鞣花酸对产肠毒素大肠杆菌K88攻毒断奶仔猪生长性能、抗氧化功能及核因子E2相关因子2/Kelch样ECH关联蛋白1信号通路的影响

赵三川; 王芳; 艾贤凯; 向永骞; 田洁; 符嘉玲; 符晨星; 贺建华

doi:10.12418/CJAN2025.414

动物营养学报 >

2025 , Vol. 37 >Issue 8: 5070 - 5083

DOI: https://doi.org/10.12418/CJAN2025.414

研究论文

鞣花酸对产肠毒素大肠杆菌K88攻毒断奶仔猪生长性能、抗氧化功能及核因子E2相关因子2/Kelch样ECH关联蛋白1信号通路的影响

赵三川 ,
王芳 ,
艾贤凯 ,
向永骞 ,
田洁 ,
符嘉玲 ,
符晨星 ^,^* ,
贺建华

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湖南农业大学动物科学技术学院,长沙 410128

^*符晨星,副教授,硕士生导师,E-mail: chenxing1110@outlook.com

赵三川(2002—),男,安徽阜阳人,硕士研究生,从事动物营养与肉质调控研究。E-mail: 905393147@qq.com

Copy editor: 靳爽

收稿日期: 2024-12-20

网络出版日期: 2025-08-14

基金资助

湖南省教育厅科学研究项目重点项目(21A0124)

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Effects of Ellagic Acid on Growth Performance, Antioxidant Function and Nuclear Factor E2 Related Factor 2/Kelch-Like ECH-Associated Protein 1 Signaling Pathway of Weaned Piglets Challenged with Enterotoxigenic Escherichia coli K88

ZHAO Sanchuan ,
WANG Fang ,
AI Xiankai ,
XIANG Yongqian ,
TIAN Jie ,
FU Jialing ,
FU Chenxing ^,^* ,
HE Jianhua

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College of Animal Science and Technology, Hunan Agricultural University, Changsha 410128, China

^*associate professor, E-mail: chenxing1110@outlook.com

Received date: 2024-12-20

Online published: 2025-08-14

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摘要

本试验以含有菌毛K88的产肠毒素大肠杆菌(ETEC)诱导建立断奶仔猪氧化应激模型,旨在研究鞣花酸(EA)对ETEC K88攻毒断奶仔猪生长性能、抗氧化功能及核因子E2相关因子2/Kelch样ECH关联蛋白1(Nrf2/Keap1)信号通路的影响。选取24头遗传背景一致、体重[(7.83±0.09) kg]接近的21日龄“杜×长×大”断奶仔猪,随机分为3组,每组8个重复,每个重复1头猪。对照组(CON组)和腹泻模型组(ETEC组)饲喂基础饲粮,EA-腹泻模型组(EA-ETEC组)饲喂在基础饲粮中添加500 mg/kg EA的饲粮。预试期3 d,正试期18 d。在正试期第16~18天,ETEC组和EA-ETEC组每头断奶仔猪每天灌服10 mL浓度为2×10⁹ CFU/mL的ETEC K88菌液,CON组断奶仔猪灌服相同体积的LB无菌培养液。结果表明:1)攻毒前,与ETEC组相比,EA-ETEC组断奶仔猪平均日采食量(ADFI)和平均日增重(ADG)显著提高(P<0.05),第15天体重有提高趋势(P=0.083);CON组与ETEC组之间以及ETEC组与EA-ETEC组之间断奶仔猪腹泻率均无显著差异(P>0.05)。攻毒期间,与CON组相比,ETEC组断奶仔猪第18天体重、ADFI和ADG均显著降低(P<0.05),腹泻率显著提高(P<0.05);与ETEC组相比,EA-ETEC组断奶仔猪第18天体重和ADFI显著提高(P<0.05),腹泻率显著降低(P<0.05)。整个试验期间,与CON组相比,ETEC组断奶仔猪ADFI和ADG显著降低(P<0.05),料重比(F/G)显著提高(P<0.05);与ETEC组相比,EA-ETEC组断奶仔猪ADFI和ADG显著提高(P<0.05),F/G显著降低(P<0.05)。2)攻毒后,与CON组相比,ETEC组断奶仔猪脾脏指数显著降低(P<0.05),肺脏指数显著提高(P<0.05);与ETEC组相比,EA-ETEC组断奶仔猪脾脏指数显著提高(P<0.05),胃指数有提高的趋势(P=0.065)。3)攻毒后,与CON组相比,ETEC组断奶仔猪空肠黏膜超氧化物歧化酶(SOD)活性和总抗氧化能力(T-AOC)显著降低(P<0.05),空肠黏膜丙二醛(MDA)含量显著提高(P<0.05);与ETEC组相比,EA-ETEC组断奶仔猪空肠黏膜SOD活性和T-AOC显著提高(P<0.05),空肠黏膜MDA含量显著降低(P<0.05)。4)攻毒后,与CON组相比,ETEC组断奶仔猪空肠黏膜SOD1、SOD2和谷胱甘肽过氧化物酶1(GSH-Px1)的mRNA相对表达量显著降低(P<0.05);与ETEC组相比,EA-ETEC组断奶仔猪空肠黏膜SOD2和GSH-Px1的mRNA相对表达量显著提高(P<0.05),空肠黏膜SOD1的mRNA相对表达量有提高趋势(P=0.083)。5)攻毒后,与CON组相比,ETEC组断奶仔猪空肠黏膜Keap1的mRNA相对表达量显著提高(P<0.05),空肠黏膜Nrf2的mRNA相对表达量显著降低(P<0.05);与ETEC组相比,EA-ETEC组断奶仔猪空肠黏膜Keap1的mRNA相对表达量显著降低(P<0.05),空肠黏膜Nrf2的mRNA相对表达量显著提高(P<0.05)。综上所述,饲粮中添加500 mg/kg EA能够缓解ETEC K88攻毒导致的断奶仔猪生长性能下降和肠道损伤。同时,EA可通过调控抗氧化信号通路,调节空肠黏膜抗氧化基因表达、抗氧化酶活性及过氧化物含量,从而有效缓解ETEC K88攻毒引发的氧化应激。

关键词： 鞣花酸; 产肠毒素大肠杆菌K88; 断奶仔猪; 生长性能; 抗氧化功能; Nrf2/Keap1信号通路

本文引用格式

赵三川 , 王芳 , 艾贤凯 , 向永骞 , 田洁 , 符嘉玲 , 符晨星 , 贺建华 . 鞣花酸对产肠毒素大肠杆菌K88攻毒断奶仔猪生长性能、抗氧化功能及核因子E2相关因子2/Kelch样ECH关联蛋白1信号通路的影响[J]. 动物营养学报, 2025 , 37(8) : 5070 -5083 . DOI: 10.12418/CJAN2025.414

Abstract

This experiment established an oxidative stress model in weaned piglets induced by enterotoxigenic Escherichia coli (ETEC) with fimbriae K88, aiming to investigate the effects of ellagic acid (EA) on growth performance, antioxidant function, and nuclear factor E2-related factor 2/Kelch-like ECH-associated protein 1 (Nrf2/Keap1) signaling pathway of weaned piglets challenged with ETEC K88. Twenty-four 21-day-old Duroc×Landrace×Yorkshire weaned piglets with consistent genetic background and similar body weight [(7.83±0.09) kg] were randomly divided into 3 groups, with 8 replicates per group and 1 pig per replicate. Piglets in the control group (CON group) and diarrhea model group (ETEC group) were fed the basal diet, while those in the EA-diarrhea model group (EA-ETEC group) were fed the basal diet supplemented with 500 mg/kg EA. The pre-trial period was 3 days, and the formal trial period was 18 days. From day 16 to 18 of the formal trial, each weaned piglet in the ETEC group and EA-ETEC group was orally administered 10 mL of ETEC K88 bacterial solution at a concentration of 2×10⁹ CFU/mL daily, while those in the CON group were administered the same volume of sterile LB medium. The results showed as follows: 1) before challenge, compared with the ETEC group, the average daily feed intake (ADFI) and average daily gain (ADG) of weaned piglets in the EA-ETEC group were significantly increased (P<0.05), and the body weight on day 15 showed a trend of increase (P=0.083); there was no significant difference in diarrhea rate between the CON group and ETEC group or between the ETEC group and EA-ETEC group (P>0.05). During the challenge period, compared with the CON group, the body weight on day 18, ADFI and ADG of weaned piglets in the ETEC group were significantly decreased (P<0.05), and the diarrhea rate was significantly increased (P<0.05); compared with the ETEC group, the body weight on day 18 and ADFI of weaned piglets in the EA-ETEC group were significantly increased (P<0.05), and the diarrhea rate was significantly decreased (P<0.05). During the whole experimental period, compared with the CON group, the ADFI and ADG of weaned piglets in the ETEC group were significantly decreased (P<0.05), and the feed to gain ratio (F/G) was significantly increased (P<0.05); compared with the ETEC group, the ADFI and ADG of weaned piglets in the EA-ETEC group were significantly increased (P<0.05), and the F/G was significantly decreased (P<0.05). 2) After challenge, compared with the CON group, the spleen index of weaned piglets in the ETEC group was significantly decreased (P<0.05), and the lung index was significantly increased (P<0.05); compared with the ETEC group, the spleen index of weaned piglets in the EA-ETEC group was significantly increased (P<0.05), and the stomach index showed a trend of increase (P=0.065). 3) After challenge, compared with the CON group, the superoxide dismutase (SOD) activity and total antioxidant capacity (T-AOC) in jejunal mucosa of weaned piglets in the ETEC group were significantly decreased (P<0.05), and the malondialdehyde (MDA) content in jejunal mucosa was significantly increased (P<0.05); compared with the ETEC group, the SOD activity and T-AOC in jejunal mucosa of weaned piglets in the EA-ETEC group were significantly increased (P<0.05), and the MDA content in jejunal mucosa was significantly decreased (P<0.05). 4) After challenge, compared with the CON group, the mRNA relative expression levels of SOD1, SOD2 and glutathione peroxidase 1 (GSH-Px1) in jejunal mucosa of weaned piglets in the ETEC group were significantly decreased (P<0.05); compared with the ETEC group, the mRNA relative expression levels of SOD2 and GSH-Px1 in jejunal mucosa of weaned piglets in the EA-ETEC group were significantly increased (P<0.05), and the relative mRNA expression level of SOD1 in jejunal mucosa showed a trend of increase (P=0.083). 5) After challenge, compared with the CON group, the mRNA relative expression level of Keap1 in jejunal mucosa of weaned piglets in the ETEC group was significantly increased (P<0.05), and the mRNA relative expression level of Nrf2 in jejunal mucosa was significantly decreased (P<0.05); compared with the ETEC group, the mRNA relative expression level of Keap1 in jejunal mucosa of weaned piglets in the EA-ETEC group was significantly decreased (P<0.05), and the mRNA relative expression level of Nrf2 in jejunal mucosa was significantly increased (P<0.05). In conclusion, dietary supplementation with 500 mg/kg EA can alleviate the decline in growth performance and intestinal damage of weaned piglets challenged with ETEC K88. Meanwhile, EA can effectively alleviate the oxidative stress induced by ETEC K88 challenge by regulating the antioxidant signaling pathway, modulating the expression of antioxidant genes, antioxidant enzyme activities and peroxide content in jejunal mucosa.

Key words： ellagic acid; enterotoxigenic Escherichia coli K88; weaned piglets; growth performance; antioxidant function; Nrf2/Keap1 signaling pathway

近年来,断奶仔猪腹泻导致的死亡量占我国生猪养殖总死亡量的50%~70%,腹泻的发生与仔猪肠道发育不完善密切相关,由于肠道的免疫防御功能尚未完全成熟,其极易受到集约化饲养过程中各种病原体及非病原性因子的侵袭^[1]。产肠毒素大肠杆菌(enterotoxigenic Escherichia coli,ETEC)是导致断奶仔猪腹泻的主要致病菌之一,它可通过食物、水源以及粪便等多种途径传播^[2]。其中,含有K88菌毛的ETEC是引起仔猪腹泻的主要菌株^[3]。研究表明,ETEC感染仔猪肠道后,会分泌黏附素和肠毒素,黏附素使得ETEC牢固地黏附于小肠黏膜,利于其进一步大量繁殖;肠毒素进入上皮细胞后,会激活腺氨酸环化酶,导致细胞内外环磷酸腺苷(cAMP)浓度失衡,抑制肠道绒毛细胞吸收钠离子(Na⁺)和氯离子(Cl^-),从而引发腹泻^[1,4]。此外,大肠杆菌侵入机体后会促使机体产生大量活性氧(reactive oxygen species,ROS),ROS具有极强的氧化性和细胞毒性,其过量产生可导致细胞发生氧化损伤^[5]。研究发现,饲粮中添加植物多酚类物质有助于缓解仔猪的氧化应激,并对降低腹泻发生率有积极作用^[6]。

鞣花酸(ellagic acid,EA)是一种天然多酚类物质,广泛存在于各种果实中。研究表明,EA在生物体内具有促生长、抗氧化以及抗炎等多种生理功能^[7-8]。Xiao等^[9]研究表明,EA可通过上调核因子E2相关因子2(nuclear factor E2 related factor 2,Nrf2)这一抗氧化转录因子,促进抗氧化酶原表达,从而缓解仔猪氧化应激。Yu等^[10]研究表明,EA通过激活Nrf2/Kelch样ECH关联蛋白1(Kelch-like ECH-associated protein 1,Keap1)信号通路,增强了NAD(P)H醌氧化还原酶1[NAD(P)H quinone oxidoreductase 1,NQO1]、过氧化氢酶(catalase,CAT)和谷胱甘肽过氧化物酶1(glutathione peroxidase 1,GSH-Px1)等抗氧化酶的活性,从而发挥缓解氧化应激的作用。Qiu等^[11]研究表明,EA可显著降低过氧化氢(H₂O₂)诱导产生的自由基和丙二醛(malondialdehyde,MDA)含量,同时显著提高超氧化物歧化酶(superoxide dismutase,SOD)活性,从而缓解氧化应激反应。刘佳佳等^[12]研究表明,给小鼠灌喂1.5 g/(d·只)石榴皮提取物[即EA灌喂量为40.72 mg/(d·只)]可显著提高小鼠血浆中SOD、CAT活性及总抗氧化能力(total antioxidant capacity,T-AOC),并显著降低MDA含量,从而增强其清除自由基和抗氧化的能力。然而,EA对ETEC K88感染引起的断奶仔猪腹泻及氧化应激反应方面影响的研究尚未见报道。因此,本研究以断奶仔猪为试验对象,探究EA对ETEC K88攻毒仔猪生长性能、抗氧化功能及Nrf2/Keap1信号通路的影响,以期为EA在猪生产中的开发利用提供理论依据。

1 材料与方法

1.1 试验材料

试验攻毒用的ETEC K88由中国科学院亚热带农业生态研究所惠赠。EA选购自西安某生物科技有限公司,分子式为C₁₄H₆O₈,化学名为2,3,7,8-四羟基-[1]-苯并吡喃并[3-CDE][1]苯并吡喃-5,10-二酮,纯度≥90%。

1.2 试验设计与饲养管理

动物试验所涉及的程序已获得湖南农业大学生物医学研究伦理委员会的批准(批准号:CACAHU 2023-00231)。

试验选取24头遗传背景一致、体重[(7.83±0.09) kg]接近的21日龄“杜×长×大”断奶仔猪,随机分为3组,每组8个重复,每个重复1头猪。对照组(CON组)和腹泻模型组(ETEC组)饲喂基础饲粮,EA-腹泻模型组(EA-ETEC组)饲喂在基础饲粮中添加500 mg/kg EA的饲粮。基础饲粮参照NRC(2012)^[13]营养标准配制,其组成及营养水平见表1。预试期3 d,正试期18 d。在正试期第16~18天,ETEC组和EA-ETEC组每头断奶仔猪每天灌服10 mL浓度为2×10⁹ CFU/mL的ETEC K88菌液,CON组断奶仔猪灌服相同体积的LB无菌培养液。ETEC K88灌服剂量和作用时间参考课题组前期研究^[14]。

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	62.12
乳清粉Whey powder	6.00
大豆油Soybean oil	2.30
豆粕Soybean meal	11.35
发酵豆粕Fermented soybean meal	10.00
鱼粉Fish meal	4.00
L-赖氨酸盐酸盐L-Lys·HCL	0.64
DL-蛋氨酸DL-Met	0.21
L-苏氨酸L-Thr	0.21
L-色氨酸L-Trp	0.08
石粉Limestone	0.93
磷酸氢钙CaHPO₄	0.79
氯化钠NaCl	0.37
预混料Premix¹⁾	1.00
合计Total	100.00
营养水平Nutrient levels²⁾
消化能DE/(MJ/kg)	14.84
粗蛋白质CP	19.01
钙Ca	0.80
有效磷AP	0.40
标准回肠可消化赖氨酸SID Lys	1.35
标准回肠可消化蛋氨酸+ 标准回肠可消化半胱氨酸 SID Met+SID Cys	0.74
标准回肠可消化苏氨酸 SID Thr	0.79
标准回肠可消化色氨酸 SID Trp	0.22

1)预混料为每千克饲粮提供 The premix provided the following per kg of the diet:VA 12 400 IU,VD₃ 2 800 IU,VE 30 IU,VK 6 mg,VB₁₂ 40 μg,VB₁ 3 mg,VB₂ 10 mg,VB₆ 8 mg,烟酸 nicotinic acid 40 mg,D-泛酸 D-pantothenic acid 15 mg,叶酸 folic acid 1 mg,生物素 biotin 0.08 mg,氯化胆碱 choline chloride 120 mg,Fe (as FeSO₄·H₂O) 120 mg,Cu (as CuSO₄·5H₂O) 16 mg,Mn (as MnSO₄·H₂O) 70 mg,Zn (as ZnSO₄·H₂O) 120 mg,I (as KI) 0.7 mg,Se (as Na₂SeO₃) 0.48 mg。
2)粗蛋白质和钙为实测值,其余均为计算值。CP and Ca were measured values, while the others were calculated values.

本试验在中国科学院亚热带农业生态研究所动物房进行,试验场地实行封闭管理,试验开始前对猪舍进行清洁、打扫及消毒。试验过程中定期对栏舍进行清洁和消毒,确保猪舍保温条件良好和空气流通。猪只实行自由采食和饮水,以确保其饮食自由和营养供应充足。

1.3 样品采集

于正试期第18天,仔猪空腹12 h后,对所有试验仔猪进行称重并记录,然后进行屠宰。颈部放血后,沿腹中线剖开胸腔及腹腔,分离出心脏、肝脏、脾脏、肺脏、肾脏、胃等器官;另取2份约6 cm长的空肠肠段,用冰浴生理盐水洗净外壁及肠内容物,用滤纸吸干生理盐水后,将肠段放置在冰砖上,迅速剪开肠管,使用无菌载玻片轻轻刮取空肠黏膜组织,分装至2 mL离心管中,编号后立即放入液氮中速冻,随后转移至-80 ℃冰箱中保存。

1.4 检测指标及方法

1.4.1 饲粮营养成分含量

饲粮粗蛋白质含量参照《饲料中粗蛋白的测定凯氏定氮法》(GB/T 6432—2018)^[15]进行测定,钙含量参照《饲料中钙的测定》(GB/T 6436—2018)^[16]进行测定;消化能、有效磷、标准回肠可消化赖氨酸、标准回肠可消化蛋氨酸+标准回肠可消化半胱氨酸、标准回肠可消化苏氨酸和标准回肠可消化色氨酸参考《中国饲料成分及营养价值表(2022年第33版)制定说明》^[17]计算得出。

1.4.2 生长性能及腹泻率

试验期间记录每天每头仔猪的采食量和腹泻情况,并在正试期第1、15和18天的相同时间点对仔猪进行称重并记录。参考Zhang等^[18]的粪便分级系统观察记录仔猪腹泻情况,试验期内每天13:00观察仔猪肛门是否红肿、肛门周围有无稀粪及粪便形态,并根据粪便稠度进行评分。评分标准为:1分,固体粪便、质地坚硬;2分,固体粪便、质地松软;3分,粪便部分成形、质地松软;4分,半液体粪便、粪水未分离;5分,水样粪便、粪水未分离。当某仔猪粪便评分大于3分时,判定该仔猪出现腹泻,记录腹泻1次。计算每头仔猪的平均日采食量(ADFI)、平均日增重(ADG)、料重比(F/G)和腹泻率。计算公式如下:

ADFI(g/d)=总采食量/试验天数;

ADG(g/d)=(终末体重-初始体重)/试验天数;

F/G=ADFI/ADG;

腹泻率(%)=100×试验期内各组仔猪腹泻

总次数/(试验天数×各组仔猪头数)。

1.4.3 器官指数

屠宰后分离出心脏、肝脏、脾脏、肺脏、肾脏和胃等器官后,吸干表面多余的血液,立即称重并记录,计算器官指数。计算公式如下:

器官指数(g/kg)=器官重/宰前活重。

1.4.4 空肠黏膜抗氧化指标

取约0.5 g空肠黏膜组织,按照1∶9(质量∶体积)的比例加入冰冷生理盐水作为匀浆介质,在冰水浴中使用电动匀浆器充分破碎组织,直至溶液中无可见组织颗粒。4 ℃下以2 200×g的离心力离心10 min后收集上清液。采用南京建成生物工程研究所生产的试剂盒测定样品中MDA含量、CAT、谷胱甘肽过氧化物酶(GSH-Px)、SOD活性及T-AOC。

1.4.5 空肠黏膜抗氧化相关基因的表达

采用实时荧光定量PCR(RT-qPCR)法测定断奶仔猪空肠黏膜中SOD1、SOD2、GSH-Px1、CAT、Keap1及Nrf2的mRNA相对表达量。

1.4.5.1 空肠黏膜RNA的提取

于-80 ℃冰箱中取出冻存的空肠黏膜,迅速称取约50 mg空肠黏膜,加入装有1 mL Trizol的1.5 mL离心管中,使用匀浆机进行匀浆处理后,置于冰水浴中静置10 min。4 ℃下以13 523×g的离心力离心10 min。离心后样品分成3层:上层为无色的RNA层;中层为白色的DNA层;下层为粉红色的蛋白层。小心吸取上层无色溶液(300~400 μL),转移至新的1.5 mL EP管中,加入等体积的异丙醇,用力振荡混匀后,室温孵育10~15 min。4 ℃下以13 523×g的离心力离心15 min,弃去异丙醇,加入1 mL 75%焦碳酸二乙酯(DEPC)乙醇,用涡旋仪振荡30 s洗涤白色RNA沉淀。4 ℃下以6 010×g的离心力离心10 min,重复洗涤步骤1次(即用75% DEPC乙醇洗涤2次)。弃去上清乙醇后,在超净工作台中开盖干燥,待75% DEPC乙醇挥发后,根据白色RNA沉淀的大小,加入适量体积的0.1% DEPC水,使RNA完全溶解。

1.4.5.2 RNA浓度测定、反转录及RT-qPCR扩增

将样品置于冰上,使用NanoDrop超微量分光光度计测定RNA浓度。吸取2 μL混匀的样品进行检测,记录RNA浓度(ng/μL)以及A260/A280(范围为1.8~2.1)。根据测定结果进行浓度计算,将所有样品RNA浓度调整至统一水平。在冰浴条件下于200 μL离心管中配制反应混合液,为确保反应液配制的准确性,进行各项反应时,先按反应数+2的量配制Master Mix,然后分装到每个反应管中,最后加入RNA样品。引物序列(表2)由上海生工生物工程有限公司合成,以β-肌动蛋白(β-actin)为内参基因,以反转录得到的cDNA作为模板,在10 μL反应体系中进行后续试验,反应体系具体组成为:cDNA模板1 μL,上、下游引物各0.2 μL,2×QuantiFast^®SYBR^®Green PCR预混液5 μL,DEPC水3.6 μL。RT-qPCR扩增程序为:95 ℃预变性30 s(单循环);95 ℃变性5 s,60 ℃退火30 s(40个循环)。采用2^-ΔΔCt法计算目的基因的mRNA相对表达量^[19]。

表2 引物序列

Table 2 Primer sequences

基因 Genes	引物序列 Primer sequences (5'—3')	产物长度 Product length/bp	登录号 Accession number
β-肌动蛋白β-actin	F:AGAGCAAGAGAGGCATCCTG R:CACGCAGCTCGTTGTAGAAG	110	NM_007393.5
超氧化物歧化酶1 SOD1	F:AAGATTCTGTGATCGCCCTCT R:ACTTCCAGCATTTCCCGTCT	128	NM_001190422.1
超氧化物歧化酶2 SOD2	F:GGCCTACGTGAACAACCTGA R:TGATTGATGTGGCCTCCACC	126	NM_214127.2
谷胱甘肽过氧化物酶1 GSH-Px1	F:AAGTGTGAGGTGAATGGCGCAAA R:TCATGAGGGCAGTGGCATCGTC	94	NM_214201.1
过氧化氢酶CAT	F:CCAAATTACTACCCCAACAGCTT R:ACCTGGGTGACATTATCTTCGTT	125	NM_214301.2
核因子E2相关因子2 Nrf2	F:AGAAACCAGTGGATCTGCCG R:AGCTCCTCCCAAACTTGCTC	201	XM_021075133.1
Kelch样ECH关联蛋白1 Keap1	F:GACAAACCGCCTCAACTCAG R:GTCTCCACGTCGTAGCGTTC	183	NM 001114671.1

1.5 数据统计分析

使用Excel 2019软件对试验数据进行初步处理,利用SPSS 26.0统计软件进行Mann-Whitney U检验,结果数据以“平均值±标准误”表示,P<0.05表示差异显著,0.05≤P<0.10表示差异具有显著趋势。

2 结果与分析

2.1 EA对断奶仔猪生长性能及腹泻率的影响

由表3可知,攻毒前(第1~15天),与ETEC组相比,EA-ETEC组断奶仔猪ADFI和ADG显著提高(P<0.05),终末体重有提高趋势(P=0.083);CON组与ETEC组之间以及ETEC组与EA-ETEC组之间断奶仔猪腹泻率均无显著差异(P>0.05)。攻毒期间(第16~18天),与CON组相比,ETEC组断奶仔猪终末体重、ADFI和ADG均显著降低(P<0.05),腹泻率显著提高(P<0.05);与ETEC组相比,EA-ETEC组断奶仔猪终末体重和ADFI显著提高(P<0.05),腹泻率显著降低(P<0.05)。整个试验期间(第1~18天),与CON组相比,ETEC组断奶仔猪ADFI和ADG显著降低(P<0.05),F/G显著提高(P<0.05);与ETEC组相比,EA-ETEC组断奶仔猪ADFI和ADG显著提高(P<0.05),F/G显著降低(P<0.05)。

表3 EA对断奶仔猪生长性能及腹泻率的影响

Table 3 Effects of EA on growth performance and diarrhea rate of weaned piglets

项目 Items		组别Groups			P值P-value
项目 Items		CON	ETEC	EA-ETEC	P1				P2
攻毒前(第1~15天) Pre-challenge period (days 1 to 15)
初始体重Initial BW/kg		7.83±0.17	7.77±0.10	7.88±0.21		0.721			0.505
终末体重Final BW/kg		11.20±0.10	10.72±0.09	11.06±0.18		0.007			0.083
平均日采食量ADFI/(g/d)		438.97±1.32	441.34±2.37	451.77±3.58		0.442			0.021
平均日增重ADG/(g/d)		224.81±9.80	196.49±1.13	211.22±5.07		<0.001			0.005
料重比F/G		1.97±0.07	2.25±0.01	2.14±0.05		<0.001			0.195
腹泻率Diarrhea rate/%		1.96±0.25	1.99±0.36	1.65±0.22		0.878			0.645
攻毒期(第16~18天) Challenge period (days 16 to 18)
终末体重Final BW/kg		11.86±0.10	11.25±0.13	11.65±0.19		0.005			0.049
平均日采食量ADFI/(g/d)		445.81±9.07	372.38±3.69	402.36±2.86		<0.001			<0.001
平均日增重ADG/(g/d)		220.16±3.89	176.66±15.00	196.25±10.48		0.049			0.442
料重比F/G		2.03±0.05	2.24±0.23	2.10±0.13		0.959			1.000
腹泻率Diarrhea rate/%		2.15±0.29	7.09±0.41	5.23±0.24		<0.001			0.001
试验全期(第1~18天) Whole experimental period (days 1 to 18)
平均日采食量ADFI/(g/d)	440.11±2.52		429.84±2.32	443.54±3.11			0.015	0.007
平均日增重ADG/(g/d)	224.04±7.99		193.18±2.62	208.91±3.13			<0.001	<0.001
料重比F/G	1.98±0.05		2.23±0.02	2.13±0.03			<0.001	0.028

P1表示CON组 vs ETEC组,P2表示ETEC组 vs EA-ETEC组。下表同。

P1 represented CON group vs ETEC group, P2 represented ETEC group vs EA-ETEC group. The same as below.

2.2 EA对断奶仔猪器官指数的影响

由表4可知,攻毒后,与CON组相比,ETEC组断奶仔猪脾脏指数显著降低(P<0.05),肺脏指数显著提高(P<0.05);与ETEC组相比,EA-ETEC组断奶仔猪脾脏指数显著提高(P<0.05),胃指数有提高趋势(P=0.065)。CON组与ETEC组之间以及ETEC组与EA-ETEC组之间断奶仔猪心脏、肝脏和肾脏指数均无显著差异(P>0.05)。

表4 EA对断奶仔猪器官指数的影响

Table 4 Effects of EA on organ indexes of weaned piglets g/kg

项目 Items	组别Groups			P值P-value
项目 Items	CON	ETEC	EA-ETEC	P1	P2
心脏指数Heart index	5.80±0.32	6.32±0.58	7.26±0.57	0.798	0.234
肝脏指数Liver index	34.31±1.81	34.42±2.82	34.04±2.19	0.878	0.959
脾脏指数Spleen index	2.69±0.19	2.08±0.14	3.24±0.32	0.028	0.007
肺脏指数Lung index	14.78±0.78	19.73±2.00	20.29±2.20	0.038	0.721
肾脏指数Kidney index	6.10±0.32	6.09±0.56	6.36±0.58	0.574	0.645
胃指数Stomach index	9.76±0.63	9.36±0.51	11.51±0.74	0.721	0.065

2.3 EA对断奶仔猪空肠黏膜抗氧化指标的影响

由表5可知,攻毒后,与CON组相比,ETEC组断奶仔猪空肠黏膜SOD活性和T-AOC显著降低(P<0.05),空肠黏膜MDA含量显著提高(P<0.05);与ETEC组相比,EA-ETEC组断奶仔猪空肠黏膜SOD活性和T-AOC显著提高(P<0.05),空肠黏膜MDA含量显著降低(P<0.05)。CON组与ETEC组之间以及ETEC组与EA-ETEC组之间断奶仔猪空肠黏膜CAT和GSH-Px活性均无显著差异(P>0.05)。

表5 EA对断奶仔猪空肠黏膜抗氧化指标的影响

Table 5 Effects of EA on jejunal mucosal antioxidant indexes of weaned piglets

项目 Items	组别Groups			P值P-value
项目 Items	CON	ETEC	EA-ETEC	P1	P2
总抗氧化能力T-AOC/(U/mg prot)	22.59±0.43	20.13±0.16	24.50±0.68	<0.001	<0.001
丙二醛MDA/(nmol/mg prot)	10.75±0.31	15.32±0.36	11.33±0.56	<0.001	<0.001
过氧化氢酶 CAT/(U/mg prot)	22.15±0.58	22.41±0.48	23.30±0.31	0.645	0.195
谷胱甘肽过氧化物酶 GSH-Px/(U/mg prot)	31.68±0.34	32.04±0.32	33.31±0.76	0.505	0.279
超氧化物歧化酶 SOD/(U/mg prot)	92.12±1.50	80.63±0.30	85.15±0.86	<0.001	<0.001

2.4 EA对断奶仔猪空肠黏膜抗氧化基因表达的影响

由表6可知,攻毒后,与CON组相比,ETEC组断奶仔猪空肠黏膜SOD1、SOD2和GSH-Px1的mRNA相对表达量显著降低(P<0.05);与ETEC组相比,EA-ETEC组断奶仔猪空肠黏膜SOD2和GSH-Px1的mRNA相对表达量显著提高(P<0.05),空肠黏膜SOD1的mRNA相对表达量有提高趋势(P=0.083)。

表6 EA对断奶仔猪空肠黏膜抗氧化基因表达的影响

Table 6 Effects of EA on jejunal mucosal antioxidant gene expression of weaned piglets

项目 Items	组别Groups			P值P-value
项目 Items	CON	ETEC	EA-ETEC	P1	P2
超氧化物歧化酶1 SOD1	1.00±0.07	0.62±0.03	0.70±0.03	<0.001	0.083
超氧化物歧化酶2 SOD2	1.00±0.02	0.79±0.03	0.93±0.04	<0.001	0.010
谷胱甘肽过氧化物酶1 GSH-Px1	1.00±0.06	0.72±0.03	0.95±0.06	<0.001	0.001
过氧化氢酶CAT	1.00±0.11	0.77±0.05	0.95±0.09	0.161	0.195

2.5 EA对断奶仔猪空肠黏膜Nrf2/Keap1信号通路基因表达的影响

由表7可知,攻毒后,与CON组相比,ETEC组断奶仔猪空肠黏膜Keap1的mRNA相对表达量显著提高(P<0.05),空肠黏膜Nrf2的mRNA相对表达量显著降低(P<0.05);与ETEC组相比,EA-ETEC组断奶仔猪空肠黏膜Keap1的mRNA相对表达量显著降低(P<0.05),空肠黏膜Nrf2的mRNA相对表达量显著提高(P<0.05)。

表7 EA对断奶仔猪空肠黏膜Nrf2/Keap1信号通路基因表达的影响

Table 7 Effects of EA on jejunal mucosal Nrf2/Keap1 pathway gene expression of weaned piglets

项目 Items	组别Groups			P值P-value
项目 Items	CON	ETEC	EA-ETEC	P1	P2
Kelch样ECH关联蛋白1 Keap1	1.00±0.04	1.43±0.04	1.17±0.25	<0.001	<0.001
核因子E2相关因子2 Nrf2	1.00±0.09	0.39±0.04	0.70±0.06	<0.001	0.003

3 讨论

3.1 EA对断奶仔猪生长性能及腹泻率的影响

仔猪在断奶期间,由于机体生长不成熟且肠道发育不完善,再加上环境因素的影响,极易出现应激反应。此时,仔猪肠道对病原菌的防御能力较弱,易受致病菌侵袭,导致肠道发育受阻,进而引发腹泻、食欲下降等一系列不利于仔猪生长的严重反应。以往的研究表明,EA在畜禽生产中能够有效发挥促进生长、改善肠道健康等作用。Qin等^[20]研究发现,饲粮中添加0.1% EA能够显著提高断奶仔猪ADFI和ADG。同时,EA能促进仔猪小肠组织生长,增加小肠绒毛高度,进而提高小肠黏膜吸收面积,使得营养物质能够更有效地被吸收,这可能是其改善仔猪消化吸收能力和促进生长的机制之一^[21]。Qin等^[22]研究发现,饲粮中添加10 mg/kg EA和10 mg/kg小檗碱能够提高仔猪ADFI和ADG,增加空肠绒毛高度与隐窝深度的比值,减少肠上皮细胞的凋亡,从而改善生产性能和肠道健康。本研究结果与上述结果相近,攻毒前,饲粮中添加500 mg/kg EA提高了断奶仔猪第15天体重,显著提高了ADFI和ADG;在攻毒期间,饲粮中添加500 mg/kg EA能够显著提高ETEC K88攻毒断奶仔猪第18天体重和ADFI;在整个试验期内,与ETEC组相比,饲粮中添加500 mg/kg EA显著提高了断奶仔猪的ADFI和ADG,显著降低了F/G,表明EA能够缓解ETEC K88攻毒导致的仔猪生长性能下降。这种缓解作用可能与EA可直接作用于ETEC菌体细胞有关,研究显示,EA可通过破坏ETEC细胞膜完整性或改变细菌形态结构的方式,降低病原菌的存活率^[23-25]。综上可知,EA通过改善机体消化吸收功能及肠道健康,促进仔猪对营养物质的消化吸收与利用。

断奶期间,仔猪肠道易受到外界刺激,引发生理性腹泻或病理性腹泻。携带K88菌毛的ETEC是导致断奶仔猪腹泻的常见致病菌,其引发的腹泻可导致仔猪严重脱水、生产性能下降、死亡率升高。ETEC可借助食物、水源、粪便等多种物质媒介进行传播,因此通过改善仔猪肠道菌群丰度、提高免疫能力来预防ETEC感染更为可取^[26]。赵铭伟等^[27]研究发现,饲粮中添加500 mg/kg EA可改善仔猪肠道屏障受损、机体稳态失衡、腹泻下痢等问题。作为植物多酚类物质,EA在动物体内难以被直接利用,主要是以肠道微生物为靶点发挥作用。Lu等^[28]研究发现,在断奶仔猪饲粮中添加500 mg/kg EA可显著提高乳杆菌属等益生菌的相对丰度。研究表明,益生菌可通过调节肠腔pH、抑制病菌黏附或产生抗菌物质等方式抑制病原菌定殖,进而保护肠道上皮屏障^[29-30]。另外,EA可通过抑制病原菌生物膜形成的方式,缓解大肠杆菌感染造成的仔猪腹泻。Bakkiyaraj等^[31]研究发现,EA具有显著的抗生物膜活性,能够抑制大肠杆菌生物膜的形成。本研究结果与上述结果相近,ETEC K88攻毒后,ETEC组断奶仔猪的腹泻率显著提高,表明ETEC K88腹泻模型建立成功;与ETEC组相比,饲粮中添加500 mg/kg EA可显著降低断奶仔猪腹泻率。综上所述,饲粮中添加500 mg/kg EA能够缓解ETEC K88攻毒导致的断奶仔猪腹泻,其机制可能与EA抑制ETEC K88定植、调节肠道菌群、增强肠道黏膜屏障功能有关。

3.2 EA对断奶仔猪器官指数的影响

器官指数在一定程度上能够反映器官的功能状态。多项研究表明,脾脏指数降低表明免疫器官成熟缓慢,提高则意味着其发育较为迅速;胃指数的变化可在一定程度上反映动物的采食量及胃部健康状况^[32-33]。断奶仔猪感染ETEC后,体内会发生氧化应激和炎症反应,进而对器官发育产生不同程度的影响。在本研究中,ETEC K88攻毒显著降低了断奶仔猪脾脏指数,显著提高了肺脏指数;而饲粮中添加500 mg/kg EA可显著提高ETEC K88攻毒断奶仔猪脾脏指数。不良应激可导致动物新陈代谢紊乱、免疫功能下降,增加患病风险,进而损害仔猪的器官结构与功能^[34]。黄诗琦等^[35]研究表明,小鼠感染大肠杆菌后会出现败血症症状,脾脏组织萎缩,脾脏指数显著下降。有研究表明,炎性反应是肺血管结构发生变化的主要因素之一,炎性因子含量与肺动脉压力呈正相关,炎性反应会导致细胞外基质成分增多,平滑肌细胞异常增殖肥大,进而导致肺血管壁增厚及管腔狭窄,且持续的炎性环境会进一步加剧肺血管病理性重塑^[36-37]。Che等^[38]研究表明,ETEC K88侵染断奶仔猪肠道后会产生大量肠毒素,刺激机体释放大量炎性因子,引发炎症反应,影响仔猪器官正常发育。Beserra等^[39]研究表明,EA能够通过调节肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、一氧化氮含量,介导大鼠的抗胃溃疡和胃保护作用。多项研究发现,连续灌胃大鼠EA能有效抑制肺脏氧化损伤,并在一定程度上保护肝脏,这可能与EA可提高组织抗氧化机能有关^[40-41]。此外,脾脏指数升高也可能是由于攻毒引发组织代偿性增生,进而导致脾脏肿大所导致的。有研究指出,ETEC K88感染小鼠和仔猪后产生的大量肠毒素,可导致脾脏中TNF-α、白细胞介素-6(interleukin-6,IL-6)、白细胞介素-8(interleukin-8,IL-8)等促炎因子含量显著提高,引发脾脏免疫细胞增殖和组织炎症,进而引起组织增生,造成脾脏肿大^[42-43]。EA干预对肠毒素诱导的器官发育变化的影响涉及多种生物学过程,其具体作用机制仍需深入研究。

3.3 EA对断奶仔猪空肠黏膜抗氧化指标的影响

ETEC感染会导致断奶仔猪体内氧化系统和抗氧化系统失衡。有研究表明,断奶仔猪感染ETEC后,会出现氧化应激,导致CAT、GSH-Px、SOD等抗氧化酶活性下降,MDA含量升高,T-AOC降低,危害仔猪健康^[44-45]。其中,CAT、SOD和GSH-Px均可对动物体内过氧化物进行分解,其活性可直接反映机体抗氧化能力^[46-50]。有研究表明,EA能够有效清除动物体内自由基,其代谢产物尿石素也具有清除自由基的能力,二者的连续反应能够持续对自由基进行清除,缓解氧化损伤,进而保护机体^[51-52]。本研究结果表明,饲粮中添加500 mg/kg EA能够显著提高ETEC K88攻毒断奶仔猪空肠黏膜SOD活性及T-AOC,显著降低MDA含量,表明EA可通过调控仔猪体内抗氧化酶活性及抗氧化物含量,缓解ETEC K88攻毒导致的仔猪氧化应激。这说明ETEC K88攻毒会加剧仔猪机体氧化应激和组织氧化损伤,而饲粮中添加EA能增强机体抗氧化酶活性,减少过氧化物生成,缓解氧化应激反应。与本研究结果相似,Qin等^[20]研究表明,饲粮中添加0.1% EA能够显著提高断奶仔猪空肠CAT活性和谷胱甘肽(GSH)含量,降低MDA含量。Xiao等^[9]研究表明,EA能够提高百草枯诱导肠道受损仔猪血清SOD活性,表明EA具有改善机体氧化应激的作用。因此,EA可能是通过增强仔猪内源性抗氧化能力,从而起到良好的抗氧化作用,但具体的机制尚不明确,有待进一步探究。

3.4 EA对断奶仔猪空肠黏膜抗氧化信号通路及相关基因表达的影响

Nrf2/Keap1信号通路是重要的抗氧化通路,在正常生理状态下,Keap1蛋白与Nrf2结合,促使Nrf2泛素化降解,使Nrf2在细胞内维持较低水平;当动物细胞受到氧化应激刺激时,Keap1的半胱氨酸残基被修饰,导致其构象改变并与Nrf2分离,Nrf2释放后进入细胞核,激活NQO1、血红素加氧酶-1(heme oxygenase-1,HO-1)、SOD、GSH-Px的表达^[53-54]。李伟^[55]研究表明,饲粮中添加400 mg/kg EA能够激活Nrf2/Keap1信号通路,增加细胞内源性抗氧化防御,提高生长猪抗氧化能力。Yang等^[56]研究表明,每天给小鼠灌服20 mg/kg EA能够激活Nrf2/Keap1信号通路,上调GSH-Px4表达,降低体内MDA含量,从而减缓氧化应激。本研究结果表明,饲粮中添加500 mg/kg EA显著降低ETEC K88攻毒断奶仔猪空肠黏膜Keap1的mRNA相对表达量,显著提高空肠黏膜Nrf2的mRNA相对表达量,表明Nrf2/Keap1信号通路被激活。同时,EA显著提高了ETEC K88攻毒断奶仔猪空肠黏膜SOD2及GSH-Px1的mRNA相对表达量,表明机体抗氧化功能得到增强,可有效应对ETEC K88导致的肠道氧化应激反应。

4 结论

综上所述,饲粮中添加500 mg/kg EA能够缓解ETEC K88攻毒导致的断奶仔猪生长性能下降和肠道损伤。同时,EA可通过调控抗氧化信号通路,调节空肠黏膜抗氧化基因表达、抗氧化酶活性及过氧化物含量,从而有效缓解ETEC K88攻毒引发的氧化应激。

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