The Role And Mechanism Of SIRT1 In Polycystic Ovary Syndrome

Polycystic ovary syndrome(PCOS)is a common reproductive endocrine disorders,often manifested as follicular non-cycle maturation leading to chronic anovulation,and often associated with infertility and glucose and lipid metabolism disorders.In addition to the pathogenesis of hypothalamus-pituitary-ovarian axis dysfunction,but also may be associated with follicular microenvironment disorders,follicular development,maturation,atresia regulation imbalance.Ovarian granulocytes have an important effect on the growth and development of follicles and follicular microenvironment,and the increase of granulocyte apoptosis leads to follicular maturation disorder.SIRT1 is a deacetylase widely found in mammals and has a wide range of functions,including energy metabolism,DNA repair,and cell life and apoptosis regulation.SIRT1 expression is regulated by a variety of factors,such as P53,FOXO and E2F1 formed by the self-feedback loop.In addition,SIRT1 is involved in the regulation of the expression of key enzymes in steroid hormone synthesis,involved in mammalian reproductive processes.RSV is the strongest specific SIRT1 agonist found so far,and its function involves maintaining the balance of energy in the body and also has antioxidant,antiinflammatory and antitumor activity.EX-527 belongs to the indole SIRT1 inhibitor and is the most active selective SIRT1 inhibitor found so far.Therefore,RSV and EX527 were selected as specific activators and inhibitors for SIRT1 in this study.Objective:PCOS women who accepted IVF or ICSI were the major object of the study,separated and purified ovarian granulosa cells and cultured in vitro,RSV and EX527 were used to treat granulosa cells.The effects of SIRT1 expression level on the apoptotic rate and the key enzymes of steroid hormone synthesis were analyzed by qPCR,Western Blot,flow cytometry and chemiluminescence method,then discussed the potential relationship between SIRT1 and PCOS.Methods:A total of 114 patients who received in vitro fertilization-embryo transfer(IVF-ET)or intracytoplasmic sperm injection(ICSI)in the First Affiliated Hospital of Hainan Medical College Reproductive Medical Center from April 1,2016 to December 1,2016,were included in this study.According to the inclusion criteria and exclusion criteria,there were two groups: PCOS group(57 cycles)and control group(57 cycles).The follicular fluid was collected from PCOS group and control group.The granulosa cells of follicular fluid were extracted by erythrocyte lysis.The cells were inoculated in 6-well plates and cultured in DMEM-F12 medium containing 15% FBS as the basal medium.After granulosa cells were adhered,use the basal medium containing 100 uM RSV or 100 uM EX527 cultured the cells,the cells were harvested for 12 h,24h and 48 h respectively.At the same time,the culture medium was collected and the untreated cells and culture medium were used as blank control.The total RNA was picked up from granulosa cells by Trizol.The total protein of granulosa cells was extracted with RIPA lysate containing PMSF.The expressionlevels of SIRT1,StAR,CYP17,CYP19 and PPARγ of granulosa cells in two groups were detected and compared by qPCR and Western Blot.Flow cytometry was used to detect the apoptotic rate of each specimen.The concentration of E2 in the culture medium was detected by chemiluminescence method.Results:1.The expression of SIRT1 was detected in both groups of granulocytes,while the expression level of SIRT1 in PCOS was lower than in control group(P<0.05),The expression of SIRT1,StAR,CYP17 and CYP19 were increased and the expression of PPARγ was decreased in the granulosa cells after treatment with RSV(P<0.05).The expression levels of SIRT1,StAR,CYP17 and CYP19 in the granulosa cells were decreased and the expression of PPARγ was increased after treated with EX527(P <0.05).2.After treated with RSV or EX527,the apoptotic rate of granulosa cells in the control group and PCOS group increased(P<0.05).The increase of apoptosis rate of granulosa cells after EX527 treatment was more significant than that of RSV(P <0.05).After treatment with RSV,the expression of Bcl-2 protein was increased and the expression of Caspase-3 protein was decreased,and that of EX527 was opposite.3.After RSV or EX527 treated cells,the concentration of E2 in the culture medium dreased first then increased(P<0.05).The concentration of E2 in the culture medium after RSV treatment was higher than that of EX527(P <0.05).Conclusions:In vitro culture of human granulosa cells,RSV and EX527 mediate the regulation of SIRT1 on granulosa cells apoptosis,SIRT1 can inhibit apoptosis and promote the expression of key enzymes in steroid hormone synthesis and the synthesis of estradiol in granulosa cell,participate in the incidence of PCOS.