Regulative Role Of Fish Oil On Intestinal And Liver Injury, And Muscle Protein Degradation Of Piglets After Lipopolysaccharide Challenge Through TLR4and NOD Signaling Pathway

This experiment was conducted to evaluate the regulative role of fish oil onintestinal and liver injury, and muscle protein degradation of piglets afterlipopolysaccharide (LPS) challenge through toll-like receptor (TLR4) andnucleotide binding oligomerization domain protein (NOD) signaling pathways.Twenty-four crossbred weaned pigs were used in a2×2factorial design, and themain factors consisted of diet (5%corn oil or5%fish oil) and immunologicalchallenge (LPS or saline). On d21, the challenged group was injectedintraperitoneally with LPS at100μg/kg BW, and the unchallenged group wasinjected with the same amount of0.9%NaCl solution. Blood samples werecollected at pre-injection (0h),2, or4h post-injection, and then pigs weresacrificed for samples collection of intestinal mucosa, liver and muscle.1. This experiment was conducted to investigate whether fish oil couldalleviate LPS-induced intestinal injury in piglets via modulation of TLR4and NODsignaling pathways. Fish oil supplementation resulted in enrichment ofeicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and total n-3polyunsaturated fatty acid (PUFA) in intestinal mucosa (P<0.001). Fish oilimproved intestinal morphology indicated by greater villus height and villusheight/crypt depth ratio (P<0.05). Fish oil improved intestinal barrier functionindicated by decreased plasma diamine oxidase (DAO) activity and increasedmucosal DAO acitivity as well as enhanced occludin protein expression in jejunumand ileum (P<0.05). Fish oil decreased jejunal tumor necrosis factor (TNF)-αconcentrations, and caspase-3protein expression in jejunum and ileum (P<0.05).Fish oil down-regulated the mRNA expression of intestinal TLR4and itsdownstream signals myeloid differentiation factor88(MyD88), IL-1receptor-associated kinase1(IRAK1), TNF-α receptor-associated factor6(TRAF6),NOD2and its adaptor molecule receptor-interacting serine/threonine-protein kinase2(RIPK2)(P<0.05). Fish oil decreased the protein expression of intestinal nuclear factor-κB (NF-κB)(P<0.05). These results indicate that fish oil supplementation isassociated with inhibition of TLR4and NOD2signaling pathways and concomitantimprovement of intestinal integrity under an inflammatory condition.2. This experiment was conducted to investigate whether fish oil couldalleviate LPS-induced liver injury in piglets via modulation of TLR4and NODsignaling pathways. Fish oil supplementation resulted in enrichment of EPA andDHA and total n-3PUFA in liver (P<0.001). To some extent, fish oilsupplementation alleviated histological liver damage induced by LPS. Fish oilimproved liver barrier function indicated by decreased serum alanineaminotransferase and alkaline phosphatase activities and total bilirubin level at2hand4h post-injection (P<0.05), increased mRNA expression of liver tight junctionproteins including occludin and claudin-1(P<0.05). Fish oil decreased hepatocyteproliferation and increased hepatocyte apoptosis indicated by lower expression ofliver PCNA and higher expression of liver caspase-3(P<0.05). Fish oil decreasedhepatic mRNA expression of TNF-α and cyclooxygenase2(COX2)(P<0.05). Fishoil reduced hepatic mRNA expression of TLR4and its downstream signals MyD88,IRAK1and TRAF6, and NOD1, NOD2and their adaptor molecule RIPK2as wellas reduced hepatic protein expression of NF-κB (P<0.05). These results indicatethat fish oil improves liver integrity partially via inhibition of TLR4and NODsignaling pathways under an inflammatory condition.3. This experiment was conducted to investigate whether fish oil couldalleviate LPS-induced muscle protein degradation in piglets via modulation ofAkt/FOXO, TLR4and NOD signaling pathways. Fish oil supplementation led toenrichment of EPA, DHA and total n-3PUFA in muscles. Fish oil increased muscleprotein mass indicated by higher protein:DNA ratio in gastrocnemius andlongissimus dorsi (LD) muscles (P<0.001). In addition, fish oil increased Akt1mRNA abundance and decreased Forkhead Box O (FOXO)1and FOXO4mRNAabundance (P<0.05). Also, fish oil increased phosphorylation of Akt and FOXO1ingastrocnemius or LD muscles(P<0.05). Fish oil decreased mRNA abundance of muscle atrophy F-box and muscle RING finger1in gastrocnemius and LD muscles(P<0.05). Moreover, fish oil reduced plasma TNF-α concentration, muscle TNF-αand prostaglandin E2concentrations, and muscle TNF-α and COX2mRNAabundance (P<0.05). Finally, fish oil down-regulated the mRNA abundance ofmuscle TLR4and its downstream signaling molecules (MyD88, TRAF6andNF-κB), and NOD1, NOD2and their adaptor molecule (RIPK2)(P<0.05). Theseresults indicate fish oil may suppress muscle pro-inflammatory cytokine productionvia regulation of TLR4and NOD signaling pathways, and therefore improve muscleprotein mass, possibly through maintenance of Akt/FOXO signaling.