ShRNA干扰核转录因子E2相关因子2基因表达对玉米赤霉烯酮诱导的IPEC-J2细胞凋亡和氧化应激的影响  

Effects of ShRNA Interference on Nuclear Transcription Factor E2 Related Factor 2 Gene Expression on Apoptosis and Oxidative Stress Induced by Zearalenone in IPEC-J2 Cells

作　　者：程群 桑艺豪 姜淑贞[2] 侯绍金 牛现琇 杨维仁[2] CHENG Qun;SANG Yihao;JIANG Shuzhen;HOU Shaojin;NIU Xianxiu;YANG Weiren(College of Animal Sciences and Technology,Qingdao Agricultural University,Qingdao 266109,China;Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention,College of Animal Sciences and Technology,Shandong Agricultural University,Tai’an 271000,China;Pingyi Agricultural and Rural Bureau,Pingyi 273300,China)

机构地区：[1]青岛农业大学动物科技学院,青岛266109 [2]山东农业大学动物科技学院学院,泰安271000 [3]平邑县农业农村局,平邑273300

出　　处：《动物营养学报》2025年第4期2661-2673,共13页CHINESE JOURNAL OF ANIMAL NUTRITION

基　　金：山东省自然科学基金青年基金项目(ZR2022QC030);山东省自然科学基金面上项目(ZR2024MC124);青岛农业大学博士启动基金(XJ2023000401);山东省现代农业产业技术体系生猪创新团队专项资金(SDAIT-08-04,SDAIT-08-05)。

摘　　要：本试验旨在通过ShRNA干扰核转录因子E2相关因子2(Nrf2)基因表达探索玉米赤霉烯酮(ZEA)诱导IPEC-J2细胞凋亡和氧化应激中Kelch样环氧氯丙烷相关蛋白1(Keap1)-Nrf2信号通路的作用。试验构建干扰Nrf2表达IPEC-J2细胞模型,分别设置空白对照组(Control组)、阴性对照组(Sh-control组)、干扰Nrf2表达转染组(Nrf2-ShRNA组),以确定Nrf2介导的信号通路对ZEA诱导的IPEC-J2细胞氧化应激的作用。当成功转染Nrf2-ShRNA的IPEC-J2细胞处于对数生长期时,分别添加10、20和40μmol/L的ZEA(Nrf2-ShRNA+ZEA10组、Nrf2-ShRNA+ZEA20组和Nrf2-ShRNA+ZEA40组)处理36 h后收集细胞,进行相关指标测定。结果显示:1)Nrf2-ShRNA组IPEC-J2细胞的凋亡率显著高于Control组和Sh-control组(P<0.05)。与Nrf2-ShRNA组相比较,ZEA以一定的剂量依赖性显著增加了干扰Nrf2表达的IPEC-J2细胞的凋亡率(P<0.05),表明干扰细胞Nrf2表达和高浓度ZEA均能诱导细胞凋亡。2)与Control组和Sh-control组相比,Nrf2-ShRNA组IPEC-J2细胞中总超氧化物歧化酶(T-SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性与Keap1、Nrf2、还原型辅酶/醌氧化还原酶1(NQO1)和血红素加氧酶-1(HO-1)mRNA和蛋白相对表达量均显著降低(P<0.05),丙二醛(MDA)含量和活性氧(ROS)荧光强度显著增加(P<0.05)。3)与Nrf2-ShRNA组相比,Nrf2-ShRNA+ZEA20组和Nrf2-ShRNA+ZEA40组IPEC-J2细胞中T-SOD和GSH-Px活性与Keap 1 mRNA和蛋白相对表达量显著降低(P<0.05),MDA含量和Nrf2荧光强度以及Nrf2、NQO1和HO-1 mRNA和蛋白相对表达量显著增加(P<0.05)。当ZEA浓度达到40μmol/L,IPEC-J2细胞中Nrf2和ROS的荧光强度最高。由此可见,ZEA能够诱导IPEC-J2细胞的氧化应激,促进细胞凋亡的发生,而干扰IPEC-J2细胞Nrf2的表达能够影响Keap1-Nrf2信号通路的激活,降低细胞抵抗ZEA诱导氧化应激能力,因此Keap1-Nrf2信号通路在ZEA诱导的肠道氧化应激中具有重要的保护作用。The aim of this experiment was to explore the role of the Kelch-like epichlorohydrin-associated protein 1(Keap1)-nuclear transcription factor E2 related factor 2(Nrf2)signaling pathway in the induction of apoptosis and oxidative stress in IPEC-J2 cells by zearalenone(ZEN)through ShRNA interference of Nrf2 expression.An cell model that IPEC-J2 cells interfered with the expression of Nrf2 was conducted,with blank control group(Control group),negative control group(Sh-control group)and Nrf2-ShRNA transfection group(Nrf2-ShRNA group)set up to determine the role of Nrf2 mediated signaling pathway in ZEA-induced IPEC-J2 cell oxidative stress.When IPEC-J2 cells successfully transfected with Nrf2-ShRNA was in the logarithmic growth phase,ZEA with the concentrations of 10,20 and 40μmol/L(Nrf2-ShRNA+ZEA10 group,Nrf2-ShRNA+ZEA20 group and Nrf2-ShRNA+ZEA40 group)were added,respectively,and cells were collected for the detection of related indexes after 36 h treatment.The results showed as follows:1)the apoptosis rate of IPEC-J2 cells in Nrf2-ShRNA group was significantly higher than that of Control group and Sh-control group(P<0.05).Compared with Nrf2-ShRNA group,ZEA significantly increased the apoptosis rate of IPEC-J2 cells(P<0.05),which interfered with Nrf2 expression in a certain dose-dependent manner,indicating that interference with Nrf2 expression and high concentration of ZEA could induce cell apoptosis.2)Compared with the Control group and Sh-control group,the IPEC-J2 cells in Nrf2-ShRNA group showed a significant decrease in total superoxide dismutase(T-SOD),glutathione peroxidase(GSH-Px)activities,Keap1,Nrf2,coenzyme/quinone oxidoreductase 1(NQO1)and heme oxygenase-1(HO-1)mRNA and protein relative expression levels(P<0.05),while malondialdehyde(MDA)content and reactive oxygen species(ROS)immunofluorescence intensity increased significantly(P<0.05).3)Compared with Nrf2-ShRNA group,the T-SOD and GSH-Px activities and the relative expression levels of Keap1 mRNA and protein in IPEC-J2 cells in Nrf2-ShRNA+ZEA20 group

关 键 词：玉米赤霉烯酮 IPEC-J2细胞 Keap1-Nrf2信号通路 Nrf2-ShRNA 氧化应激 

分 类 号：S811.2[农业科学—畜牧学]