Molecular Biological Mechanism Of Phytoestrogen In Preventing Osteoporosis

Objective: With the arrival of the aging society, more and more attention is drawn to the aging problems. Osteoporosis was an important disease affecting human health,which is related to aging. Till now about 2 billion people are infected with Osteoporosis in the world, and its incidence rate is high. A primary analysis of the main statistics of 2000 census, in China about 8800 million people(about occupide 6.6% of population) suffering from Osteoporosis,especially thoses people whose age is in 60~69.it is estimated that the medical care expenses on the treatment of Osteoporosis is about 50 billion rmb at least every year. Osteoporosis, along with it's searious complication and fractures, is severly threatening the wrinkly's health. So it is urgent affairs to find out effective drugs and methods to prevent and treat osteoporosis. Bone absorptioninhibitor drugs and bone formationacceleration drugs are two kinds of anti-osteoporosis drugs. Hormone Replacement Therapy is more commonly used , which could decrease bone mineral loss in Post-menopause Women and improve the density of bone, but promote endometrial hyperplasia, liver damage, venous embolization,vessels diseases and breast cancer.Phytoestrogen (PE) is a group of natural non steroid compound which displays estrogen like activity because of their structural similarity to human estrogens and exhibits high affinity binding to estrogen receptor beta. PE could improvement perimenopause syndrome,decrease the blood lipid level, prevent osteoprosis and urinary incontinence. Substitute for estrogen in preventing osteoprosis is a hopeful application field for PE. Traditional Chinese drugs Psoralea fruit can improve Yangqi of spleen and kidney. Modern pharmacological research shows the water soluble extractive of Psoralea has the effect on the bone turnover.The acetone-extract from Psoralea fruits can increase bone strength,improve the phosphate in blood. The model of rats osteoprosis were orally injected into the stomach by psoralea compound preparation, decrease alkaline phosphatase(ALP) leavel to normal, decrese Ca/cr and HOP/cr leavels in urine, inhibit absorbtion of bone, the rate of bone formation is eventually greater than bone absorption. Psoralen was one of active component in psoralea. Psoralen has the effects on stimulating the proliferation and maturation of cultured osteoblasts in vitro and promoting typeⅠcollagen (COLI)synthesis. The intracellular signal transduction mechanism of the psoralen to promote the growth of the osteoblasts is not clear. Based on the previous research, we explore the molecular mechanisms biology for psoralen preventing osteoprosis. In order to provide theoretical basis for clinical preventing osteoprosis. Methods:1 Cell culture: Improved tissue block culture, to demesh and cultivate osteoblast from cranial bone of newly born SD rats.By ading different concentrations of isoimperatorin (1×10-5 mol/L ~1×10-9 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 isoimperatorin 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: It can be detected that the change of the activity of ALP in osteoblast of the group of dealing with isoimperatorin in24h,48h,72h byρ-nitrophenyl phosphate(рNPP) disodium matrix dynamics methods with the contrast of the group of dealing with estrogen.4 Cell factors: It can be detected that the expression of Osteoprotegerin (OPG) mRNA, and receptor activator of nuclear factor–κB(RANKL)mRNA of the group of dealing with psoralen in 72 h via RT-PCR with the contrast of the group of dealing with estrogen and control group.5 Cell protein: It can be detected that the expression of Bone morphogenetic protein (BMP-2) of the group of dealing with psoralen in 72h via immunohistochemistry 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 cells which is inoculated is globular at first and then floats in culture fluid . Subsequently it is adherent,it will expand in 24 h.The shape of expanded cell is irregular with a appearance of triangle. After 48 h~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 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,the speed of cleavage is slower and other phenomena which are signs of aging. 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 1×10-5mol/L, 1×10-7 mol/L, 1×10-8 mol/L can promote the proliferation of osteoblast (P<0.05); After being cultivated 48 h, the concentrations of 1×10-6mol/L,1×10-8 mol/L,1×10-9mol/L can urge the proliferation of osteoblast (P<0.05); After being cultivated 72 h, the concentratons of 1×10-5 mol/L~1×10-7 mol/L estrogen can promote the proliferation of osteoblast (P<0.05) .1.3 The effect of estrogen on the ALP of osteoblastAfter 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, 1×10-5mol/L~1×10-6mol/L concentrations can urge the differentiation of osteobalst(P<0.05)2 The effect of isoimperatorin ,as one of the phytoestrogen ,on the proliferation and differentiation of the osteoblast2.1 Cell morphology observation of the isopsoralen group The phenomenon is similar to estrogen group.2.2 The effect of isoimperatorin on the proliferation of osteoblast After cultivated 24 h ,contrasted with control group, none of the concentration can promote the proliferation of osteoblast (P>0.05);After cultivated 48h,1×10-9mol/L~1×10-8mol/L concentrations can urge the proliferation of osteoblast(P<0.05);after cultivated 72h, 1×10-8mol/L~1×10-7mol/L concentration can urge the proliferation of osteoblast(P<0.05).2.3 The effect of isopsoralen on the ALP of osteoblast Contrasted with control group, after cultivated 72h 1×10-6 mol/L~1×10-5mol/L can urge the differentiation of osteobalst(P<0.05).3 The effect of psoralen on the expression of OPG mRNA in osteobalst After cultivated 72 h ,the relative quantity of OPG mRNA/GAPDH mRNA expressions in the blank group, in the psoralen group and in the estrogen group are 0.6048, 1.0794 and 1.3395. There is significant difference between psoralen group and the blank group(P<0.05). The expression of OPG mRNA in estrogen group is higher than that in black group(P<0.05). There is no significant difference between estrogen group and psoralen group.4 The effect of paoralen on the expression of RANKL mRNA in osteobalstAfter cultivated 72 h ,the relative quantity of RANKLmRNA/GAPDHmRNA expressions in the blank group ,in the psoralen group and in the estrogen group are 1.0203, 0.8374 and 0.8907. There is significant difference between psoralen group and the blank group(P<0.05). The expression of RANKLmRNA in estrogen group is higher than that in black group(P<0.05). There is no significant difference between estrogen group and psoralen group.5 The effect of paoralen on the relative quantity of OPG/RANKL in osteobalstAfter cultivated 72 h, the relative quantity of VOPG / VRANKL in the blank group ,in the psoralen group and in the estrogen group are 0.599, 1.282 and 1.513. There is significant difference between psoralen group and the blank group(P<0.05). psoralen increased VOPG / VRANKL.6 Effects of psoralen on the expression of bone morphogenetic protein 2 of osteoblastthe expression of bone morphogenetic protein 2 was tested by immunohistochemistry method.The posotive rates of osteoblast cells in psoralen group is 86.67%, 93.33% in E2 and 33.33% in black group. to compare the black group , psoralen can significantly enhance the expression of BMP-2.Conclusion:1 Certain dose of isopsoralen as phytoestrogen, can promote the proliferation of osteoblast.2 Certain dose of isopsoralen urge the differentiation of osteoblast.3 The influence of psoralen on bone may be achieved through the pathway of promoting bone formation and inhibiting bone absorption.4 The influence of psoralen on bone maybe achieved through increasing the expression of OPG mRNA , decreasing the expression of RANKL mRNA,and increasing the rate of VOPG / VRANKL.5 BMP may be the critical protein in the effection of Phytoestrogen on preventing osteoprosis. The influence of psoralen on bone maybe achieved through increasing the expression of BMP-2...