Muscone Ameliorates Ovariectomy-Induced Bone Loss and Receptor Activator of Nuclear Factor-κb Ligand-Induced Osteoclastogenesis by Suppressing TNF Receptor-Associated Factor 6-Mediated Signaling Pathways

Abstract

Postmenopausal osteoporosis is caused by the deficiency of estrogen, which breaks bone homeostasis and induces levels of pro-inflammatory cytokines. Muscone is a potent anti-inflammatory agent and is used to treat bone fracture in traditional Chinese medicine. However, its anti-osteoclastogenic effects remain unclear. For in vitro study, morphology tests of osteoclastogenesis were firstly performed. And then, factors in RANK-induced NF-κB and MAPK pathways were examined by RT-PCR and Western blot, and the binding of TNF receptor-associated factor (TRAF)6 to RANK was inspected by coimmunoprecipitation and immunofluorescence staining. For in vivo experiments, C57BL/6 ovariectomized (OVX) mice were used for detection, including H&E staining, TRAP staining, and micro CT. As a result, muscone reduced OVX-induced bone loss in mice and osteoclast differentiation in vitro, by inhibiting TRAF6 binding to RANK, and then suppressed NF-κB and MAPK signaling pathways. The expression of the downstream biomarkers was finally inhibited, including NFATc1, CTR, TRAP, cathepsin K, and MMP-9. The inflammatory factors, TNF-a and IL-6, were also reduced by muscone. Taken together, muscone inhibited the binding of TRAF6 to RANK induced by RANKL, thus blocking NF-kB and MAPK pathways, and down-regulating related gene expression. Finally, muscone inhibited osteoclastogenesis and osteoclast function by blocking RANK-TRAF6 binding, as well as downstream signaling pathways in vitro. Muscone also reduced ovariectomy-induced bone loss in vivo.

Keywords: RANK; TRAF6; muscone; osteoclasts; postmenopausal osteoporosis.

Figure 1

Muscone inhibits osteoclastogenesis in BMMs and the RAW264 cell line. (A) Chemical structure of muscone. (B) proliferation of muscone-exposed BMMs. (C, D) BMMs and RAW264.7 cells are used and treated with 10, 20, 40 μM muscone, and TRAP-positive cells are quantified (magnification ×100). *P < 0.05 vs. the control group, ^#P < 0.05 vs. the RANKL-induced group.

Figure 2

Muscone represses actin-ring formation and bone resorption by osteoclasts. (A) Actin-ring formation is observed by FITC-phalloidin staining and quantified. Muscone inhibits the actin-ring formation in a dose-dependent manner. (B) Resorption area is quantified by image analysis, and muscone significantly shrinks the resorbed range in a dose-dependent manner. *P < 0.05 vs. the control group, ^#P < 0.05 vs. the RANKL-induced group (original scale bars, 50 μm).

Figure 3

Muscone has limited impact on BMSCs differentiation. Alkaline phosphatase (ALP) staining and alizarin red (ARS) staining show that muscone at 10 μM, 20μM, and 40 μM has little influence on the mineralization of calcium nodules. And the oil red O staining shows that muscone has little consequence on the formation of fat particles in vitro.

Figure 4

Muscone inhibits osteoclastogenesis only at an early stage. After induction of M-CSF (20 ng/ml) and RANKL (50 ng/ml) on day 0, BMMs and RAW264.7 cells are divided into groups according to the different times that 40 μM muscone is added. (A, C) Besides the RANKL group, BMMs are divided into three groups: day 1-7 group, day 3-7 group, and day 5-7 group, since muscone is added on day 1, 3, and 5, respectively. (B, D) And similarly, RAW264.7 cells are divided into four groups: day 0-7 group, day 1-7 group, day 2-7 group, and day 3-7 group, as muscone is added on day 0, 1, and 3, respectively. On Day 7 after RANKL stimulation, TRAP staining is performed. (E) On day 7, mRNA expression in BMMs, including MMP-9, Cathepsin K, CTR, TRAF6, TRAP, and NFATc1, are detected by RT-PCT for each group. P < 0.05 vs. the control group, #P < 0.05 vs. the RANKL-induced group.

Figure 5

Muscone suppresses RANKL-induced signaling pathways in osteoclastogenesis. (A, B) Immunofluorescence staining shows that muscone inhibits RANKL-induced P65 nuclear translocation. (C, D) Muscone suppresses phosphorylation of factors in the NF-kB pathway, including P65, P50, and IkB protein. And muscone also blocks phosphorylation of factors in the MAPK pathways in osteoclastogenesis, including ERK, JNK, and P38. *P < 0.05 vs. the control group, #P < 0.05 vs. the RANKL-induced group.

Figure 6

Muscone suppresses the RANKL-induced RANK associated down-stream gene expression. By RT-PCR (A) and western blot (B), muscone firstly suppresses the TRAF6 and the over-expressed NFATc1. And then, muscone withdraws expression levels of MMP-9, Cathepsin K, TRAP, and CTR in RAW264.7 cells. *P < 0.05 vs. the control group, #P < 0.05 vs. the RANKL-induced group.

Figure 7

Muscone inhibits RANKL-induced TRAF6 binding to RANK. (A, B) After induction of M-CSF (20 ng/ml) and RANKL (50 ng/ml) on day 0, BMMs and RAW264.7 cells are divided into groups according to the different times that 40 μM muscone is added (day 1-7 group, day 3-7 group, and day 5-7 group, since muscone is added on day 1, 3, and 5, respectively). On day 7, RANK and TRAF6 mRNA expression in BMMs are detected by RT-PCR for each group. (C, D) RAW264.7 cells are stimulated, with or without RANKL (100 ng/ml) for 20 min, and with or without muscone (40 μM) for 6 h. Cells are then subjected to immunofluorescence staining. The nucleus percentage counting of immunofluorescence staining indicates that muscone significantly downregulated the expression of TRAF6 protein rather than RANK. (E) To verify the result, coimmunoprecipitation shows that TRAF6 protein levels of RAW264 cells treated with vehicle or muscone (40 μM) with or without RANKL, measured by Western blot analysis. Input: TRAF6 and IB: RANK was used as loading controls. (F) Schematic diagram of the mechanism by which muscone ameliorates RANKL-induced osteoclastogenesis via blocking the binding of TRAF6 to RANK. *P < 0.05 vs. the control group, #P < 0.05 vs. the RANKL-induced group.

Figure 8

Muscone prevents bone loss in OVX mice by suppressing osteoclastogenesis. (A, C) Mice are sacrificed 6 weeks after operation, and H&E staining of femoral shows that the OVX group has a significant trabecular bone loss, and the muscone group has more trabecular area. (B, D) TRAP staining shows that muscone significantly depresses osteoclasts differentiation. (E) For each femur, 100 section planes from the growth plate are scanned, and bone structure images in two-dimensional and three-dimensional version are reconstructed. (F) Bone mineral density (BMD), bone volume/total volume (BV/TV), bone surface area/total volume (BS/TV), and trabecular number (Tb.N), within the selected metaphyseal region, are showed in the charts. (G) TNF-a, IL-6, TRAcp5B, and CTX-1 are examined in serum. *P < 0.05 vs. the control group, ^#P < 0.05 vs. the OVX group.