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This study was conducted to envaluate whether glycine could alleviate Escherichia coli lipopolysaccharide (LPS)-induced intestinal injury by regulating intestinal epithelial energy status, protein synthesis, and inflammatory response via AMPK, mTOR, TLR4, and NOD signaling pathways. A total of 24 weanling piglets were randomly allotted to 1 of 4 treatments: (1) non-challenged control; (2) LPS-challenged control; (3) LPS + 1% glycine; (4) LPS + 2% glycine. After 28 days feeding, piglets were injected intraperitoneally with saline or LPS. The pigs were slaughtered and intestinal samples were collected at 4 h postinjection. The mRNA expression of key genes in these signaling pathways was measured by real-time PCR. The protein abundance was measured by Western blot analysis. Supplementation with glycine increased jejunal villus height/crypt depth ratio. Glycine also increased the jejunal and ileal protein content, RNA/DNA ratio, and jejunal protein/DNA ratio. The activities of citroyl synthetase in ileum, and α-ketoglutarate dehydrogenase complex in jejunum, were increased in the piglets fed diets supplemented with glycine. In addition, glycine decreased the jejunal and ileal phosphorylation of AMPKα, and increased ileal phosphorylation of mTOR. Furthermore, glycine downregulated the mRNA expression of key genes in inflammatory signaling. Meanwhile, glycine increased the mRNA expression of negative regulators of inflammatory signaling. These results indicate that glycine supplementation could improve energy status and protein synthesis by regulating AMPK and mTOR signaling pathways, and relieve inflammation by inhibiting of TLR4 and NOD signaling pathways to alleviate intestinal injury in LPS-challenged piglets.

This study determined whether N-acetylcysteine (NAC) could improve intestinal function through phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR), epithelial growth factor receptor (EGFR), toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB), adenosine 5'-monophosphate-activated protein kinase (AMPK), and type I interferon (IFN) signaling pathways in a piglet model of lipopolysaccharides (LPS) challenge. Thirty-two piglets (24-day-old) were randomly allocated to one of four treatments, with eight replicates per treatment and one piglet per replicate. The experiment consisted of four treatments in a 2 × 2 factorial arrangement with two diets (supplemented with 0 or 500 mg NAC/kg diet) and saline or LPS administration. On day 20 of the trial, piglets in the LPS and LPS + NAC groups were intraperitoneally injected with 0 (saline) or 100 μg LPS/kg BW. Blood samples were obtained at 3 h and intestinal mucosae were collected at 6 h post LPS or saline injection. The growth performance was not affected by dietary NAC. LPS induced intestinal dysfunction, as indicated by: (1) reductions in the small-intestinal glutathione concentrations and plasma D-xylose levels; (2) elevations in plasma diamine oxidase activity, mucosal MMP3 mRNA levels and caspase-3 protein abundance; (3) reduced the activities of the small-intestinal mucosal maltase, sucrase and lactase. The adverse effects of LPS on porcine intestinal function and redox status were mitigated by NAC supplementation through the activation of multiple signaling pathways involving PI3K/Akt/mTOR, EGFR, TLR4/NF-κB, AMPK, and type I IFN. Our findings provide novel mechanisms for beneficial effects of NAC in protecting the intestine from inflammation in animals.

This study aims to determine whether Lactobacillus casei ( L. casei ) could relieve liver injury in piglets challenged with lipopolysaccharide (LPS). Piglets were randomly allocated into one of the three groups: control, LPS, and L. casei . The control and LPS groups were fed a corn- and soybean meal-based diet, whereas the L. casei group was fed the basal diet supplemented with 6 × 10⁶ cfu/g L. casei . On Day 31 of the trial, piglets in the LPS and L. casei groups received intraperitoneal administration of LPS (100 µg/kg body weight), while the control group received the same volume of saline. Blood and liver samples were collected for analysis. Results showed that L. casei supplementation decreased the feed/gain ratio ( p = 0.027) and diarrhea incidence ( p < 0.001), and attenuated LPS-induced liver histomorphological abnormalities. Compared with the control group, LPS challenge dramatically increased glutamyl transpeptidase activity ( p = 0.001) in plasma as well as the concentrations of Interleukin 6 (IL-6) ( p = 0.048), Tumor necrosis factor-alpha (TNF-α) ( p = 0.041), and Malondialdehyde (MDA) ( p = 0.001) in the liver, while decreasing the hepatic SOD activity. LPS also increased ( p < 0.05) the mRNA levels for IL-6, IL-8, TNF-α, Toll-like receptors 4 (TLR4), Nuclear factor κB (NF-κB) and Heat shock protein 70 (HSP70) in the liver. The adverse effects of LPS challenge were ameliorated by L. casei supplementation. In conclusion, dietary L. casei alleviates LPS-induced liver injury via reducing pro-inflammatory cytokines and increasing anti-oxidative capacity., Competing Interests: The authors declare no conflict of interest.

The experiment was conducted to study the effect of the glutamate (Glu) on muscle protein loss through toll-like receptor 4 (TLR4), nucleotide-binding oligomerization domain proteins (NODs), Akt/Forkhead Box O (Akt/FOXO) and mammalian target of rapamycin (mTOR) signaling pathways in LPS-challenged piglets. Twenty-four weaned piglets were assigned into four treatments: (1) Control; (2) LPS+0% Glu; (3) LPS + 1.0% Glu; (4) LPS + 2.0% Glu. The experiment was lasted for 28 days. On d 28, the piglets in the LPS challenged groups were injected with LPS on 100 μg/kg body weight (BW), and the piglets in the control group were injected with the same volume of 0.9% NaCl solution. After 4 h LPS or saline injection, the piglets were slaughtered and the muscle samples were collected. Glu supplementation increased the protein/DNA ratio in gastrocnemius muscle, and the protein content in longissimus dorsi (LD) muscle after LPS challenge (P<0.05). In addition, Glu supplementation decreased TLR4, IL-1 receptor-associated kinase (IRAK) 1, receptor-interacting serine/threonine-protein kinase (RIPK) 2, and nuclear factor-κB (NF-κB) mRNA expression in gastrocnemius muscle (P<0.05), MyD88 mRNA expression in LD muscle, and FOXO1 mRNA expression in LD muscle (P<0.05). Moreover, Glu supplementation increased p-Akt/t-Akt ratio (P<0.05) in gastrocnemius muscle, and p-4EBP1/t-4EBP1 ratio in both gastrocnemius and LD muscles (P<0.05). Glu supplementation in the piglets' diets might be an effective strategy to alleviate LPS-induced muscle protein loss, which might be due to suppressing the mRNA expression of TLR4 and NODs signaling-related genes, and modulating Akt/FOXO and mTOR signaling pathways.

Bowel diseases are common in human and animals and are characterized by intestinal dysfunction and injury. A well-established porcine model of intestinal injury can be induced by lipopolysaccharide (LPS), an endotoxin released from the cell wall of pathogenic bacteria. LPS affects the expression of genes associated with intestinal immune response, mucosal growth, energy metabolism, absorption, mucosal barrier function, and antiviral function. Transcriptional analysis of intestinal genes reveals that the duodenum, jejunum, ileum and colon respond to LPS challenge in a similar pattern. Moreover, the jejunum and ileum exhibit greater responses to LPS challenge than the duodenum and colon with regard to gene expression. Additionally, over 85% of genes are co-expressed along the small and large intestines and there is a clear distinction in gene expression patterns amongst the different intestinal segments in pigs. These findings have important implications for underlying molecular mechanisms responsible for endotoxin-induced intestinal injury and dysfunction.

The intestine requires a high amount of energy to maintain its health and function; thus, energy deficits in intestinal mucosa may lead to intestinal damage. Asparagine (Asn) is a precursor for many other amino acids such as aspartate, glutamine and glutamate, which can be used to supply energy to enterocytes. In the present study, we hypothesise that dietary supplementation of Asn could alleviate bacterial lipopolysaccharide (LPS)-induced intestinal injury via improvement of intestinal energy status. A total of twenty-four weaned piglets were assigned to one of four treatments: (1) non-challenged control; (2) LPS+0 % Asn; (3) LPS+0·5 % Asn; (4) LPS+1·0 % Asn. On day 19, piglets were injected with LPS or saline. At 24 h post-injection, piglets were slaughtered and intestinal samples were collected. Asn supplementation improved intestinal morphology, indicated by higher villus height and villus height:crypt depth ratio, and lower crypt depth. Asn supplementation also increased the ratios of RNA:DNA and protein:DNA as well as disaccharidase activities in intestinal mucosa. In addition, Asn supplementation attenuated bacterial LPS-induced intestinal energy deficits, indicated by increased ATP and adenylate energy charge levels, and decreased AMP:ATP ratio. Moreover, Asn administration increased the activities of key enzymes involved in the tricarboxylic acid cycle, including citrate synthase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase complex. Finally, Asn administration decreased the mRNA abundance of intestinal AMP-activated protein kinase-α1 (AMPKα1), AMPKα2, silent information regulator 1 (SIRT1) and PPARγ coactivator-1α (PGC1α), and reduced intestinal AMPKα phosphorylation. Collectively, these results indicate that Asn supplementation alleviates bacterial LPS-induced intestinal injury by modulating the AMPK signalling pathway and improving energy status.

There is growing interest in glutamate as a functional amino acid in nutrition and health. This study was conducted to determine whether glutamate precursor α-ketoglutarate (AKG) could alleviate lipopolysaccharide (LPS)-induced liver injury in young pigs. Twenty-four piglets were randomly assigned to the control, LPS, or LPS + AKG group. Piglets in the control and LPS groups were fed a basal diet, whereas piglets in the NAC group were fed the basal diet supplemented with 1 % AKG. On days 10, 12, 14, and 16 of the trial, piglets in the LPS and LPS + AKG groups received intraperitoneal administration of LPS (80 μg/kg BW), whereas piglets in the control group received the same volume of saline. On day 16 of the trial, blood samples were collected 3 h after LPS or saline injection. Twenty-four hours post-administration of LPS or saline (on day 17 of the trial), piglets were killed to obtain liver for analysis. Dietary AKG supplementation alleviated LPS-induced histomorphological abnormalities and mitigated LPS-induced increases in aspartate aminotransferase (AST) activity and AST/ALT ratio (P < 0.05). Compared with the LPS group, dietary supplementation with AKG decreased plasma glutamate concentration, while increasing hepatic concentrations of glutamate, glutamine, leucine, asparagine, lysine, alanine, serine, threonine, valine, and phenylalanine (P < 0.05). LPS challenge dramatically increased concentrations of malondialdehyde and decreased glutathione peroxidase activity in the liver. Additionally, LPS challenge enhanced concentrations of AMP and total protein, as well as RNA/DNA and total protein/DNA ratios, while decreasing hepatic ADP concentrations. These adverse effects of LPS challenge were ameliorated by AKG supplementation. Collectively, dietary AKG supplementation provides a new means to ameliorate LPS-induced liver injury by increasing anti-oxidative capacity and improving energy metabolism in young pigs.

Purpose: This study was conducted to investigate whether aspartate (Asp) could improve liver energy status in the lipopolysaccharide (LPS)-challenged pigs., Methods: Twenty-four weaned pigs were assigned to four treatments: (1) nonchallenged control (control diet and saline-treated); (2) LPS-challenged control (the same control diet and LPS-challenged); (3) LPS + 0.5% Asp treatment (0.5% Asp diet and LPS-challenged); and (4) LPS + 1.0% Asp treatment (a 1.0% Asp diet and LPS-challenged). On d 19, the pigs were injected intraperitoneally with Escherichia coli LPS at 100 μg/kg body weight, and the same volume of 0.9% NaCl solution, respectively. All pigs were slaughtered at 24 h after LPS or saline injection, and the liver was collected for further analysis., Results: Dietary supplementation with Asp improved liver energy status evidenced by the increased ATP concentration and adenylate energy charges, and the decreased AMP concentration and AMP/ATP ratio (p < 0.05). Asp supplementation increased the mRNA expression of key enzymes in hepatic glycolysis and tricarboxylic acid (TCA) cycle, including pyruvate kinase and citrate synthase (p < 0.05), and had a tendency to increase hepatic pyruvate dehydrogenase and isocitrate dehydrogenase β mRNA expression (p < 0.10). In addition, Asp increased the mRNA expressions of hepatic AMP-activated protein kinase (AMPK) α1, AMPKα2, silent information regulator (Sirt1), and proliferator-activated receptor-γ coactivator 1α (PGC1α) (p < 0.05). Moreover, Asp increased AMPKα phosphorylation (p < 0.05)., Conclusions: These results indicated that dietary supplementation of Asp could improve energy status in LPS-injured liver, which might result from motivating the metabolism pathway of TCA cycle and glycolysis and stimulating the AMPK signaling pathway.

Chlorogenic acid, a natural phenolic acid present in fruits and plants, provides beneficial effects for human health. The objectives of this study were to investigate whether chlorogenic acid (CHA) could improve the intestinal barrier integrity for weaned rats with lipopolysaccharide (LPS) challenge. Thirty-two weaned male Sprague Dawley rats (21 ± 1 d of age; 62.26 ± 2.73 g) were selected and randomly allotted to four treatments, including weaned rat control, LPS-challenged and chlorogenic acid (CHA) supplemented group (orally 20 mg/kg and 50 mg/kg body). Dietary supplementation with CHA decreased (P<0.05) the concentrations of urea and albumin in the serum, compared to the LPS-challenged group. The levels of IFN-γ and TNF-α were lower (P<0.05) in the jejunal and colon of weaned rats receiving CHA supplementation, in comparison with the control group. CHA supplementation increased (P<0.05) villus height and the ratio of villus height to crypt depth in the jejunal and ileal mucosae under condictions of LPS challenge. CHA supplementation decreased (P<0.05) intestinal permeability, which was indicated by the ratio of lactulose to mannitol and serum DAO activity, when compared to weaned rats with LPS challenge. Immunohistochemical analysis of tight junction proteins revealed that ZO-1 and occludin protein abundances in the jejunum and colon were increased (P<0.05) by CHA supplementation. Additionally, results of immunoblot analysis revealed that the amount of occludin in the colon was also increased (P<0.05) in CHA-supplemented rats. In conclusion, CHA decreases intestinal permeability and increases intestinal expression of tight junction proteins in weaned rats challenged with LPS.

The intestine has a high requirement for ATP to support its integrity, function and health, and thus, energy deficits in the intestinal mucosa may play a critical role in intestinal injury. Aspartate (Asp) is one of the major sources of ATP in mammalian enterocytes via mitochondrial oxidation. We hypothesized that dietary supplementation of Asp could attenuate lipopolysaccharide (LPS)-induced intestinal damage via modulation of intestinal energy status. Twenty-four weanling piglets were allotted to one of four treatments: (1) nonchallenged control, (2) LPS-challenged control, (3) LPS+0.5% Asp treatment, and (4) LPS+1.0% Asp treatment. On day 19, pigs were injected with saline or LPS. At 24 h postinjection, pigs were killed and intestinal samples were obtained. Asp attenuated LPS-induced intestinal damage indicated by greater villus height and villus height/crypt depth ratio as well as higher RNA/DNA and protein/DNA ratios. Asp improved intestinal function indicated by increased intestinal mucosal disaccharidase activities. Asp also improved intestinal energy status indicated by increased ATP, ADP and total adenine nucleotide contents, adenylate energy charge and decreased AMP/ATP ratio. In addition, Asp increased the activities of tricarboxylic acid cycle key enzymes including citrate synthase, isocitrate dehydrogenase and alpha-oxoglutarate dehydrogenase complex. Moreover, Asp down-regulated mRNA expression of intestinal AMP-activated protein kinase α1 (AMPKα1), AMPKα2, silent information regulator 1 (SIRT1) and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC1α) and decreased intestinal AMPKα phosphorylation. These results indicate that Asp may alleviate LPS-induced intestinal damage and improve intestinal energy status., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Long-chain n-3 (ω-3) polyunsaturated fatty acids exert beneficial effects in neuroendocrine dysfunctions in animal models and clinical trials. However, the mechanism(s) underlying the beneficial effects remains to be elucidated. We hypothesized that dietary treatment with fish oil (FO) could mitigate LPS-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis through inhibition of Toll-like receptor 4 and nucleotide-binding oligomerization domain protein signaling pathways. Twenty-four weaned pigs were used in a 2 × 2 factorial design, and the main factors consisted of diet (5% corn oil vs. 5% FO) and immunological challenge (saline vs. LPS). After 21 d of dietary treatment with 5% corn oil or FO diets, pigs were treated with saline or LPS. Blood samples were collected at 0 (preinjection), 2, and 4 h postinjection, and then pigs were humanely killed by intravenous injection of 40 mg/kg body weight sodium pentobarbital for tissue sample collection. FO led to enrichment of eicosapentaenoic acid and docosahexaenoic acid and total n-3 polyunsaturated fatty acids in hypothalamus, pituitary gland, adrenal gland, spleen, and thymus. FO decreased plasma adrenocorticotrophin and cortisol concentrations as well as mRNA expressions of hypothalamic corticotropin releasing hormone and pituitary proopiomelanocortin. FO also reduced mRNA expression of tumor necrosis factor-α in hypothalamus, adrenal gland, spleen, and thymus, and of cyclooxygenase 2 in hypothalamus. Moreover, FO downregulated the mRNA expressions of Toll-like receptor 4 (TLR4) and its downstream molecules, including cluster differentiation factor 14, myeloid differentiation factor 2, myeloid differentiation factor 88, interleukin-1 receptor-associated kinase 1, tumor necrosis factor-α receptor-associated factor 6, and nuclear factor kappa-light-chain-enhancer of activated B cells p65, and also decreased the mRNA expressions of nucleotide-binding oligomerization domain 1, nucleotide-binding oligomerization domain 2, and their adaptor molecule receptor-interacting serine/threonine-protein kinase 2. These results suggested that FO attenuates the activation of the HPA axis induced by LPS challenge. The beneficial effects of FO on the HPA axis may be associated with decreasing the production of brain or peripheral proinflammatory cytokines through inhibition of TLR4 and nucleotide-binding oligomerization domain protein signaling pathways.

Long-chain (n-3) PUFA exert beneficial effects on inflammatory bowel diseases in animal models and clinical trials. In addition, pattern recognition receptors such as toll-like receptors (TLR) and nucleotide-binding oligomerization domain proteins (NOD) play a critical role in intestinal inflammation. We hypothesized that fish oil could alleviate Escherichia coli LPS-induced intestinal injury via modulation of TLR4 and NOD signaling pathways. Twenty-four weaned piglets were used in a 2 × 2 factorial design and the main factors included a dietary treatment (5% corn oil or 5% fish oil) and immunological challenge (LPS or saline). After feeding fish oil or corn oil diets for 21 d, pigs were injected with LPS or saline. At 4 h postinjection, blood samples were collected and pigs were killed. EPA, DHA, and total (n-3) PUFA were enriched in intestinal mucosa through fish supplementation. Fish oil improved intestinal morphology, indicated by greater villus height and villus height:crypt depth ratio, and intestinal barrier function, indicated by decreased plasma diamine oxidase (DAO) activity and increased mucosal DAO activity as well as enhanced protein expression of intestinal tight junction proteins including occludin and claudin-1. Moreover, fish oil decreased intestinal TNFα and PGE(2) concentrations and caspase-3 and heat shock protein 70 protein expression. Finally, fish oil downregulated the mRNA expression of intestinal TLR4 and its downstream signals myeloid differentiation factor 88, IL-1 receptor-associated kinase 1, TNFα receptor-associated factor 6, and NOD2, and its adaptor molecule, receptor-interacting serine/threonine-protein kinase 2. Fish oil decreased the protein expression of intestinal NFκB p65. These results indicate that fish oil supplementation is associated with inhibition of TLR4 and NOD2 signaling pathways and concomitant improvement of intestinal integrity under an inflammatory condition.

The neonatal small intestine is susceptible to damage by endotoxin, but effective methods for prevention and treatment are lacking. N-acetylcysteine (NAC) is a widely used precursor of L: -cysteine for animal cells and plays an important role in protecting cells against oxidative stress. This study was conducted with the lipopolysaccharide (LPS)-challenged piglet model to determine the effects of NAC on intestinal function. Eighteen piglets were randomly allocated into control, LPS and LPS + NAC groups. The control and LPS groups were fed a corn- and soybean meal-based diet, and the LPS + NAC group was fed the basal diet +500 mg/kg NAC. On days 10, 13 and 20 of the trial, the LPS and LPS + NAC groups received intraperitoneal administration of LPS (100 μg/kg BW), whereas the control piglets received saline. On day 20 of the trial, D-: xylose (0.1 g/kg BW) was orally administrated to all piglets 2 h after LPS or saline injection, and blood samples were collected 1 h thereafter. One hour blood xylose test was used to measure intestinal absorption capacity and mucosal integrity, and diamine oxidase (DAO) was used as a marker of intestinal injury. On day 21 of the trial, pigs were killed to obtain the intestinal mucosa. Compared to the control, LPS challenge reduced (P < 0.05) the concentrations of D-: xylose (a marker of intestinal absorption) in plasma, activities of DAO in the jejunal mucosa, the ratio of villus height to crypt depth in the jejunal mucosa, RNA/DNA and protein/DNA in the jejunal and ileal mucosae, while increasing (P < 0.05) DAO activity in plasma and caspase-3 expression in the intestinal mucosa. The adverse effects of LPS were partially ameliorated (P < 0.05) by NAC supplementation. Moreover, NAC prevented the LPS-induced decrease in claudin-1 and occludin expression in the jejunal and ileal mucosae. Collectively, these results indicate that dietary NAC supplementation alleviates the mucosal damage and improves the absorptive function of the small intestine in LPS-challenged piglets.

Objective: To determine whether rosiglitazone, an agonist of the peroxisome proliferator-activated receptor (PPAR) γ, could alleviate intestinal damage induced by Escherichia coli lipopolysaccharide (LPS) in weaned pigs., Animals: 18 weaned pigs (mean ± SD age, 28 ± 3 days)., Procedures: Pigs were allocated to 3 treatments (6 pigs/treatment). Control pigs were injected IP with dimethyl sulfoxide and then injected 30 minutes later with sterile saline (0.9% NaCl) solution, LPS-treated pigs were injected IP with dimethyl sulfoxide and then injected 30 minutes later with LPS (100 μg/kg, IP), and rosiglitazone plus LPS-treated pigs were injected with rosiglitazone (3 mg/kg, IP) and then injected 30 minutes later with LPS (100 μg/kg, IP). Pigs were euthanized 3 hours after challenge exposure, and samples of the small intestines were collected for histologic, biochemical, and immunohistochemical examination., Results: Rosiglitazone alleviated LPS-induced intestinal damage, which was manifested as a lower crypt depth in the duodenum and a higher villus height-to-crypt depth ratio in the duodenum, jejunum, and ileum. Rosiglitazone also mitigated inhibition of crypt cell proliferation in the jejunum and ileum induced by LPS injection. Pretreatment with rosiglitazone significantly increased the number of cells that stained for PPARγ and significantly decreased the number of cells that stained for inducible nitric oxide synthase., Conclusions and Clinical Relevance: Rosiglitazone alleviated intestinal damage induced by LPS injection in weaned pigs. The protective effects of rosiglitazone on the intestines may be associated with inhibition of intestinal proinflammatory mediators, such as inducible nitric oxide synthase.

Neonates are at increased risk for inflammatory bowel disease, but effective prevention and treatments are currently limited. This study was conducted with the lipopolysaccharide (LPS)-challenged piglet model to determine the effects of dietary supplementation with alpha-ketoglutarate (AKG) on the intestinal morphology and function. Eighteen 24-day-old pigs (weaned at 21 days of age) were assigned randomly to control, LPS, and LPS + AKG groups. The piglets in the control and LPS groups were fed a corn- and soybean meal-based diet, whereas the LPS + AKG group was fed the basal diet supplemented with 1% AKG. On days 10, 12, 14, and 16, piglets in the LPS and LPS + AKG groups received intraperitoneal administration of LPS (80 microg/kg BW), whereas piglets in the control group received the same volume of saline. On day 16, D-xylose was orally administrated to all pigs at the dose of 0.1 g/kg BW, 2 h after LPS or saline injection, and blood samples were collected 3 h thereafter. Twenty-four hours post-administration of LPS or saline, pigs were killed to obtain intestinal mucosae for analysis. Compared with the control group, LPS challenge reduced (P < 0.05) protein levels, the ratio of villus height to crypt depth, and the ratio of phosphorylated mTOR to total mTOR in duodenal, jejunal, and ileal mucosa. These adverse effects of LPS were attenuated (P < 0.05) by AKG supplementation. Moreover, AKG prevented the LPS-induced increase in intestinal HSP70 expression. Collectively, these novel results indicate that dietary supplementation with 1% AKG activates the mTOR signaling, alleviates the mucosal damage, and improves the absorptive function of the small intestine in LPS-challenged piglets. The findings not only help understand the mode of AKGs actions in the neonatal gut but also have important implications for infant nutrition under inflammatory conditions.

Peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the nuclear hormone receptor superfamily, has been implicated in regulation of immunity and inflammation in rodents and humans. The objective of the current study was to investigate whether the expression of PPARgamma was altered in the immune system of weaned pigs after Escherichia coli lipopolysaccharide (LPS) injection. PPARgamma expression was investigated in the thymus, spleen, mesenteric lymph node and peripheral white blood cells of weaned pigs (8.54+/-0.24 kg BW) after LPS injection (100 microg/kg BW, n=6) and controls (sterile saline, n=6), by using real-time polymerase chain reaction, Western blot analysis, and immunohistochemistry. Plasma pro-inflammatory cytokines and hormones were also assessed. LPS triggered PPARgamma mRNA and protein expression in the thymus (P<0.05, 4.24-fold; P<0.10, 1.46-fold), spleen (P<0.10, 2.75-fold; P<0.05, 1.84-fold), mesenteric lymph node (P<0.05, 4.32-fold; P<0.05, 1.96-fold) and peripheral white blood cells (P<0.001, 24.44-fold; P<0.001, 1.58-fold). The LPS-injected pigs showed an increase in PPARgamma staining in splenic corpuscle and periarterial lymphatic sheath of white pulp (P<0.05) and red pulp (P<0.001) of spleen, and in medullas of thymus lobule of thymus (P<0.05), and in thymus-dependent area of mesenteric lymph node (P<0.05) compared to the control pigs. Concurrent with up-regulation of PPARgamma expression, LPS induced increases in plasma interleukin-6 (P<0.001), tumor necrosis factor-alpha (P<0.001), cortisol (P<0.001), prostaglandin E(2) (P<0.01) and 15-deoxy-Delta(12,14)-prostaglandin J(2) (15 d-PGJ(2)) (P<0.05), and decreases in plasma insulin (P<0.10) and insulin-like growth factor-1 (P<0.001). These results suggest that induction of PPARgamma expression in immune system may be associated with the release of the natural PPARgamma activating ligand 15 d-PGJ(2), and play an important role in host response to immunological stress. Additionally, it is possible that PPARgamma would be a new therapeutic target in treatment of immunological stress of livestock.

Background: Skeletal muscle undergoes rapid loss in response to inflammation. α-Ketoglutarate (AKG) has been reported to enhance muscle growth in piglets, but the underlying mechanisms are largely unknown.Objectives: This study tested the hypothesis that dietary AKG supplementation activates mechanistic target of rapamycin (mTOR) signaling and improves skeletal muscle energy metabolism in lipopolysaccharide (LPS)-challenged piglets.Methods: Forty-eight male piglets (Duroc × Landrace × Yorkshire) were weaned at 21 d of age to a corn- and soybean meal-based diet. After a 3-d period of adaptation, piglets with a mean weight of 7.21 kg were randomly assigned to control, LPS (intraperitoneal administration of 80 μg LPS/kg body weight on days 10, 12, 14, and 16), or LPS plus 1% dietary AKG (LPS+AKG) groups. On day 16, blood samples were collected from 8 piglets/group 3 h after LPS administration. On day 17, piglets were killed to obtain gastrocnemius muscle from 8 piglets/group for biochemical analysis.Results: Compared with the control group, LPS administration increased (P < 0.05) plasma concentrations of globulin (by 14%) and tumor necrosis factor α (by 59%) and the intramuscular ratio of AMP to ATP (by 93%) and abundance of phosphorylated acetyl-coenzyme A carboxylase (ACC) β protein (by 64%). Compared with the control group, LPS administration reduced (P < 0.05) weight gain (by 15%); plasma concentrations of glutamine (by 20%), glucose (by 23%), insulin, insulin-like growth factor I, and epidermal growth factor; intramuscular concentrations of glutamine (by 27%), ATP (by 12%), ADP (by 22%), and total adenine nucleotides; and intramuscular ratios of phosphorylated mTOR to total mTOR (by 38%) and of phosphorylated 70-kDa ribosomal protein S6 kinase (p70S6K) to total p70S6K (by 39%). These adverse effects of LPS were ameliorated (P < 0.05) by AKG supplementation.Conclusions: Dietary AKG supplementation activated mTOR signaling, inhibited ACC-β, and improved energy status in skeletal muscle of LPS-challenged piglets. These results provide a biochemical basis for the use of AKG to enhance piglet growth under inflammatory or practical postweaning conditions. [ABSTRACT FROM AUTHOR]

In this study, the anti-inflammatory effects and mechanisms of baicalin on LPS-induced NLRP3 inflammatory pathway were investigated in piglet mononuclear phagocytes (control, LPS stimulation, LPS stimulation + 12.5 µg/ml baicalin, LPS stimulation + 25 µg/ml baicalin, LPS stimulation + 50 µg/ml baicalin and LPS stimulation + 100 µg/ml baicalin). The levels of reactive oxygen species (ROS), the secretion levels of IL-1β, IL-18 and TNF-α, mRNA expression levels of IL-1β, IL-18, TNF-α and NLRP3, as well as the protein levels of cleaved caspase-1 p20 were significantly increased after LPS-challenge in vitro. However, LPS stimulation did not influence apoptosis-associated speck-like protein and caspase-1 mRNA levels, which are also components of the NLRP3 inflammasome. Baicalin at 50 µg/ml and 100 µg/ml could inhibit the production of ROS, TNF-α, IL-1β and IL-18, and down-regulate mRNA expression of IL-1β, IL-18, TNF-α and NLRP3, as well as expression of cleaved caspase-1 p20. These results showed that the anti-inflammatory effects of baicalin occurred via the regulation of the release of ROS and mRNA expression of NLRP3. The anti-inflammatory activity of baicalin could be related to the suppression of NLRP3 inflammasome pathway under LPS stimulation. [ABSTRACT FROM AUTHOR]

This study evaluated whether fish oil exerted a hepatoprotective effect in a LPS-induced liver injury model via regulation of TLR4 and nucleotide-binding oligomerization domain protein (NOD) signaling pathways. Twenty-four piglets were used in a 2 × 2 factorial design, and the main factors included diet (5% corn oil or 5% fish oil) and immunological challenge (LPS or saline). Fish oil resulted in enrichment of eicosapentaenoic acid, docosahexaenoic acid and total (n-3) polyunsaturated fatty acids in liver. Less severe liver injury was observed in pigs fed fish oil, as evidenced by improved serum biochemical parameters and less severe histological liver damage. In addition, higher expression of liver tight junction proteins, and lower hepatocyte proliferation and higher hepatocyte apoptosis were observed in pigs fed fish oil. The improved liver integrity in pigs fed fish oil was concurrent with reduced hepatic mRNA expression of TLR4, myeloid differentiation factor 88, IL-1 receptor-associated kinase 1 and TNF-α receptor-associated factor 6, and NOD1, NOD2 and receptor-interacting serine/threonine-protein kinase 2, as well as reduced hepatic protein expression of NF-κB p65, leading to reduced hepatic pro-inflammatory mediators. These results indicate that fish oil improves liver integrity partially via inhibition of TLR4 and NOD signaling pathways under an inflammatory condition. [ABSTRACT FROM AUTHOR]

This study determined whether N-acetylcysteine (NAC) could affect intestinal redox status, proinflammatory cytokines, epidermal growth factor (EGF), EGF receptor (EGFR), Toll-like receptor-4 (TLR4), and aquaporin-8 in a lipopolysaccharide (LPS)-challenged piglet model. Eighteen piglets (35-day-old) were randomly allocated into one of the three treatments (control, LPS and NAC). The control and LPS groups were fed a basal diet, and the NAC group received the basal diet +500 mg/kg NAC. On days 10, 13, and 20 of the trial, the LPS- and NAC-treated piglets received intraperitoneal administration of LPS (100 μg/kg BW), whereas the control group received the same volume of saline. On days 10 and 20, venous blood samples were obtained at 3 h post LPS or saline injection. On day 21 of the trial, piglets were killed to obtain the intestinal mucosa for analysis. Compared with the control group, LPS challenge reduced ( P < 0.05) the activities of superoxide dismutase, catalase, and glutathione peroxidase in jejunal mucosae, while increasing ( P < 0.05) the concentrations of malondialdehyde, HO, O and the ratio of oxidized to reduced glutathione in jejunal mucosae, and concentrations of TNF-α, cortisol, interleukin-6, and prostaglandin E in both plasma and intestinal mucosae. These adverse effects of LPS were attenuated ( P < 0.05) by NAC supplementation. Moreover, NAC prevented LPS-induced increases in abundances of intestinal HSP70 and NF-κB p65 proteins and TLR4 mRNA. NAC supplementation enhanced plasma EGF concentration and intestinal EGFR mRNA levels. Collectively, these results indicate that dietary NAC supplementation alleviates LPS-induced intestinal inflammation via regulating redox, EGF, and TLR4 signaling. [ABSTRACT FROM AUTHOR]

The present study determined whether α-ketoglutarate (AKG) might affect the expression of AMP-activated protein kinase (AMPK) and energy status in the intestinal mucosa of piglets challenged with Escherichia coli lipopolysaccharide (LPS). A total of eighteen piglets (weaned at 21 d of age) were allocated to one of three treatments: (1) non-challenged (control); (2) LPS-challenged (LPS); (3) LPS+1 % AKG (LPS+AKG). Piglets in the control and LPS groups were fed a maize- and soyabean meal-based diet, and the LPS+AKG group was fed the basal diet supplemented with 1 % AKG. On days 10, 12, 14 and 16 of the trial, piglets in the LPS and LPS+AKG groups were challenged with LPS (80 μg/kg body weight), whereas piglets in the control group received the same volume of sterile saline. Pigs were euthanised 24 h after the last administration of LPS or saline to obtain intestinal mucosae for biochemical analysis. Compared with the control group, LPS administration decreased (P < 0·05) the oxidation of AKG, oleic acid, glutamine and glucose in enterocytes, decreased concentrations of ATP in the duodenal and jejunal mucosae and decreased adenylate energy charge (AMP:ATP ratio) in the jejunal and ileal mucosae. Additionally, LPS treatment reduced (P < 0·05) mucosal concentrations of phosphorylated AMPK in the jejunum and ileum as well as acetyl-CoA carboxylase in all segments of the small intestine. The adverse effects of LPS were reversed by AKG. Collectively, these results indicate that dietary supplementation with 1 % AKG beneficially modulates the AMPK signalling pathway to improve energy status in the small intestine of LPS-challenged piglets. [ABSTRACT FROM PUBLISHER]

Abstract: Pro-inflammatory cytokines play a critical role in many models of liver injury. In addition, aspartate (Asp) plays an important role in many biological and physiological processes including liver physiology. We hypothesized that Asp could alleviate lipopolysaccharide (LPS)-induced liver injury. Forty-eight weanling pigs were assigned to four treatments including: (1) non-challenged control; (2) LPS challenged control; (3) LPS+0.5% Asp; (4) LPS+1.0% Asp. After 20-d feeding with control (0% Asp), 0.5% or 1.0% Asp supplemented diets, pigs were injected with saline or LPS. At 4 (early phase) and 24 h (late phase) post-injection, blood and liver samples were obtained. Asp attenuated liver injury indicated by reduced serum aspartate aminotransferase activity and increased ratio of serum alanine aminotransferase and aspartate aminotransferase at 24 h, and less severe histological liver damage induced by LPS challenge at 4 or 24 h. In addition, Asp supplementation to LPS challenged pigs decreased mRNA expressions of tumor necrosis factor (TNF)-α and cyclooxygenase-2 linearly and quadratically at 4 h, and increased mRNA expressions of these pro-inflammatory mediators linearly and quadratically at 24 h. Finally, Asp decreased mRNA expression of toll-like receptor 4 (TLR4) signaling related genes (TLR4, myeloid differentiation factor 88, IL-1 receptor-associated kinase 1, TNF-α receptor-associated factor (6), nucleotide-binding oligomerization domain protein (NOD) signaling related genes (NOD1, NOD2 and receptor-interacting serine/threonine-protein kinase 2) and nuclear factor-κB p65 linearly or quadratically at 4 h. However, Asp increased mRNA expressions of these signaling molecules linearly or quadratically at 24 h. These results indicate that, at early and late phases of LPS challenge, Asp exerts opposite regulatory effects on mRNA expression of hepatic pro-inflammatory cytokines and TLR4 and NOD signalling related genes, and improves liver integrity. [Copyright &y& Elsevier]

Abstract: Lipopolysaccharide (LPS) is a known activator of mononuclear phagocytes. LPS activates the pro-inflammatory gene expression and induces the release of mediators/cytokines by TLR4-NF-κB signaling pathway. The purpose of this study was to investigate the effects of acetaminophen (AAP) and N-acetylcysteine (NAC), individually as well as in combination on LPS-induced cytokines production and NF-κB activation in piglets. AAP (0.125–1.0mM) and NAC (0.0625–1.0mM) down-regulate the expression of cytokines and inhibit NF-κB p65 protein transfer from the cytoplasm to the nucleus in vitro. NAC enhances the inhibition action of AAP on cytokines expression in vitro. IL-6 in piglet plasma of the AAP group (mixed feed concentration of 600mg/kg) was significantly reduced (P <0.05) at 3h after LPS-challenge as compared with the LPS control group. IL-10 also significantly reduced (P <0.05) at 24h after LPS injection. The levels of inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-10) in piglet plasma of the NAC group (mixed feeding concentration of 1200mg/kg) were significantly lower at 3h after LPS stimulation (P <0.05). IL-10 was significantly decreased in the NAC group at 24h after LPS stimulation (P <0.05). AAP or NAC treated alone could reduce the NF-κB p65 concentration ratio. The levels of cytokines (TNF-α, IL-1β, IL-6, and IL-10) in the group with piglet plasma of AAP (mixed feed concentration of 600mg/kg) plus NAC (mixed feeding concentration of 1200mg/kg) group were significantly lower (P <0.05) at 3h after LPS activation. The level of IL-10 in the group with AAP plus NAC was significantly lower (P <0.05) at 24h after LPS stimulation, while the rest of the inflammatory cytokines were returned to the original levels. The NF-κB p65 concentration ratio had significantly reduced (P <0.05) when AAP and NAC were used in combination. In summary, NAC could enhance the anti-inflammatory effects of AAP both in vitro and in vivo. [Copyright &y& Elsevier]