单宁酸缓解氟化钠诱导小鼠卵母细胞氧化应激的研究

doi:10.16431/j.cnki.1671-7236.2024.10.006

摘要/Abstract

摘要： 【目的】研究单宁酸(TA)对氟化钠(NaF)诱导小鼠卵母细胞氧化应激损伤的保护作用，从而为开发氟中毒解毒剂提供理论依据。【方法】在小鼠卵母细胞体外成熟培养液中添加0(对照)、0.25、0.50、0.75 mmol/L NaF，通过检测卵母细胞第一极体排出率，确定NaF最适作用浓度。以最佳作用浓度NaF处理小鼠卵母细胞后，通过添加不同浓度TA(0、1、10、100 μg/L)，确定最佳TA作用浓度。在上述试验基础上设置对照组、毒性组(NaF)和解毒剂组(NaF＋TA)，利用免疫荧光染色检测小鼠卵母细胞内活性氧(ROS)、谷胱甘肽(GSH)以及线粒体膜电位(MMP)水平；统计各组小鼠卵母细胞受精率、卵裂率和囊胚率，测定丙二醛(MDA)含量以及超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性。【结果】与对照组相比，0.25～0.75 mmol/L NaF组卵母细胞第一极体排出率均极显著降低(P＜0.01)，后续以0.25 mmol/L NaF作为有效毒性剂量进行试验。小鼠卵母细胞经NaF处理后，与0 μg/L TA组相比，10 μg/L TA组卵母细胞第一极体排出率极显著升高(P＜0.01)。免疫荧光检测结果显示，与对照组相比，NaF组小鼠卵母细胞ROS水平显著上升(P＜0.05)，GSH、MMP水平和SOD、GSH-Px活性显著或极显著下降(P＜0.05；P＜0.01)。与NaF组相比，NaF＋TA组小鼠卵母细胞GSH、MMP水平以及SOD和GSH-Px活性显著或极显著升高(P＜0.05；P＜0.01)，ROS水平和MDA含量显著或极显著降低(P＜0.05；P＜0.01)。早期胚胎发育统计结果显示，NaF组小鼠卵母细胞的受精率、卵裂率和囊胚率均极显著低于对照组(P＜0.01)；NaF+TA组小鼠卵母细胞受精率、卵裂率以及囊胚率均极显著高于NaF组(P＜0.01)，且与对照组相比无显著差异(P＞0.05)。【结论】 NaF通过提高卵母细胞内ROS和MDA水平诱导小鼠卵母细胞产生氧化应激，并降低细胞MMP水平，影响早期胚胎发育。添加10 μg/L TA可以缓解NaF诱导小鼠引起的氧化应激，提高小鼠卵母细胞成熟质量，从而促进体外受精和胚胎发育潜力。

关键词: 氟化钠; 单宁酸; 小鼠; 卵母细胞; 氧化应激

Abstract: 【Objective】 The aim of this experiment was to study the protective effect of tannic acid (TA) on oxidative stress injury of mouse oocytes induced by sodium fluoride (NaF),and provide theoretical basis for developing antidotes for fluorosis.【Method】 Mice oocytes were added with 0 (control),0.25,0.50 and 0.75 mmol/L NaF in vitro maturation medium.The optimal concentration of NaF was determined by detecting the first polar body excretion (PBI) rate of oocytes.After treating mouse oocytes with the optimal concentration of NaF,the optimal concentration of TA was determined by adding different concentrations of TA (0,1,10 and 100 μg/L).On the basis of the above tests,control group,toxicity group (NaF) and antidote group (NaF＋TA) were set up.The levels of reactive oxygen species (ROS),glutathione (GSH) and mitochondrial membrane potential (MMP) in mouse oocytes were detected by immunofluorescence staining.The fertilization rate,cleavage rate and blastocyst rate of mouse oocytes in each group were measured.The content of malondialdehyde (MDA) and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were measured.【Result】 Compared with control group,the excretion rate of the first polar body in 0.25-0.75 mmol/L NaF groups was extremely significantly decreased (P＜0.01).Then 0.25 mmol/L NaF was used as the effective toxic dose for testing.After NaF treatment,the excretion rate of first polar body in 10 μg/L TA group was extremely significantly higher than that in 0 μg/L TA group (P＜0.01).The results of immunofluorescence detection showed that,compared with control group,the ROS levels of oocytes in NaF group were significantly increased (P＜0.05),while the levels of GSH and MMP and the activities of SOD and GSH-Px were significantly or extremely significantly decreased (P＜0.05 or P＜0.01).Compared with NaF group,the levels of GSH and MMP and the activities of SOD and GSH-Px of mouse oocytes in NaF＋TA group were significantly or extremely significantly increased (P＜0.05 or P＜0.01),ROS level and MDA content were significantly or extremely decreased (P＜0.05 or P＜0.01).The statistical results of early embryonic development showed that the fertilization rate,cleavage rate and blastocyst rate in NaF group were extremely significantly lower than those in control group (P＜0.01).The fertilization rate,cleavage rate and blastocyst rate of mice in NaF＋TA group were extremely significantly higher than those in NaF group (P＜0.01),but had no significant difference compared with control group (P＞0.05).【Conclusion】 NaF could induce oxidative stress in mouse oocytes by increasing ROS and MDA levels in oocytes,and reduce MMP levels,which affect early embryonic development.The addition of 10 μg/L TA could relieve the oxidative stress induced by NaF in mice and improve the maturation quality of mouse oocytes,thereby promoting in vitro fertilization and embryo development potential.

Key words: sodium fluoride; tannin acid; mouse; oocyte; oxidative stress

中图分类号:

S859.82

英提扎尔·阿不力孜, 郭延华, 艾日夏提·地里夏提, 艾克拜尔·艾合麦提, 妮格尔阿依·安尼瓦尔, 王旭光, 阿布力孜·吾斯曼. 单宁酸缓解氟化钠诱导小鼠卵母细胞氧化应激的研究[J]. 中国畜牧兽医, 2024, 51(10): 4246-4254.

YINGTIZHAER Abulizi, GUO Yanhua, AIRIXIATI Dilixiati, AIKEBAIER Aihemaiti, NIGEERAYI Anniwaer, WANG Xuguang, ABULIZI Wusiman. Tannic Acid Alleviate Oxidative Stress Induced by Sodium Fluoride in Mouse Oocytes[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(10): 4246-4254.

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