Zixiang Geng,1,2,* Tiancheng Sun,1,2,* Jie Yu,1,2,* Ning Wang,1,2 Qiang Jiang,1,2 Peige Wang,1,2 Guangyue Yang,1,2 Yifei Li,3 Yue Ding,4 Jiange Zhang,5 Guoqiang Lin,5 Yongfang Zhao1,2 

1Shi’s Center of Orthopedics and Traumatology, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, People’s Republic of China; 2Institute of Traumatology and Orthopedics, Shanghai Academy of Traditional Chinese Medicine, Shanghai, 201203, People’s Republic of China; 3Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, People’s Republic of China; 4School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, People’s Republic of China; 5The Research Center of Chiral Drugs, Innovation Research Institute of Traditional, Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Zixiang Geng, Institute of Traumatology and Orthopedics, Shanghai Academy of Traditional Chinese Medicine, Shanghai, People’s Republic of China, Email gengzx@foxmail.com Yongfang Zhao, Shi’s Center of Orthopedics and Traumatology, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, People’s Republic of China, Email zhaoyongfang@shutcm.edu.cn

Background: Cinobufagin, the primary active compound in toad venom, is commonly used for anti-tumor, anti-inflammatory, and analgesic purposes. However, its specific bone-protective effects remain uncertain. This research aims to ascertain the bone-protective properties of cinobufagin and investigate underlying mechanisms.
Methods: Mice were ovariectomized to establish an osteoporosis model, followed by intraperitoneal injections of cinobufagin and cinobufagin-treated RAW.264.7-derived exosomes for therapy. MicroCT, HE staining, and TRAP staining were employed to evaluate bone mass and therapeutic outcomes, while mRNA sequencing and immunoblotting were utilized to assess markers of bone metabolism, inflammation, and lipid peroxidation. Osteoblast and osteoclast precursor cells were differentiated to observe the impact of cinobufagin-treated exosomes derived from RAW264.7 cells on bone metabolism. Exosomes characteristics were studied using transmission electron microscopy and particle size analysis, and miRNA binding targets in exosomes were determined by luciferase reporting.
Results: In ovariectomized mice, cinobufagin and cinobufagin-treated exosomes from RAW264.7 cells increased trabecular bone density and mass in the femur, while also decreasing inflammation and lipid peroxidation. The effect was reversed by an exosomes inhibitor. In vitro experiments revealed that cinobufagin-treated exosomes from RAW264.7 cells enhanced osteogenic and suppressed osteoclast differentiation, possibly linked to Upregulated miR-3102-5p in RAW-derived exosomes. MiR-3102-5p targets the 3′UTR region of alox15, thereby suppressing its expression and reducing the lipid peroxidation process in osteoblasts.
Conclusion: Overall, this study clarified cinobufagin’s bone-protective effects and revealed that cinobufagin can enhance the delivery of miR-3102-5p targeting alox15 through macrophage-derived exosomes, demonstrating anti-lipid peroxidation and anti-inflammatory effects.

Keywords: osteoporosis, cinobufagin, exosomes, miR-3102-5p