IPLA2β Prevents High Glucose-induced Injury Of Human Renal Tubular Epithelial Cells Via Regulating Ferroptosis

Background and objective:With the development of global economy and the change of human diet structure,metabolic diseases have become a key factor endangering human health,and the prevalence rate of Diabetes mellitus(DM)is rising rapidly.According to statistics,the number of people diagnosed with diabetes in the world has reached 537 million in 2021 and the International Diabetes Federation(IDF)estimates that the number of diabetics will reach a new peak of 783 million by 2045.Diabetic nephropathy(DN)is one of the main complications of diabetes and has become the main cause of End-stage renal disease ESRD in the world.DN is a microvascular complication which clinic is characterized by decreased glomerular filtration rate and macroproteinuria.In the past,the glomerulus has always been the focus of research on the pathogenesis of diabetic nephropathy,however,recent studies have found that renal tubule injury accelerates the development of DN.Therefore,exploring the mechanism of damage of renal tubular epithelial cells is of great significance for the treatment of DN.Ferroptosis is a new type of cell death that was discovered in recent years and is usually accompanied by a large amount of iron accumulation and lipid peroxidation during the cell death process,the occurrence of ferroptosis is iron-dependent.Ferroptosis is an indispensable participant in many pathological processes,including cancer,ischemia-reperfusion injury and degenerative diseases.Therefore,it is particularly important to further find out the specific mechanism of regulating ferroptosis and treat related diseases based on it.i PLA2β is a new regulator of ferroptosis which can participate in metabolism of phospholipase and protect various cells from oxidative stress.More and more studies have shown that ferroptosis plays an important role in the occurrence and development of DN.The purpose of this study is to investigate the expression of i PLA2β in human renal tubular epithelial cells(HK-2)induced by high glucose(HG),observe the protective effect of i PLA2β on HK-2 cells induced by HG,find the ameliorative effect of i PLA2β on ferroptosis and oxidative stress in HG-induced HK-2 cells,to provide new ideas for the treatment of diabetic kidney disease.Methods :In vitro experiment: Select the Human renal tubular epithelial cells for the cell experiment.Experimental groups:normal group(NC),mannitol control group(MG),high glucose group(HG),high glucose with vectors group(HG+Vectors),high glucose with i PLA2β overexpression group(HG+i PLA2β+OE),high glucose with Fer-1group(HG+Fer1).The HK-2 cells were treated with 30 m M glucose,the overexpression model was constructed by transfection of i PLA2β plasmid.Ferrostatin-1(Fer-1)(an inhibitor of ferroptosis)and erastin(an activator of ferroptosis)were used as controls.After 36 hours of intervention,the kit detected the levels of superoxide(SOD),malonaldehyde(MDA)and iron in HK-2 cells.DCF immunofluorescence was used to detect intracellular reactive oxygen species(ROS).The expression of ACSL4,GPX4,LPCAT3,TFR1 in HK-2 cells were measured by Western blot.Results :Western blot results showed that the optimal time of HG stimulation was36 h.Immunofluorescence results showed that the expression of i PLA2β downregulated in HG-induced injury of HK-2 cells.Western blot and real-time quantitative PCR results showed that the expression of KIM-1,ACSL4,LPCAT3 and TFR1 decreased,and the expression of GPX4 increased in HG-induced injury of HK-2 cells.Kit test results showed that the levels of ROS and MDA in HK-2 cells increased,while the levels of GSH and SOD decreased.However,these indexes were improved after fer-1intervention.i PLA2β overexpression can reduce the injury of HK-2 cells via attenuation of KIM-1.Further research revealed that i PLA2β overexpression inhibited oxidative stress and ferroptosis in HK-2 cells injury induced by high glucose.Meanwhile,the improvement effect of i PLA2β on HG-induced Hk-2 cells damage could be eliminated by erastin.Conclusion:i PLA2β prevents HG-induced injury of HK-2 cells via regulating ferroptosis and oxidative stress.