基于卵母细胞线粒体探究补肾养精汤改善高龄不孕症的作用机制  

Explore mechanism of Bushen Yangjing Soup in improving elderly infertility based on oocyte mitochondrion

作　　者：王昭[1] 于昕[1] 武丽丽[1] 刘鹏飞 于潇 刘金星[2] WANG Zhao;YU Xin;WU Lili;LIU Pengfei;YU Xiao;LIU Jinxing(Shandong College of Traditional Chinese Medicine,Yantai 264199,China;Affiliated Hospital of Shandong University of Traditional Chinese Medicine,Jinan 250014,China)

机构地区：[1]山东中医药高等专科学校,烟台264199 [2]山东中医药大学附属医院

出　　处：《北京中医药大学学报》2024年第1期62-69,共8页Journal of Beijing University of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(No.82104917);山东省自然科学基金项目(No.ZR2019PH053);山东省中医药科技项目(No.2020Q030,Q-2023009);山东省中医药重点学科(鲁卫中医药科教字[2022]4号)。

摘　　要：目的基于线粒体探究补肾养精汤改善高龄不孕症的机制。方法采用C57BL/6J小鼠,雌性6~8周龄15只、10月龄45只,雄性6~8周龄10只。雌性6~8周龄小鼠为青年组,10月龄小鼠随机数字表法分为高龄组、脱氢表雄酮(DHEA)组和补肾养精汤组。DHEA组和补肾养精汤组分别给予DHEA 11.26 mg/(kg·d)、补肾养精汤11.41 g/(kg·d),青年组和高龄组给予同等体积生理盐水,连续干预45 d。收集各组卵母细胞,进行体外受精,观察卵母细胞、成熟卵母细胞和受精卵数量;实时荧光PCR法检测卵母细胞线粒体DNA(mtDNA)拷贝数,采用线粒体绿色荧光探针观察线粒体分布情况,JC-1试剂盒检测卵母细胞线粒体膜电位;实时荧光PCR和蛋白质印迹法检测卵母细胞线粒体融合蛋白(Mfn)2 mRNA和蛋白表达水平。结果与青年组相比,高龄组小鼠获卵数、成熟率和体外受精率均明显下降(P<0.01);卵母细胞mtDNA拷贝数减少、分布异常率增加、膜电位下降,差异均具有统计学意义(P<0.01);Mfn2 mRNA和蛋白表达均下调(P<0.01)。与高龄组相比,补肾养精组小鼠卵母细胞、成熟卵母细胞数量和体外受精率均明显增加(P<0.05),卵母细胞mtDNA拷贝数增加(P<0.05),异常分布率降低,膜电位上调(P<0.05),Mfn2 mRNA和蛋白表达均上调(均P<0.01)。与DHEA组相比,补肾养精组小鼠卵母细胞、成熟卵母细胞数量和体外受精率增加,卵母细胞mtDNA拷贝数增加、异常分布率降低、膜电位上调,Mfn2 mRNA和蛋白表达上调,其中卵母细胞数量和Mfn2蛋白表达差异具有统计学意义(均P<0.05)。结论补肾养精汤可能通过上调Mfn2 mRNA和蛋白表达,改善卵母细胞线粒体的数量、分布和功能,从而提高卵母细胞质量,这是临床应用补肾养精汤改善卵巢功能、治疗高龄不孕症的可能机制之一。Objective We aimed to explore the mechanism by which Bushen Yangjing Soup improves elderly infertility based on oocyte mitochondrion.Methods C57BL/6J mice were used,including 15 young females(6-8 weeks old),45 elderly females(10 months old),and ten young males(6-8 weeks old).The female 6-8-week-old young mice were assigned to the young group,and the female 10-month-old elderly mice were randomly divided into the model group,the dehydroepiandrosterone(DHEA)group,and the Bushen Yangjing Soup group.Mice in the DHEA and Bushen Yangjing Soup groups were given DHEA[11.26 mg/(kg·d)]and Bushen Yangjing Soup[11.41 g/(kg·d)].Mice in the other groups were given normal saline.After 45 days,oocytes were collected,the number of oocytes and the proportion of mature oocytes were recorded,and in vitro fertilization(IVF)was carried out,followed by calculation of the successful fertilization rate.Real-time PCR was used to determine the copy number of mitochondrial DNA in oocytes,mito-tracker green was used to observe the distribution of mitochondria,and the mitochondrial membrane potential(MMP)of oocytes was determined using the JC-1 kit.The mRNA and protein levels of mitofusin 2(Mfn2)in oocytes were determined by real-time PCR and Western blotting,respectively.Results Compared with the young group,the number of oocytes,the proportion of mature oocytes,and the IVF rate were significantly reduced(P<0.01),the copy number of mitochondrial DNA in oocytes was decreased,the rate of abnormal mitochondrial distribution was increased,the MMP was decreased(P<0.01),and the mRNA and protein levels of Mfn2 were decreased(P<0.01)in the elderly model mice.Compared with the elderly model group,the number of oocytes,the proportion of mature oocytes,and the IVF rate were significantly increased(P<0.05),the copy number of mitochondrial DNA in oocytes was increased(P<0.05),the rate of abnormal mitochondrial distribution was decreased,the MMP was increased(P<0.05),and the mRNA and protein levels of Mfn2 were increased(P<0.01)in the Bushen Yangjing So

关 键 词：高龄不孕症 卵母细胞 线粒体 线粒体融合蛋白2 补肾养精汤 小鼠 

分 类 号：R285.5[医药卫生—中药学]