Protective role of glutathione against oxidative stress in Streptococcus thermophilus

Streptococcus thermophilus can grow in the presence of oxygen and survive at low concentrations of H₂O₂. Glutathione (GSH) plays an important role in living cells, but its protective roles against oxidative stress in S. thermophilus remain unclear. We assessed intracellular GSH accumulation under both anaerobic and aerobic growth conditions in 40 S. thermophilus strains. The GSH level in different strains was found to be strain-specific. A gshF gene, encoding the GSH synthesis, was inactivated in the genome of strain SDMCC18. The growth and survival of the resulting mutant SDMCC18ΔgshF were significantly reduced after exposure to oxygen and H₂O₂. However, the oxidative tolerance of the mutant strain was restored by exogenous GSH. Our findings provide a new strategy to improve the robustness of S. thermophilus in starter manufacture and industrial applications. We, for the first time, demonstrated the GSH synthesis and its transport from the culture medium in S. thermophilus.     

Effect of heat-shock protein B7 on oxidative stress in adipocytes from preruminant calves

Dairy cows with ketosis display excessive lipolysis in adipose tissue. Heat-shock protein B7 (HSPB7), a small heat-shock protein, plays important roles in mediating cytoprotective responses to oxidative stress in rodent adipose tissue. Accordingly, it is assumed that HSPB7 may also play important roles in the antioxidant response in adipose tissue of ketotic cows. Therefore, the aim of this study is to investigate (1) the redox state of adipose tissue in ketotic cows and (2) the role and mechanism of HSPB7 on the regulation of oxidative stress in adipocytes from preruminant calves. An in vivo study consisting of 15 healthy and 15 clinically ketotic cows was performed to harvest subcutaneous adipose tissue and blood samples. In addition, adipocytes isolated from calves were treated with different concentrations of H₂O₂ (0, 12.5, 25, 50, 100, or 200 μM) for 2 h, transfected with adenovirus-mediated overexpression of HSPB7 for 48 h, or transfected with small interfering RNA of HSPB7 for 48 h followed by exposure to H₂O₂ (200 μM) for 2 h. Serum concentrations of nonesterified fatty acids and β-hydroxybutyrate were greater in cows with clinical ketosis, whereas serum concentration of glucose was lower. Compared with healthy cows, the malondialdehyde content was greater but the activity of glutathione peroxidase and superoxide dismutase was lower in adipose tissue of clinically ketotic cows. The abundance of HSPB7 and nuclear factor, erythroid 2 like 2 (NFE2L2) was greater in adipose tissue of clinically ketotic cows. In vitro, H₂O₂ treatment induced the overproduction of reactive oxygen species and malondialdehyde, and inhibited the activity of antioxidant enzymes glutathione peroxidase and superoxide dismutase in adipocytes from preruminant calves. The low concentration of H₂O₂ (12.5, 25, and 50 μM) increased the abundance of HSPB7 and NFE2L2, but high concentrations of H₂O₂ (100 or 200 μM) reduced the abundance of HSPB7 and NFE2L2. The overexpression of HSPB7 improved the H₂O₂-induced oxidative stress in adipocytes via increasing the abundance of NFE2L2 and its downstream target genes heme oxygenase-1 (HMOX1) and NADH quinone oxidoreductase 1 (NQO1). Knockdown of HSPB7 markedly inhibited the expression of NFE2L2, HMOX1, and NQO1 and further exacerbated H₂O₂-induced oxidative stress. Overall, these results indicate that activation of the HSPB7-NFE2L2 pathway increases cellular antioxidant capacity, thereby alleviating oxidative stress in bovine adipocytes.     

Selenomethionine increases proliferation and reduces apoptosis in bovine mammary epithelial cells under oxidative stress

The decline in mammary epithelial cell number as lactation progresses may be due, in part, to oxidative stress. Selenium is an integral component of several antioxidant enzymes. The present study was conducted to examine the effect of oxidative stress and selenomethionine (SeMet) on morphology, viability, apoptosis, and proliferation of bovine mammary epithelial cells (BMEC) in primary culture. Cells were isolated from mammary glands of lactating dairy cows and grown for 3 d in a low-serum gel system containing lactogenic hormones and 0 or 100 μM H₂O₂ with 0, 10, 20, or 50 nM SeMet. Hydrogen peroxide stress increased intracellular H₂O₂ to 3 times control concentrations and induced a loss of cuboidal morphology, cell-cell contact, and viability of BMEC by 25%. Apoptotic cell number more than doubled during oxidative stress, but proliferating cell number was not affected. Supplementation with SeMet increased glutathione peroxidase activity 2-fold and restored intracellular H₂O₂ to control levels with a concomitant return of morphology and viability to normal. Apoptotic BMEC number was decreased 76% below control levels by SeMet and proliferating cell number was increased 4.2-fold. These findings suggest that SeMet modulated apoptosis and proliferation independently of a selenoprotein-mediated reduction of H₂O₂. In conclusion, SeMet supplementation protects BMEC from H₂O₂-induced apoptosis and increased proliferation and cell viability under conditions of oxidative stress.     

Effect of polyphenolic compounds from Coriandrum sativum on H2O2-induced oxidative stress in human lymphocytes

Polyphenolic compounds are widely distributed in plants and known to be excellent antioxidants in vitro. They have the capacity to reduce free-radical formation by scavenging free radicals and protecting antioxidant defences. The present study evaluated the antioxidant potencies of polyphenolic compounds from a spice, Coriandrum sativum against hydrogen peroxide-induced oxidative damage in human lymphocytes. Pretreatment with polyphenolic rich fractions protected human lymphocytes against H₂O₂-induced oxidative damage. H₂O₂ treatment significantly decreased the activities of antioxidant enzymes, such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione-S-transferase and caused decreased glutathione content and increased thiobarbituric acid-reacting substances (TBARS). Treatment with polyphenolic fractions (50 μg/ml) increased the activities of antioxidant enzymes and glutathione content and reduced the levels of TBARS significantly. Observed reduction in the level of lipid peroxides showed a decreased tendency of peroxidative damage. We conclude that, under these experimental conditions, polyphenolic compounds effectively suppress hydrogen peroxide-induced oxidative stress.     

Cytoprotective effects of lotus (Nelumbo nucifera Gaertner) seed extracts on oxidative damaged mouse embryonic fibroblast cell

This study was conducted to evaluate the cytoprotective activity of lotus (Nelumbo nucifera Gaertner) seed extract (LSE) on mouse embryonic fibroblast (MEF) cells. The DPPH free radical scavenging activities of LSE increased in a concentration dependent manner. The cells, damaged by oxidative stress, decreased their viability following increasing concentration of H₂O₂, but the cotreatment of ethyl acetate fraction of LSE and H₂O₂ resulted in an increase in cell growth, by about 25%, compared to the cells treated with H₂O₂. The ethyl acetate fraction of LSE inhibited the cytotoxicity induced by H₂O₂ in a concentration dependent manner. The treatment of the n-butanol fraction of LSE on MEF cell was also examined by analyzing the DNA content and apoptotic rate, using flow cytometry. The oxidative damage to the cells, measured by apoptotic and necrotic cell accumulation, was similar with the addition of the ethyl acetate fraction of LSE to H₂O₂. These results suggest that LSE inhibited the cytotoxicity which is induced by H₂O₂, and has a protective effect on MEF cell against oxidative stress.     

Glycine betaine improves oxidative stress tolerance and biocontrol efficacy of the antagonistic yeast Cystofilobasidium infirmominiatum

The effect of H₂O₂-induced oxidative stress on the viability of the yeast antagonist, Cystofilobasidium infirmominiatum, as well as the effect of exogenous glycine betaine (GB) on yeast viability under oxidative stress, was determined. GB treatment improved the tolerance of C. infirmominiatum to oxidative stress. Compared to untreated control yeast cells, GB-treated cells showed less accumulation of reactive oxygen species (ROS) and a lower level of protein oxidation in response to oxidative stress. Additionally, GB-treated yeast exhibited greater biocontrol activity against Penicillium expansum and a faster growth in wounds of apple fruits stored at 25 °C compared to the performance of untreated yeast. The activities of antioxidant enzymes, including catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPX) of C. infirmominiatum were elevated by GB treatment. Results indicate that the elicitation of antioxidant response by GB may contribute to improvements in oxidative stress tolerance, population growth in apple wounds, and biocontrol activity of C. infirmominiatum.     

Adenosine 5′-monophosphate-activated protein kinase ameliorates bovine adipocyte oxidative stress by inducing antioxidant responses and autophagy

Adipose tissue concentration of reactive oxygen species (ROS) increases in dairy cows with ketosis, suggesting that the tissue experiences oxidative stress. Autophagy, an adaptive response to cellular stress, has been shown to promote survival and plays a critical role in antioxidant responses. Dysregulation of adenosine 5′-monophosphate-activated protein kinase (AMPK) is closely related to antioxidant responses and autophagy of adipocytes in animal models of metabolic disorders, but its role in bovine adipose tissue during periods of stress is unknown. We hypothesized that AMPK may play important roles in the regulation of oxidative stress in adipose tissue of ketotic cows. Specific objectives were to evaluate autophagy status and AMPK activity in adipose tissue of ketotic cows, and their link with oxidative stress in isolated bovine adipocytes. Selection of 15 healthy and 15 clinically ketotic Holstein cows at 17 (±4) d postpartum was performed after a thorough veterinary evaluation for clinical symptoms and also based on serum β-hydroxybutyrate concentrations before collection of subcutaneous adipose tissue samples. Primary cultures of bovine adipocytes isolated from the harvested adipose tissue were stimulated with varying concentrations of H₂O₂ (0, 50, 100, 200, or 400 μM) for 2 h. In another experiment, adipocytes were cultured with the AMPK activator A769662 or adenovirus-containing small interfering RNA (ad-AMPKα-siRNA) for 3 or 48 h, respectively, followed by H₂O₂ exposure (200 μM) for 2 h. Compared with healthy cows, clinical ketosis led to increased abundance of AMPK and nuclear factor erythroid-derived 2-like 2 (NFE2L2), but lower abundance of Kelch-like ECH-associated protein 1 (KEAP1) in adipose tissue. Abundance of the key proautophagy proteins Beclin1, sequestosome 1 (SQSTM1), autophagy-related gene 7 (ATG7), ATG5, and ratio of microtubule-associated protein light chain 3 (LC3) II to LC3I were greater in adipose tissue of ketotic cows. In bovine adipocytes, treatment with H₂O₂ induced accumulation of ROS and malondialdehyde (MDA), whereas H₂O₂ stimulation inhibited activities of the antioxidant enzymes glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD). Addition of AMPK activator A769662 increased antioxidant response via activating NFE2L2 and its downstream targets heme oxygenase 1 (HMOX1), superoxide dismutase 1 (SOD1), catalase (CAT), and glutathione-S-transferase (GST) to improve H₂O₂-induced oxidative stress in adipocytes. Simultaneously, activation of AMPK increased abundance of Beclin1, SQSTM1, ATG7, ATG5, and ratio of LC3II to LC3I. In contrast, inhibition of AMPK downregulated abundance of NFE2L2, HMOX1, SOD1, CAT, Beclin1, SQSTM1, ATG7, ATG5, and ratio of LC3II to LC3I, and further aggravated H₂O₂-induced oxidative stress. Overall, these data indicate that activation of AMPK, as an adaptive mechanism for acute metabolic regulation of adipose tissue homeostasis, can induce antioxidant responses and autophagy, and further reduce oxidative stress in bovine adipocytes.     

Comparison between conjugated linoleic acid and essential fatty acids in preventing oxidative stress in bovine mammary epithelial cells

Some in vitro and in vivo studies have demonstrated protective effects of conjugated linoleic acid (CLA) isomers against oxidative stress and lipid peroxidation. However, only a few and conflicting studies have been conducted showing the antioxidant potential of essential fatty acids. The objectives of the study were to compare the effects of CLA to other essential fatty acids on the thiol redox status of bovine mammary epithelia cells (BME-UV1) and their protective role against oxidative damage on the mammary gland by an in vitro study. The BME-UV1 cells were treated with complete medium containing 50 μM of cis-9,trans-11 CLA, trans-10,cis-12 CLA, α-linolenic acid, γ-linolenic acid, and linoleic acid. To assess the cellular antioxidant response, glutathione, NADPH, and γ-glutamyl-cysteine ligase activity were measured 48 h after addition of fatty acids (FA). Intracellular reactive oxygen species and malondialdehyde production were also assessed in cells supplemented with FA. Reactive oxygen species production after 3 h of H₂O₂ exposure was assessed to evaluate and to compare the potential protection of different FA against H₂O₂-induced oxidative stress. All FA treatments induced an intracellular GSH increase, matched by high concentrations of NADPH and an increase of γ-glutamyl-cysteine ligase activity. Cells supplemented with FA showed a reduction in intracellular malondialdehyde levels. In particular, CLA isomers and linoleic acid supplementation showed a better antioxidant cellular response against oxidative damage induced by H₂O₂ compared with other FA.     

Oxidative stress prevention and anti-apoptosis activity of grape (Vitis vinifera L.) stems in human keratinocytes

To date, grape stems have been partially assessed on their content in phenolics and their radical scavenging activity, whilst the potential to modulate oxidative stress in biological models remains underexplored. In the present work, the effect of grape stems' phenolics on redox unbalance was evaluated in human keratinocytes (HaCaT cells). Grape stems' extracts were assessed on their phenolic composition by high performance liquid chromatography coupled with photodiode array detection and electrospray ionization-mass spectrometry (HPLC–PAD-ESi-MSn), besides on radical scavenging capacity (ABTS and DPPH). In addition, their protective effect against oxidative stress induced by H₂O₂ by the determination of the level of glutathione, reactive oxygen species, lipid peroxidation, and overall oxidative stress in HaCaT cells by flow cytometry was evaluated. This characterization allowed to identify five flavonols, one cinnamic acid, and one stilbene. A close correlation between the concentration of these phenolics and the capacity to scavenge free radicals and with the potential to modulate the redox balance in vitro was observed. From the analysis of correlation, the activity of malvidin-3-O-glucoside, malvidin-3-O-(6-O-caffeoyl)-glucoside, and malvidin-3-O-rutinoside with respect to the prevention of basal oxidative stress and the capacity of isorhamnetin-3-O-(6-O-feruloyl)-glucoside and kaempferol-3-O-rutinoside to prevent H₂O₂-induced redox unbalance were stated. Furthermore, grape stems' phenolics also showed an efficient capacity to modulate apoptosis in HaCaT cells, reducing the frequency of annexin V/PI double positive apoptotic cells by up to 99.5% relative to controls, which was further confirmed by the determination of the appearance of the occurrence of apoptotic bodies and the expression of activated (cleaved) caspase-3 by flow cytometry and western-blot, respectively. These results supported the potential of individual phenolics from grape stems to modulate oxidative stress, allowing to envisage dedicated combinations of single compounds for the development of efficient formulations efficient against oxidative stress.     

Protective effect of Centella asiatica extract and powder on oxidative stress in rats

The effect of Centella asiatica extract and powder in reducing oxidative stress in SpraqueDawley rats was evaluated. Lipid peroxidation was monitored by measuring malonaldehyde (MDA) level in blood. Activities of free radical-scavenging enzymes (superoxide dismutase and catalase) were determined using H₂O₂ decomposition and nitrobluetetrazolium reduction, respectively. Results showed that administration of H₂O₂ (0.1%) in drinking water of the rats, for 25 weeks, increased the malonaldehyde levels in erythrocytes of all the rats. However, rats receiving C. asiatica extract, powder and α-tocopherol had lower MDA levels than did the other rats, which indicates, decrease lipid peroxidation in these rats. Increase in catalase activity of the rats appears to be a response to H₂O₂ accumulation. The decrease in the activity of superoxide dismutase in C. asiatica- and α-tocopherol supplemented rats suggested a lower requirement for the enzyme and this indicates the protective effect of the plant in combating oxidative stress undergone by the rats. Results revealed that C. asiatica extract and powder may ameliorate H₂O₂-induced oxidative stress by decreasing lipid peroxidation via alteration of the antioxidant defence system of the rats.     

Curcumin protects mouse neuroblastoma Neuro-2A cells against hydrogen-peroxide-induced oxidative stress

Curcumin has been traditionally used in China and India for food and medicinal purposes. It has been shown to possess potent antioxidative activity both in vitro and in vivo. In the present study, the neuroprotective effects and the potential mechanisms of curcumin against H₂O₂-induced oxidative stress in mouse neuroblastoma Neuro-2A cells were investigated. Treatment with curcumin at 20 and 25 μg/mL for 1 h prior to H₂O₂ exposure significantly attenuated cell viability loss, reduced apoptosis, suppressed the elevation of intracellular reactive oxygen species (ROS) and calcium levels, and stabilised mitochondrial membrane potential. Furthermore, curcumin could block H₂O₂-mediated degradation of the protein IκBα and subsequent activation of nuclear factor κB, thus inhibiting the expression of its target gene cyclooxygenase 2. These results indicate that curcumin has potential protective effects against H₂O₂-induced oxidative stress in neuron cells, which might make curcumin a suitable therapeutic agent for prevention and treatment of neurodegenerative diseases associated with oxidative stress.     

Protective effects of the crude extracts from yam (Dioscorea alata) peel on tert-butylhydroperoxide-induced oxidative stress in mouse liver cells

Researchers have shown that yam extracts contain antioxidative activity; however, there are few reports regarding the antioxidant activities of yam peel. The effects of water and 50% ethanolic extracts from Darsan yam (Dioscorea alata) peel on the oxidative status of tert-butylhydroperoxide (t-BHP)-treated mouse Hepa 1–6 and FL83B liver cell lines were investigated. The cytosols were analysed for H₂O₂ and malondialdehyde (MDA) levels and antioxidative enzymes activities, including superoxide dismutase, glutathione peroxidase (GPx) and catalase activities. Both water and 50% ethanolic extracts from yam peel did not affect cellular MDA level in t-BHP-treated cells, but they altered the level of H₂O₂. Water extract from yam peel amplified the t-BHP-induced cytotoxicity in Hepa 1–6 whilst the ethanolic extract showed protection in FL83B cells. GPx activity might play an important role in the protective effect associated with t-BHP-induced oxidative stress.     

Sulfonation of Lactobacillus plantarum WLPL04 exopolysaccharide amplifies its antioxidant activities in vitro and in a Caco-2 cell model

Exopolysaccharide (EPS) of Lactobacillus plantarum WLPL04 and its sulfated EPS were systematically investigated for their antioxidant activities and effects on protecting the oxidative damage of Caco-2 cells from H₂O₂. Exopolysaccharide was successfully sulfonated from purified EPS as confirmed by Fourier-transform infrared spectroscopy, and the degree of sulfonation was 0.30. Both EPS and sulfated EPS showed antioxidant activities in vitro determined by 1,1-diphenyl-2-picrylhydrazyl, superoxide, and hydroxyl radical scavenging tests, and those activities of sulfated EPS were significantly enhanced at 1,000 μg/mL. Cell viabilities of Caco-2 in the range of 1 to 100 μg/mL of EPS and sulfated EPS showed no significant difference. In H₂O₂-damaged Caco-2 cells models, EPS and sulfated EPS significantly inhibited the enhancement of reactive oxygen species and malondialdehyde levels, and sulfated EPS enhanced the effects by 40.86% and 61.11% when compared with the purified EPS at the same concentration of 100 μg/mL, respectively. For the activities of antioxidant-related enzymes (superoxide dismutase, catalase, and glutathione peroxidase) and expression of genes (SOD2, GPX2, MT1M) on Caco-2 cells, strong protection abilities against the oxidative stress were displayed from both EPS and sulfated EPS, and sulfated EPS exhibited significant enhancement as compared with either EPS or control groups. In summary, sulfonation is an effective strategy for improving the antioxidant activities of EPS from L. plantarum WLPL04 in vitro and on Caco-2 cells.     

Quercitrin protects against oxidative stress-induced injury in lung fibroblast cells via up-regulation of Bcl-xL

The cytoprotective effect of quercitrin (QR) against oxidative stress induced cell damage by hydrogen peroxide (H₂O₂) in Chinese hamster lung fibroblast (V79-4) cells was investigated. QR evidenced a scavenging effect of 1,1-diphenyl-2-picrylhydrazyl (DPPH), superoxide, hydroxyl radicals and on intracellular ROS, and thus prevented lipid peroxidation. As a result, QR reduced H₂O₂-induced cell death and apoptosis in V79-4 cells. Moreover, H₂O₂ induced the cleavage of caspase-3, -9, and poly-ADP-ribose polymerase (PARP) and a reduction in Bcl-xL levels, whereas pretreatment with QR significantly inhibited caspase-3, -9, and PARP cleavage and the reduction in Bcl-xL levels, and ultimately ameliorated H₂O₂-induced apoptosis. Taken together, these results indicate that the treatment of V79-4 cells with QR can block H₂O₂-induced apoptosis via the regulation of Bcl-xL. QR may be exploited as a biopreservative in food applications or as a health supplement to alleviate oxidative stress.