活化Nrf2缓解热应激致肉鸡心肌细胞氧化损伤的研究

doi:10.16431/j.cnki.1671-7236.2022.09.008

Abstract

Abstract: 【Objective】 The study was aimed to investigate the alleviating effect of nuclear factor erythroid 2-related factor 2 (Nrf2) on oxidative damage induced by heat stress of broiler cardiomyocytes.【Method】 Primary broiler cardiomyocytes were purified through different-speed adherence method and chemical purification method.The cardiomyocytes were randomly divided into control group, heat stress model group (HS group) and Nrf2 activator pre-treatment heat stress group (TBHQ+HS group).The cardiomyocytes of the control group were cultured without any treatment, the cardiomyocytes of the HS group were treated in a high-temperature (43 ℃) incubator for 2 h, the cardiomyocytes of the TBHQ+HS group were pretreated with 50 μmol/L tert-butylhydroquinone (TBHQ) for 12 h and then heat stress treatment was carried out.The relative viability of the cells was measured by MTT method.The activity of superoxide dismutase (SOD), the content of malondialdehyde (MDA) and the activity of lactate dehydrogenase (LDH) in cell culture medium were detected by colorimetry method.Real-time PCR was used to detect the mRNA expression levels of Nrf2, heme oxygenase-1 (HO-1), glutamate cysteine ligase (GCLC) and NAD(P)H:quinone oxidoreductase 1 (NQO1).The expressions of Nrf2 total protein and HO-1 protein were detected by Western blotting.【Result】 Compared with the control group, the morphology of primary cardiomyocytes was changed and vacuolar reticular structure was shown in heat stress group, the relative activity and SOD activity of cardiomyocytes were decreased extremely significantly (P<0.01), the content of MDA was increased extremely significantly (P<0.01), and the activity of LDH in cell culture medium was increased extremely significantly (P<0.01).The difference of NQO1 and GCLC mRNA expression in the downstream of Nrf2/antioxidant response element (ARE) signal pathway was not significant (P>0.05), the expression of Nrf2 and HO-1 mRNA was remarkably increased (P<0.05), but there was no difference in Nrf2 total protein and HO-1 protein expression (P>0.05). In TBHQ pre-treatment heat stress group, compared with the heat stress group, the vacuolar reticular structure reduced in cardiomyocytes and the relative activity of cardiomyocytes was raised significantly (P<0.05), the activity of SOD was increased extremely significantly (P<0.01), the content of MDA was diminished meaningly (P<0.05), the activity of LDH enzyme in cell culture medium was decreased extremely significantly (P<0.01).The expression of NQO1 and GCLC mRNA in the downstream of Nrf2/ARE signal pathway was increased significantly(P<0.05), the expression of Nrf2 mRNA and total protein was increased significantly (P<0.05), besides, the expression of HO-1 mRNA and protein were all raised extremely significantly (P<0.01).【Conclusion】 Activation of Nrf2 could up-regulate the transcription of antioxidant genes and the expression of protein in the downstream of Nrf2 signaling pathway, improve the antioxidant capacity of heat stressed broiler cardiomyocytes, and alleviate the oxidative damage induced by heat stress.It suggested that Nrf2 might be an effective molecular target for broiler cardiomyocytes to resist oxidative damage induced by heat stress.

Key words: heat stress; broilers; cardiomyocytes; Nrf2; HO-1

CLC Number:

Q494

WU Yanan, SHI Yuxiang, ZHANG Yongying, LIU Guanhui, SONG Weiguang, ZHONG Cuihong, WANG Yongxia. Study on Nrf2 Activation Alleviating Heat Stress-Induced Oxidative Damage in Broiler Cardiomyocytes[J]. China Animal Husbandry & Veterinary Medicine, 2022, 49(9): 3343-3352.

References

[1] 徐维成.肉鸡腹水综合症的病因及防治[J].吉林畜牧兽医, 2022, 43(2):47-48. XU W C.Etiology and prevention of ascites syndrome in broilers[J] .Jilin Animal Husbandry and Veterinary Medicine, 2022, 43(2):47-48.(in Chinese)
[2] BASAKI M, TABANDEH M R, AMINLARI M, et al.Sequence and expression analysis of cardiac ryanodine receptor 2 in broilers that died from sudden death syndrome[J].Avian Pathology, 2019, 48(5):444-453.
[3] SHAO-SHUAI Z, HONG-GUANG S, YING Z, et al.Effects of sustained cold and heat stress on energy intake, growth and mitochondrial function of broiler chickens[J].Journal of Integrative Agriculture, 2016, 15(10):2336-2342.
[4] CHISTIAKOV D A, SHKURAT T P, MELNICHENKO A A, et al.The role of mitochondrial dysfunction in cardiovascular disease:A brief review[J].Annals of Medicine, 2018, 50(2):121-127.
[5] QIAN L, SONG X, REN H, et al.Mitochondrial mechanism of heat stress-induced injury in rat cardiomyocyte[J].Cell Stress and Chaperones, 2004, 9(3):281-293.
[6] 陈冬玲.HO-1过表达保护缺氧复氧诱导的H9c2细胞线粒体功能障碍[D].武汉:武汉大学, 2016. CHEN D L.HO-1 protects against hypoxia/reoxygenation-induced mitochondrial dysfunction through autophagic cytoprotection in H9c2 cardiomyocytes[D].Wuhan:Wuhan University, 2016.(in Chinese)
[7] 陈洪博, 段滇宁, 王华.银杏叶提取物对热应激致鸡心肌细胞氧化损伤的保护作用研究[J].中国畜牧兽医, 2018, 45(1):100-105. CHEN H B, DUAN D N, WANG H.Protective effects of ginkgo biloba extract on oxidative damage induced by heat stress in cardiomyocytes of chicken[J] .China Animal Husbandry & Veterinary Medicine, 2018, 45(1):100-105.(in Chinese)
[8] 韩佳良, 刘建新, 刘红云.热应激对奶牛泌乳性能的影响及其机制[J].中国农业科学, 2018, 51(16):3162-3170. HAN J L, LIU J X, LIU H Y.Effect of heat stress on lactation performance in dairy cows[J] .Scientia Agricultura Sinica 2018, 51(16):3162-3170.(in Chinese)
[9] 董馨文, 辛明, 郭秀丽.Nrf2对心肌损伤保护作用及机制的研究进展[J].生命的化学, 2016, 36(1):21-26. DONG X W, XIN M, GUO X L.Advances in the protection and mechanism of Nrf2 on myocardial damage[J] .Chemistry of Life, 2016, 36(1):21-26.(in Chinese)
[10] XU J J, CUI J, LIN Q, et al.Protection of the enhanced Nrf2 deacetylation and its downstream transcriptional activity by SIRT1 in myocardial ischemia/reperfusion injury[J].International Journal of Cardiology, 2021, 34(2):82-93.
[11] 陆灿强, 舒邓群, 臧一天.热应激诱导畜禽氧化应激、热休克反应与免疫和炎症反应的机制及营养调控措施[J].动物营养学报, 2021, 33(6):3115-3124. LU C Q, SHU D Q, ZANG Y T.Mechanism and nutritional regulation measures of oxidative stress, heat shock response, immune and inflammatory response induced by heat stress in livestock and poultry[J] .Chinese Journal of Animal Nutrition, 2021, 33(6):3115-3124.(in Chinese)
[12] 窦彩霞, 王倩, 安建勇, 等.Keap1-Nrf2/ARE信号通路及其在畜禽抗氧化中的研究进展[J].中国畜牧兽医, 2019, 46(9):2567-2574. DOU C X, WANG Q, AN J Y, et al.The reasearch progress of Keap1-Nrf2/ARE signaling pathway and Its role in the antioxidation of livestock and poultry[J] .China Animal Husbandry & Veterinary Medicine, 2019, 46(9):2567-2574.(in Chinese)
[13] ZHANG J F, BAI K W, SU W P, et al.Curcumin attenuates heat-stress-induced oxidant damage by simultaneous activation of GSH-related antioxidant enzymes and Nrf2-mediated phase Ⅱ detoxifying enzyme systems in broiler chickens[J].Poultry Science, 2018, 97(4):1209-1219.
[14] 王换换, 申正杰, 肖航, 等.热应激对肝脏中Keap1-Nrf2信号通路及下游基因表达的影响[J].南京农业大学学报, 2017, 40(1):151-156. WANG H H, SHEN Z J, XIAO H, et al.Effects of heat stress on Keap1-Nrf2 signal pathway of liver in dairy cows[J] .Journal of Nanjing Agricultural University, 2017, 40(1):151-156.(in Chinese)
[15] 秦士贞.锰对原代培养鸡胚心肌细胞的抗热应激效应及其分子机制研究[D].兰州:甘肃农业大学, 2017. QIN S Z.Anti-heat stress effect and molecular mechanisms of manganese on cultivation of chick embryo primary myocardial cells[D].Lanzhou:Gansu Agricultural University, 2017.(in Chinese)
[16] LIU Z F, JI J J, ZHENG D, et al.Calpain-2 protects against heat stress-induced cardiomyocyte apoptosis and heart dysfunction by blocking p38 mitogen-activated protein kinase activation[J].Journal of Cellular Physiology, 2019, 234(7):10761-10770.
[17] 刘辉, 刘红阳, 姜一农, 等.叔丁基对苯二酚拮抗心肌细胞氧化应激损伤机制研究[J].中国实用内科杂志, 2015, 35(8):704-707. LIU H, LIU H Y, JIANG Y N, et al.Effects of tert-butylhydroquinone (TBHQ) against oxidative stress injury of H9c2 myocardial cells[J] .Chinese Journal of Practical Internal Medicine, 2015, 35(8):704-707.(in Chinese)
[18] XU J, HUANG B, TANG S, et al.Co-enzyme Q10 protects primary chicken myocardial cells from heat stress by upregulating autophagy and suppressing the PI3K/AKT/mTOR pathway[J].Cell Stress and Chaperones, 2019, 24(6):1067-1078.
[19] 郑亚妮, 高修歌, 刘晓晓, 等.盐霉素对鸡原代心肌细胞的毒性作用及其机制的初步研究[J].畜牧兽医学报, 2017, 48(4):740-751. ZHENG Y N, GAO X G, LIU X X, et al.Preliminary research of toxic effects and mechanisms of salinomycin on chicken primary cardiomyocytes[J] .Acta Veterinaria et Zootechnica Sinica, 2017, 48(4):740-751.(in Chinese)
[20] 李光辉, NAWAB A, 吴江, 等.生姜粉与中草药复方联合作用对高温环境中蛋鸡心脏和肝脏结构与功能的影响[J].家畜生态学报, 2019, 40(12):34-39. LI G H, NAWAB A, WU J, et al.Effects of ginger powder combined with Chinese herbal medicine on the structure and function of heart and liver of laying hens in high temperature environment[J] .Acta Ecologiae Animalis Domastici, 2019, 40(12):34-39.(in Chinese)
[21] 吕秋凤, 黄帆, 杨群辉, 等.热应激对动物心肌细胞影响研究进展[J].动物医学进展, 2017, 38(1):82-85. LYU Q F, HUANG F, YANG Q H, et al.Research progress on the effects of heat stress on animal cardiomyocytes[J] .Progress in Veterinary Medicine, 2017, 38(1):82-85.(in Chinese)
[22] ZHAO L, QI Y, XU L, et al.microRNA-140-5p aggravates doxorubicin-induced cardiotoxicity by promoting myocardial oxidative stress via targeting Nrf2 and Sirt2[J].Redox Biology, 2018, 15:284-296.
[23] YE H, XU G, ZHANG D, et al.The protective effects of the miR-129-5p/Keap-1/Nrf2 axis on Ang Ⅱ-induced cardiomyocyte hypertrophy[J].Annals of Translational Medicine, 2021, 9(2):154.
[24] 张云帆.血浆肌钙蛋白Ⅰ、心肌酶谱、脑钠肽在胸部恶性肿瘤患者放射性心脏损伤诊断中的作用[J].临床医学研究与实践, 2021, 6(21):119-121. ZHANG Y F.Role of plasma troponin Ⅰ, myocardial enzyme spectrum and brain natriuretic peptide in the diagnosis of radiation-induced cardiac injury in patients with thoracic malignant tumor[J] .Clinical Research and Practice, 2021, 6(21):119-121.(in Chinese)
[25] 何方婷, 陈嘉熠, 徐佳伊, 等.氧化应激细胞模型建立的研究进展[J].食品工业科技, 2019, 40(7):341-345. HE F T, CHEN J Y, XU J Y, et al.Research progress of establishing cellular oxidative stress model[J] .Science and Technology of Food Industry, 2019, 40(7):341-345.(in Chinese)
[26] LOBODA A, DAMULEWICZ M, PYZA E, et al.Role of Nrf2/HO-1 system in development, oxidative stress response and diseases:An evolutionarily conserved mechanism[J].Cellular and Molecular Life Sciences, 2016, 73(17):3221-3247.
[27] Chen Q M, Maltagliati A J.Nrf2 at the heart of oxidative stress and cardiac protection[J].Physiological Genomics, 2018, 50(2):77-97.
[28] 李延森, 李照见, 贾潇潇, 等.环境高温诱导巴马香猪睾丸细胞凋亡并激活Nrf2/HO-1抗氧化信号通路[J].畜牧兽医学报, 2018, 49(6):1154-1162. LI Y S, LI Z J, JIA X X, et al.Ambient high temperature induced cellular apoptosis and Nrf2/HO-1 signal pathway in testes of bama miniature pigs[J] .Acta Veterinaria et Zootechnica Sinica, 2018, 49(6):1154-1162.(in Chinese)
[29] CONSOLI V, SORRENTI V, GROSSO S, et al.Heme oxygenase-1 signaling and redox homeostasis in physiopathological conditions[J].Biomolecules, 2021, 11(4):1-23.
[30] BELLEZZA I, GIAMBANCO I, MINELLI A, et al.Nrf2-Keap1 signaling in oxidative and reductive stress[J].Biochimica Biophysica Acta Molecular Cell Research, 2018, 1865(5):721-733.
[31] 李军, 毕艳兰, 孙尚德, 等.叔丁基对苯二酚及其氧化产物与牛血清蛋白的相互作用[J].中国粮油学报, 2017, 32(4):133-140. LI J, BI Y L, SUN S D, et al.Interaction of tert-butyl hydroquinone and its oxidation products with bovine serum albumin[J] .Jourmal of the Chinese Cereals and Oils Association, 2017, 32(4):133-140.(in Chinese)
[32] LAU A, ZHENG Y, TAO S, et al.Arsenic inhibits autophagic flux, activating the Nrf2-Keap1 pathway in a p62-dependent manner[J].Molecular and Cellular Biology, 2013, 33(12):2436-2446.
[33] 吕嘉诚, 金壮壮, 杨蓓.tBHQ对亚砷酸钠致胰岛β细胞氧化应激损伤的影响[J].解剖科学进展, 2018, 24(3):310-313. LYU J C, JIN Z Z, YANG B.Effects of tBHQ on the sodium arsenite- induced oxidative stress injuries of mouse pancreatic β cells[J] .Progress of Anatomical Sciences, 2018, 24(3):310-313.(in Chinese)
[34] 邓文婕, 张群子, 邓琼霞, 等.激活Nrf2-ARE通路改善尿毒症患者血清诱导的内皮细胞功能障碍及其机制[J].中山大学学报(医学科学版), 2017, 38(3):341-349. DENG W J, ZHANG Q Z, DENG Q X, et al.Investigation of effects and mechanisms of Nrf2-ARE pathway on uremic serum-mediated endothelial dysfunction in human aortic endothelial cells[J] .Journal of Sun Yat-Sen University(Medical Sciences), 2017, 38(3):341-349.(in Chinese)
[35] 胡修忠, 程蕾, 余婕, 等.siRNA抑制牛子宫内膜上皮细胞Nrf2基因的表达研究[J].中国畜牧兽医, 2019, 46(6):1567-1575. HU X Z, CHENG L, YU J, et al.Study on inhibition of Nrf2 gene expression in bovine endometrial epithelial cells by siRNA[J] .China Animal Husbandry & Veterinary Medicine 2019, 46(6):1567-1575.(in Chinese)
[36] SHI X, LI Y, HU J, et al.Tert-butylhydroquinone attenuates the ethanol-induced apoptosis of and activates the Nrf2 antioxidant defense pathway in H9c2 cardiomyocytes[J].International Journal of Molecular Medicine, 2016, 38(1):123-130.

Study on Nrf2 Activation Alleviating Heat Stress-Induced Oxidative Damage in Broiler Cardiomyocytes

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	YUAN Minggui, LI Yahui, HE Junheng, TANG Xinggang, BA Xiaomin, ZHAO Yajun, TIAN Ya, LAI Xingru, XIANG Rong, YANG Xi. Prevention Effect of Vitex negundo L.var.cannabifolia (Sieb.et Zucc.) Hand.-Mazz.Extract on Necrotic Enteritis in Broilers [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(6): 2736-2749.
[2]	ZHANG Guanfeng, SONG Xianfan, YAN Zhaoming, YANG Xiaojin, ZHENG Mengli, LIU Yating, CHEN Qinghua. Effects of Dietary Superoxide Dismutase Supplementation on Growth Performance, Immune Performance,Antioxidant Capacity and Intestinal Function of Broilers [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(5): 1987-1998.
[3]	YU Miao, LI Yanjiao, ZHAO Yanling, CHAO Zhe, WANG Feng, LI Menghan, JIANG Zhiqiang, ZHANG Ting, XUE Xinyu, REN Zili, SUN Ruiping. Effects of Weaning Stress on Intestinal Morphology, Antioxidant Capacity and Nrf2 Signaling Pathway of Wuzhishan Piglets [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(5): 2045-2055.
[4]	BIAN Zhiyao, LIU Guohua, HAN Shuaijuan, CHEN Zhimin, ZHENG Aijuan. Effect of Dietary Probiotics Supplementation on Growth Performance, Slaughter Performance,Immune Organ Index and Apparent Metabolic Rate of Nutrients in Broilers [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(5): 2140-2148.
[5]	LI Xiaoxiao, TANG Yufei, YANG Qiuli, MO Yeyun, LIN Huilyu, LIU Chengxiang, LI Li. Mechanism of Total Flavonoids from Melastoma dodecandrum Lour.in Alleviating Diabetic Nephropathy in Mice Based on Nrf2/NQO1/GPX4 Signaling Pathway [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(5): 2409-2420.
[6]	FAN Qiuli, TAO Zhengguo, LI Hui, GOU Zhongyong, WANG Yibing, LIN Xiajing, YE Jinling, JIANG Shouqun. Effects of α-mangostin on Growth Performance,Immune Function, Antioxidant Capacity and Detoxification Function of Broilers [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(3): 1123-1130.
[7]	AN Zhaoxiang, XUN Wenjuan, ZHOU Hanlin, SHI Liguang. Effects of Heat Stress on Semen Antioxidant Capacity and Seminal Plasma Metabolomics in Hainan Black Goats [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(3): 1191-1201.
[8]	DONG Liyue, FAN Zuokun, TANG Meihui, TANG Jianqiang, YU Hailiang, ZHANG Tao, ZHANG Genxi, XIE Kaizhou, ZHAO Zhenhua, DAI Guojun. Effect of Eimeria tenella on Growth Performance,Immune Organs and Intestinal Morphology of Yellow-feathered Broilers [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(3): 1263-1271.
[9]	ZHONG Ming, GENG Longjun, CUI Lianhua, XU Ziyan, YAN Changguo, FENG Jian, CAI Hongfan, ZHANG Xu. Effects of Curcumin on Growth Performance, Apparent Digestibility of Feed Nutrients and Blood Indexes of Yanbian Yellow Breeding Cattle Under Heat Stress [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(2): 655-667.
[10]	CHEN Xing, LI Xiaozhen, ZHENG Aijuan, WANG Zedong, CHEN Zhimin, LIU Guohua. Effects of Zanthoxylum bungeanum Seed Meal Replacing Soybean Meal on the Growth Performance,Slaughter Performance,Serum Biochemical Indicators,Antioxidant Capacity,and Immune Function of Broilers [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(1): 158-168.
[11]	XING Dongxu, FU Bing, WANG Junyan, LI Qingrong, ZHOU Donglai, YE Jinling, LIAO Sentai, RUAN Dong, ZOU Yuxiao. Effects of Dietary Fermented Mulberry Leaf Powder on Growth Performance, Antioxidant and Immune Capacity,and Gut Microbiota Structure of Yellow-feathered Broilers [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(1): 169-181.
[12]	ZHANG Xiaohan, SUN Lanyuan, SONG Zhuan, HOU Yongqing, WU Tao. Effect of Chebuli Extract on Intestinal and Liver Damage Induced by Lipopolysaccharide in Broilers [J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(8): 3267-3278.
[13]	WANG Changtong, YANG Liandi, YIN Li, WANG Le, ZHANG Jin, ZUO Fuyuan, HUANG Wenming. Effects of Heat Stress on the Structure and Metabolites of Fecal Flora of Goats [J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(8): 3288-3300.
[14]	E Xiaodi, ZHAO Xiaonan, ZHAO Jinbiao, HU Youjun. Research Progress on Antioxidant Mechanism of Flavonoids Regulating the Keap1-Nrf2/ARE Signaling Pathway and Its Application in Livestock and Poultry [J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(7): 2880-2889.
[15]	LIN Xiajing, GOU Zhongyong, WANG Yibing, FAN Qiuli, YE Jinling, ZHANG Sai, JIANG Shouqun, RUAN Dong. Effects of Substituting Soybean Meal with Fermented Cottonseed Meal Equivalently on Growth Performance,Meat Quality and Antioxidation Activity of Yellow-Feathered Broilers [J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(7): 2890-2898.