Shu-Hong Li

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.

Copper exposure induces oxidative injury, disturbs the antioxidant system and changes the Nrf2/ARE (CuZnSOD) signaling in the fish brain: Protective effects of myo-inositol

The brain is the center of the nervous system in all vertebrates, and homeostasis of the brain is crucial for fish survival. Copper (Cu) is essential for normal cellular processes in most eukaryotic organisms but is toxic in excess. Although Cu is indicated as a potent neurotoxicant, information regarding its threat to fish brain and underlying mechanisms is still scarce. In accordance, the objective of this study was to assess the effects and the potential mechanism of Cu toxicity by evaluating brain oxidative status, the enzymatic and mRNA levels of antioxidant genes, as well as the Nrf2/ARE signaling in the brain of fish after Cu exposure. The protective effects of myo-inositol (MI) against subsequent Cu exposure were also investigated. The results indicate that induction of oxidative stress by Cu is shown by increases in brain ROS production, lipid peroxidation and protein oxidation, which are accompanied by depletions of antioxidants, including total superoxide dismutase (T-SOD), CuZnSOD, glutathione-S-transferase (GST) and glutathione reductase (GR) activities and glutathione (GSH) content. Cu exposure increased the catalase (CAT) and glutathione peroxidase (GPx) activities. Further molecular results showed that Cu exposure up-regulated CuZnSOD, GPx1a and GR mRNA levels, suggesting an adaptive mechanism against stress. Moreover, Cu exposure increased fish brain Nrf2 nuclear accumulation and increased its ability of binding to ARE (CuZnSOD), which supported the increased CuZnSOD mRNA levels. In addition, Cu exposure caused increases of the expression of the Nrf2, Maf G1 (rather than Maf G2 gene) and PKCd genes, suggesting that de novo synthesis of those factors is required for the protracted induction of such antioxidant genes. However, the modulation of Keap1a (rather than Keap1b) of fish brain under Cu exposure might be used to turn off of the signaling cascade and avoid harmful effects. Interestingly, pre-treatment of fish with MI prevented the fish brain from Cu-induced oxidative damages mainly by increasing the GSH content and CuZnSOD and GST activities. Summarily, this study indicates that although Cu stimulates adaptive increases in the expression of some antioxidant enzyme genes through Nrf2/ARE signaling, it also induces oxidation and the depletion of most of antioxidant enzyme activities and GSH content due to the increase of ROS production, and MI protects the fish brain against Cu toxicity.

Effect of dietary arginine on growth, intestinal enzyme activities and gene expression in muscle, hepatopancreas and intestine of juvenile Jian carp (Cyprinus carpio var. Jian).

The present study was conducted to test the hypothesis that dietary arginine promotes digestion and absorption capacity, and, thus, enhances fish growth. This improvement might be related to the target of rapamycin (TOR) and eIF4E-binding protein (4E-BP). A total of 1200 juvenile Jian carp, Cyprinus carpio var. Jian, with an average initial weight of 6.33 (SE 0.03) g, were fed with diets containing graded concentrations of arginine, namely, 9.8 (control), 12.7, 16.1, 18.5, 21.9 and 24.5 g arginine/kg diet for 9 weeks. An real-time quantitative PCR analysis was performed to determine the relative expression of TOR and 4E-BP in fish muscle, hepatopancreas and intestine. Dietary arginine increased (P < 0.05): (1) glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase activities in muscle and hepatopancreas; (2) intestine and hepatopancreas protein content, folds height, and trypsin, chymotrypsin, lipase, Na⁺/K⁺-ATPase, alkaline phosphatase, γ-glutamyl transpeptidase and creatine kinase activities in intestine; (3) Lactobacillus counts; (4) relative expression of TOR in the muscle, hepatopancreas and distal intestine (DI); (5) relative expression of 4E-BP in proximal intestine (PI) and mid-intestine (MI), as compared with the control group. In contrast, dietary arginine reduced (P < 0.05): (1) plasma ammonia content; (2) Aeromonas hydrophila and Escherichia coli counts; (3) relative expression of TOR in PI and MI; (4) relative expression of 4E-BP in the muscle, hepatopancreas and DI. The arginine requirement estimated by specific growth rate using quadratic regression analysis was found to be 18.0 g/kg diet. These results indicate that arginine improved fish growth, digestive and absorptive ability and regulated the expression of TOR and 4E-BP genes.

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 graded levels of dietary methionine hydroxy analogue on immune response and antioxidant status of immune organs in juvenile Jian carp (Cyprinus carpio var. Jian).

Immune response and antioxidant status of immune organs in juvenile Jian carp (Cyprinus carpio var. Jian) fed graded levels of methionine hydroxy analogue (MHA) (0, 5.1, 7.6, 10.2, 12.7, 15.3 g kg(-1) diet) for 60 days were investigated. Results indicated that head kidney index, spleen index, red and white blood cell counts significantly increased with increasing MHA levels up to a point (P < 0.05), whereupon decreased (P < 0.05). Glutathione reductase activity in head kidney and spleen, anti-hydroxy radical and glutathione-S-transferase activities in spleen, catalase activity and GSH content in head kidney significantly increased by MHA supplement, while malondialdehyde content, anti-superoxide anion, superoxide dismutase, glutathione peroxidase activities in head kidney and spleen, protein carbonyl content and catalase activity in spleen, anti-hydroxy radical activity in head kidney significantly decreased by MHA supplement. However, protein carbonyl content and glutathione-S-transferase activity in head kidney, GSH content in spleen remained unaffected. After 60-day feeding trial, a challenge study was conducted by injection of Aeromonas hydrophila for 17 days. Results showed that survival rate, leukocytes phagocytic activity, lysozyme activity, acid phosphatase activity, total iron-binding capacity, haemagglutination titre, complement 3, 4 and immunoglobulin M contents significantly increased by optimal dietary MHA supplement (P < 0.05). These data suggested that MHA affected antioxidant status of immune organs and promoted immune response in juvenile Jian carp.

Myo-inositol prevents copper-induced oxidative damage and changes in antioxidant capacity in various organs and the enterocytes of juvenile jian carp (cyprinus carpio var. jian)

Although oxidative stress has been demonstrated to be involved in copper (Cu)-induced toxicity, information regarding the effect of antioxidants on Cu toxicity is still scarce. This study assessed the possible protective effects of myo-inositol (MI) against subsequent Cu exposure in juvenile Jian carp (Cyprinus carpio var. Jian) in vivo and in their enterocytes in vitro. First, oxidative stress was established by exposing fish to different concentrations of Cu (0-7.2 mg Cu/L water) for 4 days. Next, the protective effects of MI (administered as a dietary supplement for 60 days) against subsequent Cu exposure (0.6 mg Cu/L water for 4 days) were studied in fish. The third trial determined the effects of Cu exposure (0-6.0 mg Cu/L of medium for 24h) on enterocytes in vitro. Finally, enterocytes were pre-incubated with graded levels of MI (0-75 mg MI/L of medium) for 72 h and exposed to 6.0 mg Cu/L of medium for 24h. The results indicated that ≥ 0.6 mg Cu/L water could induce oxidative stress in fish (P<0.05). Cu exposure significantly induced increases in lipid peroxidation and protein oxidation in the gill, hepatopancreas and intestine in fish. However, these oxidative effects were prevented by MI pre-supplementation. MI also prevented the toxic effects of Cu on anti-superoxide anion (ASA), anti-hydroxyl radical (AHR), superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione peroxidase (GPx) and glutathione reductase (GR) activities and glutathione (GSH) content in these organs. In vitro, enterocytes exposed to Cu displayed a dose-dependent injury. Moreover, cell viability, protein retention (PR), alkaline phosphatase, total-SOD (T-SOD) and Cu/ZnSOD activities were all depressed by Cu (P<0.05). Interestingly, the final experiment showed that MI pre-supplementation could block the toxic effects of Cu on the antioxidant system, and thus protect enterocytes from Cu-induced oxidative damage. All of these results indicated that the induction of key antioxidant defenses by MI pre-supplementation, including SOD, CAT, GPx, GST and GSH, may play an important role in the protection of fish against oxidative stress.

Effects of dietary isoleucine on the immune response, antioxidant status and gene expression in the head kidney of juvenile Jian carp (Cyprinus carpio var. Jian)

This study was conducted to evaluate the effects of dietary isoleucine (Ile) on the immune response, antioxidant status and gene expression in the head kidney of juvenile Jian carp (Cyprinus carpio var. Jian). Six semi-purified isonitrogenous diets (4.2, 7.0, 9.5, 11.9, 13.9 and 16.9 g Ile kg(-1) diet) were fed to Jian carp (6.9 ± 0.03 g) for 60 days. The results showed that Ile supplementation improved the head kidney index, red and white blood cell counts, anti-hydroxyl radical capacity and the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione-S-transferase (P < 0.05), and decreased the malondialdehyde, protein carbonyl and glutathione contents in the head kidney (P < 0.05). After a 60 day feeding trial, an Aeromonas hydrophila challenge study was conducted for 17 days. Differences in survival rate, leucocyte phagocytic activity, serum lysozyme activity, acid phosphatase activity, haemagglutination titre, complement components 3 and 4, immunoglobulin M level and A. hydrophila agglutination antibody titre followed the same trend as that of the head kidney index (P < 0.05). Furthermore, real time polymerase chain reaction revealed that relative mRNA expression of transforming growth factor β2 and target of rapamycin (TOR) in the head kidney significantly increased with increasing Ile levels (P < 0.05). Conversely, the relative mRNA expression of tumour necrosis factor α, interleukin 10 and eIF4E-binding protein (4E-BP) in the head kidney showed a downward trend (P < 0.05). Collectively, this study indicates that dietary Ile improves the fish immune response, regulates the antioxidant status and cytokine, TOR and 4E-BP gene expression in the head kidney.

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.

Oxidative stress parameters and anti-apoptotic response to hydroxyl radicals in fish erythrocytes: Protective effects of glutamine, alanine, citrulline and proline

The present study explored the protective effects of glutamine (Gln), alanine (Ala), citrulline (Cit) and proline (Pro) on hydroxyl radical (·OH)-induced apoptosis in isolated carp erythrocytes. Hydroxyl radicals were generated by ferrous ion (Fe(2+))-mediated decomposition of hydrogen peroxide (H(2)O(2)) (Fenton reaction). In order to select an optimal ·OH concentration to induce apoptosis, cultures were treated with different concentrations of FeSO(4)/H(2)O(2) (0 μM/0 μM-50 μM/25 μM). The results showed that exposure to FeSO(4)/H(2)O(2) (0 μM/0 μM-40 μM/20 μM) increased apoptosis in a dose-dependent manner. Moreover, apoptosis was at its highest level at 40 μM FeSO(4)/20 μM H(2)O(2). We then examined the cytoprotective effects of Gln, Ala, Cit, Pro or the combination of Ala, Cit and Pro under conditions of apoptosis. Carp erythrocytes were treated with the substances listed above in the presence of 40 μM FeSO(4)/20 μM H(2)O(2) for 9 h. The controls were grown in Gln, Ala, Cit, Pro-free culture medium. The results showed that Gln, Ala, Cit, Pro and the combination of Ala, Cit and Pro effectively protected against annexin binding, decrease of forward scatter and DNA fragmentation in carp erythrocytes induced by ·OH. Furthermore, Gln, Ala, Cit, Pro and the combination of Ala, Cit and Pro effectively blocked ·OH-stimulated erythrocyte hemolysis, reduced the increase of superoxide anion and H(2)O(2) concentrations, inhibited the formation of malondialdehyde, protein carbonyls and met-hemoglobin, and prevented the decrease of superoxide dismutase, catalase and glutathione peroxidase activities and glutathione content in carp erythrocytes induced by ·OH. In addition, the results suggest that the combination of Ala, Cit and Pro produces a greater anti-apoptotic and anti-oxidative effect than their individual effects at the same concentrations. Taken together, the results showed that ·OH induces apoptosis and oxidative damage in carp erythrocytes. In addition to inhibiting apoptosis, Gln, Ala, Cit, Pro and the combination of Ala, Cit and Pro protected carp erythrocytes against oxidative damage induced by ·OH, which may be a major factor in the protection of erythrocytes from apoptosis.

Threonine Affects Intestinal Function, Protein Synthesis and Gene Expression of TOR in Jian Carp (Cyprinus carpio var. Jian)

This study aimed to investigate the effects of threonine (Thr) on the digestive and absorptive ability, proliferation and differentiation of enterocytes, and gene expression of juvenile Jian carp (Cyprinus carpio var. Jian). First, seven isonitrogenous diets containing graded levels of Thr (7.4–25.2 g/kg diet) were fed to the fishes for 60 days. Second, enterocyte proliferation and differentiation were assayed by culturing enterocytes with graded levels of Thr (0–275 mg/l) in vitro. Finally, enterocytes were cultured with 0 and 205 mg/l Thr to determine protein synthesis. The percent weight gain (PWG), specific growth rate, feed intake, feed efficiency, protein retention value, activities of trypsin, lipase and amylase, weights and protein contents of hepatopancreas and intestine, folds heights, activities of alkaline phosphatase (AKP), γ- glutamyl transpeptidase and Na+/K+-ATPase in all intestinal segments, glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) activities in hepatopancreas, and 4E-BP2 gene expression in muscle, hepatopancreas and intestinal segments were significantly enhanced by Thr (p<0.05). However, the plasma ammonia concentration and TOR gene expression decreased (p<0.05). In vitro, Thr supplement significantly increased cell numbers, protein content, the activities of GOT, GPT, AKP and Na+/K+-ATPase, and protein synthesis rate of enterocytes, and decreased LDH activity and ammonia content in cell medium (p<0.05). In conclusion, Thr improved growth, digestive and absorptive capacity, enterocyte proliferation and differentiation, and protein synthesis and regulated TOR and 4E-BP2 gene expression in juvenile Jian carp. The dietary Thr requirement of juvenile Jian carp was 16.25 g/kg diet (51.3 g/kg protein) based on quadratic regression analysis of PWG.