Experimental Study On The Effect Of Hepcdin Overexpression On Osteoclasts And Bone Mass In Iron Accumulative Mice

Part Ⅰ Changes of bone mass in "iron accumulation" mice after over-expression of hepcdinObjective: The purpose of this study was to explore the effect and changes of iron reduction and bone mass in iron accumulation male mice after the intervention of hepcidin,a key regulator of iron,and to explore a new scheme for the treatment of "iron accumulation osteoporosis"Methods: Construction of hepcidin condition over-expression mice by using Cre-lox P technology.The experiment selected 24 male mice were divided into control group(CTR group),high iron group(Fe Group)and hepcidin overexpression group(Group Hepc).CTR group and Fe Group were 8 weeks C57/bl6 male wild-type mice,Hepc group were the same week old male hepcidin gene overexpression mice.Fe group and Hepc group were intraperitoneal injection of dextran iron 0.1g/(kg·wk),continuous injection for 8weeks.Group CTR intraperitoneal injection the same volume of saline.The three groups of mice were injected with tamoxifen 10mg/ml 0.1ml,continuous injection for 5 days to induce hepcidin overexpression.The three groups of mice were sacrificed after 8 weeks to collect serum samples by enzyme-linked immunosorbent assay(ELISA)detection of serum hepcidin(hepcidin),serum ferritin(ferritin),osteoclast marker(CTX)level;bilateral femur and tibia were isolated,micro computed tomography(micro-CT)detection of mouse femur bone microstructure parameters.The Prussian blue staining of paraffin section was used to observe the accumulation of liver iron,and the hard tissue sliced was used to observe the deposition of iron.Results: Serum hepcidin levels in CTR group and Hepc group were(727.87±82.46)and(1423.06±85.32)pg/ml,respectively.Serum ferritin levels were(354.61±26.16)ng/ml,(1270.09±34.75)ng/ml and(801.34±22.82)ng/ml,respectively.CTX were(4.43±0.54)ug/ml,(8.46 ± 0.64)ug/ml and(8.46 ± 0.64)ug/ml,respectively.Compared with CTR group,the content of hepcidin(t=8.707,P=0.001),serum ferritin(t=36.46,P<0.0001),CTX(t=11.02,P=0.0004),Liver and bone iron in group Fe were significantly higher,but the bone mass was significant decreased(t=13.01,P=0.0002).Compared with Fe group,the content of hepcidin(t=20.16,P<0.0001)increased significantly,and the content of serum ferritin(t=19.53,P<0.0001),CTX(t=6.149,P=0.0035)and bone iron were decreased significantly in Hepc group.The content of liver iron and bone increased(t=4.185,P=0.0139).Conclusion: The high expression of hepcidin could inhibit the release of liver iron,regulate the redistribution of iron,reduce the content of serum ferritin and bone iron,and partially restore the bone volume of iron accumulation mouse.Part Ⅱ Effect and mechanism of hepcidin overexpression in iron accumulation mouse on the Biological activity of Primary osteoclasts in vitroObjective: To study the effects of hepcidinon the proliferation,differentiation and function of iron accumulating mouse primary osteoclast in vivo,and explored the possible mechanisms.Methods: Bone marrow macrophages were extracted from the femur and tibia of three groups of CTR,Fe and Hepc mice.Some of them were induced to proliferate and differentiate by using M-CSF and RANKL in vitro.Some of them were inoculated on the bovine bone membrane for osteoclast bone resorption test.The cells in the three groups were stained with tartrate-resistant acid phosphatase and positive osteoclasts counting,and the ability of bone resorption was detected by toluidine blue staining.The expression level of m RNA was detected by quantitative reverse transcriptase polymerase chain reaction(RT-PCR)of the osteoclast associated gene CTK,MMP9,PTK2,TRAP,CTSK.Results: Compared with the CTR group,the gene expression level of the osteoclasts was significantly increased in the Fe group,but in the hepcdin overexpression group.the Hepc group,the related gene(CTK(F=39.640,P=0.0003),MMP9(F=8.031,P=0.0201),PTK2β(F=5.353,P=0.0463),TRAP(F=19.50,P=0.0024),CTSK(F=8.800,P=0.0164))level was decreased.TRAP staining(t=4.295,P=0.0127)and bone resorption lacuna(t=7.557,P=0.0016)showed that the osteoclasts proliferation,differentiation and bone resorption in the Hepc group were significantly lower than those in the Fe group.Conclusion : In iron accumulation environment,primary osteoclast proliferation,differentiation and bone resorption were enhanced,which may be due to the increased expression of osteoclast-associated genes in iron accumulation and osteoclasts were activated.The overexpression of hepcidn could inhibit the activation of osteoclasts and the abnormal activation of osteoclasts..