Role And Mechanism Of Nrf2/keap1 In Regulating Ferroptosis In High Glucose-induced Microglial Injury

Objective: To study the role and mechanism of Nrf2/keap1 regulating ferroptosis in high glucose-induced microglia(BV2)injury.Methods: Part I:The CCK-8 method was used to detect changes in the relative viability of BV2 cells at different glucose concentrations and different intervention times;the cultured BV2 cells were divided into normal,high glucose and mannitol hyperosmolar groups and cultured for 48 hours.The changes of ROS expression in cells of each group were detected by fluorescence microplate analyzer.Westernblot was used to detect the expression changes of Nrf2,keap1,x CT and COX2 proteins in each group.RT-q PCR method was used to detect the expression changes of Nrf2,keap1,x CT and COX2 m RNA in cells of each group.The expression changes of iron ion and GSH in cells of each group were detected by iron ion and reduced GSH kit.Part II: BV2 cells were transfected using keap1 sh RNA lentivirus,and stably expressed cell lines were screened and divided into control,overexpression and no-load groups cultured for 48 hours in a high glucose environment.The changes of ROS expression in three groups of cells under high glucose environment were detected by fluorescence enzyme labeling instrument.Westernblot and RT-q PCR were used to detect the changes of protein and m RNA expression of Nrf2,keap1,x CT and COX2 in three groups of cells under high glucose environment.The expression changes of iron ion and GSH in three groups of cells under high glucose environment were detected by iron ion and reduced GSH kit.Results: Part I: Selection of 35mmol/L glucose concentration,48 hours,as the intervention condition for high glucose in subsequent experiments,based on CCK-8.The expression of ROS was significantly higher in the high glucose group compared with the normal group,and the expression of both iron ions and reduced GSH was decreased;Westernblot and RT-q PCR results showed that the expression levels of Nrf2,x CT,COX2 protein and m RNA were increased in the high glucose group compared with the normal group;the expression levels of keap1 protein and m RNA were decreased;the mannitol hyperosmolar group compared with the normal The results were not statistically different in the mannitol hypertonic group compared with the normal group.Part II: After lentivirus transfection,the expression level of keap1 gene in overexpression group was 3.6 times higher than that in control group.Compared with the control group,the expression levels of ROS and iron ions in the overexpression group increased significantly,while the expression level of reduced reduced GSH decreased.The results of Westernblot and RT-q PCR showed that the protein and m RNA expression levels of COX2 in the overexpression group increased trend,while the protein and gene expression levels of Nrf2 and x CT decreased in the same trend compared with the control group.Conclusion: In high glucose environment,Nrf2/keap1 partially ameliorates hyperglycemia-induced microglial cell injury by mediating the regulatory effect of System Xc-on reduced GSH and thereby influencing the occurrence of ferroptosis.