植物乳杆菌抑制产肠毒素型大肠杆菌诱发猪肠上皮细胞炎症反应的分子机制  

Molecular Mechanism of Lactobacillus plantarum Inhibiting Inflammatory Response Induced by Enterotoxigenic Escherichia coli in Porcine Intestinal Epithelial Cells

作　　者：李海花 梁东梅 乔家运 LI Haihua;LIANG Dongmei;QIAO Jiayun(Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry,College of Animal Science and Animal Medicine,Tianjin Agricultural University,Tianjin 300384,China;Tianjin Key Laboratory of Conservation and Utilization of Animal Diversity,College of Life Sciences,Tianjin Normal University,Tianjin 300387,China)

机构地区：[1]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 [2]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

出　　处：《动物营养学报》2021年第7期4018-4029,共12页CHINESE JOURNAL OF ANIMAL NUTRITION

基　　金：天津市自然科学基金项目(20JCZDJC00190);天津市“131”创新型人才团队(20180338)。

摘　　要：本试验旨在研究植物乳杆菌抑制产肠毒素型大肠杆菌(ETEC)诱发猪肠上皮细胞炎症反应的分子机制。试验利用植物乳杆菌预处理猪肠上皮细胞IPEC⁃J23 h,ETEC刺激细胞1、3、6、12和24 h,收集细胞及其培养上清,采用酶联免疫吸附试验(ELISA)法检测上清液中白细胞介素-1β(IL⁃1β)、白细胞介素-8(IL⁃8)和肿瘤坏死因子-α(TNF⁃α)的含量,采用实时荧光定量PCR检测细胞中Toll样受体2(TLR2)和Toll样受体4(TLR4)以及NOD样受体3(NLRP3)和NOD样受体6(NLRP6)的表达,采用Western blot检测细胞中丝裂原活化蛋白激酶(MAPK)[p38和细胞外信号调节激酶(ERK)]和核转录因子-κB(NF⁃κB)p65的磷酸化水平以及紧密连接蛋白———闭合小环蛋白-1(zonula occludens⁃1,ZO⁃1)和闭锁蛋白(occludin)的表达。分别采用p38 MAPK抑制剂、ERK⁃MAPK抑制剂和NF⁃κB p65抑制剂处理IPEC⁃J2细胞1 h,再用植物乳杆菌预处理细胞3 h,最后用ETEC处理细胞24 h,收集细胞和培养上清,采用ELISA法检测IL⁃1β、IL⁃8和TNF⁃α含量以及乳酸脱氢酶(LDH)活性。结果表明:1)与ETEC处理相比,植物乳杆菌预处理能够极显著降低ETEC感染12~24 h细胞产生IL⁃1β、IL⁃8和TNF⁃α的含量(P<0.01),显著或极显著提高ETEC感染3~12 h细胞中TLR2、TLR4、NLRP3和NLRP6的mRNA相对表达量(P<0.05或P<0.01),极显著降低ETEC感染24 h细胞中TLR2和NLRP3的mRNA相对表达量(P<0.01),极显著降低ETEC感染3~6 h细胞中p38 MAPK和NF⁃κB p65的磷酸化水平(P<0.01),显著或极显著提高ETEC感染6~24 h细胞中ZO⁃1和occludin的表达量(P<0.05或P<0.01)。2)与ETEC处理相比,抑制剂和植物乳杆菌共同作用能够极显著降低ETEC感染细胞产生IL⁃1β、IL⁃8、TNF⁃α的含量和LDH的活性(P<0.01),植物乳杆菌单独作用也可以极显著降低ETEC感染细胞产生LDH的活性(P<0.01),并且信号通路抑制剂对植物乳杆菌调控部分促炎性细胞因子的产生有协同作用。综上所述The aim of this study was to investigate the molecular mechanism of Lactobacillus plantarum(LP)inhibiting inflammatory response induced by enterotoxigenic Escherichia coli(ETEC)in porcine intestinal epithelial cells. In this experiment,LP was used to incubate porcine intestinal epithelial cells IPEC-J2 for 3 h,then ETEC was used to stimulate cells for 1,3,6,12 and 24 h. The culture supernatants and cells were collected.The contents of interleukin(IL)-1β,IL-8 and tumor necrosis factor-α (TNF-α)in the supernatant were detected by enzyme linked immunosorbent assay(ELISA),the expressions of Toll-like receptor 2(TLR2)and Toll-like receptor 4(TLR4)and NOD like receptor 3(NLRP3)and NOD like receptor 6(NLRP6)in cells were analyzed by real-time fluorescent quantitative PCR,and the phosphorylation levels of mitogen activated protein kinase(MAPK),including p38 and extracellular signal-regulated kinase(ERK),and nuclear factor-κB(NF-κB)as well as the expression of zona occludens-1(ZO-1)and occludin in cells were examined by Western blot. IPEC-J2 cells were treated with p38 MAPK inhibitor,ERK-MAPK inhibitor and NF-κB p65 inhibitor for 1 h,respectively. The inhibitor treated-cells were incubated with LP for 3 h,and then were treated with ETEC for 24 h. The culture supernatant and cells were collected for detecting the contents of IL-1β,IL-8 and TNF-αand lactate dehydrogenase(LDH)activity by enzyme-linked immunosorbent assay(ELISA). The results showed as follows:1)compared with the ETEC treatment,the LP pretreatment extremely significantly decreased the contents of IL-1β,IL-8 and TNF-αproduced by cells infected with ETEC for 12 to 24 h(P<0.01),significantly or extremely significantly increased the mRNA relative expression levels of TLR2,TLR4,NLRP3 and NLRP6 in cells infected with ETEC for 3 to 12 h(P<0.05 or P<0.01),extremely significantly decreased the mRNA relative expression levels of TLR2 and NLRP3 in cells infected with ETEC for 24 h(P<0.01),extremely significantly decreased the phosphorylation levels of p38 MAPK

关 键 词：植物乳杆菌 猪肠上皮细胞 产肠毒素型大肠杆菌 炎症反应 分子机制 

分 类 号：S811[农业科学—畜牧学]