丁酸梭菌通过激活p38丝裂原活化蛋白激酶/核因子E2相关因子信号通路减轻猪霍乱沙门氏菌导致的猪小肠上皮细胞氧化损伤  

Clostridium butyricum Alleviates Porcine Intestinal Epithelial Cells Oxidative Damage Induced by Salmonella cholerae through Activating p38 Mitogen Activated Protein Kinase/Nuclear Factor E2 Related Factor Signaling Pathway

作　　者：尚智援 窦彩霞 王克玮 刘雪姣 李海花 乔家运 SHANG Zhiyuan;DOU Caixia;WANG Kewei;LIU Xuejiao;LI Haihua;QIAO Jiayun(Tianjin Key Laboratory of Conservation and Utilization of Animal Diversity,College of Life Sciences,Tianjin Normal University,Tianjin 300387,China;Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry,College of Animal Science and Veterinary Medicine,Tianjin Agricultural University,Tianjin 300384,China)

机构地区：[1]天津师范大学生命科学学院,天津市动物多样性保护与利用重点实验室,天津300387 [2]天津农学院动物科学与动物医学学院,天津市农业动物繁育与健康养殖重点实验室,天津300384

出　　处：《动物营养学报》2021年第12期7105-7117,共13页CHINESE JOURNAL OF ANIMAL NUTRITION

基　　金：天津市自然科学基金(20JCZDJC00190);国家自然科学基金(31702147);天津市研究生科研创新项目(2020YJSS136);天津市“131”创新型人才团队(20180338)。

摘　　要：本试验旨在研究丁酸梭菌(CB)缓解猪霍乱沙门氏菌(SC)诱导的猪小肠上皮细胞(IPEC-J2细胞)氧化损伤的分子机制。选用IPEC-J2细胞为模型,采用CCK-8法检测细胞活力,酶联免疫吸附测定(ELISA)法检测细胞培养上清液中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GPX)活性和丙二醛(MDA)含量,从而筛选诱导IPEC-J2细胞氧化损伤的SC适宜剂量。CB预处理细胞后,用SC感染细胞,通过小干扰RNA(siRNA)沉默IPEC-J2细胞中核因子E2相关因子(Nrf2)的表达以及p38丝裂原活化蛋白激酶(p38 MAPK)抑制剂处理细胞,用实时荧光定量PCR(RT-qPCR)法检测细胞中p38 MAPK、SOD1、SOD2、GPX1、GPX2和Nrf2的mRNA相对表达量,蛋白印迹法(Western blotting)检测细胞中p38 MAPK和Nrf2蛋白相对表达量。结果表明,CCK-8法和ELISA法确定1×10^(3)CFU/mL为SC诱导IPEC-J2细胞氧化损伤的适宜剂量。与SC组相比,CB+SC组细胞中p38 MAPK、Nrf2及其下游基因在12 h后的mRNA相对表达量均极显著升高(P<0.01)。与对照组相比,siRNA沉默Nrf2基因后,siRNA组培养上清液GPX活性以及细胞中抗氧化酶基因的mRNA相对表达量均显著降低(P<0.05);与siRNA+SC组相比,NC+CB+SC组和siRNA+CB+SC组细胞中抗氧化酶基因的mRNA相对表达量显著升高(P<0.05)。p38 MAPK被抑制后,与p38+SC组相比,p38+CB+SC组培养上清液GPX活性以及细胞中抗氧化酶基因的mRNA相对表达量均显著或极显著升高(P<0.05或P<0.01)。综上所述,CB通过激活p38 MAPK/Nrf2信号通路抗氧化相关基因的表达,缓解SC造成的IPECJ2细胞氧化损伤。The aim of this study was to investigate the molecular mechanism of Clostridium butyricum(CB)alleviates Salmonella choleraesuis(SC)induced oxidative damage in porcine intestinal epithelial cells(iPECJ2 cells).The IPEC-J2 cells were selected as the cell model,the cell viability was detected by CCK-8 method,and the superoxide dismutase(SOD),glutathione peroxidase(GPX)activities and malondialdehyde(MDA)content in the cell culture supernatant were determined by enzyme-linked immunosorbent assay(ELISA)method,so as to select the optimal concentration of SC induced oxidative damage in IPEC-J2 cells.After CB pretreatment and SC infection and small interfering RNA(siRNA)silencing nuclear factor E2 related factor(Nrf2)gene expression and p38 mitogen activated protein kinase(p38 MAPK)inhibitor treatment of IPEC-J2 cells,the real-time quantitative PCR(RT-qPCR)was performed to determine the mRNA relative expression levels of p38 MAPK,SOD1,SOD2,GPX1,GPX2 and Nrf2 in cells,and the Western blotting was performed to determine the protein relative expression levels of p38 MAPK and Nrf2 in cells.The results showed as follows:the CCK-8 method and ELISA method were determined that 1×10^(3) CFU/mL was the optimal concentration of SC induced oxidative damage in IPEC-J2 cells.Compared with the SC group,the relative expression levels of p38 MAPK,Nrf2 and its downstream genes in the CB+SC group were significantly increased after 12 h(P<0.01).Compared with the SC group,after silencing Nrf2 gene with siRNA,the content of GPX in culture supernatant and the mRNA relative expression levels of antioxidant enzyme genes in cells in the siRNA group were significantly decreased(P<0.05);compared with the siRNA+SC group,the mRNA relative expression levels of enzyme genes in cells in NC+CB+SC group and siRNA+CB+SC group were significantly increased(P<0.05).After p38 MAPK was inhibited,compared with the p38+SC group,the content of GPX in culture supernatant and the mRNA relative expression levels of antioxidant enzyme genes in cells in the p38+CB+SC g

关 键 词：丁酸梭菌 猪霍乱沙门氏菌 IPEC-J2细胞 p38 MAPK/Nrf2信号通路 

分 类 号：S811.3[农业科学—畜牧学]