The Effect Of Phytoestrogen Supplement Zinc On Biological Activity Of Osteoblast In Rats

Objective: With the arrival of the aging society, osteoporosis is receiving increasing attention which is related to aging. It is the basic pathological mechanisms that in the process of bone metabolism bone resorption of osteoclast increased and bone formation of osteoblasts reduced, resulting in the imbalance of human body calcium and phosphorus metabolism and gradual decrease BMD (Bone mineral density). The main cause relate to age, endocrine disorders, malabsorption of calcium, as well as immunization, nutrition and other factors.Estrogen replacement therapy (ERT) can inhibit bone loss, which is a currently preferred therapy on the clinical prevention and treatment of postmenopausal osteoporosis (PMOP). However,long-term use of estrogen may increase endometrial cancer and breast cancer risk. At present, people will put the eyes that looking for estrogen replacement drug on the phytoestrogen.To study the effects of on proliferation of cultured osteoblasts in vitro,MTT were used to observe the proliferation of osteoblasts cultured in vitro with Psoraleae Injection of different concentration in it for different incubation periods.Osteoblasts which cultured by DMEM of different concentrations of psoraleae injection showed a significant effect on promoting proliferation, and affecting the bone synthesis and metabolism,with the role of prevention and treatment of osteoporosis. Isopsoralen is a kind of coumarins compounds extracted from the Psoralea corylifolia L, belong to phytoestrogen. The preliminary findings of our study group showed that isopsoralen have a better role in promote proliferation and alkaline phosphatase activity in rat osteoblast.Zinc is a necessary trace element to maintain the normal physiological function and the body environment for normal biochemical metabolism,and also is trace elements important to bone metabolism , which can effectively stimulate osteoblast bone formation and inhibit bone resorption . Zinc can maintain the normal cell cycle, promoting cell proliferation and differentiation. zinc stimulate of osteoblasts in vitro protein synthesis through ammonia synthetase ribonucleic acid acyltransferase, and promote the proliferation and differentiation in vitro rat osteoblast.A large number of studies have that reported certain drugs and nutritional factors can be used to prevented bone loss, the effects of joint compound remain obscure. Well, it may have important significance in the prevention of age-related bone loss wheather combined nutritional factors and plant estrogen has synergies on osteoporosis. Research has shown that soy isoflavone supplement calcium have preventive effects for postmenopausal bone loss in rats,the joint application of genistein and zinc have synergies on rat femur bone component organizations.The cellular mechanisms that isopsoralen and zinc on bone metabolism have combined to strengthen and/or synergistic effect have not yet been identified.This study focused on the determination whether isopsoralen and nutritional factors have synergies effect on proliferation and differentiation.To study the effects of isopsoralen plus zinc on proliferation and differentiation of cultured osteoblasts in vitro , Culture osteoblasts in vitro method were used to observe the effect of isopsoralen and zinc on proliferation and differentiation. RT-PCR technology was used to detect isopsoralen and zinc on osteoblasts secreted protein (COL I) mRNA, Smad4 mRNA, Cbfa1/Runx2 mRNA, Osterix mRNA expression. From the cellular and molecular level to explore osteoporosis mechanism applying isopsoralen- traditional Chinese medicine and zinc, with a view to improve osteoporosis clinical efficacy as well as anti-osteoporosis drug development in the future.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 isopsoralen (10^-9 mol/L-10^-8 mol/L) and zinc (10^-5 mol/L -10^-4 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 isopsoralen and zinc on the proliferation of osteoblast in 24 h ,48 h and 72 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 isopsoralen and zinc in 24 h ,48 h and 72 h byρ-nitrop-henylphosphate(рNPP) disodium matrix dynamics methods with the contrast of the group of dealing with estrogen.4 Cell factor: It can be detected that the expression of colony stimulating collagen I (COL I), Smad4 mRNA, Runx2 mRNA and Osterix mRNA of the group of dealing with isopsoralen in 48 h via RT-PCR with the contrast of the group of dealing with estrogen and 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 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 a appearance of triangle.After 7296 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 20 th generation, the volume of the cell is more larger than normal,periplast is more subtile than normal,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 24 h ,contrasted with control group, the concentrations of 10-5mol/L, 10-7 mol/L, 10-8 mol/L can promote the proliferation of osteoblast (P<0.05); after being cultivated 48 h , the concentrations of 10-6mol/L,10-8 mol/L, 10-9 mol/L can urge the proliferation of osteoblast (P<0.05);after being cultivated 72 h, the concentratons of 10-5 mol/L～10-7 mol/L estrogen can promote the proliferation of osteoblast (P<0.05).1.3 The effect of estrogen on the ALP of osteoblast After being cultivated 48 h, contrasted with control group, the concentratons of 1×10^-5 mol/L～1×10^-6 mol/L can promote the differentiation of osteoblast (P<0.05); after being cultivated 72 h , 1×10^-5 mol/L～1×10^-6 mol/L concentrations can urge the differentiation of osteobalst(P<0.05).2 The effect of isopsoralen, as one of the phytoestrogen, on the proliferation and differentiation of the osteoblast and the effect on the expression of COLI mRNA, Smad4 mRNA, Runx2/ Cbfa1) mRNA and Osterix mRNA in osteoblast 2.1 Cell morphology observation of the isopsoralen group The phenomenon is similar to estrogen group.2.2 The effect of isopsoralen zinc and the combined group on the proliferation of rat osteoblastAfter cultivated 24h, contrasted with control group, the concentrations of 10^-8 mol /L～10^-9 mol /L isopsoralen, 10^-9 mol /L isopsoralen plus 10^-5 mol /L zinc sulfate can promote the proliferation of osteoblast (P<0.05); the proliferate effect of the group of isopsoralen (10^-9 mol /L) plus zinc sulfate (10^-5 mol /L) compared to the simple isopsoralen or zinc group is significantly. Cultured 48 h, the groups of isopsoralen(10^-8 mol /L), isopsoralen (10^-8 mol /L) plus zinc sulfate (10^-4 mol /L), isopsoralen (10^-9 mol /L) plus zinc sulfate (10^-5 mol /L) compared with blank group can promote the proliferation of osteoblast, isopsoralen (10^-8 mol /L), isopsoralen (10^-9 mol /L) plus zinc sulfate (10^-5 mol /L) groups have more significant role in proliferation(P<0.01).2.3 The effect of isopsoralen zinc and the combined group on the ALP of osteoblastContrasted with control group, after cultured 48 h, the groups of isopsoralen (10^-9 mol /L), zinc sulfate (10^-4 mol /L), isopsoralen (10^-9 mol /L) plus zinc sulfate (10^-5 mol /L) enhance osteoblast alkaline phosphatase activity, the group of isopsoralen (10^-9 mol /L) plus zinc sulfate (10^-5 mol /L) is the more obvious role in promoting differentiation. Cultured 72 h isopsoralen (10^-8 and 10^-5 mol /L), isopsoralen (10^-8 mol /L) plus zine sulfate (10^-4 mol /L), isopsoralen (10^-9mol /L) plus zine sulfate (10^-5mol /L) groups can promote the alkaline phosphatase activity of osteoblast, the effects of isopsoralen (10^-9mol /L) plus zinc sulfate (10^-5mol /L) has more significantly.2.4 The effect of isopsoralen plus zinc on the expression of COLI mRNA in osteobalst on ratAfter cultivated 48 h, the relative quantity of COLI mRNA/GAPDH mRNA expressions in the blank group.The expression of COLI mRNA in isopsoralen and zinc group is higher than that in blank group (P<0.01).The expression of COLI mRNA in isopsoralen and zinc group is higher than that in black group (P<0.01).Compared to estrogen group it have statistical significance, and significantly improve the relative COLI mRNA expression (P<0.01).2.5 The effect of isopsoralen plus zinc on the expression of Smad4 mRNA in osteobalst on ratCompared with blank control group, isopsoralen, zinc and isopsoralen plus zinc group could significantly promote the Smad4 mRNA expression (P<0.01).2.6 The effect of isopsoralen plus zinc on the expression of Runx2/Cbfa1 I mRNA in osteobalst on ratIsopsoralen plus zinc group could significantly promote Runx2/Cbfa1 mRNA expression (P<0.05); its expression was higher than pure isopsoralen group.2.7 The effect of isopsoralen plus zinc on the expression of Osterix mRNA in osteobalst on ratIsopsoralen group and isopsoralen plus zinc group can enhance the expression of Osterix mRNA (respectively P*<0.05, P**<0.01). Isopsoralen plus zinc group was higher than the expression of estrogen and Isopsoralen group.Conclusion:1 Certain dose of isopsoralen and zinc, as phytoestrogen and nutritional factors, can promote the proliferation and differentiation of osteoblast. Certain dose of isopsoralen and zinc can promote the express of COLI mRNA, upgrade the express of smad4 mRNAand increase the express of Runx2/Cbfa1mRNA and Osterix mRNA. The influence of isopsoralen and zinc on bone metabolism may be achieved through the pathway of promoting bone formation.2 Phytoestrogen maybe is one of the material foundation for the Traditional Chinese Medicine to invigorate the kidney.3 The influence of isopsoralen and zinc on preventing osteoprosis bone maybe achieved through increasing the expression of the critical proteins.4 The stady of isopsoralen and zinc preventive effects for ostoeporosis provided experimental basis to develope anti-osteoporosis drug as well as improve osteoporosis clinical medication.