Spatholobus suberectus Stem-Derived Extracellular Vesicle-Like Particles Attenuate Glucocorticoid-Induced Osteoporosis via NRF2/HO-1 Signaling

Guangmou Chen,1, Hongqiang Li,1,2, Rihao Chen,1,2, Yuan Yao,2 Guan Lian,2 Liping Bai,2 Haosen Chen,1 Chong Zhang,3 Guanghua Chen1 1Orthopedic Center, Affiliated Hospital of Guangdong Medical University, Zhanjiang, Guangdong, People’s Republic of China; 2The First School of Clinical Medicine, Affiliated Hospital of Guangdong Medical University, Zhanjiang, Guangdong, People’s Republic of China; 3Zhanjiang Institute of Clinical Medicine, Central People’s Hospital of Zhanjiang, Guangdong Medical University, Zhanjiang, Guangdong, People’s Republic of ChinaThese authors contributed equally to this workCorrespondence: Guanghua Chen, Email chenguanghua@gdmu.edu.cn Chong Zhang, Email zhangchong1992@126.comPurpose: Glucocorticoid-induced osteoporosis (GIOP) is characterized by impaired osteoblast function and disrupted bone homeostasis during prolonged glucocorticoid exposure. This study investigated whether Spatholobus suberectus stem-derived extracellular vesicle-like particles (SS-EVLP) could attenuate glucocorticoid-induced osteogenic impairment and explored the potential involvement of NRF2/HO-1 signaling.Methods: SS-EVLP were isolated by ultracentrifugation and characterized by transmission electron microscopy, nanoparticle tracking analysis, and Fourier-transform infrared spectroscopy. Cellular uptake was evaluated using PKH26 labeling. In vitro, MC3T3-E1 pre-osteoblastic cells were exposed to dexamethasone (DEX) and treated with SS-EVLP. Cell viability, apoptosis, alkaline phosphatase (ALP) activity, mineralization, and protein expression were assessed by CCK-8 assay, flow cytometry, staining assays, immunofluorescence, and Western blotting. In vivo, the effects of SS-EVLP were examined in a prednisolone-induced zebrafish model by analyzing particle distribution, skeletal mineralization, osteoblast-associated signals, ALP activity, and osteogenic- and antioxidant-related gene expression.Results: SS-EVLP exhibited vesicle-like spherical morphology and were mainly distributed between approximately 100 and 200 nm, with a major peak at 146 nm. SS-EVLP were efficiently taken up by MC3T3-E1 cells and showed low cytotoxicity. In DEX-treated cells, SS-EVLP restored cell viability, reduced apoptosis, and partially recovered ALP staining and mineralized nodule formation. The reduced expression of RUNX2, SP7, HO-1, and NRF2 in DEX-treated cells was partially restored by SS-EVLP, accompanied by increased nuclear NRF2 signal. In zebrafish, SS-EVLP were detectable in vivo after microinjection, attenuated prednisolone-induced skeletal defects, restored ALP activity, and increased the expression of runx2a, sp7, nrf2, ho-1, gclc, gstp, and nqo1.Conclusion: SS-EVLP attenuated glucocorticoid-induced osteogenic impairment in vitro and in vivo, at least partly through NRF2/HO-1 signaling. These findings support the potential of SS-EVLP as a plant-derived nanovesicle candidate for GIOP intervention.Keywords: bone loss, pre-osteoblasts, antioxidant response, zebrafish model, herbal nanovesicles