The Correlations Between Serum Ferritin And Serum Hepcidin And Postmenopausal Osteoporosis And The Study On The Mechanism Of Bone Loss With Iron Accumulation

Part Ⅰ The correlations between serum ferritin and serum hepcidin and postmenopausal osteoporosisObjective:To investigate the correlations between serum ferritin(SF)and serum hepcidin(SH)and postmenopausal osteoporosis(PMOP).Methods:A total of fifty-one postmenopausal women were selected.Bone mineral density(BMD)of the femoral neck was detected using the dual energy x-ray absorptiometry.According to the result of BMD,fifty-one subjects were divided into three groups:osteoporosis group(17 cases),osteopenia group(18 cases)and normal bone mass group(16 cases).Body mass index(BMI),hemoglobin(HB)and blood biochemical indexes were detected.SF and SH were measured by enzyme-linked immunosorbent assay(ELISA).These values of indexes were compared among three groups.The correlations between the relevant factors and BMD were analyzed.Resuts:1)There were no differences in age,BMI and blood biochemical indexes among three groups(P>0.05).2)SF in osteopenia group and osteoporosis group were higher compared to normal bone mass group(P<0.05).SF in osteoporosis group was higher than that in osteopenia group(P<0.05).SH in osteopenia group and osteoporosis group were lower compared to normal bone mass group(P<0.05).SH in osteoporosis group was lower than that in osteopenia group(P<0.05).3)The results of pearson analysis showed there was a positive correlation between BMD of the femoral neck and SH(P<0.05),however,there was a negative correlation between BMD of the femoral neck and SF(P<0.05).Conclusion:SF and SH in postmenopausal women are closely associated with osteoporosis(OP).Part Ⅱ Iron accumulation inhibits the bone formation of ovariectomized mice via stimulating oxidative stressObjective:To investigate the effect of iron accumulation on bone formation of ovariectomized mice and underlying mechanism using a mouse animal model.Methods:72 mice were randomly divided into three groups:sham-operated(E+F-),ovariectomized(E-F-)and ovariectomized with iron injected(E-F+).E-F-group and E-F+group were bilaterally ovariectomized and injected intraperitoneally with normal saline(E-F-)or 60 mg·kg-1 ferric ammonium citrate(FAC)(E-F-)two times a week for one month.Blood and femurs of mice were harvested from three groups every seven days during 4 weeks of treatment.Serum ferritin(SF),osteocalcin(OC),alkaline phosphatase(ALP),superoxide dismutase(SOD)and malondialdehyde(MDA)were detected by enzyme-linked immunosorbent assay(ELISA).Bone mineral density(BMD),bone volume/total volume(BV/TV),trabecular number(Tb.N),trabecular separation(Tb.Sp)and trabecular thickness(Tb.Th)were analyzed by Micro-CT.Osteogenic-related gene expression were measured by quantitative polymerase chain reaction(q-PCR)analyses.In vitro,primary mouse osteoblasts isolated from newborn mouse calvaria were cultured,biofunction of osteoblasts were estimated by ALP staining,cell viability(CV)was tested using the Cell Counting Kit-8(CCK-8),reactive oxygen species(ROS)was measured using fluorescent probe 2’,7’-dichlorodihydrofluorescein diacetate(DCFH-DA).Results:SF in E-F+was higher than the other groups(p<0.05).Serum OC and ALP levels in E-F+group markedly reduced in the 4th week compared to those in E-F-group(OC:(88.6±8.2)vs(55.3±9.2)ng/ml;ALP:(18.1±1.6)vs(12.0±1.8)U/100ml,E-F-vs E-F+),while serum SOD and MDA levels in E-F+group were higher than those in E-F-group at every time point except SOD at the 1st week.BMD in E-F+ group further decreased compared to that in E-F-group at the 3rd and 4th week,there were significant differences between E-F-and E-F+(p<0.05).In comparison to E-F-group,the q-PCR revealed the expressions of osteogenic-related genes(Runx2,SP7 and Bglap)in the bone tissue of E-F+ group decreased to 76%,72%,74%of that in the 3rd week and 46%,49%,36%in the 4th week.In vitro,ALP staining showed ALP activity in E-F+ group was lowest,the osteoblasts viability in E-F+ group decreased to 45%of that in E-F-group at the 4th week and ROS in E-F+ group increased significaitly from the 2nd week as coVpared with E-F-group(1.43-,1.48-,1.85-fold,at 20d,3rd,and 4th week,respectively).Conclusions:The present study showed that iron accumulation could inhibit the bone formation of ovariectomized mice via stimulating oxidative stress level.Part Ⅲ Screening of microRNA and the targets prediction in mice with iron-induced osteopeniaObjective:To explore the correlation between bone loss and the differential expression of microRNA(miRNA)in mice with iron accumulation.Methods:Twelve male ICR mice(12-week-old)were randomly divided into two groups:FAC group and Control group.Intraperitoneal injections of 0.04 g·kg-1 ferric ammonium citrate(FAC)and equivalent saline three times a week for eight weeks were conducted in these two groups.Serum ferritin(SF)level was detected after 8 weeks of intervention.Micro-CT was used to detect bone volume and bone structure parameters in mice.The whole blood of mice were collected and RNA from white blood cells were extracted.Differential expression profiles of miRNA chips was conducted,and the result was verified by real-time quantitative polymerase chain reaction(RT-PCR).Targetscan and miRDB were used to predict the downstream target genes of miRNA.Bioinformatics analysis of target gene was conducted,including celluar component,biologic process,molecular function and kyoto encyclopedia of genes and genomes(KEGG)pathway.Result:SF in the FAC group(218.2±29.5)μg/L was significantly higher than that in the control group(30.6±9.9)μg/L after 8 weeks.Micro-CT analysis showed a destruction of bone structure in iron accumulation mice.Bone mass,trabecular thickness(Tb.Th)and bone volume/total volume(BV/TV)decreased significantly(P=0.001,0.005,0.021)in iron accumulation mice,while trabecular separation(Tb.Sp)increased.miRNA chip results:20 miRNAs up regulated:mmu-miR-29b-3p、mmu-miR-324-3p、mmu-miR-133a-5p、mmu-miR-214-5p、mmu-miR-22-3p、mmu-miR-34a-5p、mmu-miR-31-5p、mmu-miR-143-5p、mmu-miR-423-3p、mmu-miR-223、mmu-miR-155、mmu-miR-106a、mmu-miR-2861、mmu-miR-148a、mmu-miR-96、mmu-miR-449a-5p、mmu-miR-423-5p、mmu-miR-204-5p、mmu-miR-211、mmu-miR-23b and 7 miRNAs down regulated:mmu-miR-18a-3p、mmu-miR-223-3p、mmu-miR-199a-5p、mmu-miR-196a-5p、mmu-miR-30c-5p、mmu-miR-15b、mmu-miR-130b were screened out.The difference of mmu-miR-423-5p is the most significant.The result of RT-PCR on miR-423-5p had a high concordance with the result of microarray.91 downstream target genes of miR-423-5p were predict by Targetscan and miRDB tools.Bioinformatics analysis showed that target genes are clustered on phosphatidylinositol 3 kinase(PI3K)-protein kinase B(Akt),Rap1,Ras and mitogen-activated protein kinase(MAPK)signaling pathways.Conclusion:The iron-induced bone loss may be related to the differential expression of miR-423-5p.