Protective effect of coumarin on hydrogen peroxide induced oxidative damage in human ovarian granulosa cells by regulating the Keap1/Nrf2/ARE signaling pathway
Objective To investigate the improvement effect of coumarin on hydrogen peroxide(H2 O2)induced oxidative damage in human ovarian granulosa cells by regulating the Kelch like ECH associated protein 1(Keap1)/nuclear factor E2 related factor 2(Nrf2)/antioxidant response element(ARE)signaling pathway.Methods The human ovarian granulosa cell line COV434 was grouped into control group(normal culture),model group(0.5 mmol·L-1 H2 O2),coumarin group(0.5 mmol·L-1 H2 O2+320 μmol·L 1 coumarin),suppressor group(0.5 mmol·L-1 H2 O2+10 μmol·L-1 Keap1/Nrf2/ARE pathway inhibitor compound 20c)and combination group(0.5 mmol·L-1 H2O2+320 μmol·L-1coumarin+10 μmol·L-1 compound 20c).Cell counting kit 8(CCK-8)method was applied to detect the optical density(OD)of COV434 cells;2',7'-dichlorodihydrofluorescein diacetate(DCFH-DA)fluorescence probe method was applied to detect intracellular reactive oxygen species(ROS);the levels of superoxide dismutase(SOD)and interleukin-18(IL-18)were detected by enzyme linked immunosorbent assay(ELISA);flow cytometry was applied to detect the apoptosis rate of COV434 cells;Western blot was applied to detect the expression of Keap1,Nrf2.Results OD values of blank group,model group,coumarin group,suppressor group and combination group were 1.53±0.20,0.91±0.10,1.35±0.14,0.56±0.05 and 1.06±0.12;the ROS levels were 1.00±0.00,2.24±0.35,1.18±0.13,3.97±0.58 and 2.09±0.20;the SOD levels were(73.43±9.76),(43.32±5.88),(71.54±8.76),(27.64±3.12)and(52.46±6.45)U·mL-1;the levels of IL-18 were(205.90±20.43),(334.56±35.68),(233.24±24.58),(456.54±47.83)and(301.13±37.64)pg·mL-1;the apoptosis rates were(5.96±0.69)％,(19.62±1.77)％,(8.89±0.97)％,(30.23±3.12)％and(17.65±1.07)％;the Keap1 protein levels were 0.95±0.10,0.66±0.08,0.93±0.09,0.31±0.04 and 0.69±0.07;the Nrf2 protein levels were 1.31±0.12,0.76±0.07,1.25±0.14,0.21±0.02 and 0.90±0.11,respectively.The differences between model group with blank group,between model group with coumarin group,suppressor group,between combination group with coumarin group,were also statistically significant(all P＜0.05).Conclusion Coumarin may improve H2 O2-induced oxidative damage in human ovarian granulosa cells by up-regulating the Keap1/Nrf2/ARE signaling pathway.
coumarinKelch like ECH associated protein 1/nuclear factor E2 related factor 2/antioxidant response element signaling pathwayhydrogen peroxideovarian granulosa cellsoxidative damage
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