The role of estrogens and estrogen-like molecules in regulating bone cell activities is central to understanding the etiology and treatment of postmenopausal osteoporosis. 1,2) In the past few years, natural estrogen-like molecules, the phytoestrogens, have attracted attention for their potential roles in osteoporosis prevention and treatment. 3) In contrast to the isoflavone-type phytoestrogens such as genistein, lignan-type phytoestrogens have rarely been studied, although they are widely distributed in the plant kingdom. Cereals, grains, berries, and garlic are good dietary sources of lignans. Recently, lignans have received considerable attention for their potential role in the prevention of osteoporosis.4) Lignans are metabolized into the more biologically active mammalian lignans enterodiol and enterolactone by microbiota in the colon.
5)Osteoblasts play a crucial role in bone formation through proliferation and differentiation. Osteoblast differentiation, an important process for its function, confers marked rigidity and strength on bone while still maintaining some degree of elasticity. Thus stimulation of osteoblast differentiation has been suggested to be an important therapeutic approach for the prevention and treatment of bone disease such as osteoporosis.6,7) While the isoflavone genistein 8) and arylnaphthalene-type lignans 9) have been shown to increase the differentiation of osteoblast-like cells, the effect of enterolignans, enterodiol and enterolactone, are unknown. Therefore we carried out the present research to determine whether enterodiol and enterolactone ( Fig. 1) have stimulatory effects on osteoblast differentiation.
MATERIALS AND METHODSCell Culture MG-63 cells (purchased from the American Type Culture Collection), a human osteoblast-like cell line, were used to assess the cellular responses to enterolignans. Cells were cultured in 25 cm 2 flasks in Dulbeco's modified Eagle's medium (DMEM, Gibco) containing 10% heatinactivated fetal bovine serum, L-glutamine 2 mM, penicillin 100 U/ml, and streptomycin 100 mg/ml. Cultures were maintained at 37°C in a humidified atmosphere containing 5% CO 2 . When digested, the cells were washed with phosphatebuffered saline (PBS, Gibco), detached with trypsin-EDTA solution (0.25% trypsin, Gibco) at 37°C for 5 min, centrifuged, and resuspended for further cell tests.Cytotoxicity Assay Following incubation of cells with enterolactone and enterodiol (purchased from Sigma) for specified times in 96-well plates, 20 ml of PBS containing 5 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma) was added to each well. Plates were incubated at 37°C for 4 h, then the medium was replaced with 150 ml of dimethyl sulphoxide (DMSO, Sigma). After 10-min incubation, absorbance in control and treated wells was measured in a plate reader at 490 nm.Alkaline Phosphotase Test Alkaline phosphotase (ALP) activity was measured colorimetrically using disodium phenyl phosphate as the substrate. The enzyme ALP expressed by the cells hydrolyzed the su...