The Experimental Studies Of The Effects Of Hepcidin On Bone Metabolism

The association between iron overload and bone metabolism has been reported in many papers. Iron overload was recognized as the independent risk factor of postmenopausal osteoporosis.The studies of strategies of iron reduction used for treatment of postmenopausal osteoporosis have been reported. It is expected to be another method to treat postmenopausal osteoporosis clinically.The strategies of iron reduction include iron chelator and hepcidin. Hepcidin, a peptide hormone, is the major regulator of iron metabolism. It was first isolated from human serum and urine by Park in 2001.Few studies of osteoporosis using hepcidin were reported. In this study, hepcidin was used as intervention measure to explore the association between iron and bone metabolism.PartⅠ Effects of targeted disruption of the hepcidin 1 gene on iron metabolism and bone metabolismObjective: To analysis the effects of targeted disruption of the hepcidin 1 gene on iron metabolism and bone metabolism in C57BL/6 mice.Methods: The 7 months old mice were divided into 2 groups according to genotype: hepc-/- mice and hepc+/+ mice. The effects of iron accumulation on bone were analyzed by Micro-CT and bone turnover markers in serum. Weight, serum iron, ferritin, CTX, osteocalcin, iron contents in the liver and femur as well as microarchitecture of distal femur were analyzed. Perls’ staining was carried out on liver tissues and undecalcified tibia.Results: Serum iron and ferritin as well as iron contents in the liver and femur were significantly increased in Hep1-/- mice when compared to WT mice. In contrast, trabecular number, trabecular thickness bone mineral density and bone volume fraction were decreased in distal femur of Hepc1-/- mice. Thinner cortices and decreased cortical area were also observed in Hepc1-/- mice. Weight and serum concentrations of CTX were similar between 2 groups, while osteocalcin was significantly lower in Hepc1-/- mice.Conclusions: Our data show that Hepc-/- mice develop an iron overload and a low bone mass phenotype which probably caused by inhibiting bone formation.PartⅡ Effects of hepcidin on differentiation of mouse primary osteoblastsObjective: To explore the effects of hepcidin on differentiation, proliferation and functions of C57BL/6 mouse osteoblasts.Methods: The mesenchymal stem cells were obtained from 1 day old C57BL/6 mice, and treated with different concentrations of hepcidin. Cell viability was analyzed with CCK-8. ALP staining, Van Gieson staining and Von Kossa’s staining were also performed.Results: Hepcidin at 0-800 nmol/l possessed no effect on differentiation and proliferation of osteoblasts. The positive area of ALP staining and Van Gieson staining were observed in 400 nmol/l. No differences of Von Kossa’s staining were observed between groups with and without hepcidin.Conclusion: Hepcidin has no effect on differentiation, Proliferation and mineralization. However, hepcidin can increase type Ⅰ collagen expression.Part ⅢEffects of hepcidin on differentiation of mouse RAW264.7 monocytes into osteoclastsObjective: To investigate the effects of hepcidin on differentiation of mouse RAW 264.7 monocytes into osteoclasts.Methods: RAW264.7 cells were treated with different concentrations of hepcidin in the presence of receptor activator of NF-Kb ligand(RANKL). Cell viability, the number of tartrate-resistant acid phosphatase(TRAP)- positive cells, levels of TRAP, cathepsin K(CTK), and matrix metalloproteinase 9(MMP-9) m RNA, and levels of TRAP-5b protein in the supernatant were examined.The intracellular iron ion was measured by a confocal laser scanning microscope.Results: Hepcidin at 0-800 nmol/l could significantly increase the number of TRAP-positive MNCs, and up- regulate gene expression of TRAP, CTK, and MMP-9, and increase the concentration of TRAP-5b in the supernatant, and increase concentrations of intracellular iron of RANKL-induced RAW264.7 cells(P<0.05).Conclusion: Hepcidin can significantly facilitate RANKL-induced differentiation of RAW264.7 into osteoclasts in vitro. The mechanism behind accelerated differentiation involves increased levels of intracellular iron.