Study Of Hydrogen Sulfide On Oxidative Stress In Hypoxic Reoxygenation Injury Of Renal Tubular Epithelial Cells

Objective:To observe whether pretreatment of rat renal tubular epithelial cells(NRK-52SE)with sodium sulfide(NaHS,exogenous hydrogen sulfide donor)can reduce the occurrence of oxidative stress in renal tubular epithelial cells during hypoxia and reoxygenation.Methods:Rat renal tubular epithelial cells were randomly divided into 4 groups: Blank control group(Control group): under the environment of 37 ℃ + 95% air + 5% carbon dioxide + saturated humidity,the cell was cultured for 150 min and then collected.Hypoxic reoxygenation group(HR group): under the environment of 37 ℃ + 95% nitrogen + 1% oxygen + 4% carbon dioxide + saturated humidity,the cell was cultured for 60 min and then was cultured for 90 min under the environment of 37 ℃ + 95% air + 5% carbon dioxide + saturated humidity,at last the cell was collected.NaHS intervention group(NaHS group): in NaHS serum-free medium at a concentration of 100 μmol/L,under the environment of 37 ℃ + 95% nitrogen + 1% oxygen + 4% carbon dioxide + saturated humidity,the cell was cultured for 60 min and then was cultured for 90 min under the environment of 37 ℃ + 95% air + 5% carbon dioxide + saturated humidity,at last the cell was collected.Diphenylene iodonium intervention group(DPI group): in DPI serum-free medium at a concentration of 1 μmol/L,under the environment of 37 ℃ + 95% nitrogen + 1% oxygen + 4% carbon dioxide + saturated humidity,the cell was cultured for 60 min and then was cultured for 90 min under the environment of 37 ℃ + 95% air + 5% carbon dioxide + saturated humidity,at last the cell was collected.Western blotting was used to detect the protein expression of NADPH oxidase(NOX)2 and NOX4 in each group of cells.Real-time quantitative PCR(RT-PCR)was used to detect the expression of NOX2 and NOX4 mRNA in each group of cells.Fluorescent probe 2’,7’-dichlorofluorescent yellow diacetate(DCFH-DA)method was used to detect the content of reactive oxygen species(ROS)in each group of cells.Results:Compared with the Control group,the protein content and mRNA content of NOX2 and NOX4 in the HR group were increased significantly(all P<0.05),ROS content was increased significantly(P<0.01),NADPH oxidase activity was increased(P<0.01).Compared with HR group,the protein content and mRNA content of NOX2 and NOX4 in NaHS group and HR group were decreased(all P<0.05),ROS content of both groups was reduced(all P<0.01),NADPH oxidase activity of both groups was decreased(all P<0.01).Conclusion:Hydrogen sulfide reduce the occurrence of oxidative stress in the hypoxic reoxygenation injury of renal tubular epithelial cells by reducing the expression of mRNA and protein of NOX2 and NOX4,reducing the content of ROS,and decreasing the activity of NADPH oxidase.