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

doi:10.16431/j.cnki.1671-7236.2023.10.007

摘要/Abstract

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。

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

Abstract: 【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-α and 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-1β and TNF-α contents,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-α,IFN-γ,TLR4,NF-κB p65 and Bax,and significantly increased the mRNA and protein expressions of Occludin,Claudin-1,ZO-1,Bcl-2,PCNA and TGF-β1 in jejunum (P＜0.05).【Conclusion】 Gln alleviated ETEC K88-induced intestinal damage of mice by restoring intestinal barrier integrity,suppressing inflammatory responses and promoting epithelial cells proliferation.

Key words: glutamine(Gln); enterotoxigenic Escherichia coli (ETEC) K88; intestinal inflammation; intestinal barrier

中图分类号:

S852.61⁺2

张娜, 郭慧君, 邱建华. 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[J]. 中国畜牧兽医, 2023, 50(10): 3939-3949.

ZHANG Na, GUO Huijun, QIU Jianhua. Protective Effect of Glutamine on Intestinal Damage in Mice Infected with Enterotoxigenic Escherichia coli[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(10): 3939-3949.

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