Iron accumulation inhibits the bone formation of ovariectomized mice via stimulating oxidative stress

Abstract

Objective To observe the effect of iron accumulation on bone formation of ovariectomized mice and underlying mechanism using a mouse animal model. Methods Seventy-two mice were randomly divided into three groups: sham-operated (E+ F-), ovariectomized (E-F-) and ovariectomized with ferric ammonium citrate (FAC) injected (E-F+ ). Blood and femurs of mice were harvested from three groups every 7 days during 4 weeks of treatment. Serum ferritin (Fer), osteocalcin (OC), alkaline phosphatase (ALP), superoxide dismutase (SOD) and malondialdehyde (MDA) were measured by enzyme-linked immunosorbent assay (ELISA). Bone mineral density (BMD) was analyzed by Micro-CT. Osteogenic-related gene expressions was detected by real-time fluorescent quantitative polymerase chain reaction (FQ-PCR). In vitro, primary mouse osteoblasts were cultured, and cell viability (CV) was tested using the cell counting kit-8 (CCK-8). The reactive oxygen species (ROS) was measured using fluorescent probe dichlorodihydro-fluorescein diacetate (DCFH-DA). Results Serum OC and ALP levels in E-F+ group were markedly reduced in the 4th week compared to those in E-F- group [for OC, (88.6±8.2) vs. (55.3±9.2) ng/ml; for ALP, (18.1±1.6) vs. (12.0±1.8) U/100 mL, E-F- vs. E-F+ ). MDA and SOD were higher in E-F+ group at every time point except SOD on the 1st week compared to those in E-F- group. BMD in E-F+ group further decreased compared to that in E-F- group [(0.13±0.03) vs. (0.07±0.02) mg/mm3, E-F- vs. E-F+ ] at the 4th week) . In comparison to E-F-, the FQ-PCR revealed the expressions of osteogenic-related genes (Runx2, SP7 and Bglap) in the bone tissue of E-F+ group decreased to 76%, 72%, 74% of that in the 3rd week and 46%, 49%, 36% in the 4th week. In vitro, we found the osteoblasts viability in E-F+ group decreased to 45% of that in E-F-, and ROS in E-F+ group increased significantly from the 2nd week as compared with E-F- group (1.43-, 1.48-, 1.85-fold, at 2nd, 3rd, and 4th week, respectively). Conclusion The present study showed that iron accumulation could inhibit the bone formation of mice via stimulating oxidative stress level. Key words: Osteoporosis; Bone formation; Osteoblasts; Iron accumulation; Oxidative stress