Experimental study on the effect of iron accumulation on bonemass, intraosseous vessels and vascular endothelial cells in mice / 中华骨科杂志

ABSTRACT

Objective@#To investigate the effect of endogenous iron accumulation onbone mass, intraosseous vessels and the effect of exogenous iron on endothelial cell activity.@*Methods@#The mice were divided into control group (C57/BL6 mice without hepcidin knockout) and hepcidin-knockout group (10 mice in each group, 8 weeks old and weighing about 22 g). The mice in both groups were killed at the age of 16 weeks. Serum ferritin levels were measured by Enzyme-linked immunosorbent assay (ELISA), and iron accumulation in liver tissue was measured by Prussian blue staining, while femoral micro-structure was measured by micro-CT, and H-type vessel immunofluorescence staining was used to detect the number of H-vessels in bone. Cell experiments were divided into normal culture group (normal cell group) and intervention group (Fe group) with 200 μmol/L ammonium ferric citrate. Scratch test was used to detect the migration ability of vascular endothelial cells, and tube formation test was used to detect the function of vascular endothelial cells. The endothelial activity of vascular endothelial cells was detected by immunofluorescence.@*Results@#The level of serum ferritin (318.30±12.53 ng/ml) in the hepcidin-knockout group was significantly higher than that in control group (109.60±4.66 ng/ml). The percentage of blue area of Prussian liver iron staining in the hepcidin-knockout group (80.80%±3.156%) was significantly higher than that in control group (20.94%±2.813%). Bone mineral density in the hepcidin-knockout group (0.044±0.002 mg/m3) was significantly higher than that in control group (0.131±0.008 mg/m3). The number of intraosseous blood vessels in the hepcidin-depleted mice (17.06%±1.060%) was significantly lower than that in control group (38.76%±4.576%). There were significant differences between the two groups in each index (t=-49.367,-13.788, 35.293, 6.165; all P < 0.05). The scratches of vascular endothelial cells in Fe group were reduced by 24.300%±1.849% after 24 hours of culture, which was significantly lower than that in normal group (39.060%±3.211%). The area of vascular endothelial cells in Fe group (0.035±0.003 mm2) was significantly lower than that in normal group (0.330±0.018 mm2). The EMCN positive cells of vascular endothelial cells in Fe group were significantly lower than those in normal group. The percentage of cell number (12.000%±3.462%) was significantly lower than that of normal cell group (0.035%±0.003%). There were significant differences in cell indices between the two groups (t=9.790, 18.929, 13.922; all P< 0.05).@*Conclusion@#Endogenous iron accumulation aggravated bone loss in mice, and the number of intraosseous blood vessels decreased significantly. Vascular endothelial cells were inhibited by iron intervention in migration, tube-formation and endothelial ability.