温肾养血方改善自然衰老雌鼠卵巢颗粒细胞及卵母细胞质量的实验研究  

An Experimental Study on Improving the Quality of Ovarian Granular Cells and Oocytes in Naturally Aging Female Mice with Wenshen Yangxue Formula

作　　者：衣彦晓 黄千千 辛明蔚[1] 何军琴[1] 武颖[1] 尹晓丹[1] 杨维[1] 韩倩 YI Yanxiao;HUANG Qianqian;XIN Mingwei;HE Junqin;WU Ying;YIN Xiaodan;YANG Wei;HAN Qian(Beijing Obstetrics and Gynecology Hospital,Capital Medical University,Beijing Maternal andChild Health Care Hospital,Beijing 100026,China)

机构地区：[1]首都医科大学附属北京妇产医院/北京妇幼保健院,北京100026

出　　处：《标记免疫分析与临床》2023年第4期657-664,720,共9页Labeled Immunoassays and Clinical Medicine

基　　金：北京市自然科学基金资助(编号:7232268)。

摘　　要：目的 探究温肾养血方改善自然衰老雌鼠卵巢颗粒细胞及卵母细胞质量的作用机制。方法 使用随机数表法将40只8月龄ICR雌鼠随机分为A:自然衰老组、B:控制性超排卵组(controlled ovarian hyperstimulation, COH)组、C:温肾养血低剂量组、D:温肾养血高剂量组,连续定时给药10d, B、C、D组雌鼠于第11天腹腔注射孕马血清促性腺激素(pregnant mareserum gonadotropin, PMSG),A组注射等量0.9%生理盐水,48h后,腹腔注射人绒毛膜促性腺激素(human chorionic gonadotropin, hCG),16h后取高龄雌鼠输卵管,分离其卵巢颗粒细胞及卵母细胞,检测氧化应激及线粒体功能等各项指标,评价各组小鼠卵巢颗粒细胞及卵母细胞质量,并采用高效液相色谱-四极杆-飞行时间质谱法(UPLC-Q-TOF/MS)测定温肾养血方化学成分。结果 与自然衰老组相比,COH组卵母细胞和颗粒细胞线粒体膜电位明显下降(P<0.05),颗粒细胞α-酮戊二酸脱氢酶(α-ketoglutaric aciddehydrogenase, α-KGDH)和丙酮酸脱氢酶(pyruvate dehydrogenase, PDH)活性降低(P<0.05),卵母细胞和颗粒细胞活性氧(reactive oxygen species, ROS)水平升高(P<0.05),超氧化物歧化酶(superoxide dismutase, SOD)水平降低(P>0.05),谷胱甘肽过氧化物酶(glutathione peroxidase, GSH-px)水平降低(P<0.05)。与COH组相比,温肾养血低剂量和高剂量组卵母细胞和颗粒细胞线粒体膜电位均有所回升(P>0.05);高剂量组颗粒细胞PDH、α-KGDH活性升高并差异有统计学意义(P<0.05);低剂量和高剂量组卵母细胞和颗粒细胞ROS水平降低(P<0.05),SOD活性升高(P>0.05),GSH-px活性升高(P>0.05)。通过UPLC-Q-TOF/MS方法鉴定出78种化合物。结论 温肾养血方可通过改善线粒体功能、提高抗氧化能力从而改善自然衰老雌鼠卵母细胞和颗粒细胞质量。Objective To explore the underlying mechanism of Wenshen Yangxue Formula in improving the quality of ovarian granular cells and oocytes in naturally aging female mice.Methods Forty 8-month-old ICR female mice were randomly divided into four groups using a random number table method,respectively,as follows:A:natural aging group,B:controlled ovarian hyperstimulation(COH)group,C:low-dose group of Wenshen Yangxue Formula,and D:high-dose group of Wenshen Yangxue Formula.Mice in B,C,and D groups were continuously given timed doses for 10 days.On the 11th day,pregnant horse serum gonadotropin(PMSG)was injected intraperitoneally,while group A received an equal amount of 0.9%physiological saline.After 48 hours,human chorionic gonadotropin(hCG)was intraperitoneally injected.After 16 hours,the fallopian tubes of elderly female mice were taken,and ovarian granulosa cells and oocytes were isolated.Oxidative stress and mitochondrial function were measured,and the quality of ovarian granulosa cells and oocytes in each group of mice was evaluated.Ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry was used to determine the chemical components of Wenshen Yangxue Formula.Results The experimental results showed that:compared with the natural aging group,the mitochondrial membrane potential of oocytes and granulosa cells in the aging ovulation-promoting group decreased significantly(P<0.05).The activity of pyruvate dehydrogenase(PDH)andα-ketoglutaric aciddehydrogenase(α-KGDH)decreased(P<0.05).Levels of reactive oxygen species(ROS)in oocytes and granulosa cells increased(P<0.05),while superoxide dismutase(SOD)and glutathione peroxidase(GSH-px)levels decreased(P<0.05).Compared with the COH group,the mitochondrial membrane potential of oocytes and granulosa cells in both the low-dose and high-dose groups of the Wenshen Yangxue Formula increased(P>0.05).The activities ofα-KGDH and PDH of granular cells increased significantly(P<0.05).In the low-dose and high-dose groups of Wenshen Yangxue Formula,the l

关 键 词：温肾养血方 卵巢自然衰老 卵母细胞 颗粒细胞 线粒体 抗氧化 

分 类 号：R271[医药卫生—中医妇科学]