Bacillus licheniformis PF9 improves barrier function and alleviates inflammatory responses against enterotoxigenic Escherichia coli F4 infection in the porcine intestinal epithelial cells.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) F4 commonly colonizes the small intestine and releases enterotoxins that impair the intestinal barrier function and trigger inflammatory responses. Although Bacillus licheniformis (B. licheniformis) has been reported to enhance intestinal health, it remains to be seen whether there is a functional role of B. licheniformis in intestinal inflammatory response in intestinal porcine epithelial cell line (IPEC-J2) when stimulated with ETEC F4. METHODS: In the present study, the effects of B. licheniformis PF9 on the release of pro-inflammation cytokines, cell integrity and nuclear factor-κB (NF-κB) activation were evaluated in ETEC F4-induced IPEC-J2 cells. RESULTS: B. licheniformis PF9 treatment was capable of remarkably attenuating the expression levels of inflammation cytokines tumor necrosis factor-α (TNF-α), interleukin (IL)-8, and IL-6 during ETEC F4 infection. Furthermore, the gene expression of Toll-like receptor 4 (TLR4)-mediated upstream related genes of NF-κB signaling pathway has been significantly inhibited. These changes were accompanied by significantly decreased phosphorylation of p65 NF-κB during ETEC F4 infection with B. licheniformis PF9 treatment. The immunofluorescence and western blotting analysis revealed that B. licheniformis PF9 increased the expression levels of zona occludens 1 (ZO-1) and occludin (OCLN) in ETEC F4-infected IPEC-J2 cells. Meanwhile, the B. licheniformis PF9 could alleviate the injury of epithelial barrier function assessed by the trans-epithelial electrical resistance (TEER) and cell permeability assay. Interestingly, B. licheniformis PF9 protect IPEC-J2 cells against ETEC F4 infection by decreasing the gene expressions of virulence-related factors (including luxS, estA, estB, and elt) in ETEC F4. CONCLUSIONS: Collectively, our results suggest that B. licheniformis PF9 might reduce inflammation-related cytokines through blocking the NF-κB signaling pathways. Besides, B. licheniformis PF9 displayed a significant role in the enhancement of IPEC-J2 cell integrity.

Citral alleviates peptidoglycan-induced inflammation and disruption of barrier functions in porcine intestinal epithelial cells.

Peptidoglycan (PGN) is a major polymer in bacterial cell walls and may constrain gut functionality and lower intestinal efficiencies in livestock. Citral has been reported to exhibit antibacterial and anti-inflammatory biological activities, improving the gastrointestinal function of swine. However, the protective effect of citral against PGN-elicited cellular responses and possible underlying mechanisms are unknown. In this study, the porcine jejunal epithelial cell line (IPEC-J2) was challenged with PGN from Staphylococcus aureus (S. aureus) or Bacillus subtilis (B. subtilis) to explore PGN-induced inflammatory responses. Our data showed that the inflammatory response stimulated by PGN from harmful bacteria (S. aureus) was more potent than that from commensal bacteria (B. subtilis) in IPEC-J2 cells. Based on the inflammatory model by PGN from S. aureus, it was demonstrated that PGN could significantly induce inflammatory cytokine production and influence nutrient absorption and barrier function in a dose-dependent manner. However, the PGN-mediated immune responses were remarkably suppressed by citral. In addition, citral significantly attenuated the effect of PGN on the intestine nutrient absorption and barrier function. The expression of TLR2 was strongly induced by PGN stimulation, which was suppressed by citral. All data nominated that citral downregulated PGN-induced inflammation via TLR2-mediated activation of the NF-κB signaling pathway in IPEC-J2 cells. Furthermore, the results also indicate that the PGN degradation through the inclusion of enzymes (e.g., muramidase) as well as the inclusion of citral for attenuating inflammation may improve pig gut health and functionality.

Efficiency of Deoxynivalenol Detoxification by Microencapsulated Sodium Metabisulfite Assessed via an In Vitro Bioassay Based on Intestinal Porcine Epithelial Cells.

Deoxynivalenol (DON) contamination occurs in feeds and causes a reduction in growth performance, damage to the intestinal epithelial cells, and increased susceptibility to enteric pathogen challenge. Sodium metabisulfite (SMBS) has shown promise in reducing DON; however, SMBS quickly degrades under aqueous acidic conditions such as the environment within a stomach. Thus, protection of SMBS is required for effective delivery to the small intestine to detoxify DON. This study was to encapsulate SMBS into hydrogenated palm oil-based microparticles for its delivery to the small intestine and to evaluate its efficacy on DON detoxification in simulated intestinal fluids using IPEC-J2 cells in vitro. The diameter of the SMBS containing microparticles was 511 ± 135 µm, and the loading capacity of SMBS in the microparticles was 45.50%; 1.41% of the encapsulated SMBS (ES) was released into the simulated gastric fluid, and 66.39% of ES was progressively released into the simulated intestinal fluid within 4 h at 37 °C. In IPEC-J2 cells, when DON was treated with the simulated gastric fluid containing 0.5% ES for 2 h, then mixed with the simulated intestinal fluid (1:1) and incubated for 2 h, cytotoxicity was not observed. DON treated with 0.5 ES decreased the gene expression of inflammatory cytokines in the cells compared with DON alone and maintained the cell integrity. To conclude, the SMBS containing microparticles were stable in the simulated gastric fluid and allowed a progressive release of SMBS in the simulated intestinal fluid. The released SMBS in the simulated intestinal fluid effectively detoxified DON.

Effects of dietary energy sources on early postmortem muscle metabolism of finishing pigs.

OBJECTIVE: This study investigated the effects of different dietary energy sources on early postmortem muscle metabolism of finishing pigs. METHODS: Seventy-two barrow (Duroc×Landrace×Yorkshire, DLY) pigs (65.0±2.0 kg) were allotted to three iso-energetic and iso-nitrogenous diets: A (44.1% starch, 5.9% crude fat, and 12.6% neutral detergent fibre [NDF]), B (37.6% starch, 9.5% crude fat, and 15.4% NDF) or C (30.9% starch, 14.3% crude fat, and 17.8% NDF). After the duration of 28-day feeding experiment, 24 pigs (eight per treatment) were slaughtered and the M. longissimus lumborum (LL) samples at 45 min postmortem were collected. RESULTS: Compared with diet A, diet C resulted in greater adenosine triphosphate and decreased phosphocreatine (PCr) concentrations, greater activity of creatine kinase and reduced percentage bound activities of hexokinase (HK), and pyruvate kinase (PK) in LL muscles (p<0.05). Moreover, diet C decreased the phosphor-AKT level and increased the hydroxy-hypoxia-inducible factor-1α (HIF-1α) level, as well as decreased the bound protein expressions of HK II, PKM2, and lactate dehydrogenase A (p<0.05). CONCLUSION: Diet C with the lowest level of starch and the highest levels of fat and NDF could enhance the PCr utilization and attenuate glycolysis early postmortem in LL muscle of finishing pigs.

Alanyl-glutamine supplementation regulates mTOR and ubiquitin proteasome proteolysis signaling pathways in piglets.

OBJECTIVE: The aim of the present study was to investigate the effects of the alanyl-glutamine dipeptide (Ala-Gln) or the combination supplementation of free alanine and glutamine (Ala+Gln) on the mammalian target of rapamycin (mTOR) and ubiquitin-proteasome proteolysis (UPP) signaling pathways in piglets. METHODS: We randomly allocated 180 piglets to three treatments with three replicates of 20 piglets each, fed with diets containing 0.62% Ala, 0.5% Ala-Gln, 0.21% Ala+0.34% Gln, respectively. The duration of the experiment was 28 d. RESULTS: The results showed that Ala-Gln increased average daily gain of piglets, and decreased the ratio of feed to gain (P < 0.05). Ala-Gln supplementation increased the concentrations of Gln and glutamate and decreased the activity of glutamine synthetase in liver and skeletal muscle (P < 0.05). Ala-Gln increased the expression of glutaminase and glutamate dehydrogenate (P < 0.05). The increased phosphorylation of eIF-4 E binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1) in Ala-Gln treatment were associated with phosphorylation of the mTOR in liver and skeletal muscle. Ala+Gln did not affect the phosphorylation abundances of mTOR, 4E-BP1, or S6K1 (P > 0.05). Ala-Gln supplementation inhibited the mRNA expressions of MAFbx and MuRF1 in skeletal muscle of piglets (P < 0.05). CONCLUSION: Taken together, Ala-Gln supplementation improved the growth performance of piglets, enhanced the metabolism of Gln, upregulated protein synthetic signaling in liver and skeletal muscle and decreased protein degradative signaling in muscle of piglets. Moreover, these effects of Ala-Gln were more effective than those of Ala+Gln.

Suppression of mTOR Signaling Pathways in Skeletal Muscle of Finishing Pigs by Increasing the Ratios of Ether Extract and Neutral Detergent Fiber at the Expense of Starch in Iso-energetic Diets.

Three iso-energetic and iso-nitrogenous diets were fed to finishing pigs for 28 days to investigate the mammalian target of rapamycin (mTOR) and ubiquitin-proteasome signaling pathways of skeletal muscle by altering compositions of dietary energy sources. Diet A, diet B, and diet C contained 44.1%, 37.6%, and 30.9% starch; 5.9%, 9.5%, and 14.3% ether extract (EE); and 12.6%, 15.4%, and 17.8% neutral detergent fiber (NDF), respectively. An increase of mRNA expression of MuRF1 (1.09 ± 0.10 vs 1.00 ± 0.08) and MAFbx (1.10 ± 0.06 vs 1.00 ± 0.11) and a decrease of concentrations of plasma insulin (8.2 ± 0.8 vs 10.8 ± 1.2) and glucose (5.76 ± 0.12 vs 6.43 ± 0.33) together with mRNA expression of IRS (0.78 ± 0.19 vs 1.01 ± 0.05) and Akt (0.92 ± 0.01 vs 1.00 ± 0.05) were observed in pigs fed diet C compared with diet A. Protein levels of total and phosphorylated mTOR (0.31 ± 0.04 vs 0.48 ± 0.03 and 0.39 ± 0.01 vs 0.56 ± 0.02), 4EBP1 (0.66 ± 0.06 vs 0.90 ± 0.09 and 0.60 ± 0.12 vs 0.84 ± 0.09), and S6K1 (0.66 ± 0.01 vs 0.89 ± 0.01 and 0.48 ± 0.03 vs 0.79 ± 0.02) were decreased; however, total and phosphorylated AMPK (0.96 ± 0.06 vs 0.64 ± 0.04 and 0.97 ± 0.09 vs 0.61 ± 0.09) were increased in pigs fed diet C compared with diet A. In conclusion, diet C suppressed the mTOR pathway and accelerated the ubiquitin-proteasome pathway in skeletal muscle of finishing pigs.

Effects of Dietary Energy Sources on Post Mortem Glycolysis, Meat Quality and Muscle Fibre Type Transformation of Finishing Pigs.

Dietary energy source can influence muscle glycogen storage at slaughter. However, few studies have demonstrated whether the diet-induced change of muscle glycogen is achieved by the transformation of muscle fibre type. This study investigated the effects of dietary energy sources on meat quality, post mortem glycolysis and muscle fibre type transformation of finishing pigs. Seventy-two barrows with an average body weight of 65.0 ± 2.0 kg were selected and were allotted to three iso-energetic and iso-nitrogenous diets A, B or C, and each treatment consisted of three replicates (pens) of eight pigs each. Diet A contained 44.1% starch, 5.9% crude fat and 12.6% neutral detergent fiber (NDF); diet B contained 37.6% starch, 9.5% crude fat and 15.4% NDF; and diet C contained 30.9% starch, 14.3% crude fat and 17.8% NDF. The duration of the experiment was 28 days. After feed withdrawal 12 h, 24 pigs (eight per treatment) were slaughtered, samples from M. longissimus lumborum (LL) were collected for subsequent analysis. The results showed that pigs fed diet C had lesser average daily gain, average daily feed intake and back fat depth than those fed diet A (P<0.05). Diet C increased pH45min (P<0.05) and decreased drip loss (P<0.05) in LL muscles compared with diet A. Meat from pigs fed diet A showed increased contents of lactate and greater glycolytic potential (GP) compared with those fed diet C (P<0.05). Greater mRNA expression of myosin heavy-chain (MyHC)-I and IIa and lesser expression of MyHC-IIx and IIb (P<0.05) in LL muscles were found in pigs fed diet C, than in pigs fed diet A. In addition, pigs fed diet C resulted in downregulation of miR23a and upregulation of miR409 and miR208b (P<0.05), associated with conserved changes of their corresponding targets. These findings indicated that diets containing low starch and high fibre were beneficial in reducing muscle glycolysis, improving meat quality of finishing pigs. This reduction of GP may be partially associated with the improvement of oxidative fibre composition in LL muscle, and the change in myofibre type may be correlated with the change in the miRNA expression.

Regulation of skeletal muscle protein synthetic and degradative signaling by alanyl-glutamine in piglets challenged with Escherichia coli lipopolysaccharide.

OBJECTIVE: The aim of this study was to investigate the effects of alanyl-glutamine (Ala-Gln) on skeletal muscle protein synthetic and degradative signaling in piglets challenged with Escherichia coli lipopolysaccharide (LPS). METHODS: Piglets were arranged in a 2 × 2 factorial design and the main effects were LPS challenge (0 or 100 units) and diets (0.62% Ala or 0.5% Ala-Gln). After treatment with either Ala or Ala-Gln for 10 d, piglets were injected twice with either saline or LPS on days 11 and 15. RESULTS: During days 11 to 15 (postchallenge), LPS challenge affected the growth performance of piglets. Ala-Gln supplementation tended to alleviate the reduction of the average daily weight gain (P = 0.071) and the average daily feed intake (P = 0.087) of the LPS-challenged piglets. LPS challenge increased the concentrations of cytokines in plasma (P < 0.05), however, Ala-Gln supplementation prevented the elevation of cortisol induced by LPS challenge (P < 0.05). Moreover, Ala-Gln supplementation increased the mRNA expressions of insulin-like growth factor-1 signaling and Akt (P < 0.05). Ala-Gln supplementation also increased the phosphorylation abundance of the mammalian target of rapamycin, eIF-4 E binding protein 1 and ribosomal protein S6 kinase 1 (P < 0.05). Additionally, Ala-Gln supplementation down-regulated the mRNA abundances of toll-like receptor 4 (TLR4), muscle atrophy F-box, and muscle RING finger 1, which are associated with protein degradation induced by LPS challenge. CONCLUSION: Ala-Gln supplementation had beneficial effects in improving protein synthesis signaling of skeletal muscle, and reversed the deleterious changes of signaling molecules in muscle atrophy mainly through down-regulation of Akt/FOXO and TLR4 signaling pathways induced by LPS challenge.

L-Glutamate supplementation improves small intestinal architecture and enhances the expressions of jejunal mucosa amino acid receptors and transporters in weaning piglets.

L-Glutamate is a major oxidative fuel for the small intestine. However, few studies have demonstrated the effect of L-glutamate on the intestinal architecture and signaling of amino acids in the small intestine. The aim of this study was to investigate the effects of dietary L-glutamate supplementation on the intestinal architecture and expressions of jejunal mucosa amino acid receptors and transporters in weaning piglets. A total of 120 weaning piglets aged 35 ± 1 days with an average body weight at 8.91 ± 0.45 kg were randomly allocated to two treatments with six replicates of ten piglets each, fed with diets containing 1.21% alanine, or 2% L-glutamate. L-Glutamate supplementation increased the activity of glutamate oxaloacetate transaminase (GOT) in the jejunal mucosa. Also, the mRNA expression level of jejunal mucosa glutamine synthetase (GS) was increased by L-glutamate supplementation. The height of villi in duodenal and jejunal segments, and the relative mRNA expression of occludin and zonula occludens protein-1 (ZO-1) in jejunal mucosa were increased by dietary L-glutamate supplementation. L-Glutamate supplementation increased plasma concentrations of glutamate, arginine, histidine, isoleucine, leucine, methionine, phenylalanine and threonine. L-Glutamate supplementation also increased the relative mRNA expression of the jejunal mucosa Ca(2+)-sensing receptor (CaR), metabotropic glutamate receptor 1 (mGluR1) and metabotropic glutamate receptor 4 (mGluR4), and neutral amino acid transporter B(0)-like (SLC1A5) in the jejunal mucosa. These findings suggest that dietary addition of 2% L-glutamate improves the intestinal integrity and influences the expression of amino acid receptors and transporters in the jejunum of weaning, which is beneficial for the improvement of jejunal nutrients for digestion and absorption.