Role And Mechanism Of Iron Accumulation On Postmenopausal Osteoporosis With Hepcidin Inhibited Zebrafish By Genetic Tools

Postmenopausal women tend to appear in a decade of iron accumulation,one of the significant factors is associated with osteoporosis railway in many factors.Iron can be combined with membrane ferroportin1,promoting the internalization and degradation to indirectly regulate homeostasis.Once iron overload,iron can enhance gene expression,making increased hepcidin of liver secretion,so as to accelerate the degradation of ferroportin 1t also stop the iron transporting to the blood.This way reducing intestinal epithelial cells and macrophages of the transport of iron to the blood.While the body’s iron deficiency,the opposite change in the above process as to maintain steady state of iron.In the hepcidin knockout mice,we observed the phenomenon of iron overload.But it had disadvantages that cultivating cycle is long,genetically modified mice is hard to get,representation to difficult obtain and mechanism of further in-depth research was very hard.Cells have certain limitations in vitro experiment.It needed the arrival of the new model animal model.Zebrafish(Danio rerio)as a model organism has been widely applied in the study of genetics and developmental biology.We currently use developed knock down and knock out gene technology in recent years to observe the changes of bone metabolism of iron accumulation of zebrafish.Part I Hepcidin knocked down to study the metabolism of osteoblast in zebrafishObjective:Hepcidin was knocked down by the microinjection of oligo morpholine in zebrafish embryo,the influence of bone metabolism was observed after expression inhibited of hepcidin in zebrafish.The mechanism of hepcidin knocked down effecting on bone metabolism is explored.Methods:We treat half an hour post-fertilization zebrafish with different concentrations of oligo morpholine microinjection.Basd on identifion of the cDNA effect,the concentration of 25 PPM of oligo morpholine was applied.At 4 dph,change of iron content after the injection of larvae was observed compared with wild type,At 9 dph,Alizarin Red staining was performed for measuring vertebral bodies and the expression of bone metabolic cDNA differences was analysed with real-time PCR on 4 dph.runx2 a and sp7 in situ hybridization compared with the wild type of juvenile fish was associated part of the distribution of different gene expression.Results: At 4 dph,iron stain pigmentation was significantly darker than the wild type,alizarin red staining becomes shallow and ribs and caudal fin was not developed,al sheen blue staining did not change obviously,Quantitative PCR related metabolic gene expression was expressed down,Especially runx2 a and sp7 in the most significant,the wild type was 1.0079±0.01117 and 1.12450±0.17607 was compared with 0.25639±0.016072 and 0.016072±0.07436 after treatment.Expression of runx2 a and sp7 in situ hybridization signal was weaker than the wild type in zebrafish.Conclusion: Hepcidin knocked down in zebrafish represent iron accumulation in the body,reducing bone gene expression,bone loss and retarded bone development.Part II Research on osteogenesis metabolism of transgenic zebrafish by hepcidin green fluorescent proteinObjective To investigate the effect of hepcidin downregulation on the osteogenesis metabolism and involved mechanism based on the construction of hepcidin green fluorescent protein(GFP)in the transgenic zebrafish.Methods The eggs of zebrafish were transfected by hepcidin GFP plasmid by microinjection in half an hour after fertilization.The expression of hepcidin in the zebrafish was checked every 6 hours for 120 hours.Iron staining change was done in the larvae of zebrafish transfected by ATG morpholine and GFP plasmid compared with wild type in 4 days in the same conditions.Alizarin red staining of bone and cartilage was performed in 9 days and the expression of bone metabolic cDNA differences was analysed with real-time PCR on 4th day.Results Hepcidin expressed begin on 72 hours and strongest in 96 hours on GFP transgenic zebrafish.Iron staining was significantly darker than the GFP-control type at 4 dph.Alizarin red staining became shallow and ribs and caudal fin did not change furthermore in zebrafish transfected by ATG morpholine.PCR related metabolic gene expression was expressed down in zebrafish transfected by ATG morpholine.Conclusion Hepcidin expressed strongest on GFP transgenic zebrafish on 96 hours.The inhibition of hepcidin caused iron accumulation and lowered the osteogenesis led to retardation and reduction of bone in zebrafish.Part III Role and mechanism of iron accumulation on hepcidin knockout zebrafish by CRISPR/CAS9Objective: The bone metabolism and the correlation of iron metabolism was studied in the world’s first successful hepcidin knockout of zebrafish by CRISPR/CAS9.Methods: The eggs of zebrafish were transfected by hepcidin CRISPR/CAS9 plasmid by microinjection in half an hour after fertilization.Iron staining change was done in the larvae of zebrafish transfected by hepcidin-/-mutation compared with wild type in 4 days in the same conditions.Alizarin red staining of bone and cartilage was performed in 9 days and the expression of bone metabolic cDNA differences were analysed with real-time PCR on 4th day.Compared with the morphology of bony difference,microCT was tested after three months.Through high-throughput transcriptome sequencing show some relevant channeland quantitatively and in situ hybridization further was verificated,its related mechanism of the channelwas further found by the cell in vitro experiments.Results:We use the gene knockout technology CRISPR/CAS9 to successfully establish the hepcidin knockout zebrafish homozygous mutant.We found its iron overload through the iron staining.The mutant fish showed that caudal fin bending,gill cover is missing and maxillarymissing significant phenotypeby alizarin red staining.We found that hepcidin-/-mutation body bone lossand bone healing is not completeby Micro-CT analysis.The mutant bony part includes the skull,spinal and fin department showed different degrees of defects,and the cartilage part almost no differenceby hard bone and cartilage staining.These results suggest that iron wasessential for zebrafish hard bone development.Fluorescence quantitative analysis found that bony marker gene such as runx2 a,runx2b,sp7,alp in hepcidin-/-significantly lower than wild type.In situ hybridization experiment shows bony gene(runx2a,sp7)expression is restrainedin zebrafish,knock down the hepcidin in runx2 a andsp7 mutants zebrafish,we found it inhibit parts of the form expression.We analyzed the zebrafish’s runx2 a promoter and further found that iron adjustable element by binding the expression of bmp2 to inhibit runx2 a,failing to pass the HJVthrough the cell experiment and COIP in vivo experiment.ChIP results show that the smad1/5/8 phosphorylated proteins can bind on runx2 a promoter sites.We injected hepcidin mRNA into mutant and found its phenotype was restored.We found that the gene expression can be restored to a certain extentthrough the QPCR and in situ analysis of runx2 a.These results suggest that hepcidin directlymodulates runx2 a gene expression to regulate biomineralization of bone in zebrafish.