Peroxynitrite Anion Mediates High Glucose-Induced Injury And Ferroptosis In Mouse Embryonic Osteoblasts

BackgroundDiabetes mellitus(DM)is a systemic metabolic disease characterized by chronic hyperglycemia caused by a variety of reasons.Long-term metabolic disorders often lead to multiple system damage,such as eyes,kidneys,bones,nerves,cardiovascular and so on.Diabetic osteoporosis(DOP)is one of the common and serious complications of diabetes.To date,the mechanism causing abnormal bone metabolism in DOP has not been fully elucidated.Currently,it is now known that reactive oxygen species(ROS)are closely related to diabetes complications.The peroxynitrite anion(ONOO-),as an important member of the ROS family,plays an important role in diabetes-related complications.Osteoblast(OB)is the core link in the process of bone formation and reconstruction.But it is unclear whether ONOOparticipates in OB damage caused by hyperglycemia.Therefore,understanding the role and mechanism of ONOO-in osteoblast damage caused by high glucose(HG)is of important theoretical and clinical value for the prevention and treatment of DOP.Objective(1)To explore the effect of HG on the level of ONOO-in mouse embryonic osteoblasts(MC3T3-E1 cells).(2)To explore whether ONOO-mediates HG-induced injury and ferroptosis in MC3T3-E1 cells.Methods1.The MC3T3-E1 cells were treated with high concentration(45mmol/L)glucose(HG)to construct a model of HG-damaged osteoblasts.2.Cell count detection kit-8(CCK-8)assay was used to detect the survival rate of MC3T3-E1 cells.3.Dihydrorhodamine 123 was used to detect the level of ONOO-in MC3T3-E1 cells.4.The expression levels of 3-nitrotyrosine(3-NT)and glutathione peroxidase 4(GPX4,which reflect protein molecules that can inhibit ferroptosis)proteins were detected by Western blot.5.The level of intracellular ROS in MC3T3-E1 cells was detected by 2,7-Dichlorodihydrofluorescein diacetate(DCFH-DA)probe.6.Mitochondrial membrane potential(MMP)of MC3T3-E1 cells was determined by Rhodamine 123(Rh 123)staining.7.Alkaline phosphatase(ALP)activity of MC3T3-E1 cells were detected by ALP assay kit.8.The mineralized nodules in MC3T3-E1 cells were determined by alizarin red staining.9.The iron ion level was determined by iron colorimetry.10.Malondialdehyde(MDA)level was detected by MDA kit.11.SPSS22.0 version data analysis software was used to statistically analyze the experimental data.and the experimental data were expressed as mean± standard deviation(mean±SD).One-way analysis of variance(one-way ANOVA)was used to compare the mean of multiple samples,and the least significant difference method(LSD)test was used to compare the mean of multiple samples in pairs.The difference was considered statistically significant at P<0.05.Results1.HG increased the production of ONOO-in MC3T3-E1 cells.HG(45mmol/L)significantly decreased the survival rate of MC3T3-E1 cells,and increased the production of ONOO-and 3-NT in MC3T3-E1 cells.2.ONOO-mediated HG-induced injury and ferroptosis in MC3T3-E1 cells.2.1 ONOO-pentachloride(FeTMPyP)inhibited HG-induced a decrease of survival rate in MC3T3-E1 cells;2.2 ONOO-pentachloride inhibited intracellular increased ROS level induced by HG in osteoblasts;2.3 ONOO-pentachloride inhibited HG-induced an increase of MDA level in osteoblasts;2.4 ONOO-pentachloride inhibited HG-induced mitochondrial membrane potential(MMP)in osteoblasts;2.5 ONOO-pentachloride inhibited HG-induced an inhibitory effect of ALP activity in osteoblasts;2.6 ONOO-pentachloride inhibited HG-induced an inhibitory effect of generation of mineralized nodules in osteoblasts;2.7 ONOO-pentachloride inhibited HG-induced a promotion of iron ion in osteoblasts;2.8 ONOO-pentachloride inhibited HG-induced an inhibitory effect of the expression of GPX4 protein in osteoblasts.Conclusions1.HG increases the production of ONOO-in MC3T3-E1 cells.2.ONOO-mediates HG-induced injury and ferroptosis in MC3T3-E1 cells.