Cytoprotective Peptides from Blue Mussel Protein Hydrolysates: Identification and Mechanism Investigation in Human Umbilical Vein Endothelial Cells Injury

Cardiovascular disease represents a leading cause of mortality and is often characterized by the emergence of endothelial dysfunction (ED), a physiologic condition that takes place in the early progress of atherosclerosis. In this study, two cytoprotective peptides derived from blue mussel chymotrypsin hydrolysates with the sequence of EPTF and FTVN were purified and identified. Molecular mechanisms underlying the cytoprotective effects against oxidative stress which lead to human umbilical vein endothelial cells (HUVEC) injury were investigated. The results showed that pretreatment of EPTF, FTVN and their combination (1:1) in 0.1 mg/mL significantly reduced HUVEC death due to H₂O₂ exposure. The cytoprotective mechanism of these peptides involves an improvement in the cellular antioxidant defense system, as indicated by the suppression of the intracellular ROS generation through upregulation of the cytoprotective enzyme heme oxygenase-1. In addition, H₂O₂ exposure triggers HUVEC damage through the apoptosis process, as evidenced by increased cytochrome C release, Bax protein expression, and the elevated amount of activated caspase-3, however in HUVEC pretreated with peptides and their combination, the presence of those apoptotic stimuli was significantly decreased. Each peptide showed similar cytoprotective effect but no synergistic effect. Taken together, these peptides may be especially important in protecting against oxidative stress-mediated ED.     

Design and Synthesis of Novel Xyloketal Derivatives and Their Protective Activities against H2O2-Induced HUVEC Injury

In this work, we designed and synthesized a series of amide derivatives (1–13), benzoxazine derivatives (16–28) and amino derivatives (29–30) from xyloketal B. All 28 new derivatives and seven known compounds (14, 15, 31–35) were evaluated for their protection against H₂O₂-induced HUVEC injury. 23 and 24 exhibited more potential protective activities than other derivatives; and the EC₅₀ values of them and the leading compound 31 (xyloketal B) were 5.10, 3.59 and 15.97 μM, respectively. Meanwhile, a comparative molecular similarity indices analysis (CoMSIA) was constructed to explain the structural activity relationship of these xyloketal derivatives. This 3D QSAR model from CoMSIA suggested that the derived model exhibited good predictive ability in the external test-set validation. Derivative 24 fit well with the COMSIA map, therefore it possessed the highest activity of all compounds. Compounds 23, 24 and 31 (xyloketal B) were further to examine in the JC-1 mitochondrial membrane potential (MMP) assay of HUVECs using flow cytometry (FCM). The result indicated that 23 and 24 significantly inhibited H₂O₂-induced decrease of the cell mitochondrial membrane potential (ΔΨm) at 25 μM. Collectively, the protective effects of xyloketals on H₂O₂-induced endothelial cells may be generated from oxidation action by restraining ROS and reducing the MMP.     

The Cytoprotective Effect of Petalonia binghamiae Methanol Extract against Oxidative Stress in C2C12 Myoblasts: Mediation by Upregulation of Heme Oxygenase-1 and Nuclear Factor-Erythroid 2 Related Factor 2

This study was designed to examine the protective effects of the marine brown algae Petalonia binghamiae against oxidative stress-induced cellular damage and to elucidate the underlying mechanisms. P. binghamiae methanol extract (PBME) prevented hydrogen peroxide (H₂O₂)-induced growth inhibition and exhibited scavenging activity against intracellular reactive oxygen species (ROS) induced by H₂O₂ in mouse-derived C2C12 myoblasts. PBME also significantly attenuated H₂O₂-induced comet tail formation in a comet assay, histone γH2A.X phosphorylation, and annexin V-positive cells, suggesting that PBME prevented H₂O₂-induced cellular DNA damage and apoptotic cell death. Furthermore, PBME increased the levels of heme oxygenase-1 (HO-1), a potent antioxidant enzyme, associated with the induction of nuclear factor-erythroid 2 related factor 2 (Nrf2). However, zinc protoporphyrin IX, a HO-1 competitive inhibitor, significantly abolished the protective effects of PBME on H₂O₂-induced ROS generation, growth inhibition, and apoptosis. Collectively, these results demonstrate that PBME augments the antioxidant defense capacity through activation of the Nrf2/HO-1 pathway.     

Protective Effect of Flavonoids from a Deep-Sea-Derived Arthrinium sp. against ox-LDL-Induced Oxidative Injury through Activating the AKT/Nrf2/HO-1 Pathway in Vascular Endothelial Cells

Oxidized low-density lipoprotein (ox-LDL)-induced oxidative injury in vascular endothelial cells is crucial for the progression of cardiovascular diseases, including atherosclerosis. Several flavonoids have been shown cardiovascular protective effects. Recently, our research group confirmed that the novel flavonoids isolated from the deep-sea-derived fungus Arthrinium sp., 2,3,4,6,8-pentahydroxy-1-methylxanthone (compound 1) and arthone C (compound 2) effectively scavenged ROS in vitro. In this study, we further investigated whether these compounds could protect against ox-LDL-induced oxidative injury in endothelial cells and the underlying mechanisms. Our results showed that compounds 1 and 2 inhibited ox-LDL-induced apoptosis and adhesion factors expression in human umbilical vein vascular endothelial cells (HUVECs). Mechanistic studies showed that these compounds significantly inhibited the ROS level increase and the NF-κB nuclear translocation induced by ox-LDL. Moreover, compounds 1 and 2 activated the Nrf2 to transfer into nuclei and increased the expression of its downstream antioxidant gene HO-1 by inducing the phosphorylation of AKT in HUVECs. Importantly, the AKT inhibitor MK-2206 2HCl or knockdown of Nrf2 by RNA interference attenuated the inhibition effects of these compounds on ox-LDL-induced apoptosis in HUVECs. Meanwhile, knockdown of Nrf2 abolished the effects of the compounds on ox-LDL-induced ROS level increase and the translocation of NF-κB to nuclei. Collectively, the data showed that compounds 1 and 2 protected endothelial cells against ox-LDL-induced oxidative stress through activating the AKT/Nrf2/HO-1 pathway. Our study provides new strategies for the design of lead compounds for related cardiovascular diseases treatment.     

Enzymatic Degradation of Gracilariopsis lemaneiformis Polysaccharide and the Antioxidant Activity of Its Degradation Products

Gracilariopsis lemaneiformis polysaccharides (GLP) were degraded using pectinase, glucoamylase, cellulase, xylanase, and β-dextranase into low-molecular-weight polysaccharides, namely, GPP, GGP, GCP, GXP, and GDP, respectively, and their antioxidant capacities were investigated. The degraded GLP showed higher antioxidant activities than natural GLP, and GDP exhibited the highest antioxidant activity. After the optimization of degradation conditions through single-factor and orthogonal optimization experiments, four polysaccharide fractions (GDP1, GDP2, GDP3, and GDP4) with high antioxidant abilities (hydroxyl radical scavenging activity, DPPH radical scavenging activity, reduction capacity, and total antioxidant capacity) were obtained. Their cytoprotective activities against H₂O₂-induced oxidative damage in human fetal lung fibroblast 1 (HFL1) cells were examined. Results suggested that GDP pretreatment can significantly improve cell viability, reduce reactive oxygen species and malonaldehyde levels, improve antioxidant enzyme activity and mitochondria membrane potential, and alleviate oxidative damage in HFL1 cells. Thus, the enzyme degradation of GLP with β-dextranase can significantly improve its antioxidant activity, and GDP might be a suitable source of natural antioxidants.     

Polyphenol-Rich Extract of Fenugreek Seeds Protect Erythrocytes from Oxidative Damage

A polyphenol-rich extract from the seeds of fenugreek was evaluated for its protective effect against hydrogen peroxide(H₂O₂)-induced oxidation in normal and diabetic human erythrocytes (RBCs). RBCs, preincubated with increasing amounts of fenugreek seed extract and challenged with H₂O_2, were analyzed for hemolysis and lipid peroxidation. RBCs from diabetic subjects were more susceptible to oxidative hemolysis and lipid peroxidation than those from normal subjects. However preincubation with the polyphenol-rich extract significantly reduced the oxidative modifications in both the groups. The inhibition of lipid peroxidation was concentration-dependent up to 100 l of extract, which contained 0.75mM gallic acid equivalent (GAE) of phenolic compounds. These findings demonstrate the potent antioxidant properties of the fenugreek seeds.     

Involvement of triadimefon induced early ABA-dependent H2O2 accumulation in soybean against water stress

The present study showed that pretreatment of triadimefon (TDM), a triazole compound, could improve tolerance of soybean seedlings to subsequent water stress. TDM pretreatment resulted in early and late rise in superoxide dismutase (SOD) and catalase (CAT) activities, and upregulation of ascorbate (AsA) content in non-stressed and water-stressed seedlings, leading to late increase in net photosynthetic rate (P_n), late decrease in hydrogen peroxide (H₂O₂) and electrolyte leakage in stressed ones. These TDM-induced changes were blocked by application of abscisic acid (ABA) biosynthesis inhibitor tungstate, which inhibited early rise of ABA and H₂O₂ contents in non-stressed and stressed seedlings. However, ABA pretreatment overcomed the effects of this inhibitor. Application of NADPH oxidase inhibitor diphenyleneiodonium (DPI), polyamine oxidase (PAO) inhibitor 2-hydroxyethylhydrazine (2-HEH) and H₂O₂ scavenger dimethylthiourea (DMTU) prevented early TDM-induced rise of H₂O₂ content. DPI, 2-HEH and DMTU also decreased SOD, CAT and AsA levels, but did not affect ABA content during early and late phases in both seedlings pretreated with TDM. In addition, these chemicals decreased P_n, and increased H₂O₂ content and electrolyte leakage during late phase in TDM-pretreated stressed seedlings. Overall, these results indicated that TDM pretreatment alleviated adverse effects of water stress on soybean seedlings, which was at least in part, due to increase of antioxidant capacity and decrease of oxidative damage induced by early ABA-dependent H₂O₂ generation.     

Induction of Freezing Tolerance by the Application of Hydrogen Peroxide and Salicylic Acid as Tuber-Dip or Canopy Spraying in <Emphasis Type="Italic">Solanum tuberosum</Emphasis> L. Plants

Low temperature stress is a current challenge to plants that is associated with climate change. In plants, exposure to extreme temperatures is followed by the accumulation of reactive oxygen species, such as hydrogen peroxide (H₂O₂), leading to oxidative stress. Salicylic acid (SA) and H₂O₂ mediate the tolerance responses to stress and have been reported to induce freezing tolerance in potato microplants. The objectives of the present investigation were (1) to evaluate the short- and long-term effects of H₂O₂ and SA treatments on freezing tolerance in potato (Solanum tuberosum L.) plants grown from tubers and (2) to analyse the relationship between catalase (CAT) activity and H₂O₂ concentration associated with freezing tolerance responses. We observed the lowest freezing survival rates in 45-day-old potato plants (cv. Granate) compared to younger plants. The two treatments consisted of (1) the tuber-dip (long-term) treatment in which sprouted minitubers were saturated for 1 h in SA 10^?5 M or H₂O₂ 1 mM and planted in soil under greenhouse conditions and (2) the crop-spray (short-term) treatment in which plants 5–8 cm high were sprayed twice a week with SA 10^?5 M or H₂O₂ 1 mM until 45 days of age. In all treatments, 45-day-old plants were then exposed to ? 6?±?1 °C for 4 h. The survival rate was measured 15 days after freezing. CAT and H₂O₂ measurements were performed 1 h before and after the freezing treatment. The results showed that SA and H₂O₂ induced freezing tolerance in both the short- and long-term treatments. Survival was significantly higher in SA- and H₂O₂-treated plants than in control plants. In both the long- and short-term treatments this higher survival was associated with lower internal H₂O₂ concentrations after freezing compared with control plants and decreasing oxidative stress. SA and H₂O₂ induced different levels of CAT activity after freezing compared to that found in the control plants in the long- and the short-term treatments. These results suggest the SA and H₂O₂ function in independent pathways in terms of their induction of freezing tolerance that depends on the method the treatment was applied, by spraying the canopy or by immersion of the sprouted seed tuber.     

Study of hydrogen peroxide,potato enzymes and blackspot

The rate of oxidation of tyrosine, p-cresol and catechol by potato enzyme diminished as H₂O₂ concentration increased. By contrast, the rate of oxidation of chlorogenic acid in the presence of H₂O₂ increased. Bovine catalase destroyed H₂O₂ and thus effectively prevented either H₂O₂-induced inhibition or acceleration of oxidation of the four substrates by potato enzyme. Horseradish peroxidase in the presence of H₂O₂ did not oxidize either monophenol, but oxidized both polyphenols. Possible association of H₂O₂, peroxidase and catalase with blackspot susceptibility is discussed.     

Antioxidant Effect of trans-Resveratrol, Pterostilbene, Quercetin and Their Combinations in Human Erythrocytes In Vitro

There is evidence that a diet rich in fruit and vegetables may reduce the risk of cancer and other degenerative diseases. However, potential health impact of bioactive phytochemicals is limited by their low amount and relatively poor bioavailability. It has been suggested that the health benefits associated with fruit and red wine consumption could be due to the whole antioxidant pool of the diet microcomponents. In this study, the antioxidant activities of trans-resveratrol, pterostilbene and quercetin, and the effect of their combination were investigated in human erythrocytes in vitro. H₂O₂-induced lipid peroxidation was assessed by measuring the amount of thiobarbituric acid reactive species. Quercetin and pterostilbene protected erythrocyte membranes against lipid peroxidation (IC₅₀ values = 64 ± 8.7 μM and 44.5 ± 7.8 μM, respectively). Resveratrol was significantly less effective. However, the three compounds protected the erythocytes against hemolysis and GSH (reduced glutathione) depletion to the same extent. Combinations consisting of two compounds (molar ratio 1:1) influenced lipid peroxidation in a concentration–dependent manner. At lower concentrations, resveratrol with quercetin or pterostilbene inhibited synergistically the oxidative injury of membrane lipids At higher concentrations, an additive effect was observed. These protective effects may partially explain the health benefit of these bioactive microcomponents when together in the diet.     

Protective Effects of Fucoxanthin on Hydrogen Peroxide-Induced Calcification of Heart Valve Interstitial Cells

Cardiovascular diseases such as atherosclerosis and aortic valve sclerosis involve inflammatory reactions triggered by various stimuli, causing increased oxidative stress. This increased oxidative stress causes damage to the heart cells, with subsequent cell apoptosis or calcification. Currently, heart valve damage or heart valve diseases are treated by drugs or surgery. Natural antioxidant products are being investigated in related research, such as fucoxanthin (Fx), which is a marine carotenoid extracted from seaweed, with strong antioxidant, anti-inflammatory, and anti-tumor properties. This study aimed to explore the protective effect of Fx on heart valves under high oxidative stress, as well as the underlying mechanism of action. Rat heart valve interstitial cells under H₂O₂-induced oxidative stress were treated with Fx. Fx improved cell survival and reduced oxidative stress-induced DNA damage, which was assessed by cell viability analysis and staining with propidium iodide. Alizarin Red-S analysis indicated that Fx has a protective effect against calcification. Furthermore, Western blotting revealed that Fx abrogates oxidative stress-induced apoptosis via reducing the expression of apoptosis-related proteins as well as modulate Akt/ERK-related protein expression. Notably, in vivo experiments using 26 dogs treated with 60 mg/kg of Fx in combination with medical treatment for 0.5 to 2 years showed significant recovery in their echocardiographic parameters. Collectively, these in vitro and in vivo results highlight the potential of Fx to protect heart valve cells from high oxidative stress-induced damage.     

Anticancer Activity of a Hexapeptide from Skate (Raja porosa) Cartilage Protein Hydrolysate in HeLa Cells

In this study, the hexapeptide Phe-Ile-Met-Gly-Pro-Tyr (FIMGPY), which has a molecular weight of 726.9 Da, was separated from skate (Raja porosa) cartilage protein hydrolysate using ultrafiltration and chromatographic methods, and its anticancer activity was evaluated in HeLa cells. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay indicated that FIMGPY exhibited high, dose-dependent anti-proliferation activities in HeLa cells with an IC₅₀ of 4.81 mg/mL. Acridine orange/ethidium bromide (AO/EB) fluorescence staining and flow cytometry methods confirmed that FIMGPY could inhibit HeLa cell proliferation by inducing apoptosis. Western blot assay revealed that the Bax/Bcl-2 ratio and relative intensity of caspase-3 in HeLa cells treated with 7-mg/mL FIMGPY were 2.63 and 1.83, respectively, significantly higher than those of the blank control (p < 0.01). Thus, FIMGPY could induce apoptosis by upregulating the Bax/Bcl-2 ratio and caspase-3 activation. Using a DNA ladder method further confirmed that the anti-proliferation activity of FIMGPY was attributable to its role in inducing apoptosis. These results suggest that FIMGPY from skate cartilage protein hydrolysate may have applications as functional foods and nutraceuticals for the treatment and prevention of cancer.     

Identifying Potential Antioxidant Properties from the Viscera of Sea Snails (Turbo cornutus)

Turbo cornutus, the horned turban sea snail, is found along the intertidal and basaltic shorelines of Jeju Island, Korea. T. cornutus feeds on seaweeds (e.g., Undaria sp., and Ecklonia sp.) composed of diverse antioxidants. This study identified potential antioxidant properties from T. cornutus viscera tissues. Diverse extracts were evaluated for their hydrogen peroxide (H₂O₂) scavenging activities. T. cornutus viscera protamex-assisted extracts (TVP) were purified by gel filtration chromatography (GFC), and potential antioxidant properties were analyzed for their amino acid sequences and its peroxidase inhibition effects by in silico molecular docking and in vitro analysis. According to the results, T. cornutus viscera tissues are composed of many protein contents with each over 50%. Among the extracts, TVP possessed the highest H₂O₂ scavenging activity. In addition, TVP-GFC-3 significantly decreased intracellular reactive oxygen species (ROS) levels and increased cell viability in H₂O₂-treated HepG2 cells without cytotoxicity. TVP-GFC-3 comprises nine low molecular bioactive peptides (ELR, VGPQ, TDY, ALPHA, PAH, VDY, WSDK, VFSP, and FAPQY). Notably, the peptides dock to the active site of the myeloperoxidase (MPO), especially TDY and FAPQY showed the MPO inhibition effects with IC₅₀ values of 646.0 ± 45.0 µM and 57.1 ± 17.7 µM, respectively. Altogether, our findings demonstrated that T. cornutus viscera have potential antioxidant properties that can be used as high value-added ingredients.     

玉米大斑病菌StSte12基因对H_2O_2氧化胁迫的应激调节

通过比较野生型菌株Wt01-23与StSte12基因RNAi沉默突变体菌株StRNAi9-10和StRNAi3-6在H_2O_2胁迫下生长和发育方面的差异,分析转录因子基因StSte12对玉米大斑病菌氧化胁迫的调节能力。在不同浓度H_2O_2胁迫条件下,测定野生型菌株和突变体菌株的菌落生长速度、菌丝形态、产孢量和菌丝萌发率。结果表明,随着H_2O_2浓度的增加,玉米大斑病菌野生型菌株和突变体菌株的菌落生长速度、产孢量和菌丝萌发率均显著降低,但突变体菌株的降低程度显著高于野生型菌株,表明StSte12基因对玉米大斑病菌的氧化应激调节具有重要的调控功能。     

Bacterial Impact on H2O2 Accumulation during the Interaction between Xanthomonas and Rice

Abstract

Localization of hydrogen peroxide (H₂O₂) accumulation during the interaction between rice and Xanthomonas oryzae pv. oryzae (X. oryzae pv. oryzae) was observed by histochemical analysis and electron microscopy. The changes that occurred in an avrXa7 mutantstrain of X. oryzae pv. oryzae including the decreased production of endogenous H₂O₂, impacted on the mean level of H₂O₂ accumulation during the interaction with the plant. The results of catalase and aminotriazole treatments indicated that the changes of H₂O₂ accumulation during the interaction are induced by the impairment of endogenous H₂O₂ accumulation in X. oryzae pv. oryzae. These results suggested that bacterial pathogen is a potential source of the H₂O₂ accumulated in the interaction between rice and X. oryzae pv. oryzae.     

Chia Sprouts Elicitation with Salicylic Acid and Hydrogen Peroxide to Improve their Phenolic Content,Antioxidant Capacities In Vitro and the Antioxidant Status in Obese Rats

Elicitation is a biotechnological approach to improve phenolic compounds content and antioxidant properties of ready-to-eat functional foods. This study aimed to evaluate the chemical elicitation effects using salicylic acid (SA) and hydrogen peroxide (H₂O₂) in optimized-germination conditions on seedling vigor, phenolic content, and their antioxidant capacities in vitro and serum and urine of Wistar obese rats. Optimized-germination conditions of 26.5 °C and 178 h produced a 64% of germination and a sprout length of 56 mm. Only, the elicitation with H₂O₂ (20 mM) enhanced the germination (75%) and H₂O₂ (10 and 20 mM) the sprout length (69 and 59 mm, respectively). In contrast, both elicitors enhanced phenolic contents, being more significant total phenolic compounds content for SA (1 and 2 mM), up to 65.5–73.5%. SA and H₂O₂ improved total flavonoids content (36.5–64.1%), ABTS (19.3–61.1%), and DPPH capacities (51–86%), depending on SA and H₂O₂ concentration, compared with non-elicited chia sprouts. The QUENCHER antioxidant capacities of elicited chia sprouts increased up to three times more than extracts capacities, principally Q-ABTS, which could be attributed to phenolic bounds to dietary fiber. Rats fed with a high-fat and fructose diet (HFFD) and supplemented with chia sprouts, especially 1-mM SA, improve the obesity-related oxidative stress through an increase of antioxidant capacities, using DPPH and ABTS test, on serum (70–118%) and urine samples (80–116%). These results suggest that chia sprouts elicited with 1-mM SA are a source of antioxidant compounds that can be used to decrease obesity related oxidative stress.

     

Chitosan Nanoparticles Attenuate Hydrogen Peroxide-Induced Stress Injury in Mouse Macrophage RAW264.7 Cells

This study was carried out to investigate the protective effects of chitosan nanoparticles (CNP) against hydrogen peroxide (H₂O₂)-induced oxidative damage in murine macrophages RAW264.7 cells. After 24 h pre-incubation with CNP (25–200 μg/mL) and chitosan (CS) (50–200 μg/mL, as controls), the viability loss in RAW264.7 cells induced by H₂O₂ (500 μM) for 12 h was markedly restored in a concentration-dependent manner as measured by MTT assay (P < 0.05) and decreased in cellular LDH release (P < 0.05). Moreover, CNP also exerted preventive effects on suppressing the production of lipid peroxidation such as malondialdehyde (MDA) (P < 0.05), restoring activities of endogenous antioxidant including superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) (P < 0.05), along with increasing total antioxidant capacity (T-AOC) (P < 0.05). In addition, pre-incubation of CNP with RAW264.7 cells for 24 h resulted in the increase of the gene expression level of endogenous antioxidant enzymes, such as MnSOD and GSH-Px (P < 0.05). At the same concentration, CNP significantly decreased LDH release and MDA (P < 0.05) as well as increased MnSOD, GSH-Px, and T-AOC activities (P < 0.05) as compared to CS. Taken together, our findings suggest that CNP can more effectively protect RAW264.7 cells against oxidative stress by H₂O₂ as compared to CS, which might be used as a potential natural compound-based antioxidant in the functional food and pharmaceutical industries.     

Promotion of Seedling Growth of Seeds of Rice (Oryza sativa L. cv. Hitomebore) by Treatment with H2O2 before Sowing

Abstract

High germinability of seeds and establishment of young seedlings in rice (Oryza sativa L.) are necessary for direct seeding in paddy fields. We investigated whether germinability and seedling growth were promoted by treatment of rice seeds (cv. Hitomebore) with hydrogen peroxide solution (H₂O₂) during the imbibition for 24 h. H₂O₂ treatment with 50 mM H₂O₂ promoted seed germination, and seedling growth (shoot length, root length and shoot fresh weight) in agar culture under a low temperature condition (18°C day/14°C night). Seedling growth was promoted by H₂O₂ treatment not only under the low-temperature condition but also under a normal (23°C day/18°C night) temperature condition. Furthermore, H₂O₂ treatment promoted seedling growth under a flooding condition in a greenhouse. These results suggest that H₂O₂ treatment of rice seeds during the imbibition is advantageous for direct seeding. We discussed the relation between the promotion of the seed germinability and the seedling growth under a low-temperature condition, and the expression of some genes encoding ROS scavenger enzymes induced by H₂O₂ treatment.