Clinical And Experimental Study On Nox4-Mediated Osteoblast Ferroptosis In Iron-Accumulation-Caused Bone Loss

Part Ⅰ Iron accumulation induces osteoblasts ferroptosisObjective:The purpose of this study was to observe the effect of iron accumulation on ferroptosis of osteoblasts.We used iron agent to intervene osteoblasts and detected the changes of ferroptosis index and bone formation function in osteoblastsMethods:Different concentrations of ferroptosis inducer Erastin(0,5,10,15,20,25μm)were added as positive control and different concentrations of iron dextran(Fe;0,200,400,600,800,1000 μg/ml)as experimental group.Ferrostatin 1(Ferr-1),an ferroptosis inhibitor,was added at different concentrations(0,2,5,10,15,20 μM)and deferasirox(DFO,200 μM)to the iron intervention group for rescue intervention.The osteoblastic activity was detected by CCK-8.The content of ferroptosis marker protein GPX4 in osteoblasts was detected by Western Blot.The morphology of mitochondria in osteoblasts was observed by transmission electron microscopy.The differentiation and mineralization ability of osteoblasts were detected by ALP and alizarin red staining.The expression levels of ferroptosis marker gene PTGS2 and osteoblastic marker gene ALP,RUNX2 and OCN in osteoblasts were detected by RT-PCR.Annexin V-FITC Cell apoptosis assay kit was used to detect the apoptosis of osteoblasts.Results:The number of osteoblasts of both ferroptosis inducer Erastin and chalybeate pretreatments decreased significantly,and the intracellular GPX4 level decreased significantly with the increase of dose.we added ferroptosis inhibitor Ferr-1,and the cell number and GPX4 content were partially restored.After the intervention of Fe or Erastin,the mitochondria in osteoblasts were wrinkled from long rods to punctate,and the color was dark under transmission electron microscopy;the expression of ferroptosis marker protein GPX4 decreased,while the expression of ferroptosis marker gene PTGS2 increased;ALP and alizarin red staining showed that lighter color of the staining of osteoblasts;the expression of osteoblast marker genes ALP,RUNX2 and OCN was decreased(P<0.05).however,Ferr-1,an iron death inhibitor,and DFO,an iron chelator,can reverse the above manifestation of osteoblasts.With Annexin V-FITC apoptosis detection kit to the dyeing of osteoblast cells,then flow cytometry instrument were analyzed,there was no more apoptosis characteristics of apoptosis cells(only positive Annexin V)ratio after Fe intervention of osteoblast and Erastin intervention group was no significant difference compare with control group.Conclusion:Iron accumulation can induce the morphological changes of mitochondria and ferroptosis in osteoblasts,and the function of osteoblasts was inhibited.Part Ⅱ Bone loss in iron accumulation mouse model and ferroptosis occurred in boneObjective:In order to verify the effect of ferroptosis on bone mass,the present study used endogenous and exogenous iron accumulation mouse models to study,the relationship between ferroptosis and the changes in bone metabolism and bone mineral density.Methods:CRISPR/Cas9 technology was used to knockout systemic hepcidin in mice to construct the endogenous iron accumulation mouse model.5 months old male mice were divided into control group(WT),hepcidin knockout group(Hepc1-/-),hepcidin knockout+ferroptosis inhibitor group(Hepc1-/-+Ferr-1)and hepcidin knockout+iron chelator group(Hepc1-/-+DFO)in the study.The control group was wild-type C57B6/J mice,while the other groups were hepcidin whole-body knockout mice.Ferr-1 and DFO were interented for 8 weeks,respectively.The exogenous iron accumulation mice was constructed by intraperitoneal injection of iron.Male mice aged 5 months were divided into control group(Ctrl),iron accumulation group(Fe),iron accumulation+ferroptosis inhibitor group(Fe+Ferr-1),and iron accumulation+iron chelator group(Fe+DFO).Mice in the control group were injected intraperitoneally with normal saline,while mice in the other groups were injected intraperitoneally with ammonium ferric citrate 0.1g/(kg·wk)for 8 weeks.8 weeks later,to collect serum,liver and bone tissue samples,the mice were sacrificed.Immunohistochemistry was used to detect the expression of hepcidin in paraffin sections of liver.Prussian blue staining was used to detect the liver iron content in paraffin sections.Serum levels of hepcidin,ferritin,and MDA were detected by enzyme-linked immunosorbent assay(ELISA).Microcomputed tomography(Miceo-CT)was used to detect bone mineral density and microstructure of femur in mice.Immunohistofluorescence assay was used to detect the ferroptosis marker protein GPX4 in frozen bone tissue sections.Results:The expression of hepcidin both in liver detected by immunohistochemistry and in serum detected by ELISA were decreased in Hepc1-/-group mice.The expression of hepcidin in FAC group mice was increased,but the serum ferritin in both groups was increased(P<0.05-0.01),and the liver was darkly stained.Prussian blue staining showed obvious iron deposition in liver.The model of iron accumulation in mice was established successfully.In the two iron accumulation models,the content of MDA in serum increased(P<0.01).Hepc1-/-and FAC mice treated with ferroptosis inhibitor Ferr-1 showed reduced MDA(P<0.05).After DFO intervention,liver iron,serum ferritin(P<0.01)and MDA(P<0.05-0.01)were all decreased,although there was no significant change in hepcidin level.Micro-CT and GPX4 antibody immunohistofluorescence assay of femur of mice showed mean bone mineral density(BMD),trabecular volume percentage(BV/TV),trabecular number(Tb.P<0.05-0.001)and the total GPX4 protein content of bone tissue decreased in Hepc1-/-and FAC mice.After Ferr-1 or DFO intervention,BMD(P<0.05-0.001)and bone tissue GPX4 were partially recovered.Conclusion:Endogenous and exogenous iron accumulation can induce bone tissue ferroptosis,reduce bone mass,and inhibit ferroptosis to alleviate bone mass loss.Part Ⅲ Clinical correlation between ferroptosis and iron accumulation in postmenopausal osteoporosisObjective:To further verify the correlation between iron death and osteoblasts and bone mass,In this study,the bone tissueof postmenopausal women and the data of physical examination population were collected to observe the correlation between ferroptosis and bone metabolism in iron-accumulating osteoporosis populationResults:The study was divided into two parts.The first part:a small amount of bone tissue was collected from postmenopausal hip replacement patients without increasing iatrogenic injury,and according to whether the serum ferritin(Fer)was greater than 150μg/L,the patients were divided into two groups,namely Fer<150 μg/L and Fer>150μg/L.Dual-energy X-ray absorptiometry to determine the bone mineral density(BMD)of the contralateral hip.A part of the bone tissue was scanned by micro-CT to observe the changes of bone microstructure.Afterwards,decalcification and frozen sections were performed,and the abundance of ferroptosis marker GPX4 in bone tissue was observed by immunofluorescence.The other part of bone tissue was ground under liquid nitrogen,cracked by RIPA lysis buffer,and the supernatant was extracted to detect the content of MDA,the ferroptosis index.All patients were enrolled if the following criteria were met:trauma-induced fractures,osteoarthritis,and other patients who need hip replacement.Subjects with secondary osteoporosis,anti-osteoporosis drug intervention,malignancy,diabetes,thyroid-related diseases,or other malignant diseases were excluded.The second part:Multiple linear regression analysis of the correlation between serum ferritin and lumbar spine,femoral neck bone density,bone resorption index(3-CTX and bone formation index P1NP in 214 healthy postmenopausal women in the Second Affiliated Hospital of Soochow University physical examination center from 2015 to 2018.Results:We found that collum femoris BMD was significantly lower in patients with higher serum ferritin(Fer>150 μg/L)than with normal(p<0.05).It was also found that the bone mass of the group with high ferritin was lower than that of the group with normal.The following bone microarchitecture parameters:the relevant parameters percent bone volume(BV/TV)was reduced,and trabecular number(Tb.N)was decreased,but trabecular separation(Tb.Sp)was increased with quantitative measurements detected by micro-CT(p<0.05-0.001).Immunohistofluorescence was conducted using antibodies for GPX4 in decalcified human bone tissue,and it was found that the expression of the ferroptosis marker protein GPX4 was abundant in healthy bone tissue with low ferritin,but decreased with high ferritin bone.However,MDA was increased in bone with high ferritin compared with low ferritin bone(p<0.01).The results of 214 healthy physical examination crowd showed that serum ferritin was negatively correlated with BMD of the lumbar spine and femoral neck,and the simple correlation coefficients r=-0.17731,-0.16246,respectively.The partial correlation coefficients r’=-0.19587(P=0.0066),-0.16655(P=0.0213),respectively,when corrected for baseline age,height and other variables.The data showed that this change was not statistically significant with osteoclast activity(simple correlation coefficient r=0.03320,partial correlation coefficient r’=0.02971,P=0.6841)(Fig.2c),but it was closely related with the change of osteogenesis activity.The osteogenic activity index P1NP decreased significantly when serum ferritin increased(simple correlation coefficient r=-0.29039,partial correlation coefficient r’=-0.6963,P=0.0002).Conclusion:Ferroptosis is involved in human bone tissue metabolism and osteoblasts play an important role in bone loss caused by iron accumulation through ferroptosis.Part Ⅳ The mechanism of iron accumulation induced ferroptosis of osteoblastsObjective:In the first three sections,iron accumulation has been found to cause bone loss and ferroptosis of osteoblasts in mouse and human bone tissues,and this part focuses on the specific molecular mechanism.Methods:After the treatment of osteoblasts with iron agent,the osteoblasts were transfected with iNap1 sensor(probe plasmid that detects NADPH in living cells)to detect the level of NADPH in osteoblasts.The content of lipid peroxides in osteoblasts was detected with BODIPY 581/591 C11 dye.The expression levels of NOX1-5,DOUX1,2 in 7 members of NOXS family were detected by RT-PCR.The mechanism of iron regulation on NOX4 expression was investigated by two fluorescent reporter genes,CUT&Tag and iron-regulatory protein 1(IRP1)overexpression.The effect of iron accumulation on the mitochondrial function of osteoblasts was examined by mitochondrial pressure assay.NOX4 expression was knocked down by siRNA,and the protein expression of NOX4 in osteoblasts was detected by Western Blot.The changes of lipid peroxides in the mitochondria of osteoblasts were detected by the fluorescence probe of MitoPeDPP.The changes of mitochondria of osteoblasts were observed again under transmission electron microscope(TEM).Western Blot was used to detect NOX4 protein changes in human bone tissues and the mouse bone tissues.Results:After Fe treatment,the levels of NADPH in osteoblasts were significantly decreased as after Erastin treatment,but the levels of lipid peroxides in the cells were significantly increased.After intervention with Ferr-1 or DFO,lipid peroxides were partially decreased and NADPH was increased.NOX3 was not expressed in osteoblasts regardless of iron accumulation,and NOX1,NOX2,NOX5 and DOUX2 were low expressed,while NOX4 and DOUX1 were high expressed.However,only NOX4 expression was elevated during iron accumulation.We transfected osteoblasts with 2000 bp pre-transcription sequence of NOX4 and reporter gene ligation with iron intervention,and the fluorescence intensity of osteoblasts was significantly higher than that of the control group(P<0.001).Similar to the above results,NOX4 protein increased in osteoblasts after iron treatment,and NOX4 content decreased after siRNA knockdown.Fluorescence intensity of osteoblasts was significantly reduced after mutation of sequences similar to iron response elements(P<0.001).This sequence,which is similar to the iron response element,is referred to IRE-like sequence.The results of CUT&Tag experiment showed that regardless of the presence or not of iron,there was no enrichment of IRE-like sequence in the osteoblasts pretreated with iron regulatory protein 2(IRP2)antibody,indicating that IRE-like sequence did not bind to IRP2.However,in iron environment,the enrichment level of IRE-like in IRP1 antibody pretreated cells decreased(P<0.001).This indicates that IRP1 separates from IRE-like sequence after the action of iron with IRP1,thus promoting the transcription of NOX4.The fluorescence intensity of osteoblasts decreased after overexpression of IRP1.When iron was added,the fluorescence was enhanced again.Mitochondrial pressure test showed that basal respiration,maximum respiration,reserve respiration,ATP production and proton leakage were all increased after iron culture of osteoblasts(P<0.05-0.001).When NOX4 siRNA was added,mitochondrial respiration returned to the same level as the control group.Increased lipid peroxides in cells and mitochondria decreased,and the diminished mitochondria partially recovered.Western Blot analysis of human bone tissue showed that the expression level of NOX4 was higher in human bone tissues with increased ferritin than in normal bone tissues with increased ferritin.Similar to the results of human bone tissue,the expression of NOX4 in the Hepc1-/-and FAC groups was also increased.DFO iron reduction intervention or Ferr-1 inhibition of ferroptosis can inhibit NOX4 levels.Conclusion:Iron promotes NOX4 expression by interacting with IRP1,and activates mitochondria to produce excessive lipid peroxides,leading to iron death in osteoblasts...