Icariin promotes osteogenic differentiation by suppressing Notch signaling

Osteoporosis is a bone disease characterized by microarchitectural deterioration, low bone mass, and increased risk of fractures. Icariin (ICA), an active flavonoid glucoside isolated from Herba epimedii (HEF), is a potent stimulator of osteogenic differentiation and has potential applications for preventing bone loss in postmenopausal women. However, the molecular mechanism underlying the osteogenic effect of ICA has not yet been fully elucidated. In this study, we report that ICA treatment significantly elevated gene expression of osteogenic markers and increased alkaline phosphatase (ALP) activity in MC3T3-E1 and C3H10T1/2 cells. RNA sequencing revealed that the expression of several genes involved in the Notch pathway was decreased following ICA treatment. Real-time PCR further demonstrated that the mRNA levels of Notch ligands Jagged-1 (Jag1), lunatic fringe (Lfng), and Notch signaling downstream target gene Hey-1 were significantly decreased following ICA treatment. In addition, we found that constitutive activation of Notch signaling through overexpression of the intracellular domain of Notch (NICD) fully blocked ICA-induced osteoblast differentiation. Moreover, inhibiting Notch signaling with DAPT markedly enhanced osteogenic differentiation following ICA treatment. We found that the mRNA levels of Notch pathway molecules (Lfng, Notch1, Rbpjk and Nfatc1) were increased in ovariectomized (OVX) mice, and administration of ICA significantly decreased the expression of these genes. Our results suggest that ICA promotes osteogenic differentiation in vitro and alleviates osteoporosis in vivo through inhibition of the Notch signaling pathway.