Abstract:

Objective To discuss the regulatory effect of silent information regulator 1 （SIRT1） on ovarian granulosa cells of advanced-age women， and to preliminarily clarify the mechanism by which SIRT1 activator resveratrol （RESV） improves the function of ovarian granulosa cells of advanced-age women. Methods Sixty patients underwent in vitro fertilization-embryo transfer （IVF-ET） were enrolled， including 30 advanced-age patients （advanced age group， age ≥40 years） and 30 non-advanced-age patients （non-advanced age group， age <35 years）. Density gradient centrifugation was used to collect the granulosa cell samples from follicular fluid of female patients in advanced age group（n=30） and non-advanced age group （n=30）. The granulosa cells from each advanced-age female patient were divided into control group， RESV group， and EX527 （SIRT1 inhibitor） group. The granulosa cells in control group were treated without any drug； the granulosa cells in RESV group were cultured in medium containing 100 μmol·L^-1 RESV for 48 h； the granulosa cells in EX527 group were cultured in medium containing EX527 for 48 h. After trypsin digestion， the granulosa cells were collected. Real-time fluorescence quantitative PCR（RT-qPCR） method was used to detect the expression levels of SIRT1， steroidogenic acute regulatory protein （StAR）， cytochrome P450 family 19 （CYP19）， and cytochrome P450 family 17 （CYP17） mRNA in the cells in various groups； Western blotting method was used to detect the expression levels of cysteine-containing aspartate protease 3 （Caspase-3） and B-cell lymphoma 2 （Bcl-2） proteins in the cells in various groups； chemiluminescence method was used to detect the estradiol level in the cell culture medium of the cells in various groups on the day of human chorionic gonadotropin （HCG） injection； terminal deoxynucleotidyl transferase dUTP nick end labeling（TUNEL） method was used to detect the apoptotic rate of the cells in various groups. Results The RT-qPCR results showed that compared with non-advanced age group， the expression levels of SIRT1 mRNA and protein in the granulosa cells of the patients in advanced age group were decreased （P<0.05）. Compared with control group， the expression levels of SIRT1 mRNA and protein in granulosa cells of the patients in RESV group were increased （P<0.05）， while those in EX527 group were decreased （P<0.05）. Compared with control group， the expression levels of StAR， CYP19， and CYP17 mRNA and protein in granulosa cells of the patients in RESV group were increased （P<0.05）， while those in EX527 group were decreased （P<0.05）. The chemiluminescence method results showed that compared with control group， the estradiol level in granulosa cell culture medium of the patients in RESV group was increased （P<0.05）， while that in EX527 group was decreased （P<0.05）. The TUNEL method and Western blotting method results showed that compared with control group， the apoptotic rate of the granulosa cells of the patients in RESV group was decreased （P<0.05）， the expression level of Bcl-2 protein in the granulosa cells was increased （P<0.05）， and the expression level of Caspase-3 protein was decreased （P<0.05）； compared with control group， the apoptotic rate of granulosa cells of the patients in EX527 group was increased （P<0.05）， the expression level of Bcl-2 protein in the granulosa cells was decreased （P<0.05）， and the expression level of Caspase-3 protein was increased （P<0.05）. Conclusion The expression of SIRT1 in ovarian granulosa cells of advanced-age women is decreased. RESV inhibits granulosa cell apoptosis， increases the expression of steroidogenic enzymes in granulosa cells， and promotes estradiol synthesis in granulosa cells by promoting the expression of SIRT1 in granulosa cells of advanced-age women.

Key words: Silent information regulator 1, Advanced age, Granulosa cells, Apoptosis, Resveratrol