A method for correctly assessing hydroxyl radical scavenging activity of antioxidative chemicals and/or biological compounds/materials was proposed. This method can simultaneously assess two factors, i.e. hydroxyl radical-scavenging and 5,5-dimethyl-2-hydroxy-1-pyrrolidine-N-oxide (hydroxyl radical adduct of 5,5-dimethyl-1-pyrroline-N-oxide)-reducing ability, as antioxidative properties. In this paper, some biologically common hydrophilic molecules, cell culture media, and rat plasma were tested. X-ray-induced hydroxyl radical can be detected using the electron paramagnetic resonance spin trapping technique. Using X-ray irradiation of the reaction mixture as the hydroxyl radical source, the true hydroxyl radical-scavenging ability of the subjected antioxidant can be assessed. In addition, the method simultaneously measures the reduction of 5,5-dimethyl-2-hydroxy-1-pyrrolidine-N-oxide, to estimate the reducing ability of the antioxidant. Biological materials, such as sugars and proteins, could abolish hydroxyl radical at the biological concentration. Ascorbic acid showed reducing ability at the biological concentration. The simultaneous assessment of hydroxyl radical-scavenging and reducing ability of antioxidants can be an informative index for antioxidants.
Endoplasmic reticulum stress is related to metabolic disorders, including atherosclerosis and type 2 diabetes. It is known that inflammatory adipocytokines and oxidative stress are increased, while anti-inflammatory adipocytokines such as adiponectin are decreased in adipocytes during above conditions. Extracellular-superoxide dismutase is an anti-inflammatory enzyme that protects cells from oxidative stress. Because plasma extracellular-superoxide dismutase levels in type 2 diabetes patients were inversely related to the body mass index and homeostasis model assessment-insulin resistance index, it is speculated that the regulation of extracellular-superoxide dismutase might lead to the suppression of metabolic disorders. Here, we observed the reduction of extracellular-superoxide dismutase and adiponectin in 3T3-L1 adipocytes treated with thapsigargin, an endoplasmic reticulum stress inducer. Interestingly, tunicamycin, another endoplasmic reticulum stress inducer, did not decrease the expression of extracellular-superoxide dismutase in spite of the induction of glucose regulated protein kinase 78 kDa, an endoplasmic reticulum stress marker. Moreover, eukaryotic translation initiation factor 2α signaling cascade plays a pivotal role in the reduction of extracellular-superoxide dismutase in 3T3-L1 adipocytes during endoplasmic reticulum stress conditions.
AMP-activated protein kinase (AMPK), a crucial regulator of energy metabolic homeostasis, is suggested to regulate inflammatory responses, but its precise mechanisms are not fully understood. It has been reported that pharmacological activation of AMPK induces heme oxygenase-1 (HO-1) expression. β-Lapachone (BL), a well-known substrate of NAD(P)H:quinone oxidoreductase (NQO1), has been demonstrated to stimulate AMPK activation via NQO1 activation, and to exert anti-inflammatory effects in macrophages. Here we examined whether AMPK activation by BL would be linked to HO-1 expression in RAW264.7 macrophages and whether HO-1 expression could mediate the anti-inflammatory effects of BL. BL treatment induced concentration- and time-dependent AMPK phosphorylation and HO-1 expression. 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, an AMPK activator, also induced HO-1 expression. In contrast, compound C (CC), an inhibitor of AMPK activation, prevented the increase in BL-induced HO-1 expression. BL pretreatment reduced lipopolysaccharide-induced production of tumor necrosis factor-α, a pro-inflammatory cytokine, and expression of inducible nitric oxide synthase, a pro-inflammatory enzyme. These inhibitory effects BL were almost completely abolished by CC and partly by tin protoporphyrin-IX, a competitive inhibitor of HO-1. Accordingly, the present results indicate that BL induces anti-inflammatory HO-1 expression in macrophages via AMPK activation, providing one of possible mechanisms by which BL can exert anti-inflammatory effects.
Apurinic/apyrimidinic endonuclease-1 (APE-1), a key enzyme responsible for DNA base excision repair (BER), has been linked to cancer chemoradiosensitivity. The phosphorylation of p65 plays a role in the activation of this pathway. In this study, we investigated APE-1 expression and its interaction with p65 in esophageal squamous cell carcinoma (ESCC) tissue. The expression of APE-1, p65, p65 nuclear localization sequence (p65-NLS), and monocyte chemoattractant protein-1 (MCP-1) was assessed by immunohistochemical analysis in 67 human ESCC tissue samples. Real-time PCR and western blotting were also performed. p65 siRNA was evaluated to determine the role of p65 in the regulation of APE-1 expression. We found nuclear localization of APE-1 in 89.6% (60/67) of ESCC tissue samples. We also observed the colocalization of p65-NLS and APE-1 in esophageal cancer tissue. In KYSE220 cells, pretreatment of MG-132 significantly abrogated upregulation of p65 and APE-1 levels induced by MCP-1, and treatment with 10 and 20 nM p65 siRNA significantly inhibited APE-1 mRNA expression. siRNA for p65 treatment significantly increased the apoptotic index in 5-FU-treated KYSE220 cells. We conclude that APE-1 is overexpressed and mainly localized in the nuclear compartment of cancer cells, and partly regulated by p65 in the NF-κB pathway in ESCC tissue.
Adipose tissue functions as a key endocrine organ by releasing multiple bioactive substances, and plays a key role in the integration of systemic metabolism. We have previously shown that B cell activating factor is produced mainly in visceral adipose tissue and affects insulin sensitivity in obese individuals. In this study, we identified the signals that lead to production of B cell activating factor in adipocytes. 3T3-L1 and C3H/10T 1/2-clone 8 cells showed increased B cell activating factor expression upon exposure to hydrogen peroxide, and these changes were inhibited by treatment with the antioxidant N-acetyl-cysteine. B cell activating factor levels in both serum and visceral adipose tissue were increased in high fat diet-fed mice, and these increases were correlated with oxidative stress. In addition, serum BAFF levels in high fat diet-fed mice were reduced by N-acetyl-cysteine treatment. We also found that oxidative stress-induced B cell activating factor expression in adipocytes was regulated by NF-κB activation. These data indicate that control of the redox state in visceral adipose tissue is a potentially useful target for treating metabolic syndromes through regulation of adipokine production.
The reactions of three α-oxoaldehydes (methylglyoxal, glyoxal, and pyruvic acid) with hydroxyl radicals generated by sonolysis of water were investigated using an electron spin resonance (electron paramagnetic resonance) spin-trapping method, and their reaction kinetics were investigated. It is apparent from our experimental results that methylglyoxal exhibits the highest reactivity of the three α-oxoaldehydes. These α-oxoaldehydes can react with hydroxyl radicals faster than other well-known antioxidants can. The reactivity of hydroxyl radicals is higher than that of hydrogen peroxides.
Alzheimer’s disease (AD) is the most common cause of neurodegenerative dementia among elderly patients. A biomarker for the disease could make diagnosis easier and more accurate, and accelerate drug discovery. In this study, NMR-based metabolomics analysis in conjunction with multivariate statistics was applied to examine changes in urinary metabolites in transgenic AD mice expressing mutant tau and β-amyloid precursor protein. These mice showed significant changes in urinary metabolites throughout the progress of the disease. Levels of 3-hydroxykynurenine, homogentisate and allantoin were significantly higher compared to control mice in 4 months (prior to onset of AD symptoms) and reverted to control values by 10 months of age (early/middle stage of AD), which highlights the relevance of oxidative stress to this neurodegenerative disorder even prior the onset of dementia. The level of these changed metabolites at very early period may provide an indication of disease risk at asymptomatic stage.
Recently, we administered fish oil containing eicosapentaenoic acid and docosahexaenoic acid (DHA) to senescence-accelerated mice P8 (SAMP8), in order to investigate the effects on lifespan. Surprisingly, the lifespan of SAMP8 that were fed fish oil was shortened significantly, through a mechanism that likely involved lipid peroxidation. In this study, we investigated this phenomenon in further detail. To examine whether this phenomenon occurs only in SAMP8, we investigated the effect of fish oil on the lifespan of another organism species, Caenorhabditis elegans (C. elegans). C. elegans fed fish oil were cultured and the lifespan monitored. As a consequence of the provision of large amounts of fish oil the lifespan of C. elegans was shortened significantly, whereas an appropriate amount of fish oil extended their lifespan significantly. Lipid peroxide levels in C. elegans that were fed fish oil increased significantly in a dose-dependent manner. However, lipid peroxide levels in C. elegans were inhibited by the addition of fish oil and an antioxidant, α-tocopherol, and completely abrogated the changes in the lifespan. To further confirm whether the oxidation of n-3 polyunsaturated fatty acid in fish oil would change the lifespan of C. elegans, the effect of oxidized DHA was examined. Large amounts of oxidized DHA were found to shorten their lifespan significantly. Thus, fish oil changes the lifespan of C. elegans through lipid peroxidation.
It has been reported that α-tocopherol (α-Toc), a vitamin E analog, is effective for treatment of non-alcoholic steatohepatitis (NASH). However, it is unknown whether or not other vitamin E analogs are effective. Therefore we designed a new rat model of steatohepatitis induced by tumor necrosis factor-α (TNF-α) stimulation, and used it to investigate the effects of vitamin E analogs. The rat liver triglyceride content increased with the dosage of TNF-α/d-galactosamine (GalN), but was suppressed by intake of both tocotrienol (T3) and α-tocopherol. Moreover, lipid peroxides (thiobarbituric acid-reactive substances) level in the liver level was also lower in both groups after tocotrienol and α-Toc intake. Intake of both tocotrienol and α-tocopherol also tended to control the increase of liver damage marker activity. In the tocotrienol and α-tocopherol groups, increases of inflammatory cytokines mRNA expression in the liver were inhibited, and these effects were considered to contribute to improvement of inflammation and fibrosis. The expression of mRNAs for inflammatory cytokines in rat primary hepatocytes was increased by TNF-α stimulation, but was inhibited by addition of α-tocotrienol and γ-tocotrienol. Transforming growth factor-β1 mRNA expression in particular was significantly inhibited by γ-tocotrienol. These findings suggest that tocotrienol species are effective for amelioration of steatohepatitis, and that tocotrienol and α-tocopherol exert a synergistic effect.
Although recruited neutrophils function as first-line defense to remove bacteria, delayed apoptosis is implicated in persistent inflammation leading to organ injury. Leukotrien B4, n-6 polyunsaturated fatty acids (PUFAs) product, is one of the mediators that delay neutrophil apoptosis. The mechanism of the beneficial effects of supplementation of fish oil-based long-chain n-3 PUFAs in parenteral nutrition for critically ill patients has not been fully understood. One possible mechanism is the less inflammatory n-3 PUFAs products can compete with proinflammatory n-6 PUFAs products for access to the enzymes. The aim of this study was to determine whether n-3 PUFA rich parenteral nutrition may alter the composition of fatty acids in the neutrophil membrane and restore delay of neutrophil apoptosis during endotoxin-induced systemic inflammation in rats. The animals in group 1 were treated with 20% Hicaliq NC-N in Neoparen-2 for three days. The animals in group 2 (referred to as n-6 PUFA-rich parenteral nutrition) were given parenteral nutrition solutions containing 20% soybean oil in Neoparen-2 (n-6/n-3 = 10). The animals in group 3 (referred to as n-3 PUFA-rich parenteral nutrition) were administered parenteral nutrition consisting of 10% soybean oil and 10% fish oil emulsion (n-6/n-3 = 1.3). The n-3/n-6 ratio of the neutrophil membrane was significantly increased in group 3 and was associated with restored lipopolysaccharide-delayed-apoptosis of neutrophils in bone marrow cells and increased production of leukotriene B5 from peritoneal neutrophils stimulated by lipopolysaccharide. Our preliminary results showed that n-3 PUFA-rich parenteral nutrition regulated neutrophil apoptosis and prevented synthesis of pro-inflammatory eicosanoids, explaining the protective effects seen in the clinical setting.
Serum ceruloplasmin (CP), a marker relevant to copper metabolism, is one of famous inflammation markers with a reduction in Wilson’s disease, whereas serum ferritin is a marker relevant to iron metabolism. Recently, ferritin is pointed out to be related with oxidative stress. However, there is still no population research which showed the relation of CP and ferritin. Therefore, we investigated the relationship between CP and ferritin including oxidative stress biomarkers among healthy Japanese (n = 389). We measured serum CP, ferritin, Fe, high-sensitivity C-reactive protein (hs-CRP), and urinary oxidative stress biomarkers [H2O2, 8-hydroxy-2’-deoxyguanosine (8-OHdG), 8-isoprostane] and so on. Subjects showed that age; 41.7 ± 10.0 (year), CP; 31.9 ± 6.8 (mg/dl), ferritin; 123.5 ± 121.0 (ng/ml), hs-CRP; 0.89 ± 2.53 (mg/l), 8-OHdG; 10.2 ± 4.4 [ng/mg creatinine (Cre)] and H2O2; 6.5 ± 10.9 (µM/g Cre), (All data mentioned above were expressed as mean ± SD). CP was significantly and positively correlated with hs-CRP and inversely correlated with ferritin, Fe and 8-OHdG. By a multiple logistic regression analysis, odds ratio of CP according to quartiles of hs-CRP was 4.86, and according to quartiles of 8-OHdG was 0.39 after adjusting for age and other confounding factors. In conclusion, our findings suggest that CP was an antioxidative biomarker which controls oxidative stress, whereas ferritin was a marker which may participate in the generation of oxidative stress.
Although there is evidence that vitamin D deficiency relates to expression of chronic disease, relationship between vitamin D and cardiovascular disease predictors have not been clearly demonstrated in Korean. Our objective was to assess the correlation between vitamin D and the cardiovascular and inflammatory markers in overweight and obese people who had not been exposed to a particular disease. We enrolled 171 healthy adults (159 men and 12 pre-menopausal women) with no history of cardiovascular disease and with a body mass index >23 kg/m2 in this study. In addition, levels of serum vitamin D and concentrations of the inflammatory markers hs-CRP, interleukin-6, and adiponectin were measured. The average age of our study subjects was 48.53 years old, and 64.8% of all male subjects and 91.9% of all female subjects were in the vitamin D deficient status. Serum vitamin D levels showed a positive correlation with age (p<0.05), HDL-cholesterol (p<0.05), and adiponectin (p<0.05) levels. However, there was a negative correlation of vitamin D with triglyceride (p<0.01) and interleukin-6 levels (p<0.05). In addition, even after adjusting for factors that may affect the cardiovascular index (age, sex, body mass index, smoking, and alcohol intake), serum vitamin D levels showed a significant correlation with triglyceride (p<0.05), HDL-cholesterol (p<0.05), and adiponectin (p<0.05) levels. Therefore, the results of this study suggest that vitamin D may be a predictor of cardiovascular disease for overweight and obese people who are likely to be at a risk for cardiovascular disease.
The relationship between Helicobacter pylori (H. pylori) eradication therapy and the risk of developing gastroesophageal reflux disease (GERD) is controversial. We investigated the influence of H. pylori eradication on the risk of GERD by focusing on the quality of life (QOL) and evaluating reflux symptoms. Patients with H. pylori infection were administered triple therapy for H. pylori eradication. At 3 months and 1 year after the eradication therapy, surveys were conducted to determine the health-related QOL by quality of life in reflux and dyspepsia-Japanese version, (QOLRAD-J) and the severity of GERD symptoms by Carlsson-Dent questionnaire (CDQ). Forty patients were included in the analysis. Although no significant changes of these scores were apparent 3 months after H. pylori eradication, the QOLRAD-J and CDQ scores were significantly improved after 1 year. The degree of improvement was even more marked in cases with initially low scores. In conclusion, improved GERD-related QOL and reflux symptoms were noted 1 year after H. pylori eradication therapy. In addition, the degree of improvement was more marked in cases with severe reflux symptoms.
Gastroesophageal reflux disease (GERD) is common in patients with many chronic diseases, but has not been well recognized in rheumatoid arthritis (RA). We investigated the prevalence of GERD symptoms in 278 outpatients with RA and their association with such clinical factors as age, sex, height, weight, body mass index, medications drugs, and functional status evaluated by the Modified Health Assessment Questionnaire (MHAQ). GERD symptoms were evaluated by Frequency Scale for the Symptoms of GERD (FSSG). The mean FSSG score for all patients was 5.6, and 82 patients were considered to have GERD symptoms (FSSG score ≥8), thus the overall prevalence of GERD symptoms was 29.5%. MHAQ score and height were significantly higher and lower, respectively, and prednisolone usage was significantly more in the patients with GERD symptoms than those without. These three clinical factors were also significantly associated with GERD symptoms by univariate logistic regression. Multivariate logistic regression analysis demonstrated that MHAQ was the only clinical factor related to GERD symptoms. In conclusion, the prevalence of GERD symptoms in RA patients was high and strongly associated with decreased functional status, suggesting that physicians should pay attention to GERD symptoms in RA management, especially for patients with low functional status.