Casticin suppresses RANKL‑induced osteoclastogenesis and prevents ovariectomy‑induced bone loss by regulating the AKT/ERK and NF‑κB signaling pathways

Abstract

Postmenopausal osteoporosis is a systemic metabolic disease that chronically endangers public health and is typically characterized by low bone mineral density and marked bone fragility. The excessive bone resorption activity of osteoclasts is a major factor in the pathogenesis of osteoporosis; therefore, strategies aimed at inhibiting osteoclast activity may prevent bone decline and attenuate the process of osteoporosis. Casticin (Cas), a natural compound, has anti‑inflammatory and antitumor properties. However, the role of Cas in bone metabolism remains largely unclear. The present study found that the receptor activator of nuclear factor‑κΒ (NF‑κB) ligand‑induced osteoclast activation and differentiation were inhibited by Cas. Tartrate‑resistant acid phosphatase staining revealed that Cas inhibited osteoclast differentiation, and bone resorption pit assays demonstrated that Cas affected the function of osteoclasts. Cas significantly reduced the expression of osteoclast‑specific genes and related proteins, such as nuclear factor of activated T cells, cytoplasmic 1 and c‑Fos at the mRNA and protein level in a concentration‑dependent manner. Cas inhibited osteoclast formation by blocking the AKT/ERK and NF‑κB signaling pathways, according to the intracellular signaling analysis. The microcomputed tomography and tissue staining of tibiae from ovariectomized mice revealed that Cas prevented the bone loss induced by estrogen deficiency and reduced osteoclast activity in vivo. Collectively, these findings indicated that Cas may be used to prevent osteoporosis.

Keywords: AKT; ERK; NF‑κB; casticin; osteoclast; osteoporosis.

Figure 1

Cas inhibits receptor activator of nuclear factor κB ligand-induced osteoclast production. (A) Chemical structure of Cas. (B and C) The results of TRAP staining showed that Cas inhibited osteoclast formation in a concentration-dependent manner (osteoclasts were considered as mature when the number of nuclei was ≥3). The scale bar of the enlarged images is 500 µm. (D) The cytotoxicity of Cas on BMMs was assessed at 96 h using Cell Counting Kit-8 assay. (E and F) TRAP staining revealed the inhibitory effect of Cas in different stages of osteoclast formation. The scale bar of the enlarged images is 500 µm. (G and H) Immunofluorescence staining of F-actin belts in mature osteoclasts in the presence of different concentrations of Cas. The scale bar is 1,000 µm. ^**P<0.01 and ^***P<0.001. All data are expressed as the mean ± SD. Cas, casticin; TRAP, tartrate-resistant acid phosphatase; RANKL, receptor activator of nuclear factor κB ligand; CCK-8, Cell Counting Kit-8.

Figure 2

Cas inhibits osteoclast function. (A and B) Representative images revealed that the acidification function of osteoclasts was impaired after treatment with Cas at specified concentrations. Scale bar, 100 µm. (C and D) Representative images showed the inhibition of osteoclast resorption by Cas. Scale bar, 200 µm. ^***P<0.001. All data are expressed as the mean ± SD. Cas, casticin; RANKL, receptor activator of nuclear factor κB ligand.

Figure 3

Cas inhibits the expression of NFATc1, downstream related genes and proteins. (A-F) Cas inhibited the expression of osteoclast-related genes Nfatc1, Fos, Ctsk, Dcstamp, Mmp9 and Atp6v0d2 stimulated by RANKL. (G) Typical western blot images of NFATc1, c-Fos, CTSK and ATP6V0D2 protein expression in osteoclasts stimulated by RANKL and 2 µM Cas for 0, 1, 3 and 5 days. (H-K) The relative ratio of the gray values of NFATc1, c-Fos, CTSK and ATP6V0D2 to β-actin was quantified. ^*P<0.05, ^**P<0.01 and ^***P<0.001. All data are expressed as the mean ± SD. Cas, casticin; NFATc1, nuclear factor of activated T cells, cytoplasmic 1; Ctsk, cathepsin K; Dcstamp, dendrocyte expressed seven transmembrane protein; Mmp9, matrix metalloproteinase 9; Atp6v0d2, ATPase H⁺ transporting V0 subunit D2; RANKL, receptor activator of nuclear factor κB ligand.

Figure 4

Cas inhibits the RANKL-induced NF-κB signaling pathway. (A) Cells from BMMs treated with RANKL and Cas were stained with p65 antibody and DAPI, and then images were captured under a fluorescence microscope. Scale bar, 100 µm. (B) Quantification of the rate of p65 into the nucleus. (C) Representative phosphorylated p65 and IκBα western blot images of Cas treated-BMMs at various time points. (D and E) The ratio of phosphorylated p65 to p65 and the ratio of IκBα to β-actin were quantified. ^*P<0.05, ^**P<0.01 and ^***P<0.001. All data are expressed as the mean ± SD. Cas, casticin; RANKL, receptor activator of nuclear factor κB ligand; BMMs, bone marrow-derived macrophages; p-, phosphorylated.

Figure 5

Cas inhibits the RANKL-induced ERK/AKT/GSK3β signaling pathway. (A) Effect of Cas on RANKL-induced ERK and AKT pathways. Specific antibodies were used to evaluate the protein expression and phosphorylation of ERK, AKT and GSK3β. (B-D) The ratio of quantified phosphorylated related proteins to corresponding total proteins is presented. ^*P<0.05, ^**P<0.01 and ^***P<0.001. All data are expressed as the mean ± SD. Cas, casticin; RANKL, receptor activator of nuclear factor κB ligand; p-, phosphorylated.

Figure 6

Casticin inhibits ovariectomized-induced bone loss. (A) 3D representative images of different groups of tibial plateau of mice. (B-E) Tibial cancellous bone data of BV/TV, trabecular separation, trabecular number, and trabecular thickness (n=6). (F) Representative H&E staining results of tibial plateau sections. (G) Quantification of H&E staining BV/TV in tibial plateau. (H) Representative TRAP staining results of tibial plateau sections. (I) Quantification of the number of osteoclasts in tibial plateau after TRAP staining. ^*P<0.05, ^**P<0.01 and ^***P<0.001. All data are expressed as the mean ± SD. BV/TV, volume/tissue volume; H&E, hematoxylin and eosin; TRAP, tartrate-resistant acid phosphatase; OVX, ovariectomized; E₂, estradiol; Cas, casticin; Tb.Sp, trabecular separation; Tb.N, trabecular number; Tb.Th, trabecular thickness.

Figure 7

Working model of how Cas regulates osteoclastogenesis. Cas negatively regulates osteoclast differentiation through the receptor activator of nuclear factor κB ligand-induced AKT/ERK/NF-κB pathway to control osteoclastogenesis-related transcriptional processes and genes expression. Cas, casticin; RANKL, receptor activator of nuclear factor κB ligand; NFATc1, nuclear factor of activated T cells, cytoplasmic 1; Ctsk, cathepsin K; Atp6v0d2, ATPase H⁺ transporting V0 subunit D2; Mmp9, matrix metalloproteinase 9; Dcstamp, dendrocyte expressed seven transmembrane protein.