| Objective: Osteoporosis has became a significant issue which is faced with people with increasingly global population aging.Especially,post-menopause women with insufficient estro- gen are likely to suffer osteoporosis.Osteoporosis is a kind of general bone disease,which has the characteristic of ostopenia and microstructure degenerating and results in bone fragility increasing and inclining to bone fracture.In some clinic investin- ation hormone replacement therapy (HRT) can prevent the loss of bone mass and elevate the bone density.However,estrogen may have side-effect on people in long-term, it may promote endometrial hyperplasia and enhance the incidence of coronary heart disease ,stroke,pulmonary embolism and breast cancer.Traditionary Chinese Medicine (TCM) are experienced in treating the fractures treatment.Psoralea fruits and cuscuta se- eds are usually used to dealing with fracture via TCM ,nowadays, they are used to coping with osteoporosis. Isopsoralen and kaempferol are the monomers extracted from them. Considering these reports, it is easy to consider the possibility that isopsoralen and kaempferol could prevent osteoporostis.Our research observates that isopsoralen and kamepferol affect the proliferation and differentiation of the osteoblast derive from calvaria bone from neonate rat by alkaline phosphatase activity ,content of osteocalin and the expression of trasforming growth factor (TGF) mRNA,collagen I(COLI) mRNA,macrophase colony-stimulating factor(M-CSF) mRNA.Methods:1 Cell culture:Improved tissue block culture, to demesh and cultivate osteoblast in cranial bone of newly born SD rats.By a- ding different concentrations of isopsoralen (1×10-5 mol/L 1×10-9 mol/L ) and kaempferol (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 isopsor- alen and kaempferol 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 differentiation3.1 Alkaline phosphastase(ALP) :It can be detected that the cha- ge of the activity of ALP in osteoblast of the group of dealing wi-th isopsoralen and kaempferol in 24 h ,48 h and 72 h byÏ-nitrop- henylphosphate(Ñ€NPP) disodium matrix dynamics methods with the contrast of the group of dealing with estrogen.3.2 Osteocalcin (OC):It can be determined that the content of OC of the group of dealing with isopsoralen secreted by osteob -last in 48 h and 72 h via radiommunity method .4 Cell factor:It can be detected that the expression of macrophade colony-stimulating actor(M-CSF), transforming growth factor (TGFβ1),collagen I(COL I) of the group of dealing with isopsoralen in 48 h and 72 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 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 h96 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 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.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 1×10-5 mol/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,1×10-9 mol/L can urge the proliferation of osteoblast (P <0.05);after being cultivated 72 h, the concentratons of 1×10-5 mol/L1×10-7 mol/L estrogen can promote the proliferation of o- steoblast (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/L1×10-6 mol/L can promote the differentiation of osteoblast (P<0.05); after being cultivated 72 h ,1×10-5 mol/L1×10-6 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,TGF-β1mRNA and M-CSF mRNA in osteoblast2.1 Cell morphology observation of the isopsoralen groupThe phenomenon is similar to estrogen group.2.2 The effect of isopsoralen on the proliferation of osteoblastAfter cultivated 24 h ,contrasted with control group, the concentration of 1×10-9 mol/L can promote the proliferation ofosteoblast (P<0.05); after being cultivated 48 h ,1×10-6 mol/L1×10-9 mol/L concentrations can urge the proliferation of osteoblast(P<0.05), expecially 1×10-9 mol/L(P<0.01)is easier to be observed;after being cultivated 72 h ,contrasted with control group,the concentration of 1×10-9 mol/L can promote the proliferation of osteoblast(P<0.01).2.3 The effect of isopsoralen on the ALP of osteoblastContrasted with control group, after being cultivated 24 h ,1×10-5 mol/L concentration can promote the differentiation of osteoblast obviously compared with the blank group (P<0.05);After being cultivated 48 h , only 1×10-9 mol/L concentration can promote the differentiation(P<0.01). After being cultivated 72 h , 1×10-9 mol/L concentration can promote the differentiation of osteoblast obviously compared with the blank group(P<0.05).2.4 The effect of isopsoralen on the OC of osteobalstContrasted with control group, after being cultivated 48 h ,1×10-7 mol/L1×10-9 mol/L concentrations can promote the differentiation of osteoblast obviously compared with the blank group(P<0.05);After being cultivated 72 h,1×10-7 mol/L1×10-9 mol/L concentrations can promote the differentiation(P<0.05).2.5 The effect of isopsoralen on the expression of COLI mRNA in osteoblastAfter being cultivated 72 h ,the relative quantity of COLImRNA/ GAPDH mRNA expressions in the blank group, inin isopsoralen group and in isopsoralen group are 0.3103, 0.7323 and 0.8030.The expression of COLI mRNA in isopsoralen group is higher than that in blank group(P<0.01).The expression of COLI mRNA in estrogen group is higher than that in black group(P<0.01).There is no significant difference between estrogen group and isopsoralen group.2.6 The effect of isopsoralen on the expression of TGF-β1mRNA in osteobalstAfter being cultivated 72 h ,the relative quantity of TGF-β1 mRNA/GAPDH mRNA expressions in the blank group ,in the isopsoralen group and in the estrogen group are0.9256 ,1.0470 and 1.074.There is significant difference between isopsoralen group and the blank group. The expression of TGF-β1mRNA in estrogen group is higher than that in black group(P<0.01).The expression of TGF-β1mRNA in estrogen group is higher than that in black group(P<0.01). There is no significant difference between estrogen group and isopsoralen group.2.7 The effect of isopsoralen on the expression of M-CSFmRNA in osteoblastAfter being cultivated 72 h ,the relative quantity of M-CSF mRNA/GAPDH mRNA expression in the blank group,in the isopsoralen and in the estrogen group are is 1.0487 ,0.8877 and 0.9117. The expression of M-CSF mRNA in isopsoralen group is higher than that in black group(P<0.01).The expression of M-CSF mRNA in estrogen group is higher than that in black group(P<0.01).There is no significant difference between estrogen group and isopsoralen group.3 The effect of kaempferol ,as one of the phytoestrogen , on the proliferation and differentiation of the osteoblast3.1 Cell morphology observation of kaempferol The phenomenon is similar to estrogen group.3.2 The effect of kaempferol on the proliferation of osteoblastContrasted with control group ,after being cultivated 24 h , the concentrations of 1×10-5 mol/L can promote the proliferation of osteoblast(P<0.01) ; after being cultivated 48 h ,the concentration of 1×10-5 mol/L and 1×10-9 mol/L can urge the proliferation of osteoblast(P<0.01);after being cultivated 72 h,the concentration of 1×10-9 mol/L can urge the proliferation of osteoblast(P<0.01).3.3 The effect of kaempferol on the ALP of osteoblast:Contrasted with control group,after being cultivated 48 h ,1×10-6 mol/L and 1×10-9 mol/L concentrations can promote the differentiation of osteoblast obviously compared with the blank group(P<0.05);af- Ter cultivated 72 h, only 1×10-9 mol/L concentration can promote the differentiation(P<0.01).Conclusion:1 Certain dose of isopsoralen and kaempferol , as phytoestrogen, can promote the proliferation and differentiation of osteoblast. Certain dose of isopsoralen and kaempferol can promote the express of COLI mRNA,upgrade the express of TGF-β1mRNA and increade the express of M-CSF mRNA .All of this illustrate that isopsoralen may prevent the osteoporosis via promoting the bone forming and repress the bone resorption and kaempferol may prevent the osteoporosis by promoting the bone forming.2 The influence of isopsoralen and kaempferol,as phytoestrogen, on bone metabolism may be achieved through the pathway of promoting bone formation.3 The effective concentraton of isopsoralen and kaempferol on osteoblast is 1×10-9 mol/L.4 Phytoestrogen maybe is one of the material foundation for the Traditional Chinese Medicine to invigorate the kidney. |
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