Protective Effect Of Carnosine On Hydrogen Peroxide–induced Oxidative Stress In Human Kidney Tubular Epithelial Cells

Objective:Diabetic nephropathy（DN）endangers health and is a high financial public burden worldwide.Risk of DN is positively correlated with high levels of reactive oxygen species（ROS）.Carnosine,an antioxidant,actively regulates cell function and has the potential to reduce the occurrence of DN.Here,we explored whether carnosine could prevent oxidative stress in human kidney tubular epithelial（HK2）cells and,if so,the mechanisms underlying this effect.To explore the mechanism of apoptosis of HK2 cells under oxidative stress,and to provide a new direction for the clinical treatment of diabetic nephropathy.Methods:In this experiment,we used carnosine to interfere with H₂O₂-damaged HK2 cells.The experiments were consisted of control group,H₂O₂group,carnosine group and H₂O₂plus carnosine group,a series of experiments were performed after 24 hours of culture.In this study,the proliferation of HK2 cells treated with different concentrations of H₂O₂and carnosine was detected by CCK-8 kit.The ROS kit was used to detect the effect of carnosine and H₂O₂on the level of ROS in HK2 cells.The SOD kit was used to detect the effect of carnosine and H₂O₂on the level of SOD in HK2 cells.Western blot analyses to determine the protein expression levels of KIM-1,IL-1β,BAX,BCL-2,caspase 3,and the NADPH oxidase isoforms NOX2 and NOX4,as well as confocal laser scanning microscopy（CLSM）to assess changes in the mitochondrial membrane potential and the relative position of mitochondria to cytochrome C.Results:（1）CCK8 experiments showed that H₂O₂at different concentrations（100,200,300,400μM）all inhibited the proliferation of HK2 cells,and the optimal concentration was200μM.Different concentrations of carnosine（40,50,60,70 m M）can antagonize the toxic effect of H₂O₂on HK2 cells,,and the maximum protective effect concentration is60 m M.（2）H₂O₂significantly increased intracellular ROS level in HK2 cells,while carnosine inhibited the production of ROS in HK2 cells.（3）A significant and marked decrease in SOD activity was observed in cells treated with 200μM H₂O₂,However,this H₂O₂-induced decrease in SOD activity was completely blocked by treatment with 60 m M carnosine.（4）Western blot showed that H₂O₂significantly increased the expression levels of KIM-1,IL-1β,NOX4,BAX and caspase 3 proteins but significantly decreased the expression of BCL-2 protein.However,with the addition of carnosine,the protein expression levels of KIM-1,IL-1β,NOX4,BAX and caspase 3 were significantly decreased compared with those in the H₂O₂-damaged cells,whereas the protein expression level of BCL-2 was significantly increased.H₂O₂and carnosine had no significant effect on the expression of NOX2.（5）Using CLSM,we observed that treatment of HK2 cells with H₂O₂（200μM）significantly decreased the mitochondrial membrane potential and H₂O₂induced the translocation of cytochrome c from mitochondria,when carnosine was present,the mitochondrial membrane potential was maintained and this translocation was inhibited.Conclution:In conclusion,the results of our in vitro studies indicate that carnosine ameliorates ROS toxicity in cultured H₂O₂-damaged HK2 cells.Our findings indicate that carnosine decreased NOX4 expression and increased SOD activity,thus reducing the production of intracellular ROS,relieving the oxidative stress of cells,and ultimately inhibiting the mitochondrial pathway of apoptosis.These findings highlight that carnosine can indirectly alleviate mitochondrial oxidative stress,protect mitochondrial function,and decrease HK2 cell apoptosis,providing new mechanistic insights for the potential application of carnosine in the clinical treatment of DN.