Icariin inhibits osteoblast ferroptosis via Nrf2/HO-1 signaling and enhances healing of osteoporotic fractures

The incidence of osteoporotic fractures is increasing every year because of population aging around the world. The reduced osteoblast activity in osteoporotic fracture has been associated with ferroptosis. A recent study showed that the antioxidant icariin (ICA) reduced iron deposition in the bone marrow of osteoporotic mice, although the underlying regulatory mechanisms were not explored. The objective of present study was to assess the therapeutic effects of ICA in a rat osteoporotic fracture model, with particular focus on its impact on ferroptosis. Primary rat osteoblasts were exposed to the ferroptosis inducer erastin, and then treated with ICA or the ferroptosis inhibitor ferrostatin-1 (Fer-1) as the positive control group. The levels of Nrf2 signaling factors and osteogenesis-related factors were examined by RT-PCR and western blotting. An osteoporotic fracture model was established in rats, and the effect of ICA on bone formation was evaluated by X-ray, Micro CT analysis, histological examination and Safranin O staining. Furthermore, the levels of GPX4, Bax, Nrf2 and Runx2 proteins at the fracture site were examined by immunohistochemistry. ICA significantly reduced ROS levels in the erastin-treated osteoblasts, and downregulated glutathione peroxidase 4 (GPX4) and cystine glutamate antiporter (SLC7A11). Moreover, ICA also upregulated Nrf2, NQO-1, HO-1, Runx2, ALP, OPG and OCN in these cells, which was reversed by inhibitors of the Nrf2 signaling pathway and Nrf2 silencing. X-ray and Micro CT analysis showed that ICA increased the trabecular bone and promoted callus formation in the osteoporotic fracture model, and also enhanced the transition from fibrous to osseous callus. Furthermore, ICA upregulated GPX4, Nrf2 and Runx2 at the fracture site, and significantly reduced the expression of the apoptotic genes of Bax. Taken together, our findings indicate that ICA promotes osteoporotic fracture healing by inhibiting osteoblast ferroptosis via activation of the antioxidant Nrf2/HO-1 signaling pathway.