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

【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 1×10^(9)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。

【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 1×10^(9) 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 typeⅣcollagenα2 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.