褪黑素通过SIRT3/SOD2途径改善高龄卵巢功能减退女性颗粒细胞线粒体功能和减轻氧化应激  

Improving mitochondrial function and alleviating oxidative stress in aged women with ovarian insufficiency:the role of melatonin through the SIRT3/SOD2 pathway

作　　者：窦倩[1] 李朋粉[1] 马丽影[1] 许晓婷 张丹[1] 项云改[1] 谭丽[1] Dou Qian;Li Pengfen;Ma Liying;Xu Xiaoting;Zhang Dan;Xiang Yungai;Tan Li(Reproductive Medicine Center,the Second Affiliated Hospital of Zhengzhou University,Zhengzhou 450000,China)

机构地区：[1]郑州大学第二附属医院生殖医学中心,郑州450000

出　　处：《中华生殖与避孕杂志》2024年第4期385-393,共9页Chinese Journal of Reproduction and Contraception

基　　金：河南省医学科技攻关计划联合共建项目(LHGJ20200421,LHGJ20210367)。

摘　　要：目的研究褪黑素在体外培养中改善高龄卵巢功能减退女性颗粒细胞线粒体功能、减轻氧化应激状态的作用及可能机制。方法收集在2022年3月至6月期间在郑州大学第二附属医院生殖医学中心接受辅助生殖技术助孕患者取卵后废弃的卵泡液,提取壁层颗粒细胞,按患者年龄分为高龄组(年龄≥38岁,6例)和年轻对照组(年龄<35岁,6例)。使用透射电子显微镜观察颗粒细胞的线粒体超微结构,通过ATP检测试剂盒检测细胞内ATP,实时荧光定量PCR检测线粒体DNA(mitochondrial DNA,mtDNA)拷贝数,JC-1荧光探针检测线粒体膜电位,MitoSOX?Red线粒体氧化物指示剂检测活性氧含量,Western blotting法检测SIRT3、SOD2的蛋白表达。将高龄组的颗粒细胞样本根据随机数字表法分为褪黑素处理组和空白对照组(各5例),检测在体外培养颗粒细胞时添加褪黑素1μmol/L后线粒体上述检测指标与空白对照组的差异。转染siRNA下调颗粒细胞内SIRT3,检测褪黑素添加前后上述检测指标并与阴性对照组比较。结果高龄组颗粒细胞的线粒体超微结构与年轻对照组相比存在明显差异,线粒体结构显示不清,线粒体嵴少见且不规律排列,ATP水平(P=0.012)、mtDNA拷贝数(P=0.005)和线粒体膜电位(P=0.009)显著低于年轻对照组,活性氧含量显著增加(P=0.003),SIRT3和SOD2水平显著降低(P<0.001和P=0.002),差异均有统计学意义。经褪黑素体外培养后,线粒体超微结构有所恢复,ATP水平(P<0.001)、mtDNA拷贝数(P=0.038)和线粒体膜电位(P=0.002)高于空白对照组,SIRT3和SOD2水平高于空白对照组(P=0.011和P=0.031),活性氧含量低于空白对照组(P<0.001),差异均有统计学意义。siRNA转染后的颗粒细胞SIRT3的表达显著下调(P<0.001)。经褪黑素处理后,细胞内ATP水平、mtDNA拷贝数和线粒体膜电位均低于未下调SIRT3的阴性对照组(P<0.001、P=0.001、P<0.001),活性氧含量高于阴性对照组(P<0.001),差�Objective To investigate the potential effects of melatonin on improving mitochondrial function and reducing oxidative stress in granulosa cells in aged women with ovarian insufficiency in vitro,as well as explore the underlying mechanisms.Methods Granulosa cells were extracted from waste follicular fluid obtained from patients undergoing assisted reproductive technology at the Reproductive Medicine Center of the Second Affiliated Hospital of Zhengzhou University from March to June 2022.According to the age of the patients,they were divided into two groups:the aged group(age≥38 years old,6 cases)and the young control group(age<35 years old,6 cases).The mitochondrial ultrastructure of the granulosa cells was examined using transmission electron microscopy.Intracellular ATP levels were measured using an ATP detection kit,while mitochondrial DNA(mtDNA)copy number was assessed using real-time fluorescence quantitative PCR.Mitochondrial membrane potential was evaluated using the JC-1 fluorescent probe,reactive oxygen species(ROS)content was measured using the MitoSOX™Red mitochondrial oxide indicator,and protein expressions of SIRT3 and SOD2 were determined using Western blotting.According to the random number table method,samples from the aged group were randomly allocated to either the melatonin treatment group or blank control group(5 cases in each group)to assess the impact of in vitro melatonin treatment on the aforementioned mitochondrial parameters.SIRT3 in granular cells was down-regulated by transfection of siRNA,and the above indexes were detected before and after melatonin addition and compared with the negative control group.Results In comparison to the young group,the aged group exhibited distinct differences in the ultrastructure of granulosa cell mitochondria.Specifically,the mitochondrial structure appeared unclear,with sparse and irregularly arranged ridges.Furthermore,significant reductions were observed in ATP levels(P=0.012),mtDNA copy number(P=0.005),and mitochondrial membrane potential(P=0.00

关 键 词：褪黑素 颗粒细胞 衰老 线粒体 氧化应激 活性氧 

分 类 号：R711.75[医药卫生—妇产科学]