动物营养学报 >

2021 , Vol. 33 >Issue 10: 5817 - 5826

DOI: https://doi.org/10.3969/j.issn.1006-267x.2021.10.041

分子与细胞营养 MOLECULAR AND CELLULAR NUTRITION

致病性大肠杆菌感染小鼠肠炎模型的建立及其分子机制研究

  • 吴悦 ,
  • 王怡梦 ,
  • 王萌萌 ,
  • 周江涛 ,
  • 刘雪姣 ,
  • 李海花 ,
  • 乔家运
展开
  • 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
吴悦(2000-),女,江西鹰潭人,本科生,从事动物医学方面的研究。E-mail:xiaodui_detective@163.com

收稿日期: 2021-03-07

  网络出版日期: 2021-10-16

基金资助

天津市大学生创新训练计划项目（202010061007）；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）

收起

Establishment of Enteritis Model in Mice Infected with Enterotoxigenic Escherichia coli and Its Molecular Mechanism

  • WU Yue ,
  • WANG Yimeng ,
  • WANG Mengmeng ,
  • ZHOU Jiangtao ,
  • LIU Xuejiao ,
  • LI Haihua ,
  • QIAO Jiayun
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  • 1. Tianjin Key Laboratory of Agriculture Animal Breeding and Healthy Husbandry, College of Animal Science and Animal Medicine, Tianjin Agricultural University, Tianjin 300384, China;
    2. Tianjin Key Laboratory of Conservation and Utilization of Animal Diversity, College of Life Sciences, Tianjin Normal University, Tianjin 300387, China

Received date: 2021-03-07

  Online published: 2021-10-16

Supported by

 

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摘要

本试验旨在建立产肠毒素型大肠杆菌（ETEC）感染小鼠肠炎模型，并初步揭示其分子机制。将120只小鼠随机分为4组，每组30只，每组5个重复，每个重复6只。低、中、高剂量试验组分别经口腔灌服1×105、1×106和1×107 CFU/只ETEC，对照组灌服等体积生理盐水，各组注射剂量均为0.2 mL。分别于攻毒后2、48、72、96和120 h观察小鼠临床表现并称量其体重，同时从每个组中随机取6只采集其血液和组织样本。采用酶联免疫吸附测定（ELISA）检测血清中C反应蛋白（CRP）、肿瘤坏死因子-α（TNF-α）和白细胞介素-8（IL-8）的含量，通过组织切片和苏木精-伊红（HE）染色法观察小鼠空肠、肝脏和脾脏的病理变化，利用实时荧光定量PCR检测Toll-样受体4（TLR4）、核因子kappa B p65亚基（NF-κB p65）、髓样分化因子88（MyD88）、B细胞淋巴瘤因子3（BCl3） mRNA相对表达量。结果显示：与对照组相比，攻毒小鼠精神沉郁，进食减少，攻毒后72和96 h试验组小鼠体重均显著或极显著降低（P<0.01或P<0.05），肠道、肝脏和脾脏组织切片出现明显的炎症反应；攻毒后48和72 h试验组血清中CRP、TNF-α和IL-8的含量极显著升高（P<0.01）；攻毒后48、72和96 h试验组空肠组织中TLR4、NF-κB p65、MyD88 mRNA相对表达量极显著升高（P<0.01）；攻毒后24和120 h试验组空肠组织中BCl3 mRNA相对表达量分别极显著或显著升高（P<0.01或P<0.05），攻毒后72 h呈极显著下降（P<0.01）。综上所述，本研究成功建立了ETEC感染小鼠肠炎模型，并且该病原菌可能是通过TLR4/NF-κB信号通路诱导炎症相关因子生成和组织中炎性细胞浸润，从而引起小鼠炎症反应。

关键词： 产肠毒素型大肠杆菌; TLR4/NF-κ; B信号通路; 炎症反应; 小鼠肠炎

本文引用格式

吴悦 , 王怡梦 , 王萌萌 , 周江涛 , 刘雪姣 , 李海花 , 乔家运 . 致病性大肠杆菌感染小鼠肠炎模型的建立及其分子机制研究[J]. 动物营养学报, 2021 , 33(10) : 5817 -5826 . DOI: 10.3969/j.issn.1006-267x.2021.10.041

Abstract

The aim of this study was to establish an enterotoxigenic Escherichia coli (ETEC) infection model of mouse enteritis, and to reveal its molecular mechanism. A total of 120 mice were randomly divided into 4 groups with 30 mice in each group, and there were 5 replicates per group and 6 replicates per replicate. The mice in low, medium and high dose experimental groups were given 1×105, 1×106 and 1×107 CFU/mice by gastric gavage, respectively, while those in control group was given equal volume of normal saline by gavage, with the injection dose of 0.2 mL in each group. Clinical manifestations of mice were observed and weighed at 24, 48, 72, 96 and 120 h after challenge, respectively. Meanwhile, 6 mice were randomly selected from each treatment group and their blood and tissue samples were collected. The contents of C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), and interleukin-8 (IL-8) in serum were detected by enzyme-linked immunosorbent assay (ELISA). The pathological changes of jejunum, liver and spleen of mice were observed by tissue sections and hematoxylin-eosin staining (HE staining). The mRNA relative expression levels of Toll-like receptor 4 (TLR4), nuclear factor kappa-B (NF-κB p65), myeloid differentiation factor 88 (MyD88) and recombinant B-cell (BCl3) were detected by fluorescence quantitative PCR. The results showed as follows:compared with the control group, challenged mice were depressed and eating less, the body weight of the three experimental groups was significantly decreased at 72 and 96 h after challenge (P<0.01 or P<0.05), intestinal, liver and spleen tissue sections showed obvious inflammatory reaction; the contents of CRP, TNF-α and IL-8 in serum of test groups were significantly increased at 48 and 72 h after challenge (P<0.01). The mRNA relative expression levels of TLR4, NF-κB p65 and MyD88 in jejunum of experimental groups were significantly increased at 48, 72 and 96 h after challenge (P<0.01). The mRNA relative expression levels of BCl3 in jejunum of test groups was significantly increased at 24 and 120 h after challenge (P<0.01 or P<0.05), and was significantly decreased at 72 h after challenge (P<0.01). In conclusion, this study successfully establishes the enterotoxigenic Escherichia coli infected mouse enteritis model, and the pathogenic bacteria may induce the production of inflammatory related factors and inflammatory cell infiltration in tissues through TLR4/NF-κB signaling pathway, thus causing inflammatory response in mice.

Key words： Escherichia coli; TLR4/NF-κ; B signaling pathway; inflammatory response; colitis in mice

参考文献

[1] BULT C J, EPPIG J T, BLAKE J A, et al.Mouse genome database 2016[J].Nucleic Acids Research, 2016, 44(D1):D840-D847.
[2] 周贝, 林焱, 朱伟云.猪肠道大肠杆菌噬菌体对产肠毒素大肠杆菌的抑菌作用[J].畜牧兽医学报, 2020, 51(7):1748-1755. ZHOU B, LIN Y, ZHU W Y.Study on the bacteriostatic effect of porcine coliphages on enterotoxigenic Escherichia coli[J].Acta Veterinaria et Zootechnica Sinica, 2020, 51(7):1748-1755.(in Chinese)
[3] XI M L, QI Y, GE W P, et al.Effects of stachyose on intestinal microbiota and immunity in mice infected with enterotoxigenic Escherichia coli[J].Journal of Functional Foods, 2020, 64:103689.
[4] MIRHOSEINI A, AMANI J, NAZARIAN S.Review on pathogenicity mechanism of enterotoxigenic Escherichia coli and vaccines against it[J].Microbial Pathogenesis, 2018, 117:162-169.
[5] 齐宇, 王开, 伊淑帅, 等.产肠毒素大肠杆菌K88诱发BALB/c鼠肠炎模型的建立及评价[J].中国预防兽医学报, 2016, 38(1):19-22. QI Y, WANG K, YI S S, et al.Establishment and evaluation of bacterial enteritis model in BALB/c mice induced by enterotoxigenic Escherichia coli K88[J].Chinese Journal of Preventive Veterinary Medicine, 2016, 38(1):19-22.(in Chinese)
[6] LIN Q, SU G Q, WU A M, et al.Bombyx mori gloverin A2 alleviates enterotoxigenic Escherichia coli-induced inflammation and intestinal mucosa disruption[J].Antimicrobial Resistance and Infection Control, 2019, 8:189.
[7] LIN Q, FU Q Q, LI X, et al.Human β-defensin 118 attenuates Escherichia coli K88-induced inflammation and intestinal injury in mice[J].Probiotics and Antimicrobial Proteins, 2021, 13(2):586-597.  
[8] PU J N, CHEN D W, TIAN G, et al.Protective effects of benzoic acid, Bacillus coagulans, and oregano oil on intestinal injury caused by enterotoxigenic Escherichia coli in weaned piglets[J].BioMed Research International, 2018, 2018:1829632.
[9] WAN J, ZHANG J, WU G Z, et al.Amelioration of enterotoxigenic Escherichia coli-induced intestinal barrier disruption by low-molecular-weight chitosan in weaned pigs is related to suppressed intestinal inflammation and apoptosis[J].International Journal of Molecular Sciences, 2019, 20(14):3485.
[10] FINAMORE A, ROSELLI M, IMBINTO A, et al.Lactobacillus amylovorus inhibits the TLR4 inflammatory signaling triggered by enterotoxigenic Escherichia coli via modulation of the negative regulators and involvement of TLR2 in intestinal Caco-2 cells and pig explants[J].PloS One, 2014, 9(4):e94891.
[11] WANG X G, HARDWIDGE P R.Enterotoxigenic Escherichia coli prevents host NF-κB activation by targeting IκBα polyubiquitination[J].Infection and Immunity, 2012, 80(12):4417-4425.  
[12] 李海花, 朱琪, 王世琼, 等.产肠毒素大肠杆菌K88诱导仔猪炎症反应的分子机制[J].中国畜牧兽医, 2017, 44(1):262-267. LI H H, ZHU Q, WANG S Q, et al.Molecular mechanism of the inflammatory response induced by enterotrxigenic E.coli K88 in piglets[J].China Animal Husbandry & Veterinary Medicine, 2017, 44(1):262-267.(in Chinese)
[13] CHU C Q.Molecular probing of TNF:from identification of therapeutic target to guidance of therapy in inflammatory diseases[J].Cytokine, 2018, 101:64-69.
[14] 窦彩霞, 李海花, 孙泽阳, 等.核转录因子-κB/β-连环蛋白信号通路介导肠产毒性大肠杆菌引致IPEC-J2细胞损伤[J].动物营养学报, 2020, 32(12):5893-5902. DOU C X, LI H H, SUN Z Y, et al.Nuclear transcription factor-κB/β-catenin signaling pathway mediates enterotoxigenic Escherichia coli causing IPEC-J2 cell injury[J].Chinese Journal of Animal Nutrition, 2020, 32(12):5893-5902.(in Chinese)
[15] 于晴晴, 钟高龙, 万方, 等.高铜对大鼠肾细胞炎性因子和细胞增殖的影响[J].畜牧兽医学报, 2020, 51(11):2849-2857. YU Q Q, ZHONG G L, WAN F, Et al.Effects of high copper on inflammatory factors and cell proliferation in rat kidney cells[J].Acta Veterinaria et Zootechnica Sinica, 2020, 51(11):2849-2857.(in Chinese)
[16] SPROSTON N R, ASHWORTH J J.Role of C-reactive protein at sites of inflammation and infection[J].Frontiers in Immunology, 2018, 9:754.
[17] WU Y, POTEMPA L A, EL KEBIR D, et al.C-reactive protein and inflammation:conformational changes affect function[J].Biological Chemistry, 2015, 396(11):1181-1197.  
[18] LIU G X, LIU G L, CHEN H, et al.Involvement of Ca2+ activated Cl- channel Ano6 in platelet activation and apoptosis[J].Cellular Physiology and Biochemistry, 2015, 37(5):1934-1944.  
[19] 陈文晓, 林海华, 陈仁聘, 等.血淀粉样蛋白、CRP、降钙素在炎症性肠病小鼠模型中表达水平及其与疾病活动性的相关性研究[J].新医学, 2017, 48(3):153-159. CHEN W X, LIN H H, CHEN R P, et al. Expression level of serum amyloid protein, C-reactive protein and procalcitonin and the correlation with disease activity index in inflammatory bowel disease mouse model[J].New Medicine, 2017, 48(3):153-159.(in Chinese)
[20] 刘艳艳, 张凯, 关家伟, 等.人参皂苷对BALB/c小鼠肠道菌群的影响[J].现代生物医学进展, 2015, 15(6):1041-1045. LIU Y Y, ZHANG K, GUAN J W, et al.The effects of ginsenoside on the intestinal microbiota of mice[J].Progress in Modern Biomedicine, 2015, 15(6):1041-1045.(in Chinese)
[21] 谭婉贤, 熊晏, 赖莉敏, 等.5-氟尿嘧啶诱导小鼠化疗性肠黏膜炎模型的建立[J].中国实验动物学报, 2018, 26(3):372-377. TAN W X, XIONG Y, LAI L M, et al.Establishment of a mouse model of 5-fluorouracil-induced intestinal mucositis[J].Acta Laboratorium Animalis Scientia Sinica, 2018, 26(3):372-377.(in Chinese)
[22] 华锋.TNF-α在抗感染中的作用[J].南昌大学学报(医学版), 2012, 52(5):96-99. HUA F, LIU J H.The role of TNF-α in anti-infection[J].Journal of Nanchang University (Medical Science), 2012, 52(5):96-99.(in Chinese)
[23] 冯其麟, 邓艾平, 刘珏, 等.人参对感染后小鼠肠黏膜的干预效果及对血清CRP、TNF-α、IL-6水平的影响[J].中国医药导报, 2019, 16(4):18-21. FENG J L, DENG A P, LIU J, et al.Effects of ginseng on intestinal mucosa and the levels of serum CRP, TNF-α and IL-6 in mice after infection[J].China Medical Herald, 2019, 16(4):18-21.(in Chinese)
[24] VERNON P J, PAREDES R M, SOOTER A J, et al.Severe hemorrhagic shock induces acute activation and expansion of IL-8+/IL-10+neutrophils with enhanced oxidative reactivity in Non-Human primates[J].Shock (Augusta, Ga.), 2016, 46(3 Suppl.1):129-136.
[25] HASAN A, AKHTER N, AL-ROUB A, et al.TNF-α in combination with palmitate enhances IL-8 production via the MyD88-independent TLR4 signaling pathway:potential relevance to metabolic inflammation[J].International Journal of Molecular Sciences, 2019, 20(17):4112.
[26] TANG S C, LIAO P Y, HUNG S J, et al.Topical application of glycolic acid suppresses the UVB induced IL-6, IL-8, MCP-1 and COX-2 inflammation by modulating NF-κB signaling pathway in keratinocytes and mice skin[J].Journal of Dermatological Science, 2017, 86(3):238-248.  
[27] YAMAGUCHI R, YAMAMOTO T, SAKAMOTO A, et al.Chemokine profiles of human visceral adipocytes from cryopreserved preadipocytes:neutrophil activation and induction of nuclear factor-kappa B repressing factor[J].Life Sciences, 2015, 143:225-230.
[28] 刘灿, 王婵, 左之才, 等.黄芪多糖对腹腔注射大肠杆菌小鼠TLR信号通路MyD88、ERK及细胞因子的影响[J].浙江农业科学, 2017, 58(1):143-146. LIU C, WANG C, ZUO Z C, et al.Effects of Astragalus polysaccharides on TLR signaling pathway MyD88, ERK and cytokines in mice intraperitoneally injected with enterotoxigenic Escherichia coli[J].Journal of Zhejiang Agricultural Sciences, 2017, 58(1):143-147.(in Chinese)
[29] 李春林, 张翥, 曾荣荣, 等.黄连水提物对LPS诱导的猪肠道上皮细胞IL-1β、IL-6、TNF-α mRNA表达及NF-κB/MAPK信号通路的调控作用[J].中国兽医学报, 2020, 40(2):361-368. LI C L, ZHANG Z, ZENG R R, et al.Effect of Rhizoma coptidis water extract on expression of IL-1β, IL-6, TNF-α mRNA and regulation of NF-κB/MAPK signal pathways in porcine intestinal epithelial cells induced by LPS[J].Chinese Journal of Veterinary Science, 2020, 40(2):361-368.(in Chinese)
[30] LEMIESZEK M L, NUNES F M, MARQUES G, et al.Cantharellus cibarius branched mannans inhibits colon cancer cells growth by interfering with signals transduction in NF-κB pathway[J].International Journal of Biological Macromolecules, 2019, 134:770-780.
[31] XIE C H, KANG J, FERGUSON M E, et al.Blueberries reduce pro-inflammatory cytokine TNF-α and IL-6 production in mouse macrophages by inhibiting NF-κB activation and the MAPK pathway[J].Molecular Nutrition & Food Research, 2011, 55(10):1587-1591.  
[32] LEE S, PARK Y, DELLSPERGER K C, et al.Exercise training improves endothelial function via adiponectin-dependent and Independent pathways in type 2 diabetic mice[J].American Journal of Physiology-Heart and Circulatory Physiology, 2011, 301(2):H306-H314.
[33] LI R Y, ZHANG W Z, YAN X T, et al.Arginyl-fructosyl-glucose, a major maillard reaction product of red ginseng, attenuates cisplatin-induced acute kidney injury by regulating nuclear factor κB and Phosphatidylinositol 3-kinase/protein kinase B signaling pathways[J].Journal of Agricultural and Food Chemistry, 2019, 67(20):5754-5763.  
[34] 胡章威, 李芬, 左晶晶, 等.BCL3与免疫及肿瘤相关性的研究进展[J].中国医药生物技术, 2018, 13(6):552-555. HU Z W, LI F, ZUO J J, et al.Research progress on the correlation between BCL3 and immunity and tumor[J].Chinese Medicinal Biotechnology, 2018, 13(6):552-555.(in Chinese)
[35] WEIGHARDT H, FÖRSTER I.Bcl-3 puts the brakes on contact hypersensitivity[J].European Journal of Immunology, 2015, 45(4):971-974.  
[36] KREISEL D, SUGIMOTO S, TIETJENS J, et al.Bcl3 prevents acute inflammatory lung injury in mice by restraining emergency granulopoiesis[J].The Journal of Clinical Investigation, 2011, 121(1):265-276.  
[37] POVEDA J, SANZ A, CARRASCO S, et al. Bcl3:a regulator of NF-κB inducible by TWEAK in acute kidney injury with anti-inflammatory and antiapoptotic properties in tubular cells[J].Experimental & Molecular Medicine, 2017, 49(7):e352.
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