Effects Of Estrogen On "iron-induced Abnormal Bone Metabolism" And Its Mechanism

Part I Clinical observation of iron and bone metabolism index in young female patients undergoing hysterectomyObjective: TTo study the changes of serum iron metabolism and bone metabolism in young women with hysterectomy or normal ovarian function.Methods:41 cases of women with uterine resection due to hysteromyoma,aged 38 to 44 years old,from June 2002 to October 2010,were taken as objects.Hysterectomy time ranged from 3 to 11 years.Another 35 cases of women without uterus and ovary were randomly selected and divided into control group.Levels of estradiol(E2),follicle stimulating hormone(FSH),luteinizing hormone(LH),ferritin(FER),total iron binding capacity,type I procollagen amino terminal peptide(P1NP),collagen type C end peptide beta degradation products(beta-CTX),femoral neck and lumbar spine bone mineral density were detected.T test and multiple stepwise regression analysis were used to investigate the relationship between iron and bone metabolism.Results:The age,height,weight,hemoglobin,white blood cell count,liver function,renal function,blood lipid and blood glucose of the two groups were showed no statistical significance(P > 0.05).Women with uterine resection shared higher levels of serum ferritin(119.75 ± 43.72 ng/m L)than control group(27.66 ± 23.62 ng/m L)(t=11.004,P < 0.01).Total iron binding capacity(51.07 ± 8.82 umol/L)was significantly lower than the control group(56.99 ± 3.90 umol/L)(t=-3.673,P < 0.01).Two groups of E2,LH,FSH,P1 NP,beta-CTX,femoral neck and lumbar spine bone mineral density were in the normal range.Serum ferritin was positively correlated only with the time of hysterectomy(B=13.673,P < 0.01).Conclusion:In women,amenorrhea is one of the important causes of iron accumulation.Iron accumulation had no significant effect on bone metabolism in the presence of estrogen.Part II Effects of estrogen on bone metabolism of mice with iron accumulationObjective: To explore the effect of iron on bone mass,bone formation and bone resorption before or after ovariectomy.Methods: 8-week-old female ICR mice were randomly divided into 4 groups: Control(Con)group,Ferriion(F)group,Ovariectomy(OVX)group and Ferri ion + Ovariectomy(F+OVX)group.Mice of F group and F+OVX group were intervened by ferric ammonium citrate(FAC).Body weight and serum ferritin,alkaline phosphatase(ALP),osteocalcin,C-terminal cross linking telopeptide of type I collagen(CTX),tartrate resistant acid phosphatase-5b(TRAP-5b),malonaldehyde(MDA),superoxide dismutase(SOD)were measured.The livers and femurs were then fixed in 10% buffered formalin at 4°C for Prussian blue staining.A high resolution micro-CT was used to scan the femur for trabecular bone.Bone marrow-derived macrophages(BMM)were extracted,then operated tartrate resistant acid phosphatase(TRAP)staining and the TRAP positive cells were counted.Results: There was no significant difference between every group in body weight.The serum Ferritin(Fer)was heightened in F group and F+OVX group(P<0.001).Results of Prussian blue staining indicated that iron was deposited in both the liver and bone marrow.ROS assay results: FAC was shown to promote the generation of ROS in ovariectomized mice,whereas the presence of estrogen suppressed oxidative stress.Micro-CT analysis indicated that iron treated mice showed no significant difference of bone mineral density(BMD)between Con group and F group.After ovariotomy,BMD of OVX group was lower than that of Con group(P<0.001),but higher than that of F+OVX group(P<0.001).Iron reduced ALP and osteocalcin in both ovariectomized and Con-operated mice.We observed higher levels of two factors involved in bone resorption,Trap-5b and CTX,in the F+OVX group than in the OVX group.However,the presence of estrogen abrogated this effect.In accordance with the results of the micro-CT analysis,no significant differences were observed between Con and F groups.Results of TRAPstaining suggested that FAC increase the number of TRAP positive cells after ovariectomy(P<0.001),rather than ovariectomy before(P=0.501).Conclusion: FAC had little effect on bone resorption when estradiol existed(before ovariectomy),while resulted in osteopenia without estradiol(after ovariectomy).This effect seemed to be related with bone resorption progress,rather than bone formation progress.Part III The antagonistic effect of estrogen and iron on biological activity of osteoblasts and osteoclastsObjective: To understood the antagonistic effect of estrogen and iron on biological activity indicators of osteoblast and osteoclast,and to explore the role of reactive species and NF-κB signaling pathway in this progress.Methods: 1,The primary osteoblasts were cultured in vitro.The experiment were divided into four groups treated as follows:(1)cells received with 10 u M ferric ammonium citrate(FAC);(2)a group intervention using 10 n M estradiol(E2);(3)E2 pretreatment and using the same concentration of FAC intervention;(4)a group of normal control.ALP staining and ALP activity were detected.The expression of gene were examined through RT-PCR.2,Cell line RAW264.7 was used for osteoclasts assay.Cells were divided into 4 groups as osteoblasts research.10 n M estradiol and 50μM FAC were used for intervention.Trap staining and resorption pit assay were examined.The expression of bone resorption gene were examined through RT-PCR.Level of ROS in each group were tested using a multi-detection reader.Results: 1,The level of ALP was significantly suppressed by FAC with or without estradiol(P<0.05),which was in correspondence with the consequences of ALP staining.Meanwhile,FAC could inhibit the gene expression of Runx2、osterixin presence or absence of E2.2,FAC could only stimulate osteoclasts differentiate on in the absence of estradiol.Iron heightened trap positive cells number in the absence of E2,while there was no significant difference between FAC group and FAC+E2 group,which was in correspondence with the consequences of resorption pit assay.FAC could stimulate the gene expression of MMP9、TRAP、CTSKand CALCR only in the absence of E2.Gene expression ratio of the optical density was positively correlated with active oxygen content.Conclusion: Estrogen inhibits iron-induced osteopenia by eliminating ROS.This inhibition seemed to be non-related with osteoblasts and bone formation.