Effect Of PCBP1 On Ferroptosis Of Osteoblasts Through Ferritin In High Glucose Environment

In recent years,with the continuous development of society and the improvement of people's quality of life,the number of chronic diseases such as type 2 diabetes has increased year by year,which has brought serious harm to human health.There are hundreds of complications of diabetes,including diabetic nephropathy,diabetic osteoporosis,diabetic foot,heart,vascular and peripheral neuropathy,etc.Among them,type 2 diabetic osteoporosis is particularly common,and it occurs especially in the elderly As they get older,they have a significantly higher risk of fractures.The pain and movement disorders caused by fractures not only affect the physical and mental health of patients but also bring a lot of inconvenience to the family and society.The pathogenesis of type 2 diabetic osteoporosis is complicated,involving high glucose toxicity,inflammatory response,AGEs aggregation,and iron ion metabolism disorders.The exact cause of type 2 diabetic osteoporosis has not been fully elucidated.Iron is one of the important trace elements in the human body.The metabolic process of iron is regulated by perfect and precise mechanisms in the body to ensure that the body is in a steady state of iron.Iron homeostasis is important for cells to maintain normal physiological and biochemical functions.Ferritin is the main storage method of intracellular iron and is widely expressed in animals and plants and microorganisms.They can both protect cells from the toxic effects of excess iron,and release iron ions to meet the functional needs of cells when the iron is deficient,so they are essential for the maintenance of iron homeostasis.Ferroptosis is a new type of death pathway that depends on iron ions and ROS.When iron ions are excessively accumulated in cells,a large amount of ROS produced by the Fenton reaction attacks biological macromolecules,and the iron homeostasis in the cell is unbalanced,leading to cell death.At present,there are more studies on ferroptosis,but there are fewer articles on bone metabolism.Our previous research showed that iron overload caused by high glucose is an important factor causing osteoblast injury,and ROS levels increased significantly during this period.This shows that ferroptosis is closely related to the pathogenesis of diabetic osteoporosis,but the specific occurrence process and whether ferritin is involved need to be further explored.PCBP1 is a ferric chaperone protein that is widely present in cells.It can bind iron ions and transport iron ions to ferritin.In the PCBP family,PCBP1 is one of the more studied,and it has a wide range of functions,including regulating gene transcription,maintaining mRNA stability,promoting translation,and acting as an iron ion partner.Studies have found that mice lacking PCBP1 will exhibit small-cell iron-deficiency anemia and compensate by regulating erythropoietin.At present,no research has confirmed whether PCBP1 is involved in the occurrence and development of type 2 diabetic osteoporosis,and the relationship between changes in its level and ferritin and ferroptosis in osteoblasts remains to be studied.Therefore,the purpose of this study was to explore the role of PCBP1 in the pathogenesis of type 2 diabetic osteoporosis.We hypothesized that the compensatory increase of PCBP1 in patients with type 2 diabetic osteoporosis can lead to intracellular iron The increase of protein reduces the excessive accumulation of iron ions and slows down the occurrence and development of ferroptosis.We first detected the levels of PCBP1 and ferritin in a high glucose environment,and then tested the relevant indicators such as ferritin after lentiviral silencing and overexpression of PCBP1.This study will help explain the causes of osteoporosis in type 2 diabetes and provide reference values for the treatment of metabolic diseases that target PCBP1 to regulate ferroptosis.Methods: Part I: To investigate the iron death in osteoblasts under a high glucose environment and the effect of high glucose environment on PCBP1.CCK8 method was used to detect the changes of osteoblast activity under the action of high glucose at different concentrations and times;Western blot was used to detect the expression of PCBP1 and GPX4 in high glucose environment,and flow cytometry was used to detect ROS in osteoblasts in high glucose environment Changes in expression.Part 2: Infect osteoblasts with PCBP1 shRNA lentivirus,and screen for stable expression of cell lines;use Western blot to detect ferritin,GPX4,and osteogenic marker proteins in osteoblasts under high glucose environment or in combination with PCBP1 shRNA OPG and OCN expression;flow cytometry was used to detect changes in ROS expression in osteoblasts under the action of PCBP1 shRNA;alizarin red staining was used to detect high glucose environment or combined with PCBP1 shRNA for calcium nodule formation in osteoblasts influences.Results: Part 1: Compared with the control group,the osteoblast activity of the high-glucose group was decreased.After the ferroptosis inhibitor was added,the osteoblast activity was higher than that of the sugar group;Compared with the control group,the high glucose group had increased PCBP1 expression and reduced GPX4 expression;Flow Cytometry results showed that ROS in the high-glucose group was significantly increased compared with the control group.Part 2: After transfection of PCBP1 by lentivirus,Western blot showed that compared with the control group,ferritin in high glucose group increased with PCBP1,ferroptosis marker protein GPX4,functional indicator proteins OPG and OCN decreased.Compared with the higher glucose group,the expression of ferritin,GPX4,OPG,and OCN in the PCBP1 overexpression group was significantly increased,and the silent group was significantly reduced;Flow Cytometry results showed that compared with the higher glucose group in PCBP1 overexpression group,the ROS production was significantly reduced,while the silent group had the opposite result;The osteogenic mineralization induction results showed that the number of calcium nodule deposits in the high glucose group was reduced compared to the control group,and the number of calcium nodule deposits was significantly increased in the PCBP1 overexpression group compared with the high glucose group,while the silent group had the opposite result.Conclusions: 1.Ferroptosis exists in osteoblasts under a high glucose environment;2.PCBP1 expression in osteoblasts increased compensatory under high glucose environment;3.In a high glucose environment,PCBP1 can reduce the toxic effect on osteoblasts caused by ferroptosis by promoting ferritin expression.