Estrogen Inhibits Bone Resorption, But Not Formation, In Iron-induced Osteopenia By Suppressing NF-κB Signaling Pathway In Osteoclasts

Part I 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: con group, F group, OVX group and F+OVX group. Mice of F group and F+OVX group were intervened by ferric ammonium citrate(FAC). Body weight and serum ferritin, ALP,osteocalcin, CTX, TRAP-5b, MDA, 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 Fer was heightened in F group and F+OVX group(P<0.05). 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 BMD between con group and F group. After ovariotomy, BMD of OVX group was lower than that of con group(P<0.05),but higher than that of F+OVX group(P<0.05). 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 TRAP staining suggested that FAC increase the number of TRAP positive cells after ovariectomy(P<0.05), rather than ovariectomy before(P>0.05).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 II 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 q-PCR. 2, Cell line RAW264.7 was used for osteoclasts assay. Cells were divided into 4groups 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 q-PCR. Level of ROS in each group were tested using a multi-detection reader. Cytoplasmic and nuclear proteins of osteoclasts were extracted.Expression of nuclear proteins p50, p65, pp65, cytoplasmic proteins p50, p65, IκBα, p IκBαwere measured with western blot analysis.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 SP7, Runx2 in presence or absence of E2. 2, FAC could only stimulate osteoclasts differentiation 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 incorrespondence with the consequences of resorption pit assay. FAC could stimulate the gene expression of Ctsk, Acp5, MMP9 and Calcr only in the absence of E2. Gene expression ratio of the optical density was positively correlated with active oxygen content.Western blot result: Pretreatment with E2 significantly suppressed the activation of p65 and p50, whereas phosphorylated IκBα, p65, and p50 were increased in the presence of FAC.Conclusion: Estrogen inhibits iron-induced osteopenia by eliminating ROS and suppressing the NF-κB signaling pathway in osteoclasts. This inhibition seemed to be non-related with osteoblasts and bone formation.