Chinese Journal of Animal Nutrition >

2023 , Vol. 35 >Issue 4: 2296 - 2305

DOI: https://doi.org/10.12418/CJAN2023.216

RESEARCH PAPER

Effects of Deoxynivalenol and T-2 Toxin on Intestinal Barrier Function, Endoplasmic Reticulum Stress and Inflammatory Response of Laying Hens under Heat Stress

  • LOU Wenfang , 1, 2 ,
  • QIU Wenxin 2, * ,
  • GUO Haoneng 1, 2 ,
  • SHI Songming 3 ,
  • HUANG Bo 2, 4 ,
  • HAO Shu 2, 4 ,
  • YOU Jinming 1 ,
  • DAI Sifa , 2, 4, ** ,
  • LI Guanhong , 1, **
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  • 1 Engineering Research Center of Nutritional Feed Development, Jiangxi Province Key Laboratory of Animal Nutrition, College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang 330045, China
  • 2 Department of Pharmaceutical and Life Sciences, Jiujiang University, Jiujiang 332005, China
  • 3 Jiangxi Leadafy Bio-Pharmaceutical Co., Ltd., Jiujiang 330400, China
  • 4 Jiujiang Bozheng Institute of Biotechnology Industry, Jiujiang 332005, China
** DAI Sifa, professor, E-mail: ;
LI Guanhong, professor, E-mail:

*Contributed equally

Received date: 2022-09-21

  Online published: 2023-04-12

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Abstract

This experiment was conducted to investigate the effects of deoxynivalenol (DON) and T-2 toxin on intestinal barrier function, endoplasmic reticulum stress and inflammatory response of laying hens under heat stress. Ninety-six 36-week-old Jinghong No.1 laying hens with similar body weight were randomly divided into 4 groups with 8 replicates per group and 3 hens per replicate. Broilers in the control group (CON group) were fed a basal diet, and others in DON group, T-2 group and DON+T-2 group were fed basal diets supplemented with 3.00 mg/kg DON, 0.50 mg/kg T-2 toxin and 1.50 mg/kg DON+0.25 mg/kg T-2 toxin, respectively. The pretrial period lasted for 3 days, and the trial period lasted for 28 days. The average daytime temperature and humidity index was 84.87. The results showed as follows: 1) compared with CON group, the duodenal and jejunal crypt depth in T-2 group was significantly increased (P<0.05), the jejunal crypt depth in DON+T-2 group was significantly increased (P<0.05), the duodenal villus height/crypt depth (V/C) value in DON, T-2 and DON+T-2 groups was significantly decreased (P<0.05), the jejunal V/C value in T-2 and DON+T-2 groups was significantly decreased (P<0.05), and the ileal crypt depth in DON group was significantly increased (P<0.05). 2) Compared with CON group, the serum lipopolysaccharide (LPS) content in DON, T-2 and DON+T-2 groups was significantly increased (P<0.05), and the serum diamine oxidase (DAO) content in DON+T-2 group was significantly increased (P<0.05). 3) Compared with CON group, the ileum mucosal interleukin-6 (IL-6) content in DON, T-2 and DON+T-2 groups was significantly increased (P<0.05). 4) Compared with CON group, the ileum mucosal Occludin mRNA relative expression levels in T-2 and DON+T-2 groups was significantly decreased (P<0.05). 5) Compared with CON group, the mRNA relative expression levels of glucose regulatory protein 78 (GRP78) and X-box binding protein 1 (XBP1) in ileum mucosa in DON+T-2 group were significantly increased (P<0.05), and the ileum mucosal activated transcription factor 4 (ATF4) mRNA relative expression level in DON group was significantly increased (P<0.05). In conclusion, under the condition of summer heat stress, dietary supplementation of 3.00 mg/kg DON and 0.50 mg/kg T-2 can induce endoplasmic reticulum stress and inflammation in small intestine tissues of laying hens, and damage mucosal morphology and barrier function, and the two have detailed cumulative effects.

Key words： deoxynivalenol; T-2 toxin; heat stress; laying hens; intestinal barrier function; endoplasmic reticulum stress

Cite this article

LOU Wenfang , QIU Wenxin , GUO Haoneng , SHI Songming , HUANG Bo , HAO Shu , YOU Jinming , DAI Sifa , LI Guanhong . Effects of Deoxynivalenol and T-2 Toxin on Intestinal Barrier Function, Endoplasmic Reticulum Stress and Inflammatory Response of Laying Hens under Heat Stress[J]. Chinese Journal of Animal Nutrition, 2023 , 35(4) : 2296 -2305 . DOI: 10.12418/CJAN2023.216

我国南方地区夏季气候环境以高温高湿为普遍特征,极易造成饲料原料及配合饲料发生霉变,进而产生大量霉菌毒素。同时,南方夏季炎热潮湿,对于高密度养殖的蛋鸡,热应激问题时常存在。在热应激条件下,蛋鸡自身抵抗力下降,这时饲粮中的霉菌毒素对蛋鸡肠道的损伤会大大加重。脱氧雪腐镰刀菌烯醇(DON,又名呕吐毒素)、T-2毒素同属于单端孢霉烯族类毒素。DON属于B型单端孢霉烯族类毒素[1],能抑制蛋白质、DNA和RNA的合成,增加肠上皮的渗透性,损伤脑组织,有很强的细胞毒性、胃肠毒性和神经毒性[2-3]。T-2毒素属于A型单端孢霉烯族类毒素,具有细胞毒性,能抑制蛋白质合成,诱导细胞凋亡和程序性细胞死亡[4-5]。
家禽的肠道不仅具有消化吸收功能,同时也具有阻止肠腔内的有害物质进入机体的屏障功能[6]。肠道黏膜上皮屏障主要由机械屏障、化学屏障、免疫屏障和微生物屏障组成,其屏障功能完整性对于维持肠道稳态乃至机体健康至关重要[7]。霉菌毒素污染会作用于肠上皮细胞内质网,导致未折叠或错误折叠的蛋白质在内质网腔中过量累积,从而引发内质网应激,持续的内质网应激则会导致动物肠黏膜屏障损伤并诱发肠道炎症[8-9]。研究报道,5 μmol/L黄曲霉毒素B1可引发牛乳腺上皮细胞内质网应激并诱导细胞凋亡,3-乙酰脱氧雪腐镰刀菌烯醇可诱导巨噬细胞Raw264.7内质网应激相关蛋白表达水平升高[10-11]。用DON处理离体培养的猪空肠组织会导致肠绒毛变短、肠细胞裂解和水肿[12]。DON还可降低猪回肠组织中闭锁蛋白(Occludin)和闭合蛋白-1(Claudin-1)的基因表达[13]。DON与玉米赤霉烯酮(ZEA)联合会诱发仔猪血清和空肠炎症反应[14]。
目前,国内外对霉菌毒素的毒性研究大多局限于单一毒素,DON与T-2毒素联合摄入对蛋鸡肠道屏障功能的影响报道尚少。因此,本试验以蛋鸡为研究对象,在夏季自然高温环境下,探讨DON与T-2毒素单一及联合暴露对热应激蛋鸡肠道屏障功能及内质网应激和炎症反应的影响,并进一步明确二者之间是否存在累加效应,为霉菌毒素单一及联合暴露在家禽肠道屏障方面的研究提供试验依据。

1 材料与方法

1.1 试验材料

DON购自裕菁科技(上海)有限公司,纯度>99%;T-2毒素由北京普华仕科技发展有限公司提供,纯度>99%。

1.2 试验设计

选择36周龄体重相近的京红1号蛋鸡96只,随机分成4个组,每组8个重复,每个重复3只鸡。对照组(CON组)饲喂基础饲粮,DON组、T-2组、DON+T-2组分别在基础饲粮中添加3.00 mg/kg DON、0.50 mg/kg T-2毒素、1.50 mg/kg DON+0.25 mg/kg T-2毒素。预试期3 d,正试期28 d。霉菌毒素添加剂量参照《饲料卫生标准》(GB 13078—2017)[15]中禽配合饲料允许的最大限量值设定。

1.3 试验饲粮及饲养管理

基础饲粮组成及营养水平见表1。试验于2021年7—8月份期间进行,蛋鸡采用阶梯式笼养,舍内维持16 h光照,每天07:00、12:30各饲喂1次,自由采食和饮水,按常规免疫程序进行免疫接种。试验期间每天09:00平均温度(29.2±0.26) ℃、平均相对湿度(97.25±0.01)%、平均温湿指数82.62±0.43,14:00平均温度(32.82±0.56) ℃、平均相对湿度(77.68±0.03)%、平均温湿指数86.29±0.48,19:00平均温度(31.94±0.53) ℃、平均相对湿度(81.71±0.03)%、平均温湿指数85.69±0.52。
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) %

原料Ingredients 含量Content 营养水平Nutrient levels2) 含量Content
玉米Corn 62.07 代谢能Metabolic energy/(MJ/kg) 10.94
豆粕Soybean meal 23.65 粗蛋白质Crude protein 16.52
小麦麸Wheat bran 1.96 赖氨酸Lysine 0.85
石粉Limestone 7.39 蛋氨酸Methionine 0.34
预混料Premix1) 4.93 钙Calcium 3.60
合计Total 100.00 有效磷Available phosphorus 0.34

1)预混料为每千克饲粮提供 The premix provided the following per kg of the diet:VA 8 874~9 862 IU,VE≥16.3 IU,VD3 1 972~4 930 IU,VK3≥3.0 mg,VB1≥1.3 mg,VB6≥3.5 mg,VB2≥6.9 mg,VB12≥0.018 mg,叶酸 folic acid≥0.89 mg,烟酰胺 nicotinamide≥27.12 mg,D-生物素 D-biotin≥0.06 mg,D-泛酸 D-pantothenic acid≥11.83 mg,Fe 59.16~640.90 mg,Cu 19.72~493.00 mg,Mn 24.65~128.18 mg,Zn 39.44~98.6 mg,I 0.39~3.94 mg,Se 0.15~0.49 mg,NaCl 3 g,CaHPO4 15 g。

2)营养水平均为计算值。Nutrient levels were calculated values.

1.4 样品采集

试验结束后,各重复随机抽取2只蛋鸡翅静脉采血,4 ℃、3 000×g离心10 min,分离血清于-20 ℃保存。屠宰打开腹腔后采集十二指肠、空肠、回肠各2 cm,用4%多聚甲醛固定,用于制作切片。另取十二指肠、空肠、回肠肠段,快速刮取肠黏膜,放入冻存管于-80 ℃冰箱保存。

1.5 指标测定及方法

1.5.1 小肠组织形态的测定

取十二指肠、空肠、回肠制作石蜡切片,苏木精-伊红(HE)染色,通过光学显微镜拍照,利用Image-Pro Plus 6.0测量绒毛高度和隐窝深度,并计算绒毛高度/隐窝深度(V/C)值。

1.5.2 血清二胺氧化酶(DAO)、D-乳酸(D-LA)、脂多糖(LPS)含量的测定

采用试剂盒测定血清DAO、D-LA、LPS含量,试剂盒购自南京建成生物工程研究所,按试剂盒说明书进行操作。

1.5.3 回肠黏膜细胞因子含量的测定

采用酶联免疫吸附测定(ELISA)试剂盒测定回肠黏膜肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、白细胞介素-8(IL-8)含量,试剂盒购自上海酶联生物科技有限公司,按试剂盒说明书进行操作。

1.5.4 回肠黏膜基因表达测定

将回肠黏膜与TransZol溶液以1∶9(质量体积比)添加于2 mL离心管中,用高速组织研磨仪制备黏膜匀浆,然后提取黏膜匀浆中总RNA,将提取好的总RNA再反转录为cDNA,-20 ℃冰箱保存。测定Occludin、Claudin-1、闭锁小带蛋白-1(ZO-1)、葡萄糖调节蛋白78(GRP78)、肌醇需求酶1(IRE1)、蛋白激酶受体样内质网激酶(PERK)、活化转录因子4(ATF4)、X盒结合蛋白1(XBP1)基因表达,所用试剂购于北京全式金生物技术有限公司,试验操作步骤严格按照说明书进行。引物序列见表2。以甘油醛-3-磷酸脱氢酶(GAPDH)为内参基因,采用2-△△Ct法定量上述目的基因的mRNA相对表达量。
表2 引物序列

Table 2 Primer sequence

基因
Genes		 引物序列
Primer sequence (5'—3')		 GenBank登录号
GenBank accession No.
甘油醛-3-磷酸脱氢酶
GAPDH		 F:GACAGCCATTCCTCCACCTT
R:TTGGATGCCATGTGGACCAT		 NM_204305
闭合蛋白-1
Claudin-1		 F:CATACTCCTGGGTCTGGTTGGT
R:GACAGCCATCCGCATCTTCT		 GI:464148
闭锁蛋白
Occludin		 F:ACGGCAGCACCTACCTCAA
R:GGGCGAAGAAGCAGATGAG		 AY75089.1
闭锁小带蛋白-1
ZO-1		 F:CAGCGAAAGAAGGATTAGAGGAA
R:CAGCGAAAGAAGGATTAGAGGAA		 XM_015278981.1
葡萄糖调节蛋白78
GRP78		 F:GGAATCCTTCGTGTCACAGC
R:TAGCTCTCCAGCTCATTCCG		 NM_205491.2
肌醇需求酶1
IRE1		 F:TGTCCATCAACATCCCTGCT
R:CAGTCACCACCAGTCCATCT		 NM_001285499.2
蛋白激酶受体样内质网激酶
PERK		 F:ATCCGTTTGCCTAACAGGGA
R:GCCAATCGGTGCTTTCATCT		 XM_040671515.1
活化转录因子4
ATF4		 F:CGGATCACCCAATGACAACC
R:TTGCTGACGCTACCTTCTCA		 NM_204880.3
X盒结合蛋白1
XBP1		 F:GTGAAGGAATCCCAGGTGGA
R:AGACTGTCCAGAAAGCCGAA		 NM_001006192.2

以上引物序列均从NCBI基因文库中获取。

Above primer sequences were obtained from NCBI gene libraries.

1.6 数据统计分析

试验采用SPSS 22.0进行单因素方差分析(one-way ANOVA),Duncan氏法进行多重比较,各组数据表示为平均值和均值标准误(SEM),P<0.05为差异显著。

2 结果

2.1 DON与T-2毒素对热应激蛋鸡小肠组织形态结构的影响

由表3可知,与CON组相比,T-2组十二指肠、空肠隐窝深度显著升高(P<0.05),DON+T-2组空肠隐窝深度显著升高(P<0.05),DON、T-2、DON+T-2组十二指肠V/C值显著降低(P<0.05),T-2、DON+T-2组空肠V/C值显著降低(P<0.05),DON组回肠隐窝深度显著升高(P<0.05)。各组之间十二指肠、空肠、回肠绒毛高度及回肠V/C值均无显著差异(P>0.05)。
表3 DON与T-2毒素对热应激蛋鸡小肠组织形态结构的影响

Table 3 Effects of DON and T-2 toxin on morphological structure of small intestine of laying hens under heat stress

项目
Items		 组别Groups SEM P值
P-value
CON DON T-2 DON+T-2
十二指肠Duodenum
绒毛高度Villus height/μm 729.57 663.38 675.09 665.23 19.93 0.627
隐窝深度Crypt depth/μm 296.49b 347.31ab 380.36a 332.94ab 10.73 0.029
绒毛高度/隐窝深度V/C 2.52a 1.93b 1.80b 1.83b 0.10 0.023
空肠Jejunum
绒毛高度Villus height/μm 591.04 579.80 543.26 458.22 18.39 0.063
隐窝深度Crypt depth/μm 259.05b 299.05ab 369.23a 337.31a 14.54 0.022
绒毛高度/隐窝深度V/C 2.37a 2.01ab 1.49b 1.56b 0.11 0.007
回肠Ileum
绒毛高度Villus height/μm 449.26 445.91 395.32 369.64 12.27 0.073
隐窝深度Crypt depth/μm 233.80b 285.66a 212.16b 250.64ab 8.32 0.004
绒毛高度/隐窝深度V/C 1.87 1.59 1.91 1.55 0.07 0.256

同行数据肩标不同小写字母表示差异显著(P<0.05),相同或无字母表示差异不显著(P>0.05)。下表同。

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

2.2 DON与T-2毒素对热应激蛋鸡血清DAO、D-LA、LPS含量的影响

由表4可知,与CON组相比,DON、T-2、DON+T-2组血清LPS含量显著升高(P<0.05);DON+T-2组血清DAO含量显著升高(P<0.05)。各组之间血清D-LA含量无显著差异(P>0.05)。
表4 DON与T-2毒素对热应激蛋鸡血清DAO、D-LA、LPS含量的影响

Table 4 Effects of DON and T-2 toxin on contents of DAO, D-LA and LPS in serum of laying hens under heat stress

项目
Items		 组别Groups SEM P值
P-value
CON DON T-2 DON+T-2
脂多糖LPS/(EU/L) 0.80b 2.49a 2.66a 2.70a 0.28 0.010
二胺氧化酶DAO/(ng/mL) 0.89b 0.54b 1.56ab 2.20a 0.23 0.039
D-乳酸D-LA/(μmol/L) 2.36 2.52 2.33 2.53 0.20 0.983

2.3 DON与T-2毒素对热应激蛋鸡回肠黏膜紧密连接蛋白mRNA相对表达量的影响

由表5可知,与CON组相比,T-2、DON+T-2组回肠黏膜Occludin mRNA相对表达量显著降低(P<0.05)。各组之间回肠黏膜Claudin-1、ZO-1 mRNA相对表达量均无显著差异(P>0.05)。
表5 DON与T-2毒素对热应激蛋鸡回肠黏膜紧密连接蛋白mRNA相对表达量的影响

Table 5 Effects of DON and T-2 toxin on mRNA relative expression levels of tight junction protein in ileum mucosa of laying hens under heat stress

项目
Items		 组别Groups SEM P值
P-value
CON DON T-2 DON+T-2
闭锁蛋白Occludin 1.11a 0.88ab 0.76b 0.73b 0.05 0.025
闭合蛋白-1 Claudin-1 0.98 0.93 0.98 0.98 0.03 0.891
闭锁小带蛋白-1 ZO-1 1.01 1.04 0.92 1.12 0.05 0.465

2.4 DON与T-2毒素对热应激蛋鸡回肠黏膜炎性细胞因子含量的影响

由表6可知,与CON组相比,DON、T-2组回肠黏膜IL-6含量显著升高(P<0.05)。各组之间回肠黏膜IL-1β、IL-8、TNF-α含量均无显著差异(P>0.05)。
表6 DON与T-2毒素对热应激蛋鸡回肠黏膜炎性细胞因子含量的影响

Table 6 Effects of DON and T-2 toxin on contents of inflammatory cytokines in ileum mucosa of heat stressed laying hens pg/mL

项目
Items		 组别Groups SEM P值
P-value
CON DON T-2 DON+T-2
白细胞介素-1β IL-1β 75.590 72.380 76.659 81.747 1.616 0.186
白细胞介素-6 IL-6 3.745c 5.222ab 5.982a 4.440bc 0.235 0.001
白细胞介素-8 IL-8 18.481 17.093 17.309 17.412 0.334 0.639
肿瘤坏死因子-α TNF-α 13.743 12.308 12.121 13.667 0.349 0.205

2.5 DON与T-2毒素对热应激蛋鸡回肠黏膜内质网应激相关基因mRNA相对表达量的影响

由表7可知,与CON组相比,DON+T-2组回肠黏膜GRP78、XBP1 mRNA相对表达量均显著升高(P<0.05),DON组回肠黏膜ATF4 mRNA相对表达量显著升高(P<0.05)。各组之间回肠黏膜IRE1、PERK mRNA相对表达量均无显著差异(P>0.05)。
表7 DON与T-2毒素对热应激蛋鸡回肠黏膜内质网应激相关基因mRNA相对表达量的影响

Table 7 Effects of DON and T-2 toxin on mRNA relative expression levels of endoplasmic reticulum stress-related genes in ileum mucosa of laying hens under heat stress

项目
Items		 组别Groups SEM P值
P-value
CON DON T-2 DON+T-2
葡萄糖调节蛋78 GRP78 1.05b 1.07b 1.00b 1.54a 0.07 0.002
肌醇需求酶1 IRE1 1.05 0.98 1.01 1.12 0.03 0.494
蛋白激酶受体样内质网激PERK 1.02 0.95 0.88 0.86 0.06 0.815
活化转录因子4 ATF4 1.04b 1.42a 1.15ab 1.19ab 0.10 0.049
X盒结合蛋白1 XBP1 1.02b 1.16b 1.04b 1.58a 0.06 0.001

3 讨论

依据前人研究,产蛋鸡轻度热应激为温湿指数≥78,严重热应激为温湿指数≥83[16]。本试验中3个时间段平均温湿指数为84.87,表明本试验蛋鸡已经处于严重热应激状态。从产蛋性能分析,3.00 mg/kg DON与0.50 mg/kg T-2毒素单独暴露均无显著影响;两者联合暴露相较于对照组,36~37周龄、38~39周龄的平均日采食量分别降低了13.23%和7.50%,36~39周龄平均日采食量和平均蛋重分别降低了10.21%和6.87%,38~39周龄平均蛋重降低了11.31%。

3.1 DON与T-2对热应激蛋鸡小肠组织形态结构的影响

肠道是动物机体吸收各种营养物质的主要场所[17],其健康发育对家禽生长至关重要。小肠绒毛高度、隐窝深度及V/C值是衡量肠道机械屏障的重要指标[18-19]。绒毛高度越高与食糜接触的表面积就越多,对营养物质的吸收越充分;隐窝深度与肠上皮细胞的增殖率成反比,隐窝深度变浅表明肠黏膜上皮绒毛吸收能力增强[20]。V/C值则是小肠功能的综合体现,V/C值降低说明小肠的消化吸收能力减弱。研究表明,霉菌毒素可通过破坏肠上皮的形态和组织完整性来破坏肠黏膜的物理屏障功能[21]。DON促使猪和鸡肠道上皮病变,例如肠绒毛顶端坏死、绒毛萎缩、肠细胞出现空泡、固有层水肿等[22⇓-24],而关于T-2毒素对家禽肠道屏障功能方面的影响少见报道。在本研究中,DON组回肠、T-2组十二指肠和空肠、DON+T-2组空肠隐窝深度显著升高,T-2、DON+T-2组十二指肠、空肠V/C值显著降低,DON组十二指肠V/C值显著降低。这说明DON(3.00 mg/kg)、T-2毒素(0.50 mg/kg)以及二者剂量减半(1.50 mg/kg DON+0.25 mg/kg T-2毒素)联合暴露均会损伤热应激蛋鸡的小肠黏膜组织形态,降低小肠上皮细胞增殖率,减弱肠道上皮绒毛的吸收能力,不利于肠道对营养物质的吸收。

3.2 DON与T-2毒素对热应激蛋鸡小肠组织屏障损伤的影响

肠道黏膜屏障功能和结构的完整性对维持家禽肠道健康有重要意义。肠道上皮细胞主要是通过紧密连接蛋白来维持细胞极性和调节黏膜屏障通透性的。闭合蛋白(Claudin)、Occludin、ZO-1是几个关键的紧密连接蛋白。研究发现,Occludin、Claudin-1和ZO-1会影响肠道通透性,因此在肠道紧密连接屏障中发挥着重要作用[25]。Pinton等[26]研究发现,DON通过降低紧密连接蛋白Claudin-4的表达,导致猪肠道上皮屏障功能损伤。Osselaere等[24]利用猪肠上皮细胞研究表明,单端孢霉烯族化合物通过降低紧密连接蛋白的表达来增加肠道通透性。在本研究中,饲粮中0.50 mg/kg T-2毒素和1.50 mg/kg DON+0.25 mg/kg T-2毒素暴露使得热应激蛋鸡回肠黏膜Occludin mRNA相对表达量显著降低。这表明饲粮中0.50 mg/kg T-2毒素、1.50 mg/kg DON+0.25 mg/kg T-2毒素暴露可增加家禽肠道通透性,不利于抵御病原菌入侵,破坏了肠道紧密连接结构,进而降低产蛋鸡对营养物质的吸收效率。LPS、DAO、D-LA可作为反映肠道屏障功能损伤的理想指标。当肠道黏膜屏障功能受损时,肠道通透性会增加,血液中DAO、D-LA、LPS含量会增加。张聪[27]研究表明,4 mg/kg DON暴露会提高仔猪血清DAO含量。在本研究中,饲粮中3.00 mg/kg DON与0.50 mg/kg T-2毒素单独暴露显著提高热应激蛋鸡血清LPS含量,两者联合暴露显著提高产蛋鸡血清LPS、DAO含量。这表明DON与T-2毒素单独及联合暴露均导致热应激蛋鸡肠道通透性增加,这与本试验发现T-2组、DON+T-2组回肠黏膜Occludin mRNA相对表达量显著降低结果相一致。因此我们推测DON和T-2毒素可能通过影响肠道紧密连接蛋白表达、增加肠道通透性的方式来损伤产蛋鸡肠道机械屏障功能,从而抑制肠道对营养物质的消化吸收。

3.3 DON与T-2毒素对热应激蛋鸡小肠组织内质网应激和炎症反应的影响

霉菌毒素会引发动物机体发生内质网应激。内质网应激会激活未折叠蛋白质反应。过度的内质网应激可通过诱导肠上皮细胞凋亡和肠道促炎反应引起炎性肠病[28]。大量的研究表明,在肠上皮细胞中发生内质网应激时,常常伴随GRP78/免疫球蛋白结合蛋白(BiP)mRNA相对表达量的显著升高[29-30]。在本研究中,DON+T-2组热应激蛋鸡回肠黏膜GRP78 mRNA相对表达量显著升高,提示1.50 mg/kg DON+0.25 mg/kg T-2毒素引起热应激蛋鸡回肠发生了内质网应激。未折叠蛋白质反应共包含IRE1、PERK、ATF6 3条信号通路。细胞受到刺激诱发内质网应激时,IRE1会利用其核酸内切酶活性剪切XBP1,使其成为有功能活性的XBP1剪接异构体。在本研究中,DON+T-2组热应激蛋鸡回肠黏膜XBP1 mRNA相对表达量显著升高,提示DON与T-2毒素联合通过激活IRE1信号通路来引发肠内质网应激,从而损伤肠道黏膜。此外,肌醇需求酶1α(IRE1α)是将内质网应激与炎症反应串联起来的关键因子。IRE1自身磷酸化并与肿瘤坏死因子受体相关因子2(TRAF2)结合,以IRE1α-TRAF2的复合形式招募核因子κB抑制蛋白(IκB),使得核因子-κB(NF-κB)被解离进入细胞核,进而诱发炎症基因转录。在本研究中,DON、T-2组热应激蛋鸡回肠黏膜IL-6含量显著升高,这可能是由于毒素诱发了内质网应激,内质网应激又诱发了肠道炎症反应引起的。ATF4是PERK信号通路中真核翻译起始因子2亚基α(eIF2α)的下游信号分子,当发生内质网应激时,活化的PERK会使eIF2α磷酸化,进一步研究发现,eIF2α磷酸化后可选择性促进ATF4的翻译[31]。在本研究中,DON组热应激蛋鸡回肠黏膜ATF4 mRNA相对表达量显著升高,提示3.00 mg/kg DON通过激活未折叠蛋白质反应中PERK信号通路引发的肠内质网应激。以上结果表明,3.00 mg/kg DON、1.50 mg/kg DON+0.25 mg/kg T-2毒素分别通过激活PERK、IRE1信号通路诱发热应激蛋鸡回肠黏膜发生内质网应激,且进一步诱发了肠道炎症反应。

4 结论

① 饲粮DON与T-2毒素暴露可能通过诱发热应激产蛋鸡小肠组织细胞内质网应激和炎症反应的机制,进而增加小肠组织通透性,降低紧密连接蛋白表达,损伤黏膜组织形态结构,从而破坏夏季热应激条件下36~39周龄产蛋鸡肠道屏障功能。
② DON与T-2毒素联合作用较单独作用的负面影响更大,说明DON和T-2毒素在热应激条件下存在一定的累加效应。

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