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.     

Antioxidant enzymes in the liver of Chelidonichthys obscurus from the Montenegrin coastline

The activities of antioxidant defence enzymes — total, manganese and copper zinc containing superoxide dismutase (Tot SOD, Mn SOD, CuZn SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR) and biotransformation phase II enzyme glutathione-S-transferase (GST) — in the liver of longfin gurnard (Chelidonichthys obscurus) from the Montenegrin coastline (Adriatic sea) were investigated. The specimens were collected in winter (February) and late spring (May) at two localities: Platamuni (PL, potentially unpolluted) and the Estuary of the River Bojana (EB, potentially polluted). The obtained results show that the activities of Mn SOD, CAT, GSH-Px and GST in winter were significantly lower at EB than at PL. In spring, the activities of CAT and GST were decreased, while GR activity was increased at EB in comparison to PL. The activities of Mn SOD and GST at PL were decreased and GSH-Px, GR and GST activities at EB were increased in spring compared to winter. Our work represents the first study of liver antioxidant enzymes of longfin gurnard from the Montenegrin coastline and reveals that locality, as a variable, has a greater influence on antioxidant enzymes and biotransformation phase II enzyme GST activities compared to season.     

Antioxidant Capacity of Melatonin on Preimplantation Development of Fresh and Vitrified Rabbit Embryos: Morphological and Molecular Aspects

Embryo cryopreservation remains an important technique to enhance the reconstitution and distribution of animal populations with high genetic merit. One of the major detrimental factors to this technique is the damage caused by oxidative stress. Melatonin is widely known as an antioxidant with multi-faceted ways to counteract the oxidative stress. In this paper, we investigated the role of melatonin in protecting rabbit embryos during preimplantation development from the potential harmful effects of oxidative stress induced by in vitro culture or vitrification. Rabbit embryos at morula stages were cultured for 2 hr with 0 or 10⁻³ M melatonin (C or M groups). Embryos of each group were either transferred to fresh culture media (CF and MF groups) or vitrified/devitrified (CV and MV groups), then cultured in vitro for 48 hr until the blastocyst stage. The culture media were used to measure the activity of antioxidant enzymes: glutathione-s-transferase (GST) and superoxide dismutase (SOD), as well as the levels of two oxidative substrates: lipid peroxidation (LPO) and nitric oxide (NO). The blastocysts from each group were used to measure the expression of developmental-related genes (GJA1, POU5F1 and Nanog) and oxidative-stress-response-related genes (NFE2L2, SOD1 and GPX1). The data showed that melatonin promoted significantly (P<0.05) the blastocyst rate by 17% and 12% in MF and MV groups compared to their controls (CF and CV groups). The GST and SOD activity significantly increased by the treatment of melatonin in fresh or vitrified embryos, while the levels of LPO and NO decreased (P<0.05). Additionally, melatonin considerably stimulated the relative expression of GJA1, NFE2L2 and SOD1 genes in MF and MV embryos compared to CF group. Furthermore, melatonin significantly ameliorated the reduction of POU5F1 and GPX1 expression induced by vitrification. The results obtained from the current investigation provide new and clear molecular aspects regarding the mechanisms by which melatonin promotes development of both fresh and vitrified rabbit embryos.     

Depletion of Tip60/Kat5 affects the hepatic antioxidant system in mice

Tat-interactive protein 60 kDa (TIP60, also known as lysine acetyltransferase 5 [KAT5]) is a member of the MYST protein family with histone acetyltransferase activity. Recent studies have reported that TIP60 has multiple functions in many signal transduction mechanisms, especially p53-mediated apoptosis. Although the activation of apoptosis signaling pathways requires the presence of cellular reactive oxygen species (ROS) at a certain level, an imbalance between the production and consumption of ROS in cells results in oxidative stress (OS). In this study, we investigated for the first time how the absence of the Tip60 gene in the liver affects gene expression, enzyme activity, and protein expression of the hepatic antioxidant members localized in the cytoplasm, including superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), and glutathione S-transferase (GST). First, we successfully generated liver-specific Tip60 knockout mice (mutants) using Cre/LoxP recombination. The reduced glutathione level and nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) expression, a marker of OS, increased significantly in the Tip60 mutant liver. Gene expression, activity, and protein expression of the enzymatic antioxidant system, including SOD, CAT, GR, GPx, and GST were investigated in mutants and control groups. Despite a significant correlation between the gene, enzyme activity, and protein content for CAT and GR, this was not true for SOD and GPx. The overall results suggest that TIP60 acts on the hepatic antioxidant system both at the gene and protein levels, but the actual effect of the deletion of Tip60 is observed at the protein level, especially for SOD and GPx.     

Cardioprotective Effect of Paeonol and Danshensu Combination on Isoproterenol-Induced Myocardial Injury in Rats

Background

Traditional Chinese medicinal herbs Cortex Moutan and Radix Salviae Milthiorrhizaeare are prescribed together for their putative cardioprotective effects in clinical practice. However, the rationale of the combined use remains unclear. The present study was designed to investigate the cardioprotective effects of paeonol and danshensu (representative active ingredient of Cortex Moutan and Radix Salviae Milthiorrhizae, respectively) on isoproterenol-induced myocardial infarction in rats and its underlying mechanisms.

Methodology

Paeonol (80 mg kg⁻¹) and danshensu (160 mg kg⁻¹) were administered orally to Sprague Dawley rats in individual or in combination for 21 days. At the end of this period, rats were administered isoproterenol (85 mg kg⁻¹) subcutaneously to induce myocardial injury. After induction, rats were anaesthetized with pentobarbital sodium (35 mg kg⁻¹) to record electrocardiogram, then sacrificed and biochemical assays of the heart tissues were performed.

Principal Findings

Induction of rats with isoproterenol resulted in a marked (P<0.001) elevation in ST-segment, infarct size, level of serum marker enzymes (CK-MB, LDH, AST and ALT), cTnI, TBARS, protein expression of Bax and Caspase-3 and a significant decrease in the activities of endogenous antioxidants (SOD, CAT, GPx, GR, and GST) and protein expression of Bcl-2. Pretreatment with paeonol and danshensu combination showed a significant (P<0.001) decrease in ST-segment elevation, infarct size, cTnI, TBARS, protein expression of Bax and Caspase-3 and a significant increase in the activities of endogenous antioxidants and protein expression of Bcl-2 and Nrf2 when compared with individual treated groups.

Conclusions/Significance

This study demonstrates the cardioprotective effect of paeonol and danshensu combination on isoproterenol-induced myocardial infarction in rats. The mechanism might be associated with the enhancement of antioxidant defense system through activating of Nrf2 signaling and anti-apoptosis through regulating Bax, Bcl-2 and Caspase-3. It could provide experimental evidence to support the rationality of combinatorial use of traditional Chinese medicine in clinical practice.     

Glycogen Synthase Kinase 3β Influences Injury Following Cerebral Ischemia/Reperfusion in Rats

Abnormal activation of GSK-3β is associated with psychiatric and neurodegenerative disorders. However, no study has examined the effect of GSK-3β on cerebral ischemia/reperfusion injury. We used oxygen-glucose deprivation/reoxygenation (OGD/R) and middle cerebral artery occlusion (MCAO) as models of ischemia/reperfusion in rats in vitro and in vivo. Our study showed that knockdown of GSK-3β with a GSK-3β siRNA virus improved injury and increased viability of neurons subjected to OGD/R. Levels of total Nrf2, nuclear Nrf2, and Nrf2 downstream proteins sulfiredoxin (Srx1) and thioredoxin (Trx1) increased after transfection with the GSK-3β siRNA virus. GSK-3β siRNA increased SOD activity and decreased MDA levels. Overexpression of GSK-3β with a pcDNA-GSK-3β virus showed opposite results. We also demonstrated that intracerebroventricular injection of GSK-3β siRNA in rats ameliorated neurological deficits, reduced brain infarct volume and water content, and reduced damage to cerebral cortical neurons after MCAO. Changes in total Nrf2, nuclear Nrf2, Srx1, Trx1, SOD, and MDA were similar to those observed in vitro. Our results show for the first time that GSK-3β can influence cerebral ischemia/reperfusion injury. The effects may be due to regulating the Nrf2/ARE pathway and decreasing oxidative stress. These results suggest a potential new drug target for clinical treatment of stroke.     

Oxidative Stress in the Heart of Rats Infected with Trypanosoma evansi

This study was conducted to investigate the occurrence of oxidative stress in the heart tissue of rats infected with Trypanosoma evansi. Rats were divided into 2 groups (A and B) with 12 animals each, and further subdivided into 4 subgroups (A1 and A2, 6 animals/each; and B1 and B2, 6 animals/each). Animals in the groups B1 and B2 were subcutaneously inoculated with T. evansi. Thiobarbituric acid reactive substances (TBARS), superoxide dismutase activity (SOD), glutathione S-transferase activity (GST), reduced glutathione activity (GSH), and non-protein thiols (NPSH) in the heart tissue were evaluated. At day 5 and 15 post-infection (PI), an increase in the TBARS levels and a decrease in the SOD activity (P<0.05) were observed. GSH and GST activities were decreased in infected animals at day 15 PI (P<0.05). Considering the proper functioning of the heart, it is possible that the changes in the activity of these enzymes involved in the oxidative stress may be related, at least in part, in the pathophysiology of rats infected with T. evansi.     

Nrf2 Activates Augmenter of Liver Regeneration (ALR) via Antioxidant Response Element and Links Oxidative Stress to Liver Regeneration

Liver regeneration can be impaired by permanent oxidative stress and activation of nuclear factor erythroid 2–related factor 2 (Nrf2), known to regulate the cellular antioxidant response, and has been shown to improve the process of liver regeneration. A variety of factors regulate hepatic tissue regeneration, among them augmenter of liver regeneration (ALR), attained great attention as being survival factors for the liver with proproliferative and antiapoptotic properties. Here we determined the Nrf2/antioxidant response element (ARE) regulated expression of ALR and show ALR as a target gene of Nrf2 in vitro and in vivo. The ALR promoter comprises an ARE binding site and, therefore, ALR expression can be induced by ARE-activator tertiary butylhydroquinone (tBHQ) in hepatoma cells and primary human hepatocytes (PHH). Promoter activity and expression of ALR were enhanced after cotransfection of Nrf2 compared with control and dominant negative mutant of Nrf2. Performing partial hepatectomy in livers from Nrf2^+/+ mice compared with Nrf2^−/− knock-out (KO) mice, we found increased expression of ALR in addition to known antioxidant ARE-regulated genes. Furthermore, we observed increased ALR expression in hepatitis B virus (HBV) compared with hepatitis C virus (HCV) positive hepatoma cells and PHH. Recently, it was demonstrated that HBV infection activates Nrf2 and, now, we add results showing increased ALR expression in liver samples from patients infected with HBV. ALR is regulated by Nrf2, acts as a liver regeneration and antioxidative protein and, therefore, links oxidative stress to hepatic regeneration to ensure survival of damaged cells.     

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.     

β-Glucan from Saccharomyces cerevisiae alleviates oxidative stress in LPS-stimulated RAW264.7 cells via Dectin-1/Nrf2/HO-1 signaling pathway

β-Glucan from Saccharomyces cerevisiae has been described to be effective antioxidants, but the specific antioxidation mechanism of β-glucan is unclear. The objectives of this research were to determine whether the β-glucan from Saccharomyces cerevisiae could regulate oxidative stress through the Dectin-1/Nrf2/HO-1 signaling pathway in lipopolysaccharides (LPS)-stimulated RAW264.7 cells. In this study, we examined the effects of β-glucan on the enzyme activity or production of oxidative stress indicators in LPS-stimulated RAW264.7 cells by biochemical analysis and the protein expression of key factors of Dectin-1/Nrf2/HO-1 signaling pathway by immunofluorescence and western blot. The biochemical analysis results showed that β-glucan increased the LPS-induced downregulation of enzyme activity of intracellular heme oxygenase (HO), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) while decreasing the production of reactive oxygen species (ROS) and malondialdehyde (MDA). Furthermore, immunofluorescence results showed that β-glucan can activate the nuclear factor erythroid 2-related factor 2 (Nrf2). The antioxidant mechanism study indicated that β-glucan activated dendritic-cell-associated C-type lectin 1 (Dectin-1) receptors mediated Nrf2/HO-1 signaling pathway, thereby downregulating the production of ROS and thus produced the antioxidant effects in LPS-stimulated RAW 264.7 cells. In conclusion, these results indicate that β-glucan potently alleviated oxidative stress via Dectin-1/Nrf2/HO-1 in LPS-stimulated RAW 264.7 cells.     

The protective effect of melatonin against cypermethrin-induced oxidative stress damage in Spodoptera litura (Lepidoptera: Noctuidae)

Antioxidant enzymes form the first-line defense against free radicals damage in organisms. Their regulation depends mainly on the oxidant and antioxidant status of the cell, given that oxidants are their principal modulators. Therefore, the aim of the present study was to investigate the effect of melatonin on synthetic pyrethroid insecticide-induced antioxidative enzymes activity in Spodoptera litura larvae. In addition, activities of enzymatic antioxidants viz. superoxide dismutase (SOD), glutathione S-transferase (GST), catalase (CAT), glutathione reductase (GR), α, β-esterase, and acetylcholine esterase (AChE) were assessed. There was no significant change in GST levels in the melatonin-treated groups. Melatonin modulates cypermethrin-induced changes in the activities of esterase and AChE, whereas SOD, CAT, and GR activity was significantly increased in melatonin-treated samples when compared to control. In conclusion, the results of the current study revealed that SP toxicity activated oxidant systems in all antioxidant systems in some tissues of insects. Melatonin administration led to a marked increase in antioxidant activity and inhibited GST and AChE in most of the tissues studied.     

Prooxidant/Antioxidant Balance in Hypoxia: A Cross-Over Study on Normobaric vs. Hypobaric “Live High-Train Low”

“Live High-Train Low” (LHTL) training can alter oxidative status of athletes. This study compared prooxidant/antioxidant balance responses following two LHTL protocols of the same duration and at the same living altitude of 2250 m in either normobaric (NH) or hypobaric (HH) hypoxia. Twenty-four well-trained triathletes underwent the following two 18-day LHTL protocols in a cross-over and randomized manner: Living altitude (P_IO₂ = 111.9 ± 0.6 vs. 111.6 ± 0.6 mmHg in NH and HH, respectively); training “natural” altitude (~1000–1100 m) and training loads were precisely matched between both LHTL protocols. Plasma levels of oxidative stress [advanced oxidation protein products (AOPP) and nitrotyrosine] and antioxidant markers [ferric-reducing antioxidant power (FRAP), superoxide dismutase (SOD) and catalase], NO metabolism end-products (NOx) and uric acid (UA) were determined before (Pre) and after (Post) the LHTL. Cumulative hypoxic exposure was lower during the NH (229 ± 6 hrs.) compared to the HH (310 ± 4 hrs.; P<0.01) protocol. Following the LHTL, the concentration of AOPP decreased (-27%; P<0.01) and nitrotyrosine increased (+67%; P<0.05) in HH only. FRAP was decreased (-27%; P<0.05) after the NH while was SOD and UA were only increased following the HH (SOD: +54%; P<0.01 and UA: +15%; P<0.01). Catalase activity was increased in the NH only (+20%; P<0.05). These data suggest that 18-days of LHTL performed in either NH or HH differentially affect oxidative status of athletes. Higher oxidative stress levels following the HH LHTL might be explained by the higher overall hypoxic dose and different physiological responses between the NH and HH.     

Glutathione-dependent enzyme activities and concentrations of glutathione,vitamin E and sulfhydryl groups in barbel (Barbus barbus) and its intestinal parasite Pomphorhynchus laevis (Acanthocephala)

Barbel (Barbus barbus) is the final host of the adult worm Pomphorhynchus laevis (Acanthocephala), one of the most abundant and widespread intestinal parasites of European freshwater fish. During the course of the present study, we analyzed the activities of the glutathione-dependent enzymes glutathione peroxidase (GSH-Px), glutathione reductase (GR) and the phase II biotransformation enzyme glutathione-S-transferase (GST) as well as the concentrations of total glutathione (GSH), sulfhydryl (SH) groups and vitamin E (Vit E) in the liver and intestine of B. barbus and in its intestinal parasite P. laevis. The fish were caught from the Danube River (Serbia) in spring and summer. We detected that GSH-Px activity in fish liver was higher in spring. GR activity was significantly higher in spring in all investigated samples, while GST activity was significantly higher in spring in fish liver and in the parasite. At that time, GST activity was ten times higher in the parasite than in fish tissues. The concentration of GSH was increased in barbel liver in spring. In summer, the concentration of SH groups was significantly increased, while the concentration of Vit E was significantly decreased in fish tissues and in the parasite. We performed Canonical Discriminant Analysis, which revealed differentiation among the examined tissues during both seasons based on the all measured antioxidant components. We found that the seasonal patterns of antioxidant defense in the parasite are closely correlated with seasonal variation and physiological change in the host and represent the parasite's adaptation to changes in the host's antioxidant system. The present investigation contributes to general knowledge and provides a basis for future studies of glutathione-dependent enzymes and non-enzymatic low molecular mass antioxidants as potential biomarkers for monitoring the influence of the environment on fishes and their parasites.     

Age composition and antioxidant enzyme activities in blood of Black Sea teleosts

Age composition and age-related trends of antioxidant enzyme activities superoxide dismutase (SOD), catalase (CAT), peroxidase (PER), glutathione reductase (GR) and glutathione-S-transferase (GST) in the blood of seven Black Sea teleosts (Carangidae, Centracanthidae, Gadidae, Mullidae, Gobiidae and Scorpaenidae) collected in marine coastal area of Sevastopol (Ukraine) were studied. In the catches the animals of 1 – 2 years of age dominated while in the Scorpaena porcus population the number of relatively elder individuals belonging to classes of 3–4 years was the highest. The trends of antioxidant enzyme activities in blood were not uniform. Three types of age-dependent responses were indicated in fish blood: 1. enzymatic activity did not change with age; 2. enzymatic activity decreased with age and 3. enzyme activity increased with age or varied unclearly. The interspecies differences of age-related enzymatic activities associated with the specificity of fish biology and ecology were indicated. Despite no clear evidence of age-related differences between fish species belonging to different ecological groups both benthic forms exhibited similar age-dependent trends of SOD and PER. The correlations between blood antioxidant enzyme activities in fish belonging to suprabenthic and benthic/pelagic groups demonstrated the intermediate values as compared to the benthic and pelagic forms. The results suggest the importance of age trends for biomarkers in fish monitoring studies.     

In Vitro Protective Effect and Antioxidant Mechanism of Resveratrol Induced by Dapsone Hydroxylamine in Human Cells

Dapsone (DDS) hydroxylamine metabolites cause oxidative stress- linked adverse effects in patients, such as methemoglobin formation and DNA damage. This study evaluated the ameliorating effect of the antioxidant resveratrol (RSV) on DDS hydroxylamine (DDS-NHOH) mediated toxicity in vitro using human erythrocytes and lymphocytes. The antioxidant mechanism was also studied using in-silico methods. In addition, RSV provided intracellular protection by inhibiting DNA damage in human lymphocytes induced by DDS-NHOH. However, whilst pretreatment with RSV (10–1000 μM significantly attenuated DDS-NHOH-induced methemoglobinemia, but it was not only significantly less effective than methylene blue (MET), but also post-treatment with RSV did not reverse methemoglobin formation, contrarily to that observed with MET. DDS-NHOH inhibited catalase (CAT) activity and reactive oxygen species (ROS) generation, but did not alter superoxide dismutase (SOD) activity in erythrocytes. Pretreatment with RSV did not alter these antioxidant enzymes activities in erythrocytes treated with DDS-NHOH. Theoretical calculations using density functional theory methods showed that DDS-NHOH has a pro-oxidant effect, whereas RSV and MET have antioxidant effect on ROS. The effect on methemoglobinemia reversion for MET was significantly higher than that of RSV. These data suggest that the pretreatment with resveratrol may decrease heme-iron oxidation and DNA damage through reduction of ROS generated in cells during DDS therapy.