Specnuezhenide Alleviates Senile Osteoporosis by Activating TGR5/FXR Signaling in Bone Marrow Mesenchymal Stem Cells and RANKL-Induced Osteoclasts

Xuehui Deng,1,2,* Bingfeng Lin,1,* Wenlong Xiao,3 Fang Wang,2 Pingcui Xu,1 Nani Wang1– 3 1Department of Medicine, Zhejiang Academy of Traditional Chinese Medicine, Hangzhou, Zhejiang, People’s Republic of China; 2School of Pharmacy, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, People’s Republic of China; 3School of Pharmacy, Hangzhou Medical College, Hangzhou, Zhejiang, People’s Republic of China*These authors contributed equally to this workCorrespondence: Nani Wang, Department of Medicine, Zhejiang Academy of Traditional Chinese Medicine, 132 Tianmushan Road, Hangzhou, Zhejiang, 310007, People’s Republic of China, Tel/Fax +86-571-88849089, Email [email protected]: Specnuezhenide (SPN) is an iridoid glycoside isolated from Fructus Ligustri Lucidi, an herb prescribed for the treatment of senile osteoporosis. However, the direct role of SPN on bone metabolism remains unclear. In this study, the effects of SPN on d-galactose (d-gal)-induced mice, bone marrow mesenchymal stem cells (BMSCs), and nuclear factor-κB ligand-induced osteoclasts were examined.Methods: Micro-computed tomography was used to observe the bone microstructure. Osteogenesis was examined using Western blotting and alkaline phosphatase staining. Osteoclastogenesis was examined using Western blotting and F-actin ring staining. Senescence-associated β-galactosidase was used to detect cell senescence. In addition, the expression of Takeda G protein-coupled receptor 5 (TGR5)/farnesoid X receptor (FXR) signaling pathway-related genes and proteins was determined through quantitative real-time polymerase chain reaction and immunofluorescence.Results: Oral administration of SPN improved the bone microstructure in d-gal-induced mice and increased bone mineral density, bone volume, trabecular thickness, and trabecular number. SPN also upregulated the expression of the osteogenesis markers osteocalcin, bone morphogenetic protein 2, and runt-related transcription factor 2 and downregulated the expression of the osteoclasis markers tartrate-resistant acid phosphatase, nuclear factor-κB, and nuclear factor of activated T-cells in the d-gal-induced bone. Furthermore, SPN increased alkaline phosphatase staining, inhibited F-actin ring formation, and reduced the activity of senescence-associated β-galactosidase in vitro. Mechanistically, SPN activated the TGR5/FXR pathway in d-gal-induced BMSCs and osteoclasts. The protective effects of SPN were abolished after addition of the TGR5 inhibitor SBI-115 or FXR inhibitor DY268. Moreover, SPN could elevate the protein and mRNA levels of TGR5, FXR, and the downstream small heterodimer partner in d-gal-induced bone.Conclusion: SPN alleviated senile osteoporosis and cell senescence by activating the TGR5/FXR pathway.Keywords: specnuezhenide, Takeda G protein-coupled receptor 5, farnesoid X receptor, osteoporosis