Puerarin specifically disrupts osteoclast activation via blocking integrin-β3 Pyk2/Src/Cbl signaling pathway

Zuocheng Qiu; Ling Li; Yuying Huang; Keda Shi; Lizhong Zhang; Cuishan Huang; Jiechao Liang; Qingqiang Zeng; Jiali Wang; Xiangjiu He; Ling Qin; Xinluan Wang

doi:10.1016/j.jot.2022.01.003

Puerarin specifically disrupts osteoclast activation via blocking integrin-β3 Pyk2/Src/Cbl signaling pathway

J Orthop Translat. 2022 Feb 16:33:55-69. doi: 10.1016/j.jot.2022.01.003. eCollection 2022 Mar.

Authors

Zuocheng Qiu^{1

2}, Ling Li¹, Yuying Huang³, Keda Shi¹, Lizhong Zhang¹, Cuishan Huang¹, Jiechao Liang¹, Qingqiang Zeng¹, Jiali Wang⁴, Xiangjiu He³, Ling Qin^{1

5}, Xinluan Wang^{1

5}

Affiliations

¹ Translational Medicine R&D Center, Institute of Biomedical and Health Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518057, China.
² Guangzhou Key Laboratory of Formula-Pattern of Traditional Chinese Medicine, Formula-Pattern Research Center, School of Traditional Chinese Medicine, Jinan University, Guangzhou, 510632, China.
³ School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, 510006, China.
⁴ School of Biomedical Engineering, Sun Yat-sen University, Guangzhou, 510275, China.
⁵ Musculoskeletal Research Laboratory of Department of Orthopaedics & Traumatology, Innovative Orthopaedic Biomaterial and Drug Translational Research Laboratory of Li Ka Shing Institute of Health, The Chinese University of Hong Kong, Hong Kong SAR, China.

Abstract

Objective: Given the limitations of current anti-resorption agents for postmenopausal osteoporosis, there is a need for alternatives without impairing coupling crosstalk between bone resorption and bone formation ie. osteoclastogenesis. Puerarin, a unique C-glycoside isoflavonoid, was found to be able to prevent bone loss by inhibiting bone resorption, but the underlying mechanism was controversial. In this study, we investigated the effects of puerarin on osteoclastic differentiation, activation and bone resorption and its underlying molecular mechanism in vitro, and then evaluated the effects of puerarin on bone metabolism using an ovariectomized (OVX) rat model.

Methods: In vitro, the effect of puerarin on osteoclastic cytotoxicity, differentiation, apoptosis, activation and function were studied in raw 264.7 cells and mouse BMMs. Mechanistically, osteoclast-related makers were determined by RT-PCR, western blot, immunofluorescence, and kinase activity assay. In vivo, Micro-CT, histology, serum bone biomarker, and mechanical testing were used to evaluate the effects of puerarin on preventing osteoporosis.

Results: Puerarin significantly inhibited osteoclast activation and bone resorption, without affecting osteoclastogenesis or apoptosis. In terms of mechanism, the expressions of protein of integrin-β3 and phosphorylations of Src, Pyk2 and Cbl were lower in puerarin group than those in the control group. Oral administration of puerarin prevented OVX-induced trabecular bone loss and significantly improved bone strength in rats. Moreover, puerarin significantly decreased trap positive osteoclast numbers and serum TRAP-5b, CTx1, without affecting bone formation rate.

Conclusions: Collectively, puerarin prevented the bone loss in OVX rat through suppression of osteoclast activation and bone resorption, by inhibiting integrin-β3-Pyk2/Cbl/Src signaling pathway, without affecting osteoclasts formation or apoptosis.

Translational potential of this article: These results demonstrate the unique mechanism of puerarin on bone metabolism and provide a novel agent for prevention of postmenopausal osteoporosis.

Keywords: Amino-terminal propeptide of type I collagen, RANKL; Atp6v0d2, ATPase H + Transporting V0 Subunit D2; BMD, Bone mineral density; BMMs, Bone marrow monocyte cells; BS, Bone surface; BV/TV, Bone volume/tissue volume; Carbonic anhydrase II, Cs. Th; Cathepsin K, E2: 17-β estradiol; Charge-coupled Cl-channel, CA II; Connectivity density, CTSK; Cortical area, CTx1; Cortical thickness, Conn. D; ER, Estrogen receptor; Ec. Pm, Endocortical perimeter; F-actin ring; Integrin-β3; Luminescence, SD; M-CSF, Macrophage colony-stimulating factor; Marrow area, MRM; Matrix metalloproteinase-9, N. Oc; Multiple reaction monitoring, MMP9; Nuclear factor of activated T cells 1, TRAP-5b; Number of trap positive osteoclasts, NFATc1; OVX, ovariectomized; Osteoclast activation; Osteoclast stimulatory transmembrane protein, OPG; Osteoclasts surface, P1NP; Osteoprotegerin, Oc. S; Puerarin; Pyk2/Cbl/Src; Receptor activator of nuclear factor κ-B ligand, RLU; Sprague–Dawley, rBMSC; Src: Proto-oncogene tyrosine-protein kinase Src, Ma. Ar; TRAF6, TNF-receptor-associated factor 6; TRAP, Tartrate resistant acid phosphatase; TUNEL, Terminal deoxynucleotidyl transferase dUTP nick end labeling; Tartrate-resistant acid phosphatase 5b, OC-STAMP; Tb. Sp, Trabecular separation; Tb. Th, Trabecular thickness; c-Fos: Proto-oncogene C-Fos, Ct. Ar; carboxyl-terminal telopeptides of type I collagen, CLCN7; rat bone marrow-derived mesenchymal stem cells. Tb. N, Trabecular number.