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.

Keywords: 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.