Dose-dependent Effects Of Daidzein In Regulating Bone Formation Through Estrogen Receptors And Peroxisome Proliferator-activated Receptor Gamma

BackgroundThe incidence of postmenopausal osteoporosis is increasing year by year, although estrogen replacement therapy can reduce the bone loss of postmenopausal women,and reduce the incidence of fracture, due to high risk of breast cancer and endometrial carcinoma caused by long-term use of estrogen, its application is restricted. Daidzein is a common kind of phytoestrogens, has estrogen-like effects and no estrogen-like side effects, caused widespread concern in recent years.Recent studies suggest that phytoestrogen can protect bone mass,and shows biphasic dose-dependent effect, the mechanism is still unclear. Some researches have revealed that estrogen receptors and peroxisome proliferator-activated receptor gamma may mediate the dose-dependent bone-protective effect,but related reports are less,and the specific mechanism is not clear yet.Part?Dose-dependent effects of daidzein on osteoporosis in the ovariectomized rats,and its effects on the expression of mRNA and protein of ERs and PPAR?in bone marrow cellsObjectiveTo investigate the effects of daidzein on the serum levels of E2?ALP?adiponectin?leptin, bone mineral density, and the expressions of mRNA and protein of ER??ER?and PPAR?in bone marrow cells of the ovariectomized rats, and discuss the dose-dependent effects and the molecular mechanisms of daidzein on osteoporosis in the ovariectomized rats. Materials and methodsSeventy three-month-old Sprague-Dawley (SD) female rats were randomly allotted to one of the following six groups:ovariectomized group, sham-ovariectomized group, E2 group, low-dose daidzein group, medium-dose daidzein group and high-dose daidzein group. The OVX operation was believed success when there were no Keratinocytes in vaginal smear in the next five days from the 7th day after operation. Four weeks after reproducing the osteoporosis model of ovariectomized SD rats, sham group and control OVX were administrated with distilled water, whereas the other groups continuously received valerate estriol (800?g/kg·d) or different doses of daidzein (10,50,200 mg/kg·d) by gavage respectively for three months. These parameters were measured after the sacrifice of the rats.The concentrations of E2, ALP, adiponectin and leptin were detected from serum;bone mineral density of the lumbar spine L1-5 and left femur was measured with a dual energy x-ray absorptiometer. By H-E staining of decalcified bone slices,we made bone histomorphometric analysis.The expressions of mRNA of ER?, ER?and PPAR?in bone marrow cells were measured by realtime-RT-PCR, while proteins were measured by western blot.Results1?The serum concentrations of E2?ALP?adiponectin and leptin in E2 and L-DAI group were significantly higher than in OVX group (P<0.05);while the serum concentrations of E2?ALP?adiponectin in L-DAI group were significantly higher than in E2 group (P<0.05).The serum concentrations of E2?ALP?adiponectin and leptin in H-DAI and M-DAI group showed no significant difference (P>0.05),and were significantly lower than in E2 and L-DAI group (P<0.05)2?The BMD of lumbar spine and left femur in sham group increased significantly than in other groups (P<0.05); these indexes in L-DAI group increased significantly than in OVX group (P<0.05),and there was no significant difference with in E2 group.The BMD of left femur in M-DAI group increased significantly than in OVX group (P<0.05); and showed little difference than in E2 and L-DAI?(P>0.05).The BMD of lumbar spine in M-DAI group has no significant difference than in OVX group (P>0.05).The BMD of lumbar spine and left femur in H-DAI group decreased significantly than in L-DAI group (P<0.05),which showed no significant difference than in OVX group (P>0.05)3?Bone morphology of rat's lumbar spine and left femur:The trabecular bone volume decreased and adipocyte increased significantly.E2 could increase trabecular bone volume and reduce adipocyte number.In the three daidzein groups,L-DAI group had a significant increase in trabecular number and inhibit fat formation;with increasing doses of daidzein, the trabecular bone volume decreased and adipocyte increased significantly.4?The expressions of mRNA and protein of ERa of bone marrow cells in L-DAI group-were higher than M-DAI group (P<0.05),H-DAI group (P<0.05),E2 group (P>0.05), OVX group (P>0.05) and sham group (P>0.05).The expressions of mRNA and protein of PPARy of bone marrow cells in H-DAI group were significantly higher than M-DAI and L-DAI group (P<0.05).The expressions of mRNA and protein of ER?in each group were low and showed no significant difference (P>0.05)Conclusions1?Daidzein shows estrogen-like effect on bone, and shows preventive and curative effect on osteoporosis in ovariectomized rats. The effect is dose-dependent,low-dose of daidzein is as effective as E2,while high-dose shows no bone preventive effect.2?Both ERa and ER?are expressed in bone marrow cells,while ERa is predominant, which implied that the bone-protective effect of daidzein through ERa mainly.3?When daidzein in level of low dose, the level of expression of ERa in bone marrow cells was significantly higher,which implied that the low-dose of daidzein give play to the function of bone protection mainly through ERa.When daidzein in level of high dose, the level of expression of PPARy in bone marrow cells was significantly higher,which implied that the high-dose of daidzein mainly acts on PPARy, causing bone loss and adipocyte hyperplasia in bone marrow cavity. It implied that to regulate the balance between bone formation and adipogenic can become a new target of prevention and treatment of osteoporosis.4?The serum levels of adiponectin and leptin play a protective role on the prevention of osteoporosis.Part?Dose-dependent effects of daidzein on human fetal osteoblast in regulating bone formation through estrogen receptors and peroxisome proliferator-activated receptor gamma.ObjectiveTo investigate the different effect of daidzein at different dosage in regulating bone formation in osteoblasts, and the regulation mechanism mediated by estrogen receptors and peroxisome proliferator-activated receptor gamma.Materials and methods?The human fetal osteoblast (hFOB) incubated without any treatment were served as controls (control group,Ctrl).The hFOB cells were exposed to daidzein for 72 hours at different concentrations of 10-9mol/L?10-7mol/L or 10-5 mol/L, and to E2 at 10-8mol/L as positive control, respectively. MTT assay was employed to determine the proliferation ability of osteoblasts, and nitrophenyl phosphate salt method was applied to determine the activity of alkaline phosphatase (ALP).?Estrogen receptors (ERs) antagonist ICI182780, Estrogen receptor?(ER?) antagonist MPP and PPAR?antagonist GW9662 were used to block the corresponding receptor, while hFOB cells were exposed to E2 or different concentrations of daidzein for 48 hours. MTT assay and PNPP were used separately to observe the proliferation and the activity of ALP of osteoblasts cultured in vitro.?One high concentration and one low concentration of daidzein were chosen to culture hFOB respectively,the expressions of mRNA of ER?, ER?and PPAR?were measured by realtime-RT-PCR, while proteins were measured by western blot method.Results1. The proliferation rate of osteoblasts decreased progressively as the dose of daidzein increasing. Compared with controls, the group of daidzein at the dose of 10-9mol/L could stimulate the proliferation rate significantly, while the group of daidzein at the dose of 10-5mol/L cut down the proliferation rate significantly (P< 0.05). The level of alkaline phosphatase decreased progressively as the dose of daidzein increasing, but the difference was not significant (P> 0.05).2. When estrogen receptors (ERs) were blocked by ICI182780, the proliferation rates in E2 group?daidzein 10-9mol/L?10-7mol/L and 10-5mol/L group were 88.16%?76.30%?81.18% and 83.19% respectively, which were significantly lower than the before block group(P< 0.05). After MPP was used to block ERa purely, the corresponding proliferation rates were 88.16%?76.30%?81.18% and 83.19% respectively, which were also significantly lower than the before block group(P< 0.05). The level of alkaline phosphatase in daidzein 10-9mol/L group decreased significantly when ERa was blocked alone.3. When peroxisome proliferator-activated receptor-?(PPARy) inhibitor GW9662 (GW) was added to the culture system, the proliferation rates in E2 group?daidzein 10-9mol/L?10-7mol/L and 10-5mol/L group were 103.14%?96.99%?112.88% and 122.22%,respectively. Compared to the before block group, the proliferation rate increased significantly (P< 0.05) at daidzein concentration of 10-7mol/L and 10-5mol/L, and the level of alkaline phosphatase increased significantly (P< 0.05) at daidzein concentration of 10-5mol/L.4. The expressions of ER?mRNA in E2 group and daidzein 10-9mol/L group showed no significant difference (P>0.05), either was significantly higher than daidzein 10-5mol/L group (P<0.05).The expression of PPARy mRNA in daidzein 10-5mol/L group was significantly higher than that in E2 group (P<0.05)5. Compared with other groups,the protein expression of ERa in daidzein 10-9mol/L group was significantly higher;while the protein expression of PPARy in daidzein 10-5mol/L group was significantly higher.6. The expressions of mRNA and protein of ER?in control group?daidzein 10-9mol/L and 10-5mol/L group were low,there was no significant difference among the groups.Conclusions1. Daidzein showed biphasic effect on prevention and treatment of osteoporosis, and the effect was dose-dependent:Low-dose of daidzein stimulated the proliferation and differentiation of osteoblasts, while high-dose of daidzein inhibited the proliferation and differentiation of osteoblasts.2. Low-dose of daidzein mainly acted on ERa to stimulate the proliferation and differentiation of osteoblasts,and showed little effect on PPARy.3. High-dose of daidzein mainly acted on PPARy to inhibit the proliferation of osteoblasts, and to some extent, it acted on ERs to promote the proliferation of osteoblasts.4. E2 had no effect on PPAR?. Low-dose of daidzein may act on ER?to inhibit the proliferation of osteoblasts.