| Objective:As one of the common complications of type 2 diabetes,osteoporosis can cause pain,deformity,and difficulty walking,which seriously affects the live quality of the elderly and causes a heavy economic burden.As the pathogenesis of type 2diabetic osteoporosis(T2DOP)is relatively complicated and is not yet fully understood,the pathogenesis and prevention measures of T2DOP urgently need to be further explored and studied.Studies have reported that oxidative stress can cause the destruction of bone homeostasis and ultimately lead to the occurrence of osteoporosis,and the accumulation of reactive oxygen species(ROS)and lipid peroxides is considered to be an important feature of ferroptosis.In recent years,a large number of preliminary studies have found that high glucose can lead to ferroptosis of osteoblasts and weakened osteogenic function,the ferroptosis-inhibitors can significantly improve their osteogenic function and improve osteoporosis,and this has been fully confirmed in animal experiments.However,as a newly discovered form of cell death in recent years,the mechanism and regulatory pathways of ferroptosis in osteoblasts under high glucose have not been studied clearly.Activation transcription factor 3(ATF3)is a member of the ATF/CREB family of transcription factors,which has dual roles of transcriptional repression and promotion,and plays an important role in the regulation of apoptosis,proliferation and cell cycle.Upregulation of ATF3 has been implicated in cellular damage,including apoptosis of pancreaticβcells,production of reactive oxygen species,and high concentrations of glucose or fatty acids associated with diabetic complications.Studies have confirmed that the high expression of ATF3 is related to stress-induced apoptosis of pancreaticβcells.At the same time,ATF3-mediated pathological angiogenesis was reported to be involved in the occurrence of reactive oxygen species-related diabetic vascular complications.A recent study reported that ATF3played a role in promoting ferroptosis in human fibrosarcoma cells and retinal pigment epithelial cells induced by erastine,followed by reports that ATF3 was involved in brucin-induced ferroptosis of glioma cells,which revealed an entirely new role for ATF3 in the occurrence of ferroptosis.However,the involvement of ATF3 in the process of high glucose-induced ferroptosis in osteoblasts and its related regulatory mechanism have not been studied.In our previous experiments,we found that ferroptosis occurred in osteoblasts under high glucose condition,and the expression of ATF3 was up-regulated.Based on the results of previous experiments and literature reports,we speculate that ATF3 is implicated in the process of ferroptosis in osteoblasts under high glucose condition,which may be one of the pathogenesis of T2DOP.Therefore,this study intend to explore the effect of ferroptosis on T2DOP through in vitro experiments and animal models,and clarify the role of ATF3 in the pathogenesis of T2DOP,then further explore its specific regulatory mechanism,providing a new targets for the prevention and treatment of T2DOP.Methods:Firstly,we constructed the human osteoblast cell line h FOB 1.19,which was divided into normal glucose group,high glucose group,normal glucose+ATF3-sh RNA group and high glucose+ATF3-sh RNA group(culturing the cells with 35mmol/L glucose to obtain high glucose group cells,and obtained ATF3-sh RNA group cells by lentivirus infection using sh RNA).Western blot was used to detect the expression of ATF3,solute carrier family 7 member 11(SLC7A11),glutathione peroxidase 4(GPX4),osteocalcin(OCN)and osteoprotegerin(OPG)in each group of cells,and the morphological changes of osteoblasts was detected using transmission electron microscopy,then JC-1 staining was used to detect the level of cell mitochondrial membrane potential.We used flow cytometry to detect intracellular ROS level,and CCK8 to detect cell viability.The lipid peroxidation level of osteoblasts was detected by the interaction of malondialdehyde(MDA)and thiobarbituric acid through a microplate reader,and the content of intracellular glutathione and extracellular glutamate and alkaline phosphatase(ALP)activity were detected using corresponding kit.Next,we constructed normal T2DOP rat models and ATF3-interference T2DOP rat models using si RNA,and detected the expression of ATF3,SLC7A11 and GPX4 in femur tissue by immunohistochemistry,and the serum levels of Fe2+,MDA,GSH and the contents of bone metabolism markers ALP,PINP andβ-CTX were detected by Elisa,then micro-CT was used for the detection of femur bone mineral density(BMD),trabecular bone volume per tissue volume(BV/TV),number of trabeculae(Tb.N)and trabecular thickness(Tb.Th)to evaluate the bone microarchitecture of rats femur.Results:(1)After treatment with high glucose,we found morphological changes of ferroptosis in osteoblasts determined by electron microscope,and JC-1 staining showed a decreased level of mitochondrial membrane potential.There was ferroptosis-related changes in osteoblasts,such as accumulation of ROS and lipid peroxides and decreased expression of GPX4.(2)High glucose can lead to a decrease of osteoblast viability and a decrease expression of OCN and OPG and ALP activity,The above indexes were significantly improved with ferroptosis inhibitor(3)The expression of ATF3 in osteoblasts increased under high glucose condition.(4)After knockdown of ATF3 by lentiviral transfection,the expression of GPX4 in osteoblasts increased,and the accumulation of ROS and lipid peroxides in the cells decreased,which inhibited the occurrence of ferroptosis in osteoblasts and improved osteogenic function.(5)After high glucose-induced up-regulation of ATF3 expression in osteoblasts,the expression of SLC7A11 decreased,and the contents of intracellular glutathione and extracellular glutamate decreased,indicating that the cystine glutamate transport receptor(system Xc~-)activity was inhibited.(6)Femoral BMD,BV/TV,Tb.N and Tb.Th of T2DOP rats were decreased,and the levels of ALP,PINP andβ-CTx in serum were decreased.(7)Immunohistochemical detection showed that the expression of GPX4 in bone tissue of T2DOP rats was significantly decreased,The levels of Fe2+and MDA in serum increased significantly,while the levels of GSH decreased,indicating that there was ferroptosis in bone tissue.(8)The expression of ATF3 in bone tissue of T2DOP rats was increased,while the expression of SLC7A11 was decreased.(9)After interfering with ATF3 by si RNA,the expressions of GPX4 and SLC7A11 in the bone tissue of the rats were increased,and the BMD,BV/TV,Tb.N and Tb.Th values of the femur were increased,and the levels of ALP,PINP andβ-CTx in the serum were increased,The levels of Fe2+and MDA in serum decreased,while the levels of GSH increased.Conclusion:(1)ATF3 mediates the ferroptosis of osteoblasts induced by high glucose.(2)ATF3-mediated ferroptosis of osteoblasts is associated with reduced osteogenic function of osteoblasts under high glucose conditions.(3)ATF3-mediated ferroptosis is involved in the pathogenesis of T2DOP in rats.(4)ATF3 knockdown and function inhibition can effectively inhibit the ferroptosis of osteoblasts induced by high glucose,thereby improving the osteogenic function of osteoblasts and improving osteoporosis.(5)ATF3 induces ferroptosis by inhibiting the expression of SLC7A11,attenuating the activity of system Xc~-,thereby hindering cystine/glutamate transport,resulting in the inactivation of GPX4. |
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