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Dextromethorphan inhibits osteoclast differentiation by suppressing RANKL-induced nuclear factor-κB activation

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Abstract

Summary

Dextromethorphan (DXM), a commonly used antitussive, is a dextrorotatory morphinan. Here, we report that DXM inhibits the receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis and bone resorption by abrogating the activation of NF-κB signalling in vitro. Oral administration of DXM ameliorates ovariectomy (OVX)-induced osteoporosis in vivo.

Introduction

DXM was reported to possess anti-inflammatory properties through inhibition of the release of pro-inflammatory factors. However, the potential role and action mechanism of DXM on osteoclasts and osteoblasts remain unclear. In this study, in vitro and in vivo studies were performed to investigate the potential effects of DXM on osteoclastogenesis and OVX-induced bone loss.

Methods

Osteoclastogenesis was examined by the TRAP staining, pit resorption, TNF-α release, and CCR2 and CALCR gene expression. Osteoblast differentiation was analyzed by calcium deposition. Osteogenic and adipogenic genes were measured by real-time PCR. Signaling pathways were explored using Western blot. ICR mice were used in an OVX-induced osteoporosis model. Tibiae were measured by µCT and serum markers were examined with ELISA kits.

Results

DXM inhibited RANKL-induced osteoclastogenesis. DXM mainly inhibited osteoclastogenesis via abrogation of IKK-IκBα-NF-κB pathways. However, a higher dosage of DXM antagonized the differentiation of osteoblasts via the inhibition of osteogenic signals and increase of adipogenic signals. Oral administration of DXM (20 mg/kg/day) partially reduced trabecular bone loss in ovariectomized mice.

Conclusion

DXM inhibits osteoclast differentiation and activity by affecting NF-κB signaling. Therefore, DXM at suitable doses may have new therapeutic applications for the treatment of diseases associated with excessive osteoclastic activity.

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Acknowledgement

The work was supported by a grant from Far Eastern Memorial Hospital—National Taiwan University Hospital Joint Research Program. We thank the NRPB (National Research Program for Biopharmaceuticals) Core Service Center (NSC 100-2312-B-002-007) for μCT analysis. We also thank Chung Gung University Animal Center and Taiwan Mouse Clinic for technical support in μCT experiments.

Conflicts of interest

None.

Author information

Authors and Affiliations

  1. Department of Orthopedics, Far Eastern Memorial Hospital, No. 21, Sec. 2, Nanya S. Rd., New Taipei City 220, Taiwan, Republic of China

    Karl Wu

  2. Department of Pharmacology, College of Medicine, National Taiwan University, No. 1 Sec. 1, Jen-Ai Rd., Taipei 100, Taiwan, Republic of China

    Tzu-Hung Lin, Houng-Chi Liou, Dai-Hua Lu, Yi-Ru Chen & Wen-Mei Fu

  3. Department of Orthopedics, College of Medicine, College of Medicine, National Taiwan University Hospital, No. 7, Zhong-Shang South Road, Taipei 100, Taiwan, Republic of China

    Rong-Sen Yang

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Correspondence to Wen-Mei Fu or Rong-Sen Yang.

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Karl Wu and Tzu-Hung Lin have equally contributed to this study

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Fig. S1

Inhibition of high concentration of RANKL-induced NF-κB activation by dextromethorphan in RAW264.7 cells RAW264.7 cells were incubated with DXM (50 μM) for 30 min and then treated with high concentration of RANKL (100 ng/ml) for several time intervals. Western blot analysis showed that treatment with RANKL rapidly and markedly increased the phosphorylation of IKK and IκBα and enhanced IκBα degradation time dependently. Pretreatment with DXM decreased the activation of IKK and IκBα and inhibited the degradation of IκBα (n = 3) (TIFF 156 kb)

Fig. S2

Induction of adipogenic signals by dextromethorphan in osteoblasts cultured in adipogenic medium osteoblasts were treated with different concentrations of DXM in the presence of adipogenic medium for 3 days. mRNA was extracted by TriZol kit and was then analysed by RT-qPCR. a It was shown that administration of DXM higher than 1 μM significantly increased mRNA of LPL (b) and PPARgamma2 (c) (n = 4). Each value represents mean ± S.E.M. *, p < 0.05 as compared with control group without DXM (TIFF 872 kb)

Fig. S3

Effect of dextromethorphan on OVX-induced osteoclast activation. Bone resorption was determined by the serum level of c-terminal telopeptides of type-I collagen (CTX). The serum was collected from left ventricle 5 weeks after OVX surgery. DXM was put in drinking water and the dosage was evaluated by the quantity of water consumption. a DXM (20 mg/kg/day) significantly antagonized OVX-induced increase of serum level of osteoclastic marker. In addition, DXM also antagonized the increase of osteoclast number in proximal tibia. Representative TRAP staining images were shown in b and the quantitative data were shown in c. Scale bar: 100μm (n = 4–9). Each value represents mean ± S.E.M. *p < 0.05 as compared with Sham group. #p < 0.05 as compared with OVX alone group (TIFF 2,915 kb)

Fig. S4

Dextromethorphan suppresses RANKL-induced TNF-α production in RAW264.7 cells. RAW264.7 cells were pretreated with different concentrations of DXM for 2 h before the addition of RANKL (10 ng/ml). The levels of TNF-α in the supernatant were measured by using TNF-α ELISA kit 16 h later. Each value represents the mean ± SEM of three independent experiments with triplicate cultures. *p < 0.05 as compared with RANKL-free control. #p < 0.05 as compared with RANKL-treatment alone group (TIFF 206 kb)

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Wu, K., Lin, TH., Liou, HC. et al. Dextromethorphan inhibits osteoclast differentiation by suppressing RANKL-induced nuclear factor-κB activation. Osteoporos Int 24, 2201–2214 (2013). https://doi.org/10.1007/s00198-013-2279-8

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