Icariin (ICA) is the main active flavonoid glucoside from herbs of the genus Epimedium; in traditional Chinese medicine, these herbs have long been prescribed for the treatment of bone fractures and osteoporosis. Several studies have shown that treatment with ICA can increase osteogenic differentiation and reduce bone loss in vivo and in vitro. However, the definite signaling pathway of this osteogenic effect remains unclear. In this study, we selected bone morphogenetic protein 2 (BMP2)-induced osteoblastic differentiation of multipotent mesenchymal progenitor C2C12 cells as a model of osteoblast differentiation. We investigated the effects of ICA on C2C12 cells osteogenic differentiation and the underlying molecular mechanisms. We found that ICA could enhance BMP2-mediated osteoblastic differentiation of C2C12 cells in a dose-dependent manner. Treatment with ICA activated the cAMP/PKA/CREB signaling axis in a time-dependent manner. Blocking cAMP signaling using the PKA selective inhibitor H89 significantly inhibited the stimulatory effect of ICA on osteogenesis. Therefore, the osteoinductive potential and the low cost of ICA indicate that it is a promising alternative treatment or promoter for enhancing the therapeutic effects of BMP2.

Neurofibromatosis type 1 is a rare autosomal dominant genetic disorder, with up to 50% of patients clinically displaying skeletal defects. Currently, the pathogenesis of bone disorders in NF1 patients is unclear, and there are no effective preventive and treatment measures. In this study, we found that knockout of the NF1 gene reduced cAMP levels and osteogenic differentiation in an osteoblast model, and icariin activated the cAMP/PKA/CREB pathway to promote osteoblast differentiation of the NF1 gene knockout cell model by increasing intracellular cAMP levels. The PKA selective inhibitor H89 significantly impaired the stimulatory effect of icariin on osteogenesis in the NF1 cell model. In this study, an osteoblast model of NF1 was successfully constructed, and icariin was applied to the cell model for the first time. The results will help to elucidate the molecular mechanism of NF1 bone disease and provide new ideas for the clinical prevention and treatment of NF1 bone disease and drug development in the future.