Clinical Observation And Molecular Mechanism Of The Influence Of Iron Accumulation On Bone Metabolism

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Clinical correlation between "iron metabolism" and "bone metabolism" in patients with postmenopausal brittle fractureObjective: To observe the clinical correlation of serum ferritin,bone iron content,bone mineral density(hip,lumbar)and bone turnover markers in patients with postmenopausal hip brittle fracture.Methods: 304 cases of postmenopausal women with hip fracture treated by total hip arthroplasty were reviewed from 2014-01 to 2017-05.The femoral head specimens were collected during the operation for the determination of bone iron content.All patients serum were collected 1 day before the operation and related indexes were detected,7 days after operation,hip and lumbar vertebra bone density were detected.Exclusion criteria:multiple fractures(n=6);chronic liver disease(n=2);diabetes and thyroid related diseases(n=31);postoperative infection(n=4);bisphosphonate application history(n=1).In the end,260 patients were included,and the related clinical data were statistically analyzed.Results: Serum iron metabolism markers(ferritin,Fer;Bone iron;transferrin,TRF;serum iron ion,Fe)was analyzed and presentes as matrix diagrams.Ferritin was the strongest marker correlatied to bone iron.Pearson correlation analysis indicated that all clinical indicators were not correlated with bone iron except Fer(r=0.696,p=0.000).Correlation between the clinical indicators and BMD(hip and lumbar)was observed using Pearson correlation analysis.Age,Fer,bone iron,BMI and TRF were significantly correlated with BMD.Age,Fer,and BMI could be incorporated into multiple linear regression equation of hip BMD.Age,Fer and bone iron could be incorporated into multiple linear regression equation of lumbar BMD.Partial correlation analysis indicated that there was a significant negative correlation between the bone iron and the BMD of the hip and lumbar.Three-dimensional scatter points and mesh graphs were drawn for age,bone iron and bone density.The contour map was used as a result to show that bone mineral density decreased significantly with the increase of bone iron content and age.260 patients were divided into osteoporosis group and non osteoporosis group.OP diagnosis was used as dependent variable.Bone iron was used as the independent variable to draw the receiver operating characteristic curve(ROC).The area under the curve(AUC)was0.790(p<0.05),and the cut-point of bone iron was 139.7 ?g/g.Taking this as the boundary value,the population was divided into high bone iron group and low bone iron group.The odds ratio of OR was12.143(95%CI=5.406-27.777),the risk of osteoporosis in the patients with high bone iron was 12.143 times as much as that of the low bone iron patients.The correlation between bone iron and bone turnover index(BTMs)was observed by Peason correlation analysis.The results showed that there was a significant positive correlation between bone iron and bone resorption index(TRAP-5b and B-CTX)(r=0.202,0.209,p=0.012,0.009).Conclusion: The index of bone iron is associated with serum ferritin,which may be a potential value in the diagnosis of iron metabolism in fracture patients.The accumulation of bone iron may be an independent risk factor for the decrease of bone mass.The effect of bone accumulation on bone mass may be related to the process of bone absorptionPart ? Iron accumulation effect on osteoporosis in mice model of bone metabolism,osteoblast and osteoclast activity.Objective: To observe bone mass,bone turnover markers,osteoblast and osteoclast related functional changes in normal mice and ovariectomized osteoporosis mice model after iron intervention.Methods: A preliminary simulation of the basic experiment on the first part of the clinical conclusion was conducted.1.Animal experiment: 3 month old female ICR mice were randomly divided into 4 groups(CON group,F group,OVX group,F+OVX group),two groups underwent bilateral ovariectomy.Con group and OVX group were injected with NS,the two other groups injected with iron ammonium citrate(FAC).After 4 weeks,body weight was tested.The liver and bone tissue were stained with Prussian blue iron.Three-dimensional reconstruction of femurs were analyzed.ELISA was used to detect serum Fer,alkaline phosphatase(ALP),osteocalcin(osteocalcin),type I collagen C terminal peptide(CTX),tartrate resistant acid phosphatase 5b(TRAP-5b),malondialdehyde(MDA)and superoxide dismutase(SOD).Mouse bone marrow was added to macrophage colony-stimulating factor(M-CSF)and NF kappa B receptor activating factor ligand(RANKL)to induce osteoclasts,and TRAP staining was used to count positive cells.2.Cell experiment:(1)primary osteoblasts were divided into control group,FAC group,E2 group,FAC+E2 group.FAC group and FAC+E2 group were intervened by 10 ?M FAC,E2 group,FAC+E2 group were treated with 10 n M estradiol.Cells were stained with ALP and alizarin red staining.Bone related genes expressions(RUNX2,SP7,BGLAP)were observed by q-PCR.(2)RANKL induced RAW264.7 to differentiate osteoclasts.TRAP staining was used to count osteoclasts.The reactive oxygen species(ROS)fluorescence intensity of osteoblasts and osteoclasts after DCFH-DA intervention was observed.Osteoclast related genes(TRAP5,CTSK,MMP9,CALCR)were tested.Results: 1.Animal experiments: there was no significant difference in weight between the 4 groups(p>0.05).After 4 weeks,the serum levels of Fer in group F and F+OVX increased significantly(p<0.05),and the deposition of Prussian blue iron in the liver and distal femur was significant.Micro-CT results showed that the bone density was not affected by the normal level of estrogen and the accumulation of iron,and the decrease of bone mass in the iron accumulation group was more significant after the ovariectomy.Serum protein ELISA results showed that normal estrogen levels in mice(F group)compared with the control group,bone absorption index(TRAP-5b,CTX)and oxidative stress index(MDA,SOD)had no significant difference.Bone formation index(ALP and osteocalcin)of F group decreased compared with control group.After estrogen deficiency,iron accumulation significantly increased MDA and bone resorption index,and reduced bone formation index and SOD,and the number of osteoclasts was significantly higher than that in ovariectomy group(p<0.05).2,Cell experiment: osteoblasts ALP detection showed that iron can inhibit osteoblast ALP activity,q-PCR activity test results were consistent with the results of ALP.The RAW264.7 cell TRAP staining showed: after the intervention of iron,osteoclast like cells significantly increased.There wad no significant difference of TRAP positive cells number between E2 group and FAC+E2 group.DCFH-DA test results: iron can significantly increase the ROS level of osteoblasts and osteoclasts(p<0.05).FAC significantly increased the level of expression of TRAP-5,CTSK,MMP9 and CALCR.This up-regulated effect was inhibited when estrogen was pre intervened.Conclusion: In the presence of estrogen,iron accumulation has no obvious effect on bone metabolism.In the absence of estrogen,it promotes the decrease of bone mass,which is related to the up-regulation of bone resorption and osteoclast activity.ROS may regulate the process.Part ? The changes of bone metabolism of osteoporosis mice model in different times after intervention of ironObjective: To observe bone mass,bone turnover markers,osteoblast and osteoclast related functional changes of osteoporosis mice model at different time points after the intervention of iron.Methods: According to the conclusions of the second part,the March old female ICR mice were randomly divided into 3 groups(E+F-group,E-F-group,E-F+ group),E-F-group and E-F+ group underwent bilateral ovariectomy.Group E+F-and group E-F-were injected with NS,the remaining group was injected with equal amount of FAC,and the intervention was 4 weeks.6 mice were killed each group per week,and all the mice were collected to carry out the cell test.The weight of the mice was measured.Three dimensional reconstruction of Micro-CT was performed on the distal femur.The serum Fer,ALP,osteocalcin,CTX,TRAP-5b,MDA and SOD were detected by ELISA.The bone marrow cells in the femur and tibia of all mice were induced to osteoclasts.TRAP staining was used to observe the differentiation of osteoclasts.The primary osteoblasts from newborn mice were extracted and cultured with the serum collected from every mouse.Therefore,the mice were divided into three groups.ALP staining was performed on the 3 day and alizarin red staining on the 14 day.Results: There were statistically significant differences of serum Fer 1 week after the FAC intervention(p<0.05).The serum bone formation related indexes ALP and osteocalcin were statistically different in 4th week after the FAC intervention(p<0.05).There was a statistical difference of CTX in the 2nd week and TRAP-5b in the 3rd week(p<0.05).SOD decreased(p<0.05)at second weeks after FAC intervention,and MDA increased at first weeks(p<0.05).The results of Micro-CT showed that there was a statistical difference of BMD in the 4th week(p<0.05),and the three dimensional reconstruction and the other parameters were consistent with the results.TRAP staining: osteoclast differentiation increased in the 2nd week,reaching the peak in 4th week.ALP staining showed that FAC intervention significantly inhibited osteoblast activity from the second week.Calcium nodule staining suggested that the mineralization of calcium nodules was significantly inhibited in the 3rd week.Conclusion: Iron accumulation first affects the level of reactive oxygen species(1-2weeks),then affects osteoclast differentiation(2 weeks)and bone resorption(2-3 weeks),then affects the mineralization of osteoblasts(3 weeks)and bone formation(4 weeks),and ultimately affects bone mass(4 weeks).All the indexes of each group showed the greatest difference in the 4th week.Part ? The role of MAPK and NF-kappa B signaling pathway in "iron-induced stimulation of osteoclasts"Objective: To observe the changes of MAPK and NF-kappa B signaling pathway in bone tissue and osteoclast,and to explore the role of oxidative stress in this process.Methods: All specimens of the 4th week in previous study were collected.The bone tissue sections were stained with H&E and TRAP.RNA and protein were extracted from bone tissue,gene and protein expressions of bone and osteoclasts were detected,and the level of oxidative stress protein(GSH-Px)was measured.The primary osteoclasts were extracted and induced,the number of positive cells was counted by TRAP staining,and the level of intracellular ROS was detected by DCFH-DA.The bone tissue protein of MAPK(ERK1/2,p38,JNK and corresponding phosphorylated protein)and NF-kappa B(nucleus P50,P65 and cytoplasmic I kappa B alpha)were detected.The primary osteoclasts in group E-F-and group E-F+ were collected.Cells were intervened by inhibitors of MAPK pathway and NF-kappa B pathway,and the corresponding protein expression and osteoclast differentiation were detected.The gel electrophoresis mobility test(EMSA)was used to detect the binding capacity of DNA and NF-kappa B.The cells in group E-F-were interfered with H2O2.We used BAY11-7082 to inhibit the NF-?B pathway.The expression of pathway related protein and osteoclast differentiation of three groups were detected.Denosumab(Damb)was treated in E-F-group and E-F+ group of mice to explore the upstream target of FAC.Micro-CT 3D reconstruction,extraction and induction of primary osteoclasts and TRAP staining were conducted in bone tissue.Western blot eas used to detect the RANKL protein expression in bone tissue.Results: The results of q-PCR suggest that iron accumulation significantly up-regulated the expression of osteoclast related gene(p<0.05).The expression level of osteoclast related protein TRAP and Cathepsin K also increased significantly in the high iron environment(p<0.05).FAC had no obvious effect on the gene and protein expression of osteogenesis.The expression of GSH-Px in bone tissue was obviously decreased by iron accumulation.H&E staining showed that the trabecular bone was sparse after ovariectomy,and the bone trabecular space was further increased after FAC intervention.Bone tissue and primary osteoclast TRAP staining showed: the number of osteoclasts in bone tissue was significantly increased after iron intervention.DCFH-DA fluorescence showed that the intervention of iron increased osteoclast number and intracellular ROS fluorescence intensity.Bone tissue Western blot results: MAPK signaling pathway related protein detection results showed that FAC significantly activated ERK and JNK signaling pathway,and had no significant effect on p38 signal pathway protein(p>0.05).The detection of NFkappa B signaling pathway related proteins showed that FAC significantly activates the expression of P65/P50 two polymer in the nucleus.The MAPK pathway was suppressed in the primary osteoclasts under the high iron environment.There was no significant difference in the differentiation of osteoclast,and the results of q-PCR were in accordance with the results of TRAP staining.The NF-kappa B pathway was suppressed in the primary osteoclasts under the high iron environment and the number of TRAP positive cells decreased significantly(p<0.05).EMSA results showed that the binding ability of NF-kappa B to DNA decreased.The intervention of H2O2 decreased osteoclast GSH-Px expression,increased IKK expression and stimulated osteoclast differentiation.IKK expression and activity of osteoclasts decreased when NF-?B pathway was inhibited.The bone mass recovery was obvious,and the differentiation of osteoclast was obviously inhibited after the treatment of Damb.There was no statistical difference of activity of osteoclasts between E-F-group and E-F+ group.Conclusion: The enhancement of iron accumulation on osteoclast differentiation is related to the up regulation of ROS and the further activation of the NF-kappa B signaling pathway,which is not related to the MAPK signaling pathway.IKKs protein may be the upstream target of iron to influence bone absorption.