The Demethylase FTO-mediated M6A Methylation Affects Osteoblast Ferroptosis In The Pathogenesis Of Diabetic Osteoporosis

Objectives:As a country with a large diabetes population,the incidence of diabetes in China is increasing year by year,and it is a common disease in endocrine system diseases.Osteoporosis caused by diabetes is characterized by a decrease in bone strength,which is very easy to cause fractures and greatly reduce the quality of life.The pathogenesis of diabetic osteoporosis(DOP)compared with the pathogenesis of other types of osteoporosis is not well understood.In previous studies,our group has found the ferroptosis phenotype during the pathogenesis of diabetic osteoporosis osteoblasts.Divalent metal transporter 1(DMT1)is the main protein that regulates cell uptake of iron,and the upregulation of DMT1 causes the accumulation of ferrous Fe2+in cells and causes ferroptosis.As an important component in RNA methylation modifications,m6A methylation has been shown to be associated not only with bone development,but also in osteoporosis.Among them,fat mass and obesity-associated protein(FTO)were found to be associated with the osteoporosis phenotype.During osteogenic differentiation,downregulation of FTO expression may promote cell adipogenic differentiation,leading to the formation of osteoporosis.However,the mechanism of action of m6A methylation,especially demethylase FTO,in osteoblastic ferroptosis during diabetic osteoporosis is unclear.Methods:In this study,HFOB1.19 cells were cultured in groups,and the glucose and DFO concentrations were based on the previous research methods of the research group(35mM,72h)and(50 μ M,72h),respectively.RtPCR and western blot detected the expression of FTO and DMT1 in osteoblasts under different induction conditions.The m6A RNA methylation assay kit detects the total m6A level in osteoblasts under different induction conditions.CCK8 experiments detected the effects of FTO inhibitors and DFO on osteoblast viability after high glucose induction,and screened out the concentration and time of FTO inhibitors.Flow cytometry detected the changes of intracellular ROS levels of HFOB1.19 cells under different induction conditions,and the changes in the activities of the lipid malondialdehyde peroxide(MDA),glutathione(GSH),and total superoxide dismutase(SOD)in HFOB1.19 cells were detected by the ferroptosis related indicator kit.Western blot was used to detect the expression of glutathione peroxidase4(GPX4)protein in the nebrophil marker of ferroptosis in osteoblasts under different culture conditions.In animal experiments,stable diabetic osteoporosis model mice were constructed by continuous multiple injections of low-dose Streptozocin(STZ)after long-term feeding on high-fat feed.Micro-CT instrument was used to detect the microstructure changes of femur bone in each group.HE staining should be applied to observe the microstructure of bone tissue;Finally,immunohistochemistry(IHC)was used to detect the expression of DMT 1,GPX4 and F TO in the femur.Results:The expression of m6A methylation modification and demethylase FTO was downregulated to participate in the process of high glucose-induced ferroptosis in osteoblasts,and the changes in iron content in osteoblasts after high sugar induction pointed to the occurrence of ferroptosis,while DFO could reverse this phenomenon,and the osteoblast induced by FTO m6A demethylase activity inhibitor showed similar performance to that of high glucose inducer.Moreover,FTO inhibitors induced upregulation of DMT1 expression in osteoblasts after induction of high glucose.In conclusion,the demethylase FTO may cause iron accumulation in osteoblasts leading to cellular ferroptosis by acting on the upregulation of DMT 1 expression.In animal models,bone tissue morphology and microstructure of diabetic osteoporosis mice were inferior to those in the control group,and DFO treatment partially improved this result.The expression of FTO and ferrozois marker GPX4 in femoral tissue of diabetic mice was reduced,while DMT1 expression was upregulated,and deferoxamine DFO could reverse the above changes of GPX4 and DMT1.Conclusion:The m6A methylation modification of osteoblasts and the demethylase FTO are involved in the high glucose-induced ferroblastic dygoticosis.FTO may mediate DMT1 expression through m6A demethylase activity,resulting in iron accumulation in osteoblasts and inducing ferroptosis.Demethylase FTO expression down-regulated in diabetic osteoporosis mice and DMT1 expression up-regulated.Deferoxamine DFO has a certain effect on improving the bone microstructure of diabetic osteoporosis mice.