The Mechanism Analysis And Study Of Oxidative Stress In Granulosa Cells Of Patients With Polycystic Ovary Syndrome

Objective:To investigate the expression level of ROS in ovarian granulosa cells of patients with polycystic ovary syndrome（PCOS）,and to explore the effect of oxidative stress on the growth and development of human ovarian granulosa cells by using H2O2-mediated oxidative damage model in KGN cells.Methods:1.Granulosa cells were extracted from follicular fluid of PCOS group and control group,and the expression level of ROS in granulosa cells of PCOS group and control group was detected by flow cytometry.2.Human ovarian granulosa cells KGN were cultured in vitro,treated with different concentrations of H₂O₂ to construct oxidative stress model,and cell proliferation activity was detected by CCK8.3.ROS levels and apoptosis rate in granular cells were detected by flow cytometry after H₂O₂ treatment,and expression levels of LC3-Ⅱ,cleaved caspase3,LC3-Ⅰ,BCL-2,and P62 were detected by WB.4.The H₂O₂mediated KGN cells were pretreated with NAC,and the ROS levels and apoptosis rate were detected by flow cytometry,while the expression levels of LC3-Ⅱ,cleaved caspase3,LC3-Ⅰ,BCL-2,and P62 were detected by WB.Results:1.According to flow cytometry analysis,ROS expression level in granular cells of PCOS patients was significantly increased compared with normal control group.2.With the increase of H₂O₂ concentration,the proliferation of granulosa cells was significantly inhibited in a dose-dependent manner.The effect of 100μM H₂O₂ concentration was significant.ROS was released from the cell with the increase of H₂O₂ concentration,and the autophagy and apoptosis rates were increased.3.After H₂O₂ treatment,LC3-Ⅱand cleaved caspase3 protein expression increased in KGN cells.The decreased expressions of LC3-Ⅰ,BCL-2 and P62 proteins indicated that H₂O₂ could induce autophagy and apoptosis of KGN cells.4.After the intervention of antioxidant NAC,H₂O₂ mediated the recovery of ROS levels in KGN cells,and the rates of autophagy and apoptosis decreased,which further verified that autophagy and apoptosis were related to ROS production.Conclusion:1.Compared with normal women,the ovarian granulosa cells of PCOS women showed a higher level of ROS,suggesting that the ovary of PCOS patients may have oxidative damage.2.H₂O₂ can inhibit the decrease of human ovarian granulosa cell proliferation,oxidative stress and early autophagy apoptosis.NAC intervention can improve the H₂O₂ mediated decline in KGN cell proliferation,improve the oxidative stress state,and improve the oxidative stress-induced apoptosis of KGN cells.