Ala-Gln通过PPARγ/NF-κB信号通路对LPS免疫应激诱导的泥鳅肠道损伤的保护作用

In high-density farming, loach is often affected by the environment, producing immune response, causing intestinal damage and intestinal barrier function decreased. Research shows that, Glutamine (Gln) is the main energy source of some rapidly dividing cells and plays an important role in maintaining the intestinal normal morphology and immune system. In stressful conditions, endogenous Gln can't meet the body needs. The integrity of intestinal tissue structure and function both need exogenous Gln to sustain. Because of the low absorption rate and being easily transformed to pyroglutamic acid and ammonia, Gln is frequently substituted by Ala-Gln. However, few researches focus on the regulatory effect of Ala-Gln on barrier function of intestine in loach. This project plans to establish an immune stress model: (1) to research the effect of Ala-Gln on growth performance, intestinal morphology and immune function in loach; (2) to define the effect of Ala-Gln on the effects of immunological indices, inflammatory cytokines and antioxidant indices in the plasma, liver and intestinal tract of loach after LPS immune stress; (3) to reveal protection mechanism of Ala-Gln on intestinal damage of loach induced by immune-stress through PPARγ/NF-κB signaling pathway. The results can provide a necessary theory evidence for illustrating mechanism of regulatory effect of Ala-Gln on intestinal barrier function of loach.

在高密度养殖过程中，泥鳅常受到环境因素影响，产生免疫应激，导致肠道损伤，肠道屏障功能下降。研究表明，谷氨酰胺（Gln）是机体细胞快速分裂的主要能量来源，对维持肠道的正常形态和免疫系统有着重要的作用。但应激状态下，内源性Gln无法满足需要，需靠外源补充来维持肠道功能的完整。Gln吸收率低且易转化为焦谷氨酸和氨，常用丙胺酰谷氨酰胺（Ala-Gln）代替。但关于Ala-Gln对泥鳅肠道屏障功能的探讨还很少。本项目拟在前期工作基础上，建立免疫应激模型：（1）探明不同浓度Ala-Gln对泥鳅生产性能、肠道形态结构及免疫功能的影响；（2）明确Ala-Gln对脂多糖（LPS）免疫应激后泥鳅血浆、肝脏和肠道中免疫指标、炎性细胞因子和抗氧化指标的影响；（3）从 PPARγ/NF-κB 信号通路揭示Ala-Gln对泥鳅免疫应激诱导的肠道损伤的保护作用，为通过营养调控缓解或预防泥鳅免疫应激提供思路和理论依据。

本项目在饲料中添加不同浓度梯度的Ala-Gln（0、0.25%、0.50%、0.75%、1.00%、1.25%），研究了Ala-Gln对大鳞副泥鳅黏膜屏障功能、免疫功能的影响及对嗜水气单胞菌诱导的大鳞副泥鳅肠道炎症的缓解作用机制。结果表明，大鳞副泥鳅饲料中添加0.75%Ala-Gln，可促进生长，提高氧化能力，改善肠道组织形态。Ala-Gln添加量在0.75%~1.25%时能够改善大鳞副泥鳅幼鱼的肠道组织结构，提高机体的非特异性免疫性能。基于RNA-seq 测序技术，分析了大鳞副泥鳅感染嗜水气单胞菌后4个不同时间（0 h、6 h、12 h、24 h）的肠道转录组差异。通过KEGG富集对筛选的差异基因进行分析，获得了Toll 样受体信号通路、RIG-I 样受体信号通路、IL-17信号通路、C型凝集素受体信号通路和补体和凝血级联等与免疫相关通路，并筛选出TLR5、MyD88、TNF-α、C3、CD59等与免疫有关的差异表达基因，明确了大鳞副泥鳅对嗜水气单胞菌感染的免疫应答机制。并对 4种不同Ala-Gln添加量组（0.00%、0.25%、0.75%、1.25%）的大鳞副泥鳅进行嗜水气单胞菌感染试验，并在感染后0 h、6 h、12 h、24 h取肠道、肝脏进行免疫和抗氧化指标测定以及组织切片观察，并对肠道进行转录组分析。结果表明0.75%~1.25%Ala-Gln显著提高了嗜水气单胞菌感染后大鳞副泥鳅的抗氧化、非特异性免疫性能，维持了嗜水气单胞菌感染后大鳞副泥鳅的肝脏和肠道组织结构。Ala-Gln能够通过调控抗原处理和呈递、补体凝血和级联等通路上基因的表达来缓解嗜水气单胞菌感染引起的大鳞副泥鳅肠道炎症反应。结合16S rRNA基因测序，转录组，代谢组、肠道微生物、组织切片等数据，采用多组学手段初步阐释了Ala-Gln对对嗜水气单胞菌诱导的大鳞副泥鳅肠道炎症的缓解作用机理。项目执行期间发表第一标注论文3篇，第二标注4篇。培养硕士研究生2人。