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Here, the potential mechanisms of the protective effects of fish oil against LPS-induced liver injury in a piglet model were investigated by using RNA sequencing. 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, on d 19). All piglets were slaughtered at 4 h after challenge, and liver samples were collected. Fish oil improved liver morphology and reduced TNF-α, IL-1β and IL-6 productions after LPS challenge. RNA sequencing analysis showed fish oil had significant effect on the expressions of genes involved in immune response during LPS-induced inflammation. Selected gene expression changes were validated using quantitative RT-PCR. Fish oil reduced the expressions of pro-inflammatory genes IL1R1, IL1RAP, CEBPB and CRP, and increased that of anti-inflammatory genes IL-18BP, NFKBIA, IFIT1, IFIT2 and ATF3. Moreover, fish oil restored the expressions of some lipid metabolism-related genes, such as ACAA1, ACACA, ACADS and ACADM, which were only decreased in pigs fed a corn oil diet after LPS challenge. Our RNA sequencing reveals novel gene-nutrient interactions following fish oil supplementation and evoked inflammation, which add to the current understanding of the benefits of n-3 polyunsaturated fatty acids against liver injury. [ABSTRACT FROM AUTHOR]

Stress causes intestinal inflammation and barrier dysfunction. Corticotrophin-releasing factor (CRF)/CRF receptor (CRFR) signaling pathway has been shown to be important for stress-induced intestinal mucosal alteration. L-Asparagine (ASN) is a powerful stimulator of ornithine decarboxylase and cell proliferation in a variety of cell types, including colonic cells. In the present study, we investigated whether dietary ASN supplementation could alleviate the damage of intestinal barrier function caused by LPS through modulation of CRF/CRFR signaling pathway. Twenty-four weaned pigs were randomly divided into one of four treatments: (1) non-challenged control; (2) Escherichia coli LPS challenged control; (3) LPS + 0.5% ASN; (4) LPS + 1.0% ASN. LPS stress induced villous atrophy, intestinal morphology disruption and decreased claudin-1 expression. ASN supplementation increased intestinal claudin-1 protein expression and alleviated villous atrophy and intestinal morphology impairment caused by LPS stress. In addition, ASN supplementation increased the number of intestinal intraepithelial lymphocytes and reversed the elevations of intestinal mast cell number and neutrophil number induced by LPS stress. Moreover, ASN decreased the mRNA expression of intestinal CRF, glucocorticoid receptors and tryptase. These results indicate that ASN attenuates intestinal barrier dysfunction induced by LPS stress, and regulates CRF/CRFR1 signaling pathway and mast cell activation. [ABSTRACT FROM AUTHOR]

Infection and inflammation can result in the rapid loss of muscle mass and myofibrillar proteins (muscle atrophy). In addition, aspartate (Asp) is necessary for protein synthesis in mammalian cells. We hypothesized that Asp could attenuate LPS-induced muscle atrophy in a piglet model. Twenty-four weaning piglets were allotted to four treatments, including non-challenged control, LPS challenged control, LPS+0.5% Asp and LPS+1.0% Asp. On d 21, the piglets were injected with i.p. injection of LPS (100 ug/kg BM) or saline. At 4 h post-injection, blood, gastrocnemius and longissimus dorsi muscles samples were collected for biochemical and molecular analyses. Asp decreased the concentrations of cortisol and glucagon in plasma. In addition, Asp increased protein and RNA contents in muscles, and decreased mRNA expression of muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1). Moreover, Asp decreased phosphorylation of AMPKα but increased phosphorylation of Akt and Forkhead Box O (FOXO) 1 in the muscles. Our results indicate that Asp suppresses LPS-induced MAFbx and MuRF1 expression via activation of Akt signaling, and inhibition of AMPKα and FOXO1 signaling. [ABSTRACT FROM AUTHOR]

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]

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. [ABSTRACT FROM PUBLISHER]

Pro-inflammatory cytokines play a key role in many models of hepatic damage. In addition, asparagine (Asn) plays an important role in immune function. We aimed to investigate whether Asn could attenuate lipopolysaccharide (LPS)-induced liver damage. Forty-eight castrated barrows were allotted to four groups including: (1) non-challenged control; (2) LPS-challenged control; (3) LPS+0·5 % Asn; and (4) LPS+1·0 % Asn. After 19 d feeding with control, 0·5 or 1·0 % Asn diets, pigs were injected with LPS or saline. Blood and liver samples were obtained at 4 h (early stage) and 24 h (late stage) post-injection. Asn alleviated liver injury, indicated by reduced serum aspartate aminotransferase and alkaline phosphatase activities linearly and quadratically; it increased claudin-1 protein expression linearly and quadratically at 24 h, and less severe liver morphological impairment at 4 or 24 h. In addition, Asn decreased mRNA expression of TNF-α and heat shock protein 70 (HSP70) linearly and quadratically at 4 h; it increased TNF-α mRNA expression, and HSP70 protein expression linearly and quadratically at 24 h. Moreover, Asn increased inducible NO synthase activity linearly and quadratically. Finally, Asn down-regulated the mRNA expression of Toll-like receptor 4 (TLR4) signalling molecules (TLR4, IL-1 receptor-associated kinase 1 (IRAK1), TNF-α receptor-associated factor 6), nucleotide-binding oligomerisation domain protein (NOD) signalling molecules (NOD1, NOD2 and their adaptor molecule receptor-interacting serine/threonine-protein kinase 2 (RIPK2)), and NF-κB p65 linearly or quadratically at 4 h. Oppositely, Asn up-regulated mRNA expressions of TLR4 and NOD signalling molecules (TLR4, myeloid differentiation factor 88, IRAK1, NOD2 and RIPK2), and their negative regulators (radioprotective 105, single Ig IL-1R-related molecule, Erbb2 interacting protein and centaurin β1) linearly or quadratically at 24 h. These results indicate that, in early and late stages of LPS challenge, Asn improves liver integrity and exerts different regulatory effects on mRNA expression of TLR4 and NOD signalling molecules. [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 was conducted to evaluate whether dietary supplementation with L-arginine (Arg) could attenuate Escherichia coli LPS-induced liver injury through the TLR4 signaling pathway in weaned pigs. Eighteen weaned pigs were allotted to three treatments: non-challenged control, LPS challenged control and LPS + 0.5% Arg. On d 18, pigs were injected with LPS at 100 µg/kg of body weight (BW) or sterile saline. Blood samples were obtained at 4 h post-injection. Pigs were then sacrificed for the collection of liver samples. Arg supplementation (0.5%) alleviated liver morphological impairment, including hepatocyte caryolysis, karyopycnosis and fibroblast proliferation induced by LPS challenge; it mitigated the increase of serum aspartate aminotransferase and alkaline phosphatase activities induced by LPS (P < 0.05); it prevented the increase of hepatic TNF-α, malondialdehyde contents and mast cell number induced by LPS administration (P < 0.05); and it attenuated the elevation of hepatic NF-κB and TLR4-positive cell percentages (P < 0.05). These results indicate that Arg supplementation has beneficial effects in attenuating hepatic morphological and functional injury induced by LPS challenge in piglets. Additionally, it is possible that the protective effects of Arg on the liver are associated with a decreased release of liver pro-inflammatory cytokines and free radicals through inhibiting 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]

This study evaluated whether arginine (Arg) supplementation could attenuate gut injury induced by Escherichia coli lipopolysaccharide (LPS) challenge through an anti-inflammatory role in weaned pigs. Pigs were allotted to four treatments including: (1) non-challenged control; (2) LPS-challenged control; (3) LPS+0·5 % Arg; (4) LPS+1·0 % Arg. On day 16, pigs were injected with LPS or sterile saline. At 6 h post-injection, pigs were killed for evaluation of small intestinal morphology and intestinal gene expression. Within 48 h of challenge, 0·5 % Arg alleviated the weight loss induced by LPS challenge (P = 0·025). In all three intestinal segments, 0·5 or 1·0 % Arg mitigated intestinal morphology impairment (e.g. lower villus height and higher crypt depth) induced by LPS challenge (P < 0·05), and alleviated the decrease of crypt cell proliferation and the increase of villus cell apoptosis after LPS challenge (P < 0·01). The 0·5 % Arg prevented the elevation of jejunal IL-6 mRNA abundance (P = 0·082), and jejunal (P = 0·030) and ileal (P = 0·039) TNF-α mRNA abundance induced by LPS challenge. The 1·0 % Arg alleviated the elevation of jejunal IL-6 mRNA abundance (P = 0·053) and jejunal TNF-α mRNA abundance (P = 0·003) induced by LPS challenge. The 0·5 % Arg increased PPARγ mRNA abundance in all three intestinal segments (P < 0·10), and 1·0 % Arg increased duodenal PPARγ mRNA abundance (P = 0·094). These results indicate that Arg supplementation has beneficial effects in alleviating gut mucosal injury induced by LPS challenge. Additionally, it is possible that the protective effects of Arg on the intestine are associated with decreasing the expression of intestinal pro-inflammatory cytokines through activating PPARγ expression. [ABSTRACT FROM PUBLISHER]

Simple Summary: The biological characteristics of E. faecium R1 and the effect of dietary supplementation with E. faecium R1 on the growth performance of weaned piglets were studied. The results showed that E. faecium R1 had the characteristics of effective bacteriostatic activity, acid resistance, bile salt resistance, and high-temperature resistance. Dietary supplementation with E. faecium R1 (6.5 × 106 CFU/g) improved intestinal function of weaning piglets by decreasing diarrhea incidence. Further research found that dietary supplementation with E. faecium R1 (6.5 × 106 CFU/g) attenuated intestinal and liver injury in piglets challenged by lipopolysaccharide. In this study, a strain of E. faecium R1 with effective bacteriostatic activity, acid resistance, bile salt resistance, high-temperature resistance was screened. To study the effect of E. faecium R1 on lipopolysaccharide (LPS)-induced intestinal and liver injury in piglets, twenty-four weaned female piglets were randomly assigned into one of three groups (8 piglets per group). Piglets in the control group and LPS group were fed a basal diet, piglets in the E. faecium group were fed the basal diet supplemented with E. faecium R1 (6.5 × 106 CFU/g). On day 21 of the trial, piglets in the LPS group and E. faecium group were intraperitoneally administered LPS (100 μg/kg), piglets in the control group were administered the same volume of saline. Subsequently, blood samples were collected at 3 h, and intestinal, liver, and pancreas samples were collected at 6 h. Results showed that E. faecium R1 supplementation significantly decreased the diarrhea rate and feed to gain ratio, and dramatically reduced LPS-induced intestinal and liver injury in piglets. Compared with the LPS group, E. faecium R1 supplementation significantly increased the content of glucagon in plasma and IL-1β in the liver, and the mRNA levels of villin in jejunum and ileum and Bcl-xL and pBD-L in the ileum, and significantly decreased the contents of prostaglandin 2 and malondialdehyde in the liver and the activities of myeloperoxidase and aspartate aminotransferase in plasma in piglets. Moreover, E. faecium R1 improved the pancreatic antioxidant capacity in piglets, which was indicated by a significant increase in catalase activity and a decrease in total nitric oxide synthase activity. In summary, dietary supplementation with E. faecium R1 alleviates intestinal and liver injury in LPS-challenged piglets. [ABSTRACT FROM AUTHOR]

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: 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 &y& Elsevier]

Abstract: Peroxisome proliferator-activated receptor γ (PPARγ), 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 PPARγ was altered in the immune system of weaned pigs after Escherichia coli lipopolysaccharide (LPS) injection. PPARγ expression was investigated in the thymus, spleen, mesenteric lymph node and peripheral white blood cells of weaned pigs (8.54±0.24kg BW) after LPS injection (100μg/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 PPARγ 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 PPARγ 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 PPARγ expression, LPS induced increases in plasma interleukin-6 (P <0.001), tumor necrosis factor-α (P <0.001), cortisol (P <0.001), prostaglandin E2 (P <0.01) and 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) (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 PPARγ expression in immune system may be associated with the release of the natural PPARγ activating ligand 15d-PGJ2, and play an important role in host response to immunological stress. Additionally, it is possible that PPARγ would be a new therapeutic target in treatment of immunological stress of livestock. [Copyright &y& Elsevier]