[Experimental study on the effect of hepcidin overexpression on osteoclasts and bone mass in iron accumulation mice]

Objective: To investigate the effects of hepcidin on iron, osteoclasts and bone mass in iron accumulation mice. Methods: Eight-week old C57/BL6 male mice were divided into control group (CTR group), high iron group (Fe group) and iron reduction group (Hepc group). CTR group and Fe group were wild-type mice, Hepc group were hepcidin gene overexpression mice.Fe group and Hepc group were injected intraperitoneally with iron, and the mice in the CTR group were injected intraperitoneally with the same amount of saline.Three groups of mice were injected with tamoxifen to induce hepcidin overexpression.Eight weeks later, specimens of three groups of mice were collected and the serum levels of hepcidin, ferritin and carboxy-terminal telopeptides (CTX) were measured by enzyme linked immunosorbent assay (ELISA). Frozen section and Prussian blue stain were carried out to detect bone iron.Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect osteoclast related gene expression[cytokinin (CTK), matrix metalloprotein (MMP9), protein tyrosine kinase 2β(PTK2β), cathepsin K (CTSK)]. Tartrate resistant acid phosphatase (TRAP) staining was used to observe the proliferation and differentiation of osteoclasts.The osteoclast activity was examined by lacunar resorption pits.The microstructure of the femur was measured by micro-computed tomography (Micro-CT). Variance analysis was applied to compare the differences among groups. Results: The levels of serum hepcidin in CTR, Fe and Hepc group were (508±42), (728±82) and (1 423±85) ng/L, respectively; serum ferritin were (355±26), (1 270±35) and (801±23) μg/L, respectively; CTX were (4.4±0.5), (13.9±1.4) and (8.5±0.6) mg/L, respectively; there were significant differences in the up-mentioned indexes among the three groups (F=129.6, 781.7, 77.3, all P<0.05). The genes expression level of CTK, MMP9, PTK2β, TRAP, CTSK in the Hepc and CTR group were all significantly lower than those in Fe group (F=39.6, 8.0, 5.4, 19.5, 8.8, all P<0.05). The results of TRAP staining and pits formation also showed that osteoclast proliferation and activity in group Hepc reduced significantly when compared with those in Fe group (t=4.295, 7.557, both P<0.05). Conclusion: The overexpression of hepcidin can down-regulate the content of ferritin in iron-accumulating mice and inhibit the proliferation, differentiation and activity of osteoclasts, and improve the bone mass.