Effects of estrogen on bone metabolism of mice with iron accumulation

Abstract

Objective 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 ℃ 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 [(335.20±44.10), (324.80±38.60) μg/L, P=0.000]. 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=0.000), but higher than that of F+ OVX group (P=0.000). 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=0.000), 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. Key words: Estrogen; Ironaccumulation; Bone metabolism; Oxidativestress