Protective Effect And Mechanism Of Action Of Coenzyme Q10 On Oxidative Stress Injury In Human Ovarian Granulosa Cells

Objective(s):To explore the protective effect of CoQ10 on oxidative stress injury of human ovarian granulosa cells and its possible mechanism,in order to provide experimental and theoretical basis for the use of CoQ10 in clinic.Method(s):1.Isolation,extraction and identification of human ovarian granulosa cells:Selection in October 2021 and August 2020-the First People’s Hospital of Yunan Province,Department of Reproductive Medicine for infertility in patients undergoing assisted reproductive help pregnant,collecting aged 22 to 35,ovarian reserve function in patients with normal follicular fluid,by density gradient centrifugation separation from the follicular fluid extraction ovarian granulosa cells,cells by immunofluorescence identification.2.Establish H2O2-induced oxidative stress injury model of ovarian granulosa cells:The granulosa cells were treated with different concentrations of H2O2 for 2 hours,and the optimal concentration of H2O2 was screened by CCK8.The expression of apoptosis-related proteins(Caspase3,Bax,Bcl2)was detected by Western Blot to verify whether H2O2 could induce apoptosis of ovarian granulosa cells.3.To screen CoQ10 concentration and verify whether CoQ10 has protective effect on H2O2-induced granulosa cell injury:granulosa cells were pretreated with different concentrations of CoQ10 for 24 hours,and then H2O2 solution was added for 2 hours to screen the optimal concentration of CoQ10.The cells were divided into control group,H2O2 group and CoQ10 pretreatment group.Western Blot was used to detect the expression of apoptosis-related proteins to verify whether CoQ10 had protective effect on H2O2-induced granulosa cell damage.4.To explore the effect of CoQ10 on H2O2-induced mitochondrial function of granulosa cells:The production of reactive oxygen species,the change of mitochondrial membrane potential and the production of ATP of granulosa cells in the control group,H2O2 group and CoQ10 pretreatment group were respectively detected to evaluate whether CoQ10 can alleviate H2O2 induced mitochondrial function decline.5.The specific protective mechanism of CoQ10 was further explored by using human ovarian granulosa cell line COV434,which has similar functions to human ovarian granulosa cells:qPCR and Western Blot were used to detect the Nrf2/Keap1 signaling pathway and the expression of SOD1 and HO-1 downstream of antioxidant stress.Results:1.Human ovarian granulosa cells extracted and cultured from follicular fluid were fusiform after adherence,and the cells contained granulosa substances under microscope;Immunofluorescence was used to identify the specific expression of FSHR in human ovarian granulosa cells.2.Compared with the control group,the cell survival rate decreased gradually with the increase of H2O2 concentration,and the cell survival rate reached IC50(P<0.05).Western Blot analysis showed that compared with the control group,the levels of pro-apoptotic protein Caspase3 and Bax protein were increased and the levels of anti-apoptotic protein Bcl2 protein were decreased in H2O2 group(P<0.05).3.Compared with 0.5mmol/L H2O2 treatment group,the survival rate of cells pretreated with 2.5umol/L and 5umol/L CoQ10 solution was significantly increased(P<0.05),but there was no statistical difference in the survival rate of cells pretreated with 2.5umol/L and 5umol/L CoQ10 solution.Western Blot analysis showed that compared with H2O2 group,the expression levels of proapoptotic protein Caspase3 and Bax in CoQ10 pretreatment group were significantly decreased,and the expression levels of anti-apoptotic protein Bcl2 were significantly increased(P<0.05).4.Compared with the control group,the mitochondrial function of H2O2 group decreased,which was manifested as increased reactive oxygen species production level,decreased mitochondrial membrane potential and ATP level(P<0.05);CoQ10 pretreatment alleviated H2O2-induced mitochondrial function decline,which was manifested as decreased reactive oxygen species production level,increased mitochondrial membrane potential and ATP level(P<0.05).5.qPCR detection of Nrf2/Keap1 pathway showed that compared with the control group,Nrf2 mRNA expression was increased and Keap1 mRNA expression was decreased in H2O2 group,but the differences were not statistically significant,while HO-1 and SOD1 mRNA expression was increased(P<0.05).Compared with H2O2 group,Nrf2 mRNA expression in CoQ10 pretreatment group was increased but the difference was not statistically significant,Keap1 mRNA expression was decreased(P<0.01),HO-1 and SOD1 mRNA expression was increased(P<0.05).6.Western Blot analysis of Nrf2/Keap1 pathway showed that:Compared with the control group,total Nrf2 protein expression in H2O2 group was increased but the difference was not statistically significant,nuclear Nrf2 expression was increased(P<0.05),cytoplasmal Nrf2 expression was decreased but not statistically significant,Keap1 protein expression was decreased(P<0.05),HO-1 protein expression was increased(P<0.05).The expression of SOD1 protein increased but the difference was not statistically significant.Compared with H2O2 group,total Nrf2 protein expression in CoQ10 pretreatment group was increased but the difference was not statistically significant,the protein expression of nuclear Nrf2,HO-1 and SOD1 was increased(P<0.05),and the protein expression of cytoplasmic Nrf2 and Keapl was decreased(P<0.05).Conclusion:H2O2 induced oxidative damage of ovarian granulosa cells,which showed decreased mitochondrial function and increased apoptosis;CoQ10 has a protective effect on oxidative stress injury of ovarian granulosa cells,and its mechanism may be related to improving mitochondrial function and activating Nrf2/Keap1 pathway.