Osthole promote differentiation and inhibit proliferation of osteoblast by activating wnt signaling and endoplasmic reticulum stress

Background: Osthole is extracted from Fructus Cnidii and is proved to be effective in the treatment of osteoporosis in rats. However, data are still scarce and the mechanism remains elusive. Objective: To investigate the effect of Osthole on proliferation and differentiation of osteoblast. Materials and Methods: Cells were divided into five groups: control group, β-estradiol group (10 −8 M), and Osthole groups (10 −6 M, 10 −5 M, and 10 −4 M). Osteoblast proliferation was evaluated by Cell Counting Kit-8 (CCK-8) assay. Alkaline phosphatase (ALP) activity was detected by ALP staining and enzymatic measurement. Mineralization was detected by alizarin-red staining. The level of osteocalcin was measured by enzyme-linked immunosorbent assay (ELISA). The expression of key proteins of Wnt/β-catenin signaling pathway and endoplasmic reticulum stress (ERS) was analyzed by Western blot. Results: Cell proliferation was retarded in moderate-dose and high-dose Osthole group at 1d, 2d and 3d and in low-dose Osthole group at 1d and 2d ( P P P P Conclusion: It can be concluded that the effect of Osthole on inhibition of proliferation is relevant with activation of ERS, and activation of Wnt/β-catenin signaling pathway is one of the mechanisms how Osthole promotes osteoblast differentiation. In summary, this study provided more evidence for Osthole as a potential anti-osteoporosis medicine. Abbreviations used: ALP: Alkaline phosphatase; ELISA: Enzyme-linked immunosorbent assay; CCK-8: Cell Counting Kit-8; ERS: endoplasmic reticulum stress; DMEM: Dulbecco's Modified Eagle Medium; α-MEM: α-minimum essential medium; EDTA: trypsin/ethylenediaminetetraacetic acid; DMF: N,N-dimethylformamide; TBS-T: Tris-buffered saline-Tween 20; RIPA: Radio Immuno Precipitation Assay; HRP: Horseradish peroxidase; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; BCA: Bicinchoninic acid; ANOVA: Analysis of variance.