that osthole (0.1 nM, 1 nM, 10 nM, 100 nM, 1000 nM, 10000 nM) significantly enhanced ALP activity in comparison with the control group after 2 days, 4 days, and 6 days, reaching a maximum at 10 nM (Figure 2B).The alizarin red staining assay revealed that osthole (1 nM, 10 nM, 100 nM, 1000 nM) significantly augmented the number of mineralized nodules in comparison with the control group after 18 days, reaching a maximum at 10 nM (Figure 2C). Furthermore, the present study investigated the ability of osthole to inhibit cisplatin-induced cell apoptosis in MG-63 cells. The results of the flow cytometry analysis demonstrated that cisplatin dramatically increased the number of apoptotic cells, which was attenuated by pretreatment with 0.1 and 1 nM osthole (Figure 2D).

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.

Effect of osthole on the expression of bone-related factors

The process of osteoclast formation and maturation is subject to precise regulation by a number of factors, including macrophage colony stimulating factor, RANKL, and OPG, which can be secreted by osteoblasts, mesenchymal cells, and mast chondrocytes[26]. Among them, RANKL has been demonstrated to promote osteoclastogenesis, whereas OPG has been shown to inhibit osteoclastogenesis. RT-qPCR and western blot results demonstrated that osthole (0.1 nM, 1 nM, 10 nM) significantly promoted the expression of OPG and inhibited the expression of RANKL in comparison to the control, reaching a maximum effect at 1 nM (Figure 3A and B). Osterix has been identified as a crucial transcription factors in the differentiation of osteoblasts[27]. It was established that the levels of both gene and protein of Osterix were elevated by osthole (0.1 nM, 1 nM, 10 nM) in comparison with the control group, reaching a maximum at 1 nM (Figure 3A and B). In light of the experimental findings outlined above, the decision was made to utilise 1 nM osthole in subsequent experiments.

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.

Effects of osthole on PI3K/AKT, MEK/ERK, and Wnt/β-catenin signaling pathways

To investigate the effect of osthole on the PI3K/AKT, MEK/ERK, and Wnt/β-catenin signaling pathways, we analyzed the key molecules in these signal pathways: AKT, ERK, and β-catenin. As phosphorylation of AKT and ERK is the process of activation[28], we measured the protein levels of ERK, p-ERK, AKT, and p-AKT at 5 minutes, 15 minutes, 30 minutes, 2 hours, 6 hours, 12 hours, and 24 hours. The results of the Western blot analysis indicated that osthole significantly increased the protein expression of p-ERK (Figure 3C) and p-AKT (Figure 3D). The activation of the classical Wnt signaling pathway is accompanied by an increase in β-catenin protein, and β-catenin is then translocated from the cytoplasm to the nucleus. In the nucleus, β-catenin binds to the co-activators T-cell factor/Lymphoid enhancer factor, thereby activating target genes[29]. In order to ascertain measure the effect of osthole on β-catenin signaling, we examined the β-catenin protein expression levels in cytoplasm and nucleus. Western blot results indicated that the levels of β-catenin increased in the nucleus and decreased in the cytoplasm following osthole administration, reaching a maximum at 24 hours (Figure 3E). The present study set out to investigate the effects of osthole on the PI3K/AKT, MEK/ERK, and Wnt/β-catenin signaling pathways, specific inhibitors of AKT, ERK, and Wnt were administrated respectively. Western blot results indicated that the increased p-ERK and p-AKT proteins induced by osthole, were reduced by pretreatment with the specific inhibitors PD98059 (Figure 3F) and Wortmanin (Figure 3G), and the increased β-catenin in the nucleus was decreased by DKK-1 (Figure 3H).

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

The present study set out to investigate the hypothesis that osthole affects bone metabolism by regulating ER, PI3K/AKT, MEK/ERK, and Wnt/β-catenin signaling pathways. To this end, specific inhibitors of ER, AKT, ERK, and Wnt were administrated, respectively. The results indicated that all of the substances inhibited the increased cell proliferation (Figure 4A), ALP activity (Figure 4B), and up-regulated OPG/RANKL and Osterix (Figure 4C-F) induced by osthole. The results of the present study demonstrate that osthole exerts osteoprotective effects by activating ER, PI3K/AKT, MEK/ERK, and Wnt/β-catenin signaling pathways.

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.

Osthole inhibits osteoclastogenesis via osteoblast-derived CM

In accordance with the observations made in MG-63 cells, osthole was found to promote the viability of MC3T3-E1 cells in a biphasic manner. Low concentrations (0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1000 nM, 10000 nM, 20000 nM, 50000 nM, or 80000 nM) significantly enhanced cell viability, with 1 nM and 10 nM showing the most potent effects after 48 hours of treatment (Figure 5A). The selection of these two concentrations (designated as osthole A: 1 nM; osthole B: 10 nM) was made for the subsequent experimentation with conditioned media. In the following section, the direct effect of osthole on RAW264.7 cells was evaluated (Figure 5B): Direct treatment with osthole (0.01 nM-50 μM) slightly elevated RAW264.7 viability at low concentrations (0.01 nM-1 μM, all ^aP < 0.05 vs control). However, no significant changes were observed at higher concentrations, indicating limited direct regulation of osthole on these osteoclast precursors. We then proceeded to collect CM from MC3T3-E1 cells that had been treated with 1 nM/10 nM osthole [CM (1 nM Ost), CM (10 nM Ost)] and subsequently cultured RAW264.7 cells in these CMs. Following a 48-hour period, it was evident that RAW264.7 viability was significantly reduced in the osthole-CM groups in comparison to the blank CM group (Figure 5C, ^aP < 0.05 vs control; ^cP < 0.05 vs blank CM). Moreover, ELISA analysis revealed that MC3T3-E1 cells treated with 1 nM or 10 nM osthole secreted significantly lower levels of RANKL (Figure 5D, both ^aP < 0.05 vs control) - a key paracrine factor driving osteoclast differentiation. Following a 48-hour culture period in CM culture, RT-qPCR results demonstrated a significant decrease in the mRNA expression of osteoclastogenic genes (TRAP, CTR, RANK, MMP-9, CatK) in RAW264.7 cells within the osthole-CM groups (Figure 5E, ^aP < 0.05 vs control; ^cP < 0.05 vs blank CM). After a 6-day culture period, TRAP activity (a functional marker of mature osteoclasts) was markedly reduced (Figure 5F, ^aP < 0.05 vs control; ^cP < 0.05 vs blank CM), and the number of viable osteoclasts per microscopic field (counted post-TRAP staining) was also reduced (Figure 5G, ^aP < 0.05 vs control; ^cP < 0.05 vs blank CM). Morphological observation via TRAP staining (Figure 5H, × 100 magnification; 100 μm scale bar) further validated these effects: The control and blank CM groups contained abundant TRAP-positive multinucleated cells (marked by blue arrows), whereas the osthole-CM groups exhibited fewer, smaller TRAP-positive cells, confirming osthole’s indirect inhibitory effect on osteoclastogenesis via osteoblast-derived paracrine signals.

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.

Signaling pathways mediate the inhibitory effect of osthole on osteoclastogenesis

Firstly, the investigation will be conducted to ascertain whether osthole exerts a comparable effect on the bone metabolism of MC3T3-E1 cells through the modulation of ER, PI3K/AKT, MEK/ERK, and Wnt/β-catenin signaling pathways, in accordance with the observations made in MG-63 cells. In order to achieve this objective, specific inhibitors targeting ER, AKT, ERK, and Wnt will be administered, respectively. The results demonstrated that none of these inhibitors alone exerted cytotoxic effects on MC3T3-E1 cell viability in isolation; however, all four inhibitors significantly attenuated the osthole-induced enhancement of cell viability (Figure 5I and J). Then, we assessed RAW264.7 cell viability and cytotoxicity after 48 hours of CM culture (Figure 6A and B): Compared with the control group, CM from osthole-treated MC3T3-E1 cells (CM + Ost) significantly reduced RAW264.7 viability (P < 0.05) and elevated cytotoxicity (P < 0.05); however, these effects were notably reversed when MC3T3-E1 cells were pretreated with any of the four inhibitors (CM + Ost + DKK, CM + Ost + ICI, CM + Ost + PD, CM + Ost + Wort), with viability recovering and cytotoxicity decreasing relative to the CM + Ost group (^aP < 0.05 vs control; ^cP < 0.05 vs blank CM ^dP < 0.05 vs CM (1 nM Ost)). We then proceeded to quantify RANKL secretion in MC3T3-E1 cells under different pretreatment conditions (Figure 6C and D). Osthole treatment alone significantly reduced RANKL protein expression in MC3T3-E1 cells (P < 0.05 vs control), Pretreatment with DKK-1, ICI 182780, PD98059, or wortmannin attenuated this inhibitory effect, leading to higher RANKL levels compared to the osthole treatment alone group (^bP < 0.05 vs osthole alone). Following a 48-hour culture period in CM culture, we detected the relative mRNA expression levels of osteoclast-specific genes (TRAP, CTR, RANK, MMP-9, CatK) in RAW264.7 cells were detected (Figure 6E and F). CM + Ost significantly downregulated the expression of all these genes (^cP < 0.05 vs blank CM). However, CM from inhibitor-pretreated, osthole-exposed MC3T3-E1 cells (CM + Ost + inhibitor) reversed this downregulation. Indeed, gene expression levels in these groups were significantly higher than those in the CM + Ost group (^dP < 0.05 vs CM (1 nM Ost)), and partially restored to levels comparable to the blank CM group. Morphological observation via TRAP staining (Figure 6G, original magnification × 100) further corroborated these results: The control group and the blank CM group exhibited abundant large, TRAP-positive multinucleated osteoclasts (blue arrows), while CM + Ost significantly reduced the number and size of these cells. Notably, when MC3T3-E1 cells were pretreated with inhibitors, the inhibitory effect of osthole-CM on TRAP-positive cell formation was attenuated, with more and larger multinucleated cells appearing in the CM + Ost + DKK, CM + Ost + ICI, CM + Ost + PD, and CM + Ost + Wort groups compared to CM + Ost. Quantitative analysis of osteoclast viability per field (Figure 6H and I) and TRAP activity (Figure 6J and K) after 6 days of CM culture provided confirmation of the morphological findings: The CM + Ost significantly decreased the number of viable osteoclasts per field and TRAP activity (^cP < 0.05 vs blank CM), while each pathway inhibitor reversed these effects - both osteoclast count and TRAP activity in the CM + Ost + inhibitor groups were significantly higher than those in the CM + Ost group (^dP < 0.05 vs CM (1 nM Ost)). Collectively, these results demonstrate that osthole relies on activating the ER, Wnt/β-catenin, MEK/ERK, and PI3K/AKT pathways in osteoblasts to exert its paracrine inhibitory effect on osteoclastogenesis.

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.

DISCUSSION

Osteoporosis is defined as a systemic metabolic bone disease characterised by an imbalance between bone resorption and formation, leading to decreased bone mass and microarchitectural deterioration, which consequently increases fracture risk[30,31]. Whilst contemporary pharmacological interventions, such as bisphosphonates and RANKL inhibitors, have exhibited efficacy in relevant models, concerns remain regarding their long-term safety, as their use is associated with adverse effects including osteonecrosis of the jaw, atypical femoral fractures, and cardiovascular concerns[32,33]. Consequently, natural compounds from traditional medicine, with their multi-target potential and favourable safety profiles, have garnered significant interest for the management of osteoporosis[34,35]. Osthole, a natural coumarin derivative, has demonstrated encouraging osteoprotective effects. The present study systematically elucidates the dual mechanisms through which osthole exerts its bone-protective actions. Firstly, osthole activates ER, PI3K/AKT, MEK/ERK, and Wnt/β-catenin signaling pathways in osteoblasts. Secondly, osthole suppresses osteoclastogenesis via modulation of the osteoblast secretome.

Osthole enhances osteoblast function through coordinated activation of multiple signaling pathways

The molecular docking analysis predicted a strong binding affinity between osthole and key signaling proteins, including ERα, AKT, ERK, and β-catenin, suggesting potential interactions. In terms of functionality, osthole, at concentrations of between 0.1 nM and 10 nM, demonstrated a significant stimulatory effect on the proliferation, ALP activity, and mineralisation of both MG-63 and MC3T3-E1 osteoblastic cells. The concentration-dependent effects, with 1 nM being most potent for proliferation and OPG/RANKL ratio modulation, and 10 nM for ALP activity and mineralization, may reflect differential engagement of signaling pathways at various phenotypic endpoints. A key finding of this study is the systematic demonstration that osthole activates the PI3K/AKT pathway in osteoblasts. A significant increase in AKT phosphorylation was observed upon osthole treatment, which was effectively abolished by the PI3K inhibitor wortmannin. The PI3K/AKT pathway, a pivotal regulator of cell survival and metabolism, exerts its influence on osteoblast differentiation through the action of downstream targets such as mammalian target of rapamycin and glycogen synthase kinase-3β[36].

Furthermore, the data obtained confirm the activation of MEK/ERK and Wnt/β-catenin pathways by osthole, as evidenced by increased ERK phosphorylation and β-catenin nuclear translocation, respectively. These effects were specifically reversed by PD98059 (MEK/ERK inhibitor) and DKK-1 (Wnt inhibitor). These results are consistent with prior reports by Kuo et al[14], who described osthole-induced osteoblast differentiation via the ERK pathway, and Jin et al[37], who implicated the Wnt/β-catenin pathway in osthole-mediated bone formation. Moreover, the present study corroborates the estrogen-like activity of osthole via ER activation, thereby supporting the findings of Jia et al[7] in MCF-7 cells and highlighting its potential relevance for postmenopausal osteoporosis.

Osthole indirectly suppresses osteoclastogenesis by modulating the osteoblast secretome

It is widely accepted that the maintenance of bone homeostasis is critically regulated by the communication between osteoblasts and osteoclasts[38], primarily mediated by the OPG/RANKL/RANK system. The present study found that treatment of osteoblasts with osthole resulted in a significant upregulation of OPG expression, alongside a downregulation of RANKL, thus leading to an increased OPG/RANKL ratio at both the gene and protein levels. In order to ascertain the functional impact of this altered secretome on osteoclasts, we employed a CM co-culture system. CM collected from osthole-treated MC3T3-E1 osteoblasts demonstrated the capacity to inhibit osteoclast formation and function, as evidenced by a reduction in TRAP-positive multinucleated cell formation, a decreased in TRAP activity, and the downregulation of key osteoclastogenic genes (TRAP, CTR, RANK, CatK, MMP-9) in RAW264.7 cells. These inhibitory effects were found to correlate with reduced RANKL levels in the CM. It is of particular significance that when osteoblasts were pretreated with specific inhibitors targeting the ER, PI3K/AKT, MEK/ERK, or Wnt/β-catenin pathways, the anti-osteoclastogenic effect of the osthole-CM was significantly attenuated. This not only establishes the necessity of these pathways in regulating OPG/RANKL expression but also directly links their activation in osteoblasts to the indirect suppression of osteoclast differentiation via intercellular communication. This indirect mechanism serves to complement the direct inhibitory effects of osthole on osteoclastogenesis, as reported by others. For instance, Wang et al[39] and Ma et al[40] demonstrated that osthole directly inhibits osteoclast differentiation by suppressing the P38/MAPK and nuclear factor kappa B pathways. The findings of this study demonstrate a distinct, osteoblast-mediated indirect mechanism, thereby emphasising the multi-targeted nature of osthole’s action in bone remodeling.

Synergistic potential of multi-pathway activation and therapeutic implications

The results of this study indicate that the ER, PI3K/AKT, MEK/ERK, and Wnt/β-catenin pathways, which are known to be activated by osthole in osteoblasts, likely function in a coordinated network rather than in isolation. For instance, the Wnt/β-catenin pathway has been identified as a regulator of OPG expression, and ER signaling has been demonstrated to exert an indirect influence on RANKL transcription. It has been hypothesised that this coordinated activation may produce additive or synergistic effects, leading to a more robust promotion of osteoblast function and suppression of osteoclastogenesis[41,42]. This multi-target profile could be a therapeutic advantage for osthole. In comparison with single-target osteoporosis drugs[43,44] (e.g., teriparatide primarily acting on the PTH pathway, denosumab targeting RANKL), osthole’s coordinated activation of multiple anabolic pathways and its simultaneous regulation of the OPG/RANKL ratio may offer a more effective approach to addressing the complex, multifactorial pathogenesis of osteoporosis. However, the precise molecular interplay and potential synergism between these pathways in response to osthole warrant further investigation[34].

Contrast with previous findings and clinical perspective

Whilst the majority of studies have indicated that osthole exerts a positive effect on bone formation, a contradictory report by Hu et al[45] demonstrated that osthole inhibited the proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells by blocking the Wnt/β-catenin and ERK pathways[46]. This discrepancy necessitates a more profound examination that extends beyond the confines of mere disparities in cell models or concentrations. Firstly, celltype specificity: Mesenchymal stromal cells (MSCs) are undifferentiated multipotent cells, whereas MG63 and MC3T3E1 are committed osteoblasts. The surface receptor profiles (e.g., ER subtypes) and baseline signaling activities of these cells differ, and osthole may promote function in osteoblasts but suppress expansion in MSCs. Secondly, concentrationdependent hormesis: Hu et al[45] used 50-200 μM osthole, while our effective concentrations were 1-10 nM. Many natural compounds show biphasic effects - low doses activate, high doses inhibit. Osthole might inhibit MSCs only at suprapharmacological levels, while low nanomolar doses selectively promote osteoblast function. Thirdly, metabolic capacity: It is hypothesised that MSCs and osteoblast lines may express different levels of drugmetabolizing enzymes (e.g., CYP450) and efflux transporters, leading to distinct intracellular osthole concentrations or active metabolites. Fourthly, divergent pathway regulation: Hu et al[45] reported that osthole inhibited Wnt/βcatenin and ERK pathways in MSCs. However, our observations revealed the activation of these pathways in osteoblasts. This paradox could arise from celltypespecific expression of negative feedback regulators (e.g., DKK-1, sclerostin) or differences in basal phosphatase activity. Despite these discrepancies, our consistent findings across multiple osteoblastic models and the CM system support the hypothesis that osthole exerts osteoprotective effects by activating ER, PI3K/AKT, MEK/ERK and Wnt/βcatenin pathways in osteoblasts, thereby indirectly suppressing osteoclastogenesis. In order to resolve this discrepancy, it is necessary to conduct direct comparative studies of MSCs and osteoblasts under identical conditions. The present study utilised established osteoblast models and incorporated the osteoclast communication aspect, thereby providing comprehensive evidence supporting the osteoprotective role of osthole via pathway activation. In consideration of the findings, osthole is identified as a promising multi-target candidate for the prevention and treatment of osteoporosis. Its potential for estrogen-like activity via ER activation could be particularly relevant for postmenopausal osteoporosis, potentially offering an alternative to conventional hormone replacement therapy. Nevertheless, translational challenges persist. Beyond the in vitro nature of our study, the translational potential of osthole is constrained by its welldocumented pharmacokinetic limitations. Osthole is a hydrophobic coumarin with poor aqueous solubility, which results in low and erratic oral bioavailability. Furthermore, it is subject to rapid metabolism primarily via CYP3A4/3A5 and CYP1A2, resulting in a short plasma halflife. These shortcomings - namely, low solubility, extensive firstpass metabolism, and rapid clearance - pose significant obstacles to clinical translation. Fortunately, emerging formulation strategies offer promising solutions. For instance, nanoparticle encapsulation [e.g., poly(butyl cyanoacrylate) nanoparticles] has been shown to increase osthole bioavailability by more than threefold. Enteric nanocarriers and bonetargeted delivery systems (e.g., bisphosphonateconjugated nanoparticles) could further enhance skeletal accumulation. Coadministration with CYP inhibitors or incorporation into traditional Chinese medicine formulas, has been demonstrated to enhance systemic exposure. It is recommended that future studies place a priority on the development of such formulations and the subsequent evaluation of their pharmacokinetics and therapeutic efficacy in animal models of osteoporosis. It is imperative to address these delivery challenges if the clinical potential of osthole, as suggested by our mechanistic findings, is to be realised.

Study limitations and future directions

The present study is subject to several limitations. Firstly, it is important to note that all experiments were conducted in vitro; future validation using in vivo osteoporosis models (e.g., ovariectomized rats) is essential. Secondly, while we confirmed the activation of multiple pathways, the potential for crosstalk between them was not fully explored. Thirdly, the precise primary molecular target(s) of osthole in osteoblasts remain to be identified. Fourthly, while molecular docking predicted high binding affinity between osthole and several signaling proteins (ERα, AKT, ERK), this computational evidence does not conclusively prove physical interaction. Direct binding assays such as surface plasmon resonance or cellular thermal shift assay are necessary to confirm whether osthole directly binds to these targets within the cellular environment. Consequently, the utilisation of terminology that alludes to “direct activation” should be regarded as a generator of hypotheses, awaiting experimental validation. The employment of biophysical methodologies in future studies is recommended, with the aim of identifying the precise molecular targets of osthole. Future research should focus on: (1) Validating the osteoprotective efficacy of osthole in robust animal models of osteoporosis; (2) Employing genetic approaches (e.g., small interfering RNA and clustered regularly interspaced short palindromic repeats-associated protein 9) to dissect the interplay between the identified pathways; (3) The optimization of the pharmacokinetic and bone-targeting properties of osthole through structural modification or novel formulation strategies; and (4) The exploration of potential synergistic effects of osthole in combination with existing osteoporosis drugs at lower, safer doses.

CONCLUSION

In conclusion, the present study has demonstrated that osthole exerts osteoprotective effects via the activation of the ER, PI3K/AKT, MEK/ERK, and Wnt/β-catenin signaling pathways in osteoblasts. This not only enhances osteoblast proliferation, differentiation, and mineralization but also indirectly suppresses osteoclast differentiation by modulating the osteoblast secretome, particularly via the OPG/RANKL axis (Figure 7). These results suggest that osthole exerts osteoprotective effects via multiple pathways, supporting its future clinical application.

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.