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50μmol/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.
【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
作者
武亚南
石玉祥
张永英
刘冠慧
宋伟光
钟翠红
王永霞
WU Yanan;SHI Yuxiang;ZHANG Yongying;LIU Guanhui;SONG Weiguang;ZHONG Cuihong;WANG Yongxia(College of Life Sciences and Food Engineering,Hebei University of Engineering,Handan 056038,China;Chenguang Biotech Group Co.,Ltd.,Handan 056038,Chin)
出处
《中国畜牧兽医》
CAS
北大核心
2022年第9期3343-3352,共10页
China Animal Husbandry & Veterinary Medicine
基金
河北省重大科技成果转化专项(19046637Z)。
