Sheng-Yao Kuang

Dietary tryptophan modulates intestinal immune response, barrier function, antioxidant status and gene expression of TOR and Nrf2 in young grass carp (Ctenopharyngodon idella)

The present research evaluated the effects of dietary tryptophan (Trp) on growth performance, intestinal mucosal immune, barrier function and antioxidant capacity and gene expression of young grass carp (Ctenopharyngodon idella). Fish were fed six different experimental diets containing graded levels of Trp at 0.7(control), 1.7, 3.1, 4.0, 5.2 and 6.1 g kg(-1) diet for 8 weeks. The results showed that Trp supplementation significantly enhanced the percent weight gain (PWG), feed intake and feed efficiency (P < 0.05), and decreased the plasma ammonia content (PAC) (P < 0.05). After the 8-week feeding trail, an environmental copper exposure trail was conducted for 4 days. Results from the copper exposure trail showed that dietary Trp enhanced the lysozyme, acid phosphatase activities and complement 3 contents in the intestine of young grass carp (P < 0.05). In addition, Trp supplementation increased the copper/zinc superoxide dismutase (SOD1), glutathione peroxidase (GPx) activities and glutathione contents (P < 0.05), and decreased the protein carbonyl and malondialdehyde contents (P < 0.05). Furthermore, the relative gene expression levels of interleukin 10, transforming growth factor-β1, occludin, zonula occludens 1, claudin-b, -c, and -3, SOD1, GPx and NF-E2-related factor 2 in the intestine were significantly up-regulated with increasing of dietary Trp up to a certain level (P < 0.05). Conversely, the mRNA levels of tumor necrosis factor α, interleukin 8, target of rapamycin, Kelch-like-ECH-associated protein 1, claudin-12 and -15a in the intestine were significantly down-regulated by Trp (P < 0.05). Collectively, appropriate dietary Trp level improves fish growth, intestinal immune response, barrier function and antioxidant status, and regulated the mRNA levels of related signal molecules of young grass carp. Based on the quadratic regression analysis of the PWG and PAC, the dietary Trp requirement of young grass carp (287-699 g) was estimated to be 3.81 g kg(-1) diet (12.7 g kg(-1) protein) and 3.89 g kg(-1) diet (13.0 g kg(-1) protein), respectively.

The impaired intestinal mucosal immune system by valine deficiency for young grass carp (Ctenopharyngodon idella) is associated with decreasing immune status and regulating tight junction proteins transcript abundance in the intestine.

This study investigated the effects of dietary valine on the growth, intestinal immune response, tight junction proteins transcript abundance and gene expression of immune-related signaling molecules in the intestine of young grass carp (Ctenopharyngodon idella). Six iso-nitrogenous diets containing graded levels of valine (4.3-19.1 g kg(-)(1) diet) were fed to the fish for 8 weeks. The results showed that percentage weight gain (PWG), feed intake and feed efficiency of fish were the lowest in fish fed the valine-deficient diet (P < 0.05). In addition, valine deficiency decreased lysozyme, acid phosphatase activities and complement 3 content in the intestine (P < 0.05), down-regulated mRNA levels of interleukin 10, transforming growth factor β1, IκBα and target of rapamycin (TOR) (P < 0.05), and up-regulated tumor necrosis factor α, interleukin 8 and nuclear factor κB P65 (NF-κB P65) gene expression (P < 0.05). Additionally, valine deficiency significantly decreased transcript of Occludin, Claudin b, Claudin c, Claudin 3, and ZO-1 (P < 0.05), and improved Claudin 15 expression in the fish intestine (P < 0.05). However, valine did not have a significant effect on expression of Claudin 12 in the intestine of grass carp (P > 0.05). In conclusion, valine deficiency decreased fish growth and intestinal immune status, as well as regulated gene expression of tight junction proteins, NF-κB P65, IκBα and TOR in the fish intestine. Based on the quadratic regression analysis of lysozyme activity or PWG, the dietary valine requirement of young grass carp (268-679 g) were established to be 14.47 g kg(-1) diet (4.82 g 100 g(-1) CP) or 14.00 g kg(-1) diet (4.77 g 100 g(-1) CP), respectively.

Soybean β-Conglycinin Induces Inflammation and Oxidation and Causes Dysfunction of Intestinal Digestion and Absorption in Fish

β-conglycinin has been identified as one of the major feed allergens. However, studies of β-conglycinin on fish are scarce. This study investigated the effects of β-conglycinin on the growth, digestive and absorptive ability, inflammatory response, oxidative status and gene expression of juvenile Jian carp (Cyprinus carpio var. Jian) in vivo and their enterocytes in vitro. The results indicated that the specific growth rate (SGR), feed intake, and feed efficiency were reduced by β-conglycinin. In addition, activities of trypsin, chymotrypsin, lipase, creatine kinase, Na+,K+-ATPase and alkaline phosphatase in the intestine showed similar tendencies. The protein content of the hepatopancreas and intestines, and the weight and length of the intestines were all reduced by β-conglycinin. β-conglycinin increased lipid and protein oxidation in the detected tissues and cells. However, β-conglycinin decreased superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione peroxidase (GPx) and glutathione reductase (GR) activities and glutathione (GSH) content in the intestine and enterocytes. Similar antioxidant activity in the hepatopancreas was observed, except for GST. The expression of target of rapamycin (TOR) gene was reduced by β-conglycinin. Furthermore, mRNA levels of interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), and transforming growth factor-β (TGF-β) genes were increased by β-conglycinin. However, β-conglycinin increased CuZnSOD, MnSOD, CAT, and GPx1b gene expression. In conclusion, this study indicates that β-conglycinin induces inflammation and oxidation, and causes dysfunction of intestinal digestion and absorption in fish, and finally reduces fish growth. The results of this study provide some information to the mechanism of β-conglycinin-induced negative effects.

Effects of dietary arginine supplementation on growth performance, flesh quality, muscle antioxidant capacity and antioxidant-related signalling molecule expression in young grass carp (Ctenopharyngodon idella)

Growth performance, flesh quality, antioxidant status and antioxidant-related signalling molecule expression in the muscle of young grass carp, which were fed graded levels of arginine (6.9-24.5 g/kg diet) for eight weeks, were investigated. Muscle protein, lipid and nitric oxide contents, shear force, hydroxyproline concentration, and pH were significantly improved by appropriate arginine. Cooking loss, lactate content, cathepsins activities, malondialdehyde and protein carbonyl contents exhibited an opposite tendency. Additionally, optimum arginine significantly enhanced glutathione content and the activities and gene expression of copper/zinc superoxide dismutase, catalase and glutathione peroxidase in muscle. Moreover, the expression levels of glutamate-cysteine ligase, target of rapamycin, ribosome protein S6 kinase 1, casein kinase 2 and NF-E2-related factor 2 in muscle were significantly elevated by appropriate arginine. However, optimum arginine significantly decreased Kelch-like ECH-associated protein 1 mRNA levels in muscle. In conclusion, arginine improved the flesh quality and muscle antioxidant capacity and regulated antioxidant-related signalling molecule expression.

Dietary riboflavin deficiency decreases immunity and antioxidant capacity, and changes tight junction proteins and related signaling molecules mRNA expression in the gills of young grass carp (Ctenopharyngodon idella).

This study investigated the effects of dietary riboflavin on the growth, gill immunity, tight junction proteins, antioxidant system and related signaling molecules mRNA expression of young grass carp (Ctenopharyngodon idella). Fish were fed six diets containing graded levels of riboflavin (0.63-10.04 mg/kg diet) for 8 weeks. The study indicated that riboflavin deficiency decreased lysozyme and acid phosphatase activities, and complement component 3 content in the gills of fish (P < 0.05). Moreover, riboflavin deficiency caused oxidative damage, which might be partly due to decrease copper, zinc superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase and glutathione-S-transferase activities and reduced glutathione content in the gills of fish (P < 0.05). Furthermore, the relative mRNA levels of antimicrobial peptides (liver expressed antimicrobial peptide 2 and Hepcidin), anti-inflammatory cytokines (interleukin 10 and transforming growth factor β1), tight junction proteins (Occludin, zonula occludens 1, Claudin-c and Claudin-3), signaling molecules (inhibitor of κBα, target of rapamycin and NF-E2-related factor 2) and antioxidant enzymes (copper, zinc superoxide dismutase and glutathione reductase) were significantly decreased (P < 0.05) in the gills of fish fed riboflavin-deficient diet. Conversely, the mRNA levels of pro-inflammatory cytokines (tumor necrosis factor α, interleukin 8, interferon γ2, and interleukin 1β), signaling molecules (nuclear factor kappa B p65, IκB kinase β, IκB kinase γ, Kelch-like-ECH-associated protein 1b and myosin light chain kinase) and tight junction protein Claudin-12 were significantly increased (P < 0.05) in the gills of fish fed riboflavin-deficient diet. In addition, this study indicated for the first time that young fish fed a riboflavin-deficient diet exhibited anorexia and poor growth. In conclusion, riboflavin deficiency decreased growth and gill immunity, impaired gill antioxidant system, as well as regulated mRNA expression of gill tight junction proteins and related signaling molecules of fish. Based on percent weight gain, gill lysozyme activity and reduced glutathione content, the dietary riboflavin requirements for young grass carp (275-722 g) were estimated to be 5.85, 7.39 and 6.34 mg/kg diet, respectively.

Exogenous phospholipids supplementation improves growth and modulates immune response and physical barrier referring to NF-κB, TOR, MLCK and Nrf2 signaling factors in the intestine of juvenile grass carp (Ctenopharyngodon idella).

This study was conducted to investigate the effects of dietary phospholipids (PL) on the growth performance, intestinal enzyme activity and immune response and intestinal physical barrier of juvenile grass carp (Ctenopharyngodon idella). A total of 1080 juvenile grass carp with an average initial weight of 9.34 ± 0.03 g were fed six semi-purified diets containing 0.40% (unsupplemented control group), 1.43%, 2.38%, 3.29%, 4.37% and 5.42% PL for 2 months. Results indicated that 3.29% PL increased lysozyme (LZ) and acid phosphatase (ACP) activities and complement component 3 (C3) content (P < 0.05), up-regulated the mRNA relative expression levels of interleukin 10, transforming growth factor β1 (TGF-β1), inhibitor protein κBα (IκBα), target of rapamycin (TOR) and casein kinase 2 (CK2) (P < 0.05), and down-regulated tumor necrosis factor α (TNF-α), interleukin 1β, nuclear factor κB p65 (NF-κB p65), IκB kinase β (IKKβ) and IκB kinase γ (IKKγ) mRNA relative expression levels (P < 0.05) in the intestine, suggesting that optimum PL could improve fish intestinal immunity. In addition, 3.29% PL increased the activities of anti-superoxide anion (ASA), anti-hydroxyl radical, copper/zinc superoxide dismutase (SOD1), glutathione peroxidase (GPx) and glutathione reductase (GR), the content of glutathione (P < 0.05), and the mRNA relative expression levels of occludin, zonula occludens 1 (ZO-1), claudin 3, claudin 12, claudin b, claudin c, SOD1, GPx, GR and NF-E2-related factor 2 (Nrf2) and decreased malondialdehyde (MDA), protein carbonyl (PC) and ROS content (P < 0.05), the mRNA relative expression levels of Kelch-like-ECH-associated protein 1a (Keap1a), myosin light chain kinase (MLCK) and p38 mitogen-activated protein kinase (p38 MAPK) in the intestine, indicating that the optimum PL could improve fish intestinal physical barrier. Finally, based on the PWG, C3 content in the DI, ACP activity in the DI, intestinal PC content and intestinal ASA activity, the optimal dietary PL levels for juvenile grass carp (9.34-87.50 g) were estimated to be 3.46%, 3.79%, 3.93%, 3.72%, and 4.12%, respectively.

Effect of dietary arginine on the immune response and gene expression in head kidney and spleen following infection of Jian carp with Aeromonas hydrophila

This study was conducted to test the hypothesis that elevated dietary arginine enhances immunity of juvenile Jian carp (Cyprinus carpio var. Jian). Fish were fed graded levels of dietary arginine for 9 weeks. Subsequently, a challenge test was conducted by injection of Aeromonas hydrophila. Head kidney and spleen weights, as well as erythrocyte and leukocyte counts were significantly influenced by dietary arginine levels. A similar trend was also observed for hemagglutination titre, serum lysozyme activity, IgM concentration, C3 and C4 content. The highest survival rates following A. hydrophila infection were obtained in fish fed the diets containing arginine at 16.1-21.9 g/kg diet. Phagocytic activity of leukocytes was significantly enhanced by dietary arginine supplementation. In contrast, acid phosphatase activity significantly decreased with dietary arginine levels. Dietary arginine levels did not have a significant effect on the total iron-binding capacity. Gene expression of TNF-α and TGF-β in head kidney significantly increased with dietary arginine levels up to 21.9 g/kg diet, and decreased thereafter. Fish fed the basal diet exhibited the highest IL-10 mRNA expression level. Gene expression of IL-1β and TOR increased with dietary arginine addition, reaching a plateau at 18.5 and 21.9 g arginine/kg diet, respectively. In spleen, higher IL-1β and TNF-α gene expressions were obtained in fish fed the diets containing 24.5 g arginine/kg diet than in fish fed the other dietary treatments. TGF-β mRNA expression levels were significantly lower in fish fed the diets containing ≤21.9 g arginine/kg diet. IL-10 and TOR mRNA expression levels were lower in fish fed 16.1 g arginine/kg diet, while 4E-BP mRNA expression levels increased with dietary arginine levels up to 12.7 g/kg diet and decreased thereafter. Our results indicate that arginine has beneficial effects on regulating mRNA expression of inflammatory cytokines, as well as TOR and 4E-BP and improving humoral and cellular immunity, therefore enhancing disease resistance of fish.

Intestinal immune function, antioxidant status and tight junction proteins mRNA expression in young grass carp (Ctenopharyngodon idella) fed riboflavin deficient diet.

This study investigated the effects of riboflavin on intestinal immunity, tight junctions and antioxidant status of young grass carp (Ctenopharyngodon idella). Fish were fed diets containing graded levels of riboflavin (0.63-10.04 mg/kg diet) for 8 weeks. The study indicated that riboflavin deficiency decreased lysozyme, acid phosphatase, copper/zinc superoxide dismutase, glutathione reductase and glutathione peroxidase activities, and contents of complement component 3 and reduced glutathione in the intestine of fish (P < 0.05). Meanwhile, riboflavin deficiency increased reactive oxygen species, malondialdehyde and protein carbonyl contents and catalase activity (P < 0.05) in the intestine of fish. Furthermore, real-time polymerase chain reaction analysis was used to investigate mRNA expression patterns and found that the mRNA levels of interleukin 10 and transforming growth factor β1, Occludin, zonula occludens 1, Claudin-b and Claudin-c, inhibitor protein κBα, target of rapamycin, ribosomal S6 protein kinase 1 and NF-E2-related factor 2, copper/zinc superoxide dismutase, glutathione peroxidase and glutathione reductase were decreased (P < 0.05) in the intestine of fish fed riboflavin-deficient diet. Conversely, the mRNA levels of tumor necrosis factor α, interleukin 1β, interleukin 8, nuclear factor kappa B p65, Ikappa B kinase β, Ikappa B kinase γ, Kelch-like-ECH-associated protein 1b, p38 mitogen-activated protein kinase, myosin light chain kinase and Claudin-12 were increased (P < 0.05) in the intestine of fish fed riboflavin-deficient diet. In conclusion, riboflavin deficiency decreased immunity and structural integrity of fish intestine. The optimum riboflavin level for intestinal acid phosphatase activity of young grass carp was estimated to be 6.65 mg/kg diet.

Dietary vitamin C deficiency depresses the growth, head kidney and spleen immunity and structural integrity by regulating NF-κB, TOR, Nrf2, apoptosis and MLCK signaling in young grass carp (Ctenopharyngodon idella).

This study investigated the effects of dietary vitamin C on the growth, and head kidney, spleen and skin immunity, structural integrity and related signaling molecules mRNA expression levels of young grass carp (Ctenopharyngodon idella). A total of 540 grass carp (264.37 ± 0.66 g) were fed six diets with graded levels of vitamin C (2.9, 44.2, 89.1, 133.8, 179.4 and 224.5 mg/kg diet) for 10 weeks. Subsequently, a challenge test was conducted by injection of Aeromonas hydrophila and the survival rate recorded for 14 days. The results indicated that compared with optimal vitamin C supplementation, vitamin C deficiency (2.9 mg/kg diet) decreased lysozyme (LA) and acid phosphatase (ACP) activities, and complement 3 and complement 4 (C4) contents (P < 0.05), down-regulated the mRNA levels of antimicrobial peptides [liver expressed antimicrobial peptide (LEAP) 2A, LEAP-2B, hepcidin, β-defensin] and anti-inflammatory cytokines-related factors, interleukin (IL) 4/13A, IL-4/13B (only in head kidney), IL-10, IL-11, transforming growth factor (TGF) β1, TGF-β2, inhibitor of κBα and eIF4E-binding protein 1 (P < 0.05), and up-regulated pro-inflammatory cytokines-related factors, tumor necrosis factor α, interferon γ2, IL-1β, IL-6, IL-8, IL-12 P35 (only in spleen), IL-12 P40, IL-15, IL-17D, nuclear factor κB p65, IκB kinases (IKKα, IKKβ, IKKγ), target of rapamycin and ribosomal protein S6 kinase 1 mRNA levels (P < 0.05) in the head kidney and spleen under injection fish of A. hydrophila, suggesting that vitamin C deficiency could decrease fish head kidney and spleen immunity and cause inflammation. Meanwhile, compared with optimal vitamin C supplementation, vitamin C deficiency decreased the activities and mRNA levels of copper/zinc superoxide dismutase, manganese superoxide dismutase (MnSOD), catalase, glutathione peroxidase, glutathione S-transferases and glutathione reductase (P < 0.05), and down-regulated zonula occludens (ZO) 1, ZO-2, Claudin-b, -c, -3c, -7a, -7b, B-cell lymphoma-2, inhibitor of apoptosis protein, NF-E2-related factor 2 mRNA levels (P < 0.05), increased reactive oxygen species (ROS), malondialdehyde (MDA) and protein carbonyl contents (P < 0.05), and up-regulated Claudin-12, 15a, -15b, Fas ligand, mitogen-activated protein kinase kinase 6, p38 mitogen-activated protein kinase, B-cell lymphoma protein 2 associated X protein, apoptotic protease activating factor-1, caspase-3, -7, -8, -9, Kelch-like ECH-associating protein (Keap) 1a and Keap 1b mRNA levels (P < 0.05) in the head kidney and spleen under injection fish of A. hydrophila, suggesting that vitamin C deficiency could decrease fish head kidney and spleen structural integrity through depression of antioxidative ability, induction of apoptosis and disruption of tight junctional complexes. In addition, except the activities of ACP and MnSOD, and mRNA expression levels of TGF-β1, Occludin and MnSOD, the effect of vitamin C on fish head kidney, spleen and skin immunity and structural integrity other indicators model are similar under infection of A. hydrophila. Finally, the vitamin C requirement for the growth performance (PWG) of young grass carp was estimated to be 92.8 mg/kg diet. Meanwhile, the vitamin C requirement for against skin lesion morbidity of young grass carp was estimated to be 122.9 mg/kg diet. In addition, based on the biochemical indices [immune indices (LA activity in the head kidney and C4 content in the spleen) and antioxidant indices (MDA content in the head kidney and ROS content in the spleen)] the vitamin C requirements for young grass carp were estimated to be 131.2, 137.5, 135.8 and 129.8 mg/kg diet, respectively.

Dietary pantothenic acid deficiency and excess depress the growth, intestinal mucosal immune and physical functions by regulating NF-κB, TOR, Nrf2 and MLCK signaling pathways in grass carp (Ctenopharyngodon idella).

This study investigated the effects of dietary pantothenic acid (PA) on the growth, intestinal mucosal immune and physical barrier, and relative mRNA levels of signaling molecules in the intestine of grass carp (Ctenopharyngodon idella). A total of 540 grass carp (253.44 ± 0.69 g) were fed six diets with graded levels of PA (PA1, PA15, PA30, PA45, PA60 and PA75 diets) for 8 weeks. The results indicated that compared with PA deficiency (PA1 diet) and excess (PA75 diet) groups, optimal PA supplementation increased (P < 0.05): (1) percent weight gain (PWG), feed intake and feed efficiency; (2) lysozyme activity, complement 3 content, liver-expressed antimicrobial peptide 2 and hepcidin, interleukin 10, transforming growth factor β1 and inhibitor of κBα mRNA levels in some intestinal segments; (3) activities and mRNA levels of copper/zinc superoxide dismutase, manganese superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferases and glutathione reductase, and NF-E2-related factor 2 (Nrf2) mRNA level in the whole intestine; (4) Claudin b, Claudin 3, Claudin c, Occludin and ZO-1 mRNA levels in some intestinal segments of grass carp. Conversely, optimal PA supplementation decreased (P < 0.05): (1) tumor necrosis factor α, interleukin 1β, interferon γ2, interleukin 8, nuclear factor κB P65 (NF-κB P65), IκB kinase α, IκB kinase β, IκB kinase γ and target of rapamycin (TOR) mRNA expression levels in some intestinal segments; (2) reactive oxygen species, malondialdehyde and protein carbonyl contents, and Kelch-like ECH-associating protein 1a, Kelch-like ECH-associating protein 1b in the intestine; (3) Claudin 12, Claudin 15a and myosin light-chain kinase (MLCK) mRNA levels in some intestinal segments of grass carp. In conclusion, optimum PA promoted growth, intestinal mucosal immune and physical function, as well as regulated mRNA levels of signaling molecules NF-κB P65, TOR, Nrf2 and MLCK in grass carp intestine. Based on the quadratic regression analysis of PWG and intestinal lysozyme activity, the optimal PA levels in grass carp (253.44-745.25 g) were estimated to be 37.73 mg/kg and 41.38 mg/kg diet, respectively.