谷氨酰胺对肠产毒素性大肠杆菌感染小鼠肠道损伤的保护作用  

Protective Effect of Glutamine on Intestinal Damage in Mice Infected with Enterotoxigenic Escherichia coli

作　　者：张娜[1] 郭慧君 邱建华[2,3] ZHANG Na;GUO Huijun;QIU Jianhua(Weifang Economic School,Weifang Business Vocational College,Weifang 262200,China;Shandong Province Key Laboratory of Animal Biotechnology and Disease Control and Prevention,Tai’an 271018,China;College of Animal Science and Veterinary Medicine,Shandong Agricultural University,Tai’an 271018,China)

机构地区：[1]潍坊工商职业学院/潍坊市经济学校,潍坊262200 [2]山东省动物生物工程与疾病防治重点实验室,泰安271018 [3]山东农业大学动物科技学院/动物医学院,泰安271018

出　　处：《中国畜牧兽医》2023年第10期3939-3949,共11页China Animal Husbandry & Veterinary Medicine

基　　金：山东省现代农业产业技术体系特种经济动物创新团队岗位科学家专项(SDAIT-21-10);国家林业局中央本级专项(2130211);山东省“双一流”奖补资金。

摘　　要：【目的】探究谷氨酰胺(glutamine,Gln)对肠产毒素性大肠杆菌(enterotoxigenic Escherichia coli,ETEC)感染小鼠肠道损伤的保护作用,为Gln缓解大肠杆菌性肠损伤提供理论依据。【方法】选取健康的SPF级昆明小鼠24只,随机分为3组:对照组(Con)、模型组(ETEC)、Gln干预组(ETEC+Gln)。Con和ETEC组小鼠采用灌胃方式每天给予0.2 mL生理盐水,ETEC+Gln组小鼠给予0.2 mL 1%Gln,于试验第7天,ETEC和ETEC+Gln组小鼠采用灌胃方式给予0.2 mL 8.6×109 CFU/mL ETEC K88,试验期10 d。每天对小鼠进行称重;采用HE和PAS染色观察空肠病理变化;采用ELISA法检测血清炎症因子的浓度;采用实时荧光定量PCR和Western blotting分别检测空肠炎症因子(IL-1β、IL-6、TNF-α、IFN-γ)、紧密连接蛋白(Occludin、Claudin-1、ZO-1)、肠上皮细胞增殖(TGF-β1、PCNA、EGF)和凋亡(Bax、Bcl-2)相关指标的mRNA和蛋白表达水平;采用免疫组化检测空肠Toll样受体4(Toll-like receptor 4,TLR4)和核因子κB(nuclear factor kappa-B,NF-κB)的蛋白表达情况。【结果】与Con组相比,ETEC组小鼠体重显著下降(P<0.05),而Gln处理缓解了体重下降的趋势;ETEC组小鼠脾脏指数显著高于Con和ETEC+Gln组(P<0.05);与ETEC组相比,Gln处理显著提高了小鼠空肠绒毛高度、绒毛高度/隐窝深度比值、杯状细胞数量和黏液层厚度(P<0.05)。与Con和ETEC+Gln组相比,ETEC组小鼠血清IL-1β和TNF-α含量、D-乳酸(D-lac)浓度、二胺氧化酶(DAO)活性均显著升高(P<0.05);与ETEC组相比,Gln干预显著降低了小鼠空肠IL-1β、IL-6、TNF-α、IFN-γ、TLR4、NF-κB p65和Bax的mRNA表达水平(P<0.05),显著增加了小鼠空肠Occludin、Claudin-1、ZO-1、Bcl-2、PCNA和TGF-β1的mRNA和蛋白表达水平(P<0.05)。【结论】Gln干预可通过抑制肠道炎症反应、促进肠道上皮细胞增殖和提高肠上皮细胞紧密连接等缓解ETEC K88感染引起的小鼠肠道损伤。【Objective】This experiment was conducted to investigate the protective effect of glutamine(Gln)on intestinal damage in mice infected with enterotoxigenic Escherichia coli(ETEC),and provide a theoretical basis and rationale for Gln to alleviate E.coli intestinal injury.【Method】Twenty-four health SPF KM mice were randomly allocated to 3 groups:Control group(Con),model group(ETEC)and Gln intervention group(ETEC+Gln).Mice in Con and ETEC groups were given 0.2 mL saline daily by gavage,and mice in ETEC+Gln group were given 0.2 mL 1%Gln.On days 7,the mice in ETEC and ETEC+Gln groups were challenged with 0.2 mL 8.6×109 CFU/mL ETEC K88 by gavage.The trial lasted for 10 days.Mice were weighed daily.The pathological changes of jejunum were observed by hematoxylin-eosin(HE)and periodic acid-schiff(PAS)staining.The concentrations of serum inflammatory factors were determined by ELISA.The mRNA and protein expressions of inflammatory factors(IL-1β,IL-6,TNF-αand IFN-γ),tight junction proteins(Occludin,Claudin-1 and ZO-1),intestinal epithelial cell proliferation(TGF-β1,PCNA and EGF)and apoptosis(Bax and Bcl-2)in jejunum were detected by Real-time quantitative PCR and Western blotting,respectively.The protein expression of TLR4 and NF-κB p65 in jejunum were detected by immunohistochemistry.【Result】Compared with Con group,the body weight of mice in ETEC group was significantly decreased(P<0.05),while Gln treatment prominently reversed that.The spleen index of mice in ETEC group was significantly higher than that in Con and ETEC+Gln groups(P<0.05).Compared with ETEC group,treatment with Gln significantly improved the villus height,the ratio of villus height/crypt depth,the number of goblet cells and the mucus thickness in jejunum(P<0.05).Compared with Con and ETEC+Gln groups,the IL-1βand TNF-αcontents,D-lac concentration and DAO activity in serum of mice in ETEC group were significantly increased(P<0.05).Compared with ETEC group,treatment with Gln significantly decreased the mRNA expressions of IL-1β,IL-6,TNF-�

关 键 词：谷氨酰胺 肠产毒素性大肠杆菌(ETEC)K88 肠道炎症 肠道屏障 

分 类 号：S852.612[农业科学—基础兽医学]