An iron-based beverage, HydroFerrate fluid (MRN-100), alleviates oxidative stress in murine lymphocytes in vitro

BackgroundSeveral studies have examined the correlation between iron oxidation and H2O2 degradation. The present study was carried out to examine the protective effects of MRN-100 against stress-induced apoptosis in murine splenic cells in vitro. MRN-100, or HydroFerrate fluid, is an iron-based beverage composed of bivalent and trivalent ferrates.MethodsSplenic lymphocytes from mice were cultured in the presence or absence of MRN-100 for 2 hrs and were subsequently exposed to hydrogen peroxide (H2O2) at a concentration of 25 μM for 14 hrs. Percent cell death was examined by flow cytometry and trypan blue exclusion. The effect of MRN-100 on Bcl-2 and Bax protein levels was determined by Western blot.ResultsResults show, as expected, that culture of splenic cells with H2O2 alone results in a significant increase in cell death (apoptosis) as compared to control (CM) cells. In contrast, pre-treatment of cells with MRN-100 followed by H2O2 treatment results in significantly reduced levels of apoptosis. In addition, MRN-100 partially prevents H2O2-induced down-regulation of the anti-apoptotic molecule Bcl-2 and upregulation of the pro-apoptotic molecule Bax.ConclusionOur findings suggest that MRN-100 may offer a protective effect against oxidative stress-induced apoptosis in lymphocytes

S-allylmercaptocysteine reduces carbon tetrachloride-induced hepatic oxidative stress and necroinflammation via nuclear factor kappa B-dependent pathways in mice

Purpose To study the protective effects and underlying molecular mechanisms of SAMC on carbon tetrachloride (CCl4)-induced acute hepatotoxicity in the mouse model. Methods Mice were intraperitoneally injected with CCl4 (50 μl/kg; single dose) to induce acute hepatotoxicity with or without a 2-h pre-treatment of SAMC intraperitoneal injection (200 mg/kg; single dose). After 8 h, the blood serum and liver samples of mice were collected and subjected to measurements of histological and molecular parameters of hepatotoxicity. Results SAMC reduced CCl4-triggered cellular necrosis and inflammation in the liver under histological analysis. Since co-treatment of SAMC and CCl4 enhanced the expressions of antioxidant enzymes, reduced the nitric oxide (NO)-dependent oxidative stress, and inhibited lipid peroxidation induced by CCl4. SAMC played an essential antioxidative role during CCl4-induced hepatotoxicity. Administration of SAMC also ameliorated hepatic inflammation induced by CCl4 via inhibiting the activity of NF-κB subunits p50 and p65, thus reducing the expressions of pro-inflammatory cytokines, mediators, and chemokines, as well as promoting pro-regenerative factors at both transcriptional and translational levels. Conclusions Our results indicate that SAMC mitigates cellular damage, oxidative stress, and inflammation in CCl4-induced acute hepatotoxicity mouse model through regulation of NF-κB. Garlic or garlic derivatives may therefore be a potential food supplement in the prevention of liver damage

Phosphorylation of p65(RelA) on Ser547 by ATM Represses NF-κB-Dependent Transcription of Specific Genes after Genotoxic Stress

The NF-κB pathway is involved in immune and inflammation responses, proliferation, differentiation and cell death or survival. It is activated by many external stimuli including genotoxic stress. DNA double-strand breaks activate NF-κB in an ATM-dependent manner. In this manuscript, a direct interaction between p65(RelA) and the N-terminal extremity of ATM is reported. We also report that only one of the five potential ATM-(S/T)Q target sites present in p65, namely Ser547, is specifically phosphorylated by ATM in vitro. A comparative transcriptomic analysis performed in HEK-293 cells expressing either wild-type HA-p65 or a non-phosphorylatable mutant HA-p65S547A identified several differentially transcribed genes after an etoposide treatment (e.g. IL8, A20, SELE). The transcription of these genes is increased in cells expressing the mutant. Substitution of Ser547 to alanine does not affect p65 binding abilities on the κB site of the IL8 promoter but reduces p65 interaction with HDAC1. Cells expressing p65S547A have a higher level of histone H3 acetylated on Lys9 at the IL8 promoter, which is in agreement with the higher gene induction observed. These results indicate that ATM regulates a sub-set of NF-κB dependent genes after a genotoxic stress by direct phosphorylation of p65

Butyrate Attenuates Lipopolysaccharide-Induced Inflammation in Intestinal Cells and Crohn's Mucosa through Modulation of Antioxidant Defense Machinery

Oxidative stress plays an important role in the pathogenesis of inflammatory bowel disease (IBD), including Crohn's disease (CrD). High levels of Reactive Oxygen Species (ROS) induce the activation of the redox-sensitive nuclear transcription factor kappa-B (NF-κB), which in turn triggers the inflammatory mediators. Butyrate decreases pro-inflammatory cytokine expression by the lamina propria mononuclear cells in CrD patients via inhibition of NF-κB activation, but how it reduces inflammation is still unclear. We suggest that butyrate controls ROS mediated NF-κB activation and thus mucosal inflammation in intestinal epithelial cells and in CrD colonic mucosa by triggering intracellular antioxidant defense systems. Intestinal epithelial Caco-2 cells and colonic mucosa from 14 patients with CrD and 12 controls were challenged with or without lipopolysaccaride from Escherichia Coli (EC-LPS) in presence or absence of butyrate for 4 and 24 h. The effects of butyrate on oxidative stress, p42/44 MAP kinase phosphorylation, p65-NF-κB activation and mucosal inflammation were investigated by real time PCR, western blot and confocal microscopy. Our results suggest that EC-LPS challenge induces a decrease in Gluthation-S-Transferase-alpha (GSTA1/A2) mRNA levels, protein expression and catalytic activity; enhanced levels of ROS induced by EC-LPS challenge mediates p65-NF-κB activation and inflammatory response in Caco-2 cells and in CrD colonic mucosa. Furthermore butyrate treatment was seen to restore GSTA1/A2 mRNA levels, protein expression and catalytic activity and to control NF-κB activation, COX-2, ICAM-1 and the release of pro-inflammatory cytokine. In conclusion, butyrate rescues the redox machinery and controls the intracellular ROS balance thus switching off EC-LPS induced inflammatory response in intestinal epithelial cells and in CrD colonic mucosa

Dual Beneficial Effects of (-)-Epigallocatechin-3-Gallate on Levodopa Methylation and Hippocampal Neurodegeneration: In Vitro and In Vivo Studies

A combination of levodopa (L-DOPA) and carbidopa is the most commonly-used treatment for symptom management in Parkinson's disease. Studies have shown that concomitant use of a COMT inhibitor is highly beneficial in controlling the wearing-off phenomenon by improving L-DOPA bioavailability as well as brain entry. The present study sought to determine whether (-)-epigallocatechin-3-gallate (EGCG), a common tea polyphenol, can serve as a naturally-occurring COMT inhibitor that also possesses neuroprotective actions.Using both in vitro and in vivo models, we investigated the modulating effects of EGCG on L-DOPA methylation as well as on chemically induced oxidative neuronal damage and degeneration. EGCG strongly inhibited human liver COMT-mediated O-methylation of L-DOPA in a concentration-dependent manner in vitro, with an average IC50 of 0.36 microM. Oral administration of EGCG moderately lowered the accumulation of 3-O-methyldopa in the plasma and striatum of rats treated with L-DOPA+carbidopa. In addition, EGCG also reduced glutamate-induced oxidative cytotoxicity in cultured HT22 mouse hippocampal neuronal cells through inactivation of the nuclear factor kappaB-signaling pathway. Under in vivo conditions, administration of EGCG exerted a strong protective effect against kainic acid-induced oxidative neuronal death in the hippocampus of rats.These observations suggest that oral administration of EGCG may have significant beneficial effects in Parkinson's patients treated with L-DOPA and carbidopa by exerting a modest inhibition of L-DOPA methylation plus a strong neuroprotection against oxidative damage and degeneration

Nrf2 protects against pulmonary fibrosis by regulating the lung oxidant level and Th1/Th2 balance

<p>Abstract</p> <p>Background</p> <p>Pulmonary fibrosis is a progressive and lethal disorder. Although the precise mechanisms of pulmonary fibrosis are not fully understood, oxidant/antioxidant and Th1/Th2 balances may play an important role in many of the processes of inflammation and fibrosis. The transcription factor Nrf2 acts as a critical regulator for various inflammatory and immune responses by controlling oxidative stress. We therefore investigated the protective role of Nrf2 against the development of pulmonary fibrosis.</p> <p>Methods</p> <p>To generate pulmonary fibrosis, both wild-type C57BL/6 mice and Nrf2-deficient mice of the same background were administered bleomycin intratracheally.</p> <p>Results</p> <p>The survival of Nrf2-deficient mice after bleomycin administration was significantly lower than that of wild-type mice. The degree of bleomycin-induced initial pulmonary inflammation and pulmonary fibrosis was much more severe in Nrf2-deficient mice than in wild-type mice. The expression of antioxidant enzymes and phase II detoxifying enzymes was significantly reduced in the lungs of Nrf2-deficient mice, concomitant with an elevation of lung 8-isoprostane level, compared with wild-type mice. The expression of Th2 cytokines, such as interleukin-4 and interleukin-13, was significantly elevated in the lungs of Nrf2-deficient mice with an increase in the number of Th2 cells that express GATA-binding protein 3.</p> <p>Conclusions</p> <p>The results indicated that Nrf2 protects against the development of pulmonary fibrosis by regulating the cellular redox level and lung Th1/Th2 balance. Thus, Nrf2 might be an important genetic factor in the determination of susceptibility to pulmonary fibrosis.</p

Dysfunction of Nrf-2 in CF Epithelia Leads to Excess Intracellular H2O2 and Inflammatory Cytokine Production

Cystic fibrosis is characterized by recurring pulmonary exacerbations that lead to the deterioration of lung function and eventual lung failure. Excessive inflammatory responses by airway epithelia have been linked to the overproduction of the inflammatory cytokine IL-6 and IL-8. The mechanism by which this occurs is not fully understood, but normal IL-1β mediated activation of the production of these cytokines occurs via H2O2 dependent signaling. Therefore, we speculated that CFTR dysfunction causes alterations in the regulation of steady state H2O2. We found significantly elevated levels of H2O2 in three cultured epithelial cell models of CF, one primary and two immortalized. Increases in H2O2 heavily contributed to the excessive IL-6 and IL-8 production in CF epithelia. Proteomic analysis of three in vitro and two in vivo models revealed a decrease in antioxidant proteins that regulate H2O2 processing, by ≥2 fold in CF vs. matched normal controls. When cells are stimulated, differential expression in CF versus normal is enhanced; corresponding to an increase in H2O2 mediated production of IL-6 and IL-8. The cause of this redox imbalance is a decrease by ∼70% in CF cells versus normal in the expression and activity of the transcription factor Nrf-2. Inhibition of CFTR function in normal cells produced this phenotype, while N-acetyl cysteine, selenium, an activator of Nrf-2, and the overexpression of Nrf-2 all normalized H2O2 processing and decreased IL-6 and IL-8 to normal levels, in CF cells. We conclude that a paradoxical decrease in Nrf-2 driven antioxidant responses in CF epithelia results in an increase in steady state H2O2, which in turn contributes to the overproduction of the pro-inflammatory cytokines IL-6 and IL-8. Treatment with antioxidants can ameliorate exaggerated cytokine production without affecting normal responses

Reconciling conflicting clinical studies of antioxidant supplementation as HIV therapy: a mathematical approach

<p>Abstract</p> <p>Background</p> <p>Small, highly reactive molecules called reactive oxygen species (ROS) play a crucial role in cell signalling and infection control. However, high levels of ROS can cause significant damage to cell structure and function. Studies have shown that infection with the human immunodeficiency virus (HIV) results in increased ROS concentrations, which can in turn lead to faster progression of HIV infection, and cause CD4⁺T-cell apoptosis. To counteract these effects, clinical studies have explored the possibility of raising antioxidant levels, with mixed results.</p> <p>Methods</p> <p>In this paper, a mathematical model is used to explore this potential therapy, both analytically and numerically. For the numerical work, we use clinical data from both HIV-negative and HIV-positive injection drug users (IDUs) to estimate model parameters; these groups have lower baseline concentrations of antioxidants than non-IDU controls.</p> <p>Results</p> <p>Our model suggests that increases in CD4⁺T cell concentrations can result from moderate levels of daily antioxidant supplementation, while excessive supplementation has the potential to cause periods of immunosuppression.</p> <p>Conclusion</p> <p>We discuss implications for HIV therapy in IDUs and other populations which may have low baseline concentrations of antioxidants.</p

Disulfiram modulated ROS–MAPK and NFκB pathways and targeted breast cancer cells with cancer stem cell-like properties

BACKGROUND: Previous studies indicate that disulfiram (DS), an anti-alcoholism drug, is cytotoxic to cancer cell lines and reverses anticancer drug resistance. Cancer stem cells (CSCs) are the major cause of chemoresistance leading to the failure of cancer chemotherapy. This study intended to examine the effect of DS on breast cancer stem cells (BCSCs). METHODS: The effect of DS on BC cell lines and BCSCs was determined by MTT, western blot, CSCs culture and CSCs marker analysis. RESULTS: Disulfiram was highly toxic to BC cell lines in vitro in a copper (Cu)-dependent manner. In Cu-containing medium (1 mu M), the IC50 concentrations of DS in BC cell lines were 200-500 nM. Disulfiram/copper significantly enhanced (3.7-15.5-fold) cytotoxicity of paclitaxel (PAC). Combination index isobologram analysis demonstrated a synergistic effect between DS/Cu and PAC. The increased Bax and Bcl2 protein expression ratio indicated that intrinsic apoptotic pathway may be involved in DS/Cu-induced apoptosis. Clonogenic assay showed DS/Cu-inhibited clonogenicity of BC cells. Mammosphere formation and the ALDH1(+VE) and CD24(Low)/CD44(High) CSCs population in mammospheres were significantly inhibited by exposure to DS/Cu for 24 h. Disulfiram/copper induced reactive oxygen species (ROS) generation and activated its downstream apoptosis-related cJun N-terminal kinase and p38 MAPK pathways. Meanwhile, the constitutive NF kappa B activity in BC cell lines was inhibited by DS/Cu. CONCLUSION: Disulfiram/copper inhibited BCSCs and enhanced cytotoxicity of PAC in BC cell lines. This may be caused by simultaneous induction of ROS and inhibition of NF kappa B. British Journal of Cancer (2011) 104, 1564-1574. doi: 10.1038/bjc.2011.126 www.bjcancer.com Published online 12 April 2011 (C) 2011 Cancer Research U