Chinese Journal of Animal Nutrition >

2024 , Vol. 36 >Issue 12: 7754 - 7769

DOI: https://doi.org/10.12418/CJAN2024.661

RESEARCH PAPER

Effects of Callicarpa nudiflora Extract on Growth Performance, Immune Function, Anti-Inflammatory Ability and Intestinal Barrier Function of Chickens Infected with Escherichia coli

  • HUANG Gengxiong , 1 ,
  • LIU Mengjie 1 ,
  • MA Ming 1 ,
  • ZHANG Wenbo 1 ,
  • JIN Wenxin 1 ,
  • ZHU Yongqi 1 ,
  • QU Qian 1 ,
  • LYU Weijie 1 ,
  • GUO Shining , 1, 2, *
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  • 1 College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China
  • 2 Traditional Chinese Veterinary Medicine and Natural Medicine Engineering Technology Research Center of Guangdong, Guangzhou 510642, China
*professor, E-mail:

Received date: 2024-03-29

  Online published: 2024-12-12

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Abstract

This experiment was conducted to study the effects of Callicarpa nudiflora extract on growth performance, immune function, anti-inflammatory ability and intestinal barrier function of chickens infected with Escherichia coli. A total of 300 healthy 1-day-old white feather chickens were randomly divided into 4 groups with 3 replicates per group and 25 chickens per replicate. Chickens in control group (CON group) and Escherichia coli infection group (EC group) were fed basal diets, and others in low and high dose Callicarpa nudiflora extract groups were fed basal diets supplemented with 0.3 (CNEL group) and 0.5 g/kg (CNEH group) Callicarpa nudiflora extract, respectively. On 19 days of age, chickens in EC, CNEL and CNEH groups were intraperitoneally injected with 1.0 mL Escherichia coli with a concentration of 1×108 CFU/mL, and chickens in the CON group were intraperitoneally injected with 1.0 mL normal saline with a concentration of 0.9%. The experiment lasted for 21 days. The results showed as follows: 1) after infected with Escherichia coli, compared with the CON group, the average daily gain (ADG) of EC group was significantly decreased (P<0.05), and the feed conversion rate (FCR) and mortality rate (DR) were significantly increased (P<0.05). Compared with the EC group, the ADG of CNEL and CNEH groups was significantly increased (P<0.05), and the FCR and DR were significantly decreased (P<0.05). 2) Compared with the CON group, the liver index and spleen index of EC group were significantly increased (P<0.05), and the bursa of Fabricius index was significantly increased (P<0.05). Compared with the EC group, the liver index and spleen index of CNEL and CNEH groups were significantly decreased (P<0.05), and the bursa of Fabricius index of CNEH group was significantly increased (P<0.05). 3) Compared with the CON group, the serum interleukin-6 (IL-6) content of EC group was significantly increased (P<0.05), and serum interleukin-10 (IL-10) content was significantly decreased (P<0.05). Compared with the EC group, the serum IL-6 and tumor necrosis factor-α (TNF-α) contents of CNEL and CNEH groups were significantly decreased (P<0.05), and the serum IL-10 content of CNEH group was significantly increased (P<0.05). 4) Compared with the CON group, the mRNA relative expression levels of IL-10 in jejunum and interleukin-4 (IL-4), IL-10, B-cell lymphoma-2 (Bcl-2) in spleen of EC group were significantly decreased (P<0.05), and the mRNA relative expression levels of TNF-α, myeloid differentiation protein 88 (MyD88), B-cell lymphoma 2-associated X protein (Bax), cysteinyl aspartate specific proteinase 3 (Caspase 3), cysteinyl aspartate specific proteinase 8 (Caspase 8) in jejunum and IL-6, TNF-α, interferon-γ (IFN-γ), Toll-like receptor 4 (TLR4), Bax, Caspase 8 in spleen were significantly increased (P<0.05). Compared with the EC group, the mRNA relative expression levels of IL-1β, TNF-α, Caspase 3, Caspase 8 in jejunum and MyD88 in spleen of CNEL and CNEH groups were significantly decreased (P<0.05), and the mRNA relative expression levels of IL-4, IL-10 in jejunum and IL-4, IL-10 in spleen were significantly increased (P<0.05). 5) Compared with the CON group, the serum D-lactic acid (D-LA) and lipopolysaccharide (LPS) contents of EC group were significantly increased (P<0.05). Compared with the EC group, the serum D-LA and LPS contents of CNEH group were significantly decreased (P<0.05), and the serum diamine oxidase (DAO) content of CNEL and CNEH groups were significantly decreased (P<0.05). 6) Compared with the CON group, the mRNA relative expression levels of zonula occludens protein-1 (ZO-1) and Claudin in jejunum of EC group were significantly decreased (P<0.05). Compared with the EC group, the mRNA relative expression levels of ZO-1 and Occludin in jejunum of CNEL and CNEH groups were significantly increased (P<0.05), and the mRNA relative expression level of Claudin in jejunum of CNEH group was significantly increased (P<0.05). 7) Compared with the CON group, the liver Escherichia coli number of EC group was significantly increased (P<0.05). Compared with the EC group, the liver Escherichia coli number of CNEL and CNEH groups was significantly decreased (P<0.05). In conclusion, dietary Callicarpa nudiflora extract can improve the effects of Escherichia coli infection on growth performance of chickens, and reduce the DR. In addition, it can improve the organ indexes of chickens, enhance the body anti-apoptosis ability, regulate the serum immune indexes and partially regulate the expression of TLR4/MyD88/nuclear factor-κB (NF-κB) pathway and related inflammatory factors to resist the inflammatory response caused by Escherichia coli infection, and improve the intestinal morphology and bacterial translocation. The Callicarpa nudiflora extract can be used as a potential substitute for antimicrobials in poultry production, and the optimal dose is 0.5 g/kg.

Key words： Callicarpa nudiflora extract; chickens; Escherichia coli; immune; anti-inflammatory; intestinal barrier function

Cite this article

HUANG Gengxiong , LIU Mengjie , MA Ming , ZHANG Wenbo , JIN Wenxin , ZHU Yongqi , QU Qian , LYU Weijie , GUO Shining . Effects of Callicarpa nudiflora Extract on Growth Performance, Immune Function, Anti-Inflammatory Ability and Intestinal Barrier Function of Chickens Infected with Escherichia coli[J]. Chinese Journal of Animal Nutrition, 2024 , 36(12) : 7754 -7769 . DOI: 10.12418/CJAN2024.661

鸡大肠杆菌病是由禽致病性大肠杆菌引起的局部或全身性感染[1],受感染病禽的免疫器官受损,免疫功能降低,易继发其他病原菌[2];同时肠道菌群平衡会被破坏,肠道结构受损,吸收能力降低[3],导致畜禽的生长性能下降[4],严重阻碍家禽业的发展。抗菌药物对禽大肠杆菌病有良好的治疗效果,但其不科学使用导致了耐药菌株的出现以及人类食物(如鸡肉和鸡蛋)中的药物残留。因此,寻找一种安全有效的抗菌药物替代品是家禽业和公共卫生研究的重点问题之一[5]。
裸花紫珠为马鞭草科、紫珠属植物,属于常用的海南传统民族药材,又称为黎药,目前收录在《中国药典》2020年版,现代药理研究表明,裸花紫珠的干燥叶和带叶的嫩枝中含有黄酮类、挥发油类、三萜类、倍半萜类、二萜类、环烯醚萜类、苯丙酸类、木脂素类和苯乙醇苷类等化学成分,在祛风祛湿[6-7]、抗氧化[8]、抗病毒、抗菌、止血凝血、抗炎、促进伤口愈合[9]及抗肿瘤[10]等方面具有显著的功能。裸花紫珠在人类医学临床应用主要用于治疗呼吸科、消化科和泌尿科疾病,在抗炎、止血和伤口愈合、皮肤炎症等方面取得了明显的功效。在兽医临床应用方面,裸花紫珠在鸡和猪抗炎、止血、促生长方面的应用开始具有一定的关注和研究[11-12],并批准裸花紫珠为三类新兽药。目前,裸花紫珠在肉鸡的应用上还相对较少,对感染大肠杆菌肉鸡的临床指标改善情况犹未可知。因此,本研究旨在研究裸花紫珠提取物(Callicarpa nudiflora extract,CNE)对大肠杆菌感染肉鸡生长性能、免疫功能、抗炎能力及肠道屏障功能的影响,为裸花紫珠提取物作为家禽生产中抗菌药物的潜在替代品提供理论依据。

1 材料与方法

1.1 试验设计

选择300只1日龄爱拔益加(AA)白羽肉鸡,体重(37±2) g,购自佛山市高明区新广农牧有限公司,随机分成4组,每组3个重复,每个重复25羽。对照组(CON组)和大肠杆菌攻毒组(EC组)饲喂基础饲粮,低、高剂量裸花紫珠提取物组分别在基础饲粮中添加0.3(CNEL组)和0.5 g/kg(CNEH组)裸花紫珠提取物。19日龄时,EC、CNEL和CNEH组试验鸡腹腔注射1.0 mL浓度为1×108 CFU/mL的大肠杆菌,CON组试验鸡腹腔注射1.0 mL浓度为0.9%的生理盐水。试验期21 d。所有试验程序均经华南农业大学动物伦理委员会批准(许可证号:SYXK,2022-0136)。

1.2 裸花紫珠提取物的制备

取裸花紫珠粉200 g,加蒸馏水2 000 mL(10倍),保持微沸煎煮120 min,放冷,过滤,收集滤液,复用1 600 mL(8倍)蒸馏水保持微沸煎煮60 min,合并滤液。蒸发浓缩,然后使用冷冻干燥器干燥滤液,所得水提物冻干粉末即为裸花紫珠提取物,-20 ℃储存备用。

1.3 大肠杆菌的活化与培养

首先将大肠杆菌O78(CVCC1569)保存液从-80 ℃冰箱中取出,然后用灭菌的接种环蘸取少量菌液在LB琼脂平板划线,随后置于37 ℃恒温培养箱中培养12 h。挑取单菌落置于200 mL的LB肉汤中,置于摇床中,37 ℃培养12 h。培养好的大肠杆菌菌液经离心后,用生理盐水调整浓度为1×108 CFU/mL。

1.4 饲养管理与基础饲粮

试验于华南农业大学动物实验中心进行,试验开展前清理消毒鸡舍,所有鸡只自由采食和自由饮水,鸡舍通风和环境符合卫生要求。基础饲粮参照《鸡饲养标准》(NY/T 33—2004)配制,其组成及营养水平见表1。饲粮中粗蛋白质、钙和总磷含量分别参考国家标准GB/T 6432—2018、GB/T 6436—2018和GB/T 6437—2018中的方法进行测定;赖氨酸和DL-蛋氨酸含量按照国家标准GB/T 18246—2019中的方法,采用塞卡姆氨基酸自动分析仪(S-433D,德国Sykam公司)进行测定。代谢能参考《中国饲料成分及营养价值表(2017年第28版)》计算得出[13]。
表1 基础饲粮组成及营养水平(干物质基础)

Table 1 Composition and nutrient levels of the basal diets (DM basis) %

项目Items 含量Content
原料Ingredient
玉米Corn 66.90
豆粕Soybean meal 24.00
豆油Soybean oil 3.50
石粉Limestone 0.80
碳酸氢钙Ca(HCO3)2 0.50
食盐NaCl 0.30
预混料Premix1) 4.00
合计Total 100.00
营养水平Nutrient levels2)
粗蛋白质Crude protein 18.00
钙Calcium 1.00
总磷Total phosphorus 0.40
赖氨酸Lysine 0.87
DL-蛋氨酸DL-methionine 0.68
代谢能Metabolic energy/(MJ/kg) 13.17

1)预混料为每千克饲粮提供 The premix provided the following per kg of the diet:VA 2 700 IU,VD 400 IU,VE 10 IU,VK 0.5 mg,VB12 0.007 mg,硫胺素 thiamine 2 mg,核黄素 riboflavin 5 mg,泛酸 pantothenic acid 10 mg,烟酸 niacin 30 mg,吡哆醇 pyridoxine 3 mg,生物素 biotin 0.1 mg,叶酸 folic acid 0.5 mg,胆碱 choline 750 mg,Fe 11.8 mg,Cu 0.15 mg,Mn 69.4 mg,Zn 56.3 mg,I 0.7 mg,Se 0.27 mg。

2)代谢能为计算值,其余为实测值。Metabolic energy was a calculated value, while the others were measured values.

1.5 样品采集

试验第21天,每个重复选取2只接近于平均体重的肉鸡,颈静脉采血5 mL,37 ℃斜放静置1 h,900×g离心10 min,取上清于500 μL离心管中,置于-20 ℃保存待测。采血后宰杀鸡只,去除相关器官表面结缔组织和脂肪,称重,取脾脏组织保存;同时采集空肠中部5 cm左右的肠段,用预冷的生理盐水轻轻冲洗干净食糜后,取一部分(1~2 cm)迅速置于4%多聚甲醛溶液中常温固定,使用苏木精-伊红(HE)染色,用于空肠黏膜形态学结构观察;余下部分纵向剖开肠段,用载玻片刮取空肠黏膜至2 mL冻存管中,置于-80 ℃保存,用于肠道紧密连接基因的测定。

1.6 测定指标及方法

1.6.1 裸花紫珠提取物中毛蕊花糖苷和木犀草素含量

1.6.1.1 高效液相色谱(HPLC)条件

色谱柱为Agilent C18分析柱(4.6 mm×250 mm,5 μm);流动相A为甲醇,流动相B为0.3%磷酸水溶液,按以下程序进行梯度洗脱(表2);流速为1 mL/min;进样量为10 μL;紫外检测波长为350 nm;柱温为30 ℃。
表2 梯度洗脱程序

Table 2 Gradient elution procedure %

时间
Time/min		 流动相A
Mobile phase A		 流动相B
Mobile phase B
0~10 40~50 60~50
10~15 50~60 50~40
15~25 60~70 40~30

1.6.1.2 对照品溶液的制备

毛蕊花糖苷购自成都瑞芬思生物科技有限公司(纯度≥98%),木犀草素购自成都瑞芬思生物科技有限公司(纯度≥98%)。取适量对照品毛蕊花糖苷和木犀草素,精密称定,加50%甲醇溶解制成每1 mL各含毛蕊花糖苷为1.460 mg、木犀草素为0.055 mg的混合溶液。

1.6.1.3 供试品溶液的制备

将50 mg的裸花紫珠提取物粉末放入离心管中,并加入10 mL的50%甲醇。称定质量,超声处理(功率300 W,频率40 kHz)40 min,放冷,再次称定质量,用50%甲醇补足减失的质量,混匀,离心,然后用0.22 μm微孔滤膜过滤,取续滤液作为供试品溶液。

1.6.1.4 精密度试验

精密吸取1.6.1.3项下的同一供试品溶液,按1.6.1.1项下色谱条件,重复进样6次,测定毛蕊花糖苷和木犀草素含量。

1.6.1.5 重复性试验

按1.6.1.3项下方法制备供试品溶液6份,按1.6.1.1项下色谱条件测定毛蕊花糖苷和木犀草素含量。

1.6.2 生长性能

分别于试验第19和21天记录死亡鸡只,并对每个重复进行称重,详细记录每个重复的饲料采食量,计算各组试验鸡的平均日增重(ADG)、平均日采食量(ADFI)、饲料转化率(FCR)和死亡率(DR)。

1.6.3 免疫相关指标

记录肝脏、胸腺、脾脏、法氏囊等器官的重量,计算器官指数,计算公式如下:
器官指数(%)=100×器官重量(g)/体重(kg)。
按照相应试剂盒(上海酶联生物科技有限公司生产)说明书的方法,采用酶标仪测定血清白细胞介素-6(IL-6)、白细胞介素-10(IL-10)、肿瘤坏死因子-α(TNF-α)和干扰素-γ(IFN-γ)含量。

1.6.4 肠道形态

空肠组织经石蜡包埋、HE染色后,使用Axio Scope A1显微镜(德国Zeiss公司)观察切片并拍照,使用Image-Pro软件测量肠道绒毛高度(villus height,VH)和隐窝深度(crypt depth,CD),并计算绒毛高度/隐窝深度(VH/CD)。

1.6.5 肠道通透性指标

按照相应试剂盒(上海酶联生物科技有限公司生产)说明书的方法,采用酶标仪测定血清二胺氧化酶(DAO)、脂多糖(LPS)和D-乳酸(D-LA)含量。

1.6.6 空肠和脾脏基因表达检测

通过TRIzol试剂(南京诺唯赞生物科技股份有限公司)法提取空肠和脾脏组织总RNA,用微量紫外可见分光光度计(NanoDrop-2000,青岛聚创环保集团有限公司)测定RNA浓度和纯度,参照反转录试剂盒[HiScript III RT SuperMix for qPCR(+gDNA wiper)]说明,对总RNA进行反转录合成cDNA,储存在-20 ℃冰箱中备用。实时荧光定量PCR检测采用SYBR Green染料法,在Light Cycler® 480Ⅱ Systems仪器上进行,按照ChamQ Universal SYBR qPCR Master Mix试剂盒配制反应体系并进行反应。以甘油醛-3-磷酸脱氢酶(GAPDH)为内参基因,采用2-△△Ct法计算目的基因[髓样分化因子88(MyD88)、Toll样受体4(TLR4)、核因子-κB(NF-κB)、白细胞介素-4(IL-4)、IL-6、IL-10、TNF-α、白细胞介素-1β(IL-1β)、IFN-γ、B淋巴细胞瘤-2相关X蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)、半胱氨酸蛋白酶3(Caspase 3)、半胱氨酸蛋白酶8(Caspase 8)、闭锁蛋白(Occludin)、封闭蛋白(Claudin)、闭锁小带蛋白-1(ZO-1)]mRNA相对表达量。引物由北京擎科生物技术有限公司合成,目的基因及内参基因引物序列见表3。
表3 目的基因及内参基因引物序列

Table 3 Primer sequences of target genes and reference gene

基因
Genes		 引物序列
Primer sequence (5'—3')		 登录号
Accession number
甘油醛-3-磷酸脱氢酶
GAPDH		 F:GGTGGTGCTAAGCGTGTTAT
R:ACCTCTGTCATCTCTCCACA		 K01458
髓样分化因子88
MyD88		 F:CTCCTAAACCTTCAGACTTCCA
R:CTTGTAGGAAGGCACTAATGG		 NM_001030962.1
Toll样受体4
TLR4		 F:CCGATATCTGAGGAGGACA
R:CTTCCAAGCACCAAACATCA		 KP410249.1
核因子-κB
NF-κB		 F:TCAACGCAGGACCTAAAGACAT
R:GCAGATAGCCAAGTTCAGGATG		 NM 205134.1
白细胞介素-4
IL-4		 F:CTTCCTCAACATGCGTCAGC
R:TGAAGTAGTGTTGCCTGCTGC		 AJ621735
白细胞介素-6
IL-6		 F:AGAAATCCCTCCTCGCCAAT
R:AAATAGCGAACGGCCCTCA		 NM_204628.1
白细胞介素-10
IL-10		 F:CGCTGTCACCGCTTCTTCA
R:TCCCGTTCTCATCCATCTTCTC		 NM_001004414.2
肿瘤坏死因子-α
TNF-α		 F:ATGAACCCTCCGCAGTACTC
R:AAGAGGCCACCACACGACA		 NM 204267.1
白细胞介素-1β
IL-1β		 F:GGGCTACAAGCTCTACATGT
R:GTAGAAGATGAAGCGGGTCA		 NM 204524.1
干扰素-γ
IFN-γ		 F:AGCTGACGGTGGACCTATTATT
R:GGCTTTGCGCTGGATTC		 Y07922.1
B淋巴细胞瘤-2相关X蛋白
Bax		 F:GTGATGGCATGGGACATAGCTC
R:TGGCGTAGACCTTGCGGATAA		 XM_422067.2
B淋巴细胞瘤-2
Bcl-2		 F:ATCGTCGCCTTCTTCGAGTT
R:ATCCCATCCTCCGTTGTCCT		 Z11961.1
半胱氨酸蛋白酶3
Caspase 3		 F:AAGGCTCCTGGTTTATTC
R:CTGCCACTCTGCGATTTA		 NM 204725.1
半胱氨酸蛋白酶8
Caspase 8		 F:TGGGAAAGTGGACAAGAGCC
R:ACAGTTGCCCAGAGAATCGG		 NM_204592.4
闭锁蛋白
Occludin		 F:CCGTAACCCCGAGTTGGAT
R:ATTGAGGCGGTCGTTGATG		 NM_205128.1
封闭蛋白
Claudin		 F:CCTGCTCACCCTCATTGGAG
R:GCTGAACTCACTCTTGGGCT		 NM_001277622.1
闭锁小带蛋白-1
ZO-1		 F:TGTAGCCACAGCAAGAGGTG
R:CTGGAATGGCTCCTTGTGGT		 XM_413773.4

1.6.7 肝脏细菌数量

每个重复随机选取2只试验鸡,采集肝脏,并称取适量肝脏样品,用无菌生理盐水按重量稀释10倍后组织匀浆,每种匀浆从10-1到10-6进行梯度稀释。用麦康凯琼脂平板在37 ℃恒温培养箱中孵育每个稀释的样品(100 μL),选择含有30~300个细菌菌落的平板进行计数。

1.7 统计分析

试验数据采用SPSS 26.0软件进行单因素方差分析(one-way ANOVA)和Duncan氏法多重比较,采用GraphPad Prism 9.1软件绘图,以P<0.05作为差异显著水平,数据分析结果用平均值±标准差表示。

2 结果

2.1 裸花紫珠提取物有效活性成分含量

以1.6.1.1的方法,毛蕊花糖苷的回归方程为:y=9 057.2x-17 203,决定系数(R2)=0.999 9,表明毛蕊花糖苷进样量在29.2~730 μg时与峰面积线性关系良好;木犀草素的回归方程为:y=25 552x-19 503,R2=0.999 3,表明木犀草素进样量在1.1~27.5 μg时与峰面积线性关系良好。以1.6.1.4的方法,获得毛蕊花糖苷和木犀草素峰面积的相对标准偏差(RSD)分别为0.17%和0.73%,表明仪器精密度良好。以1.6.1.5的方法,获得毛蕊花糖苷和木犀草素峰面积的RSD分别为0.47%和0.48%,表明该方法重复性良好。裸花紫珠提取物在350 nm处的色谱图如图1所示,测得裸花紫珠提取物中毛蕊花糖苷和木犀草素含量分别为28.36和0.34 mg/g。
图1 裸花紫珠提取物在350 nm处的色谱图

a:基线;b:对照品;c:供试品;1:毛蕊花糖苷;2:木犀草素。

Fig.1 Chromatogram of Callicarpa nudiflora extract at 350 nm

a: baseline; b: control product; c: test product; 1: verbascoside; 2: luteolin.

2.2 裸花紫珠提取物对大肠杆菌感染肉鸡生长性能的影响

由表4可见,饲粮中添加裸花紫珠提取物对1~19日龄肉鸡的ADFI和FCR均无显著影响(P>0.05),但显著影响1~19日龄肉鸡的ADG(P<0.05);与CON组相比,CNEL和CNEH组的ADG显著升高(P<0.05)。感染大肠杆菌后(19~21日龄),与CON组相比,EC组的ADG显著降低(P<0.05),FCR和DR显著升高(P<0.05);与EC组相比,CNEL和CNEH组的ADG显著升高(P<0.05),FCR和DR显著降低(P<0.05)。
表4 裸花紫珠提取物对大肠杆菌感染肉鸡生长性能的影响

Table 4 Effects of Callicarpa nudiflora extract on growth performance of chickens infected with Escherichia coli

项目
Items		 组别Groups P值
P-value
CON EC CNEL CNEH
1~19日龄1 to 19 days of age
平均日增重ADG/(g/d) 15.17±0.07b 15.22±0.15b 15.58±0.19a 15.42±0.21ab 0.02
平均日采食量ADFI/(g/d) 20.48±0.38 20.63±0.25 20.48±0.20 20.73±0.28 0.56
饲料转化率FCR 1.35±0.02 1.36±0.03 1.32±0.02 1.34±0.03 0.25
死亡率DR/% 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00
19~21日龄19 to 21 days of age
平均日增重ADG/(g/d) 16.05±0.07a 15.16±0.22b 16.30±0.19a 16.13±0.11a <0.01
平均日采食量ADFI/(g/d) 22.48±0.38ab 22.09±0.20b 22.48±0.20ab 22.73±0.28a 0.04
饲料转化率FCR 1.40±0.03b 1.46±0.02a 1.38±0.03b 1.41±0.01b <0.01
死亡率DR/% 0.00±0.00b 32.00±2.00a 00.00±0.00b 00.00±0.00b <0.01

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.

2.3 裸花紫珠提取物对大肠杆菌感染肉鸡器官指数的影响

由表5可见,与CON组相比,EC组的肝脏指数和脾脏指数显著升高(P<0.05),法氏囊指数显著降低(P<0.05);与EC组相比,CNEL和CNEH组的肝脏指数和脾脏指数显著降低(P<0.05),CNEH组的法氏囊指数显著升高(P<0.05)。各组之间胸腺指数无显著差异(P>0.05)。
表5 裸花紫珠提取物对大肠杆菌感染肉鸡器官指数的影响

Table 5 Effects of Callicarpa nudiflora extract on organ indexes of chickens infected with Escherichia coli %

项目
Items		 组别Groups P值
P-value
CON EC CNEL CNEH
脾脏指数Spleen index 0.95±0.17c 2.17±0.38a 1.72±0.27b 1.66±0.23b <0.01
法氏囊指数Bursa of Fabricius index 1.92±0.37a 1.25±0.11c 1.30±0.22bc 1.60±0.15b <0.01
胸腺指数Thymus index 1.60±0.33 1.47±0.10 1.63±0.21 1.49±0.17 0.64
肝脏指数Liver index 24.84±2.87c 39.16±4.08a 34.58±3.43b 31.37±3.32b <0.01

2.4 裸花紫珠提取物对大肠杆菌感染肉鸡血清炎性因子含量及空肠和脾脏炎性因子和TLR4、MyD88、NF-κB表达的影响

由图2可见,与CON组相比,EC组的血清IL-6含量显著升高(P<0.05),血清IL-10含量显著降低(P<0.05)。与EC组相比,CNEL和CNEH组的血清IL-6和TNF-α含量显著降低(P<0.05),CNEH组的血清IL-10含量显著升高(P<0.05)。
图2 裸花紫珠提取物对大肠杆菌感染肉鸡血清炎性因子含量及空肠和脾脏炎性因子表达的影响

A:血清 serum;B:空肠 jejunum;C:脾脏 spleen。

CON:对照组;EC:大肠杆菌攻毒组;CENL:低剂量裸花紫珠提取物组;CENH:高剂量裸花紫珠提取物组。数据柱标相同小写字母或无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)。下图同。

Fig.2 Effects of Callicarpa nudiflora extract on serum inflammatory factor contents and expression of inflammatory factor in jejunum and spleen of chickens infected with Escherichia coli

CON: control group; EC: Escherichia coli infection group; CENL: low dose Callicarpa nudiflora extract group; CENH: high dose Callicarpa nudiflora extract group. Value columns with the same small letter or no letter mean no significant difference (P>0.05), while with different small letters mean significant difference (P<0.05).The same as below.

与CON组相比,EC组的空肠IL-10 mRNA相对表达量显著降低(P<0.05),空肠TNF-α mRNA相对表达量显著升高(P<0.05)。与EC组相比,CNEL和CNEH组的空肠IL-1β、TNF-α mRNA相对表达量显著降低(P<0.05),空肠IL-4、IL-10 mRNA相对表达量显著升高(P<0.05);CNEH组的空肠IL-6 mRNA相对表达量显著降低(P<0.05)。各组之间空肠IFN-γ mRNA相对表达量无显著差异(P>0.05)。
与CON组相比,EC组的脾脏IL-4、IL-10 mRNA相对表达量显著降低(P<0.05),脾脏IL-6、TNF-α和IFN-γ mRNA相对表达量显著升高(P<0.05)。与EC组相比,CNEL和CNEH组的脾脏IL-4、IL-10 mRNA相对表达量显著升高(P<0.05),CNEL组的脾脏IL-1β mRNA相对表达量显著降低(P<0.05);CNEH组的脾脏IL-6、TNF-α mRNA相对表达量显著降低(P<0.05)。
由图3可见,与CON组相比,EC组的空肠MyD88 mRNA相对表达量显著升高(P<0.05)。与EC组相比,CNEL组的空肠TLR4 mRNA相对表达量显著降低(P<0.05)。各组之间空肠NF-κB mRNA相对表达无显著差异(P>0.05)。
图3 裸花紫珠提取物对大肠杆菌感染肉鸡空肠和脾脏TLR4、MyD88、NF-κB表达的影响

A:空肠 jejunum;B:脾脏 spleen。

Fig.3 Effects of Callicarpa nudiflora extract on expression of TLR4, MyD88 and NF-κB in jejunum and spleen of chickens infected with Escherichia coli

与CON组相比,EC组的脾脏MyD88和TLR4 mRNA相对表达量显著升高(P<0.05)。与EC组相比,CNEL和CNEH组的脾脏MyD88 mRNA表达量显著降低(P<0.05)。各组之间脾脏NF-κB mRNA相对表达无显著差异(P>0.05)。

2.5 裸花紫珠提取物对大肠杆菌感染肉鸡空肠和脾脏凋亡相关基因表达的影响

由图4可见,与CON组相比,EC组的空肠Bax、Caspase 3、Caspase 8 mRNA相对表达量显著升高(P<0.05)。与EC组相比,CNEL和CNEH组的空肠Caspase 3、Caspase 8 mRNA相对表达量显著降低(P<0.05),CNEL组的空肠Bax mRNA相对表达量显著降低(P<0.05)。各组之间空肠Bcl-2 mRNA相对表达无显著差异(P>0.05)。
图4 裸花紫珠提取物对大肠杆菌感染肉鸡空肠和脾脏凋亡相关基因表达的影响

A:空肠 jejunum;B:脾脏 spleen。

Fig.4 Effects of Callicarpa nudiflora extract on expression of apoptosis related gene in jejunum and spleen of chickens infected with Escherichia coli

与CON组相比,EC组的脾脏Bax和Caspase 8 mRNA相对表达量显著升高(P<0.05),脾脏Bcl-2 mRNA相对表达量显著降低(P<0.05)。与EC组相比,CNEL组的脾脏Bax和Caspase 8 mRNA相对表达量显著降低(P<0.05)。各组之间脾脏Caspase 3 mRNA相对表达无显著差异(P>0.05)。

2.6 裸花紫珠提取物对大肠杆菌感染肉鸡空肠形态的影响

由图5可见,与CON组相比,EC组的空肠VH和VH/CD显著降低(P<0.05)。与EC组相比,CNEH组的空肠VH显著升高(P<0.05)。各组之间空肠CD无显著差异(P>0.05)。
图5 裸花紫珠提取物对大肠杆菌感染肉鸡空肠形态的影响

Fig.5 Effects of Callicarpa nudiflora extract on jejunal morphology of chickens infected with Escherichia coli (40×)

2.7 裸花紫珠提取物对大肠杆菌感染肉鸡血清肠道通透性指标和空肠屏障基因表达的影响

由图6可见,与CON组相比,EC组的血清D-LA和LPS含量显著升高(P<0.05)。与EC组相比,CNEH组的血清DL-A和LPS含量显著降低(P<0.05),CNEL和CNEH组的血清DAO含量显著降低(P<0.05)。
图6 裸花紫珠提取物对大肠杆菌感染肉鸡血清肠道通透性指标和空肠屏障基因表达的影响

A:血清 serum;B:空肠 jejunum。

Fig.6 Effects of Callicarpa nudiflora extract on serum intestinal permeability indexes and jejunal barrier gene expression of chickens infected with Escherichia coli

与CON组相比,EC组的空肠ZO-1和Claudin mRNA相对表达量显著降低(P<0.05)。与EC组相比,CNEL和CNEH组的空肠ZO-1和Occludin mRNA相对表达量显著升高(P<0.05),CNEH组的空肠Claudin mRNA相对表达量显著升高(P<0.05)。

2.8 裸花紫珠提取物对大肠杆菌感染肉鸡肝脏细菌数量的影响

由图7可见,与CON组相比,EC组的肝脏大肠杆菌数量显著升高(P<0.05)。与EC组相比,CNEL和CNEH组的肝脏大肠杆菌感染数量显著降低(P<0.05)。
图7 裸花紫珠提取物对大肠杆菌感染肉鸡肝脏细菌数量的影响

Fig.7 Effects of Callicarpa nudiflora extract on liver bacterial number of chickens infected with Escherichia coli

3 讨论

3.1 裸花紫珠提取物有效活性成分含量

目前裸花紫珠中成药的法定工艺质量标准主要是以水为溶媒进行煎煮[14],常用黄酮类成分木犀草素和苯乙醇苷类成分毛蕊花糖苷作为质量评估指标[15-16]。本试验采用了HPLC法同时测定裸花紫珠提取物中木犀草素和毛蕊花糖苷含量,发现裸花紫珠水提物中毛蕊花糖苷含量为28.36 mg/g,木犀草素含量为0.34 mg/g。这与前人的研究结果[17⇓-19]一致,为后续进行动物试验研究提供了依据。

3.2 裸花紫珠提取物对大肠杆菌感染肉鸡生长性能的影响

禽致病性大肠杆菌常通过突破血气屏障引起败血症和多器官功能衰竭,是家禽的主要细菌性疾病之一,对全球家禽生产和公共卫生构成了重大威胁[5]。裸花紫珠提取物可以减轻大肠杆菌的不良影响。研究表明,感染大肠杆菌出现的临床症状包括食欲不振、嗜睡、脱水、腹泻等[20-21]。在本研究中,在1~14日龄,饲粮中添加裸花紫珠提取物可提高肉鸡的ADG;肉鸡在感染大肠杆菌后,ADG降低,DR和FCR均升高,而饲粮中添加裸花紫珠提取物后,可缓解大肠杆菌产生的不良影响,且相关数据与CON组相当,表明裸花紫珠提取物对大肠杆菌具有一定的预防作用。这与一些黄酮类化合物如黄芩苷、染料木黄酮等对大肠杆菌影响的研究结果[22]一致的。由此推测,裸花紫珠提取物对大肠杆菌感染肉鸡的生长性能具有促进作用。

3.3 裸花紫珠提取物对大肠杆菌感染肉鸡器官指数的影响

禽类免疫器官的生长发育状态直接影响其免疫功能,器官指数可反映其生长发育状态[2]。本研究发现,大肠杆菌感染肉鸡的肝脏指数和脾脏指数显著升高。肝脏指数和脾脏指数病理状态下的升高可能意味着免疫抑制以及器官的出血坏死[20]。在本研究中,饲粮中添加裸花紫珠提取物可以降低大肠杆菌导致的肝脏指数和脾脏指数升高,这可能减少大肠杆菌感染对器官带来的损伤[23]。蒋光阳[24]研究发现,木犀草素可提高肉鸡免疫器官指数;高莉等[25]研究指出,毛蕊花糖苷可拮抗D-半乳糖致亚急性衰老小鼠的免疫器官萎缩。木犀草素和毛蕊花糖苷为裸花紫珠提取物的活性成分,因此推测裸花紫珠提取物具有促进肉鸡主要器官生长的功效。法氏囊是肉鸡的体液免疫器官,在增强其抗菌能力方面起着至关重要的作用[26]。大肠杆菌感染导致肉鸡法氏囊指数显著下降,说明大肠杆菌会引起肉鸡免疫力及抗菌能力的降低,但在饲粮中添加裸花紫珠提取物没有表现出改善的优势。

3.4 裸花紫珠提取物对大肠杆菌感染肉鸡抗炎能力的影响

在肠道屏障的调节中,炎症细胞因子具有重要意义,它们被认为是机体对有害刺激的防御机制。促炎细胞因子具有刺激和加剧炎症反应的能力,而抗炎细胞因子具有阻碍促炎细胞因子产生的能力[27]。Toll样受体(Toll-like receptor,TLR)在对抗病原体的先天防御机制中发挥关键作用,在适应性免疫的发展中发挥关键作用,并且可能是对感染易感性的主要决定因素。研究发现,通过MyD88依赖性和Toll样受体结构域衔接蛋白(TRIF)依赖性途径激活TLR信号,对鸡单核细胞介导的先天免疫更有利[28]。TLR还激活NF-κB和丝裂原活化蛋白激酶(MAPK)通路,在TRIF依赖通路中反应延迟。TLR4信号传导还导致许多趋化因子和细胞因子的产生,在感染或损伤期间介导炎症中发挥作用。在本研究中,大肠杆菌感染后脾脏和空肠MyD88和TLR4 mRNA相对表达量升高,而饲粮中添加裸花紫珠提取物能够缓解这一不利因素。此外,本试验还研究了相关抗炎因子的表达。TNF-α可以与肿瘤坏死因子受体结合,激活NF-κB通路,并活化Caspase 3进而诱导肠上皮细胞凋亡,与肠屏障功能障碍有关[29-30]。本研究结果表明,大肠杆菌感染导致血清中促炎因子TNF-α和IL-6含量升高,抗炎因子IL-10含量降低;而饲粮中添加裸花紫珠提取物可不同程度地降低血清TNF-α和IL-6含量,提高血清IL-10含量,提示裸花紫珠提取物可减轻大肠杆菌引起的炎症损伤。在脾脏和空肠中也观察到类似的结果。因此,推测裸花紫珠提取物可能通过TLR4/MyD88/NF-κB通路增强机体的适应性免疫反应,从而增强机体抗炎能力,对抗大肠杆菌引起的炎症反应。

3.5 裸花紫珠提取物对大肠杆菌感染肉鸡抗凋亡能力的影响

Bcl-2家族在细胞凋亡信号的传递中起着至关重要的作用,其表达和调控是影响细胞凋亡的关键因素。降低Bcl-2的表达或增加Bax的表达均可促进细胞凋亡。此外,Bax和Bcl-2可以在膜内形成调控线性,导致细胞色素C的释放,Caspase的激活,最终导致凋亡[31]。先前的研究表明,大肠杆菌感染可导致大鼠凋亡相关基因的激活[32]。在本研究中,大肠杆菌感染显著上调了肉鸡的脾脏和空肠Caspase 8、Caspase 3和Bax的mRNA相对表达量,而饲粮中添加裸花紫珠提取物下调了这些基因的mRNA相对表达量。刘志龙等[33]研究发现,毛蕊花糖苷可降低凋亡细胞的数量,相应的凋亡基因mRNA相对表达量也降低。在血清中也观察到同样的趋势,其中Caspase 8含量也降低了。上述结果表明,裸花紫珠提取物具有调控肉鸡细胞凋亡基因表达的能力。

3.6 裸花紫珠提取物对大肠杆菌感染肉鸡肠道通透性和肠道屏障功能的影响

肠道机械屏障主要由肠上皮细胞和细胞间的紧密连接蛋白构成,它不仅是保护肠道免受外界病原体或有害物质侵入的关键,而且是维持肠道上皮屏障功能的基础[34]。DAO存在于肠黏膜和纤毛细胞中,肠道屏障受损时可以释放大量的DAO到血液循环中,导致血液DAO含量升高,因此DAO可以作为肠道通透性的生物标志物之一[35]。D-LA中毒可导致肠道菌群的破坏,在严重的情况下,可导致脑病[36]。LPS是革兰氏阴性菌(如大肠杆菌)细胞壁的主要成分。当病原体感染宿主时,它会引发炎症反应,导致血清LPS含量升高,生长性能下降[37]。本试验中,大肠杆菌感染导致肉鸡的血清LPS和D-LA回来了显著升高,DAO含量也有升高的趋势,表明大肠杆菌感染对肉鸡肠黏膜屏障造成了损伤,导致了肠道通透性增加,这与前人的研究结果[38]一致。然而,在饲粮中添加高剂量(0.3 g/kg)裸花紫珠提取物可降低血清DAO、LPS和D-LA含量,这表明裸花紫珠提取物可能具有直接的抗菌和抗炎作用,可能是由于其活性成分残留在胃肠道或其各种水解产物。由此可见,裸花紫珠提取物可能对肉仔鸡肠道通透性具有调节作用。
本试验在肉鸡19日龄时用大肠杆菌攻毒,21日龄时观察肉鸡空肠形态,结果表明,大肠杆菌感染肉鸡后,其肠道大量肠绒毛萎缩、断裂、脱落,进一步表明致病菌的入侵对肉鸡早期肠道结构产生了负面影响,导致肠黏膜严重损伤和屏障功能障碍。通过病理切片显示,低剂量(0.3 g/kg)裸花紫珠提取物组的绒毛长度和完整性较好,提示饲粮中添加裸花紫珠提取物可有效对抗大肠杆菌感染造成的肠道屏障完整性的损伤。此外,肠道屏障功能主要受紧密连接蛋白的调控,一般包括Occludin、Claudin和ZO-1[39-40]。本试验结果表明,大肠杆菌感染显著下调了肉鸡空肠Claudin和ZO-1的mRNA相对表达量,而饲粮中添加裸花紫珠提取物上调了Occludin、Claudin和ZO-1的mRNA相对表达量,这与前人的研究结果[27,41-42]一致。综上所述,裸花紫珠提取物可通过上调空肠紧密连接蛋白的mRNA表达来改善空肠的屏障功能,减轻肠道炎症,这可能进一步改善大肠杆菌定植对空肠造成的肠道损伤。

3.7 裸花紫珠提取物对大肠杆菌感染肉鸡的肝脏细菌数量的影响

肠道黏膜是抵御有害物质入侵的重要防御屏障[43]。肠道通透性的微观损伤会导致肠道细菌从管腔转移到肠系膜或其他器官,这一过程被称为细菌易位[44],细菌易位被认为是评估肠道结构完整性的一个指标[22]。在本研究中,大肠杆菌感染导致肉鸡肝脏大肠杆菌数量显著增加,而饲粮中添加裸花紫珠提取物有效地减少了感染大肠杆菌肉鸡的肝脏大肠杆菌数量。研究表明,肠腔内的黏液层是由肠上皮细胞及上皮间的连接所构成的屏障,其中最为重要的即为上皮间的紧密连接,它是由细胞间的多种紧密连接蛋白组成的复合物,这些蛋白包括Claudin、Occludin和闭锁小带蛋白(ZO)等,可以限制大于2 ku的分子(如细菌或LPS)从细胞旁途径通过[45]。同时肝脏和肠道与门静脉相连,门静脉含有来自肠道的营养物质、食物抗原、细菌产物和环境毒素。当肝脏受损时,肝脏中库普弗细胞(KCs)的功能会受到抑制。肠道中的细菌和内毒素侵入血液循环系统,导致肠源性感染。内毒素激活KCs并释放多种炎症因子。这些炎症因子的相互作用加剧了肠道黏膜屏障的损伤[5]。本研究结果表明,饲粮中添加裸花紫珠提取物可以不同程度地提高肠道紧密连接蛋白和抗炎基因的mRNA相对表达量。所以肠道大肠杆菌数量的减少可能是通过提高紧密连接蛋白的表达,提高肠道的机械屏障,从而限制了细菌进入肠道内,减少了炎症的发生。

4 结论

大肠杆菌感染影响肉鸡的生长性能,而饲粮中添加裸花紫珠提取物(0.3和0.5 g/kg)均能改善大肠杆菌感染对肉鸡生长性能的影响,并能降低DR;此外,还可以改善肉鸡的器官指数、提高机体抗凋亡能力,调节血清免疫指标和部分通过调控TLR4/MyD88/NF-κB通路及相关炎性因子表达来抵抗大肠杆菌感染引起的炎症反应,改善肠道形态结构和细菌易位。裸花紫珠提取物可作为家禽生产中抗菌药物的潜在替代品,且适宜剂量为0.5 g/kg。

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