补肾调经方对体外培养氧化应激模型小鼠卵母细胞质量及抗氧化能力的影响  

Effects of Bushen Tiaojing Formula on the quality and antioxidant ability of oocytes cultured in vitro with oxidative stress model mice

作　　者：管凤丽 杜惠兰[1] GUAN Fengli;DU Huilan(Hebei University of Chinese Medicine Hebei,Shijiazhuang 050200,China)

机构地区：[1]河北中医学院,石家庄050200

出　　处：《北京中医药大学学报》2022年第4期407-413,共7页Journal of Beijing University of Traditional Chinese Medicine

基　　金：国家自然科学基金面上项目(No.81774359)。

摘　　要：目的 通过体外添加H;O;建立氧化应激模型,探讨补肾调经方对小鼠卵母细胞质量及抗氧化能力的影响。方法 选用D24-26健康雌性昆明小鼠,每只小鼠注射血促性素(10 IU)48 h后,体式显微镜下分离卵巢,获取GV期卵母细胞,将细胞随机分为正常组、模型组、补肾调经方高剂量组、补肾调经方低剂量组,置于体外成熟卵母细胞(IVM)培养液中培养14 h。收集MⅡ期卵母细胞,在倒置显微镜下观察卵母细胞第一极体(first polar body, PB1)排出情况,透射电镜观察线粒体超微结构,Mito-Tracker Red CMXRos与DCHF-DA试剂盒检测线粒体分布及活性氧(ROS)水平,化学比色法与RT-qPCR法检测超氧化物歧化酶(SOD)与谷胱甘肽过氧化物酶(GSH-Px)活性及mRNA表达。结果 与正常组比较,模型组卵母细胞PB1排出率降低,线粒体结构完整性被破坏、分布异常及ROS水平升高,抗氧化酶SOD、GSH-Px活性及mRNA表达均降低;与模型组比较,补肾调经方高、低剂量组线粒体结构较完整,仅有部分线粒体嵴断裂,线粒体呈均匀分布,ROS水平降低,PB1排出率、抗氧化酶SOD、GSH-Px活性及mRNA表达均升高(P<0.05)。结论 补肾调经方可提高卵母细胞质量,其机制可能与保护线粒体结构和功能、调节抗氧化酶活性、降低氧化损伤有关。Objective The oxidative stress model was established by adding H;O;to the culture medium in vitro to explore the effect of Bushen Tiaojing Formula(Kidney-supplementing Menstruation-regulating formula, BSTJF) on the quality and antioxidant capacity of oocytes. Methods Eighty SPF female Kunming mice aged 3-4 weeks(25±3 g) were selected. Each mouse was injected with serum gonadotrophin(10 IU). After 48 hours, the ovaries were isolated under a stereo microscope to obtain GV-phase oocytes. The cells were randomly divided into normal group, model group, high-dose BSTJF group, low-dose BSTJF group, and incubated in IVM for 14 hours. MⅡ stage oocytes were then collected. The first polar body(PB1) extrusion rate of the oocytes was observed under an inverted microscope while the ultrastructure of mitochondria observed under a transmission electron microscope. The distribution of mitochondria and the level of reactive oxygen species(ROS) were evaluated with the Mito-Tracker Red CMXRos and DCHF-DA kits. Chemistry Colorimetric method and RT-qPCR method for detection of superoxide dismutase(SOD) and glutathione peroxidase(GSH-px) activity and mRNA expression. Results Compared with the normal group, the PB1 excretion rate of oocytes in the model group was significantly reduced;the integrity of the mitochondrial structure was destroyed with abnormal distribution, and the level of ROS increased. The antioxidant enzymes SOD and GSH-px activity and mRNA expression were all decreased;Compared with the model group, the mitochondrial structure of the high-and low-dose groups of BSTJF was relatively complete, with only part of the mitochondrial cristae(MC) destroyed. Mitochondria were uniformly distributed, the level of ROS decreased, and the PB1 extrusion rate, antioxidant enzyme SOD, GSH-px activity and mRNA expression were all increased(P<0.05). Conclusion Bushen Tiaojing Formula can improve the quality of oocytes, and its mechanism may be related to protecting the structure and function of mitochondria, regulating the activity o

关 键 词：补肾调经方 线粒体 氧化应激 卵母细胞质量 小鼠 

分 类 号：R285.5[医药卫生—中药学]