Nuclear factor erythroid 2-related factor 2 antioxidant response element pathways protect bovine mammary epithelial cells against H2O2-induced oxidative damage in vitro
Nuclear factor erythroid 2-related factor 2 antioxidant response element pathways protect bovine mammary epithelial cells against H2O2-induced oxidative damage in vitro
复制标题
核因子红细胞2相关因子2抗氧化反应元件通路保护牛乳腺上皮细胞免受H2O2诱导的体外氧化损伤
DOI：
10.3168/jds.2017-14128
复制
发表时间：
2018-06-01
影响因子：
3.5
通讯作者：
Loor, J. J.
中科院分区：
文献类型：
--
作者：
Ma, Y. F.;Wu, Z. H.;Loor, J. J.
本实验旨在研究核因子（红细胞衍生2）样因子2（NFE 2L 2，Nrf 2）抗氧化反应元件（ARE）通路在保护牛乳腺上皮细胞（BMEC）免受H2 O2诱导的氧化应激损伤中的作用。转染NFE 2L 2小干扰RNA（siRNA）干扰或pCMV 6-XL 5-NFE 2L 2质粒片段以独立地下调或上调NFE 2L 2的表达。将分离的BMEC一式三份暴露于H2 O2（600 μ M）6小时以诱导氧化应激，然后用乱序siRNA、NFE 2L 2-siRNA、pCMV 6-XL 5和pCMV 6-XL 5-NFE 2L 2瞬时转染。检测细胞增殖、凋亡和坏死率、抗氧化酶活性、活性氧（ROS）和丙二醛（MDA）的产生、NFE 2L 2及其下游靶基因的蛋白和mRNA表达以及ARE的荧光活性。结果显示，与对照组相比，转染NFE 2L 2-siRNA 3的BMEC在无H2 O2或有H2 O2的情况下增殖率分别降低了9%和65%。这些细胞的凋亡和坏死率分别是对照组的27倍和3.5倍。与此相反，转染的pCMV 6-XL 5-NFE 2L 2的增殖率分别比对照组高64.3%或低17%。与对照组相比，H2 O2的细胞凋亡率低1.8倍。与对照组相比，转染pCMV 6-XL 5-NFE 2L 2的细胞中ROS和MDA的产生以及超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）、过氧化氢酶（CAT）和谷胱甘肽-S-转移酶（GST）的活性显著增加。与对照组相比，转染pCMV 6-XL 5-NFE 2L 2的细胞经H2 O2孵育后，细胞内ROS和MDA的产生量、CAT和GSH-Px的活性显著增加，而SOD和GST的活性显著降低。与对照组相比，转染NFE 2L 2-siRNA 3的细胞无论是否存在H2 O2，其ROS和MDA的产生量以及SOD、CAT、GSH-Px和GST的活性均明显降低。与对照孵育相比，用pCMV 6-XL 5-NFE 2L 2转染或不转染H2 O2的细胞具有显著更高的NFE 2L 2、血红素加氧酶-1（HMOX-1）、NADH醌氧化还原酶1、谷氨酸半胱氨酸连接酶催化亚基和谷氨酰胱氨酸连接酶调节亚基的蛋白和mRNA表达。与对照孵育相比，用NFE 2L 2-siRNA 3转染的细胞在无H2 O2或有H2 O2的情况下具有显著较低的NFE 2L 2、HMOX-1、NADH醌氧化还原酶1、谷氨酰胱氨酸连接酶调节亚基和谷氨酸-半胱氨酸连接酶催化亚基的蛋白和mRNA表达。此外，HMOX-1的表达是对照组的5.3倍。总体而言，结果表明NFE 2L 2通过控制HMOX-1在NFE 2L 2-ARE途径中起重要作用。体内相关机制值得进一步研究。
The experiment was conducted to determine the role of nuclear factor (erythroid-derived 2)-like factor 2 (NFE2L2, formerly Nrf2) antioxidant response element (ARE) pathway in protecting bovine mammary epithelial cells (BMEC) against H2O2-induced oxidative stress injury. An NFE2L2 small interfering RNA (siRNA) interference or a pCMV6-XL5-NFE2L2 plasmid fragment was transfected to independently downregulate or upregulate expression of NFE2L2. Isolated BMEC in triplicate were exposed to H2O2 (600 mu M) for 6 h to induce oxidative stress before transient transfection with scrambled siRNA, NFE2L2-siRNA, pCMV6-XL5, and pCMV6-XL5-NFE2L2. Cell proliferation, apoptosis and necrosis rates, antioxidant enzyme activities, reactive oxygen species (ROS) and malondialdehyde (MDA) production, protein and mRNA expression of NFE2L2 and downstream target genes, and fluorescence activity of ARE were measured. The results revealed that compared with the control, BMEC transfected with NFE2L2-siRNA3 had proliferation rates that were 9 or 65% lower without or with H2O2, respectively. These cells also had apoptosis and necrosis rates that were 27 and 3.5 times greater with H2O2 compared with the control group, respectively. In contrast, transfected pCMV6-XL5-NFE2L2 had proliferation rates that were 64.3% greater or 17% lower without or with H2O2 compared with the control group, respectively. Apoptosis rates were 1.8 times lower with H2O2 compared with the control. In addition, compared with the control, production of ROS and MDA and activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and glutathione-S-transferase (GST) increased markedly in cells transfected with pCMV6- XL5-NFE2L2 and without H2O2. However, compared with the control, production of ROS and M DA and activity of CAT and GSH-Px increased markedly, whereas activities of SOD and GST decreased in cells transfected with pCMV6-XL5-NFE2L2 and incubated with H2O2. Compared with the control, cells transfected with NFE2L2-siRNA3 with or without H2O2 had lower production of ROS and MDA and activity of SOD, CAT, GSH-Px, and GST. Cells transfected with pCMV6-XL5-NFE2L2 with or without H2O2 had markedly higher protein and mRNA expression of NFE2L2, heme oxygenase-1 (HMOX-1), NADH quinone oxidoreductase 1, glutamate cysteine ligase catalytic subunit, and glutamyl cystine ligase modulatory subunit compared with the control incubations. Cells transfected with NFE2L2-siRNA3 without or with H2O2 had markedly lower protein and mRNA expression of NFE2L2, HMOX-1, NADH quinone oxidoreductase 1, glutamyl cystine ligase modulatory subunit, and glutamate-cysteine ligase catalytic subunit compared with the control incubations. In addition, expression of HMOX-1 was 5.3-fold greater with H2O2 compared with the control. Overall, results indicate that NFE2L2 plays an important role in the NFE2L2-ARE pathway via the control of HMOX-1. The relevant mechanisms in vivo merit further study.