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Carnosic acid attenuates RANKL-induced oxidative stress and osteoclastogenesis via induction of Nrf2 and suppression of NF-κB and MAPK signalling

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Abstract

Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor, which plays an important role in the cellular defense against oxidative stress by induction of anti-oxidant and cytoprotective enzymes. In the current study, we sought to investigate the osteoprotective effect of carnosic acid (CA), a phenolic (catecholic) diterpene. It is widely identified for its electrophilic nature under oxidative stress conditions and thus anticipated to counter osteoporosis by facilitation of Nrf2 signalling. Osteoclast differentiation was induced by incubation of RAW 264.7 (mouse macrophage) cells and mouse bone marrow macrophages (BMMs) in the presence of receptor activator of NF-κB ligand (RANKL) (100 ng/ml). After treatment, osteoclastogenesis was assessed using tartrate-resistant acid phosphatase (TRAP) assay. We observed that 6 h pretreatment with CA (1.25, 2.5, 5 μM) decreased RANKL-induced osteoclast formation and abolished RANKL-induced ROS generation by activating Nrf2 and its transcriptional targets. Further, CA also inhibited RANKL-induced activation of NF-κB and MAPK signalling. RANKL-induced mRNA expression of osteoclast related genes and transcription factors was also diminished by CA. In vivo osteolysis was developed in C57BL/6 male mice using lipopolysaccharide (LPS). Consistent with in vitro results, in vivo μ-CT analysis of femurs showed that bone mineral density (BMD), bone mineral content (BMC), and bone architecture parameters such as trabecular thickness (Tb.Th) and trabecular space (Tb.Sp) were positively modulated by CA in LPS-injected mice. The results obtained in this study support that CA inhibits RANKL-induced osteoclastogenesis by maintaining redox homeostasis through modulation of Nrf2 and NF-κB pathways.

Key Messages

  • Carnosic acid (CA) inhibits RANKL-induced osteoclastogenesis.

  • CA inhibits RANKL-induced oxidative stress by upregulating Nrf2 transcriptional targets.

  • CA attenuates RANKL-induced NF-κB and MAPK signalling activation.

  • CA decreases NFATc1 and c-Fos expression.

  • In vivo μ-CT analysis reveals that CA prevents bone loss in LPS-injected mice.

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Acknowledgements

Financial support from DBT (Sanction order number: BT/SBIRI-1188/SBIRI-23/13) and Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, Government of India is acknowledged.

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Authors and Affiliations

  1. Department of Pharmacology & Toxicology, National Institute of Pharmaceutical Education and Research-Hyderabad, Balanagar, Hyderabad, Andhra Pradesh, 500 037, India

    Dinesh Thummuri & V. G. M. Naidu

  2. Department of Pharmacology & Toxicology, National Institute of Pharmaceutical Education and Research-Guwahati, Guwahati, Assam, 781 032, India

    V. G. M. Naidu

  3. Comparative Oncology Program & Small Animal Imaging Facility, Advanced Center for Treatment, Research & Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai, 410 210, India

    Pradip Chaudhari

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  1. Dinesh Thummuri
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Correspondence to V. G. M. Naidu.

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Thummuri, D., Naidu, V.G.M. & Chaudhari, P. Carnosic acid attenuates RANKL-induced oxidative stress and osteoclastogenesis via induction of Nrf2 and suppression of NF-κB and MAPK signalling. J Mol Med 95, 1065–1076 (2017). https://doi.org/10.1007/s00109-017-1553-1

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  • DOI: https://doi.org/10.1007/s00109-017-1553-1

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