Orexin Regulates Osteogenic Differentiation of MC3T3-E1 Cells Through TGFβ/Smad Signal Pathway

Osteoblast cell secretes bone matrix to facilitate mineral formation for osteogenesis and is thus of critical importance for bone homeostasis. Orexin (Ox) is a neuropeptide secreted by the hypothalamus to stimulate appetite. However, whether orexin participates in osteoclast development has not been illustrated. In vitro cultured osteoblast cell line MC3T3-E1 was randomly assigned into control and Ox group, in which 50 mu g/ml ascorbic acid was added for 2-week osteogenic induction, followed by treatment with 20 mu g/ml Ox. After 6 h and 12 h incubation, MTT method was used to measure proliferation of MC3T3-E1 cells, and alizarin red staining analyzed the formation of mineral nodules. Activity of alkaline phosphatase (ALP) was measured by enzyme linked immunosorbent assay (ELISA). Real-time PCR detected the expression of osteogenic genes RUNX2 and OPN mRNA, and Western blot quantified change of TGF beta/Smad signal pathway. Ox facilitated MC3T3-E1 cell proliferation and enhanced ALP activity, with potentiated formation of mineral nodules, accompanied with elevated mRNA expression of RUNX2 and OPN, plus up-regulation of TGF beta 1, Smad2 and Smad7 proteins (P < 0.05 comparing to control group). These effects were more significant with extended culture time (P < 0.05). In conclusion, Ox facilitates osteogenic differentiation of MC3T3-E1 cells possibly via mediating TGF beta/Smad signal pathway.