姜黄素通过Nrf2信号通路对H2O2诱导奶牛乳腺上皮细胞氧化应激的缓解

doi:10.3864/j.issn.0578-1752.2021.08.017

摘要/Abstract

摘要：

【目的】验证姜黄素是否能够通过转录因子E2相关因子2（Nuclear factor E2-related factor 2,Nrf2）信号通路减轻H₂O₂诱导的奶牛乳腺上皮细胞的氧化应激,为防治围产期奶牛代谢紊乱导致的氧化损伤提供理论依据。【方法】培养奶牛乳腺上皮细胞（MAC-T）,用500 μmol·L^-1H₂O₂处理MAC-T细胞24 h后加入不同浓度的姜黄素（0、5、15或30 μmol·L^-1）处理3 h;MAC-T细胞转染Nrf2 siRNA 48 h后,用500 μmol·L ^-1 H₂O₂处理奶牛乳腺上皮细胞系MAC-T细胞24 h后加入不同浓度的姜黄素（0、5、15或30 μmol·L^-1）处理3 h。采用实时定量PCR（Quantitative real-time PCR,qPCR）、蛋白免疫印迹（Western blot,WB）和试剂盒等方法,检测Nrf2的蛋白表达量及下游靶基因NAD（P）H醌氧化还原酶1（NAD(P)H quinone oxidoreductase 1,NQO1）和血红素加氧酶1（Heme oxygenase 1,HO-1）的mRNA及蛋白表达量和超氧化物歧化酶（Superoxide dismutase,SOD）、谷胱甘肽过氧化物酶（Glutathione peroxidase,GSH-Px）、过氧化氢酶（Catalase,CAT）和丙二醛（Malondialdehyde,MDA）等氧化应激相关指标的活性及含量。【结果】（1）H₂O₂处理组MAC-T细胞中MDA含量显著高于对照组（P<0.01）,而CAT、SOD和GSH-Px的活性显著低于对照组（P<0.01）。15 μmol·L^-1姜黄素+H₂O₂共同处理组和30 μmol·L^-1姜黄素+H₂O₂共同处理组MAC-T细胞中MDA的含量显著低于H₂O₂处理组（P<0.01）,而CAT、SOD和GSH-Px的活性显著高于H₂O₂处理组（P<0.05,P<0.01）。（2）H₂O₂处理组总Nrf2蛋白水平显著低于对照组（P<0.01）,H₂O₂处理组Nrf2下游基因HO-1和NQO1的mRNA及蛋白表达量也显著低于对照组（P<0.01）。姜黄素处理组总Nrf2蛋白表达水平显著高于对照组（P<0.01）,姜黄素处理组HO-1和NQO1的mRNA及蛋白表达量也显著高于对照组（P<0.01）。姜黄素+H₂O₂处理组总Nrf2蛋白表达水平显著高于H₂O₂处理组（P<0.01）,姜黄素+H₂O₂处理组HO-1和NQO1的mRNA及蛋白表达量也显著高于H₂O₂处理组（P<0.01）。（3）si-Nrf2组与对照组相比,Nrf2的mRNA表达水平显著降低（P<0.01）。si-Control+H₂O₂+姜黄素组与si-Control+H₂O₂组相比,MDA的含量显著降低（P<0.01）,而CAT、SOD和GSH-Px的活性显著升高（P<0.01）。si-Nrf2+H₂O₂+姜黄素处理组与si-Control+H₂O₂+姜黄素组相比,MDA的含量显著升高（P<0.01）,而CAT、SOD和GSH-Px活性显著降低（P<0.01）。【结论】姜黄素可以通过增加Nrf2的表达,诱导下游抗氧化分子的转录从而缓解H₂O₂诱导奶牛乳腺上皮细胞的氧化应激。本研究为防治围产期奶牛代谢紊乱导致的奶牛乳腺上皮细胞氧化损伤提供理论依据。

关键词: 姜黄素, 奶牛, 乳腺上皮细胞, 氧化应激, Nrf2

Abstract:

【Objective】The aim of this study was to investigate whether curcumin alleviated oxidative stress in bovine mammary epithelial cells induced by H₂O₂ via the nuclear factor E2-related factor 2 (Nrf2) signaling pathway. 【Method】Bovine mammary epithelial cells MAC-T cells were treated with H₂O₂ (500 μmol·L^-1) for 24 h, followed by incubation of curcumin (0, 5, 15 or 30 μmol·L ^-1) for an additional 3 h; the MAC-T cells were transfected with Nrf2 siRNA for 48 h, followed by incubation of H₂O₂ (500 μmol·L^-1) for 24 h and then treated with curcumin (30 μmol·L ^-1) for an additional 3 h. Real-time quantitative PCR (qPCR), Western blot (WB) were used to detect the protein abundance of Nrf2 and the mRNA and protein abundance of NAD(P)H quinone oxidoreductase 1 (NQO1) and Heme oxygenase 1 (HO-1), the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and the content of malondialdehyde (MDA). 【Result】 (1) Compared with the control group, H₂O₂ treatment significantly increased MDA content (P<0.01), while it decreased the activity of SOD, GSH-Px, and CAT (P<0.01). Compared with the H₂O₂ group, the content of MDA in MAC-T cells in the 15 μmol·L^-1 or 30 μmol·L ^-1 curcumin with H₂O₂ treatment groups was significantly decreased (P<0.01), while the activity of SOD, GSH-Px, and CAT were significantly increased (P<0.05, P<0.01). (2) Compared with the control group, H₂O₂ treatment significantly decreased Nrf2 protein abundance (P<0.01) and decreased HO-1 and NQO1 their mRNA and protein abundance (P<0.01). However, compared with the control group, curcumin treatment significantly increased Nrf2 protein abundance (P<0.01) and increased HO-1 and NQO1 mRNA and protein abundance (P<0.01). Compared with the H₂O₂ group, H₂O₂+curcumin treatment significantly increased Nrf2 protein abundance (P<0.01) and increased HO-1 and NQO1 mRNA and protein abundance (P<0.01). (3) Compared with the control group, si-Nrf2 treatment group significantly decreased Nrf2 mRNA abundance (P<0.01). Compared with si-Control+H₂O group, the content of MDA was significantly decreased in si-Control+H₂O₂+curcumin treatment group (P<0.01), while the activity of SOD, GSH-Px, and CAT were significantly increased (P<0.01). However, compared with si-Control+H₂O₂+curcum group, the content of MDA was significantly increased in si-Nrf2+H₂O₂+curcumin treatment group (P<0.01), while the activity of SOD, GSH-Px, and CAT were significantly decreased (P<0.01). 【Conclusion】These results suggested that curcumin could alleviate the oxidative stress of bovine mammary epithelial cells induced by H₂O₂ through increasing the expression of Nrf2 and inducing the transcription of downstream antioxidant molecules. This study provided a theoretical basis for the prevention and treatment of oxidative damage of mammary epithelial cells caused by metabolic disorderd in perinatal dairy cows.

Key words: curcumin, bovine, mammary epithelial cells, oxidative stress, Nrf2

姜春晖,孙旭东,唐燕,罗胜缤,徐闯,陈媛媛. 姜黄素通过Nrf2信号通路对H₂O₂诱导奶牛乳腺上皮细胞氧化应激的缓解[J]. 中国农业科学, 2021, 54(8): 1787-1794.

JIANG ChunHui,SUN XuDong,TANG Yan,LUO ShengBin,XU Chuang,CHEN YuanYuan. Curcumin Alleviates H₂O₂-Induced Oxidative Stress in Bovine Mammary Epithelial Cells Via the Nrf2 Signaling Pathway[J]. Scientia Agricultura Sinica, 2021, 54(8): 1787-1794.

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参考文献 30

[1]	KAWASHIMA C, MUNAKATA M, SHIMIZU T, MIYAMOTO A, KIDA K, MATSUI M. Relationship between the degree of insulin resistance during late gestation and postpartum performance in dairy cows and factors that affect growth and metabolic status of their calves. Journal of Veterinary Medical Science, 2016,78(5):739-745. doi: 10.1292/jvms.15-0583
[2]	GROSS J J, SCHWARZ F J, EDER K, Dorland H A, Bruckmaier R M. Liver fat content and lipid metabolism in dairy cows during early lactation and during a mid-lactation feed restriction. Journal of Dairy Science, 2013,96(8):5008-5017. doi: 10.3168/jds.2012-6245
[3]	GROSS J J, BRUCKMAIER R M. Invited review: Metabolic challenges and adaptation during different functional stages of the mammary gland in dairy cows: Perspectives for sustainable milk production. Journal of Dairy Science, 2019,102(4):2828-2843. doi: 10.3168/jds.2018-15713 pmid: 30799117
[4]	SUN X D, JIA H D, XU Q S, ZHAO C X, XU C. Lycopene alleviates H₂O₂-induced oxidative stress, inflammation and apoptosis in bovine mammary epithelial cells via the NFE2L2 signaling pathway. Food & Function, 2019,10(10):6276-6285. pmid: 31576860
[5]	NGUYEN T, NIOI P, PICKETT C B. The Nrf2-antioxidant response element signaling pathway and its activation by oxidative stress. Journal of Biological Chemistry, 2009,284(20):13291-13295. doi: 10.1074/jbc.R900010200
[6]	张倩. 金丝桃苷对Nrf2-ARE途径的影响及其对肝细胞氧化应激损伤的保护作用[D]. 重庆: 西南大学, 2014.
	ZHANG Q. Effect of hyperoside on Nrf2-ARE pathway and its protective effect on oxidative stress injury of hepatocytes[D]. Chongqing: Southwest University, 2014. (in Chinese)
[7]	MA Y F, WU Z H, GAO M, LOOR J J. Nuclear factor erythroid 2-related factor 2 antioxidant response element pathways protect bovine mammary epithelial cells against H₂O₂-induced oxidative damage in vitro. Journal of Dairy Science, 2018,101(6):5329-5344. doi: 10.3168/jds.2017-14128 pmid: 29573798
[8]	宋志华, 毕小宝. 姜黄素对脂多糖诱导腹腔巨噬细胞释放炎症因子的影响. 现代医院, 2014,14(10):7-8.
	SONG Z H, BI X B. Effect of curcumin on lipopolysaccharide induced release of inflammatory factors from peritoneal macrophages. Modern Hospital, 2014,14(10):7-8. (in Chinese)
[9]	SURH Y J, CHUN K S. Cancer chemopreventive effects of curcumin. Advances in Experimental Medicine & Biology, 2007,595:149-172. doi: 10.1007/978-0-387-46401-5_5 pmid: 17569209
[10]	MOTTERLINI R, FORESTI R, BASSI R, GREEN C J. Curcumin, an antioxidant and anti-inflammatory agent, induces heme oxygenase-1 and protects endothelial cells against oxidative stress. Free Radical Biology & Medicine, 2000,28(8):1303-1312. doi: 10.1016/s0891-5849(00)00294-x pmid: 10889462
[11]	周代营, 杜志云, 郑希, 张焜. 姜黄素的生物学活性研究进展. 广东化工, 2013,40(19):91-92.
	ZHOU D Y, DU Z Y, ZHENG X, ZHANG K. Research progress in the biological activity of curcumin. Guangdong Chemical Industry, 2013,40(19):91-92. (in Chinese)
[12]	RUBY A J, KUTTAN G, BABU K V D, RAJASEKHARAN K N, KUTTAN R. Anti-tumour and antioxidant activity of natural curcuminoids. Cancer Letters, 1995,94(1):79-83. doi: 10.1016/0304-3835(95)03827-j pmid: 7621448
[13]	PIPER J T, SINGHAL S S, SALAMEH M S, TOEMAN R T, AWASTHI Y C, AWASTHI S. Mechanisms of anticarcinogenic properties of curcumin: The effect of curcumin on glutathione linked detoxification enzymes in rat liver. International Journal of Biochemistry and Cell Biology, 1998,30(4):445-465. doi: 10.1016/s1357-2725(98)00015-6 pmid: 9675878
[14]	SOETIKNO V, SARI F R, LAKSHMANAN A P, ARUMUGAM S, HARIMA M, SUZUKI K, KAWACHI H, WATANABE K. Curcumin alleviates oxidative stress, inflammation, and renal fibrosis in remnant kidney through the Nrf2-keap1 pathway. Molecular Nutrition & Food Research, 2013,57(9):1649-1659. doi: 10.1002/mnfr.201200540 pmid: 23174956
[15]	宋永周, 关健, 李明, 马维, 袁鹤, 王斌, 童九辉. Nrf2介导姜黄素对软骨细胞氧化应激损伤的保护作用. 中国组织工程研究, 2015,19(51):8218-8222.
	SONG Y Z, GUAN J, LI M, MA W, YUAN H, WANG B, TONG J H. Nrf2 mediates the protective effect of curcumin on oxidative stress injury of chondrocytes. Chinese Journal of Tissue Engineering Research, 2015,19(51):8218-8222. (in Chinese)
[16]	郑媛媛, 刘震雄, 赵曙光, 王旭霞, 崔曼丽, 闻勤生. 姜黄素对氧化应激中肝星状细胞活化及细胞外基质分泌的影响. 胃肠病学和肝病学杂志, 2011(4):22-25.
	ZHENG Y Y, LIU Z X, ZHAO S G, WANG X X, CUI M L, WEN Q S. Effect of curcumin on hepatic stellate cell activation and extracellular matrix secretion during oxidative stress. Chinese Journal of Gastroenterology and Hepatology, 2011(4):22-25. (in Chinese)
[17]	LOOR J J, BIONAZ M, DRACKLEY J K. Systems physiology in dairy cattle: nutritional genomics and beyond. Annual Review of Animal Biosciences, 2012,1(1):365-392. doi: 10.1146/annurev-animal-031412-103728
[18]	Castillo C, Hernandez J, Bravo A, Lopez-Alonso M, Pereira V, Benedito J L. Oxidative status during late pregnancy and early lactation in dairy cows. Veterinary Journal, 2005,169(2):286-292. doi: 10.1016/j.tvjl.2004.02.001
[19]	HOLBEN D H, SMITH A M. The diverse role of selenium within selenoproteins: a review. Journal of the American Dietetic Association, 1999,99(7):836-843. doi: 10.1016/S0002-8223(99)00198-4 pmid: 10405682
[20]	乔青青, 任顺成, 吕真真. 姜黄色素稳定性研究. 江西食品工业, 2011(4):46-48.
	QIAO Q Q, REN S C, LÜ Z Z. Study on stability of curcumin. Jiangxi Food Industry, 2011(4):46-48. (in Chinese)
[21]	MENON V P, SUDHEER A R. Antioxidant and anti-inflammatory properties of curcumin. Advances in Experimental Medicine & Biology, 2007,595(1):105-125.
[22]	NITURE S K, KASPAR J W, SHEN J, JAISWAL A K. Nrf2 signaling and cell survival. Toxicology & Applied Pharmacology, 2010,244(1):37-42. doi: 10.1016/j.taap.2009.06.009 pmid: 19538984
[23]	COHLY H H P, TAYLOR A, ANGEL M F, SALAHUDEEN A K. Effect of turmeric, turmerin and curcumin on H₂O₂-induced renal epithelial (LLC-PK1) cell injury. Free Radical Biology & Medicine, 1998,24(1):49-54. doi: 10.1016/S0891-5849(97)00140-8
[24]	林健, 文富强. Nrf2-Keap1抗氧化系统与慢性阻塞性肺疾病. 西部医学, 2009,21(9):1590-1592.
	LIN J, WEN F Q. Nrf2-keap1 antioxidant system and chronic obstructive pulmonary disease. Western Medicine, 2009,21(9):1590-1592. (in Chinese)
[25]	HAYES J D, MCMAHON M. Molecular basis for the contribution of the antioxidant responsive element to cancer chemoprevention. Cancer Letters, 2001,174(2):103-113. doi: 10.1016/s0304-3835(01)00695-4 pmid: 11689285
[26]	KANSANEN E, KUOSMANEN S M, LEINONEN H, LEVONEN A. The Keap1-Nrf2 pathway: Mechanisms of activation and dysregulation in cancer. Redox Biology, 2013,1(1):45-49. doi: 10.1016/j.redox.2012.10.001
[27]	SCHOGOR A L B, PALIN M F, DOS SANTOS G T, BENCHAAR C, LACASSE P, PETIT H V. Mammary gene expression and activity of antioxidant enzymes and oxidative indicators in the blood, milk, mammary tissue and ruminal fluid of dairy cows fed flax meal. British Journal of Nutrition, 2013,110(10):1743-1750. doi: 10.1017/S0007114513001220
[28]	BALOGUN E, HOQUE M, GONG P, KILLEEN E, GREEN C, FORESTI R, ALAM J, MOTTERLINI R. Curcumin activates the haem oxygenase-1 gene via regulation of Nrf2 and the antioxidant- responsive element. Biochemical Journal, 371(3):887-895. doi: 10.1042/bj20021619
[29]	黎珊珊, 徐兆发. 姜黄素激活Nrf2-ARE通路抑制镉肾毒性的研究进展. 环境与健康杂志, 2014 (1):94-97.
	LI S S, XU Z F. Research progress of curcumin activating Nrf2-ARE pathway to inhibit cadmium nephrotoxicity. Journal of Environment and Health, 2014(1):94-97. (in Chinese)
[30]	展平. 姜黄素激活Nrf2系统缓解线粒体损伤和胰岛素抵抗作用机制研究[D]. 福州: 福建中医药大学, 2017. doi: 10.1186/1749-8546-6-27 pmid: 21777476
	ZHAN P. Study on the mechanism of curcumin-activates Nrf2 System to Alleviate mitochondrial damage and Insulin Resistance[D]. Fuzhou: Fujian University of Traditional Chinese Medicine, 2017. (in Chinese) doi: 10.1186/1749-8546-6-27 pmid: 21777476

基因 Gene	引物序列 Primer sequences (5′-3′)	产物大小 Length (bp)
Nrf2	F GCCCTCACTGGATAAAGAA	202
Nrf2	R CATGCCGTTGCTGGTAC	202
NQO1	F AACCAACAGACCAGCCAATC	154
NQO1	R CACAGTGACCTCCCATCCTT	154
HO-1	F CAAGGAGAACCCCGTCTACA	225
HO-1	R CCAGACAGGTCTCCCAGGTA	225
β-Actin	F GCCCTGAGGCTCTCTTCCA	101
β-Actin	R GCGGATGTCGACGTCACA	101
GAPDH	F TCTTCACTACCATGGAGAAGG	197
GAPDH	R TCATGGATGACCTTGGCCAG	197

姜黄素通过Nrf2信号通路对H₂O₂诱导奶牛乳腺上皮细胞氧化应激的缓解

Curcumin Alleviates H₂O₂-Induced Oxidative Stress in Bovine Mammary Epithelial Cells Via the Nrf2 Signaling Pathway

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