The Basic And Clinical Study Of The Effects And Related Mechanism Of Iron Overload On Bone Metabolism

Part Ⅰ The effect of iron excess on the biological activities of osteoblastObjective:To examine the effects of iron excess on the biological activities of osteoblast in vitro.To explore the mechanism behind effects of iron excess on the biological activities of osteoblast in vitro.Method:Human osteoblast cells(hFOB 1.19)were incubated in a medium supplemented with 50,100,200μmol/l ferric ammonium citrate(FAC).The intracellular iron was measured by a confocal laser scanning microscope.Proliferation of osteoblasts was evaluated by MTT assay.Apoptotic cells 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 of calcified nodules and mineral area were evaluated by Von-Kossa staining assay.The expressions of type Ⅰ collagen(COL-I)and osteocalcin(OC)of cultured osteoblasts were detected by reverse transcriptase polymerase chain reaction(RT-PCR)and Western blot.To explore the mechanism behind effects of iron excess on the biological activities of osteoblast in vitro,detection of osteoblast iron metabolism related genes membrane transferrin 1(FPN1)mRNA,transferrin receptor(TfR)mRNA and divalent metal transport protein 1(DMT1)mRNA expression,Osteoblast reactive oxygen species(ROS),malondialdehyde(Maleic Dialdehyde,MDA),Superoxide Dismutase(SOD),Superoxide Dismutase,SOD)and Glutathione peroxidase(Glutathione peroxidase,gsh-px)levels,further at the same time using tendency for 2.5L antioxidant n-acetyl cysteine(NAC)intervention,detection of intracellular ROS level,cell viability,cell OPG,OC and COL1 mRNA expression and activity of ALP.Results:FAC decreased the proliferation,ALP activity,mineralization function,and expressions of COL-I and OC at both mRNA and protein levels in cultured osteoblasts in a concentration-dependent manner.FAC increased percentage of apoptotic osteoblasts.After treated with different concentrations of FAC,the expression of FPN1 was up-regulated,while TfR and DMT1 were down-regulated significantly(P<0.05),the level of Intracellular ROS was increased significantly(P<0.05),the level of MDA was increased significantly with concentration-dependently,and the activity of SOD and GSH-PX were decreased significantly with concentration-dependently(P<0.05).After NAC 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,but higher than the NAC group;The level of ROS was negatively related to osteoblast biological activities including cell viability,OPG,OC and COL1mRNA expression and ALP activity(P<0.05).Conclusion:Excessive iron inhibited osteoblast activity in a concentration-dependent manner.The mechanism may be related to oxidative stress injury caused by the increase of iron concentration in cells.Part Ⅱ The effect of iron excess on the biological activities of osteoclastObjective:To examine the effects of iron excess on the biological activities of osteoclast in vitro,and to explore the NF-κB signaling pathway in this progress.Methods:The mouse monocytes were selected as RAW264.7 and differentiated into osteoclast cells under the action of RANKL.Using the TRAP staining and the osteoclasts to detect the differentiation of the osteoclast by 50 uM FAC intervention.q-PCR was used to detect the expression of broken bone related genes.The ROS level in osteoclast were detected by luciferase marker.The differences in protein expression of NF-κB pathway were detected by Western-Blot.Results:The number of TRAP staining positive cells and the bone pit can significantly increase when iron excess(P<0.05);MMP9,Acp5,Ctsk,Calcr gene expression levels can significantly increase(P<0.05);The activity of intracellular ROS can increase(P<0.05),high iron environment make the nucleoprotein p50 and p65,cytoplasm protein expression pIκBα predominate the volume increased significantly,and cut the cell nucleoprotein p65,cytoplasm protein p50,IκBα predominate expression level(P<0.05).Conclusion:Excessive iron can promote the differentiation and bone absorption of osteoclasts,which may be related to the activation of NF-κB signaling pathway.Part Ⅲ Effects of iron excess on bone metabolism of miceObjective:To investigate the effects and related mechanism of iron excess on bone metabolism of mice.Methods:24 male ICR mice were randomly divided into three group(8 in each)and were treated intraperitoneally with normal saline,FAC,or FAC plus NAC respectively.Bone marrow-derived monocyte(BMMs)were separated from the femurs of mice model and were further incubated to observe the osteoclast formation.Longitudinal femoral sections were stained with hematoxylin and eosin(HE)to observe the trabecular structure change.Micro-CT was used to analyse the trabecular and cortical structure changes in femurs of mice model.The Ferritin、MDA、8-OH-DG、TRAP-5b、CTX、RANKL/OPG、BALP and BGP in mice serum were determined by enzyme linked immunosorbent assay(ELISA).Results:HE staining showed that mice treated with FAC had less trabecular density at distal ends of femurs than mice in placebo group,and NAC treatment attenuated this effect.Micro-CT pictures and analysis revealed prominent reduction both in bone trabeculars and cortical bone in the femur of FAC-challenged mice,whereas the reduction was much lower in NAC treated mice.(P<0.05).Serum levels of MDA、8-OH-DG、TRAP-5b、CTX and RANKL/OPG increased after FAC treatment and decreased in mice treated with NAC(P<0.05).While BALP and BGP in mice serum decreased after FAC treatment and increased after NAC treatment(P<0.05).Conclusion:Iron excess may reduce the bone mass of the mice,and the mechanism may be related to the increase of bone absorption in mice after the increase of oxidative stress levels.Part Ⅳ Relationship between serum ferritin and bone density in healthy womenObjective:To study how increased iron storage in women influenced bone metabolism.Methods:This study composed of 435 women from January 2015 to December 2016,whose serum levers of ferritin(Fer)were measured and remarked.Bone mineral density(BMD)in femur and lumbar spine were measured by dual-energy X-ray absorptiometry.We observed serum Fer and BMD alteration with age.Then we categorized subjects into several groups according to the level of ferritin,and Non-conditional Logistic Regression Analysis was used to evaluate the risk of osteopenia.At last,associations between Fer and BMD in all areas were measured by multiple regression analyses and partial correlation.Results:The serum Fer level was low and stable in women before the age of 40,gradually increased in women aged 41-49,rapidly rose in women aged 50-65,and was remaining steady high after the age of 65.However,the BMD was high before the age of 50,rapidly fall in women aged 50-65,and kept low after the age of 65.In analysis by quintiles,after adjusting confounding factors,compared with the individuals in the lowest quintile,those in the highest quintile were more than twice as likely to suffer osteopenia in femur neck and L1-L4(OR:2.83 and 2.09).Age,weight,serum Fer and BMI were associated with BMD after using multiple regression analyses.Then adjusting age,weight and BMI,serum Fer also showed negative correlation with BMD of all regions(P<0.05).Conclusion:The concentration of serum Fer increased and BMD decreased with age.With increasing with the concentrations of Fer,the BMD decreased and the risk of osteopenia was higer and higer.Part ⅤStudy on the correlation between serum ferritin and bone density in patients with osteoporosisObjective:To study the correlation between bone and iron metabolism index in osteoporosis patients.Methods:117 cases of postmenopausal women were divided into normal group,bone loss(BL)and osteoporosis group according to BMD.All patients were measured the serum biochemical indicators,including calcium(Ca),phosphorus(P),and 25-hydroxyvitamin D(25-OH),hemoglobin,C-reactive protein(CRP)and white blood cell count(WBC),creatinine,uric acid,alanine aminotransferase(ALT),aspertate aminotransferase(AST),glucose;All patients were determined to include FER and TRF,as well as serum bone metabolism indicators including beta CTX and PINP.Results:Compared with Normal group,the serum ferritin(Fer)in BL and Osteoporosis group was significantly increased(p<0.05).The iron protein was positively correlated with CRP level(P<0.05).As women age,the risk of osteoporosis increases significantly.Fer level was negatively correlated with BMD(p<0.01).TRACP levels in Osteoporosis group were significantly higher than Normal group(p<0.05).ALP levels in Osteoporosis group were significantly upregulated compared with Normal group(p<0.05).β-CTX levels in Osteoporosis group were significantly increased compared with Normal group(p<0.05).PINP levels in Osteoporosis group were significantly higher than Normal group(p<0.01).More importantly,there was a positive correlation between serum Fer and PINP(p<0.01).Serum Fer showed a positive correlation of serum β-CTX.Conclusion:The overloaded iron improved the degradation of type I collagen,which is associated with inflammation,and collagen degradation may accelerate the process of osteoporosis.