收稿日期: 2023-07-12
网络出版日期: 2023-12-19
基金资助
海南省自然科学基金项目(821RC702);海南省省级临床医学中心项目
Regulation of ovarian granulosa cell by SIRT1 in women with poor ovarian response
Received date: 2023-07-12
Online published: 2023-12-19
目的 探讨卵巢低反应女性中沉默信息调节因子1（SIRT1）对卵巢颗粒细胞的调控。 方法 本研究纳入行IVF/ICSI-ET助孕的女性共60例，其中卵巢低反应（POR）组和卵巢正常反应（NOR）组各30例。采用密度梯度离心法分离纯化卵泡液中的颗粒细胞。分别使用SIRT1激动剂白藜芦醇（RESV）或抑制剂EX527处理POR组颗粒细胞，对照组为不使用药物处理的颗粒细胞。采用qPCR检测颗粒细胞SIRT1和类固醇激素合成关键酶（StAR、CYP19、CYP17）的mRNA水平。采用Western blot检测颗粒细胞SIRT1、类固醇激素合成关键酶蛋白和凋亡相关蛋白（Bcl-2、Caspase-3）的水平。TUNEL法检测颗粒细胞的凋亡率。化学发光法检测颗粒细胞培养液中雌二醇水平。 结果 POR组颗粒细胞SIRT1 mRNA和蛋白水平低于NOR组（P = 0.003，P < 0.001）。RESV上调SIRT1 mRNA和蛋白水平（P < 0.001），上调类固醇激素合成关键酶mRNA和蛋白水平（P < 0.05），EX527下调SIRT1 mRNA和蛋白水平（P = 0.023、0.001），下调类固醇激素合成关键酶mRNA和蛋白水平（P < 0.05）。RESV作用颗粒细胞后，细胞凋亡率降低（P = 0.000），Bcl-2蛋白水平上升（P < 0.001），Caspase-3蛋白水平下降（P < 0.001）；EX527作用颗粒细胞后，细胞凋亡率升高（P < 0.001），Bcl-2蛋白水平下降（P = 0.003），Caspase-3蛋白水平上升（P < 0.001）。 结论 POR女性颗粒细胞SIRT1水平低于NOR女性。RESV增加SIRT1表达。SIRT1抑制颗粒细胞凋亡，提高类固醇激素合成关键酶水平，促进颗粒细胞合成雌二醇。因此，SIRT1激活剂如RESV可能成为改善POR女性卵巢反应性，增加获卵数的治疗药物。
柯慧 , 颜礼征 , 张宇 , 毛记龙 , 姚宇飞 . 卵巢低反应女性中沉默信息调节因子1对卵巢颗粒细胞的调控[J]. 实用医学杂志, 2023 , 39(21) : 2709 -2717 . DOI: 10.3969/j.issn.1006-5725.2023.21.004
Objective To investigate the regulation of SIRT1 on ovarian granulosa cell in women with poor ovarian response. Methods A total of 60 women who underwent IVF/ICSI-ET were included in this study， 30 women in poor ovarian response （POR） group and 30 women in normal ovarian response （NOR） group. The granulosa cells in follicular fluid were isolated and purified by density gradient centrifugation. The granulosa cells of POR group were treated with SIRT1 agonist resveratrol （RESV） or inhibitor EX527， respectively， and the control group was granulosa cells treated without drugs. mRNA levels of SIRT1 and key enzymes of steroid hormone synthesis （StAR， CYP19， CYP17） in granulosa cells were detected by qPCR. The levels of SIRT1， steroid hormone synthesis key enzymes protein and apoptosis-related protein （Bcl-2， Caspase-3） in granulosa cells were detected by Western blot. The apoptosis rate of granulosa cells was detected by TUNEL method. Determination of estradiol in granulosa cell culture medium by chemiluminescence. Results The mRNA and protein levels of SIRT1 in granulosa cells in POR group were lower than those in NOR group （P = 0.003， P < 0.001）. RESV up-regulated SIRT1 mRNA and protein levels （P < 0.001）， up-regulated mRNA and protein levels of steroid hormone synthesis key enzyme （P < 0.05）. EX527 down-regulated SIRT1 mRNA and protein levels （P = 0.023， 0.001）， down-regulated mRNA and protein levels of steroid hormone synthesis key enzymes （P < 0.05）. After RESV treatment， the apoptosis rate of granulosa cells was decreased （P < 0.001）， the Bcl-2 protein level was increased （P < 0.001）， and the Caspase-3 protein level was decreased （P < 0.001）. After EX527 treatment， the apoptosis rate of granulosa cells was increased （P < 0.001）， the Bcl-2 protein level was decreased （P = 0.003）， and the Caspase-3 protein level was increased （P < 0.001）. Conclusion The SIRT1 level in granulosa cells of POR women was lower than that of NOR women. RESV up-regulated SIRT1 expression in granulosa cells. SIRT1 inhibited granulosa cell apoptosis， increased the level of steroid hormone synthesis key enzyme， and promoted estradiol synthesis in granulosa cell. Therefore， SIRT1 activators such as RESV may be a therapeutic agent to improve ovarian reactivity and increase the number of oocytes obtained in women with POR.
| 1 | MARUTHINI D， HARRIS S E， BARTH J H， et al. The effect of metformin treatment in vivo on acute and long-term energy metabolism and progesterone production in vitro by granulosa cells from women with polycystic ovary syndrome［J］. Hum Reprod， 2014，29（10）：2302-2316. |
| 2 | HAO C， ZHU P， YANG X， et al. Overexpression of SIRT1 promotes metastasis through epithelial-mesenchymal transition in hepatocellular carcinoma［J］. BMC Cancer， 2014，14（1）：978. |
| 3 | PENRALZ S. Resveratrol： from grapevines to mammalian biology［J］. FASEB J， 2003， 17 （14）： 1975-1985. |
| 4 | MA S， FENG J， ZHANG R， et al ．SIRT1 activation by resveratrol alleviates cardiac dysfunction via mitochondrial regulation in diabetic cardiomyopathy mice［J］. Oxid Med Cell Longe， 2017， 2017： 4602715. |
| 5 | NISHIGAKI A， TSUBOKURA H， TSUZUKI-NAKAO T， et al. Hypoxia： Role of SIRT1 and the protective effect of resveratrol in ovarian function［J］．Reprod Med Biol， 2022， 2l （1）： e12428. |
| 6 | SAI M， JING F， RAN Z， et al ．SIRT1 activation by resveratrol alleviates cardiac dysfunction via mitochondrial regulation in diabetic cardiomyopathy mice［J］. Oxid Med Cell Longev， 2017， 2017： 4602715. |
| 7 | SAUVE A A， CELIC I， AVALOS J. Chemistry of gene silencing： The mechanism of NAD+- dependent deacetylation reactions［J］. J Biochem， 2001， 40 （51）：15456-15463. |
| 8 | KUNDU A， RICHA S， DEY P， et al. Protective effect of EX-527 against high-fat diet-induced diabetic nephropathy in Zucker rats ［J］. Toxicol Appl Pharmacol， 2020， 390（C）： 114899. |
| 9 | GERTZ M， FISCHER F， NGUYEN G T， et al. Ex-527 inhibits Sirtuins by exploiting their unique NAD+-dependent deacetylation mechanism ［J］. Proc Natl Acad Sci USA， 2013， 110（30）： E2772-E2781. |
| 10 | YANG X， WEI J， HE Y， et al. SIRT1 inhibition promotes atherosclerosis through impaired autophagy ［J］. Oncotarget， 2017， 8（31）： 51447-51461. |
| 11 | SHI J， YOSHINO O， OSUGA Y， et al. Bone morphogenetic protein 7 （BMP-7） increases the expression of follicle-stimulating hormone （FSH） receptor in human granulosa cells［J］. Fertil Steril， 2010， 93（4）， 1273-1279. |
| 12 | UBALDI F， VAIARELLI A， D'ANNA R， et al. Management of poor responders in IVF： is there anything new？ ［J］. Biomed Res Int， 2014， 2014： 352098. |
| 13 | 柯慧，颜礼征.博洛尼亚标准卵巢低反应者在促排卵体外受精/卵胞质内单精子注射周期中的卵巢反应性［J］. 中华生殖与避孕杂志，2018，38（1）：5-10. |
| 14 | 柯慧，颜礼征.博洛尼亚标准卵巢低反应者IVF/ICSI妊娠结局的预测因子［J］. 生殖与避孕，2016，（2）：106-111. |
| 15 | DOU X， SUN Y， LI J， et al. Short term rapamycin treatment increases ovarian lifespan in young and middle-aged female mice［J］. Aging Cell， 2017， 16：825. |
| 16 | ZHOU X L， XU J J， NI Y H， et al. SIRT1 activator （SRT1720） improves the follicle reserve and prolongs the ovarian lifespan of diet-induced obesity in female mice via activating SIRT1 and suppressing mTOR signaling［J］. J Ovarian Res， 2014， 7： 97. |
| 17 | ILJAS J D， WEI Z， HOMER H A. Sirt1 Sustains Female Fertility by Slowing Age-Related Decline in Oocyte Quality Required for Post-Fertilization Embryo Development［J］. Aging Cell， 2020， 19（9）：e13204. |
| 18 | TIWARI M， TRIPATHI A， CHAUBE S K. Presence of encircling granulose cells protects against oxidative stress-induced apoptosis in rat eggs cultured in vitro ［J］. Apoptosis， 2017， 22（1）：98-107. |
| 19 | HAN Y， LUO H N， WANG H， et al. SIRT1 induces resistance to apoptosis in human granulosa cells by activating the ERK pathway and inhibiting NF-κB signaling with anti-infammatory functions［J］. Apoptosis， 2017， 22：1260-1272. |
| 20 | AHMAD I， HODA M. Molecular mechanisms of action of resveratrol in modulation of diabetic and non-diabetic cardiomyopathy［J］. Pharmacol Res，2020， 161： 105112. |
| 21 | ZHANG L X， LI C X， KAKAR M U， et al. Resveratrol （RV）： A pharmacological review and call for further research［J］. Biomed Pharmacother， 2021， 143： 112164. |
| 22 | FAIZA A， MAHEEN S， SUMAIRA R， et al. SIRT1 and antioxidants in infertile females： Exploration of the role of vitamin D［J］. PLoS One， 2023， 18（7）： e0287727. |
| 23 | ZHOU D D， LUO M， HUANG S Y， et al. Effects and Mechanisms of Resveratrol on Aging and Age-Related Diseases［J］. Oxid Med Cell Longe， 2021， 2021： 9932218. |
| 24 | SIROTKIN A V. Effects of Resveratrol on Female Reproduction： A Review［J］. Phytother Res， 2021， 35， 5502–5513. |
| 25 | SHOJAEI-ZARGHANI S， RAFRAF M. Resveratrol and Markers of Polycystic Ovary Syndrome： a Systematic Review of Animal and Clinical Studies［J］. Reprod Sci， 2022，29（9）：2477-2487. |
| 26 | PARK S J， ABROAD F， PHILP A， et al. Resveratrol ameliorates aging-related metabolic phenotypes by inhibiting cAMP phosphodiesterases［J］. Cell， 2012， 148（3）： 421-433. |
| 27 | KOLTAI E， SZABO Z， ATALAY M， et al. Exercise alters SIRTI， SIRT6， NAD and NAMPT levels in skeletal muscle of aged rats［J］. Mech Ageing Dev， 2010， 131（1）： 21-28. |
| 28 | NIE Z， HUA R， ZHANG Y N， et al. Resveratrol protects human luteinised granulosa cells against hydrogen peroxide-induced oxidative injury through the Sirt1［J］. Reprod Fertil Dev， 2021， 33（15-16）： 831-840. |
| 29 | HOLLOWAY K R， BARBIERI A， MALYARCHUK S， et al. SIRT1 positively regulates breast cancer associated human aromatase （CYP19A1） expression［J］. Mol Endocrinol， 2013， 27（3）： 480-490. |
| 30 | MORITA Y， WADA-HIRAIKE O， YANO T， et al. Resveratrol promotes expression of SIRT1 and StAR in rat ovarian granulosa cells： an implicative role of SIRT1 in the ovary［J］. Reprod Biol Endocrinol， 2012， 10：14. |
| 31 | XIONG F， HU L， ZHANG Y， et al. miR -22 inhibits mouse ovarian granulosa cell apoptosis by targeting SIRT1［J］. Biol Open， 2016， 5（3）： 367-371. |
| 32 | ITAMI N， SHIRASUNA K， KUWAYAMA T， et al. Resveratrol improves the quality of pig oocytes derived from early antral follicles through sirtuin 1 activation［J］. Theriogenology， 2015， 83（8）： 1360-1367. |
| 33 | KALE J， OSTERLUND E J， ANDREWS D W. BCL-2 family proteins： changing partners in the dance towards death［J］. Cell Death Differ， 2018， 25（1）： 65-80. |
| 34 | YU C L， QIU M H， YIN H D， et al. miR-138-5p promotes chicken granulosa cell apoptosis via targeting SIRT1［J］. Anim Biotechnol， 2022，1-10. |
| 35 | PARK S A， JOO N R， PARK J H， et al. Role of the SIRT1/p53 regulatory axis in oxidative stress mediated granulosa cell apoptosis［J］. Mol Med Rep， 2021， 23（1）： 20. |
| 36 | PARK J H， PARK S A， LEE Y J， et al. TOPK inhibition accelerates oxidative stress induced granulosa cell apoptosis via the p53/SIRT1 axis［J］. Int J Mol Med， 2020， 46（5）： 1923-1937. |
| 37 | CAI M H， WANG J， SUN H J， et al. Resveratrol attenuates hydrogen peroxide-induced injury of rat ovarian granulosa-lutein cells by resisting oxidative stress via the SIRT1/Nrf2/ARE signaling pathway［J］. Curr Pharm Des， 2023， 29（12）： 947-956. |
| 38 | 潘毅， 陈军喜， 闫智杰. 白术内酯Ⅲ调节AMPK/SIRT1/NF?κB信号通路对脓毒症大鼠肝损伤的影响［J］. 实用医学杂志， 2023， 39（7）： 827-832. |
| 39 | ZHAO H， CHEN S， GAO K， et al. Resveratrol protects against spinal cord injury by activating autophagy and inhibiting apoptosis mediated by the SIRT1/AMPK signaling pathway［J］. Neuroscience， 2017， 348： 241-251. |
| 40 | JACKSON S， MAGDALENA S， RINA M. Diverse actions of sirtuin-1 on ovulatory genes and cell death pathways in human granulosa cells［J］. Reprod Biol Endocrinol， 2022， 20（1）： 104. |
| 41 | YU E J， KIM S H， HEO K， et al. Reciprocal roles of DBC1 and SIRT1 in regulating estrogen receptor α activity and co-activator synergy［J］. Nucleic Acids Res， 2011， 39（16）： 6932-6943. |
| 42 | LIU Z， RUDD M D， HERNANDEZ-GONZALEZ I， et al. FSH and FOXO1 regulate genes in the sterol/steroid and lipid biosynthetic pathways in granulosa cells［J］. Mol Endocrinol， 2009， 23（5）： 649-661. |
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