Immunolocalization of Jian Carp (Cyprinus Carpio Var. Jian) Cathepsin B: Cloning, Expression, Characterization, and Antibody Preparation.

Cathepsin B (CatB) cDNA of 759 bp from Jian carp (Cyprinus carpio var. Jian) with amino acid similarity of 99.6% to common carp was cloned. The mature CatB was expressed in Escherichia coli BL21 transferred with vector CatB-pET-30a. It was purified and identified as a single band (29 kDa) on SDS-PAGE. Optimum CatB activity was observed at 40 °C and pH 5.5. Mouse anti-CatB polyclonal antibody with a high titer of 1:256000 was prepared successfully and shown to specifically recognize the antigen both in prokaryotic cells and in the tissues of Jian carp according to western blotting and immunohistochemistry results. Immunolocation analysis showed that CatB distribution at protein level varied among the tested tissues. The results presented in this study may provide a significant reference for future research on the inherent relationship between CatB and the quality of fish or fish products at both the gene and protein levels.

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.

Flesh quality loss in response to dietary isoleucine deficiency and excess in fish: a link to impaired Nrf2-dependent antioxidant defense in muscle.

The present study explored the impact of dietary isoleucine (Ile) on fish growth and flesh quality and revealed a possible role of muscle antioxidant defense in flesh quality in relation to dietary Ile. Grass carp (weighing 256.8±3.5 g) were fed diets containing six graded levels of Ile (3.8, 6.6, 9.3, 12.5, 15.2 and 18.5 g/kg) for eight weeks. The results indicated that compared with Ile deficiency (3.8 g/kg diets) and excess (18.5 g/kg diets) groups, 9.3-15.2 g Ile/kg diet supplementations promoted fish growth and muscle fat deposition, whereas 6.6-15.2 g Ile/kg diets supplementation enhanced muscle nutrients (protein and total EAAs) deposition. Furthermore, muscle shear force, pH value, and hydroxyproline concentration were improved by 9.3-12.5, 9.3 and 9.3 g Ile/kg diet supplementations, respectively. However, muscle cooking loss, lactate content, and activities of cathepsin B and L were decreased by 6.6-15.2, 9.3-12.5, 9.3-12.5 and 9.3-15.2 g Ile/kg diet supplementations, respectively. Additionally, 6.6-15.2 and 6.6-12.5 g Ile/kg diet supplementations attenuated malondialdehyde and protein carbonyl contents, respectively. The activities of copper/zinc superoxide dismutase (Cu/Zn-SOD) and glutathione peroxidase (GPx), and glutathione content were enhanced by 6.6-9.3, 6.6-12.5 and 6.6-15.2 g Ile/kg diet supplementations, respectively. Moreover, the relative mRNA expressions of antioxidant enzymes, including Cu/Zn-SOD (6.6-12.5 g/kg diets) and GPx (12.5 g/kg diets), as well as antioxidant-related signaling molecules, including NF-E2-related factor 2 (Nrf2) (6.6-12.5 g/kg diets), target of rapamycin (6.6-12.5 g/kg diets), ribosomal S6 protein kinase 1 (9.3-12.5 g/kg diets) and casein kinase 2 (6.6-12.5 g/kg diets), were up-regulated when Ile diet supplementations were administered at these levels, respectively, whereas the relative mRNA expression of Kelch-like ECH-associated protein 1 was down-regulated with 9.3 g Ile/kg diet supplementations. Collectively, the present study indicated that optimum isoleucine improved flesh quality, partly due to the activation of antioxidant defense through the Nrf2 signaling pathway.

Effect of choline on antioxidant defenses and gene expressions of Nrf2 signaling molecule in the spleen and head kidney of juvenile Jian carp (Cyprinus carpio var. Jian).

The present work evaluates the effects of various levels of dietary choline on antioxidant defenses and gene expressions of Nrf2 signaling molecule in spleen and head kidney of juvenile Jian carp (Cyprinus carpio var. Jian). Fish were fed with six different experimental diets containing graded levels of choline at 165 (choline-deficient control), 310, 607, 896, 1167 and 1820 mg kg(-1) diet for 65 days. At the end of the feeding trail, fish were challenged with Aeromonas hydrophila and mortalities were recorded over 17 days. Dietary choline significantly decreased malondialdehyde and protein carbonyl contents in spleen and head kidney. However, anti-superoxide anion and anti-hydroxyl radical activities in spleen and head kidney also decreased. Interestingly, activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and glutathione reductase (GR) in spleen, GPx activity in head kidney, and glutathione contents in spleen and head kidney were decreased with increase of dietary choline levels up to a certain point, whereas, activities of SOD, GST and GR in head kidney showed no significantly differences among groups. Similarly, expression levels of CuZnSOD, MnSOD, CAT, GPx1a, GPx1b and GR gene in spleen and head kidney were significantly lower in group with choline level of 607 mg kg(-1) diet than those in the choline-deficient group. The relative gene expressions of Nrf2 in head kidney and Keap1a in spleen and head kidney were decreased with increasing of dietary choline up to a certain point. However, the relative gene expression of Nrf2 in spleen were not significantly affected by dietary choline. In conclusion, dietary choline decreased the oxidant damage and regulated the antioxidant system in immune organs of juvenile Jian carp.

[Effect of pungent dispersion bitter purgation method on the esophageal mucosal intercellular space of reflux esophagitis model rats].

OBJECTIVE: To observe the effect of pungent dispersion bitter purgation method (PDBPM) on the esophageal mucosal intercellular space of reflux esophagitis (RE) model rats. METHODS: Totally 100 Wistar rats were randomly divided into the control group, the model group, the Western medicine group (WM), the Chinese medicine group (CM), 25 rats in each group. Rats in the control group only received switch operation. Rats in the rest three groups received modified partial cardia muscle incision combined pylorus ligation of external parts to prepare the RE rat model. Starting from the 3rd day after operation, WM mixture (Motilium 3. 2 mg/kg + Omeprazole Capsule 4.3 mg/kg + Hydrotalcite Tablet 161.4 mg/kg) was administered by gastrogavage to rats in the WM group. Rats in the CM group was administered by gastrogavage with Modified Banxia Xiexin Decoction (5.7 g/kg), 2.5 mL each time, twice daily for 14 consecutive days. Equal volume of normal saline was administered by gastrogavage to rats in the control group and the model group. On day 7 and 14, the lower esophagus pH value, general specimen of mucosa and histopathologic changes were observed. Intercellular spaces of esophageal epithelium were measured for a control study. RESULTS: Compared with the same group at day 7, the lower esophagus pH value increased at day 14 (P < 0.01); the naked eye integral of esophageal mucosa and intercellular spaces of esophageal epithelium also decreased at day 14 in the CM group and the WM group (P < 0.05). Compared with the control group at the same time point, the lower esophagus pH value decreased in the model group (P < 0.01). The naked eye integral of esophageal mucosa, and intercellular spaces of esophageal epithelium increased in the model group with increased intercellular spaces (P < 0.01). Compared with the model group at the same time point, the lower esophagus pH value increased and the naked eye integral of esophageal mucosa decreased in the CM group and the WM group at day 7 and 14 (P < 0.01). Intercellular spaces of esophageal epithelium of RE model rats at day 14 was lower in the CM group and the WM group than in the model group (P < 0.01). Compared with the WM group, the lower esophagus pH value decreased at day 7 in the CM group (P < 0.05); the naked eye integral of esophageal mucosa and intercellular spaces of esophageal epithelium decreased at day 14 in the CM group (P < 0.05). CONCLUSIONS: PDBPM had favorable treatment effect on RE model rats. The therapeutic effect was more obvious along with the therapeutic course went by. Its mechanism might be achieved through good repair effect on damaged mucosa, increasing the pressure of esophageal sphincter, and inhibiting gastric acid.

Effect of dietary lysine on growth, intestinal enzymes activities and antioxidant status of sub-adult grass carp (Ctenopharyngodon idella).

The dietary lysine requirement of sub-adult grass carp (460 ± 1.5 g) was assessed by feeding diets supplemented with grade levels of lysine (6.6, 8.5, 10.8, 12.9, 15.0 and 16.7 g kg(-1) diet) for 56 days. The test diets (28% CP) contained fish meal, casein and gelatin as sources of intact protein, supplemented with crystalline amino acids. Weight gain (WG), feed intake and feed efficiency were significantly improved with increasing levels of lysine up to 12.9 g kg(-1) diet and thereafter declined (P < 0.05). Quadratic regression analysis of WG at 95% maximum response indicated lysine requirement was 10.9 g kg(-1) diet. Activities of trypsin, chymotrypsin, lipase, Na(+), K(+)-ATPase and alkaline phosphatase in intestine, creatine kinase activity in proximal and mid-intestine responded similar to WG (P < 0.05). In addition, lipid and protein oxidation decreased with increasing levels of lysine up to certain values and increased thereafter (P < 0.05); the anti-hydroxyl radical capacity, dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione-S-transferase (GST) activities and glutathione content were increased with increasing dietary lysine levels up to certain values in the detected tissues, except for hepatopancreatic GST. Requirement estimated on the basis of malondialdehyde content in intestine and hepatopancreas was 10.6 and 9.53 g lysine kg(-1) diet, respectively.

Effect of tea polyphenol on oxidative injury in S180 cells induced hepatocarcinoma mice.

The purpose of this study was to evaluate the antioxidant nature of tea polyphenol on S180 cells induced liver cancer in mice. In the present study, hepatocellular carcinoma was induced by tumor transplantation of liver in situ. The antitumor activity of tea polyphenol has been determined in vivo in hepatocellular carcinoma mice after treatment of drug (50, 100, 150 mg/kg body weight) by gavage for 20 days. Results showed that a significant increase in serum aspartate transaminase (AST), alkaline phosphatase (ALP), alanine aminotransfere (ALT), malondialdehyde (MDA) level, decrease in serum white blood cells (WBC), serum total protein (TP), albumin (ALB), A/G, tumor necrosis factor-α (TNF-α) and interferon-gamma (IFN-γ), liver reduced glutathione (GSH) levels were observed. In addition, the levels of enzymic and non-enzymic antioxidants were decreased when subjected to S180 cells induction. These altered enzyme levels were ameliorated significantly by administration of tea polyphenol at the concentration of 50, 100, 150 mg/kg body weight in drug-treated animals. These results indicate that the protective effect of tea polyphenol was associated with inhibition of MDA induced by S180 cells and to maintain the antioxidant enzyme levels.

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.

Novel cardioprotective effects of tetrahydrobiopterin after anoxia and reoxygenation: Identifying cellular targets for pharmacologic manipulation.

OBJECTIVES: Contemporary cardioprotective strategies to prevent perioperative ischemia-reperfusion injury have focused on the l-arginine nitric oxide pathway. Tetrahydrobiopterin is an absolute cofactor required for the enzyme nitric oxide synthase and is thus a critical determinant of nitric oxide production. We hypothesized that ischemia-reperfusion results in diminished levels of tetrahydrobiopterin, which might represent a key cellular defect underlying endothelial and myocyte dysfunction after ischemia-reperfusion. To this aim, we examined the effects of tetrahydrobiopterin supplementation in (1) an in vivo experimental model of global ischemia-reperfusion and (2) an in vitro human ventricular heart cell model of simulated ischemia-reperfusion. Measures of endothelial function, oxidant production, cell survival, and cardiac function were used to assess outcome. METHODS: In study 1 Wistar rats were divided into one of 2 groups (n = 10 per group). One group received tetrahydrobiopterin (25 mg x kg(-1) x d(-1) for 7 days), and the other group served as the control group. Hearts were subjected to 30 minutes of ischemia followed by 30 minutes of reperfusion, and left ventricular developed pressure, left ventricular systolic pressure, and left ventricular end-diastolic pressure were determined by using the modified Langendorff technique. In study 2 we quantitated myocardial malondialdehyde, a marker of lipid peroxidation, in ventricular tissues from both groups of animals using butanol phase extraction and spectrophotometric analysis. In study 3 coronary vascular responses were determined in vascular segments of the left coronary artery in both groups of animals after ischemia-reperfusion. Endothelium-dependent and endothelium-independent vasodilatation to acetylcholine and sodium nitroprusside, respectively, were compared between groups. In study 4, using a human ventricular heart cell model of simulated ischemia-reperfusion, we studied the effects of tetrahydrobiopterin (20 micromol/L) on cellular injury (as assessed by means of trypan blue uptake). RESULTS: After ischemia-reperfusion, myocardial dysfunction was evidenced by a decrease in left ventricular developed pressure and an increase in left ventricular end-diastolic pressure (P =.01 compared with baseline). Hearts from tetrahydrobiopterin-treated rats exhibited protection against ischemia-reperfusion injury (left ventricular developed pressure: 74 +/- 4 vs control 42 +/- 8 mm Hg, P =.01; left ventricular end-diastolic pressure: 12 +/- 3 vs 34 +/- 7 mm Hg, P =.01). Furthermore, tetrahydrobiopterin treatment attenuated the rise in malondialdehyde levels after ischemia-reperfusion (P =.01). After reperfusion, coronary endothelial function to acetylcholine was attenuated (P =.003 vs sham-treated mice), whereas responses to sodium nitroprusside remained unchanged. Tetrahydrobiopterin-treated rats exhibited an improvement in acetylcholine-mediated vasorelaxation (P =.01 vs ischemia-reperfusion group). Cellular injury, as assessed by means of trypan blue uptake, was higher in human ventricular heart cells subjected to simulated ischemia-reperfusion; this effect was prevented with tetrahydrobiopterin treatment (P =.001). CONCLUSIONS: Supplemental tetrahydrobiopterin provides a novel cardioprotective effect on left ventricular function, endothelial-vascular reactivity, oxidative damage, and cardiomyocyte injury after ischemia-reperfusion injury and might represent an important cellular target for future operative myocardial protection strategies.