Effects And Related Mechanism Of Iron Overload On Bone Metabolism

Part I A comparison of the biological activities of osteoblast betweeniron excess and iron deficiencyObjective: To examine the effects of iron excess and iron deficiency on the biologicalactivities of osteoblast in vitro.Method: human osteoblast cells (hFOB1.19) were incubated in a mediumsupplemented with0,50,100,200μmol/l ferric ammonium citrate (FAC) and0,5,10,20μmol/L deferoxamine (DFO). The intracellular iron was measured by a confocal laserscanning microscope. Proliferation of osteoblasts was evaluated by MTT assay. Apoptoticcells were detected using Annexin intervention V/PI staining with a flow cytometry.Alkaline phosphatase (ALP) activity was measured using an ALP assay kit. The number ofcalcified nodules and mineral area were evaluated by Von-Kossa staining assay. Theexpressions of type I collagen (COL-I) and osteocalcin (OC) of cultured osteoblasts weredetected by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot.Results: FAC decreased the proliferation, ALP activity, mineralization function, andexpressions of COL-I and OC at both mRNA and protein levels in cultured osteoblasts in aconcentration-dependent manner. FAC increased percentage of apoptotic osteoblasts.These activity indexes were increased by DFO at low concentration, but were decreased athigh concentration.Conclusion: Excessive iron inhibited osteoblast activity in a concentration-dependentmanner. Low iron concentrations, in contrast, produced a biphasic manner on osteoblasts:mild low iron promoted osteoblast activity, but serious low iron inhibited osteoblastactivity. Osteogenesis was optimal in certain iron concentrations.Part II Mechanism of effects of iron excess on the biological activities ofosteoblastObjective: To explore the mechanism behind effects of iron excess on the biologicalactivities of osteoblast in vitro. Methods: Human osteoblast cells (hFOB1.19) were cultured at34℃in vitro. Cellswere incubated in media supplemented with different concentrations (0,50,100,200μmol/l) of FAC. The genes expression of transferring receptor(TfR), divalent metaltransporter1(DMT1) and ferroportin1(FPN1) were detected by RT-PCR; Intracellularreactive oxygen species(ROS) was measured using the oxidation sensitive dye DCF-DA byflow cytometry; Maleic dialdehyde(MDA) content, superoxide dismutase(SOD) andglutathione peroxidase(GSH-PX) activity were measured with MDA, SOD and GSH-PXcommercial kits; To investigate mechanisms of oxidative stress, antioxidant N-acety lcysteine(NAC) was added to medium. Intracellular ROS was measured by flow cytometry,CCK-8assay detected the cell viability, the expression of OPG, OC and COL1mRNAdetected by RT-PCR, ALP activity was measured using ALP viability kit.Results: After treated with different concentrations of FAC, the expression of FPN1was up-regulated, while TfR and DMT1were down-regulated significantly (P<0.05), thelevel of Intracellular ROS was increased significantly (P<0.05), the level of MDA wasincreased significantly with concentration-dependently, and the activity of SOD andGSH-PX were decreased significantly with concentration-dependently (P<0.05). AfterNAC was added to medium, the level of ROS was significantly different in virous groups(P<0.05), and the FAC+NAC group was significantly lower than the FAC group, buthigher than the NAC group; The level of ROS was negatively related to osteoblastbiological activities including cell viability, OPG, OC and COL1mRNA expression andALP activity(P<0.05).Conclusion: The mechanism of iron excess on the biological activity of osteoblasts maybe related to the increasing of intracellular iron concentration and oxidative stress.Part III Effects of iron excess on differentiation of osteoblast andosteoclastObjective: To investigate the effects of iron excess on differentiation of osteoblastand osteoclast in vitro.Methods: Mouse MC3T3-E1cells were incubated in a medium supplemented withdifferent concentrations (0,50,100,200μmol/l) of FAC under induction of of10mmol/lβ-Glycerophosphate and50μg/ml L-Ascorbic acid. The expression of Runx2、Osterrix、bone sialoprotein(BSP) and osteocalcin(OC) mRNA detected by RT-PCR. ALP activitywas measured using ALP viability kit. Mouse monocyte RAW264.7cells were incubated in a medium supplemented with different concentrations (0,12,25,50μmol/l) of FAC underinduction of20ng/ml receptor activator of NF-Kb ligand (RANKL). Cell viability, thenumber of tartrate-resistant acid phosphatase (TRAP)-positive cells, levels of TRAP,cathepsin K (CTK), and matrix metalloproteinase9(MMP-9) mRNA were examined.Results: The expression of Runx2, Osterrix, BSP and OC mRNA was down-regulatedand ALP activity was decreased by FAC in a concentration-dependent manner (p<0.05) inMC3T3-E1cells. The number of TRAP-positive cells was increased and the expression ofTRAP, CTK, and MMP-9mRNA was up-regulated by FAC in a concentration-dependentmanner (p<0.05) in RAW264.7cells.Conclusion: Iron excess can significantly inhibit L-Ascorbic acid andβ-Glycerophosphate-induced differentiation of MC3T3-E1cells into osteoblasts, andfacilitate RANKL-induced differentiation of RAW264.7into osteoclasts in vitro.Part IV Effects of iron overload on bone metabolismin ovariectomized ratsObjective: To explore the effects of iron overload on bone metabolism inovariectomized rats.Methods:40three-month-old female SD rats were randomly divided into5groups:Sham group, OVX group，OVX+high dose of FAC(FAC1), OVX+medium dosa ofFAC(FAC2), and OVX+low dose of FAC(FAC1) with8in each. Rats in3groups of FACtreatment received throurh enterocoelia injections of different dose (45,90,180mg/kg) ofFAC every other day for9weeks, while rats in groups of Sham and OVX received throurhenterocoelia injections of saline simultaneously. All of the rats were executed, iron ion inserum were measured with an automatic biochemistry analyzer, and the iron content of thetibia was measured by ICP tester, and iron in the liver was observed by Perl’s staining, andthe trabecular architecture of the right femur were observed by histomorphology, and theleft femur was scaned with a high resolution micro-CT, and β-CTX and BGP in serumwere measured with an ELISA kits.Results: Serum iron and tibia iron in OVX group were decreased significantlycompared to Sham group(p<0.05); Serum iron and tibia iron in FAC1, FAC2and FAC3groups were increased significantly with increasing of dose of FAC treatment(p<0.05);Perl’s staining in liver was positive for FAC1, FAC2, and FAC3groups. Micro-CT andbone histopathology showed the bone trabeula in Sham group lined up in order, and the bone trabeula in OVX group was sparse, and the bone trabeula in FAC1, FAC2and FAC3groups become thinner, even distorted or broken with increasing of dose of FAC treatment.ELISA showed the levels of serum β-CTX and BGP in OVX, FAC1, FAC2, and FAC3groups were increased significantly compared to Sham group(p<0.05), but the level ofserum BGP were no significant difference in later fore groups(p>0.05), but the level ofserum β-CTX were increased significantly with increasing of dose of FACtreatment(p<0.05).Conclusion: iron overload can aggravate osteoporosis in ovariectomized rats, and themechanism may be attributed to enhanced bone resorption reduced by exssive iron.