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The Journal of Nutrition. First published ahead of print November 23, 2011 as doi: 10.3945/jn.111.147074. The Journal of Nutrition Genomics, Proteomics, and Metabolomics
Dietary Supplementation with Probiotic Lactobacillus fermentum I5007 and the Antibiotic, Aureomycin, Differentially Affects the Small Intestinal Proteomes of Weanling Piglets1–3 Xiaoqiu Wang,4 Fang Yang,4 Chuang Liu,4 Huaijun Zhou,5* Guoyao Wu,4,6 Shiyan Qiao,4 Defa Li,4 and Junjun Wang4* 4 State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, China; and 5Department of Poultry Science, and 6Department of Animal Science, Texas A&M University, College Station, TX
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Abstract Antibiotics have long been used in animal production and medication to alleviate weaning stress. However, due to the concerns over food safety and human health, its use in animal production has been prohibited in many countries. Therefore, there is growing interest in developing alternative additives, such as the probiotic Lactobacillus. In this study, a proteomic approach coupled with biochemical analysis was applied to investigate alterations of proteomes in the small intestinal mucosa of weanling piglets after a 13-d period of feeding with supplemental L. fermentum I5007 or aureomycin (an antibiotic). We indentified 27 differentially expressed protein spots that participated in 7 key biological processes, including: 1) energy metabolism; 2) lipid metabolism; 3) protein synthesis; 4) cell structure and mobility; 5) cellular proliferation and apoptosis; 6) immune response; and 7) stress response and detoxification. Both L. fermentum I5007 and aureomycin decreased the expression of proteins related to apoptosis, stress response, and increased the expression of proteins related to detoxification in the gastrointestinal (GI) tract of weanling piglets. L. fermentum I5007 exhibited additional effects in alleviating weaning stress syndrome by enhancing the levels of proteins involved in energy metabolism, lipid metabolism, cell structure and mobility, protein synthesis, and immune response, thereby facilitating cellular proliferation and depressing apoptosis. In contrast, aureomycin reduced the levels of proteins related to energy metabolism, protein synthesis, cell structure, motility, and immunity. These novel findings have important implications for understanding the mechanisms whereby L. fermentum I5007 can improve the GI health of postweaning piglets. J. Nutr. doi: 10.3945/jn.111.147074.
Introduction Weaning is a critical event for piglets due to dramatic changes in both diets and the environment (1,2). Furthermore, weaning stress results in GI7 dysfunction in piglets, which negatively
1 Supported by the National Natural Science Foundation of China (nos. 30828024, 30972156, 30930066, and 31129006), the Thousand-People-Talent program at China Agricultural University, and the Texas AgriLife Research. 2 Author disclosures: X. Q. Wang, F. Yang, C. Liu, H. J. Zhou, G. Y. Wu, S. Y. Qiao, D. F. Li, and J. J. Wang, no conflicts of interest. 3 Supplemental Figures 1–3 are available from the “Online Supporting Material” link in the online posting of the article and from the same link in the online table of contents at jn.nutrition.org. 7 Abbreviations used: ABX, antibiotic treatment; CON, control; 2-DE, 2-dimensional gel electrophoresis; GI, gastrointestinal; IEF, isoelectric focusing; LF, Lactobacillus fermentum treatment. * To whom correspondence may be addressed. E-mail: [email protected] or [email protected]
affects the luminal microflora, the integrity of mucosal barrier of the digestive tract, thereby increasing the susceptibility to pathogenic infections and inducing inflammatory bowel disease, acute diarrhea, and colitis (3–6). This problem is even more severe in piglets with intrauterine growth retardation (7). Antibiotics have long been used in animal production and medication worldwide to alleviate weaning stress (8). Unfortunately, the use of antibiotics in animal feeds has caused a resistance of pathogenic microbes to antibiotics in both humans and animals (8). Therefore, many nations (e.g., members of the European Union) have prohibited the use of subtherapeutic levels of antibiotics in animal production. There is growing interest in developing alternative additives (9), one of which is a probiotic Lactobacillus (i.e., L. fermentum I5007) because of its beneficial effects on the health of the GI tract without any adverse action (10,11). Lactobacillus, one of the lactic acidproducing bacteria, has been widely used in the fermented food
ã 2012 American Society for Nutrition. Manuscript received June 30, 2011. Initial review completed July 18, 2011. Revision accepted October 3, 2011. doi: 10.3945/jn.111.147074.
Copyright (C) 2011 by the American Society for Nutrition
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Materials and Methods Bacterial strain and growth conditions. The L. fermentum I5007, originally isolated from the GI mucosa of healthy weanling piglets in our laboratory (15) and identified by the Institute of Microbiology, Chinese Academy of Sciences, were grown in DeMan Rogosa Sharp broth at 378C under anaerobic conditions for 20 h. After incubation and counting of CFU, 1.0 L cultured medium was mixed with 0.25 kg bran and then freeze-dried (6). For the long-term storage, L. fermentum I5007 were harvested and kept at 2708C in 30% glycerol (17). Piglets and tissue collection. Eighteen crossbred (Large White sires 3 Landrace dams; n = 18 litters) barrows (Dalland, Sino-Dutch Animal Husbandry Training and Demonstration Center, Beijing, China) were weaned at d 18 (5.20 6 0.12 kg BW) and allotted to one of the following three dietary treatments (n = 6) based on body weight and litter of origin: 1) a corn- and soybean meal-based diet as control (CON) (5); 2) the basal diet supplemented with 150 mg × kg21 aureomycin as the antibiotic treatment (ABX); and 3) the basal diet supplemented with 108 CFU × g21 L. fermentum I5007 as the probiotic treatment (LF). Weanling piglets were individually housed in stainless steel (1.4 3 0.5 m) pens over a totally slotted floor. Each pen was equipped with a self-feeder and a nipple waterer in an environmentally controlled building (temperature maintained at 26–288C with 16-h-light and 8-h-dark cycles). Piglets had free access to water and their respective diets during the 13-d experimental period. At d 31 (total 13 d of feeding treatment), all piglets were weighed, killed by CO2 asphyxiation, and then exsanguinated. A 20-cm tissue section was rapidly excised at 50% of the length of the small intestine, rinsed with cold physiologic saline, and blotted dry on paper. Mucosa from this small intestine section was sequentially obtained by careful scraping of the mucosal layer using a glass microscope slide as previously described (19), immediately frozen in liquid nitrogen, and stored at 2808C for proteome analysis. The animaluse protocol was reviewed and approved by the China Agricultural University Animal Care and Use Committee. Protein extraction. Proteins were extracted from the small intestinal mucosa as previously described (17,20). Briefly, small intestinal mucosa samples were homogenized in a lysis buffer [7 mol/L urea, 2 mol/L thiourea, 4% 3-[3-(-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 50 mmol/L DTT] containing 1% protease inhibitors (1003) (GE Healthcare). Mucosal samples were ruptured at 08C using an 2 of 7
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Ultrasonicater Model VCX 500 (Sonics and Materials) at 20% power output for 10 min with 2-s-on and 8-s-off cycles. After adding 1% (v:v) nuclease mix (1003) (GE Healthcare), the lysed cell suspension was kept at room temperature for 1 h to solubilize tissue followed by resonication as described above to completely break up cell membranes. The homogenate was subsequently centrifuged for 10 min at 13,000 3 g at 158C. The supernatant fluid was collected and its protein concentration was determined using a PlusOne 2-D Quant kit (GE Healthcare). Protein extracts were stored in aliquots (1 mg of protein) at 2808C for both proteomic and Western-blotting analyses. 2-DE. With one gel for each piglet’s mucosal sample in each of the three treatments (LF, ABX, and CON), a total of 18 gels were run for the 2-DE using commercial Immobilized pH gradient strips (pH 3–10 NL, 24 cm) (GE Healthcare) for IEF and then standard vertical SDS-PAGE (12.5%) for second dimension, according to the manufacturer’s instructions (21). Briefly, mucosal extracts (1 mg protein/sample) were loaded onto DryStrips using the in-gel sample rehydration technique. After rehydration for 12 h, the first-dimensional IEF was carried out at 208C for 100,000 Vh (300 V, 1.5 h, gradient; 500 V, 1.5 h, gradient; 1000 V, 2 h, gradient; 1000 V, 1 h, Step; 10,000 V, 3 h, gradient; 10,000 V, step until reaching 100,000 Vh) in the Ettan IPGphor II IEF system (GE Healthcare) (19,22). Sequentially, IPG strips were equilibrated for 15 min in 4 mL equilibration buffer-1 (6 mol/L urea, 1% DTT, 30% glycerol, and 50 mmol/L Tris-Cl, pH 8.8) and then in 4 mL equilibration buffer-2 (6 mol/L urea, 2.5% iodoacetamide, 30% glycerol, and 50 mmol/L Tris-Cl, pH 8.8) for 15 min. The second dimension was carried out on an Ettan DALT six (GE Healthcare) at 30 mA/gel for 30 min and then at 50 mA/gel for ~5 h. In the second-dimensional procedure, the temperature was set at 108C. The gels were then stained with colloidal Coomassie Brilliant Blue G-250 (Amresco). Image analysis. High-resolution gel images (400 dpi) were obtained using an ImageScanner Model Powerlook 2100XL (UMAX Technologies) and image analysis was performed using an Image-Master 2D Platinum version 6.01 according to the manufacturer’s protocol (GE Healthcare). After normalizing the quantity of each spot by total valid spot intensity, differentially expressed protein spots (P , 0.05) with a fold-change .1.5 in the relative volume (% vol) and P # 0.05 were selected for identification by MS. Protein identification by MS and database search. The selected protein spots of interest were manually obtained by in-gel digestion with 3 mL of 10 mg × L21 trypsin (Amresco) as we previously described (19). Subsequently, peptide fragments produced from each in-gel digested proteins were individually mixed with a matrix solution (a-cyano-4hydroxycinnamic acid in 0.1% Trifluoroacetic acid and 50% acetonitrile). Matrix assisted laser desorption ionization-time of flight MS was performed on a Bruker Daltonics ultraflex instrument. Protein identification was achieved through peptide mass fingerprint data searches using the online searching engine Mascot by Matrix Science Ltd. and the searching taxonomy of Other mammalian against the NCBInr database (NCBInr 20100120). Search parameters included: 1) trypsin, as the enzyme of protein digestion; 2) monoisotopic, as mass value; 3) unrestricted, as peptide mass; 4) 6 0.2 Da, as peptide mass tolerance; 5) oxidation (M) and carbamidomethyl (C), as variable modifications; and 6) 1, as maximum missed cleavages (23). In this procedure, a protein match with a score .65 was considered significant (P , 0.05). Additionally, protein functional groups and subcellular locations were obtained through the ExPASy proteomics server of the Swiss Institute of Bioinformatics. Western blotting for protein analysis. Extracted small intestine proteins (30 mg/sample) were separated by electrophoresis (Bio-Rad) on 12.5% SDS-PAGE before being transferred electrophoretically to a polyvinylidene fluoride membrane (Millipore). After blocking with Trisbuffered saline Tween-20 containing 5% fat-free dry milk at 48C overnight, the membranes were incubated with primary antibodies, i.e., anti-Keratin 8 (KRT8), anti-Stathmin (STMN1), anti-Gelsolin (GSN), and anti-HSP27 (Beijing Biosynthesis Biotechnology) in dilutions of
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and beverage industry (e.g., cheese, yogurt, wine, and beer) not only because of its flavoring and preservation benefits (12) but also due to its activation on the host immune system (13,14). L. fermentum I5007 was initially isolated from the colonic mucosa of healthy weaning piglets in our laboratory (15). Compelling evidence shows that L. fermentum I5007 has several important characteristics, including: 1) resistance to heat; 2) a high ability of adhesion to Caco-2 cells (16,17); 3) competitive exclusion against Salmonella and E. coli (15,16); 4) positive regulation of immune function and redox status in piglets (3,18); and 5) improvement in growth performance of postweaning piglets (6,15). Furthermore, we recently demonstrated an interaction between GI mucosal cells (Caco-2 cells) and L. fermentum I5007 in vitro (17). At present, little is known about the alterations of proteomes in the GI mucosa of weanling piglets supplemented with L. fermentum I5007. Based on previous findings, we hypothesized that L. fermentum I5007 and antibiotics confer beneficial effects on the host primarily through affecting the proteome of the GI mucosa via different mechanisms. The current work, which involved a proteomic approach coupled with biochemical analysis, was conducted to investigate the proteomes of the GI mucosa in weaning piglets supplemented with the probiotic L. fermentum I5007 or the antibiotic aureomycin.
1:500, 1:300, 1:300, and 1:500, respectively, for 2 h. The membranes were then rinsed in Tris-buffered saline Tween-20 and incubated with a secondary antibody (HRP-labeled anti-rabbit IgG diluted in 1:1000) (Beijing Biosynthesis Biotechnology) for 2 h. The protein bands were visualized with the chemiluminescent HRP substrate (Beijing Biosynthesis Biotechnology) using a gel-imaging system (Tanon Science and Technology) with image analysis software (GE Healthcare). Statistical analysis. The normality of the data and homogeneity of variance were tested using the Shapiro-Wilk test and Brown-Forsythe test in SAS, respectively (version 8.1; SAS Institute). Data were analyzed by 1-way ANOVA and orthogonal contrasts (both the Fisher’s least significant difference and the Tukey’s honestly significant difference) with each piglet as an experimental unit. All analyses were performed using SAS. Data are expressed as means and pooled SEM. P # 0.05 was considered significant.
Proteomic analysis of small intestine. Twenty-seven protein spots were found to be differentially expressed in the small intestine among LF, ABX, and CON piglets after the 13-d feeding. Related information about these protein spots is summarized in Tables 2 and 3 and their appearances on the 2-DE gel image are marked in Supplemental Figures 1 and 2. Based on their biological functions, these proteins were classified in seven groups: energy metabolism, lipid metabolism, protein synthesis, cell structure and mobility, cellular proliferation and apoptosis, immune response, and stress response and detoxification. Furthermore, these proteins occur in different subcellular locations, including the cytoplasm, mitochondrion, cytoskeleton, Golgi apparatus, membrane, and endoplasmic reticulum. Proteins whose abundances in the small intestinal mucosa were similarly affected by probiotic and antibiotic supplementation. Nine proteins were similarly affected by both LF and ABX (Supplemental Fig. 3A). Among them, six proteins [ankyrin repeat and BTB/POZ domain-containing protein 1 (ABTB1, Spot C019); heat shock 27 kDa protein (HSP27, Spot A011); heat shock 90 kDa protein (HSP90, Spot
Proteins whose abundances in the small intestinal mucosa were differentially affected by probiotic and antibiotic supplementation. The abundance of 10 proteins in the small intestinal mucosa was differentially affected by probiotic and antibiotic supplementation (Supplemental Fig. 3B). These proteins included: V-ATPase (ATP6V1A, Spot A001); mitochondrial succinate dehydrogenase complex subunit A (SDHA, Spot B011); GTP-binding protein SAR1b (SAR1B, Spot A004); eukaryotic initiation factor 4A-I (EIF4A, Spot B004); keratin 10 (KRT10, Spot B008); Rho-associated kinase (ROCK, Spot B010); LIM-kinase 1 (LIMK1, Spot C006); megakaryocyte-associated tyrosine-protein kinase (MATK, Spot A008); MHC class II antigen (DRB1, Spot B002); and maspardin (spastic paraplegia 21 protein) (SPG21, Spot A006). The levels of all these proteins were higher (P , 0.05) in LF pigs but lower (P , 0.05) in ABX pigs compared with the CON group. Proteins whose abundances in the small intestinal mucosa were affected only by probiotic supplementation. The abundance of eight proteins in the small intestinal mucosa were affected only by probiotic supplementation (Supplemental Fig. 3C). They included: creatine kinase (CK, Spot A009); isocitrate dehydrogenase 3 (NAD+) alpha (IDH3A, Spot A010); eukaryotic translation initiation factor 4E (EIF4E, Spot A013); spectrin alpha chain (SPTA1, Spot C011); keratin 8 (KRT8, Spot A005); heparan-sulfate 6-O-sulfotransferase (HS6ST, Spot C010); serine/threonine-protein phosphatase 2A catalytic subunit beta isoform (PP2AB, Spot A012); and stathmin (STMN1, Spot A007). Interestingly, the levels of all these proteins were higher (P , 0.05) in the probiotic-supplemented than in the CON pigs. Validation of proteomic data by Western blot. Results of the Western-blot analysis of four proteins (KRT8, STMN1, GSN, and HSP27) randomly selected from Table 2 for the validation of proteomic data are illustrated in Figure 1. The Western blotting results are consistent with the findings from the proteomics analysis.
Discussion TABLE 1
Initial and final body weights of piglets from ABX, CON, and LF groups1
Body weight at d 18
Body weight at d 28
LF ABX CON
kg 5.24 6 0.24 5.24 6 0.21 5.13 6 0.19
kg 8.89 6 0.33a 8.67 6 0.28ab 8.18 6 0.09b
g/d 374 6 10.9a 343 6 17.8ab 306 6 15.0b
g/d 524 6 15.2 511 6 13.6 487 6 12.4
1 Values are means 6 SEM, n = 6. Means in a column with superscripts without a common letter differ, P , 0.05. ABX, antibiotic treatment, CON, control; LF, Lactobacillus fermentum treatment.
GI health is of importance for growth and development (24) but is impaired by weaning stress (25,26). Results of the published studies have shown that dietary supplementation with L. fermentum I5007 positively affects GI health and growth performance of weanling piglets (3,6,9,15–18). However, the underlying mechanisms are largely unknown. The present study extended this work to early-weanling piglets in vivo and further identified 27 differentially expressed proteins in small intestinal mucosa among LF, ABX, and CON groups. Based on their known functions in metabolic pathways, these proteins affect the metabolism of nutrients, cell structure and mobility, and antioxidative responses. Lactobacillus, probiotics, and weanling piglets
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Body weights of piglets. In this study, all piglets (LF, ABX, and CON) started at the same age (d 18) and similar body weights (P = 0.91) (Table 1). At the end of the experimental period (d 31), LF piglets had an 8.7% greater (P , 0.05) body weight and a 22.2% higher (P , 0.05) daily weight gain than CON piglets. Neither body weights nor weight gains differed between the LF and ABX groups or between the ABX and CON groups. During the entire experimental period, daily feed intake did not differ among the LF, ABX, and CON piglets.
B005); protein disulfide-isomerase (PDI, Spot A002); catalase (CAT, Spot B009); and phospholipid hydroperoxide glutathione peroxidase, mitochondrial (GPX4, C002)] were less abundant (P , 0.05) in both LF and ABX groups than in the CON group. In contrast, three proteins [gelsolin (GSN, Spot A003); trans1,2-dihydrobenzene-1,2-diol dehydrogenase (DHDH, Spot B013); D-amino-acid oxidase (DAAO, Spot A014)] were upregulated (P , 0.05) in both the LF and ABX groups compared with the CON group.
Differentially expressed proteins in the small intestinal mucosa of ABX, CON, and LF piglets1 Accession no.
ABX, antibiotic treatment, CON, control; LF, Lactobacillus fermentum treatment; ND, not detectable. Means in a row with superscripts without a common letter differ, P , 0.05. Spot number refers to protein spot numbers that correspond to the labels in Supplemental Figures 1 and 2. Protein score generated by MS identification platform; a score .65 is considered significant. 4 Protein abundance quantity, calculated from spot integrated density (volume), expressed as mean 6 SEM, n = 6 gels for each group. 1 2 3
Same changes for small intestinal mucosal proteins in response to probiotic and antibiotic supplementation. This study identified nine mucosal proteins that responded with the same changes (either increase or decrease) to probiotic and antibiotic supplementation. GSN inhibits apoptosis through stabilizing mitochondria and impeding the release of cytochrome c (27). Higher levels of GSN in both the LF and ABX groups indicate reduced apoptosis of enterocytes. Moreover, ABTB1 mediates the PTEN growth-suppressive signaling pathway. The other differentially expressed proteins that are related to stress response and detoxification include HSP27, HSP90, PDI, CAT, and GPX4. The changes in these proteins can enhance 4 of 7
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antioxidative reactions while directly scavenging harmful lipid hydroperoxides and free hydrogen peroxide in enterocytes (18,19). Reduced levels of HSP27, HSP90, PDI, CAT, and GPX4 in both the LF and ABX piglets attenuated oxidative stress in the small intestine (3). DHDH catalyzes the concomitant generation of NADPH from NADP+, thereby reducing reactive carbonyl compounds and preventing protein glycation (28). Likewise, DAAO acts as a detoxifying agent, degrading D-amino acids to ammonia and ketoacids. These ketoacids can be used for the formation of L-amino acids in both the host and L. fermentum I5007 (24). Thus, along with DHDH, high levels of DAAO in both LF and
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ATP synthesis coupled proton transport Creatine pathway Tricarboxylic acid cycle Tricarboxylic acid cycle Lipid transport Protein biosynthesis Protein biosynthesis Regulation of cell shape Cytoskeleton organization Cytoskeleton organization Protein amino acid phosphorylation Rho protein signal transduction; protein amino acid phosphorylation Carbohydrate biosynthetic process T cell receptor signaling pathway Cell differentiation; intracellular signaling cascade Actin filament severing Protein biosynthesis Signal transduction Antigen processing and presentation of peptide or polysaccharide antigen via MHC class II Antigen receptor-mediated signaling pathway Stress response Stress response Cell redox homeostasis Cell redox homeostasis Cell redox homeostasis; stress response Oxidation reduction Arginine and proline metabolism
ABX, antibiotics; CON, control; LF, Lactobacillus fermentum I5007. Spot number refers to protein spots that correspond to the labels in Supplemental Figures 1 and 2.
ABX pigs may facilitate the use of bacteria-derived acids by the small intestine.
Proteins that respond differently to probiotic and antibiotic supplementation. A novel finding of this study is that probiotic and antibiotic supplementation differentially affected the intestinal levels of 10 proteins that participate in nutrient metabolism, cell structure and mobility, and immune response. For example, V-ATPase serves as an energizer in proton-motive force generation for ligand trafficking and nutrient uptake in vacuolar and plasma membranes of animal cells (29). The increased abundance of V-ATPase and SDHA in L. fermentum I5007supplemented pigs can contribute to enhanced ATP production in the gut. In contrast, aureomycin acts differently than probiotics, which may explain why chronic use of antibiotics could impair the function of the small intestine (8). SAR1B, which is a major mediator in the transport of chylomicron and VLDL from the endoplasmic reticulum to the Golgi apparatus (30), is defective in weanling pigs (5,6,31). Compared with the CON, the increased abundance of SAR1B in probiotic-supplemented pigs could result in enhanced absorption of dietary lipids. In contrast, reduced abundance of mucosal SAR1B in antibiotic-supplemented pigs may predispose the neonates to high risk for fat malabsorption. Likewise, a higher abundance of EIF4A in L. fermentum I5007-supplemented pigs indicates an increased capacity for protein synthesis to improve overall gut function and integrity (32). In support of this notion, the abundance of KRT10 was higher in LF but lower in ABX
pigs compared with the CON pigs. In contrast, a reduced concentration of KRT10 may lead to dysmotility-dependant intestinal permeability. Furthermore, increased levels of both ROCK and LIMK1 may be beneficial for stabilization of cell shape in LF piglets by the Rho-ROCK-LIMK1-cofilin singling transduction pathway (33). On the contrary, this effect of the probiotic was not observed in ABX piglets. Three proteins, MATK, DRB1, and SPG21, participate in the immune response. MATK regulates the activity of Src family protein tyrosine kinases (34) and DRB1 plays a central role in the immune system by presenting peptides derived from extracellular proteins in favor of a Th1 immune response, whereas their reduction promotes a Th2 response (35). In contrast, SPG21 downregulates CD4-dependent T-cell activation by modulating the stimulatory activity of CD4. Compared with the CON group, increased levels of MATK, DRB1, and SPG21 in probiotic-supplemented pigs may enhance the immunity of the small intestine, whereas antibiotics may result in an opposite effect. Exclusive changes in the small intestinal mucosa of probiotic-supplemented pigs. Eight proteins in the piglet small intestinal mucosa were altered exclusively in response to probiotic supplementation. The abundance of CK, which plays a vital role in catalyzing interconversion between creatine plus ATP and phosphocreatine plus ADP, was higher in the small intestine of weanling piglets supplemented with L. fermentum I5007. Likewise, increased abundance of IDH3A may further Lactobacillus, probiotics, and weanling piglets
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Acknowledgments H.J.Z., S.Y.Q., D.F.L., and J.J.W. designed the research; X.Q. W., F.Y., and C.L. conducted the research; X.Q.W, F.Y., and J.J. W. analyzed the data; X.Q.W., H.J.Z., G.Y.W., and J.J.W. wrote the paper; and H.J.Z. and J.J.W. had primary responsibility for the final content. All authors read and approved the final manuscript.
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FIGURE 1 Western-blotting analysis of small intestinal mucosal proteins KRT8 (A), STMN1 (B), GSN (C), and HSP27 (D) in piglets fed a LF, ABX, or CON diet for 13 d. b-Actin was used as an internal standard to normalize the signal. Data are mean + SEM, n = 6. Means without a common letter differ, P , 0.05. ABX, antibiotics; CON, control; LF, Lactobacillus fermentum 15007
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stimulate ATP production by the small intestine of LF piglets to meet the metabolic needs (36), including an mRNA-ribosome binding step (37). Additionally, the abundance of SPTA1 (an actin crosslinking and molecular scaffold protein that links the plasma membrane to the actin cytoskeleton) (38) and KRT8 (the major intermediate filament protein in the intestinal epithelia) was increased in the mucosal cells of probiotic-supplemented piglets. These two proteins are crucial for maintaining intestinal cell structure and motility. Finally, we identified three exclusively altered proteins in the small intestine of probiotic-supplemented piglets that are essential for cell proliferation. These proteins are: 1) HS6ST, which regulates fibroblast growth factor-dependent signaling pathways (39,40) and Wnt-dependent signaling pathways (41); 2) PP2AB, which is a positive regulator of the Wnt/bcatenin pathway (42); and 3) STMN1, which is a key regulator of microtubule dynamics in the intestinal epithelium (43). In conclusion, the results of this study provide the first line of evidence for altered abundances of proteomes in the small intestine of weanling pigs supplemented with probiotics or antibiotics. Piglets responded to both L. fermentum I5007 and aureomycin by similarly changing the level of nine mucosal proteins that can inhibit cellular apoptosis, modulate stress response, and enhance detoxification capacity. Such effects were greater in probiotic- than in antibiotic-supplemented neonates. Supplementation with L. fermentum I5007 resulted in additional benefits to alleviate weaning stress by improving intestinal-mucosal energy metabolism, lipid absorption, cell structure and mobility, protein synthesis, immune response, and cell proliferation. In contrast, the opposite was observed for antibiotics. These novel findings aid in understanding the mechanisms whereby dietary supplementation with L. fermentum I5007 improve the GI health of postweaning piglets and also have important implications for feeding human infants.
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