Chinese Journal of Animal Nutrition >

2026 , Vol. 38 >Issue 2: 1332 - 1342

DOI: https://doi.org/10.12418/CJAN2026.106

RESEARCH PAPER

Mechanistic Study of Glutamine in Alleviating Acute Cold Stress Injury in Lung Tissue of Chicks

  • QIU Wenxin , 1 ,
  • LI Guanhong 1 ,
  • PENG Youfeng 2 ,
  • ZENG Tingxia 2 ,
  • SHAO Longyi 2 ,
  • LI Yanmei 3 ,
  • DAI Sifa 2, 3, *
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  • 1 College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang 214000, China
  • 2 Department of Pharmaceutical and Life Sciences, Jiujiang University, Jiujiang 332000, China
  • 3 Jiujiang Bozheng Biotechnology Industrial Research Institute, Jiujiang 332000, China
* professor, E-mail: daisifa-004@163.com

Received date: 2025-07-17

  Online published: 2026-02-12

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Abstract

This experiment investigated the mechanism by which glutamine alleviates acute cold stress-induced lung injury in chicks, focusing on the cold-inducible RNA-binding protein (CIRP)-Toll-like receptor 4 (TLR4)-inositol-requiring enzyme 1 (IRE1) signaling pathway. Ninety-six 1-day-old Shengze 901 chicks were randomly divided into 4 groups: the acute cold stress group (ACS group), the glutamine intervention group (Gln group), the inhibitor intervention group (TAK-242 group), and the combined intervention group (Gln/TAK-242 group). Each group consisted of 6 replicates with 4 chicks per replicate. Chicks in the Gln group and Gln/TAK-242 group were supplemented with 1.2% glutamine in drinking water from 1 day of age. Chicks in the TAK-242 group and Gln/TAK-242 group were intraperitoneally injected with 200 μL/bird TAK-242 working solution (equivalent to 3 mg/kg BW of TAK-242) at 6 days of age, while those in the ACS group and Gln groups were injected with an equal volume of normal saline. Immediately after injection, all four groups were subjected to 12 h of acute cold stress at (12±1) ℃, followed by sample collection and analysis. The results showed as follows: 1) the combined intervention of glutamine and TAK-242 showed significant or highly significant interaction effects on the activities of catalase (CAT) and glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), malondialdehyde (MDA) content, the levels of diamine oxidase (DAO) and interleukin-6 (IL-6), as well as the mRNA relative expression levels of glucoseregulated protein 78 (GRP78), Creactive protein (CRP), DAO, IL-6, and C/EBP-homologous protein (CHOP) in lung tissue (P<0.05 or P<0.01). 2) Main effect analysis revealed that glutamine intervention significantly or extremely significantly elevated the activities of CAT and superoxide dismutase (SOD) (P<0.05 or P<0.01), while it significantly or extremely significantly reduced the levels of CIRP, DAO, IL-6 and tumor necrosis factor-α (TNF-α), as well as the mRNA relative expression levels of CIRP, TLR4, IRE1, GRP78, nuclear factor-κB (NF-κB), CRP, DAO and IL-6 in lung tissue (P<0.05 or P<0.01). TAK-242 intervention extremely significantly increased the activity of SOD and the level of interleukin-10 (IL-10) (P<0.01), and significantly or extremely significantly reduced the levels of DAO and TNF-α, together with the mRNA relative expression levels of TLR4, IRE1, GRP78, NF-κB, CRP, DAO, IL-6 and CHOP in lung tissue (P<0.05 or P<0.01). 3) Compared with the Gln group, the Gln/TAK-242 group showed an extremely significant increase in SOD activity and IL-6 level (P<0.01), and an extremely significant decrease in TNFα level as well as in the mRNA relative expression levels of TLR4, GRP78, DAO and IL-6 in lung tissue (P<0.01). Relative to the TAK-242 group, the Gln/TAK-242 group exhibited a significant or extremely significant elevation in T-AOC and SOD activity (P<0.05 or P<0.01), and a significant or extremely significant reduction in TNF-α level and the mRNA relative expression levels of CIRP, TLR4 and NF-κB in lung tissue (P<0.05 or P<0.01). It is suggested that the mechanism of glutamine alleviating acute cold stress injury in lung tissue of chicks may be mediated by CIRP-TLR4-IRE1 signaling pathway to mediate NF-κB pathway.

Key words： chicks; cold stress; glutamine; signal pathway; cold-inducible RNA-binding protein; Toll-like receptor 4

Cite this article

QIU Wenxin , LI Guanhong , PENG Youfeng , ZENG Tingxia , SHAO Longyi , LI Yanmei , DAI Sifa . Mechanistic Study of Glutamine in Alleviating Acute Cold Stress Injury in Lung Tissue of Chicks[J]. Chinese Journal of Animal Nutrition, 2026 , 38(2) : 1332 -1342 . DOI: 10.12418/CJAN2026.106

在鸡的早期生命阶段,环境温度应激(尤其是冷应激)可引发肠道神经化学物质浓度以及肠道微生物组成与功能的改变[1]。低温会直接影响机体脂肪、蛋白质代谢,并造成不同程度的组织损伤[2]。雏鸡由于体温调节中枢尚未发育完全,对环境冷应激尤为敏感,常表现为采食量与饲料利用率下降,进而影响其生长性能[3]。然而,现有研究多集中于冷应激对肠道或免疫器官的作用,关于其对呼吸系统影响的分子机制,仍存在许多未解之处。
冷诱导RNA结合蛋白(CIRP)是较晚在哺乳动物冷应激研究中被发现的一种应激反应蛋白,参与多种细胞功能相关的信号转导途径[4]。该蛋白在包括家禽在内的脊椎动物组织中基础表达水平较低,但在低温刺激下显著上调,并从细胞核转移至细胞质,进而进入循环系统[5]。Toll样受体4(TLR4)是先天免疫系统中的一类模式识别受体,能够识别病原体相关分子模式(PAMP)和损伤相关分子模式(DAMP)等特定配体。近年的研究发现,在肺成纤维细胞炎症表型[6]及脓毒症[7]等哺乳动物模型中,CIRP/TLR4信号通路可通过独立于病原微生物的机制诱导炎症反应,调控增殖相关细胞因子的表达,且CIRP的细胞外促炎作用依赖于TLR4。内质网作为蛋白质合成与折叠的重要场所,在维持细胞内稳态及相关信号协调中起关键作用。一项关于肠道组织的新近研究揭示,冷应激可促使CIRP从细胞核向胞质/胞外释放,作为DAMP激活TLR4,继而通过内质网应激(ERS)-肌醇需求激酶1(IRE1)信号轴,驱动核因子-κB(NF-κB)依赖的炎症级联反应[8]。
谷氨酰胺(Gln)是体内快速增殖细胞所需的关键营养物质,与多种黏膜组织的炎症反应密切相关。前期研究表明,谷氨酰胺可通过抑制内质网应激,减轻气道炎症及气道重塑[9];能够下调TLR4的基因与蛋白表达,缓解脂多糖(LPS)诱导的牛胚胎气管细胞炎症反应[10];还可通过内质网应激通路,减轻高氧诱导的新生大鼠肺组织水肿与炎症[11]。本研究拟从DAMP作用途径、TLR4非依赖性诱导及内质网应激机制等角度,探讨CIRP-TLR4-IRE1信号通路是否通过介导NF-κB及其下游基因表达,参与雏鸡肺组织急性冷应激引起的炎症损伤,并评估谷氨酰胺对该过程的干预作用,以期为相关机制解析和生产实践提供新思路。

1 材料与方法

1.1 试验材料

TAK-242(纯度99.1%)购自MedChemExpress公司(美国),产品编号HY-11109。谷氨酰胺(项目号G970167,CAS号3918-84-1,纯度98%)购自上海麦克林生化科技股份有限公司。

1.2 试验设计

本研究经江西农业大学实验动物福利伦理委员会批准(JXAULL-2023-09-10),于2023年9月在江西农业大学试验基地进行。
选取96只1日龄健康的圣泽901白羽公鸡,采用2×2因子设计,随机分为4组,每组6个重复,每个重复4只鸡。具体分组如下:急性冷应激组(ACS组)、谷氨酰胺干预组(Gln组,急性冷应激+1.2%谷氨酰胺)、抑制剂干预组(TAK-242组,急性冷应激+TAK-242)以及联合干预组(Gln/TAK-242组,急性冷应激+1.2%谷氨酰胺+TAK-242)。4组均接受为期12 h的急性冷应激处理。鸡舍初始环境温度为(33±1) ℃,相对湿度为55%~65%,此后每日降温约0.5 ℃。ACS组和TAK-242组全程饮用自来水;Gln组和Gln/TAK-242组则从1日龄起,在饮用水中添加1.2%谷氨酰胺。至6日龄20:00,对各组雏鸡进行腹腔注射:ACS组与Gln组每只注射200 μL生理盐水;TAK-242组与Gln/TAK-242组每只注射200 μL TAK-242工作液。TAK-242给药剂量参考前人研究[12]设定为3 mg/kg BW,TAK-242工作液浓度为2.7 mg/mL,实际注射量依据6日龄雏鸡平均体重(约180 g)计算得出。注射完成后,立即将全部雏鸡转移至预设的人工气候室[温度(12±1) ℃,相对湿度60%]内,进行连续12 h的急性冷应激处理,结束后立即开展采样与样品制备工作。整个试验期为7 d。

1.3 基础饲粮配制

基础饲粮参照圣泽901白羽公鸡营养需要和《鸡饲养标准》(NY/T 33—2004)配制,为颗粒料,基础饲粮组成及营养水平见表1。其中,代谢能、氨基酸含量参照《中国饲料成分及营养价值表(2022年第33版)》计算得出,粗蛋白质、钙、总磷含量分别依据GB/T 6432—2018、GB/T 6436—2018、GB/T 6437—2018进行测定。
表1 基础饲粮组成及营养水平(风干基础)

Table 1 Composition and nutrient levels of the basal diet (air-dry basis) %

项目Items 含量Content
原料Ingredients
玉米Corn 58.34
豆粕Soybean meal 27.84
玉米蛋白粉Corn gluten meal 4.82
面粉Flour 3.00
大豆油Soybean oil 1.79
磷酸氢钙CaHPO4 1.70
石粉Limestone 1.36
氯化钠NaCl 0.30
L-赖氨酸硫酸盐L-lysine·H2SO4 0.26
DL-蛋氨酸DL-methionine 0.16
L-苏氨酸L-threonine 0.10
维生素预混料Vitamin premix1) 0.04
矿物质预混料Mineral premix2) 0.15
氯化胆碱Choline chloride 0.13
植酸酶Phytase 0.01
合计Total 100.00
营养水平Nutrient levels3)
代谢能Metabolic energy/(MJ/kg) 12.57
粗蛋白质Crude protein 21.64
钙Calcium 1.01
总磷Total phosphorus 0.73
赖氨酸Lysine 1.21
蛋氨酸+半胱氨酸Methionine+cysteine 0.88
苏氨酸Threonine 0.84
色氨酸Tryptophan 0.25

1)维生素预混料为每千克饲粮提供 Vitamin premix provided the following per kilogram of the diet:VA 10 800 IU,VD3 4 500 IU,VE 45.0 IU,VK3 2.5 mg,VB12 0.02 mg,硫胺素 thiamine 2.0 mg,核黄素 riboflavin 6.5 mg,烟酰胺 nicotinamide 50.0 mg,泛酸 pantothenate acid 12.0 mg,吡哆醇 pyridoxine 4.0 mg,生物素 biotin 0.18 mg,叶酸 folic acid 1.5 mg。

2)矿物质预混料为每千克饲粮提供 Mineral premix provided the following per kilogram of the diet:Fe 20.00 mg,Cu 10.00 mg,Mn 120.00 mg,Zn 100.00 mg,I 1.00 mg,Se 0.35 mg。

3)代谢能、氨基酸为计算值,其余为实测值。Metabolic energy and amino acids were calculated values, while the others were measured values.

1.4 样品采集与处理

在冷应激处理指定时间后,从每个重复中随机选取1只雏鸡,颈动脉放血处死,分离肺组织至2 mL冻存管中,液氮速冻后于-80 ℃冰箱保存,用于后续指标测定。

1.5 指标测定及方法

1.5.1 肺组织中抗氧化指标的测定

取适量肺组织,按1∶9[重量(g)∶体积(mL)]的比例加入生理盐水,于冰上充分研磨后,离心取上清液使用试剂盒(购自南京建成生物工程研究所)并严格按照说明书操作测定以下指标:过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性及丙二醛(MDA)含量和总抗氧化能力(T-AOC)。

1.5.2 肺组织中炎症和损伤相关指标的测定

取适量肺组织,按1∶9[重量(g)∶体积(mL)]的比例加入磷酸盐缓冲液(0.01 mol/L,pH=7.4),于冰上充分研磨后,离心取上清液使用酶联免疫吸附测定(ELISA)试剂盒(购自上海酶联生物科技有限公司)并严格按照试剂盒说明书操作步骤检测肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、白细胞介素-10(IL-10)、二胺氧化酶(DAO)和CIRP水平。

1.5.3 肺组织中CIRP-TLR4-IRE1信号通路及下游靶基因表达的测定

将肺组织按1∶9[重量(g)∶体积(mL)]的比例加入TransZol裂解液,置于2 mL离心管中,用组织研磨仪充分匀浆。随后提取总RNA,并将其反转录为cDNA,保存于-20 ℃冰箱备用。使用北京全式金生物技术有限公司提供的试剂,严格按照说明书步骤,通过实时荧光定量PCR检测以下目的基因的表达量:CIRP、TLR4、IRE1、X盒结合蛋白1(XBP1)、葡萄糖调节蛋白78(GRP78)、NF-κB、C反应蛋白(CRP)、DAO、IL-6及C/EBP同源蛋白(CHOP)。相关引物序列见表2。以甘油醛-3-磷酸脱氢酶(GAPDH)作为内参基因,采用2-ΔΔCt法计算各目的基因的mRNA相对表达量。
表2 实时荧光定量PCR分析所用引物序列

Table 2 Primer sequences for real-time quantitative PCR analysis

基因名称
Gene names		 引物序列
Primer sequences (5’—3’)		 登录号
Accession No.		 产物大小
Product size/bp		 退火温度
Tm/℃
甘油醛-3-磷酸脱氢酶
GAPDH		 F:GTGAAAGTCGGAGTCAACGG
R:CGTTCTCAGCCTTGACAGTG		 NM_204305.2 184 56.0
冷诱导RNA结合蛋白
CIRP		 F:CAGATGAGGGCAAGCTGTTC
R:ATGGCATCCTTTGCATCGTC		 KC905084.1 163 56.0
Toll样受体4
TLR4		 F:AGAACCTCAATGCGATGCAC
R:CAGTCCACTCAAGAGTCCGT		 KF697090.1 128 56.0
肌醇需求激酶1
IRE1		 F:TGTCCATCAACATCCCTGCT
R:CAGTCACCACCAGTCCATCT		 XM_046929413.1 137 56.0
X盒结合蛋白1
XBP1		 F:GTGAAGGAATCCCAGGTGGA
R:AGACTGTCCAGAAAGCCGAA		 NM_001006192.2 193 56.0
葡萄糖调节蛋白78
GRP78		 F:GGAATCCTTCGTGTCACAGC
R:TAGCTCTCCAGCTCATTCCG		 NM_205491.2 189 56.0
核因子-κB
NF-κB		 F:CTCTCCCAGCCCATCTATGA
R:CCTCAGCCCAGAAACGAAC		 NM_001396038.1 156 56.0
C反应蛋白
CRP		 F:CCAAAGCTCAGGACAACGAG
R:CGCGGAAGGTGACGTATTTC		 DQ374639.1 101 56.0
二胺氧化酶
DAO		 F:GACCACAAGTCCATCTGGGA
R:GTGGATGGTGATCCCAAAGC		 XM_040684680.2 112 56.0
白细胞介素-6
IL-6		 F:GGCTTCGACGAGGAGAAATG
R:AGAGACTCGACGTTCTGCTT		 NM_204628.2 165 56.0
C/EBP同源蛋白
CHOP		 F:GCCTCATGGCTCTGGATACT
R:CACAAACTCTGCCACCAACA		 NM_001031060.2 135 56.0

以上引物序列均从NCBI Gene数据库中获取。

Above primer sequences were obtained from NCBI gene library.

1.6 数据处理与分析

试验数据用Excel 2021进行整理和计算,结果以平均值和均值标准误(SEM)表示。采用SPSS 27.0软件进行统计分析:首先通过单因素方差分析(one-way ANOVA)结合Duncan氏法进行4组间的多重比较;然后运用一般线性模型(GLM)中的UNIVARIATE过程,对2×2因子设计进行主效应及互作效应分析。显著性水平设定为:P<0.05表示差异显著,P<0.01表示差异极显著,P>0.05表示差异不显著。

2 结果

2.1 谷氨酰胺和TAK-242对急性冷应激雏鸡肺组织中抗氧化指标的影响

如表3所示,互作效应分析表明,冷应激条件下,谷氨酰胺和TAK-242联合干预对雏鸡肺组织中CAT与GSH-Px活性、MDA含量、T-AOC存在显著或极显著的互作效应(P<0.05或P<0.01)。与ACS组相比,Gln组、TAK-242组和Gln/TAK-242组中CAT活性显著升高(P<0.05),SOD活性极显著升高(P<0.01),MDA含量显著下降(P<0.05);同时,Gln/TAK-242组中SOD活性极显著高于Gln组和TAK-242组(P<0.01),T-AOC显著高于TAK-242组(P<0.05)。主效应分析表明,谷氨酰胺干预显著或极显著提升CAT、SOD活性(P<0.05或P<0.01);TAK-242干预极显著升高SOD活性(P<0.01)。
表3 谷氨酰胺和TAK-242对急性冷应激雏鸡肺组织中抗氧化指标的影响

Table 3 Effects of Gln and TAK-242 on antioxidant indexes in lung tissue of chicks under acute cold stress

项目
Items		 过氧化氢酶
CAT/(U/mg prot)		 谷胱甘肽过氧化物酶
GSH-Px/(U/mg prot)		 丙二醛
MDA/(nmol/mg prot)		 总抗氧化能力
T-AOC/(mmol/g prot)		 超氧化物歧化酶
SOD/(U/mg prot)
组别Groups
ACS 25.704b 655 27.429a 1.592ab 515.1Cc
Gln 34.084a 777.2 18.361b 1.498ab 687.8Bb
TAK-242 31.215a 785 18.113b 1.361b 690.9Bb
Gln/TAK-242 32.006a 764.7 21.998b 1.823a 804.0Aa
SEM 1.014 4 18.17 1.114 2 0.065 0 24.71
主效应Main effects
谷氨酰胺Gln/%
0 28.459 720 22.771 1.476 603
1.2 33.045 771 20.179 1.661 745.9
P值P-value 0.013 0.139 0.167 0.117 <0.001
TAK-242/(mg/kg)
0 29.894 716.1 22.895 1.545 601.4
3 31.61 774.8 20.055 1.592 747.5
P值P-value 0.373 0.091 0.132 0.676 <0.001
互作效应(谷氨酰胺×TAK-242) Interaction effect (Gln×TAK-242)
P值P-value 0.035 0.044 0.002 0.024 0.32

同列数据肩标无字母或相同字母表示差异不显著(P>0.05),不同小写字母表示差异显著(P<0.05),不同大写字母表示差异极显著(P<0.01)。下表同。

In the same column, values with no letter or the same letter superscripts mean no significant difference (P>0.05), while with different small letter superscripts mean significant difference (P<0.05), and with different capital letter superscripts mean significant difference (P<0.01). The same as below.

2.2 谷氨酰胺和TAK-242对急性冷应激雏鸡肺组织中炎症和损伤相关指标的影响

如表4所示,互作效应分析表明,冷应激条件下谷氨酰胺和TAK-242联合干预对雏鸡肺组织中DAO、IL-6水平存在极显著的互作效应(P<0.01)。与ACS组相比,Gln组和Gln/TAK-242组中CIRP水平显著降低(P<0.05),且DAO、IL-6、TNF-α水平极显著降低(P<0.01),TAK-242组中DAO、IL-6水平极显著降低(P<0.01),TAK-242组和Gln/TAK-242组中IL-10水平均显著升高(P<0.05);同时,Gln/TAK-242组中TNF-α水平极显著低于Gln组和TAK-242组(P<0.01)。主效应分析表明,谷氨酰胺干预显著或极显著降低CIRP、DAO、IL-6、TNF-α水平(P<0.05或P<0.01);TAK-242干预显著降低DAO、TNF-α水平(P<0.05),极显著升高IL-10水平(P<0.01)。
表4 谷氨酰胺和TAK-242对急性冷应激雏鸡肺组织中炎症和损伤相关指标的影响

Table 4 Effects of Gln and TAK-242 on indicators associated with inflammation and injury in lung tissue of chicks with acute cold stress

项目
Items		 冷诱导RNA
结合蛋白
CIRP/(pg/
mg prot)		 二胺氧化酶
DAO/(ng/
mg prot)		 白细胞
介素-1β
IL-1β/(pg/
mg prot)		 白细胞
介素-6
IL-6/(pg/
mg prot)		 白细胞
介素-10
IL-10/(pg/
mg prot)		 肿瘤坏
死因子-α
TNF-α/(pg/
mg prot)
组别Groups
ACS 6.578a 5.636Aa 311.7 8.270Aa 27.083b 27.844Aa
Gln 4.319b 3.577Bb 251.4 4.520Bb 27.831ab 23.228Bb
TAK-242 5.067ab 3.849Bb 281.4 5.540Bb 33.008a 23.743ABab
Gln/TAK-242 4.864b 3.904Bb 277.4 6.009Bb 33.300a 19.049Cc
SEM 0.303 8 0.217 2 11.78 0.374 2 1.020 5 0.899 9
主效应Main effects
谷氨酰胺Gln/%
0 5.822 4.742 296.6 6.905 30.046 26.293
1.2 4.591 3.741 264.4 5.265 30.566 21.138
P值P-value 0.032 0.004 0.197 0.015 0.781 0.002
TAK-242/(mg/kg)
0 5.448 4.606 281.5 6.395 27.457 25.536
3 4.965 3.877 279.4 5.775 33.154 21.896
P值P-value 0.376 0.026 0.930 0.321 0.007 0.018
互作效应(谷氨酰胺×TAK-242) Interaction effect (Gln×TAK-242)
P值P-value 0.068 0.002 0.256 0.003 0.903 0.703

2.3 谷氨酰胺和TAK-242对急性冷应激雏鸡肺组织中CIRP-TLR4-IRE1信号通路及下游靶基因表达的影响

如表5所示,互作效应分析表明,冷应激条件下,谷氨酰胺和TAK-242联合干预对冷应激雏鸡肺组织中GRP78、CRP、DAO、IL-6、CHOP的mRNA相对表达量存在显著或极显著的互作效应(P<0.05或P<0.01)。与ACS组相比,Gln组、TAK-242组和Gln/TAK-242组中TLR4、GRP78、NF-κB、CRP、DAO、IL-6、CHOP的mRNA相对表达量均极显著下降(P<0.01),Gln组和Gln/TAK-242组中CIRP mRNA的相对表达量极显著下降(P<0.01),Gln/TAK-242组中IRE1的mRNA相对表达量显著下降(P<0.05);同时Gln/TAK-242组中TLR4、GRP78、DAO、IL-6的mRNA相对表达量极显著低于Gln组(P<0.01),CIRP、TLR4、NF-κB的mRNA相对表达量极显著低于TAK-242组(P<0.01)。主效应分析表明,谷氨酰胺干预显著或极显著降低CIRP、TLR4、IRE1、GRP78、NF-κB、CRP、DAO、IL-6的mRNA相对表达量(P<0.05或P<0.01);TAK-242干预显著或极显著降低TLR4、IRE1、GRP78、NF-κB、CRP、DAO、IL-6、CHOP的mRNA相对表达量(P<0.05或P<0.01)。
表5 谷氨酰胺和TAK-242对急性冷应激雏鸡肺组织中CIRP-TLR4-IRE1信号通路及下游靶基因表达的影响

Table 5 Effects of Gln and TAK-242 on CIRP-TLR4-IRE1 signal pathway and downstream target gene expression in lung tissue of chicks under acute cold stress

项目
Items		 冷诱导RNA
结合蛋白
CIRP		 Toll样受体4
TLR4		 肌醇需求
激酶1
IRE1		 X盒结合
蛋白1
XBP1		 葡萄糖调
节蛋白78
GRP78		 核因子-κB
NF-κB		 C反应蛋白
CRP		 二胺氧化酶
DAO		 白细胞
介素-6
IL-6		 C/EBP同
源蛋白
CHOP
组别Groups
ACS 1.022Aa 1.082Aa 1.004a 1.006 1.012Aa 1.015Aa 1.027Aa 1.012Aa 0.992Aa 0.946Aa
Gln 0.702Bb 0.557Bb 0.864ab 1.038 0.744Bb 0.749BCbc 0.565Bb 0.663Bb 0.547Bb 0.671Bb
TAK-242 1.096Aa 0.488Bb 0.849ab 1.025 0.393Cc 0.785Bb 0.439Bb 0.366Cc 0.444BCbc 0.303Cc
Gln/TAK-242 0.725Bb 0.088Cc 0.752b 0.954 0.349Cc 0.619Cc 0.406Bb 0.441Cc 0.328Cc 0.523BCbc
SEM 0.048 6 0.091 2 0.032 0 0.030 6 0.071 2 0.038 4 0.059 1 0.061 1 0.080 3 0.065 3
主效应Main effects
谷氨酰胺Gln/%
0 1.059 0.785 0.927 1.016 0.702 0.900 0.733 0.689 0.718 0.624
1.2 0.714 0.323 0.808 0.996 0.546 0.684 0.486 0.552 0.438 0.597
P值P-value <0.001 <0.001 0.034 0.769 0.007 <0.001 <0.001 0.037 <0.001 0.729
TAK-242/(mg/kg)
0 0.862 0.820 0.934 1.022 0.878 0.882 0.796 0.837 0.769 0.809
3 0.911 0.288 0.801 0.990 0.371 0.702 0.422 0.404 0.386 0.413
P值P-value 0.405 <0.001 0.019 0.627 <0.001 0.002 <0.001 <0.001 <0.001 <0.001
互作效应(谷氨酰胺×TAK-242) Interaction effect (Gln×TAK-242)
P值P-value 0.662 0.185 0.684 0.444 0.041 0.312 0.002 0.003 0.016 0.005

3 讨论

3.1 谷氨酰胺和TAK-242对急性冷应激雏鸡肺组织中氧化反应的影响

冷应激作为一种物理性环境应激源,会增加禽类在亚临床状态下对坏死性肠炎的易感性,并可引起多种组织器官损伤[13]。在冷应激过程中,动物体内的抗氧化水平会受到抑制[14]。本试验结果显示,冷应激条件下,单独饮水添加谷氨酰胺或腹腔注射TAK-242后,肺组织中CAT活性分别升高32.60%、21.44%,SOD活性分别升高33.53%、34.13%,同时MDA含量分别降低33.06%、33.96%。这表明谷氨酰胺能够有效调节肺组织中的氧化还原平衡,通过提高抗氧化酶CAT的活性,减少自由基的产生,从而降低MDA含量,同时对SOD活性的调节机制产生影响。已有研究表明,谷氨酰胺可使小肠中的氧化还原状态和增殖/细胞死亡率正常化[15]。而TAK-242作为TLR4的特异性抑制剂,可能通过阻断TLR4介导的信号通路,影响抗氧化酶活性来减轻损伤。对比急性冷应激组,谷氨酰胺和TAK-242联合使用后,肺组织中CAT、SOD活性分别升高24.52%、56.09%,MDA含量下降19.80%,与单独饮水添加谷氨酰胺或腹腔注射TAK-242相比,两者联合使用又能使SOD活性进一步极显著升高。这表明谷氨酰胺和TAK-242联合使用具有协同抗氧化作用,可能通过共同的机制调节冷应激诱发的氧化损伤。该结果可为开发针对冷应激的抗氧化干预措施提供理论依据。

3.2 谷氨酰胺和TAK-242对冷应激雏鸡肺组织中炎症和损伤相关指标的影响

冷应激会诱导胃肠道疾病,导致机体炎症反应加剧[16],补充谷氨酰胺能适度改善低体重仔猪的生长和牛奶摄入量,提示谷氨酰胺对肠道和肝脏功能有潜在影响[17]。
研究证实,饮水补充1.2%谷氨酰胺在很大程度上改善了冷应激对雏鸡的影响,促进了肠道和免疫系统的发育[18]。在本研究中,与急性冷应激组相比,单独饮水添加谷氨酰胺使肺组织中CIRP、DAO、IL-6、TNF-α水平分别降低34.34%、36.53%、45.34%、16.58%,从而有效缓解了冷应激引起的肺组织炎症反应。这一结果与Zhang等[19]的研究结果一致。TAK-242已被广泛用于抑制炎症反应的研究[20]。研究表明,TAK-242可通过抑制TLR4/NF-κB信号通路改善大鼠脓毒症相关急性肾损伤[21]。此外,TAK-242与粒细胞集落刺激因子(G-CSF)联合使用时可抑制炎症反应,促进肝脏再生[22]。本试验中,对比急性冷应激组,单独腹腔注射TAK-242使肺组织中DAO、IL-6水平分别降低31.71%、33.01%,同时IL-10水平升高21.88%。这表明谷氨酰胺可能通过抑制CIRP的表达从而减少炎症反应,而TAK-242直接抑制TLR4介导的促炎因子生成,促进抗炎细胞因子的产生,从而缓解肺组织炎症反应;TAK-242与谷氨酰胺联合使用时肺组织中IL-10水平升高22.96%,CIRP、IL-6和TNF-α水平分别降低26.06%、27.34%、31.59%;与单独饮水添加谷氨酰胺或腹腔注射TAK-242相比,TAK-242与谷氨酰胺联合使用又进一步显著降低肺组织中TNF-α水平。上述结果表明,TAK-242和谷氨酰胺联合使用具有协同抗炎效应,能有效调控炎症因子表达,缓解冷应激炎症。

3.3 谷氨酰胺和TAK-242对冷应激雏鸡肺组织中CIRP-TLR4-IRE1信号通路及下游基因表达的影响

Zhang等[23]研究发现,CIRP可介导脓毒症小鼠的炎症反应及肺、肾组织损伤。TLR4作为先天免疫与炎症反应的关键调控分子,其下调可通过抑制炎症和氧化应激减轻小鼠急性肺损伤[24]。研究表明,气道上皮细胞在体内外均组成性表达CIRP,且CIRP可能通过直接作用于TLR4/NF-κB信号通路,介导冷空气诱导的黏液产生[25]。NF-κB是TLR4信号转导的主要下游转录因子,在炎症基因调控中发挥核心作用[26-27]。此外,在低氧、能量耗竭或病原感染等应激条件下,内质网稳态失衡,可引发内质网应激,进而激活一系列信号通路,统称为未折叠蛋白反应(UPR);该反应通过3个信号通路分支[IRE1、活化转录因子6(ATF6)、蛋白激酶R样内质网激酶(PERK)]启动细胞适应性调控和保护机制,其中IRE1在炎症反应中起关键作用,以维持细胞和组织稳态[28]。本试验发现,与急性冷应激组相比,单独饮水添加谷氨酰胺使肺组织中CIRP、TLR4、GRP78、NF-κB、CRP、DAO、IL-6、CHOP的mRNA相对表达量降低26.21%~48.52%,这一结果表明谷氨酰胺可能通过抑制CIRP的表达,进而阻断TLR4介导的内质网应激发生,最终抑制下游NF-κB信号通路的激活,从而减轻炎症反应。这提示谷氨酰胺对肺组织的保护效应可能是通过调控CIRP-TLR4-IRE1信号轴实现的。单独腹腔注射TAK-242后,肺组织中TLR4、GRP78、NF-κB、CRP、DAO、IL-6、CHOP的mRNA相对表达量降低22.66%~67.97%,但CIRP、IRE1、XBP1的mRNA相对表达量无显著变化。这表明TAK-242可能通过抑制下游信号分子(如GRP78和NF-κB)来缓解冷应激引起的炎症反应,而不直接影响IRE1和XBP1的表达,这与前人研究结果[12,29]一致。此外,TAK-242与谷氨酰胺联合使用时,肺组织中CIRP、TLR4、IRE1、GRP78、NF-κB、CRP、DAO、IL-6、CHOP的mRNA相对表达量降低25.10%~91.87%,且TLR4的mRNA相对表达量显著低于单独饮水添加谷氨酰胺或腹腔注射TAK-242时。进一步分析发现,主效应TAK-242通过直接抑制TLR4基因表达(未影响CIRP基因表达)进而影响下游IRE1信号通路及炎症相关基因表达;谷氨酰胺在降低CIRP基因表达(表5)及其蛋白水平(表4)的前提下,抑制了TLR4、IRE1及下游基因表达,然而谷氨酰胺和TAK-242在影响TLR4基因表达方面不存在显著的互作效应,由此可以初步推测,谷氨酰胺是通过直接干预CIRP,并进而影响到TLR4-IRE1,从而发挥介导下游炎症相关因子作用的。然而,在此需要特别说明的是,由于当前国内外缺乏鸡属种的TLR4特异性抗体,本研究仅从基因表达水平初步揭示了谷氨酰胺缓解雏鸡肺组织急性冷应激损伤的CIRP-TLR4-IRE1信号通路机制,尚缺少相关信号分子蛋白表达水平分析(Western Blotting法)的进一步验证,此为本研究的一个局限性。随着今后相关抗体试剂的上市,我们团队将及时完善本研究项目。

4 结论

通过饮水补充谷氨酰胺、腹腔注射TLR4抑制剂TAK-242均能缓解冷应激导致的雏鸡肺组织炎症损伤,两者联合使用效果更为明显。谷氨酰胺缓解雏鸡肺组织冷应激炎症损伤的机制可能是通过调控CIRP-TLR4-IRE1信号通路,降低内质网应激效应介导的NF-κB及其下游靶基因表达,从而发挥其保护性干预作用。

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