Research Paper Volume 12, Issue 21 pp 21706—21729

Puerarin alleviates osteoporosis in the ovariectomy-induced mice by suppressing osteoclastogenesis via inhibition of TRAF6/ROS-dependent MAPK/NF-κB signaling pathways

Long Xiao1,2, *, , Mengdan Zhong3, *, , Yu Huang4,5, , Jie Zhu1, , Wenkai Tang1, , Danyong Li1, , Jiandong Shi1, , Aiqing Lu1, , Huilin Yang2, , Dechun Geng2, , Hong Li3, , Zhirong Wang1, ,

  • 1 Department of Orthopedics, Zhangjiagang TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Zhangjiagang 215600, China
  • 2 Department of Orthopedics, The First Affiliated Hospital of Soochow University, Suzhou 215006, China
  • 3 Department of Endocrinology, Zhangjiagang TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Zhangjiagang 215600, China
  • 4 Department of Gynecology, The First Affiliated Hospital of Soochow University, Suzhou 215006, China
  • 5 Department of Gynecology, The First People's Hospital of Zhangjiagang, Soochow University, Zhangjiagang 215600, China
* Equal contribution

Received: January 15, 2020       Accepted: August 1, 2020       Published: November 7, 2020      

https://doi.org/10.18632/aging.103976
How to Cite

Copyright: © 2020 Xiao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

In this study, we investigated the mechanisms by which puerarin alleviates osteoclast-related loss of bone mass in ovariectomy (OVX)-induced osteoporosis model mice. Puerarin-treated OVX mice exhibited higher bone density, fewer tartrate-resistant acid phosphatase (TRAcP)-positive osteoclasts, and levels of lower reactive oxygen species (ROS) within bone tissues than vehicle-treated OVX mice. Puerarin suppressed in vitro osteoclast differentiation, hydroxyapatite resorption activity, and expression of osteoclastogenesis-related genes, such as NFATc1, MMP9, CTSK, Acp5 and c-Fos, in RANKL-induced bone marrow macrophages (BMMs) and RAW264.7 cells. It also reduced intracellular ROS levels by suppressing expression of TRAF6 and NADPH oxidase 1 (NOX1) and increasing expression of antioxidant enzymes such as heme oxygenase-1 (HO-1). Puerarin inhibited TRAF6/ROS-dependent activation of the MAPK and NF-κB signaling pathways in RANKL-induced RAW264.7 cells, and these effects were partially reversed by HO-1 silencing or TRAF6 overexpression. These findings suggest puerarin alleviates loss of bone mass in the OVX-model mice by suppressing osteoclastogenesis via inhibition of the TRAF6/ROS-dependent MAPK/NF-κB signaling pathway.

Abbreviations

Acp5: Acid phosphatase 5, tartrate resistant; BMD: Bone mass density; BMMs: bone marrow macrophages; BV/TV: bone volume per tissue volume; BS/BV: bone surface/bone volume; BS/TV: bone surface/total volume; c-Fos: proto-oncogene C-Fos; CTSK: cathepsin K; CCK-8: Cell Counting Kit-8; Conn.Dn: connectivity density; DAPI: 4,6-diamidino-2-phenylindole; DCFH-DA: 2’,7’-dichlorodihydrofluorescein diacetate; DHE: dihydroethidium; DMSO: Dimethylsulfoxide; DMEM: Dulbecco's Modified Eagle Medium; EDTA: Ethylenediaminetetraacetic acid; HO-1: Heme oxygenase-1; H&E: Hematoxylin and eosin; IκB-α: Inhibitor kappa B-alpha; IHC: Immunohistochemistry; MAPKs: Mitogen-activated protein kinases; M-CSF: Macrophage-Colony Stimulating Factor; MMP9: matrix metallopeptidase 9; NF-κB: Nuclear factor-κB; NOX: NADPH oxidase; NFATc1: Nuclear factor of activated T cells 1; OD: Optical density; OPG: Osteoprotegerin; OVX: Ovariectomized; PBS: phosphate buffered saline; PCR: Polymerase Chain Reaction; ROS: Reactive Oxygen Species; RANKL: Receptor Activator of Nuclear Factor-κB Ligand; Tb.N: trabecular number; Tb.Th: trabecular thickness; TRAcP: Tartrate Resistant Acid Phosphatase; TRAF6: TNF Receptor-Associated Factor 6; α-MEM: Alpha-modified Minimal Essential Medium.