Iron overload increases osteoclastogenesis and aggravates the effects of ovariectomy on bone mass

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   Figure 1

   Body weight and iron accumulation in our in vivo mouse model. Mice received i.p. injections of normal saline or FAC with or without ovariotomy for 2 months. (A) Body weight was noted to exclude this interference factor. (B) Livers were decalcified and embeded in paraffin and stained with prussian blue. (C) Undecalcified distal femurs were embeded in resin. Iron accumulation level was observed with Prussian blue staining. Iron was deposited in the cortical surface (arrows). (D) The serum ferritin was examined using an ELISA kit. The bar graph represents means±s.d. The asterisks (*) indicate significant differences at P<0.05. A full colour version of this figure is available at http://dx.doi.org/10.1530/JOE-14-0657.
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   Figure 2

   Micro-CT three-dimensional reconstruction images and evaluation of oxidative stress, bone resorption, and bone formation markers in vivo. (A) Micro-CT images of mice belonging to the Con, F, OVX, and F+OVX groups. The trabecular bone of the distal femur and cortical bone of the mid-diaphysis femur are shown. (B and C) Levels of the oxidative stress markers superoxide dismutase (SOD) and malondialdehyde (MDA). (D and E) Serum levels of the bone resorption markers tartrate resistant acid phosphatase (Trap5b) and C-terminal telopeptide of type 1 collagen (CTX). (F and G) Serum levels of osteocalcin and alkaline phosphatase (ALP) were evaluated as markers of bone formation. The bar graphs show the means±s.d. The asterisks (*) indicate significant differences at P<0.05.
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   Figure 3

   Relative biological function of OBs. (A) ALP staining of osteoblasts (OBs). (B) The level of ALP activity in OBs. ALP activity and protein concentration in supernatants were measured using an ALP activity assay kit. (C) Alizarin red staining of OBs (40× magnification). (D, E and F) Quantitative real-time polymerase chain reaction (RT-PCR) analysis of the expression of bone formation markers including runt related transcription factor 2 (RUNX2), transcription factor SP7, and bone γ gamma carboxyglutamate acid containing protein (BGLAP). (G and H) The effect of FAC on the generation of reactive oxygen species (ROS) in OBs in the presence and absence of estradiol (E₂) (100× magnification). The bar graphs show the means±s.d. *P<0.05 indicates a statistically significant difference. A full colour version of this figure is available at http://dx.doi.org/10.1530/JOE-14-0657.
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   Figure 4

   Cell viability and the effects of E₂ and FAC on RAW264.7 cells pretreated with RANKL. (A and B) Cell Counting kit 8 (CCK8) was used to test cell viability. (C and D) Trap staining of RAW264.7 cells in medium containing the indicated concentrations of E₂ and FAC and 100 ng/ml receptor activator of NF-kB ligand (RANKL) (200× magnification). Wine red cells (Trap-positive cells) containing three or more nuclei were considered to be osteoclasts. The numbers of wine red cells were quantified. The bar graph shows the means±s.d. *P<0.05 indicates a statistically significant difference. A full colour version of this figure is available at http://dx.doi.org/10.1530/JOE-14-0657.
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   Figure 5

   E₂ abrogated the stimulatory effect of FAC on osteoclast differentiation. (A and B) RAW264.7 cells were treated with FAC and E₂ as indicated and Trap stained (200× magnification). Wine red Trap-positive cells were scored. (C, D and E) The effect of FAC and E₂ on bone resorption. The eroded area and the number of pits were shown. (F and G) Bone-marrow-derived macrophages (BMMs) were cultured in the presence of 50 ng/ml RANKL and 30 ng/ml macrophage colony-stimulating factor (M-CSF) for 5 days and subjected to Trap staining to observe osteoclast formation (100× magnification). Wine red cells were considered to be mononucleated cells (MNCs). The bar graph shows the means±s.d. *P<0.05 indicates a statistically significant difference. A full colour version of this figure is available at http://dx.doi.org/10.1530/JOE-14-0657.
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   Figure 6

   FAC increased and E₂ decreased RANKL-induced gene expression, ROS levels, and NF-κB activation. (A and B) FAC enhanced the production of ROS in RAW264.7 cells only in the absence of E₂ (100× magnification). (C, D, E and F) Quantitative RT-PCR measuring the expression levels of bone formation markers including TRAP5, cathepsin K (CTSK), calcitonin receptor (CALCR), and matrix metallopeptidase 9 (MMP9). (G and H) Nuclear and cytoplasmic protein levels were analyzed by western blot. The bar graph shows the means±s.d. *P<0.05 indicates a statistically significant difference. A full colour version of this figure is available at http://dx.doi.org/10.1530/JOE-14-0657.

• © 2015 Society for Endocrinology