Experimental Study Of The Relationship Between The Cyclins During The G1 Phase In Osteonlast And Postmenopausal Osteoporosis And The Effect Of Jiangu Granula On The Cyclins

ObjectiveTo reveal the relationship between Cyclin D1,CDK4,p21 and the experimental postmcnopause osteoporosis,and research the effects of Jiangu granula(JGG) on the cyclins during the G1 phase in osteoblast(OB),so that to probe into the detailed mechanism of prevention and cure of postmenopause osteoporosis with traditional Chinese medicine.Methods1.To study the change of the cyclins during the G1 phase in OB of the ovariectomized rats and the effect of JGG on the cyclins.(1) Establishing the experimental animal model:SD female(6-month-old) rats were ovariectomized as the postmenopausal osteoporosis animal model.(2) Grouping and treatment:170 SD female rats were randomly divided into 2 groups:sham(50 rats),ovariectomy(OVX) group(120 rats).Rats in OVX group were bilaterally ovariectomied and sham group were subjected to sham surgeries.OVX group were divided into two groups then:model group(70 rats) and prevent group(50 rats). The rats belong to the prevent group perfused with JGG 2g·kg^-1·d^-1 on the second day after opertion.Then after 13 weeks the rats of model group were divided into two group again:momel group and treatment group,20 rats each group.At the beginning of 13 weeks after operation,the rats of treatment group perfused with JGG 2g·kg^-1·d^-1,and the model group and sham group with normal saline 2ml·d^-1.JGG were dissolved in solution of normal saline.10 rats each of the rats remaided belong to the 4 group were sacrificed respectively 4,8,12,18 and 24 weeks after operation. (3) Detective index:Bone mineral density(BMD) were measured by dual-energy X-ray absorptiometry(DXA) for the whole body.Serum concentration of E2 were measured by radioimmunoassay(RIA).The expression of Cyclin D1,CDK4,p21 proteinum in OB in bone tissue were measured by immunohistochemistry.2.To study the effects of JGG rat serum on the cyclins during the G1 phase in OB which in vitro culture.(1) Establishing the culture in vitro system of OB:Primary cells were isolated from calvaria of newborn SD rats by enzymatic digestion.OB characteristics were identified by alkaline phosphatase(AKP) stain of azo dye method and calcification scobination of alizarin bordeaux dyeing.Cells at the third passage were selected for experiment.(2) Preparation for JGG rat serum:20 male SD rats(3-month-old) were randomly divided into 2 groups:JGG-group and normal saline(NS)group,10 rats each group.The rats of JGG-group perfused with JGG 2g.kg^-1·d^-1,and NS-group with normal saline 2ml·d^-1.The serum containing JGG or NS were extracted after 7 days.(3) Detective index:Cell proliferation of OB cultured in differenent concentration serum was measured by MTT Method,and the optimization concentration of the serum containing JGG would be obtained.Then the serum containing JGG or NS were respectively added and cultured the cells belong to JGG-group or NS-group for 24h, 48h and 72h.The cell cycle were analyzed and Cyclin D1 measured by flow cytometry(FCM).Immunocytochemical(ICC) method was applyed to measure the expression of Cyclin D1,CDK4,p21,while RT-PCR applyed to measure Cyclin D1,CDK4,p21 mRNA.Results1.12 weeks after operation,BMD for whole body in the model group decreased significantly(P＜0.01),while BMD in the prevent group and treament group were higher than that in the model group,but than that in the sham group.Serum concentration of E2 in the rats from the model group decreased significantly after opertion(P＜0.01),and Serum concentration of E₂ from the prevent group and treament group were higher than that in the model group in the same period,while that in the prevent group higher than treament group,but low than sham group.Cyclin D1,CDK4 in OB positive expression were major located at the superficies of bone trabecula.There was Cyclin D1,CDK4 positive expression in the bone tissue from the sham group,but that from the model group,treament group and prevent group were all obviously higher than the sham group.The positive expression of the prevent group was the highest one among the 4 group,and the treament group was the second one.p21 positive expression location was similar to Cyclin D1.Obviously positive expression of p21 appeared in the bone tissue from all four groups,in which that of the sham group was the lowest,while the positive expression of the model group maintained at a high level,and higher than that of the rest 3 groups in different period after operation.2.(1) It showed that cell proliferation rate of OB was the fastest when the concentra-tion of the serum containing JGG was 20%,which was added to the OB of JGG-group.And it was significantly faster than that of NS-group(P＜0.05).(2) The measure by FCM on cell cycle of OB showed that with the action time lasting,the cell percentage of G0/G1 phase decreased,while that of S and G2/M phases and proliferation index(PI) rising.The change of the JGG-group was more obviously:the cell percentage of S phase and PI were higher than NS-group at the 24h,48h and 72h point(P＜0.05).The result of Cyclin D1 measured by FCM showed that the positive expression of Cyclin D1 in the JGG-group was significantly higher than that in NS-group.(3) ICC showed that positive expression of Cyclin D1 and CDK4 were locatet at cell nucleus in OB in vitro culture.With the action time lasting,the positive expression of Cyclin D1 and CDK4 increased,and that of JGG-group was more significant(P＜0.05 or P＜0.01).Positive expression of p21 in OB decreased With the action time lasting,and the expression of JGG-group was lower than the other group,which there were significant difference at 48h and 72h points(P＜0.05).(4) The results measured by RT-PCR were mainly coincident.Cyclin D1,CDK4 mRNA in OB of JGG-group was significantly higher than that in NS-group(P＜0.05 or P＜0.01) at 48h and 72h points.On the contrary,p21 mRNA of JGG-group was significantly lower than that in NS-group(P＜0.05).The distinction were even obviously at 48h and 72h.Conclusion1 With the serum concentration of E₂ decreasing,the Cyclin D1,CDK4 and p21 in OB in the process of osteoporosis in OVX model rats are hyper-expressed, speeding up the cell multiplication and bone formation;while the blockage of the cell cycle and the Apoptosis are excessive,so that the relative insufficience of OB and bone formation cause the high transform osteoporosis.2 JGG can increase the expression of Cyclin D1,CDK4 and inhibit p21 expres-sion in OB in OVX osteoporosismodel rats,so that to stimulate cell proliferation of OB. It is,perhaps one of mechanisms that JGG can prevention and cure postmenopausal osteoporosis.3 JGG can increase the Cyclin D1,CDK4 expression and inhibit the p21 expres-sion in OB cultured in vitro,so that to accelerate the proceeding cell generation cycle and stimulate cell proliferation of OB.The thesis deeply approaches the mechanism that JGG stimulate cell proliferation of OB by the effect of the cyclins during the G1 phase.