Chinese Journal of Animal Nutrition >

2023 , Vol. 35 >Issue 4: 2196 - 2208

DOI: https://doi.org/10.12418/CJAN2023.207

RESEARCH PAPER

Effects of Fermented Ginkgo biloba-Eucommia Leaves on Intestinal Function and Immune Response in Stressed Broilers Challenged by Lipopolysaccharide

  • ZHANG Xuhui , 1 ,
  • CAO Yindi 1, 2 ,
  • SUN Zhiyuan 3 ,
  • CHEN Yulian 1 ,
  • CAO Fuliang , 1, *
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  • 1 Co-Innovation Center for the Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China
  • 2 Science and Technology Office, Qingdao Agricultural University, Qingdao 266109, China
  • 3 Institute of Animal Husbandry and Veterinary Medicine, Jiangsu Vocational College of Agriculture and Forestry, Jurong 212400, China
*professor, E-mail:

Received date: 2022-05-05

  Online published: 2023-04-12

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Abstract

This experiment was conducted to investigate the effects of dietary fermented Ginkgo binkgo-Eucommia leaves (FGEL) on growth performance, intestinal structure, digestion and absorption function and immune response of broilers under lipopolysaccharide (LPS) stress, and to compare them with unfermented Ginkgo binkgo-Eucommia leaves (GEL). A 2×3 two-factor experimental design was used, namely, two immune stress treatments (intraperitoneal injection of LPS or injection of the same volume of normal saline) and three dietary treatments (basal diet and experimental diets supplemented with 0.40% GEL or 0.40% FGEL, respectively). A total of 360 one-day-old broiler chicks were randomly divided into 6 treatments with 6 replicates of 10 birds per cage. The experiment lasted for 21 d. On days 13, 15, 15, 17, 19 and 21 of the trial, LPS 500 μg/kg BW was injected intraperitoneally in the three stress treatments, and the same amount of normal saline was injected in the other three groups. Slaughtering and sampling were carried out between 2 to 4 h after the last injection at day 21 of the trial. The results showed as follows:1) compared with the control diet, the feed gain ratio (F/G) of broilers fed FGEL diet was significantly improved (P=0.024); dietary FGEL prevented LPS-induced reductions in average daily weight gain (ADG) (P=0.023) and serum D-xylose content (P=0.011). 2) Compared with the control diet, dietary FGEL supplementation dramatically alleviated the LPS-induced decrease of duodenal (P=0.011) and jejunal (P=0.030) relative weight, duodenal (P=0.043) and jejunal (P=0.014) villus height (VH), as well as duodenal (P=0.012), jejunal (P=0.018) and ileal (P=0.014) alkaline phosphatase (AKP) activity. Additionally, FGEL supplementation significantly mitigated the LPS-induced increases in the duodenal (P=0.024) and jejunal (P=0.018) crypt depth (CD), duodenal sodium/glucose cotransporter 1 (SGLT1) mRNA relative expression level (P=0.013). 3) Compared with NGEL diet and control diet, significant decrease of duodenal and jejunal inducible nitric oxide synthase (iNOS) (P=0.031), nuclear transcription factor-κB (NF-κB) (P=0.029), interferon-γ (IFN-γ) (P=0.017), interleukin-1β (IL-1β) (P=0.020), interleukin-6 (IL-6) (P=0.048) and interleukin-10 (IL-10) (P=0.037) mRNA relative expressional levels were found in LPS-challenged birds pretreated with FGEL. In conclusion, under the conditions of this study, dietary FGEL can improve the growth performance of broilers by improving intestinal structure, digestion and absorption function and alleviating the excessive activation of NF-κB-related inflammatory factors.

Key words： fermented Ginkgo biloba-Eucommia leaves; broiler; intestinal morphology; digestion and absorption; immune stress

Cite this article

ZHANG Xuhui , CAO Yindi , SUN Zhiyuan , CHEN Yulian , CAO Fuliang . Effects of Fermented Ginkgo biloba-Eucommia Leaves on Intestinal Function and Immune Response in Stressed Broilers Challenged by Lipopolysaccharide[J]. Chinese Journal of Animal Nutrition, 2023 , 35(4) : 2196 -2208 . DOI: 10.12418/CJAN2023.207

近年来,我国养禽业已从传统生产方式向高密度、规模化、集约化养殖模式转变,但新生肉仔鸡的免疫系统发育尚不成熟,在饲养过程中极易受到外源因素(如环境污染、免疫接种程序复杂以及多种病原菌)的侵袭,这些外源性刺激会间接诱导动物机体处于免疫应激状态[1]。而肉鸡肠上皮组织位于肠道与外界环境的交界处,更易受肠腔中有害物质的直接影响[2]。肠道受到外源刺激时,活性氧(ROS)等自由基大量产生,引发的肠炎性腹泻又进一步加剧肠道黏膜组织的损伤程度,极易引发肉鸡死亡,给养殖行业带来了严重的经济损失。由于胃肠道对肉鸡的整体健康和生产性能的贡献已越来越受到重视,因此,研究和开发能增强和调节肠道黏膜免疫功能的天然物质迫在眉睫。
我国木本植物资源丰富,木本饲料添加剂的开发利用潜力很大。银杏和杜仲是我国特有的药用植物。其中,银杏叶的活性成分主要是类黄酮(黄酮苷,主要由槲皮素组成)、多糖(葡萄糖、鼠李糖、阿拉伯糖、甘露糖、半乳糖和木糖的聚合物)和萜类化合物(银杏内酯)。杜仲叶富含绿原酸、槲皮素、京尼平苷酸、多种氨基酸、微量元素、维生素等。若干体内外研究表明,银杏叶和杜仲叶均具有抗氧化、抗炎、调节免疫、抗感染的功能[3⇓⇓⇓-7]。最新研究表明,杜仲绿原酸可通过调节肠道菌群平衡保护小鼠免受镉诱导的肝肾损伤[8]。Hussain等[9]通过猪的抗炎试验结果表明,杜仲黄酮可介导磷脂酰肌醇-3-羟激酶/核转录因子-κB(PI3K/NF-κB)信号通路缓解由脂多糖(LPS)诱导的猪肠道细胞的损伤,降低肠道炎症反应。Yao等[10]研究表明,银杏叶提取物通过抑制NF-κB p65从而发挥对LPS诱导的免疫应激损伤的保护作用。然而,复杂的提取过程会导致提取物的价格较高。此外,提取剩余物中纤维含量高、消化率低,这限制了银杏叶中营养物质的吸收和利用[11]。
发酵是一种简单、廉价且有效的方法,减少抗营养因子,并可以破坏细胞壁结构,释放其中的酚类化合物等有效成分,同时将其中的大分子活性成分转化为小分子,从而促进动物的肠道吸收[12]。但目前,有关银杏-杜仲叶发酵物(FGEL)对动物肠道功能和免疫应激方面的研究还较少。因此,本研究旨在探讨富含黄酮和绿原酸活性成分的FGEL对LPS应激爱拔益加(AA)肉仔鸡的肠道结构、功能和免疫反应的影响,并同时与未发酵银杏-杜仲叶(GEL)进行比较,以期为银杏叶和杜仲叶作为木本饲料资源应用于肉鸡生产提供科学依据。

1 材料与方法

1.1 FGEL的制备

银杏叶片来源于10月份南京林业大学银杏园的银杏树采摘,杜仲叶片来源于12月份河南理工大学杜仲园的杜仲落叶。分别将银杏叶和杜仲叶在60 ℃烘干,然后将干燥的叶片在电动研磨机中研磨并通过40目筛,自封袋密封备用。将银杏叶和杜仲叶按照2∶1的比例复配,具体的发酵参数详见文献[13]。FGEL主要营养成分及活性成分见表1。
表1 FGEL主要营养成分及活性成分

Table 1 Main nutritional components and active components of FGEL %

项目
Items		 粗蛋白质
Crude protein		 必需氨基酸
Essential
amino acids		 总氨基酸
Total amino
acids		 总黄酮
Total
flavonoids		 绿原酸
Chlorogenic
acid
发酵前Before fermentation 12.41±0.97 3.74±0.13 9.76±0.29 1.67±0.02 0.42±0.03
发酵后After fermentation 25.23±1.28* 6.34±0.23* 17.49±0.94* 2.13±0.04* 0.50±0.02*
增量Increment 103.30 69.52 79.20 27.55 19.48

*表示与发酵前对应值相比差异显著(P<0.05)。

*indicated that the differences were significant compared with the corresponding values before fermentation (P<0.05).

1.2 试验设计与试验饲粮

本研究基于前期试验,综合生长性能、抗氧化功能(血清、肝脏和胸肌)、血清免疫和肠道功能指标的数据[13-14],确定FGEL在肉仔鸡饲粮中的最佳添加剂量为0.40%。在正式试验开始之前,通过测定应激指标来确定LPS注射的剂量和途径,最终,本试验通过腹腔注射的方法来模拟肠道的应激状态,注射剂量(500 μg/kg BW)参考前期研究[15-16]。采用2×3双因子试验设计,即2种免疫应激处理(腹腔注射LPS或注射等量生理盐水)和3种饲粮处理(基础饲粮及在基础饲粮中添加0.40% GEL或0.40% FGEL的试验饲粮)。选取360只1日龄健康AA肉仔鸡(购自山东烟台苏佳丽禽业有限公司),体重差异不显著(P>0.05),随机分为6个处理,每个处理均设6个重复,每个重复10只鸡。试验期为21 d。LPS的大肠杆菌血清型为O55∶B5(Sigma, L2880)。试验鸡饲养至13 d时,3个应激处理的试鸡腹腔注射500 μg/kg BW的LPS(溶解于1 mL生理盐水),并在试验第13、15、17、19和21天按首次剂量在相同时间内重复注射,其他3个处理注射等量的生理盐水。基础饲粮为玉米-豆粕型,其组成及营养水平见表2。在不同的饲粮处理中,用等量麸皮来替代试验添加物的量。
表2 基础饲粮组成及营养水平(风干基础)

Table 2 Composition and nutrient levels of the basal diet (air-dry basis) %

项目Items 含量Content
原料Ingredients
玉米Maize 60.74
豆粕Soybean meal 30.82
玉米蛋白粉Corn gluten meal 2.00
豆油Soybean oil 2.03
石粉Limestone 1.10
磷酸氢钙CaHPO4 1.39
麸皮Wheat bran 0.50
预混料Premix1) 1.50
食盐NaCl 0.20
L-赖氨酸L-Lys 0.07
DL-蛋氨酸DL-Met 0.15
合计Total 100.00
营养水平Nutrient levels2)
代谢能ME/(MJ/kg) 12.27
粗蛋白质CP 21.20
赖氨酸Lys 1.08
钙Ca 1.00
蛋氨酸Met 0.50
蛋氨酸+半胱氨酸Met+Cys 0.82
有效磷AP 0.43

1)预混料为每千克饲粮提供 The premix provided the following per kg of the diet:VA 8 500 IU,VB1 2.2 mg,VB2 10 mg,VB5 30 mg,VB6 10 mg,VB12 0.03 mg,VD3 3 000 IU,VE 15 IU,VK3 1.5 mg,生物素 biotin 0.10 mg,叶酸 folic acid 1.3 mg,D-泛酸 D-pantothenic acid 9 mg,烟酸 nicotinic acid 10 mg,Cu (as copper sulfate) 7.5 mg,Fe (as ferrous sulfate) 60 mg,Mn (as manganese sulfate) 110 mg,Zn (as zinc sulfate) 65 mg,I (as potassium iodide) 1.10 mg,Se (as sodium selenite) 0.40 mg。

2)粗蛋白质为测定值,其他为计算值。Crude protein was a measured value, while the others were calculated values.

1.3 饲养管理

进雏前,彻底清扫、冲洗鸡舍地面及育雏设施,用甲醛和高锰酸钾熏蒸消毒。饲养遵循少喂勤添的原则,自由采食、饮水,采用3层笼养的管理模式。
房间温度在试验第1~7天控制在(35±1) ℃,试验第7天后逐步下调温度。试验第1~7天采用每天24 h光照,之后逐步降到每天12 h光照。及时通风并清洗饮水管道,每日清洁鸡舍,确保鸡舍内干净、卫生。采用颈背侧皮下注射方式,于试验第10天逐只接种禽流感疫苗和新城疫(La Sota株)三联多价灭活疫苗,进行免疫。试验期间注意观察试鸡,记录每个重复每天的死亡数量。

1.4 样品采集

于试验第20天的20:00断料,自由饮水,于试验第21天的08:00注射LPS或者生理盐水后2~4 h进行采样。从每个处理的每个重复中随机选取1只鸡,称重后由颈动脉放血致死,迅速剖开腹腔,将不同的肠段(十二直肠、空肠和回肠)进行分离,并进行称重记录。各肠段的相对重量表示为:肠段重与体重之比(g/kg)。之后分离十二指肠、空肠和回肠,于每段肠开始部位截取1 cm左右肠段,并用生理盐水冲洗肠道,分别立即置于4%的多聚甲醛固定液和2.5%的戊二醛溶液中,用于制作石蜡切片和扫描电镜(Quqnta 200)。同时再截取相同部位肠段,轻轻挤出肠道食糜,剪开肠道黏膜,用载玻片轻轻刮去肠道表层食糜,再刮取黏膜,立即置于液氮保存待测。

1.5 检测指标

1.5.1 生长性能测定

分别在试验第1和21天的07:00空腹称重并结料,称重和计算以重复为单位进行,计算平均日采食量(ADFI)、平均日增重(ADG)及料重比(F/G)。

1.5.2 肠道组织切片

将在4%多聚甲醛固定液中固定24 h的肠道组织,经乙醇逐级脱水,二甲苯透明,石蜡包埋,用轮转式切片机(浙江YD-1508R)连续切厚度为5 μm片3张,之后经摊片烘干,苏木精-伊红(HE)染色,中性树胶封固后,使用低倍镜选择典型视野观测切片,再采用高倍镜观测典型视野中所有的完整绒毛(Nikon YS100纤维摄像系统),记录绒毛高度(VH)和隐窝深度(CD)。

1.5.3 肠道扫描电镜

将在2.5%戊二醛溶液中固定的十二指肠和空肠肠段切成1 mm3的小块,用pH 7.4的磷酸缓冲液漂洗5 min,转入4%戊二醛溶液中,于4 ℃下固定48 h,再用pH 7.4的磷酸盐缓冲液清洗3次,再用1%的锇酸固定。用梯度浓度至100%浓度的乙醇逐级进行脱水,再用醋酸异戊酯置换2次。二氧化碳临界点干燥后,把样品粘贴在样品台上,喷金镀膜(真空镀膜离子溅射仪),电镜扫描(日本明石1400)观察并拍照。

1.5.4 肠黏膜碱性磷酸酶(AKP)活性测定

用10倍体积生理盐水制备十二指肠、空肠和回肠黏膜组织匀浆,4 ℃,4 000 r/min离心20 min,取上清液,采用南京建成生物工程研究所的试剂盒测定肠黏膜中AKP活性。组织匀浆上清液中蛋白质含量采用Lowry等[16]方法进行测定。

1.5.5 血清D-木糖含量测定

采用南京建成生物工程研究所试剂盒测定血清D-木糖含量。从各处理的每个重复中重新选取1只鸡,按0.5 g/kg·BW的剂量给其灌服D-木糖溶液,1 h之后,翅静脉采血,分离的血清采用Doerfler等[17]的方法测定D-木糖的含量。

1.5.6 十二指肠黏膜相关基因mRNA相对表达量测定

取冻存的十二指肠组织,Trizol(TaKaRa)一步提取法提取总RNA。用核酸蛋白仪测定总RNA浓度,确保OD260/OD280值均在1.8~2.0,并通过1%琼脂糖凝胶进行水平电泳(电压:120 V,20 min)测定RNA的完整性。使用PrimeScriptTM RT(TaKaRa)试剂盒,按照说明书将RNA合成cDNA。利用β-肌动蛋白(β-actin)对cDNA在普通PCR仪的扩增,检测反转录结果。根据GenBank相应的cDNA序列,进行BLAST分析后,用Premier 5.0软件设计目的基因钠/葡萄糖协同转运蛋白1(SGLT1)、诱导型一氧化氮合酶(iNOS)、核转录因子-κB(NF-κB)、干扰素-γ(IFN-γ)、白细胞介素-1β(IL-1β)、白细胞介素-4(IL-4)、白细胞介素-6(IL-6)、白细胞介素-13(IL-13)和内参基因β-actin的引物。采用双标准曲线法在荧光定量PCR仪对以上所述基因进行PCR扩增,同时扩增内参基因β-actin校正加样误差。实时荧光定量PCR反应程序如下:95 ℃ 30 s,40个循环的95 ℃ 5 s(变性阶段),60 ℃ 30 s(退火和延伸阶段),以及95 ℃ 15 s,60 ℃ 1 min,95 ℃ 15 s和60 ℃ 15 s(熔化阶段)。每当循环退火结束时收集1次荧光信号。cDNA的PCR扩增结果采用Stratagene's Mx3000P荧光分析系统得出,根据2-ΔΔCt方法计算各处理十二指肠中各基因的mRNA相对表达量。相关基因的引物序列见表3。
表3 引物序列

Table 3 Primer sequences

基因名称
Gene names		 GenBank登录号
GenBank accession No.		 引物序列
Primer sequence (5'—3')
β-肌动蛋白
β-actin		 NM_205518 F:CCACCGCAAATGCTTCTAAAC
R:AAGACTGCTGCTGACACCTTC
钠/葡萄糖转运载体1
SGLT1		 AJ236903 F:GATGTGCGGATACCTGAAGC
R:AGGGATGCCAACATGACTG
诱导型一氧化氮合酶
iNOS		 D85422 F:CCTGTACTGAAGGTGGCTATTGG
R:AGGCCTGTGAGAGTGTGCAA
核转录因子-κB
NF-κB		 D13721 F:TCAACGCAGGACCTAAAGACAT
R:GCAGATAGCCAAGTTCAGGATG
干扰素-γ
IFN-γ		 NM_205149 F:GCTCCCGATGAACGACTTGA
R:TGTAAGATGCTGAAGAGTTCATTCG
白细胞介素-1β
IL-1β		 NM_204524 F:TGCCTGCAGAAGAAGCCTCG
R:GACGGGCTCAAAAACCTCCT
白细胞介素-4
IL-4		 NM_001007079 F:GCTCTCAGTGCCGCTGATG
R:GAAACCTCTCCCTGGATGTCAT
基因名称
Gene names		 GenBank登录号
GenBank accession No.		 引物序列
Primer sequence (5'—3')
白细胞介素-6
IL-6		 HM179640 F:TGCAGTGTTACCTGGGAGAA
R:CGGTGTGATTTAGACCCGTAA
白细胞介素-13
IL-13		 NM_001007085 F:CATGACCGACTGCAAGAAGGA
R:CCGTGCAGGCTCTTCAGACT

1.6 统计分析

数据采用SPSS 16.0统计软件的GLM程序进行2×3两因子方差分析,模型中的因素包括免疫应激处理(应激)和饲粮处理(饲粮)以及两者之间的交互效应(应激×饲粮)。如果处理结果有显著影响,则通过LSD多重比较确定处理间的差异显著性。以重复为统计单位进行生长性能指标分析,以鸡只个体为统计单位进行血清和组织指标分析。结果用平均值和均值标准误(SEM)表示,以P<0.05为差异显著性判断标准。

2 结果与分析

2.1 FGEL对LPS应激肉仔鸡生长性能与肠道发育的影响

由表4可见,与LPS(-)相比,LPS应激对肉仔鸡的F/G和死亡率无显著影响(P>0.05);LPS诱导的免疫应激肉仔鸡的ADG(P=0.015)和ADFI(P=0.046)显著降低。与对照饲粮相比,饲粮中添加FGEL的肉仔鸡F/G(P=0.024)和ADG(P=0.023)有显著改善。
表4 FGEL对LPS应激肉仔鸡生长性能的影响

Table 4 Effects of FGEL on growth performance in stressed broilers challenged by LPS

处理
Treatments		 死亡率
Mortality/%		 料重比
F/G		 平均日增重
ADG/g		 平均日采食量
ADFI/g
LPS (-) Cont. 1.70 1.68 23.98 40.31
GEL 1.62 1.61 24.94 40.09
FGEL 1.51 1.57 25.56 40.00
LPS (+) Cont. 1.73 1.72 21.94 37.71
GEL 1.64 1.62 23.99 38.83
FGEL 1.60 1.59 24.53 38.92
SEM 0.031 0.021 0.251 0.345
应激Stress LPS (-) 1.61 1.62 24.83a 40.13a
LPS (+) 1.66 1.64 23.49b 38.49b
饲粮Diet Cont. 1.72 1.70b 22.96b 39.01
GEL 1.63 1.62ab 24.47ab 39.46
FGEL 1.56 1.58a 25.05a 39.46
主效应P值
P-value of
main effect		 应激Stress 0.703 0.516 0.015 0.046
饲粮Diet 1.060 0.024 0.023 0.789
应激×饲粮Stress×diet 0.914 0.561 0.257 0.684

LPS:脂多糖;LPS (-):为未注射LPS;LPS(+):注射LPS;Cont.:对照饲粮,GEL:饲粮添加0.4% GEL;FGEL:饲粮添加0.4% FGEL。同列中同一种主效应间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)。下表同。

LPS: lipopolysaccharide; LPS (-):without LPS injection; LPS (+): LPS injection; Cont.: control diet, GEL: the diet supplemented with 0.4% GEL; FGEL: the diet supplemented with 0.4% FGEL. In the same column between the same main effect, 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). The same as below.

由表5可见,与LPS(-)相比,LPS诱导的免疫应激显著降低了肉仔鸡十二指肠(P=0.002)和空肠(P=0.002)的相对重量。而与对照饲粮相比,饲粮中添加FGEL的肉仔鸡十二指肠(P=0.011)和空肠的(P=0.030)相对重量有显著改善,肉仔鸡的回肠相对重量影响不显著(P=0.598)。
表5 FGEL对LPS应激肉仔鸡肠道相对重量的影响

Table 5 Effects of FGEL on relative intestinal weight in stressed broilers challenged by LPS g/kg

处理Treatments 十二指肠Duodenum 空肠Jejunum 回肠Ileum
LPS (-) Cont. 11.87 14.95 10.42
GEL 12.06 15.92 10.91
FGEL 12.58 16.19 10.97
LPS (+) Cont. 10.76 13.42 9.31
GEL 11.48 14.85 9.83
FGEL 11.95 15.61 10.47
SEM 0.141 0.212 0.336
应激Stress LPS (-) 12.17a 15.69a 10.77
LPS (+) 11.40b 14.62b 9.87
饲粮Diet Cont. 11.32b 14.19b 9.87
GEL 11.77ab 15.39ab 10.37
FGEL 12.27a 15.90a 10.72
主效应P值
P-value of
main effect		 应激Stress 0.002 0.002 0.197
饲粮Diet 0.011 0.030 0.598
应激×饲粮Stress×diet 0.783 0.392 1.311

2.2 FGEL对LPS应激肉仔鸡肠道结构和形态的影响

由表6可见,与LPS(-)相比,LPS(+)处理的肉仔鸡十二指肠(P=0.014)和空肠(P=0.019)的VH显著降低;与对照饲粮和添加GEL饲粮相比,饲喂添加FGEL饲粮的肉仔鸡在十二指肠和空肠绒毛营养障碍方面有显著改善(P=0.043和P=0.014)。与LPS(-)相比,LPS刺激了十二指肠(P=0.015)和空肠(P=0.016)隐窝深度的增加;而与对照饲粮相比,饲粮中添加FGEL的肉仔鸡的十二指肠(P=0.024)和空肠(P=0.018)隐窝深度显著降低,回肠隐窝深度也显著降低(P=0.037)。
表6 FGEL对LPS应激肉仔鸡肠道结构和形态的影响

Table 6 Effects of FGEL on intestinal structure and morphology in stressed broilers challenged by LPS μm

处理
Treatments		 绒毛高度VH 隐窝深度CD
十二指肠
Duodenum		 空肠
Jejunum		 回肠
Ileum		 十二指肠
Duodenum		 空肠
Jejunum		 回肠
Ileum
LPS (-) Cont. 811.34 566.09 417.82 119.62 97.23 53.27
GEL 830.29 570.91 420.64 116.93 94.92 51.98
FGEL 875.41 627.75 428.69 110.51 89.48 49.29
LPS (+) Cont. 756.05 517.68 408.68 128.47 99.89 56.36
GEL 763.79 523.95 409.45 120.27 98.11 52.99
FGEL 839.58 609.89 412.80 115.97 92.09 52.32
SEM 8.71 10.37 5.49 1.31 0.82 1.71
应激Stress LPS (-) 839.00a 594.90a 422.38 115.69b 93.88b 51.18
LPS (+) 786.47b 557.17b 410.31 121.57a 96.70a 53.56
处理
Treatments		 绒毛高度VH 隐窝深度CD
十二指肠
Duodenum		 空肠
Jejunum		 回肠
Ileum		 十二指肠
Duodenum		 空肠
Jejunum		 回肠
Ileum
饲粮Diet Cont. 783.70b 541.89b 413.25 124.05a 98.56a 54.82a
GEL 797.04b 547.43b 415.05 118.60ab 96.52ab 52.49ab
FGEL 857.50a 618.82a 420.75 113.24b 90.79b 50.80b
主效应P值
P-values of
main effect		 应激Stress 0.014 0.019 0.307 0.015 0.016 0.079
饲粮Diet 0.043 0.014 0.892 0.024 0.018 0.037
应激×饲粮Stress×diet 0.945 0.738 0.310 1.023 0.874 0.347
如图1、图2所示,与对照组相比,LPS注射明显影响了十二指肠和空肠的肠道绒毛的有序性和规则性,与电镜扫描结果图3、图4所显示肠道黏膜的形态相对应,LPS+FGEL组的损伤程度有显著缓解。图1至图4与表5的结果吻合,从不同角度说明,LPS应激会破坏肠道绒毛的形态结构,LPS+FGEL组的肠道损伤程度降低。
图1 LPS和FGEL对十二指肠肠道结构与形态的影响

Fig.1 Effects of LPS and FGEL on structure and morphology of duodenum (40×)

图2 LPS和FGEL对空肠肠道结构与形态的影响

Fig.2 Effects of LPS and FGEL on structure and morphology of jejunal (40×)

图3 LPS和FGEL对十二指肠肠道黏膜完整性的影响(电镜扫描)

Fig.3 Effects of LPS and FGEL on intestinal mucosal integrity of duodenum (electron microscope scanning)

图4 LPS和FGEL对空肠肠道黏膜完整性的影响(电镜扫描)

Fig.4 Effects of LPS and FGEL on intestinal mucosal integrity of jejunal (electron microscope scanning)

2.3 FGEL对LPS应激肉仔鸡肠道吸收功能的影响

由表7可见,与LPS(-)相比,LPS诱导免疫应激显著降低了十二指肠(P=0.011)和空肠(P=0.012)黏膜AKP活性及血清D-木糖的含量(P=0.028)。饲粮中添加FGEL不仅能够抑制LPS刺激所诱导的负面作用,而且与对照饲粮和添加GEL饲粮相比,能够显著增加血清D-木糖含量(P=0.011)和十二指肠(P=0.012)﹑空肠(P=0.018)及回肠(P=0.014)黏膜AKP活性,并显著降低十二指肠SGLT1 mRNA相对表达量(P=0.003)。十二指肠SGLT1 mRNA相对表达量在LPS刺激下显著增加(P=0.001),同时,饲粮与应激对肉仔鸡十二指肠SGLT1的mRNA相对表达量(P=0.011)和回肠黏膜AKP活性(P=0.015)存在显著的互作效应。
表7 FGEL对LPS应激肉仔鸡肠道吸收功能的影响

Table 7 Effects of FGEL on intestinal absorptive function in stressed broilers challenged by LPS

处理
Treatments		 碱性磷酸酶活性AKP activity/(U/g) 血清D-木糖含量
Serum D-xylose
content/(mmol/L)		 十二指肠SGLT1
mRNA相对表达量
Relative expression of
duodenal SGLT1 mRNA
十二指肠
Duodenum		 空肠
Jejunum		 回肠
Ileum
LPS (-) Cont. 22.04 14.64 12.37 2.57 1.44c
GEL 23.19 14.69 12.41 2.62 1.46c
FGEL 27.44 15.47 12.92 3.27 1.49c
LPS (+) Cont. 17.63 13.07 11.40 2.23 2.69a
GEL 17.87 13.16 11.50 2.59 2.61a
FGEL 23.11 14.31 12.40 3.04 1.75bc
SEM 0.641 0.204 0.220 0.070 0.092
应激Stress LPS (-) 24.22a 14.93a 12.90 2.82a 1.46c
LPS (+) 19.54b 13.51b 11.77 2.62b 2.35a
处理
Treatments		 碱性磷酸酶活性AKP activity/(U/g) 血清D-木糖含量
Serum D-xylose
content/(mmol/L)		 十二指肠SGLT1
mRNA相对表达量
Relative expression of
duodenal SGLT1 mRNA
十二指肠
Duodenum		 空肠
Jejunum		 回肠
Ileum
饲粮Diet Cont. 19.84b 13.89b 11.89b 2.40b 2.07b
GEL 20.53b 13.93b 11.96b 2.61b 2.04b
FGEL 25.28a 14.89a 12.66a 3.16a 1.62
主效应P值
P-values of
main effect		 应激Stress 0.011 0.012 0.760 0.028 0.001
饲粮Diet 0.012 0.018 0.014 0.011 0.013
应激×饲粮Stress×diet 0.172 0.241 0.015 1.452 0.011

2.4 FGEL对LPS应激肉仔鸡肠黏膜炎性因子mRNA相对表达量的影响

由表8可见,与LPS(-)相比,LPS诱导的免疫应激显著增加了肉仔鸡肠黏膜中iNOS、NF-κB、IFN-γ、IL-1β、IL-6、IL-4和IL-10的mRNA相对表达量(P<0.05)。然而,与对照饲粮相比,饲粮中添加FGEL的肉仔鸡肠黏膜中iNOS、NF-κB、IFN-γ、IL-1β、IL-6和IL-10的mRNA相对表达量显著降低(P<0.05)。与添加GEL饲粮和对照饲粮相比,饲粮中添加FGEL能够在一定程度上减缓LPS诱导的肉仔鸡肠黏膜中iNOS(P=0.031)、NF-κB(P=0.029)、IFN-γ(P=0.017)、IL-1β(P=0.020)、IL-6(P=0.048)和IL-10(P=0.037)的mRNA相对表达量的过度增加。同时,饲粮与应激在肠道黏膜IFN-γ(P=0.012)、IL-1β(P=0.025)和IL-10(P=0.012)的mRNA相对表达量上存在互作效应。
表8 FGEL对LPS应激肉仔鸡肠道黏膜炎性因子mRNA相对表达量的影响

Table 8 Effects of FGEL on mRNA relative expression levels of intestinal mucosal inflammatory factor in stressed broilers challenged by LPS

处理
Treatments		 诱导型一氧
化氮合酶
iNOS		 核转录
因子-κB
NF-κB		 干扰素-γ
IFN-γ		 白细胞
介素-1β
IL-1β		 白细胞
介素-6
IL-6		 白细胞
介素-4
IL-4		 白细胞
介素-10
IL-10
LPS (-) Cont. 1.00 1.00 1.00 1.00 1.00 1.00 1.00
GEL 0.98 1.00 1.00 0.97 1.00 0.99 1.00
FGEL 0.97 0.99 0.98 0.98 0.98 0.97 0.99
LPS (+) Cont. 1.54 1.85 1.90 1.82 1.70 1.44 1.81
GEL 1.43 1.75 1.79 1.76 1.67 1.34 1.75
FGEL 1.22 1.23 1.51 1.53 1.42 1.29 1.58
SEM 0.08 0.05 0.13 0.12 0.06 0.09 0.17
应激Stress LPS (-) 0.98b 1.00b 0.99b 0.98b 0.99b 0.99b 1.00b
LPS (+) 1.40a 1.61a 1.73a 1.68a 1.60a 1.36a 1.69a
饲粮Diet Cont. 1.27a 1.43a 1.45a 1.41a 1.35a 1.22 1.41a
GEL 1.21a 1.38ab 1.40a 1.37ab 1.34a 1.17 1.38a
FGEL 1.10b 1.11b 1.25b 1.22b 1.20b 1.13 1.25b
主效应P值
P-values of
main effect		 应激Stress 0.013 0.010 0.015 0.011 0.022 0.032 0.014
饲粮Diet 0.031 0.029 0.017 0.020 0.048 0.055 0.037
应激×饲粮Stress×diet 0.065 0.059 0.012 0.025 0.074 0.064 0.012

3 讨论

3.1 双菌发酵对银杏叶-杜仲叶中营养成分的影响

发酵已被证明是提高中药生物活性的可行策略,可以刺激中草药的主要活性成分转化为其代谢产物,并会增强其治疗潜力和效果[18⇓⇓⇓-22]。本试验采用了黑曲霉菌和产朊假丝酵母菌进行混合发酵,并添加了20%的麸皮作为发酵基料,发酵产物中的粗蛋白质、氨基酸、总黄酮类和绿原酸含量皆得到提高,这可能归因于:1)植物细胞壁的结构破坏并释放酚类化合物;2)真菌分泌的蛋白酶水解FGEL中的大分子蛋白质,纤维素降解导致蛋白质从细胞壁释放;3)黑曲霉会产生大量的纤维素酶,分解纤维素产生大量还原糖,这些还原糖除了供应黑曲霉自身利用外,大部分将积累在培养基,从而阻遏黑曲霉继续分泌纤维素酶[8],而产朊假丝酵母菌恰恰可以利用这些还原糖,从而使发酵持续进行,直至发酵更为彻底,而且还产生了大量的菌体蛋白。

3.2 FGEL对LPS应激肉仔鸡生长性能的影响

本课题组前期研究表明,黑曲霉、芽孢杆菌和酿酒酵母等益生菌可在林源植物叶固态发酵产物中产生包括单宁酸、酚酸在内的多酚类化合物,从而显著提高发酵产物的生物利用价值[23],作为添加剂可以有效提高动物的生长性能和饲料效率[14,24]。黄酮类化合物的生物学作用主要归功于其所含的甙元[25],发酵后的黄酮甙元能够更容易且更迅速地被肠道吸收[26]。前期的若干研究表明,中药发酵产物能促进肉鸡的生长[27-28]。本试验中,LPS刺激后,肉仔鸡有明显的食欲减退及精神萎靡,致肉仔鸡的ADFI和ADG有显著的降低,而饲粮添加FGEL的肉仔鸡ADG和F/G有显著改善。Huang等[29]和Wang等[30]的研究表明,发酵中药能够通过调节肉仔鸡的肠道菌群,增强肠道免疫,改善肉仔鸡的生长。

3.3 FGEL对LPS应激肉仔鸡肠道形态结构的影响

肠道黏膜的结构是肠道健康的一个指标。本试验中,LPS刺激后,十二指肠和空肠的相对重量显著降低,而肉仔鸡的F/G并未受到LPS刺激的影响。原因可能是小肠是一个活跃的器官,它能在应激期间优先利用有限的可用营养物质。VH与小肠功能有关,更大的VH表明小肠吸收营养的能力更强[31]。而LPS可导致肠道VH的显著降低,这表明LPS导致肉仔鸡肠道上皮层通透性增加、炎症反应和肠道运动功能障碍[32]。本试验中,肉仔鸡饲粮中添加FGEL或GEL能够增加十二指肠和空肠的VH并降低CD。原因可能是总黄酮和绿原酸均具有抗炎作用,能够一定程度上减轻LPS致炎作用[9-10]。这也与前期关于发酵银杏叶对肉仔鸡肠道绒毛结构改善作用的结果[12]一致。Gao等[33]试验表明,发酵中药与益生菌复合能调节肉仔鸡肠道形态和功能,如增加VH和VH/CD,促进养分的吸收和利用。Pluske等[34]报道,VH的增加和CD的降低,是一种更加适合于消化的肠道结构,改善吸收和水解能力,且直接用于肠道维持的营养素也较少。因此,FGEL有利于肉仔鸡十二指肠和空肠结构的改善,这也能解释生长阶段肉仔鸡ADG和ADFI有所改善。

3.4 FGEL对LPS应激肉仔鸡肠道吸收功能的影响

D-木糖通过被动扩散在小肠上端被吸收,是不参加代谢的戊糖,与葡萄糖的活性转运系统不同[16]。在家禽中,D-木糖的吸收试验被广泛应用,特异性强,方法简单,常作为测定营养不良和肠吸收的敏感试验[18]。在若干LPS诱导的幼禽肠道机能障碍中,营养吸收不良常用血清D-木糖含量的降低来标示。从组织形态学来看,肠道吸收营养不良反映在绒毛萎缩和隐窝肥大[35]。忽略LPS刺激,正如预期,饲粮FGEL处理的肉仔鸡血清D-木糖含量显著升高,这可能是由于饲喂添加FGEL饲粮的肉仔鸡肠道形态学的改善及肠黏膜AKP活性的提高。
肠道葡萄糖主要是通过位于肠细胞膜上SGLT1进行主动转运的,SGLT1蛋白一般在十二指肠中表达[36]。本试验结果显示,LPS刺激十二指肠SGLT1表达上调,这与之前的研究结果[16]吻合。禁食或生理应激能够增加SGLT1及其他营养转运基因的表达,在营养不足时,是机体的一种补偿效应[37]。本试验结果表明,饲粮FGEL处理能够部分地减弱LPS诱导的肉仔鸡十二指肠SGLT1 mRNA相对表达量的增加。

3.5 FGEL对LPS应激肉仔鸡肠道黏膜炎性因子mRNA相对表达量的影响

LPS被广泛应用于建立鸡肠道损伤模型,模拟动物细菌感染和诱导炎症性细胞因子为特征的炎症反应[38-39]。若干报道表明,LPS刺激能够诱导鸡NF-κB及其下游的IFN-γ、IL-6及IL-1β等对LPS敏感基因表达量的增加。IL-1β和IL-6由单核细胞和巨噬细胞产生,并作为重要的促炎细胞因子在炎症早期发挥相关作用[40]。IL-10是一种关键的抗炎细胞因子[41]。当促炎细胞因子大量表达时,机体可以通过上调抗炎细胞因子的表达来调节炎症反应[42]。本试验结果表明,FGEL可以缓解肉仔鸡体内促炎因子(IL-1β和IL-6)和抗炎因子(IL-10)的释放。微生物发酵过程中产生的微生物酶分解植物细胞壁并释放的酚类等功能性成分[43],如黄酮、绿原酸和多糖类,已被证明可调控NF-κB,并下调其下游的炎性细胞因子的表达,调节免疫平衡[44-45]。
应激和炎症时肠道iNOS的表达也会增加[46]。本试验中,LPS诱导的免疫应激致使肠黏膜iNOS的表达上调,而饲喂FGEL饲粮肉仔鸡的iNOS表达有所下调。研究发现,FGEL中富含的黄酮、绿原酸等酚类化合物是iNOS的天然抑制剂,可能有益于一氧化氮过量表达的炎症性疾病的治疗[47],因此,这可能也是FGEL具有抗炎特性的原因。

4 结论

肉仔鸡饲粮中添加FGEL,可以缓解LPS应激诱导的生长抑制、肠道形态及功能损伤;并能通过NF-κB信号途径缓解LPS诱导的肠道炎症反应。

References

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