口服鼠李糖乳杆菌GG影响哺乳仔猪肠道屏障 功能的研究

Oral administration of Lactobacillus rhamnosus GG to newborn piglets augments gut barrier function in pre-weaning piglets # # 口服鼠李糖乳杆菌GG影响哺乳仔猪肠道屏障 功能的研究 1 first-author 1 1 1 2 1 3 4 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:zhangxiaoping403@gmail.comwfli@zju.edu.cn †E-mail:zhangxiaoping403@gmail.comwfli@zju.edu.cn zhangxiaoping403@gmail.com 1 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:zhangxiaoping403@gmail.comwfli@zju.edu.cn †E-mail:zhangxiaoping403@gmail.comwfli@zju.edu.cn zhangxiaoping403@gmail.com Key Laboratory of Molecular Animal Nutrition and Feed Science, Key Laboratory of Animal Feed and Nutrition of Zhejiang Province, College of Animal Science, Zhejiang University, Hangzhou 310058, China Key Laboratory of Molecular Animal Nutrition and Feed Science, Key Laboratory of Animal Feed and Nutrition of Zhejiang Province, College of Animal Science, Zhejiang University, Hangzhou 310058, China Department of Animal Sciences, Oregon State University, Corvallis, OR 97330, USA Department of Animal Sciences, Oregon State University, Corvallis, OR 97330, USA College of Animal Science and Technology, Guangxi University, Nanning 530005, China College of Animal Science and Technology, Guangxi University, Nanning 530005, China China National Bamboo Research Center, Key Laboratory of High Efficient Processing of Bamboo of Zhejiang Province, Hangzhou 310012, China China National Bamboo Research Center, Key Laboratory of High Efficient Processing of Bamboo of Zhejiang Province, Hangzhou 310012, China To understand the effects of Lactobacillus rhamnosus GG (ATCC 53103) on intestinal barrier function in pre-weaning piglets under normal conditions, twenty-four newborn littermate piglets were randomly divided into two groups. Piglets in the control group were orally administered with 2 mL 0.1 g/mL sterilized skim milk while the treatment group was administered the same volume of sterilized skim milk with the addition of viable L. rhamnosus at the 1st, 3rd, and 5th days after birth. The feeding trial was conducted for 25 d. Results showed that piglets in the L. rhamnosus group exhibited increased weaning weight and average daily weight gain, whereas diarrhea incidence was decreased. The bacterial abundance and composition of cecal contents, especially Firmicutes, Bacteroidetes, and Fusobacteria, were altered by probiotic treatment. In addition, L. rhamnosus increased the jejunal permeability and promoted the immunologic barrier through regulating antimicrobial peptides, cytokines, and chemokines via Toll-like receptors. Our findings indicate that oral administration of L. rhamnosus GG to newborn piglets is beneficial for intestinal health of pre-weaning piglets by improving the biological, physical, and immunologic barriers of intestinal mucosa. 目的 2 探究新生仔猪口服鼠李糖乳杆菌GG对肠道屏障功能的影响. 创新点 2 新生仔猪早期口服鼠李糖乳杆菌GG可明显改善其断奶前肠道菌群结构及免疫屏障功能. 方法 2 二十四头新生仔猪分为对照组和实验组:对照组仔猪在出生后第1、3、5天口服2 mL 0.1 g/mL 的脱脂牛奶;而实验组仔猪口服等体积的含有活鼠李糖乳杆菌GG的脱脂牛奶.饲喂25天后,收集仔猪血清、肠道粘膜和盲肠内容物等样品.通过分析肠道菌群、紧密连接蛋白和细胞因子等指标,评价鼠李糖乳杆菌对肠道屏障功能的影响. 结论 2 在正常生理条件下,新生仔猪口服鼠李糖乳杆菌GG可明显改变肠道菌群结构.此外,鼠李糖乳杆菌GG还可增加仔猪肠道的通透性,并通过调控抗菌肽、细胞因子和趋化因子的分泌以改善肠道的免疫屏障功能. Lactobacillus rhamnosus Gut microbiota Intestinal physical barrier Intestinal immunological barrier Piglet 鼠李糖乳杆菌 肠道菌群 肠道物理屏障 肠道免疫屏障 仔猪 Introduction 1 Diarrhea, an important cause of morbidity and mortality in livestock, is one of the most common diseases of suckling piglets worldwide (Toledo et al., 2012 2012 ). In October 2010, the severe outbreak of porcine epizootic diarrhea in southern China caused the death of more than 1 000 000 piglets (Sun et al., 2012 2012 ). Diarrheal disease was also reported in European countries (Hanke et al., 2015 2015 ; Theuns et al., 2015 2015 ) and spread rapidly to approximately 50% of the US swine breeding herds from July 2013 to July 2014 (Goede and Morrison, 2015 2015 ). To fight against diarrhea, antibiotics have been applied widely in neonatal piglets to enhance gut function and prevent diarrhea (Hermann-Bank et al., 2015 2015 ; Ngamwongsatit et al., 2016 2016 ). However, the promiscuous use of antibiotics could influence the balance of gut bacteria and result in the emergence and spread of resistant bacteria (Kemper, 2008 2008 ). Since bacterial colonization of the mammalian gastrointestinal tract begins at birth (Hermann-Bank et al., 2015 2015 ), it is necessary to establish a healthy intestinal microbiota of newborn suckling piglets. Gut microbiota constitutes the intestinal biological barrier (Natividad and Verdu, 2013 2013 ; Kelly et al., 2015 2015 ), prevents pathogen colonization, and converts indigestible substances into digestible components that benefit the host and help in the maturation of the gastrointestinal tract as well as of the immune system (Guarner and Malagelada, 2003 2003 ; Bauer et al., 2006 2006 ; Ley et al., 2008 2008 ; Koenig et al., 2011 2011 ). As well as the biological barrier, the physical barrier and immune mediators are also important in defending against pathogens (McCracken and Lorenz, 2001 2001 ). In the presence of an intact epithelial cell layer, the paracellular pathway between cells must be sealed. This function is achieved by a physical barrier, especially tight junctions, which are multi-protein complexes to limit solute flux along the paracellular pathway. The tight junction is, therefore, the rate-limiting step in transepithelial transport and the principal determinant of mucosal permeability (Turner, 2009 2009 ). Additionally, the cytokines and chemokines secreted by enterocytes and the antimicrobial peptides secreted by Paneth cells could regulate bacterial interactions with the mucosal surface, so as to maintain the intestinal immune homeostasis (Rescigno, 2011 2011 ). Probiotics are nonpathogenic bacteria that exert a beneficial influence on host health, physiology, or both (Rajput et al., 2013 2013 ), and further improve intestinal structure, aid in the development of immunity to defend against pathogens, and subsequently improve growth performance (Lei et al., 2013 2013 ). In animal production, several selected probiotics have been applied to improve animal health and performance, such as Lactobacillus , Streptococcus , Saccharomyces , Aspergillus , and Bacillus species (Tannock, 2001 2001 ). As a prominent probiotic member, Lactobacillus could augment intestinal health by modulating the gut microbiota of mice with metabolic syndrome (Wang et al., 2015 2015 ), attenuating the negative effects of alcohol on mouse tight junction expression (Chen et al., 2016 2016 ), and activating the gut mucosal immune system of mice (Galdeano and Perdigón, 2006 2006 ). Although there are some reports on probiotics’ effects on pig intestinal barrier functions (Zhang et al., 2010 2010 ; Li et al., 2012 2012 ; Deng et al., 2013 2013 ; Hou et al., 2015 2015 ), few studies have administered Lactobacillus rhamnosus to newborn piglets in the first few days after birth to determine its protective effects on intestinal functions without challenging with pathogens. Therefore, this study was conducted to determine the effects of orally administered L. rhamnosus on the intestinal barrier function in pre-weaning piglets under normal conditions. Materials and methods 1 Bacterial preparation 2 The L. rhamnosus GG (ATCC 53103) in freeze-dried powder form was purchased from the China Center of Industrial Culture Collection (CICC), and cultured in de Man-Rogosa-Sharpe (MRS) medium (Oxoid, Basingstoke, UK) in anaerobic condition at 37 °C until reaching the logarithmic phase. Then the bacterial strain was separated by centrifugation (15 min at 3000 g ) and washed twice with sterile phosphate-buffered saline (pH 7.4). Thereafter, the bacteria were re-suspended in 0.1 g/mL sterile skim milk made from cows’ milk powder to prepare the required concentration (5×10 8 CFU/mL). Animals and treatments 2 Six sows (Duroc×Landrace×Yorkshire) were participants in this experiment. Four newborn piglets with similar initial weights were selected from each sow’s offspring. The twenty-four newborn piglets were then randomly divided into two groups. Each group had three litters with four pigs per litter (half male and half female). The basal diet was supplemented with minerals and vitamins to meet or exceed the requirements for pigs (National Research Council, 1998 1998 ). All pigs were fed ad libitum. The experiment was approved by and performed in accordance with the guidelines of the Zhejiang University Animal Care and Use Committee, Hangzhou, China. Piglets in the control group were orally administered with 2 mL of sterile skim milk, while the treatment group ( L. rhamnosus group) received the same volume of sterile skim milk suspended with viable L. rhamnosus (5×10 8 CFU/mL) by gavage at the 1st, 3rd, and 5th days after birth. From Day 12, piglets were provided with free access to water and a supplemented pre-starter feed. Body weights were recorded at the beginning and the weaning day (the 25th day) in this experiment. Diarrhea was observed every day. Composition and nutrient levels in the diets of sows are listed in Tables S1 S1 and S2 S2 , respectively. Composition and nutrient levels in the diets of the pre-starter feed were listed in Tables S3 S3 and S4 S4 , respectively. No antibiotic was added throughout the trial. The schematic flow diagram of the study design can be found in Fig. S1 S1 . Sample collection 2 On Day 25, six piglets (half male and half female) were randomly picked from each group to collect intestine samples. Briefly, ketamine (11 mg/kg) and xylazine (1.5 mg/kg) were injected to minimize stress. Thereafter, chemical euthanasia was performed using an overdose of intravenous pentobarbital via a catheterized ear vein (Li et al., 2012 2012 ). The mid-jejunum segments were carefully dissected and rinsed with sterilized saline. Mucosae were gently scraped off and cecal contents were carefully squeezed out, then placed in liquid nitrogen immediately, and stored at −80 °C till further analysis. ELISA 2 The mucosa samples were diluted 1:2 (v/v) in sterile saline solution and homogenized with a glass homogenizer. Then the homogenates were centrifuged at 3000 g for 15 min at 4 °C and supernatants were collected for the measurement of the concentrations of interleukin-6 (IL-6), IL-8, IL-10, IL-12, transforming growth factor-β1 (TGF-β1), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) by porcine enzyme-linked immunosorbent assay kit (ELISA kit; R & D Systems, Inc., Minneapolis, USA) as per the manufacturer’s instructions. Data are presented as pg/g wet weight (Li et al., 2012 2012 ). Diamine oxidase assay 2 The mucosa samples were homogenized with ice-cold physiologic saline (1:10, w/v) and centrifuged at 2000 g for 10 min (Centrifuge, Eppendorf, Germany). Supernatant was collected for determination of the diamine oxidase (DAO) activity. The kit for DAO was obtained from Nanjing Bioengineering Institute, Nanjing, China, and the DAO value was determined by spectrophotometry according to the manufacturer’s instructions. RNA extraction and qRT-PCR 2 Total RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) were performed according to Wang et al. ( 2017 2017 ). The primer sequences used for qRT-PCR are listed in Table 1 1 . The 2 −∆∆ C T method was used to estimate mRNA abundance. Relative gene expression levels were normalized by the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ). F: forward; R: reverse Gene symbol Gene name GenBank accession No. Primer sequence Product size (bp) GAPDH Glyceraldehyde-3-phosphate dehydrogenase NM_001206359.1 F: 5'-ATGGTGAAGGTCGGAGTGAAC-3' R: 5'-CTCGCTCCTGGAAGATGGT-3' 235 MUC2 Mucin 2 XM_002347185.2 F: 5'-ACGACTTTGACGGACACTGCT-3' R: 5'-AGGGGACGTTCTCGGTGAT-3' 99 pBD-1 Porcine β-defensin-1 NM_213838.1 F: 5'-CGCCTCCTCCTTGTATTCCTC-3' R: 5'-GGTGCCGATCTGTTTCATCTT-3' 144 PMAP-37 Porcine myeloid antimicrobial peptide 37 NM_213863.1 F: 5'-CACCTGCAATGAGGGTGTCA-3' R: 5'-GTCGCAACCGTGGTCTTCG-3' 68 LYZ Lysozyme C-2 precursor NM_214392 F: 5'-CCGCTACTGGTGTAATGATGG-3' R: 5'-ATGCTTTAACGCCTAGTGGAT-3' 143 OCLN Occludin NM_001163647 F: 5'-AATGCTTTCTCAGCCAGCGTAT-3' R: 5'-GCAAGCGTGGAGGCAACA-3' 153 ZO-1 Zonula occludens-1 XM_003121673.1 F: 5'-GCCTCCTGAGTTTGATAGTGG-3' R: 5'-CTCGGCAGACCTTGAAATAGA-3' 287 MCP-1 Monocyte chemoattractant protein-1 NM_214214.1 F: 5'-AGAAGAGTCACCAGCAGCAAG-3' R: 5'-TAGGGCAAGTTAGAAGGAAATG-3' 206 TNF-α Tumor necrosis factor-α NM_214022.1 F: 5'-CATCGCCGTCTCCTACCA-3' R: 5'-CCCAGATTCAGCAAAGTCCA-3' 199 IFN-γ Interferon-γ NM_213948.1 F: 5'-GAGCCAAATTGTCTCCTTCTAC-3' R: 5'-CGAAGTCATTCAGTTTCCCAG-3' 140 TGF-β1 Transforming growth factor-β1 NM_214015.1 F: 5'-GGACCTTATCCTGAATGCCTT-3' R: 5'-TAGGTTACCACTGAGCCACAAT-3' 133 IL-8 Interleukin-8 NM_213867.1 F: 5'-ATGCCAGTGCATAAATACGC-3' R: 5'-TTGGGAGCCACGGAGAAT-3' 251 TLR2 Toll-like receptor 2 NM_213761.1 F: 5'-GGTCCGATGCTGGTCTTTAT-3' R: 5'-GCAAGTCACCCTTTATGTTATTCA-3' 83 TLR9 Toll-like receptor 9 NM_213958.1 F: 5'-CCCACGACAGCCGAATAG-3' R: 5'-GGAACAGGGAGCAGAGCA-3' 122 TLR6 Toll-like receptor 6 NM_213760.1 F: 5'-TCTGCTCAAGGACTTCCGTGT-3' R: 5'-CAGCCCAGTGACTCCGATG-3' 79 TLR8 Toll-like receptor 8 NM_214187.1 F: 5'-GGATACCATTGCGGCGATAA-3' R: 5'-CCAGGGCAGCCAACATAACT-3' 71 DNA extraction 2 Genomic DNA was extracted from every cecal content sample using a TIANamp Stool DNA kit according to the manufacturer’s protocols (Tiangen Biotech, Beijing, China). 454 pyrosequencing 2 The 454 pyrosequencing was performed according to Zhang et al. ( 2014 2014 ). Amplification of the bacterial V3 region of the 16S rRNA gene was conducted using a primer set (Casserly and Erijman, 2003 2003 ), and the 5' terminus of each forward and reverse primer contained an 8-bp barcode sequence to tag specific samples (Table 2 2 ). Group Primer Barcode sequence Primer sequence (5'→3') 6 Control V3-60F 2 AGATACTG AGATACTGCCTACGGGAGGCAGCAG V3-60R AGATACTGATTACCGCGGCTGCT V3-61F 2 AGAGTATG AGAGTATGCCTACGGGAGGCAGCAG V3-61R AGAGTATGATTACCGCGGCTGCT V3-63F 2 AGAGTGAT AGAGTGATCCTACGGGAGGCAGCAG V3-63R AGAGTGATATTACCGCGGCTGCT 6 L. rhamnosus V3-55F 2 AGATATGT AGATATAGCCTACGGGAGGCAGCAG V3-55R AGATATAGATTACCGCGGCTGCT V3-58F 2 AGATAGAT AGATAGTGCCTACGGGAGGCAGCAG V3-58R AGATAGTGATTACCGCGGCTGCT V3-62F 2 AGATACAT AGAGTAGTCCTACGGGAGGCAGCAG V3-62R AGAGTAGTATTACCGCGGCTGCT Statistical analysis 2 Data are presented as means with their standard deviations (SDs). They were analyzed with SPSS 16.0 for Windows, using independent samples t -test. Differences were considered statistically significant at P < 0.05 or P < 0.01. Different superscripts denote significant difference between groups. Results 1 L. rhamnosus improved growth performance 2 Compared with the control group, L. rhamnosus significantly increased the final body weight and daily weight gain, while decreasing the diarrhea rate from 6.41% to 4.44% (Table 3 3 ). The data followed by the different small letters in the same column are significantly different ( P < 0.05). Diarrhea rate (%)=(number of piglets with diarrhea in seven days/total number of piglets in the group)×100%. Data are expressed as mean±SD ( n =12) Group Body weight at birth (kg) Final body weight (kg) Daily weight gain (kg) Diarrhea rate (%)  Control 1.43±0.03 6.67±0.13 b 0.22±0.01 b 6.41±0.72 a   L . rhamnosus 1.38±0.04 7.62±0.13 a 0.25±0.01 a 4.44±0.34 b L. rhamnosus regulated bacterial diversity and composition in cecal contents 2 The phylogenetic differences within the intestinal microbiota were assessed by principal component analysis (PCoA) (Fig. 1 1 ). L. rhamnosus treatment had a distinct microbiota composition that clustered separately from control diet-fed piglets. The α-diversity (richness and evenness) of the communities was measured by Shannon, chao1, PD_whole_tree, observed_species, and goods_coverage’s indices (Table 4 4 ). However, no significant differences were observed between the two groups. To assess specific changes in the gut microbiota, we compared the relative abundance of the phylum identified from sequencing (Fig. 2 2 ). Results showed that Firmicutes, Bacteroidetes, and Fusobacteria phyla were dominant in the control group, comprising 33.56%, 35.89%, and 15.20% of the sequences, respectively; however, in L. rhamnosus group, Firmicutes percentage was dominant and the Bacteroidetes proportion was decreased. Noticeably, Fusobacteria almost disappeared in the L. rhamnosus group (Fig. 2 2 ). Data are expressed as mean±SD ( n =3) Group Shannon Chao1 PD_whole_tree Observed_species Goods_coverage Control 5.15±0.71 251.13±105.56 11.02±3.05 178.67±62.74 0.98±0.00 L . rhamnosus 5.17±1.18 195.65±149.13 9.36±2.17 127.00±66.47 0.97±0.02 The abundance is presented in terms of a percentage of the total effective bacterial sequences in the sample, which were classified using the RDP Classifier at a confidence threshold of 50% L. rhamnosus regulated mRNA expression of genes related to physical barrier function 2 The results of expression of genes related to tight junctions in jejunal mucosa showed that the transcript level of occludin ( OCLN ) was significantly down-regulated by 48.12% with L. rhamnosus treatment, while no significant difference was found in the zonula occludens-1 ( ZO-1 ) mRNA expression between the two groups (Fig. 3 3 ). L. rhamnosus increased mucin-2 ( MUC-2 ) gene expression slightly (Fig. 3 3 ), but DAO activity was significantly decreased (Fig. 4 4 ). The different capital letters on the bar mean that the values are very significantly different ( P < 0.01). Data are expressed as mean±SD ( n =6) The different capital letters on the bar mean that the values are very significantly different ( P < 0.01). Data are expressed as mean±SD ( n =6) L. rhamnosus improved intestinal immune barrier function 2 Oral administration of L. rhamnosus significantly up-regulated the mRNA levels of porcine β-defensin-1 ( pBD-1 ) and porcine myeloid antimicrobial peptide 37 ( PMAP-37 ), but decreased the expression of lysozyme C-2 precursor ( LYZ ) in jejunal mucosa of the suckling piglets (Fig. 5a 5a ). Results in Fig. 5b 5b illustrate that L. rhamnosus markedly up-regulated the mRNA levels of both pro-inflammatory cytokines ( TNF-α , IFN-γ ) and the anti-inflammatory cytokine ( TGF-β1 ). In the L. rhamnosus treatment group, chemokine MCP-1 mRNA expression was almost quadrupled compared with that in the control group (1.00 vs. 3.88). Despite this, most of the pro-inflammatory cytokine concentrations in jejunal mucosa were significantly decreased in the L. rhamnosus group, including IL-1β, IL-6, IL-12, IL-8, and TNF-α, whereas the IFN-γ concentration was increased (Table 5 5 ), which was in line with the qRT-PCR result (Fig. 5b 5b ). Additionally, production of anti-inflammatory cytokines, such as IL-10 and TGF-β1, was markedly increased with oral administration of L. rhamnosus (Table 5 5 ). The data followed by different capital letters in the same column are very significantly different ( P < 0.01). Data are expressed as mean±SD ( n =6) Group IL-1β (pg/g) IL-6 (pg/g) IL-12 (pg/g) TNF-α (pg/g) Control 3244.33±90.22 A 164.43±9.30 A 263.24±16.01 A 96.95±11.76 A L. rhamnosus 2133.55±46.21 B 89.78±5.22 B 161.14±4.39 B 31.62±3.63 B 5 5 Group IFN-γ (pg/g) IL-8 (pg/g) IL-10 (pg/g) TGF-β1 (pg/g) 5 Control 4721.51±268.08 B 25 102.68±468.97 A 199.16±14.92 B 149.19±3.78 B L. rhamnosus 6733.47±254.09 A 14 776.40±512.04 B 242.55±9.01 A 200.97±7.44 A (a) Relative expression of pBD-1 , PMAP-37 , and LYZ in jejunal mucosa; (b) Relative expression of cytokine and chemotactic factor genes in jejunal mucosa; (c) Relative expression of TLR genes in jejunal mucosa; (d) Relative expression of innate immune signaling relative genes in jejunal mucosa. The different small letters or different capital letters on the bar mean that the values are significantly different ( P < 0.05) or very significantly different ( P < 0.01), respectively. Data are expressed as mean±SD ( n =6) L. rhamnosus regulated transcript levels of key genes involved in innate immune signaling pathways in jejunal mucosa 2 In the L. rhamnosus group, Toll-like receptor 2 ( TLR2 ), TLR9 , and myeloid differentiation-2 ( MD-2 ) mRNA expression levels were dramatically up-regulated. However, no significant difference was observed in TLR6 or TLR8 expression between the two groups (Fig. 5c 5c ). To determine whether L. rhamnosus played a role in innate immune signaling pathways, we detected mRNA expression of TGF-β activated kinase 1 ( TAK1 ), TNF receptor associated factor 6 ( TRAF6 ), c-Jun N-terminal kinase ( JNK ), p38 and peroxisome proliferator activated receptor γ ( PPARγ ) (Fig. 5d 5d ), but only TRAF6 expression was significantly increased by L. rhamnosus . Discussion 1 Immediately after birth, the gastrointestinal tract of neonates is involved in a process of microbiota colonization and succession (Mackie et al., 1999 1999 ; Gaskins et al., 2008 2008 ). Increasing evidence has suggested that early exposure with desirable microbiota could alter the pattern of microbial succession as well as immunological maturation (Rakoff-Nahoum and Medzhitov, 2008 2008 ; Hou et al., 2015 2015 ). The use of probiotics in piglet nutrition has been increasingly discussed in recent years (Taras et al., 2006 2006 ; Ayala et al., 2015 2015 ; Patil et al., 2015 2015 ). Many studies have found beneficial effects of probiotics supplementation on the growth performance and gut health of suckling piglets (Shim et al., 2005 2005 ; Zeyner and Boldt, 2006 2006 ; Hayakawa et al., 2016 2016 ). However, little is known about the piglets’ intestinal functions following L. rhamnosus treatment in the first few days after birth under normal conditions. Thus, the objective of this experiment is to determine whether L. rhamnosus administration to newborn piglets in the first few days after birth can enhance the intestinal barrier functions, including the intestinal immune function, intestinal permeability, and gut microbial composition of pre-weaning piglets. As is known, L. rhamnosus can improve the daily weight gain of piglets (Bocourt et al., 2004a 2004a , 2004b 2004b ) and ameliorate diarrhea (Guarino et al., 2009 2009 ). In the present study, L. rhamnosus also effectively increased the growth performance of pre-weaning piglets and alleviated the diarrhea, which implied that L. rhamnosus might improve the function of the intestinal barrier to defend against enteric pathogens (Nalle and Turner, 2015 2015 ). Recently, accumulating evidence has suggested that gut microbiota plays a key role in gut health (Gareau et al., 2010 2010 ; Flint et al., 2012 2012 ). Intestinal bacterial communities are comprised of more than 1000 different species (Lozupone et al., 2012 2012 ) and constitute an exceptionally diverse microbial ecosystem, which is critical for numerous physiologic processes (Stappenbeck et al., 2002 2002 ; Hooper, 2004 2004 ; Mazmanian et al., 2005 2005 ). Genera of the phylum Firmicutes, which comprise the dominant microflora in the gut (Eckburg et al., 2005 2005 ), including Bacillus , Enterococcus , Lactobacillus , and Lactococcus , play an important role in human and animal health (Haakensen et al., 2008 2008 ). Although Bacteroidetes species also boost animal growth and health (Salyers, 1984 1984 ), enterotoxigenic B. fragilis , a human colonic commensal, has been demonstrated to increase colonic thickness, inflammation and visible colonic tumors (Wu et al., 2009 2009 ). Zhu et al. ( 2014 2014 ) also found that Bacteroidetes species were more abundant in rats with colorectal cancer. Fusobacteria species have long been recognized in a wide spectrum of human infections (Klinge et al., 2002 2002 ; Gmür et al., 2006 2006 ; Allen-Vercoe et al., 2011 2011 ). In the analysis of bacterial composition in cecal contents in this study, we observed that at the phylum level, the most remarkable result was the increase in Firmicutes and the decrease in Bacteroidetes as well as Fusobacteria with L. rhamnosus treatment. Similarly, the study of Angelakis and Raoult ( 2010 2010 ) also noticed that Lactobacillus inoculation improved the weight gain via increasing the Firmicutes/Bacteroidetes ratio in the gut flora of newborn chicks and ducks. Hence, we speculate that L. rhamnosus could exert beneficial effects on piglets’ health via modulating the bacterial composition, especially the abundance of Firmicutes, Bacteroidetes, and Fusobacteria. Microflora colonization can also be influenced by intestinal MUC-2, which is a major gel-forming mucin and is in direct contact with gut bacteria (Zhang et al., 2015 2015 ). It was reported that dietary Lactobacillus supplementation increased MUC-2 concentration (Mao et al., 2016 2016 ). Here, MUC-2 expression was also up-regulated by L. rhamnosus , but not to a statistically significant extent. DAO is an intracellular enzyme in the mucosal villous epithelial cells of humans and all mammals; the highest activity is detected in the jejunum and ileum (Meng et al., 2016 2016 ). Following damage, necrosis, and exfoliation of the intestinal mucosal cells, DAO translocates into blood, which implies the destruction of the intestinal mucosal barrier and changes in intestinal permeability (Swank and Deitch, 1996 1996 ; Sun et al., 2011 2011 ). Compared with the control group, oral administration of L. rhamnosus significantly decreased DAO in jejunal mucosa. In addition, unlike the results in other papers (Mennigen et al., 2009 2009 ; Liu et al., 2013 2013 ), the tight junction OCLN in the present study was down-regulated when exposed to L. rhamnosus . As far as we are concerned, when the gut epithelium is damaged, the expression of tight junction proteins can also be increased in order to repair the damage; when there is no damage or in healthier conditions, they may remain at baseline or lower expression levels. As the biggest immune organ, the intestine can secrete some bioactive substances to defend against foreign antigens, toxins, and macromolecules. Paneth cells in the intestinal villi are secretory cells that are specialized in the production of antimicrobial peptides (Rescigno, 2011 2011 ). In this study, L. rhamnosus increased the mRNA expression of antimicrobial peptides pBD-1 and PMAP-37 . On the other hand, LYZ expression decreased dramatically. LYZ hydrolyzes N -acetylmuramic acid and N -acetylglucosamine, which are constituents of the peptidoglycan layer of bacterial cell walls (Balcázar et al., 2007 2007 ). Gram-negative bacteria, such as Escherichia coli , are more resistant to LYZ than Gram-positive bacteria, such as Lactobacillus , because their outer membranes hinder the access of lysozyme to peptidoglycan (Callewaert and Michiels, 2010 2010 ). Therefore, we conjecture that due to the elevated antibacterial ability caused by pBD-1 and PMAP-37 , there is no need for intestinal mucosa to express more LYZ to exert its bactericidal capacity and protect intestinal symbiotic bacteria in suckling piglets. Innate immunological responses have evolved specifically to defend the intestinal mucosal interface by limiting direct bacterial contact with the epithelial surface. Enterocytes are known to serve as immuno-effector cells and are capable of secreting cytokines and chemokines to regulate inflammation (Kagnoff and Eckmann, 1997 1997 ). Different strains of Lactobacillus species can elicit a wide range of cytokine responses in immune cells (Meijerink et al., 2010 2010 , 2012 2012 ). In our findings, except for IFN-γ, levels of all the tested pro-inflammatory cytokines and chemokine were decreased, whereas levels of IL-10 and TGF-β1, the well-known anti-inflammatory cytokines, were increased in the L. rhamnosus group. The result of IFN-γ secretion was in line with previous studies in which Lactobacillus augmented the number of IFN-γ producing cells and increased the synthesis of IFN-γ in the small intestine of mice (Wen et al., 2014 2014 , 2015 2015 ), as IFN-γ plays a key role in the maturation of immune cells and regulates their cellular proliferation in the intestine (Rumbo et al., 2004 2004 ). As for qRT-PCR results, the IFN-γ and TGF-β transcript levels were in accordance with the ELISA results. Although mRNA expression is predictive for protein expression, in the present study, the TNFα and IL-8 transcription levels were not consistent with the protein secretions. However, in previous studies, the same inconsistency also existed (Brundel et al., 2001 2001 ; Guo et al., 2008 2008 ), implying that there may be some post-transcriptional regulation (Brundel et al., 2001 2001 ). In addition, MCP-1 transcript level was markedly elevated by L. rhamnosus . Similar to our results, probiotic L. rhamnosus GG and E. coli Nissle 1917 have also been reported to enhance MCP-1 expression in other studies (Lan et al., 2005 2005 ; Ukena et al., 2005 2005 ). MCP-1 is a proinflammatory chemokine produced by many cells, including epithelial, endothelial, and smooth muscle cells (Cushing et al., 1990 1990 ; Standiford et al., 1991 1991 ). These cells are important for antiviral immune responses in the peripheral circulation and in tissue (Deshmane et al., 2009 2009 ). Moreover, MCP-1 shows chemotactic activity for monocytes, basophils, natural killer cells, and T lymphocytes (Ukena et al., 2005 2005 ). Taken together, in this study, L. rhamnosus could regulate immune responses and increase the antibacterial ability in the gut of piglets through altering the production of cytokines and chemokines. Activation of TLRs provides information about the bacterial census in the intestine and activates expression of secreted antimicrobial proteins to maintain mucosal surface-associated bacterial populations at homeostatic levels (Duerkop et al., 2009 2009 ). Several studies have showed that strain-specific Lactobacillus up-regulated TLR2 and TLR9 transcript levels in vitro (Cammarota et al., 2009 2009 ; Vizoso Pinto et al., 2009 2009 ). Dogi et al. 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All institutional and national guidelines for the care and use of laboratory animals were followed. 10.1631/jzus.B1800022 Project supported by the National Natural Science Foundation of China (No. 31472128), the Special Research Fund for the PhD Program of University, China (No. 20110101110101), and the Key Project of Science and Technology of Zhejiang Province, China (No. 2006C12086) 2018-01-15 2018-06-06 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2019 2019 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 02 20