Effects Of Phytoestrogens On Biological Activity Of Cultured Osteoblasts In Rats

Objective:Osteoporosis is receiving increasing attention which is related to aging. The main clinical manifestations of osteoporosis is back or hip pain, bent over, humpback, be low, and minor trauma can cause fracture. The basic pathological mechanisms are that at the process of bone metabolism,osteoclasts (OC) bone resorption increase in and osteoblasts (OB) reduction in bone formation, that resulting in an imbalance in human body's calcium and phosphorus metabolism, bone mineral density(Bone mineral density,BMD)gradually decreased and osteoporosis is formation. Osteoporosis can be divided into two categories: postmenopausal osteoporosis and senile osteoporosis, postmenopausal osteoporosis is a more serious public health problem than senile osteoporosis.Estrogen deficiency can lead to postmenopausal osteoporosis, on clinical the classic treatment is estrogen replacement therapy, positive effect. But long-term use has increased coronary heart disease, endometrial cancer and breast cancer incidence of side-effects. To find more desirable drug to treat of postmenopausal osteoporosis has become an urgent need. In recent years, phytoestrogens had been found that it had effective in preventing postmenopausal osteoporosis and not associated with complications. Phytoestrogens can be found in plants, fruits and vegetables in a class of natural. Phytoestrogen is a nonsteroidal compounds, its structure and biological activity like estrogen and has weak estrogenic activity, it is a heterocyclic polyphenols. PE can bind with estrogen receptor, Plays two-ways regulation role to the endogenous estrogen. PE can improve the symptoms of perimenopausal period, lower blood lipid levels, prevent osteoporosis. PE can be bind to receptor which is inside of osteoclasts. It can inhibit the activity of osteoclasts when combined with osteoblast receptor, which also can stimulate osteoblast to secrete collagenase, release of growth factors and cytokines, enhance the activity of osteoblasts, promote bone growth and accelerate bone formation and effective in preventing the formation of osteoporosis.Phytoestrogens can be divided into four kinds:Isoflavones, Coumarin, Lignans, Flavonol. Based on previous experimental study and reported in the literature we can found that phytoestrogens have effects on promoting proliferation and differentiate. However, a comprehensive comparison of various types of phytoestrogens on osteoblasts has been not reported .To study the effects of 4 kinds of phytoestrogens on proliferation and differentiation of cultured osteoblasts in vitro. Used RT-PCR method to detect the differences of phytoestrogens gene expression at the molecular level, in order to compare the effect of different types of phytoestrogens on osteoblasts at cell and molecular level, with the view to improve the clinical efficacy of osteoporosis, as well as the screening of anti-osteoporosis drug, also for development and provide the experimental basis for combination therapy.Methods:1 Cell culture: Improved tissue block culture, to demesh and cultivate osteoblast in cranial bone of newly born SD rats. By adding different concentrations of phytoestrogens (1×10-9mol/L and 1×10-7 mol/L) to the nutrient medium of osteoblast it can be observed the alteration of the shape and quantity of cells using inverted phase contrast microscope.2 Cell proliferation: It can be inspected that the effect of phytoestrogens on the proliferation of osteoblast in 12 h ,24 h and 36 h by MTT methods with the contrast of the group of dealing with estrogen and control group.3 Cell differentiation Alkaline phosphastase(ALP): It can be detected that the change of the activity of ALP in osteoblast of the group of dealing with phytoestrogens in 24 h, 48h and 72h byρ-nitrophenylphosphate (рNPP) disodium matrix dynamics methods with the contrast of the group of dealing with estrogen. 4 Cell differentiation BPG: It can be detected that the Concentration of BPG in osteoblast of the group of dealing with phytoestrogens in 24 h ,48 h and 72 h by RIA.5 Cell factor: It can be detected the expression of colony stimulating collagen I (COL I), smad4 mRNA and runx2 mRNA of the group of dealing with phytoestrogens in 48 h RT-PCR with the contrast of the group of dealing with control group.Results:1 The effect of estrogen, as positive control, on the proliferation and differentiation of the osteoblast1.1 Cell morphology observation of estrogen group, as positive controlThe osteoblast cell which is inoculated is globular at first and then floats in culture fluid. Sub sequently it is adherent, it will expand in 24 h. The shape of expanded cell is irregular with an appearance of triangle. After 72~96 h, osteoblast shows on long fusiform and trabs shape and mixes together and the boundary is vague between cells. Osteoblast shows slabstone shape after 96~120 h. If you keep on culturing cell, cells will form cell nodule and opaque mineral nodus with accumulating of collagen and calcium salts. When you culture the 20th generation, the volume of the cell is larger than normal, periplast is more subtile than normal, and the speed of cleavage is more slower and other phenomena which are signs of aging. After 72~96 h, osteoblasts are more intensive in estrogen than the blank group. Also its intercellular space is smaller than those of the blank.1.2 The effect of estrogen, as positive control on the proliferation of osteoblastAfter being cultivated, contrasted with control group, at the concentrations of 1×10-7mol/L, 24h can promote the proliferation of osteoblast (P<0.05) at the concentrations of 1×10-9mol/L 12 h, 24 h can urge the proliferation of osteoblast (P<0.05).1.3 The effect of estrogen on the ALP of osteoblastAfter being cultivated for 24 h, 48 h, 72h, contrasted with control group, both of the two concentrations can not urge the differentiation of osteobalst (p>0.05).1.4 The effect of estrogen on the BGP of osteoblastAfter being cultivated, contrasted with control group, at the concentrations of 1×10-7mol/L, estrogen had the effects on promoting the expression of BPG cultured for 48 h and 72 h(P<0.05). At the concentrations of 1×10-9mol/L estrogen had the effects on promoting the expression of BPG cultured for 24 h and 72 h (P<0.05).2 The effect of phytoestrogens on the proliferation and differentiation of the osteoblast and the effect on the expression of COLI mRNA, Smad4 mRNA, Runx2/Cbfa1 mRNA in osteoblast2.1 Cell morphology observation of the phytoestrogens group The phenomenon is similar to estrogen group.2.2 The effect of phytoestrogen on the proliferation of rat osteoblastAfter cultivated, contrasted with control group, at the concentrations of 1×10-7mol/L, Daidzein and Psoralen can promote the proliferation of osteoblast for 12 h. Daidzein, Psoralen and Kaempferol can promote the proliferation of osteoblast for 24 h, Deoxyschizandrin for 36 h. At the concentrations of 1×10-9mol/L, Deoxyschizandrin and Psoralen can promote the proliferation of osteoblast for 12 h, Psoralen for 24 h, and Daidzein, Psoralen, Kaempferol, Deoxyschizandrin for 36h.2.3 The effect of phytoestrogens on the ALP of osteoblastAfter cultivated, contrasted with control group, when at the concentrations of 1×10-7mol/L, Deoxyschizandrin enhance osteoblast alkaline phosphatase activity for 24 h and 48 h, Daidzein and Kaempferol for 72 h. At the concentrations of 1×10-9mol/L, Deoxyschizandrin, Daidzein and Kaempferol enhance osteoblast alkaline phosphatase activity for 24 h, Daidzein, Psoralen, Kaempferol and Deoxyschizandrin for 48 h, Kaempferol, Deoxyschizandrin for 72 h.2.4 The effect of phytoestrogens on the BGP of osteoblast After cultivated, contrasted with control group, when at the concentration of 1×10-7mol/L, Daidzein and Deoxyschizandrin had the effects on promoting the expression of BPG for 24 h, Deoxyschizandrin for 48 h and Psoralen, Deoxyschizandrin for 72 h. At the concentrations of 1×10-9mol/L Kaempferol, Deoxyschizandrin and Psoralen had the effects on promoting the expression of BPG for 48 h, Deoxyschizandrin and Psoralen for 72 h.2.5 The effect of phytoestrogens on the expression of COL I mRNA in osteobalst on ratAfter cultivated 48 h, the relative quantity of COLImRNA/GAPDH mRNA expression in the blank group.The expression of COLI mRNA in control group is 0.209. The expression of COLI mRNA in Daidzein is 0.855. The expression of COLI mRNA in Kaempferol is 0.514. The expression of COLI mRNA in Deoxyschizandrin is 0.465. The expression of COLI mRNA in Psoralen is 0.612, both of them is higher than that in blank group (P<0.05), Daidzein is highest than others.2.6 The effect of phytoestrogens on the expression of Smad4 mRNA in osteobalst on ratAfter cultivated 48 h, the relative quantity of Smad4 mRNA/GAPDH mRNA expression in the blank group .The expression of Smad4 mRNA in control group is 0.372. The expression of Smad4 mRNA in Daidzein is 2.741. The expression of Smad4 mRNA in Kaempferol is 2.845. The expression of Smad4 mRNA in Deoxyschizandrin is 1.317. The expression of Smad4 mRNA in Psoralen is 4.112, both of them is higher than that in blank group (P<0.05), Psoralen is highest than others.2.7 The effect of phytoestrogens on the expression of Runx2/Cbfa1 mRNA in osteobalst on ratAfter cultivated 48 h, the relative quantity of Runx2 mRNA/GAPDH mRNA expression in the blank group. The expression of Runx2/Cbfa1 mRNA in control group is 0.886.The expression of Runx2/Cbfa1 mRNA in Daidzein is 1.090. The expression of Runx2/Cbfa1 mRNA in Kae-mpferol is 1.223. The expression of Runx2/Cbfa1 mRNA in Deoxyschiz-andrin is 1.135. The expression of Runx2/Cbfa1 in Psoralen is 0.476, both of them is higher than that in blank group (P<0.05).Conclusion:1 All of the four kinds of phytoestrogens had effect on the proliferation and differentiation of osteoblast at the level of cell, but the effective time and stage were different.2 On the proliferation of rat osteoblast,Deoxyschizandrin and Psoralen 's effective are better than Daidzein and Kaempferol. On the ALP of osteoblast, Deoxyschizandrin and Kaempferol more utility than the other two. On the BGP of osteoblast, Deoxyschizandrin and Psoralen more effective than others.3 All in all, Deoxyschizandrin and psoralen effective are better than Daidzein even close to or more bettle than estrogen at the level of cell.4 Four kinds of phytoestroge had differences mechanism at the molecular level.5 At the molecular level, Daidzein can enhance the expressions of COLI mRNA, the effective was bettle than other PE, Psoralen can enhance the expression of smad4 mRNA, both of Daidzein, Deoxyschizandrin and Kaempferol can effect the expression of Runx2/Cbfa1 mRNA and the effictive was same.6 Four kinds of phytoestroge had differences mechanism at the molecular level, which provide experimental basis for compatibility in order to treatment of osteoporosis.