引用本文
崔百磊, 王丽, 郭乾鹏, 黄怡, 胡胜兰. 屎肠球菌对仔猪小肠紧密连接蛋白、细胞因子应答以及Toll样受体基因表达的影响[J]. 动物营养学报, 2022, 34(12): 7616-7627.
CUI Bailei, WANG Li, GUO Qianpeng, HUANG Yi, HU Shenglan. Effects of Enterococcus faecium on Expression of Intestinal Tight Junction Proteins, Cytokine Responses and Toll-Like Receptors Genes in Piglets[J]. Chinese Journal of Animal Nutrition, 2022, 34(12): 7616-7627.
屎肠球菌对仔猪小肠紧密连接蛋白、细胞因子应答以及Toll样受体基因表达的影响
1. 广西大学动物科学技术学院, 南宁 530004;
2. 广东省农业科学院动物科学研究所, 畜禽育种国家重点实验室, 农业农村部华南动物营养与饲料重点实验室, 广东省畜禽育种与营养研究重点实验室, 岭南现代农业科学与技术广东省实验室茂名分中心, 广州 510640
2. 广东省农业科学院动物科学研究所, 畜禽育种国家重点实验室, 农业农村部华南动物营养与饲料重点实验室, 广东省畜禽育种与营养研究重点实验室, 岭南现代农业科学与技术广东省实验室茂名分中心, 广州 510640
摘要: 本研究旨在比较屎肠球菌活菌和灭活菌对仔猪小肠紧密连接蛋白、细胞因子应答以及Toll样受体（TLR）基因表达的影响。试验选用健康的杜×长×大新生仔猪9窝，窝产仔数10~11头，随机分为对照组（CON组）、屎肠球菌活菌组（LB组）和屎肠球菌灭活菌组（HB组），每组3窝仔猪。从出生开始至6日龄，CON组、LB组和HB组仔猪分别经口灌服无菌脱脂乳、含屎肠球菌活菌（1×108 CFU/mL）的脱脂乳和含屎肠球菌灭活菌（1×108 CFU/mL）的脱脂乳，口服剂量为10 mL/（头·d）。仔猪于7日龄开始饲喂教槽料，21日龄断奶，并分别于这2个日龄从每窝中选出1头接近平均体重的仔猪屠宰取样，检测小肠黏膜紧密连接蛋白和细胞因子应答基因表达及分泌量，以及Toll样受体基因的表达。结果表明：1）与CON组相比，LB组7日龄仔猪空肠黏膜闭锁小带蛋白-1（ZO-1）基因相对表达量有提高趋势（P=0.08），而回肠黏膜ZO-1基因相对表达量则显著提高（P < 0.05）；LB组和HB组21日龄仔猪空肠和回肠黏膜ZO-1基因相对表达量均无显著变化（P>0.05）。2）与CON组相比，仔猪7日龄时，LB组空肠黏膜白细胞介素-8（IL-8）基因相对表达量有降低趋势（P=0.052），白细胞介素-10（IL-10）基因相对表达量呈提高趋势（P=0.063），但肿瘤坏死因子-α（TNF-α）和转化生长因子-β（TGF-β）基因相对表达量均无显著变化（P>0.05）；HB组空肠黏膜IL-8基因相对表达量显著降低（P < 0.05），但其他细胞因子基因相对表达量均无显著变化（P>0.05）；LB组和HB组回肠黏膜IL-8、IL-10、TNF-α和TGF-β基因相对表达量均无显著变化（P>0.05）。与CON组相比，仔猪21日龄时，LB组空肠黏膜IL-8基因相对表达量显著降低（P < 0.05），回肠黏膜IL-8基因相对表达量也显著降低（P < 0.05），且IL-10和TGF-β基因相对表达量显著提高（P < 0.05）；HB组空肠黏膜IL-8基因相对表达量显著降低（P < 0.05），TGF-β基因相对表达量显著提高（P < 0.05），但回肠黏膜的4种细胞因子基因相对表达量无显著差异（P>0.05）。3）与CON组相比，LB组7日龄仔猪空肠黏膜TNF-α分泌量有降低趋势（P=0.087），而回肠黏膜IL-10分泌量则显著提高（P < 0.05），且21日龄仔猪空肠黏膜IL-8分泌量显著减少（P < 0.05），对TGF-β的分泌量无显著影响（P>0.05）；HB组7日龄仔猪空肠和回肠黏膜细胞因子分泌量均无显著变化（P>0.05）。4）与CON组相比，LB组和HB组7日龄仔猪空肠和回肠黏膜TLR-2、TLR-4、TLR-9基因相对表达量均无显著变化（P>0.05）；LB组和HB组21日龄仔猪空肠黏膜TLR-2、TLR-4、TLR-9基因相对表达量也无显著变化（P>0.05），但HB组回肠黏膜TLR-4基因相对表达量显著提高（P < 0.05）。综上所述，屎肠球菌活菌比灭活菌更有利于增强小肠黏膜紧密连接蛋白基因表达，调节细胞因子基因表达和分泌，并稳定小肠黏膜TLR基因表达，从而改善仔猪肠道健康。
关键词:
屎肠球菌 仔猪 紧密连接蛋白 细胞因子 Toll样受体
Effects of Enterococcus faecium on Expression of Intestinal Tight Junction Proteins, Cytokine Responses and Toll-Like Receptors Genes in Piglets
1. College of Animal Science and Technology, Guangxi University, Nanning 530004, China;
2. Maoming Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Guangdong Key Laboratory of Animal Breeding and Nutrition, Key Laboratory of Animal Nutrition and Feed Science in South China Ministry of Agriculture, State Key Laboratory of Livestock and Poultry Breeding, Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
2. Maoming Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Guangdong Key Laboratory of Animal Breeding and Nutrition, Key Laboratory of Animal Nutrition and Feed Science in South China Ministry of Agriculture, State Key Laboratory of Livestock and Poultry Breeding, Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
Abstract: The objective of this study was to compare the effects of live and heat-inactivated bacteria of Enterococcus faecium on expression of small intestinal tight junction proteins, cytokine responses, and Toll-like receptor (TLR) genes of piglets. Nice litters (10 to 11 piglets per litter) of healthy neonatal piglets (Duroc×Landrace×Yorkshire) were selected and randomly divided into three groups: control group (CON group), E. faecium group (LB group) and heat-inactivated E. faecium group (HB group), with 3 litters in each group. From birth to 6 days of age, piglets from 3 groups were orally fed sterile skimmed milk, skimmed milk containing E. faecium, and skimmed milk containing heat-inactivated E. faecium (concentration 1×108 CFU/mL), 10 mL/(head·d), respectively. Creep feed was added for piglets from 7 days of age to 21 days of age (weaned day). On 7 and 21 days of age, one piglet from each litter was selected for slaughter sampling to detect small intestine mucosa tight junction protein gene expression, cytokine gene expression and secretion, and gene expression of TLR. The results showed as follows: 1) compared with the CON group, the relative expression level of zonula occludens-1 (ZO-1) gene in jejunal mucosa of 7-day-old piglets in LB group had a trend to improve (P=0.08), while the ZO-1 gene relative expression level in ileal mucosa was significantly increased (P < 0.05); There were no significant changes in relative expression level of ZO-1 gene in the jejunal and ileum of 21-day-old piglets in the LB and HB groups (P>0.05). 2) At 7 days of age, the jejunal mucosal cytokine interleukin (IL)-8 gene relative expression level had a trend to decrease (P=0.052), and the IL-10 gene relative expression level had a trend to increase (P=0.063), however, neither tumor necrosis factor-alpha (TNF-α) nor transforming growth factor-beta (TGF-β) changed significantly (P>0.05). The relative expression level of IL-8 gene in the jejunal mucosa was significantly decreased in the HB group (P < 0.05), however, there was no significant change in the relative expression of any other cytokines (P>0.05). There were no significant changes in IL-8、IL-10、TNF-α and TGF-β gene relative expression levels in the ileal mucosa in both LB and HB groups (P>0.05). At 21 days of age, the jejunal and ileal mucosa IL-8 gene relative expression level was significantly decreased (P < 0.05), and the ileal mucosa cytokine IL-10 and TGF-β gene relative expression levels were significantly increased (P < 0.05). The relative expression level of IL-8 gene in jejunal mucosa in HB group decreased significantly (P < 0.05), the relative expression level of TGF-β gene was significantly increased (P < 0.05), and the relative expression level of 4 cytokines gene in the ileal mucosa was not significantly changed (P>0.05). 3) The secretion of TNF-α in the jejunum mucosa of 7-day-old piglets in LB group had a trend to decrease (P=0.087), while the secretion of IL-10 in the ileal mucosa was significantly increased (P < 0.05), and the content of IL-8 in the jejunal mucosa of 21-day-old piglets was significantly reduced (P < 0.05), which had no significant effect on the content of TGF-β (P>0.05). There were no significant changes in mucosal cytokine secretion in the jejunum and ileum of 7-day-old piglets in the HB group (P>0.05). 4) The relative expression levels of TLR-2, TLR-4 and TLR-9 genes were not significantly changed in jejunal of 7-day-old piglets in LB and HB group (P>0.05), and those of TLR-2, TLR-4 and TLR-9 genes were not significantly changed in jejunum of 21-day-old piglets in LB and HB group (P>0.05), however, the relative expression level of TLR-4 gene was significantly increased in HB group (P < 0.05). All the above-mentioned results indicate that compared with heat-inactivated E. faecium, live bacteria are more efficient to regulate the intestinal health of piglets by enhancing the expression of tight junction, mucosal cytokines, and stable TLR expression.
Key words:
Enterococcus faecium piglets tight junction cytokines Toll-like receptors
益生菌是一类对宿主有益的活性微生物[1-3]。对肠上皮细胞具有较强的黏附力且定植后能大量繁殖成优势菌群是微生物作为益生菌的重要前提[4-5]。屎肠球菌(Enterococcus faecium)是自然界中常见的一种益生菌，属于肠球菌属，革兰氏阳性，兼性厌氧，一般呈圆形或者椭圆形，无鞭毛及芽孢[6-9]。由于屎肠球菌具有优良的耐酸碱特性，并且可以定植于动物肠道内，因此被作为益生菌广泛用于饲料添加剂中[10-13]。在实际生产中添加适当剂量的屎肠球菌往往可以改善机体健康状态，提高生长性能[14-18]。屎肠球菌与肠道黏膜接触后产生免疫应答可以对动物机体产生免疫刺激作用，进而影响免疫系统和宿主健康[19-21]。研究表明，新生仔猪灌服屎肠球菌EF1可有效抑制小肠黏膜肿瘤坏死因子-α(TNF-α)、γ-干扰素(IFN-γ)和白细胞介素-l(IL-1)等炎症细胞因子的产生，提高相关抗炎细胞因子的分泌，进而促进肠黏膜稳态[19-20, 22]。屎肠球菌HDRsEF1能保护猪小肠上皮细胞(IPEC-J2)细胞膜的完整性，并且能降低IPEC-J2细胞白细胞介素-8(IL-8)的分泌[21, 23]。研究发现，灌喂屎肠球菌能显著提高结肠固有层中免疫球蛋白A(IgA)+细胞和CD11b+IgA+细胞的数量增加[24]。灌服屎肠球菌AL41可显著缓解空肠弯曲杆菌CCM6191攻毒诱导的肉鸡盲肠转化生长因子-β4(TGF-β4)表达降低，同时提高IgA+细胞的免疫应答[25]。
为了保证益生菌的安全使用，并避免造成公共污染，目前市场上使用的益生菌类制剂除了严格选用保证菌种质量的活菌制剂外，也采用灭活菌制剂。灭活处理的益生菌制剂相较于活菌制剂不需要满足数量的要求就能具有显著的疗效，并且便于运输和储存[26]，也能很好解决活菌时会出现的公共安全问题。然而灭活处理的菌种会因理化特性的改变[27]，从而影响到其生物学功能，因此对活菌与灭活菌的益生特性进行对比研究是非常有必要的[22]。本课题组前期研究结果表明，屎肠球菌EF1(Enterococcus faecium EF1)活菌和灭活菌均不影响0~21日龄仔猪的体重和平均日增重，活菌组可显著降低仔猪腹泻率[28-29]。因此，在此基础上，本研究拟深入开展屎肠球菌EF1活菌和灭活菌对仔猪肠道紧密连接蛋白、细胞因子应答以及Toll样受体(TLR)基因表达的影响，对比活菌和灭活菌在仔猪体内作用效果差异，以期更全面地了解益生屎肠球菌的益生作用，为屎肠球菌在仔猪养殖中的高效使用提供充分的科学依据和实践基础。
1 材料与方法 1.1 试验材料屎肠球菌EFI菌株来自浙江大学动物科学学院微生态实验室。
1.2 试验设计试验选用窝产仔数和体重相近且胎次和出生时间相同的杜×长×大新生健康仔猪9窝，每窝10~11头，随机分成3组，每组3窝仔猪。对照组(CON组)仔猪灌服10%脱脂乳溶液10 mL；活菌组(LB组)仔猪灌服含益生屎肠球菌活菌(1×108 CFU/mL)的10%脱脂乳溶液10 mL；灭活菌组(HB组)仔猪灌服含热灭活屎肠球菌(1×108 CFU/mL)的10%脱脂乳溶液10 mL。每天定时灌服1次，持续6 d。7日龄时饲喂仔猪教槽料，至21日龄断奶。饲养期间严格按照规定进行仔猪的日常管理，仔猪和母猪饲粮均无抗生素。
1.3 样品采集在7和21日龄时，从每窝中选出1头接近平均体重的仔猪屠宰，取空肠和回肠肠段，去除肠道内容物后，用手术刀片刮取黏膜放入1.5 mL离心管内，液氮暂存，之后放-80 ℃保存备用。
1.4 RNA抽提、cDNA制备和实时荧光定量PCR(RT-PCR)检测选用RNAiso Plus试剂盒提取空肠、回肠黏膜组织总RNA，测定RNA浓度时吸光度(OD)260/OD280比值在1.8~2.2，表示RNA纯度较高。参考TaKaRa公司PrimeScriptTM RT reagent Kit说明书完成cDNA制备，-80 ℃保存，用于后续基因表达的检测。根据TaKaRa相应试剂盒说明配制反应体系，以反转录的cDNA为模板扩增基因，进行RT-PCR，反应程序为：95 ℃ 30 s→95 ℃ 5 s→54 ℃ 10 s→72 ℃ 30 s(39个循环)；熔解曲线95 ℃ 10 s→60 ℃ 5 s→95 ℃ 5 s，基因相对表达量采用2-ΔΔCt法计算，以甘油醛-3-磷酸脱氢酶(GAPDH)作内参，引物序列见表 1。
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表 1 实时荧光定量PCR引物序列 Table 1 Primer sequences for real-time PCR |
采用SPSS 25.0统计软件one-way ANOVA程序进行方差分析，LSD法进行组间多重比较，P < 0.05为差异显著，0.05≤P < 0.10为差异有显著趋势。试验数据用“平均值±标准差”表示。
2 结果 2.1 屎肠球菌对仔猪小肠紧密连接蛋白基因表达的影响由表 2可知，与CON组相比，LB组有提高7日龄仔猪空肠黏膜闭锁小带蛋白-1(ZO-1)基因相对表达量的趋势(P=0.08)，且显著提高回肠黏膜ZO-1基因相对表达量(P < 0.05)。
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表 2 屎肠球菌对7日龄仔猪空肠和回肠黏膜紧密连接蛋白基因表达的影响 Table 2 Effects of E. faecium on expression of tight junction genes in jejunal and ileal mucosa of 7-day-old piglets |
由表 3可知，21日龄时，与CON组相比，屎肠球菌活菌和灭活菌对空肠回肠黏膜咬合蛋白(Occludin)和ZO-1的基因相对表达量均无显著影响(P＞0.05)。
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表 3 屎肠球菌对21日龄仔猪空肠和回肠黏膜紧密连接蛋白基因表达的影响 Table 3 Effects of E. faecium on expression of tight junction genes in jejunal and ileal mucosa of 21-day-old piglets |
由表 4可知，与CON组相比，屎肠球菌活菌处理有降低7日龄仔猪空肠黏膜IL-8基因相对表达量的趋势(P=0.052)，白细胞介素-10(IL-10)基因相对表达量呈现提高的趋势(P=0.063)；与CON组相比，仔猪灌喂屎肠球菌后降低空肠IL-8基因相对表达量，其中HB组IL-8基因相对表达量显著降低(P < 0.05)；屎肠球菌活菌和灭活菌对7日龄仔猪回肠黏膜各细胞因子基因相对表达量无显著影响(P＞0.05)。
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表 4 屎肠球菌对7日龄仔猪空肠和回肠黏膜细胞因子基因表达的影响 Table 4 Effects of E. faecium on expression of cytokine genes in jejunal and ileal mucosa of 7-day-old piglets |
由表 5可知，与CON组相比，21日龄仔猪灌喂屎肠球菌后，结果显示活菌可显著降低空肠和回肠黏膜IL-8基因相对表达量(P < 0.05)，显著提高回肠黏膜IL-10和转化生长因子-β(TGF-β) 基因相对表达量(P < 0.05)；灭活菌处理显著降低空肠黏膜IL-8基因相对表达量(P < 0.05)，显著提高TGF-β基因相对表达量(P < 0.05)，对回肠黏膜的4种细胞因子基因相对表达量无显著影响(P＞0.05)。
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表 5 屎肠球菌对21日龄仔猪空肠和回肠黏膜细胞因子基因表达的影响 Table 5 Effects of E. faecium on expression of cytokine genes in jejunal and ileal mucosa of 21-day-old piglets |
由表 6可知，与CON组相比，7日龄仔猪灌服活菌显著提高回肠黏膜IL-10的分泌量(P < 0.05)，有降低空肠肿瘤坏死因子-α(TNF-α)分泌量的趋势(P=0.087)；与对照CON组相比，屎肠球菌活菌和灭活菌处理均降低了空肠和回肠黏膜的TNF-α分泌量(P>0.05)，提高了TGF-β分泌量(P>0.05)。
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表 6 屎肠球菌对7日龄仔猪空肠和回肠黏膜细胞因子分泌量的影响 Table 6 Effects of E. faecium on cytokines secretion in jejunum and ileum mucosa of 7-day-old piglets |
由表 7可知，屎肠球菌处理后21日龄仔猪空肠和回肠黏膜的IL-8和TNF-α的分泌量均低于CON组，其中活菌处理显著降低空肠黏膜的IL-8分泌量(P < 0.05)；此外，屎肠球菌处理对肠道黏膜IL-10和TGF-β的分泌量均无显著影响(P>0.05)。
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表 7 屎肠球菌对21日龄仔猪空肠和回肠黏膜细胞因子分泌量的影响 Table 7 Effects of E. faecium on cytokines secretion in jejunum and ileum mucosa of 21-day-old piglets |
由表 8和表 9可知，屎肠球菌活菌和灭活菌对7和21日龄仔猪空肠和TLR基因相对表达量均无显著影响(P>0.05)；屎肠球菌处理没有显著影响21日龄仔猪空肠TLR基因相对表达量(P>0.05)，但检测回肠相关受体基因相对表达量时发现，灭活菌显著提高回肠TLR-4基因相对表达量(P < 0.05)。
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表 8 屎肠球菌对7日龄仔猪空肠和回肠黏膜Toll样受体基因表达的影响 Table 8 Effects of E. faecium on expression of Toll-like receptor genes in jejunal and ileal mucosa of 7-day-old piglets |
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表 9 屎肠球菌对21日龄仔猪空肠和回肠黏膜Toll样受体基因表达的影响 Table 9 Effects of E. faecium on expression of Toll-like receptor genes in jejunal and ileal mucosa of 21-day-old piglets |
紧密连接蛋白主要包括ZOs、闭合蛋白(Claudin)和Occludin 3种跨膜蛋[30-31]。其中ZO-1分布于肠细胞内，Claudin和Occludin为跨膜蛋白，3种跨膜蛋白彼此关联，将相邻的2个肠上皮细胞连接起来，形成1个结构性屏障，将抗原物质阻隔在肠上皮细胞之外[32]，这也是肠黏膜防御系统中的第1道防线，可见增强紧密连接蛋白的表达有利于维护肠上皮细胞对外界抗原物质的屏障作用。已有研究表明，益生菌对动物肠道黏膜TLR具有调节作用[33-36]，孔雨昕等[37]在乌鳢饲料中分别添加108 CFU/g乳酸乳球菌L21、植物乳杆菌W21、粪肠球菌L2饲喂8周后，检测发现试验组乌鳢肠道Claudin-3及ZO-1的mRNA相对表达量显著高于与对照组。研究发现，给仔猪饲喂罗伊氏乳杆菌I5007后，空肠和回肠上皮Occludin和ZO-1表达显著提高[35]，另有研究发现，给断奶仔猪灌服弗氏乳杆菌后，十二指肠、空肠和回肠黏膜的Occludin和ZO-1基因相对表达量也显著提高[36]。本研究中，灌服屎肠球菌活菌有提高7日龄仔猪空肠黏膜ZO-1基因相对表达量的趋势，并显著提高回肠ZO-1基因相对表达量，与前人的研究结果相似。然而本研究中，屎肠球菌灭活菌对肠道紧密连接蛋白基因相对表达量无显著影响。因此，屎肠球菌活菌在提高肠道紧密连接蛋白表达、维护肠道物理屏障完整性的方面优于灭活菌的作用。
3.2 屎肠球菌对仔猪小肠黏膜细胞因子基因表达和分泌的影响细胞因子是一类由免疫细胞和某些非免疫细胞经过刺激作用而生成的具有生物活性物质的小分子蛋白[38]，细胞因子主要包含IL、IFN、TNF、TGF等[39]。相关研究表明，益生菌作用机体后，有利于促进抗炎因子表达，抑制炎症发生[22, 40-43]。双歧三联活菌治疗处理结肠炎的大鼠后，显著降低脾细胞上清液中的TNF-α的表达水平，显著提高IL-10的表达水平[40]。攻毒大肠杆菌O157 ∶ H7的小鼠灌喂嗜酸乳杆菌和瑞士乳杆菌后，IL-2、IL-4和IL-6表达水平显著提高，缓解了大肠杆菌的感染[41]。断奶仔猪饲粮中添加丁酸梭菌后，血清中抗炎因子IL-2含量显著提高，并且显著降低血清促炎因子IL-6和TNF-α含量[42]，丁酸梭菌刺激人外周血单核细胞和树突状细胞也同样观察到抗炎性细胞因子IL-10的表达上调[43]。此外，团队前期研究发现，屎肠球菌活菌较灭活菌可显著提高RAW264.7细胞TNF-α的分泌[22]。本研究中，7日龄仔猪灌服屎肠球菌活菌可在一定程度上降低空肠黏膜IL-8基因相对表达量，提高IL-10基因相对表达量，灭活菌处理后IL-8基因相对表达量显著降低。21日龄仔猪空肠和回肠黏膜在屎肠球菌活菌作用后，IL-8基因相对表达量显著降低，回肠黏膜IL-10和TGF-β基因相对表达量表达显著提高；灭活菌显著降低空肠黏膜IL-8基因相对表达量，同时提高TGF-β基因相对表达量，对回肠黏膜的4种细胞因子基因相对表达量无显著影响。酶联免疫吸附测定(ELISA)发现，活菌处理可降低7日龄仔猪空肠TNF-α的分泌量，提高其回肠黏膜IL-10的分泌量。屎肠球菌活菌处理后，21日龄仔猪空肠IL-8的分泌量显著降低，灭活菌对于空肠和回肠黏膜因子分泌量无显著影响，该结果与基因表达结果基本一致。因此，屎肠球菌的添加可以诱导仔猪肠道黏膜产生免疫应答，提高抗炎因子表达，降低炎症因子分泌，从而保护肠道健康，并且活菌效果优于灭活菌。
3.3 屎肠球菌对仔猪小肠黏膜TLR基因表达的影响TLR基因在非特异免疫中具有重要作用，可以精准识别突破肠道黏膜屏障的微生物，产生细胞因子和趋化因子，激活免疫应答[44]。研究表明，在饲粮中添加乳酸菌显著提高鸡肠道中TLR-2和TLR-4的表达[45]。健康小鼠灌服益生菌后，TLR基因的表达显著提高[46-48]。断奶仔猪饲喂鼠李糖乳杆菌LR6001达到108 CFU/mL时可显著提高肠道TLR-2和TLR-9的表达量[49]。本研究结果发现，屎肠球菌活菌和灭活菌对7日龄仔猪肠道TLR表达无显著影响，在21日龄仔猪肠道中，活菌和灭活菌对于空肠黏膜TLR基因相对表达量无显著影响，而灭活菌显著提高回肠TLR-4的基因相对表达量，由此可见，屎肠球菌灭活菌对于肠道TLR基因调节作用优于活菌，两者对于TLR基因的分泌均有一定的促进作用，表明益生菌屎肠球菌有利于调节仔猪肠黏膜屏障的应答。
前期试验表明，给新生仔猪灌服屎肠球菌后，在整个哺乳期(1~21日龄)益生菌处理组均有效降低了仔猪腹泻率，且活菌组效果更显著[29]，表明屎肠球菌作用新生仔猪后影响了肠道的通透性，缓解了肠道的炎性症状，对于紧密连接蛋白、炎症细胞因子及TLR基因表达测定结果进一步表明，屎肠球菌通过提高肠道紧密连接蛋白基因表达，抑制炎症因子IL-8和TNF-α基因的表达，促进抗炎因子IL-10和TGF-β基因的表达，有效改善了仔猪肠道健康状况。相关研究表明，在受损的肠道上皮中，TLR-4和TLR-9具有拮抗作用，TLR-9的表达可以促进分子抑制信号白细胞介素-1相关激酶M(IRAK-M)的产生，减轻由TLR-4表达后产生的一系列炎症反应的伤害[50-51]，本试验中，肠道TLR基因在屎肠球菌作用后几乎没有产生显著影响，但灭活菌提高了TLR-4基因相对表达量，可能会诱发仔猪肠道炎症反应，进而体现了屎肠球菌活菌对仔猪肠道产生的健康保护作用。
4 结论屎肠球菌活菌和灭活菌对仔猪均有益生作用。本研究使用浓度为1×108 CFU/mL屎肠球菌活菌较其热灭活菌更有利于促进仔猪小肠紧密连接蛋白基因表达，抑制促炎细胞因子并提高抗炎细胞因子表达和分泌，稳定TLR基因的表达，从而改善仔猪肠道健康。
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