三丁酸甘油酯对产肠毒素大肠杆菌K88诱发的小鼠肠道损伤的缓解作用

Alleviation Effects of Triglyceride on Intestinal Injury of Mice Induced by Enterotoxigenic Escherichia coli K88

本试验旨在研究三丁酸甘油酯(TB)对产肠毒素大肠杆菌K88(ETEC K88)诱导的小鼠肠道损伤的缓解作用。试验1:选用(20±2)g的BALB/c小鼠50只,随机分为5组,每组10只。各组均饲喂基础饲粮,在第4天对照组腹腔注射0.5 mL无菌磷酸盐缓冲液(PBS),试验组腹腔分别注射浓度为5.5×10^(8)、4.9×10^(7)、4.0×10^(6)、6.5×10^(5) CFU/mL的ETEC K88悬液0.5 mL,连续观察3 d临床症状,统计腹泻率和死亡率,剖检、观察肠道病理变化,确定ETEC K88最佳造模剂量。试验2:选择(20±2)g的BALB/c小鼠105只,随机分为7组,每组3个重复,每个重复5只。空白组、阴性对照组饲喂基础饲粮,阳性对照组饲喂添加0.16%金霉素的饲粮,试验1、2、3、4组分别饲喂添加0.05%、0.10%、0.20%、0.30%TB的饲粮。连续饲喂14 d,在第15天空白组小鼠腹腔注射0.5 mL无菌PBS,阴性对照组、阳性对照组和试验组小鼠按照试验1中确定的最佳造模剂量腹腔注射ETEC K88悬液0.5 mL,继续观察3 d,采血、剖检,检测血清中二胺氧化酶(DAO)活性与内毒素(ET)、C-反应蛋白(CRP)和分泌型免疫球蛋白A(SIgA)含量,制作空肠组织切片,观察肠道组织形态变化。结果显示:小鼠腹腔注射不同浓度ETEC K88悬液后出现不同程度的腹泻和死亡情况,根据腹泻率和死亡率,确定4.9×10^(7) CFU/mL为ETEC K88建立小鼠腹泻模型的最佳剂量。阳性对照组和试验3、4组的平均日增重与空白对照组差异不显著(P<0.05),但显著高于阴性对照组(P>0.05)。小鼠的腹泻率表现为阴性对照组(53.33%)>试验1组(33.33%)>阳性对照组(26.67%)=试验2组(26.67%)>试验3组(20.00%)=试验4组(20.00%)>空白对照组(无腹泻发生)。肠道损伤程度:阴性对照组>试验1组>阳性对照组>试验2组>空白对照组>试验4组>试验3组。与阴性对照组相比,各试验组以及阳性对照组小鼠血清CRP、ET含量和DAO活性均有不同程度降低,以试验3组降低幅度最大,其次为试验4组。与阳性对照组相比,试验3、4组小鼠血清CRP、ET含量和DAO活性均有不同程度降低,但差异不显著(P>0.05)。与阴性对照组相比,试验3、4组小鼠血清SIgA含量分别增加了169.00%(P<0.05)、114.33%(P<0.05)。与阳性对照组相比,试验3、4组小鼠血清SIgA含量分别增加了72.81%(P<0.05)和37.69%(P<0.05)。由此得出,在小鼠基础饲粮中添加0.20%或0.30%的TB可以对抗大肠杆菌感染,缓解ETEC K88诱导的肠道损伤,且效果与添加0.16%的金霉素相当。

This experiment was conducted to study the alleviation effect of triglyceride(TB)on intestinal injury of mice induced by enterotoxigenic Escherichia coli K88(ETEC K88).Experiment 1:fifty BALB/c mice with the body weight of(20±2)g were selected and randomly divided into 5 groups with 10 mice per group.All mice fed the same basal diet,and the mice in control group were intraperitoneal injection of 0.5 mL aseptic phosphate buffer solution(PBS),while those in experimental groups were intraperitoneal injection of 0.5 mLETEC K88 suspension with the concentrations of 5.5×10^(8),4.9×10^(7),4.0×10^(6) and 6.5×10^(5)CFU/mL,respectively.The clinical symptoms were observed and recorded within three days,calculated the diarrhea rate and mortality.Then,necropsied the mice to observe their intestinal pathological changes to determine the best dose of ETEC K88 for modeling.Experiment 2:one hundred and five mice with the body weight of(20±2)g were selected and randomly divided into 7 groups with 3 replicates per group and 5 mice per replicate.The negative control group intraperitoneal injection of the ETEC K88 suspension(best dose determined in experiment 1,the same as below)after fed the basal diet for 14 days,the blank control group intraperitoneal injection of aseptic PBS after fed the basal diet for 14 days,the positive control group intraperitoneal injection of ETEC K88 suspension after fed the basal diet added 0.16%chlortetracycline for 14 days,and the four experimental groups intraperitoneal injection of ETEC K88 suspension after fed the basal diet added 0.05%,0.10%,0.20%and0.30%TB for 14 days.After observation for 3 days,collected blood and necropsied the mice,then detected the changes of diamine oxidase(DAO)activity,endotoxin(ET),C-reactive protein(CRP),and secretory immunoglobulin A(SIgA)contents in serum and made intestinal pathology sections to observe the intestinal histological changes.The results showed that,after intraperitoneal injection of different concentrations of ETECK88 suspension,the mice developed diarrhea and death of varying degrees.The best dose of ETEC K88 to build mice diarrhea model was determined according to the diarrhea rate and mortality,it was 4.9×10^(7) CFU/mL.The average daily gain of positive control group and experimental groups 1 and 2 had no significant difference compared with the blank control group(P<0.05),but significantly higher than that of negative control group(P>0.05)The diarrhea rate of mice was negative control group(53.33%)>experimental group 1(33.33%)>positive control group(26.67%)=experimental group 2(26.67%)>experimental group 3(20.00%)=experimental group 4(20.00%)>blank control group(no diarrhea).Degree of intestinal injury manifested as:negative control group>experimental group 1>positive control group>experimental group 2>blank control group>experimental group 4>experimental group 3.Compared with the negative control group,the serum CRP and ET content and DAO activity of experimental groups 3,4 and positive control group were reduced in different degrees,and experimental group 3 had the biggest reduction,followed by experimental group 4.the serum CRP and ET content and DAO activity of experimental groups 3,4 were reduced in different degrees compared with the positive control group,but there were no significant differences(P>0.05).Serum SIgA content of experimental groups 3,4 were increased by 169.00%(P<0.05)and114.33%(P<0.05)compared with the negative control group,respectively,and they were increased by72.81%(P<0.05)and 37.69%(P<0.05)compared with the positive control group,respectively.In conclusion,adding 0.20%or 0.30%TB to the basal diet of mice can combat Escherichia coli infection,alleviate intestinal injury induced by ETEC K88,and the effect can be comparable to 0.16%chlortetracycline.