产肠毒素大肠杆菌对断奶仔猪肠道形态、紧密连接蛋白及细胞外基质的影响

doi:10.16431/j.cnki.1671-7236.2023.07.011

中国畜牧兽医 ›› 2023, Vol. 50 ›› Issue (7): 2697-2706.doi: 10.16431/j.cnki.1671-7236.2023.07.011

产肠毒素大肠杆菌对断奶仔猪肠道形态、紧密连接蛋白及细胞外基质的影响

刘春艳^1,2, 唐青松¹, 杨必婧¹, 吴绮雯¹, 熊云霞¹, 黄艳娜², 杨雪芬¹, 王丽¹, 蒋宗勇¹, 易宏波¹

1. 广东省农业科学院动物科学研究所, 畜禽育种国家重点实验室, 农业农村部华南动物营养与饲料重点实验室, 岭南现代农业科学与技术广东省实验室茂名分中心, 广东省畜禽育种与营养研究重点实验室, 广州 510640;
2. 广西大学动物科学技术学院, 南宁 530004

收稿日期:2022-11-22 发布日期:2023-06-30
通讯作者: 易宏波 E-mail:yihongbo@gdaas.cn
作者简介:刘春艳,E-mail:1838799235@qq.com。
基金资助:
茂名实验室科研启动项目（2021TDQD002）；茂名实验室重点项目（2022ZD003）；广州市科技计划项目（202201011372）；国家现代农业产业技术体系（CARS-35）；科技创新战略专项资金-高水平农科院建设（R2019PY-QF005、202106TD）

Effects of Enterotoxigenic Escherichia coli on Intestinal Morphology, Tight Junction Protein and Extracellular Matrix of Weaned Piglets

LIU Chunyan^1,2, TANG Qingsong¹, YANG Bijing¹, WU Qiwen¹, XIONG Yunxia¹, HUANG Yanna², YANG Xuefen¹, WANG Li¹, JIANG Zongyong¹, YI Hongbo¹

1. Institute of Animal Science, Guangdong Academy of Agricultural Sciences, State Key Laboratory of Livestock and Poultry Breeding, Key Laboratory of Animal Nutrition and Feed Science in South China, Ministry of Agriculture and Rural Affairs, Maoming Branch of Guangdong Laboratory for Lingnan Modern Agriculture, Guangdong Key Laboratory of Animal Breeding and Nutrition, Guangzhou 510640, China;
2. College of Animal Science and Technology, Guangxi University, Nanning 530004, China

Received:2022-11-22 Published:2023-06-30

摘要/Abstract

摘要： 【目的】探究产肠毒素大肠杆菌（ETEC）对断奶仔猪肠道形态、紧密连接蛋白及细胞外基质（ECM）表达的影响。【方法】选取35日龄杜长大断奶仔猪12头，随机分成2组（每组6个重复，每个重复1头）：对照组灌喂20 mL生理盐水；产肠毒素大肠杆菌组（K88组）灌喂20 mL 1×10⁹ CFU/mL ETEC K88悬浮液，连续灌喂3 d。试验期间所有仔猪自由采食和饮水，在试验第1和4天，以重复为单位称量仔猪体重，记录耗料量。计算每组的平均日增重（ADG）、平均日采食量（ADFI）和料重比（F/G）；在试验第4天屠宰并采集肠道组织样品，切片观察空肠和回肠的组织形态以及肠道内胶原纤维的含量；采用实时荧光定量PCR分析肠道紧密连接蛋白基因的表达；采用实时荧光定量PCR和蛋白质印迹法测定ECM中胶原蛋白分子以及调控酶系的表达。【结果】与对照组相比，K88组仔猪平均日增重显著降低（P<0.05），平均日采食量极显著降低（P<0.01）；空肠绒毛高度及绒毛高度与隐窝深度的比值极显著降低（P<0.01），回肠的绒毛高度也显著降低（P<0.05）；空肠闭锁蛋白（Occludin） mRNA表达量显著降低（P<0.05），闭合蛋白-1（Claudin-1） mRNA表达量极显著降低（P<0.01），回肠闭锁小带蛋白-1（ZO-1）、Occludin mRNA表达量极显著降低（P<0.01），Claudin-1 mRNA表达量也显著降低（P<0.05）；空肠、回肠胶原纤维含量显著减少（P<0.05）；空肠Ⅳ型胶原蛋白α2链（COL4A2）、COL5A1、COL6A1的mRNA表达量极显著降低（P<0.01），空肠COL4A2以及COL6A1蛋白表达量均显著降低（P<0.05）；空肠基质金属蛋白酶9（MMP9） mRNA相对表达量显著增加（P<0.05），纤溶酶原激活物（PAs）的mRNA相对表达量则极显著降低（P<0.01）；MMP9蛋白含量极显著提高（P<0.01）。【结论】灌喂ETEC K88降低了断奶仔猪平均日增重以及平均日采食量，损伤肠道形态并降低肠道紧密连接蛋白的表达。并且可通过减少肠道中胶原纤维的含量、降低胶原蛋白分子以及ECM调控酶系的表达进而造成肠道中ECM的变化。

关键词: 产肠毒素大肠杆菌; 断奶仔猪; 肠道紧密连接蛋白; 细胞外基质

Abstract: 【Objective】 To explore the effects of enterotoxigenic Escherichia coli (ETEC) on intestinal morphology, tight junction protein and extracellular matrix (ECM) expression of weaned piglets.【Method】 Twelve Duroc weaned piglets aged 35 days were randomly divided into 2 groups (6 replicates per group, 1 pig per replicate):Control group (fed with 20 mL normal saline) and enterotoxin-producing Escherichia coli group (K88 group, fed with 20 mL of 1×10⁹ CFU/mL ETEC K88 suspension), and continuous feeding for 3 days. All piglets were free to eat and drink.On day 1 and day 4, each pig was weighed by repetition and the feed consumption was recorded.The average daily gain (ADG), average daily feed intake (ADFI) and feed to gain ratio (F/G) of each group were calculated.On day 4, the piglets were slaughtered and the intestines were collected, and sliced to observe the intestinal tissue morphology of jejunum and ileum and the content of collagen fiber in the intestines.Real-time quantitative PCR was used to analyze the expression of intestinal compact junction protein gene.Real-time quantitative PCR and western blotting were used to determine the expression of collagen molecules and regulatory enzymes in ECM.【Result】 Compared with control group, the average daily gain of K88 group was significantly decreased (P<0.05), and the average daily feed intake was extremely significantly decreased (P<0.01).The villus height in jejunum and the ratio of villus height to crypt depth in K88 group were extremely significantly decreased (P<0.01), and the villus height in ileum was also significantly decreased (P<0.05).The mRNA expression of Occludin was significantly decreased (P<0.05), and the mRNA expression of Claudin-1 was extremely significantly decreased (P<0.01) in jejunum.At the same time, the mRNA expressions of Occludin and ZO-1 were extremely significantly decreased (P<0.01), and the mRNA expression of Claudin-1 was also significantly decreased (P<0.05) in ileum.The content of collagen fiber in both jejunum and ileum was significantly reduced (P<0.05).The mRNA expressions of type Ⅳ collagen α2 chain (COL4A2), COL5A1 and COL6A1 in jejunum were extremely significantly decreased (P<0.01), and the protein expression of COL4A2 and COL6A1 in the jejunum of K88 group were significantly decreased (P<0.05).The relative mRNA expression of MMP9 in jejunum was significantly increased (P<0.05), while the relative mRNA expression of PAs was highly significantly decreased (P<0.01), and the MMP9 protein content was extremely significantly increased in K88 group (P<0.01).【Conclusion】 In conclusion, feeding ETEC K88 reduced average daily gain and average daily feed intake, damaged intestinal morphology and reduced the expression of intestinal tight junction protein in weaned piglets.In addition, ECM changes could be induced by reducing the content of collagen fibers and the expression of collagen molecules and ECM regulatory enzyme system in the intestine.

Key words: enterotoxigenic Escherichia coli; weaned piglets; intestinal health; extracellular matrix

中图分类号:

S852.61

刘春艳, 唐青松, 杨必婧, 吴绮雯, 熊云霞, 黄艳娜, 杨雪芬, 王丽, 蒋宗勇, 易宏波. 产肠毒素大肠杆菌对断奶仔猪肠道形态、紧密连接蛋白及细胞外基质的影响[J]. 中国畜牧兽医, 2023, 50(7): 2697-2706.

LIU Chunyan, TANG Qingsong, YANG Bijing, WU Qiwen, XIONG Yunxia, HUANG Yanna, YANG Xuefen, WANG Li, JIANG Zongyong, YI Hongbo. Effects of Enterotoxigenic Escherichia coli on Intestinal Morphology, Tight Junction Protein and Extracellular Matrix of Weaned Piglets[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2697-2706.

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产肠毒素大肠杆菌对断奶仔猪肠道形态、紧密连接蛋白及细胞外基质的影响

Effects of Enterotoxigenic Escherichia coli on Intestinal Morphology, Tight Junction Protein and Extracellular Matrix of Weaned Piglets

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