Oxyresveratrol attenuates bone resorption by inhibiting the mitogen-activated protein kinase pathway in ovariectomized rats

Abstract

Background: Bone is continuously produced by osteoblasts and resorbed by osteoclasts to maintain homeostasis. Impaired bone resorption by osteoclasts causes bone diseases such as osteoporosis and arthritis. Most pharmacological treatment of osteoporosis focuses on inhibiting osteoclast differentiation, often to restore osteoclast/osteoclast balance. However, recent osteoporosis treatments have various side effects. According to a recent study, resveratrol, known as a stilbenoid family, is known to increase bone density, and the osteoclast inhibitory effect was confirmed using oxyresveratrol, a stilbenoid family. Here, we investigated the effect of oxyresveratrol on osteoclast differentiation and an ovariectomized mouse model.

Methods: Mouse leukemia monocyte/macrophage cell line RAW 264.7 was treated with oxyresveratrol, and cell cytotoxicity was confirmed by measuring MTT assay. Tartrate-resistant acid phosphatase (TRAP), an enzyme marker for osteoclasts, was confirmed by staining. In addition, osteoclast differentiation markers and MAPK-related markers were confirmed at the mRNA level and protein expression. The effect of oxyresveratrol was confirmed using ovariectomized mice. Deoxypyridinoline (DPD) was measured using mouse urine and TRAP activity was observed using serum. Bone mineral density was also measured using Micro-CT.

Results: The polyphenol oxyresveratrol inhibited receptor activator of nuclear factor kappa-Β ligand (RANKL)-induced osteoclast differentiation of RAW 264.7 cells. Furthermore, oxyresveratrol inhibited TRAP activity and actin-ring formation. Moreover, oxyresveratrol suppressed the phosphorylation of the RANKL-induced mitogen-activated protein kinases (MAPKs) p38, JNK, and ERK and significantly reduced the expression of bone differentiation markers (NFATc1, cathepsin K, and TRAP).

Conclusion: Oxyresveratrol inhibits osteoclast differentiation via MAPK and increases bone density in ovariectomized rats, suggesting it has therapeutic potential for bone diseases such as osteoporosis. We confirmed the osteoporosis prevention effect of OR in Raw 264.7 cells, and future studies should confirm the effect of OR using rat bone marrow-derived cells.

Keywords: Mitogen-activated protein kinase; Osteoclast; Osteoporosis; Ovariectomized rat; Oxyresveratrol.

Fig. 1

Inhibition of osteoclast differentiation by oxyresveratrol (OR) at non-cytotoxic concentrations. (a) Structure of OR. (b) RAW 264.7 cells were treated with OR for 3 days and subjected to MTT assay. Data represent percentages versus control (CTL). Ordinary one-way ANOVA; Dunnett’s multiple comparisons; n = 3; *** p < 0.001. (c) TRAP staining and the formation of TRAP-positive multinucleated RAW 264.7 cells. Cells were seeded at 5 × 10⁴/mL in a 96-well plate and cultured for 3 days in the presence of OR (10 nM, 100 nM, 1 μM, and 10 μM). RANKL induced the formation of TRAP-positive multinucleated RAW 264.7 cells. Original magnification, × 200. (d) ROC number of TRAP-positive multinucleated cells. (e) TRAP activity of TRAP-positive multinucleated cells. Data are percentages versus RANKL. **p < 0.01, ***p < 0.001, n = 3. Error bars, standard deviations. OR, oxyresveratrol; RANKL, receptor activator of nuclear factor kappa-Β ligand; TRAP, tartrate-resistant acid phosphatase; ROC, round osteoclast

Fig. 2

Effect of oxyresveratrol (OR) on actin ring formation in RAW 264.7 cells. (a) Osteoclasts were stained with rhodamine phalloidin to observe actin rings; nuclei were visualized with DAPI. RAW 264.7 cells were seeded onto two-well plates at a density of 1 × 10⁵/mL. (b) Quantitative analysis of the number of actin rings in RAW 264.7 cells. Error bars indicate standard deviation. Ordinary one-way ANOVA; Tukey’s test for multiple comparisons; n = 3; *p < 0.05; **** p < 0.0001. Original magnification, × 200

Fig. 3

Effect of oxyresveratrol (OR)-mediated mitogen-activated protein kinase (MAPK) inhibition on RANKL-induced osteoclast differentiation. pERK, pJNK, and pP38 levels in RAW 264.7 cells by western blotting. Levels of (a) pERK, (c) pP38, and (e) pJNK after treatment with RANKL and OR (100 nM, 1 μM, and 10 μM). Cells were harvested after 15, 240, and 30 min for pERK, pP38, and pJNK, respectively. Protein levels of (b) pERK, (d) pJNK, and (f) pP38. ERK, P38, and JNK were used as loading controls. Data are percentages versus RANKL (***p < 0.001, n = 3)

Fig. 4

Effect of oxyresveratrol (OR) on RANKL-induced bone-differentiation and bone-resorption marker genes. (a) Effect of OR on the NFATc1 mRNA level during osteoclast differentiation of RAW 264.7 cells. RAW 264.7 cells were seeded onto 35-mm culture dishes at 2 × 10⁶/mL. Data are percentages versus RANKL (*p < 0.05, **p < 0.01, n = 3). (b–g) Effect of RANKL and OR (1 μM, or 10 μM) on NFATc1 (b, c), cathepsin K (d, e), and TRAP (f, g) protein levels in RAW 264.7 cells by western blotting. β-actin was used as the loading control. Data are percentages versus RANKL (*p < 0.05, ***p < 0.001, n = 3)

Fig. 5

Effects of bone density in oxyresveratrol (OR) treatment on OVX mouse model. (a) The body weight of the rat was measured weekly after the start of the experiment. It was confirmed that the weight increased in all groups except the sham group. (b) After normalization to creatine in the group treated with 10 and 20 mg/kg of OR, DPD activity was decreased, but not as much as in the ADN group (n = 5); *p < 0.05, **p < 0.01, ***p < 0.001 compared with OVX group. Error bars, standard deviations. (c) Serum TRAP enzyme activity (mean ± SD) in rats in the OVX group was compared to the OR group. The decrease was confirmed in the OR groups of 10 and 20 mg/kg. (n = 5); **p < 0.01, ***p < 0.001 compared with OVX group. Error bars, standard deviations

Fig. 6

Micro-CT analysis of the femurs in oxyresveratrol (OR)-administered rats. The femurs were analyzed after oral administration of the OR to rats of each group for 8 weeks. (a) It was confirmed that bone density was significantly lower in the OVX group, and an increase in bone density was observed at 10 and 20 mg/kg (b) Bone volume per total volume(BV/TV) result obtained by oral administration of OR. Data represent the mean ± SEM of experiments, n = 5; *p < 0.05, **p < 0.01 compared with the OVX group. Error bars indicate standard deviation. BV/TV; trabecular separation