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Glycyrrhizin Suppresses RANKL-Induced Osteoclastogenesis and Oxidative Stress Through Inhibiting NF-κB and MAPK and Activating AMPK/Nrf2

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Abstract

The treatment for osteoporosis involves inhibiting bone resorption and osteoclastogenesis. Glycyrrhizin (GLY) is a triterpenoid saponin glycoside known to be as the most medically efficacious component of the licorice plant. It has strong anti-inflammatory, antioxidant, and antitumor properties. We investigated the effect of GLY on osteoclastogenesis, bone resorption, and intracellular oxidative stress and its molecular mechanisms. In vitro osteoclastogenesis assays were performed using bone marrow monocytes with and without glycyrrhizin. We also evaluated the effects of glycyrrhizin on the secretion of TNF-α, IL-1β, and IL-6 in LPS-stimulated RAW 264.7 cells using ELISA. The effects of glycyrrhizin on the expression of osteoclast-related genes, such as Nfatc1, c-fos, Trap, and cathepsin K (CK), were investigated by RT-PCR. Intracellular reactive oxygen species (ROS) were detected in receptor activator of nuclear factor kappa-Β ligand (RANKL)-stimulated osteoclasts in the presence and absence of glycyrrhizin. During the inhibition of osteoclastogenesis by glycyrrhizin, phosphorylation of AMPK, Nrf2, NF-κB, and MAPK was analyzed using western blotting. Our results showed that glycyrrhizin significantly inhibited RANKL-induced osteoclastogenesis, downregulated the expression of NFATc1, c-fos, TRAP, CK, DC-STAMP, and OSCAR, and inhibited p65, p38, and JNK. Glycyrrhizin was found to significantly decrease the secretion of inflammatory cytokines (TNF-α, IL-1β, and IL-6). Additionally, glycyrrhizin reduced the formation of ROS in osteoclasts by inducing AMPK phosphorylation and nuclear transfer of NRF2, resulting in an upregulation of antioxidant enzymes, such as HO-1, NQO-1, and GCLC. In summary, we found that glycyrrhizin inhibited RANKL-induced osteoclastogenesis. It was also indicated that glycyrrhizin could reduce oxidative stress by inhibiting the MAPK and NF-κB pathways and activating the AMPK/NRF2 signaling. Therefore, glycyrrhizin may be used as an effective therapeutic agent against osteoporosis and bone resorption.

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Abbreviations

AMPK:

AMP-activated protein kinase

ROS:

Reactive oxygen species

NFATc1:

Nuclear factor of activated T cells 1

JNK:

c-Jun N-terminal kinase

NF-κB:

Nuclear factor kappa B

Nrf2:

Nuclear factor-erythroid 2-related factor 2

HO-1:

Heme oxygenase-1

TNF-α:

Necrosis factor-α

RANKL:

Receptor activator of nuclear factor kappa-B (NF-κB) ligand

MAPK:

Mitogen-activated protein kinase

M-CFS:

Macrophage colony-stimulating factor

TRAP:

Tartrate-resistant acid phosphatase

BMM:

Bone marrow monocytes

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

CK:

Cathepsin K

CTR:

Calcitonin receptor

DC-STAMP:

Dendrocyte expressed seven transmembrane protein

OSCAR:

Osteoclast-associated immunoglobulin-like receptor

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Acknowledgements

We thank the staff at Department of Orthopedic, Tongren Hospital, Shanghai Jiao Tong University School of Medicine.

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Correspondence to Wei Xu.

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Conflict of interest

Zhikun Li, Chao Chen, Xiaodong Zhu, Yifan Li, Ronghua Yu, Wei Xu declare that they have no conflict of interest.

Human and Animal Rights and Informed Consent

This study was ethically approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University School of Medicine and performed in accordance with the criteria defined by the rules of the committee.

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Li, Z., Chen, C., Zhu, X. et al. Glycyrrhizin Suppresses RANKL-Induced Osteoclastogenesis and Oxidative Stress Through Inhibiting NF-κB and MAPK and Activating AMPK/Nrf2. Calcif Tissue Int 103, 324–337 (2018). https://doi.org/10.1007/s00223-018-0425-1

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