Calycosin-7-O-β-D-glucopyranoside Stimulates Differentiation Of ST2Cells Into Osteoblast Through BMP/Wnt Signaling Pathway

Bone is a connective tissue which contains two distinct cell lineages, osteoblast and osteoclast, by which maintain the mineral homeostasis inside our body. Any abnormal interaction between osteoblast and osteoclast could cause imbalance between the formation and resorption of bone, thus induced various bone-related diseases including osteoporosis. To date, osteoporosis has been the major health concerns in the aging society.Astragali radix (AR) has a wide range of pharmacological properties which include immunostimulant effects, anti-bacterial and antiviral properties, and so on. It was reported that AR extracts, polysaccharide aqueous solution and flavonoids extracts also were useful for osteoporosis. Calycosin-7-O-β-D-glucopyranoside (CG) is one of the main flavonoids derived from AR, and it was reported that CG had an antiviral effect, but there has been no report on the relationship between CG and osteogenic activity.This paper investigated the affect and mechanism of CG on differentiation of mouse bone marrow stromal cell line ST2into osteoblast. The experimental result showed that:(1) CG (4-32μM) had no effect of the cell proliferation and had no toxic effect for the cells.(2)Alkaline phosphatase (ALP) is an early marker of osteoblast differentiation, and mineralized nodules are the osteoblast differentiation marker in the late period. CG (4-32μM) could not only significantly increase the ALP activity of culture supernatant, but also promote osteoblast mineralization in a dose-dependent manner.(3) BMP and Wnt signaling pathways are two tightly interacted pathways of osteoblast differentiation. CG could significantly up-regulated the mRNA expression of bone morphogenetic protein-2which was an important factor in BMP signaling, and could cause rapid phosphorylation of Smadl/5/8. Noggin (the inhibitor of BMP pathway) could inhibit the above-mentioned effects of CG inducing. Accordingly, CG also significantly up-regulated the mRNA and protein expression of β-catenin which was an important factor in Wnt signaling. DKK-1(the inhibitor of Wnt pathway) could inhibit this effect of CG inducing.(4) Runx2is an important regulatory factor of osteoblast differentiation. CG significantly up-regulated the mRNA and protein expression of Runx2. OCL is an importantly osteogenic gene in the downstream of Runx2, and CG also significantly up-regulated the mRNA expression of OCL.Above all, the research of this paper showed that CG could stimulate differentiation of ST2cells into osteoblast through BMP/Wnt signaling pathway.