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Scope: Phosphoserine-containing peptides have been shown to exert antioxidative stress effects, by lowering lipid peroxidation, increasing intracellular glutathione, and increasing the expression of antioxidant enzymes in human intestinal epithelial cells. However, the role of phosphoserine residues in antioxidative stress activity, and their mechanism of action, remains unknown., Methods and Results: The antioxidative stress activity of phosphoserine and phosphoserine peptides was examined using an in vitro model of hydrogen peroxide (H2 O2 )-induced oxidative stress in Caco-2 cells. Phosphoserine dimers (2PS) reduced IL-8 secretion in H2 O2 -treated Caco-2 cells, and reduced H2 O2 -induced expression of genes involved in inflammation and generation of reactive oxygen species (ROS), including chemokine (C-C motif) ligand 5 (CCL5), lactoperoxidase (LPO), myeloperoxidase (MPO), neutrophil cytosolic factor 1/2 (NCF1/2), and nitric oxide synthase 2A (NOS2), and upregulated metallothionein 3 (MT3), peroxiredoxin 3 (PRDX3), and surfactant, pulmonary-associated protein D (SFTPD), which are involved in protection against oxidative stress and activation of the Nrf2 signaling pathway. At the protein level, 2PS reduced H2 O2 -induced phosphorylation of the ERK1/2 and JNK MAPKs, and increased Nrf2 expression. Moreover, the ability of 2PS to reduce H2 O2 -induced IL-8 secretion, a marker of inflammation and oxidative stress, was abrogated in Nrf2 knockdown cells., Conclusion: These results suggest that 2PS reduce H2 O2 -induced oxidative stress via the Nrf2 signaling pathway, and reveal a potential mechanism for the antioxidative stress activity of phosphoserine-containing peptides., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Oxidative stress is involved in the initiation and propagation of chronic intestinal pathologies. Bioactive peptides such as egg yolk-derived phosvitin phosphopeptides (PPP3) have been previously shown to reduce in vitro oxidative stress by up-regulating glutathione synthesis and antioxidant enzyme activities. Peptide and gene expression profile analysis of the PPP3 peptides can provide insight into structures involved in signal transduction mechanisms in the antioxidative stress response. The objectives of this research were to identify the PPP3 amino acid sequences before and after simulated gastrointestinal digestion and to assess the genes influenced by PPP3. Peptide sequences were analyzed using ESI Q-TOF-MS/MS, and the expression profile of 84 human oxidative stress and antioxidant defense genes were analyzed. Undigested PPP3 was composed of three main peptides: GTEPDAKTSSSSSSASSTATSSSSSSASSPNRKKPMDE (phosvitin-PV residues 4-41), NSKSSSSSSKSSSSSSRSRSSSKSSSSSSSSSSSSSSKSSSSR (PV residues 155-197), and EDDSSSSSSSSVLSKIWGRHEIYQ (PV residues 244-257) and their fragments. There was limited degradation of PPP3 after gastrointestinal digestion as deduced from the fragment sizes of digested PPP3, which ranged from 5 to 32 amino acids. These fragments were rich in contiguous serines and, in some cases, monoesterified with phosphate. Both undigested and digested PPP3 significantly reduced IL-8 secretion in H(2)O(2)-induced Caco-2 cells, indicating that antioxidative stress bioactivity is retained upon digestion. After PPP3 pretreatment, antioxidant genes associated with oxygen and reactive oxygen species (ROS) metabolism and cellular responses to chemical stimulus, oxidative stress, and ROS are up-regulated in the presence and absence of oxidative stress, thereby contributing to the prevention of intestinal oxidative stress and the promotion of gut health.

Long-term oxidative stress in the gastrointestinal tract can lead to the development of chronic intestinal disorders. Many food-derived antioxidants are effective in vitro, but the variable reports of in vivo efficacy and the pro-oxidant nature of some antioxidants necessitate alternative strategies for the reduction of in vivo oxidative stress. Compounds that up-regulate the production of endogenous antioxidants such as glutathione (GSH) and antioxidant enzymes provide novel approaches for the restoration of redox homeostatis. Egg yolk peptides (EYP) prepared from Alcalase and protease N digestion of delipidated egg yolk proteins were found to exhibit antioxidative stress properties. The effect of EYP supplementation was examined in a hydrogen peroxide-induced human colon cell line and in an animal model of intestinal oxidative stress. EYP significantly reduced the pro-inflammatory cytokine, IL-8, in Caco-2 cells. In piglets given intraperitoneal infusions of hydrogen peroxide, EYP treatment increased GSH and gamma-glutamylcysteine synthetase mRNA expression and activity, significantly increased antioxidant enzyme activities, in particular catalase and glutathione S-transferase activities, and reduced protein and lipid oxidation in the duodenum, jejunum, ileum, and colon. Furthermore, EYP boosted the systemic antioxidant status in blood by increasing the GSH concentration in red blood cells. These results suggest that EYP supplementation is a novel strategy for the reduction of intestinal oxidative stress.

The protective effects of amino acids against H 2O 2-induced oxidative stress were investigated in an in vitro assay using human intestinal epithelial cells. Caco-2 cells were pretreated with amino acids (1, 2, and 5 mM) for 2 h and then stimulated with 1 mM H 2O 2 for 6 h. The secretion of IL-8, a proinflammatory mediator, was determined by ELISA as an indicator of tissue oxidative stress. The inhibition of H 2O 2-induced IL-8 secretion from Caco-2 cells was observed by pretreatment with Cys, Val, Ile, Leu, Trp, His, Lys, and Ala. Cys enhanced glutathione (GSH) biosynthesis enzyme activity and increased cellular GSH levels. Branched-chain amino acids such as Val, Ile, and Leu elevated activities of GSH S-transferase (GST) and catalase. Trp, His, and Lys caused increases in GST activity. Ala enhanced GSH reductase activity. These data suggest that specific amino acids exert protective effects against tissue oxidative stress in intestinal epithelial cells based on the structure.

Previously, we have found phosphopeptides (PPPs) from hen egg yolk phosvitin possess a potent antioxidative activity against oxidative stress in human intestinal epithelial cells, Caco-2. However, their biological activity at the cellular level has not yet fully understood. The objective of this study is to evaluate the regulation of glutathione (GSH) biosynthesis-associated and antioxidant enzymes against oxidative stress in Caco-2 cells using an in vitro model. Treatment of 1 mM H2O2-induced Caco-2 cells with PPPs increased cellular GSH levels, concomitant with a significant increase in gamma-glutamylcysteine synthetase (gamma-GCS) activity and the expression of gamma-GCS heavy subunit mRNA. Furthermore, intracellular glutathione reductase, glutathione S-transferase, and catalase activities were elevated by PPPs. In addition, PPPs with high content of phosphorus showed higher induction of these enzyme activities than PPPs without phosphorus. These data indicate that oligophosphopeptides from hen egg yolk phosvitin can up-regulate cellular GSH biosynthesis-associated enzymes activity and antioxidative activities, which play key roles against tissue oxidative stress in the human intestinal epithelial cells.

The protective effects of hen egg yolk phosvitin phosphopeptides (PPPs) against hydrogen peroxide (H2O2)-induced oxidative stress were evaluated in an in vitro assay using human intestinal epithelial cells. Caco-2 cells were stimulated with 1 mM H2O2 for 6 h, and the secretion of IL-8, a proinflammatory mediator, was determined by ELISA as a biomarker of oxidative stress. The inhibition of H2O2-induced IL-8 secretion from Caco-2 cells was observed by pretreatment for 2 h with PPPs, but not with phosvitin. PPPs also suppressed the formation of malondialdehyde in H2O2-treated Caco-2 cells. Furthermore, intracellular glutathione levels and glutathione reductase activity were elevated by the addition of PPPs. The protective effects of PPPs against H2O2-induced oxidative stress were almost the same as that of glutathione, and PPPs with a high content of phosphorus exhibited higher protective activity than PPPs without phosphorus; however, phosphoserine itself did not show any significant antioxidative stress activity. These findings suggest that oligophosphopeptides from hen egg yolk phosvitin possess novel antioxidative activity against oxidative stress in intestinal epithelial cells and that phosphorus and peptide structure seem to have a key role in the activity.

Excess reactive oxygen species (ROS) cause oxidative stress that is associated with the development of oxidative damage and chronic inflammation. The objective of this study was to evaluate the bioactivity of eggshell membrane (ESM) peptides digested with Alcalase and Protease S (AL-PS) at the cellular level. Effects were tested for inhibition of lipid and protein oxidation, synthesis of glutathione (GSH), and cellular antioxidant enzyme activity in H 2 O 2 -stimulated Caco-2 cells. AL-PS and its ultrafiltered fraction AL-PS-I (MWCO < 5 kDa) significantly suppressed the formation of H 2 O 2 -induced malondialdehyde (MDA) and protein carbonyl derivatives. AL-PS and AL-PS-I also increased glutathione peroxidase (GPx), glutathione S transferase (GST), and glutathione reductase (GR) activity. The peptides also elevated cellular GSH levels via up-regulation of γ-glutamylcysteine synthetase (γ-GCS) activity and its mRNA expression. This study confirms that ESM peptides are able to reduce intestinal oxidative stress and thus validates their use as a valuable source material of ESM waste. [ABSTRACT FROM AUTHOR]

Eggshell membrane (ESM) hydrolysate was prepared by Alcalase and Protease S (AL-PS) sequentially and was ultrafiltered as fractions AL-PS-I (<5 kDa), AL-PS-II (5-10 kDa), and AL-PS-III (>10 kDa). The antioxidant activity of ESM hydrolysate was evaluated using chemical assays and its protective ability against oxidative stress was investigated using human intestinal epithelial Caco-2 cell-based assay. AL-PS-I possessed the highest scavenging activity against DPPH and hydroxyl radicals. AL-PS-III demonstrated the best Fe 2+ chelating activity. AL-PS and AL-PS-I had higher cellular antioxidant activity to inhibit the formation of DCF. The secretion of IL-8 was inhibited significantly (P < 0.05) at 2 h post pretreatment with AL-PS, AL-PS-I and AL-PS-II (0.1 and 0.5 mg/mL) and at 6 h post stimulation with 1 mM H 2 O 2 . In addition, AL-PS, AL-PS-I and AL-PS-II (0.5 mg/mL) increased glutathione (GSH) level significantly (P < 0.05) in H 2 O 2 -treated Caco-2 cells. [ABSTRACT FROM AUTHOR]

We examined responses of the maximal specific activity (Vmax) of key carbohydrate digestive enzymes in piglets with hydrogen peroxide (H2O2)-induced systemic oxidative stress. Liquid formula-fed piglets at the age of d 5 were fitted with an intraperitoneal catheter and infused with either 5 mmol H2O2/kg BW (PER, n=8) or physiologic saline (CON, n=8) in six injections daily for a period of 10 d. Jejunal enzyme activity experiments for lactase, sucrase, maltase and maltase-glucoamylase were conducted at 37°C with substrate concentrations over two-fold of Km values. Piglets experiencing oxidative stress had lower (P<0.05) jejunal Vmax for lactase and maltase (estimates±SE: PER, 6.54±0.68 vs. CON, 12.65±0.69; and PER, 57.39±1.02 vs. CON, 75.6±1.04 µmol/mg protein.min). However, there were no differences (P>0.05) in Vmax of maltase-glucoamylase and sucrase (estimates±SE: PER, 0.71±0.08 vs. CON, 0.70±0.07; and PER, 10.50±1.37 vs. CON, 12.40±1.55 µmol/mg protein.min). We conclude that oxidative stress reduced intestinal lactase and maltase functions without affecting maltase-glucoamylase and sucrase activities. [ABSTRACT FROM AUTHOR]

Chronic fatigue syndrome (CFS) is characterized by persistent and relapsing fatigue that involves oxidative stress in its pathogenesis. In this study we report on the prevalence of CFS-associated symptoms in an oxidative stress pig model. Piglets of 5 d of age were fitted with an intraperitoneal catheter and infused with either H202 at 5 mmol/kg BW (PER, n=8) or the same volume of saline (CON, n=8) in six 20-ml doses daily for a period of 10 d. During this time, animal behavior was monitored and blood samples taken. At the end of the study, CON piglets had higher (P<0.05) BW gain in comparison to the PER group (CON, 111.46±9.00 vs. PER, 74.78±9.62 g/d). Since there were no differences (P>0.05) in the formula intake between PER and CON groups, the CON piglets had lower (P<0.05) feed to gain ratios in comparison to the PER piglets (CON, 8.72±0.85 vs. PER, 15.82±0.83 ml/g). Plasma GSH levels remained similar (P>0.05) to the pre-infusion level over the study duration in the CON group, whereas this was 65% lower (P<0.05) than the pre-infusion level in the PER' group. Additionally, the PER treatment piglets were more lethargic, less mobile and less responsive to new environmental stimuli. These piglets (PER group) also consumed their meals at a slower pace and all developed diarrhea in comparison to the CON group. We conclude that chronic fatigue syndrome could be established by infusion of a suitable dose of hydrogen peroxide to investigate mechanisms and modulations of CFS in association with oxidative stress in the pig. Supported by OMAFRA of Canada. [ABSTRACT FROM AUTHOR]

Abstract: Conventional therapies for the treatment of inflammatory bowel disease (IBD) have demonstrated limited efficacy and potential toxicity; therefore, there is a need for novel therapies that can safely and effectively treat IBD. Recent evidence has indicated that amino acids may play a role in maintaining gut health. l-Tryptophan has been shown to reduce oxidative stress and improve neurological states. The objective of this study was to assess the therapeutic effects of l-tryptophan in a porcine model of dextran sodium sulfate (DSS)-induced colitis. DSS was administered to piglets via intragastric catheter for 5 days followed by tryptophan administration at 80% of the daily recommended intake. The severity of colitis was assessed macroscopically and histopathologically, and intestinal permeability was monitored in vivo by d-mannitol analysis. The effect of tryptophan on the local expression of key mediators of inflammation and IBD pathogenesis was examined at the protein and gene expression levels. Supplementation with tryptophan ameliorated clinical symptoms and improved weight gain to feed intake conversion ratios. Histological scores and measurements were also improved, and gut permeability was notably reduced in tryptophan-supplemented animals. Moreover, tryptophan reduced the expression of the pro-inflammatory cytokines tumor necrosis factor-α, interleukin (IL)-6, interferon (IFN)-γ, IL-12p40, IL-1β and IL-17, as well as IL-8 and intracellular adhesion molecule-1, and resulted in increased expression of apoptosis initiators caspase-8 and Bax. These results demonstrate that l-tryptophan supplementation can reduce inflammation and enhance the rate of recovery in DSS-induced colitis and may be an effective immunomodulating agent for the treatment of IBD. [Copyright &y& Elsevier]