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

摘要/Abstract

摘要： 【目的】探究活化核因子红系2相关因子2（nuclear factor erythroid 2-related factor 2，Nrf2）对热应激肉鸡心肌细胞氧化损伤的缓解作用及其机制。【方法】利用差速贴壁法结合化学纯化法制备肉鸡原代心肌细胞。将心肌细胞随机分为对照组（Control组）、热应激组（HS组）和Nrf2激活剂叔丁基对苯二酚（tert-butylhydroquinone，TBHQ）预处理热应激组（TBHQ+HS组）。对照组心肌细胞正常培养不做任何处理，HS组心肌细胞置于高温（43 ℃）培养箱处理2 h，TBHQ+HS组心肌细胞用50 μmol/L TBHQ预处理12 h，再进行热应激处理。采用四甲基偶氮唑蓝（MTT）法测定各组心肌细胞相对活力；用比色法测定超氧化物歧化酶（SOD）活性、丙二醛（MDA）含量和细胞培养液中乳酸脱氢酶（LDH）活性；用实时荧光定量PCR检测Nrf2、血红素氧合酶-1（heme oxygenase 1，HO-1）、谷氨酰半胱氨酸连接酶（glutamate cysteine ligase catalytic，GCLC）和NAD （P） H：醌氧化还原酶1（NAD （P） H：quinone oxidoreductase 1，NQO1） mRNA表达水平；用蛋白质免疫印迹法（Western blotting）检测Nrf2总蛋白和HO-1蛋白表达量。【结果】与对照组相比，热应激组肉鸡原代心肌细胞形态发生改变，出现空泡网状结构，心肌细胞的相对活力和SOD活性极显著降低（P<0.01），MDA含量极显著升高（P<0.01），细胞培养液中LDH活性极显著升高（P<0.01），Nrf2/抗氧化反应原件（ARE）信号通路下游NQO1和GCLC mRNA表达升高，但差异不显著（P>0.05），Nrf2和HO-1 mRNA表达显著增加（P<0.05），但Nrf2总蛋白和HO-1蛋白表达量增加不显著（P>0.05）；与热应激组相比，TBHQ预处理热应激组心肌细胞内空泡网状结构减少，心肌细胞的相对活力显著升高（P<0.05），SOD活性极显著升高（P<0.01），MDA含量显著降低（P<0.05），细胞培养液中LDH活性极显著降低（P<0.01），Nrf2/ARE信号通路下游NQO1和GCLC基因的表达水平显著上调（P<0.05），Nrf2基因和总蛋白表达量显著增加（P<0.05），HO-1基因和蛋白表达量极显著增加（P<0.01）。【结论】活化Nrf2可以上调Nrf2/ARE信号通路下游相关抗氧化基因和蛋白表达，提高热应激肉鸡心肌细胞的抗氧化能力，缓解热应激诱导的氧化损伤，提示Nrf2可能是肉鸡心肌细胞抗热应激氧化损伤的有效分子靶点。

关键词: 热应激; 肉鸡; 心肌细胞; 核因子红系2相关因子2(Nrf2); 血红素氧合酶-1(HO-1)

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

中图分类号:

Q494

武亚南, 石玉祥, 张永英, 刘冠慧, 宋伟光, 钟翠红, 王永霞. 活化Nrf2缓解热应激致肉鸡心肌细胞氧化损伤的研究[J]. 中国畜牧兽医, 2022, 49(9): 3343-3352.

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 and Veterinary Medicine, 2022, 49(9): 3343-3352.

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