研究论文 RESEARCH PAPER

小鼠卵巢颗粒细胞氧化应激损伤及对沉默信息调节因子2相关酶1信号通路的影响

  • 弓浩杰 ,
  • 丁雪梅 ,
  • 白世平 ,
  • 曾秋凤 ,
  • 张克英 ,
  • 王建萍
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  • 四川农业大学动物营养研究所, 动物抗病营养教育部重点实验室, 动物抗病营养与饲料农业农村部重点实验室, 动物抗病营养四川省重点实验室, 成都 611130
弓浩杰(1997-),男,山西太原人,硕士,动物营养与饲料科学专业。E-mail:ghjie2904@163.com

收稿日期: 2021-08-23

  网络出版日期: 2022-03-14

基金资助

国家自然科学基金面上项目(31872792);四川省科技厅国际合作项目(2019YFH0062);四川省蛋鸡产业链项目(2018NZ20009)

Oxidative Stress Injury of Mouse Ovarian Granulosa Cells and Its Effects on Silencing Information Regulator 2-Related Enzyme 1 Signaling Pathway

  • GONG Haojie ,
  • DING Xuemei ,
  • BAI Shiping ,
  • ZENG Qiufeng ,
  • ZHANG Keying ,
  • WANG Jianping
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  • Sichuan Key Laboratory of Animal Disease-Resistance Nutrition, Key Laboratory of Animal Disease-Resistance Nutrition and Feed, Ministry of Agriculture and Rural Affairs, Key Laboratory of Animal Disease-Resistance Nutrition, Ministry of Education, Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, China

Received date: 2021-08-23

  Online published: 2022-03-14

摘要

本试验旨在通过探究不同浓度过氧化氢(H2O2)对小鼠卵巢颗粒细胞(MOGC)氧化应激损伤的影响,以构建MOGC氧化应激模型,并探究氧化应激损伤对沉默信息调节因子2相关酶1(SIRT1)信号通路的影响。试验选用MOGC,采用单因素试验设计,分别用0(对照组)、100、200、400和800 μmol/L H2O2处理24 h,测定细胞凋亡、细胞内活性氧(ROS)生成量、相关抗氧化酶活性和丙二醛(MDA)含量以及凋亡相关基因表达。结果表明:1)与对照组相比,不同浓度H2O2组细胞活力均显著降低(P<0.05),当H2O2浓度大于200 μmol/L并且作用时间超过24 h时,细胞活力低于50%。2)与对照组相比,不同浓度H2O2组细胞凋亡率显著提高(P<0.05)。3)与对照组相比,200、400和800 μmol/L H2O2组细胞内ROS生成量显著提高(P<0.05)。4)与对照组相比,400 μmol/L H2O2组细胞内超氧化物歧化酶活性显著降低(P<0.05);400、800 μmol/L H2O2组细胞内过氧化氢酶、谷胱甘肽过氧化物酶(GSH-Px)活性和还原型谷胱甘肽含量显著降低(P<0.05),细胞内MDA含量显著提高(P<0.05);200 μmol/L H2O2组细胞内GSH-Px活性显著降低(P<0.05),细胞内MDA含量显著提高(P<0.05)。5)与对照组相比,800 μmol/L H2O2组细胞中B细胞淋巴瘤-2相互作用的细胞死亡中介物(Bim)、半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)和半胱氨酸天冬氨酸蛋白酶-9(Caspase-9)mRNA相对表达量显著提高(P<0.05),细胞中B细胞淋巴瘤-xL(Bcl-xL)mRNA相对表达量显著降低(P<0.05);400 μmol/L H2O2组细胞中Caspase-3 mRNA相对表达量显著提高(P<0.05),细胞中Bcl-xL mRNA相对表达量显著降低(P<0.05);此外,200、400和800 μmol/L H2O2组细胞中SIRT1 mRNA相对表达量显著降低(P<0.05),而细胞中叉头盒蛋白O1(FoxO1)、p53 mRNA相对表达量显著提高(P<0.05)。结果提示,200 μmol/L H2O2处理24 h为建立MOGC氧化应激模型的理想条件,并且SIRT1-FoxO1/p53通路参与了H2O2诱导的MOGC凋亡。

本文引用格式

弓浩杰 , 丁雪梅 , 白世平 , 曾秋凤 , 张克英 , 王建萍 . 小鼠卵巢颗粒细胞氧化应激损伤及对沉默信息调节因子2相关酶1信号通路的影响[J]. 动物营养学报, 2022 , 34(3) : 1942 -1954 . DOI: 10.3969/j.issn.1006-267x.2022.03.053

Abstract

The aim of this study was to explore the effects of different concentrations of hydrogen peroxide (H2O2) on oxidative stress injury of mouse ovarian granulosa cells (MOGC), to establish the MOGC oxidative stress model, and to explore the effects of oxidative stress injury on silencing information regulator 2-related enzyme 1 (SIRT1) signaling pathway. A single factor design was adopted and MOGC were treated with 0 (control group), 100, 200, 400 and 800 μmol/L H2O2 for 24 h, respectively, and the apoptosis, intracellular reactive oxygen species (ROS) production, related antioxidant enzyme activities, malondialdehyde (MDA) content and apoptosis-related gene expression were determined. The results showed as follows:1) compared with the control group, the cell viability in different concentrations of H2O2 groups was significantly decreased (P<0.05), and the cell viability was lower than 50% when H2O2 concentration was more than 200 μmol/L and treated for more than 24 h. 2) Compared with the control group, the apoptosis rate in different concentrations of H2O2 groups was significantly increased (P<0.05). 3) Compared with the control group, the intracellular ROS production in 200, 400 and 800 μmol/L H2O2 groups was significantly increased (P<0.05). 4) Compared with control group, the intracellular superoxide dismutase activity in 400 μmol/L H2O2 group was significantly decreased (P<0.05); the intracellular catalase and glutathione peroxidase (GSH-Px) activities and reduced glutathione content in 400 and 800 μmol/L H2O2 groups were significantly decreased (P<0.05), while the intracellular MDA content was significantly increased (P<0.05); the intracellular GSH-Px activity in 200 μmol/L H2O2 group was significantly decreased (P<0.05), and the intracellular MDA content was significantly increased (P<0.05). 5) Compared with the control group, the intracellular mRNA relative expression levels of B-cell lymphoma-2 interacting mediator of cell death (Bim), cysteine-aspartic protease-3 (Caspase-3) and cysteine-aspartic protease-9 (Caspase-9) in 800 μmol/L H2O2 group were significantly increased (P<0.05), while the intracellular mRNA relative expression level of B-cell lymphoma-xL (Bcl-xL) was significantly decreased (P<0.05); the intracellular mRNA relative expression levels of Caspase-3 in 400 μmol/L H2O2 group was significantly increased (P<0.05), while the intracellular mRNA relative expression level of Bcl-xL in 400 μmol/L H2O2 group was significantly decreased (P<0.05); in addition, the intracellular mRNA relative expression level of SIRT1 in 200, 400 and 800 μmol/L H2O2 groups was significantly decreased (P<0.05), while the intracellular mRNA relative expression levels of Forkhead box protein O1 (FoxO1) and p53 were significantly increased (P<0.05). The results suggest that 200 μmol/L H2O2 treatment for 24 h is the ideal condition for establishing MOGC oxidative stress model, and SIRT1-FoxO1/p53 pathway is involved in H2O2-induced MOGC apoptosis.

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