Iron Accumulation Affects Osteogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells By Inhibiting Mitophagy

BackgroundOsteoporosis is an important manifestation of bone degeneration in postmenopausal women.In recent years,studies have shown that iron accumulation is a new pathogenic factor of osteoporosis.In the Guidelines for the Diagnosis and Treatment of Primary Osteoporosis issued in 2022,iron accumulation has been regarded as one of the pathogenesis of osteoporosis.At present,the studies on iron accumulation-induced osteoporosis mainly focus on osteoblasts and osteoclasts,with less research on upstream cells such as bone marrow mesenchymal stem cells(Bone mesenchymal stem cells,BMSCs)is less.ObjectiveThis study aims to explore the effects of iron accumulation on bone marrow mesenchymal stem cells and explore the pathogenesis and new therapeutic targets of iron accumulation-induced osteoporosis.Method1.Explored the effect of iron accumulation on BMSCs in vitro.In vitro,BMSCs intervened with different concentrations(0,50,100,200 μM)of ferric ammonium citrate(FAC)were set as the experimental group,and the non-intervened group was set as the control group.Three days after the intervention,detected the osteogenic function,senescence and mitochondria function indicators.(1)To detect whether iron accumulation affected osteogenic differentiation and mineralization abilities of BMSCs:BMSCs were subjected to osteogenic differentiation.Detected the osteogenic mineralization ability by ALP staining and alizarin red staining,detected the expression of osteogenic related proteins by Western Blot,and detected the expression of osteogenesis related genes by Quantitative Real-time PCR(qPCR).At the same time,to further explore the mechanism of iron accumulation affecting BMSCs,transcriptome sequencing was performed on the control group and the experimental group(FAC concentration:50 μM);(2)To detect whether iron accumulation accelerates the senescence of BMSCs:Detected the expression of y-H2AX and H3k9me3 by confocal fluorescence microscopy and β-galactosidase staining of senescence cells,and detected the expression of senescence-related proteins P16,P21,and P53 by Western Blot;(3)To detect whether iron accumulation has an impact on the mitochondrial function of BMSCs:Flow cytometry detected the level of reactive oxygen species(ROS)in cells,and the level of reactive oxygen species in mitochondria;Detected ATP production by microplate reader;Detected mitochondrial membrane potential and mitophagy level by confocal microscope;Detected the expression of mitophagy-related proteins Parkin and PINK1 and autophagy-related proteins LC3 and P62 by Western Blot.2.Detected the changes of bone mass and BMSCs in the iron accumulation mice.Eight-week-old mice were selected and iron dextran was intermittently injected intraperitoneally for 8 weeks to construct an in vivo iron accumulation mouse model.The intervented mice were used as the experimental group,and unintervented mice were used as the control group.(1)Detection of bone mass in mice:Micro CT was performed on the femur to detect bone mass;(2)Detection of whether the BMSCs of iron-accumulating mice were senescent:Extracted BMSCs of iron-accumulating mice and detected β-galactosidase,the expression of y-H2AX and H3K9me3,and the expression of aging-related proteins P16,P21,and P53;(3)Detection of whether the mitochondrial function of BMSCs in iron accumulation mice was damaged:Detected the level of reactive oxygen species(ROS)in cells and the level of reactive oxygen species in mitochondria,ATP production,mitochondrial membrane potential,mitophagy level and expression of mitophagy-related proteins and autophagy-related proteins.3.To detect whether mitophagy agonists can restore BMSCs cell senescence,mitochondrial function damage and osteogenic function decline caused by iron accumulation.BMSCs were divided into control group(no intervention),FAC group,CCCP(mitochondrial uncoupler)group(positive control group)and FAC+CCCP group(rescue group).(1)To detect whether the mitophagy agonist CCCP can rescue cell senescence in the state of iron accumulation:Detected the expression of γ-H2AX and H3K9me3 and the expression of aging-related proteins P16,P21,and P53;(2)To detect whether the mitophagy agonist CCCP whether can rescue the damaged mitochondrial function caused by iron accumulation:Detected intracellular reactive oxygen species level(ROS),mitochondrial reactive oxygen species level,ATP production,mitochondrial membrane potential;(3)To detect whether mitophagy agonist CCCP can rescue the impaired osteogenic function caused by iron accumulation:ALP staining,alizarin red staining,and detection of osteogenesis related genes and proteins.Result1.In vitro iron accumulation inhibit the osteogenic differentiation of BMSCs,accelerate the senescence of BMSCs,and inhibit the mitochondrial function and other mitochondria-related functions of BMSCs.(1)Iron accumulation in vitro can inhibit the osteogenic differentiation of BMSCs:the results of alizarin red staining and ALP staining suggested that the osteogenic mineralization ability of BMSCs gradually decreased with the increase of iron concentration;the results of qPCR suggested that the expression of osteogenesis-related genes Sp7,Alp,Runx2,and Colla1 decreased;the results of Western Blot suggested that with the increase of iron concentration,the expression of osteogenesis-related proteins RUNX-2 and ALP gradually decreased.Transcriptome sequencing analysis suggested that the differentially expressed genes in the control group and the experimental group were enriched in the cellular senescence pathway and mitophagy pathway.(2)In vitro iron accumulation would accelerate senescence of BMSCs:with the increase of iron concentration,the number of senescent cells increased,the expression of γ-H2AX and H3k9me3 increased,and the expression of senescence-related proteins P16,P21,and P53 increased.(3)Iron accumulation inhibited mitophagy and other mitochondria functions:with the increase of iron concentration,the ATP production,the level of mitochondrial membrane potential,the level of mitophagy,and the expression of autophagy-related and mitophagy-related proteins decreased.But the intracellular reactive oxygen species levels and mitochondrial reactive oxygen species levels increased.2.The bone mass of iron accumulation mice decreased,BMSCs in vivo showed an senescent state,and mitochondrial functions including mitophagy were damaged.(1)Bone mass in iron-accumulating mice were decreased:Micro CT showed that compared with control mice,iron-accumulating mice had significantly decreased bone mass.(2)BMSCs in iron-accumulating mice showed an senescent state:compared with control group,the expressions of γ-H2AX and H3k9me3,β-galactosidase and senescence-related proteins P16,P21 and P53 in the iron accumulation group were increased;(3)Iron-accumulating mice’s mitochondrial functions including mitophagy were disrupted:compared with the control group,the ATP production in BMSCs of iron accumulation mice,the level of mitochondrial membrane potential,the level of mitophagy,and the expression of autophagy-related and mitophagy-related proteins decreased.But the intracellular reactive oxygen species levels and mitochondrial reactive oxygen species levels increased.3.Whether mitophagy agonists can rescue BMSCs senescence,mitochondrial function damage and osteogenic function decline caused by iron accumulation:(1)Mitophagy agonists can rescue BMSCs senescence:Compared with the FAC group,the expression of y-H2AX and H3k9me3 decreased,and the expression of aging-related proteins P16,P21,and P53 decreased in rescue group.(2)Mitophagy agonists could rescue mitochondrial function:compared with the FAC group,the membrane potential level of mitochondria and the ATP production increased in rescue group,while the levels of reactive oxygen species in cells and mitochondria decreased in rescue group.(3)Mitophagy agonists could rescue the impaired osteogenic mineralization function of BMSCs:compared with the FAC group,alizarin red staining and ALP staining suggested that the bone mineralization ability increased;the expression of osteogenesis related genes Sp7,Alp,Runx2,and Colla1 increased;the expression of osteogenesis related proteins RUNX-2 and ALP increased in rescue group.Conclusion1.Iron accumulation in vitro can inhibit the osteogenic mineralization ability of BMSCs,accelerate the senescence of BMSCs,and inhibit their mitochondria functions including mitophagy.2.Iron accumulation in mice can lead to decreased bone mass,accelerate the senescence of BMSCs,and inhibit mitochondria functions including mitophagy.3.Mitophagy agonists can rescue BMSCs senescence and mitochondrial function impairment caused by iron accumulation,as well as decreased osteogenic mineralization ability.Iron accumulation can affect the osteogenic differentiation of BMSCs by inhibiting mitophagy.