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Chronic Ethanol Consumption Alters SOD2 Dynamics through Lysine Acetylation
by AAS abuse.
doi: 10.1016/j.freeradbiomed.2016.10.244 204 Characterising the Reactions of Diselenides with Reactive Oxidants Generated During Chronic Inflammation Kelly A Gardiner1,2, Michael J Davies1,2,3, and David I Pattison1,2 1 The Heart Research Institute, Sydney, Australia, 2Sydney Medical School, Australia, 3Panum Institute, Copenhagen, Denmark Introduction: Activated leukocytes present at sites of injury or infection releases enzymes that produce reactive oxidants including hydrogen peroxide (H2O2), hypochlorous acid (HOCl), singlet oxygen (1O2), various peroxides and free radicals. These oxidants play a key role in killing pathogens but excessive or inappropriate formation can give rise to host tissue damage and oxidative stress, and this been associated with multiple human pathologies. Recent kinetic data have shown selenium-containing compounds (selenols RSeH, and selenides RSeR) react rapidly with multiple oxidants and are readily recycled (by GSH and enzymes) to their reduced form, allowing catalytic activity. Hypothesis: Readily available diselenides (RSeSeR) may act as highly efficient oxidant scavengers of biologically-relevant oxidants. Results: The diselenides selenocystamine (SeCA), diselenodipropionic acid (DSePA) and glutathione diselenide (GSeSeG) react rapidly with HOCl, H2O2 and a range of organic hydroperoxides as assessed via UV/vis spectroscopy. These reactions are more rapid with HOCl than with H2O2, with the second order rate constants for reaction with H2O2 being in the range 102 (for DSePA) to ~10-4 M-1 s-1 (SeCA and GSeSeG). The oxidation products of the fluorescently-labeled diselenide, Fmocselenocystine, have been characterised using UPLC and LCMS/MS. Oxidation by HOCl and H2O2 results in cleavage of the diselenide bond (with a loss of absorption at 300 nm) and formation of low molecular mass species in a dose- and time-dependent manner. The seleninic acid (RSeO2H) is a major oxidation product. Conclusions: Readily available and stable diselenides are rapidly oxidised by biologically-relevant oxidants, and may act as efficient oxidant scavengers in vivo, thereby ameliorating oxidative damage associated with inflammatory diseases.
doi: 10.1016/j.freeradbiomed.2016.10.245 205 Gambusia Punctata (Poeciliidae) as Alternative Biomodel in Oxidative Stress Evaluation for Ecotoxicological and Pharmaceutical Applications Frenkel Guisado Bourzac1, Juan Carlos Moreno Guisado1, Alina González Marañón1, Jesús Rafael Rodríguez Amado2, and Raimundo Nonato Picanço Souto2 1 University of Oriente, Santiago, Cuba, 2Universidade Federal do Amapá, Macapá, Brazil Background: Fish are sensitive organisms to changes caused by oxidative stress inducing agents, due to expression of biological changes based on the quality of the environment at cellular,
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individual, populations or community level. Objective: To evaluate Gambusia punctata as an alternative biomodel for evaluating physiological response facing oxidative stress and changes in cell REDOX potential. Method: Different criteria were analyzed: ubiquitous distribution, easy taxonomic identification, appropriate size, limited mobility, relatively long life cycle, trophic chain position, ecological relevance, tolerance to contamination and acceptable survival facing environmental variations, easy capture and handling for laboratory studies and proven correlation between environmental pollution and physiological response due to metals, inorganic and organic pollutants bioaccumulation, among others. Results: Molecular biomarkers response assessed for G. punctata is consistent for regular assessment of cell damage and antioxidant response determinations. Increase in multi-pollutants levels for the initial “in situ” evaluation, followed by Conductivity and Total Dissolved Solids (TDS), was positively correlated with molecular determinations (total dissolved proteins, NAD(P)H, H2O2, ascorbate, like-MDA reaction products) and physiological changes. Ionic strength increase in historic records is mainly based in freshwater bodies content of Na+, K+, Cl–, Mg2+, SO42–, HCO3–, NO3– , NO2–, NH4+, PO43–, and significant and persistent content in metals through water course such as Al, Ni and Mo due to anthropic effects. Biomarkers response reflects the cell damage for G. punctata and its antioxidant response. These initial results reveal this fish potentiality as alternative oxidative stress biomodel. Molecular criteria are useful for both, “in situ” evaluation for biomonitoring ecosystems health, “in vitro” early analytical stages of toxicological studies for drugs and chemical exposure and pharmaceutical studies. Conclusion: G. punctata fulfil basic standards for its use as alternative biomodel, allowing oxidative stress evaluation due to its adaptative response.
doi: 10.1016/j.freeradbiomed.2016.10.246 206 Chronic Ethanol Consumption Alters SOD2 Dynamics through Lysine Acetylation Mohammed Assiri1, Peter S Harris1, Hadi Ali1, Yongliang Liang2, Colin T Shearn1, James R Roede1, Donald S Backos1, and Kristofer S Fritz1 1 University of Colorado, Aurora, USA, 2Emory University, Atlanta, USA Chronic ethanol consumption is a significant source of morbidity and mortality worldwide and contributes to approximately 6% of global deaths. Understanding mechanisms of hepatic mitochondrial dysfunction resulting from ethanol metabolism remains a key area of interest. Our findings demonstrate that liver physiology is altered by ethanol-induced mitochondrial protein hyperacetylation. Since oxidative stress is a critical marker of earlystage ethanol toxicity in the liver, scrutiny is focused on the impact of ethanol toxicity on the acetylation status of mitochondrial antioxidant proteins and overall antioxidant status. We used a mouse model that employs a Lieber-DeCarli liquid diet to examine the impact of chronic ethanol consumption in a high-fat Western diet. Towards the goal of elucidating mechanisms of alcohol toxicity, we integrated a multi-omics approach utilizing techniques such as 1D- and 2D-SDS-PAGE followed by Western blot, immunohistochemistry (IHC), enzyme activity assays, quantitative acetylomics via HPLC-MS/MS, and computational modeling. These proteomic techniques were employed to assess changes in the acetylation status of individual lysine residues on mitochondrial antioxidant proteins from control and ethanol-fed mice. The results of our studies indicate that, as a result of chronic ethanol
SfRBM / SFRRI 2016
consumption, the global acetylation of SOD2 lysine residues was increased in liver by 270% and 400%, as quantified by Western blot and LC-MS/MS, respectively. Western blot analysis also showed an approximate 400% increase in SOD2 K68 acetylation, which is known to inhibit enzyme activity. IHC analysis revealed that ethanol-induced acetylation of SOD2-K68 is significantly increased in the zone 3 in liver, which is generally known to be hypoxic. Our findings also correlate with a decreased ratio of GSH/GSSG as a consequence of ethanol toxicity. These acetyl-related changes were then correlated with alterations in SOD2 enzymatic structure and activity. Interestingly, chronic ethanol consumption shifted the isoelectric point of SOD2 toward a more negative state as demonstrated by 2D- electrophoresis. Ethanol-induced acetylation in vivo also decreased SOD2 activity by 40%. Through the integration of enzyme assays, mass spectrometry, and computational biology, our goal is to further elucidate mechanisms of nutrient disruption and oxidative stress due to alcohol toxicity.
doi: 10.1016/j.freeradbiomed.2016.10.247 207 Redox Activity Induced by Metal Coordination with Prion Peptide Wakako Hiraoka1, Naoya Matsunaga1, Asuka Kato1, and Shinnosuke Kondo1 1 Meiji University, Tokyo, Japan Background: Physiological function of prion protein (PrP) is still unknown, however its redox and metal-reservoir potentials are viewed as meaningful. We also showed the conformational change of the human PrP binding to Cu2+ [1]. In this report, we have examined the redox ability and the stability of metals at binding site using octarepeat peptide, which is a primary site for Cu2+ uptake in the N-terminal region of PrP. Materials and Methods: An eight-residue peptide (PHGGGWGQ) of prion protein was used for the spectroscopic affinity assay for metal ions (Cu2+, Ni2+, Co2+, Zn2+, and Mn2+). For electron spin resonance (ESR), the peptide and Cu2+ were prepared as 0.1 mM in 10 mM HEPES buffer (pH 7.6) containing 30% glycerol. The ESR spectrum was acquired at 20 K. For circular dichroism (CD), the peptide and Cu2+ were prepared as 0.8 mM in 15 mM HEPES buffer. To verify competitive metal binding, octapeptide-Cu2+ was titrated with other divalent metal ions such as Ni2+, Co2+, Zn2+, and Mn2+. The reactivity of metal-containing peptide with ROS was analyzed by ESR spin trapping with CYPMPO. Results: The ESR at 20K showed two types of coordination modes for the binding of octapeptide-Cu2+. The CD spectrum of octapepide-Cu2+ indicated the characteristic positive peak of molecular ellipticity at visible wavelengths, which was also assigned to Cu2+ coordination complexes. The CD peak of octapeptideCu2+was reduced by titrating with other divalent metal ions, such as Ni2+, Co2+, Zn2+, and Mn2+. ESR spin trapping showed that the copper-peptide complex converted superoxide into hydroxyl radical. References: [1] Biochem. Biophys. Res. Commun., Vol. 366, 244, 2008. & Vol. 394, 522, 2010.
doi: 10.1016/j.freeradbiomed.2016.10.248
208 SOD1-Knockout Mice Are Resistant to Lethal Effects of Thioacetamide-Induced Hepatotoxicity Takujiro Homma1, Takaya Shirato1, Jaeyong Lee1, Toshihiro Kurahashi1,2, and Junichi Fujii1 1 Graduate School of Medical Science, Yamagata University, Japan, 2Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Japan We investigated the responses of mice that are defective for superoxide dismutase 1 (SOD1) to thioacetamide (TAA)-induced hepatotoxicity. When a lethal dose of TAA (500 mg/kg) was intraperitoneally injected, the wild-type (WT) mice all died within 36h, but all of the SOD1-knockout (KO) mice survived. To elucidate the mechanism responsible for this, we examined the acute effects of a sublethal dose of TAA (200 mg/kg) on the livers of WT and KO mice. The extent of TAA-induced liver damage was less in the KO mice. The levels of proteins modified with acetyl-lysine, a marker for TAA metabolism, were lower in the KO mice than the WT mice upon the TAA treatment. The KO mice, which were under oxidative stress per se, exhibited a lower CYP2E1 activity. Thus, we conclude that elevated TAA metabolites and ROS that are produced by CYP-mediated drug metabolism trigger lipogenesis as well as liver damage and determine the fate of the mice.
doi: 10.1016/j.freeradbiomed.2016.10.249 209 Luteolin Exerts Anti-Inflammatory Effects through Regulating Functional Diversity of Macrophage Phenotypes Hiroshi Ichikawa1, Mayuko Osada-Oka2, Keiko Kobayashi2, and Yukiko Minamiyama2 1 Doshisha University, Japan, 2Kyoto Prefectural University, Japan Objectives: Macrophages (Mφ) play major roles in innate immunity for host defense against infection with bacteria. Activated Mφ are classified into M1 and M2 phenotypes. Pro-inflammatory M1-Mφ have the function of phagocytosis and bactericidal activity. In contrast, M2-Mφ play an anti-inflammatory role and can decrease M1-derived immune reaction. However, an excessive or prolonged M1 polarization leads to tissue injury and chronic inflammation. Thus, it is an important to regulate M1/M2 balance. In this study, we investigated whether luteolin, including in many vegetables and fruit, affects M1 or M2 polarization, and this functional diversity. Materials & Methods: Polarization of mice bone marrow-derived Mφ (BMDMs) was induced by lipopolysaccharide (LPS) and interferon (IFN)-γ to M1-Mφ) or by interleukin (IL) -4 and IL-13 to M2-Mφ. Additionally, BMDMs were cultured with luteolin during activation to M1 or M2 phenotype. The expressions of hypoxia inducible factor-1α (HIF-1α), inducible nitric oxide synthase (iNOS), and tumor necrosis factor-α (TNF-α) for M1-marker, and mannose receptor (MR), arginase-1 and Ym-1 for M2-marker were determined by Western blot or quantitative real-time PCR. Nitrogen oxides (NOx) and phorbol 12-myristate 13-acetate-induced reactive oxygen species (ROS) production were measured by Griess reaction and chemiluminescence assay, respectively. Results: Mφ activated by LPS and IFN-γ increased the protein and mRNA levels for M1-marker and the productions of NOx and ROS. Mφ activated by IL-4 and IL-13 were up-regulated the expressions for M2-marker. Luteolin inhibited the increased in the expressions
SfRBM / SFRRI 2016
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doi: 10.1016/j.freeradbiomed.2016.10.244 204 Characterising the Reactions of Diselenides with Reactive Oxidants Generated During Chronic Inflammation Kelly A Gardiner1,2, Michael J Davies1,2,3, and David I Pattison1,2 1 The Heart Research Institute, Sydney, Australia, 2Sydney Medical School, Australia, 3Panum Institute, Copenhagen, Denmark Introduction: Activated leukocytes present at sites of injury or infection releases enzymes that produce reactive oxidants including hydrogen peroxide (H2O2), hypochlorous acid (HOCl), singlet oxygen (1O2), various peroxides and free radicals. These oxidants play a key role in killing pathogens but excessive or inappropriate formation can give rise to host tissue damage and oxidative stress, and this been associated with multiple human pathologies. Recent kinetic data have shown selenium-containing compounds (selenols RSeH, and selenides RSeR) react rapidly with multiple oxidants and are readily recycled (by GSH and enzymes) to their reduced form, allowing catalytic activity. Hypothesis: Readily available diselenides (RSeSeR) may act as highly efficient oxidant scavengers of biologically-relevant oxidants. Results: The diselenides selenocystamine (SeCA), diselenodipropionic acid (DSePA) and glutathione diselenide (GSeSeG) react rapidly with HOCl, H2O2 and a range of organic hydroperoxides as assessed via UV/vis spectroscopy. These reactions are more rapid with HOCl than with H2O2, with the second order rate constants for reaction with H2O2 being in the range 102 (for DSePA) to ~10-4 M-1 s-1 (SeCA and GSeSeG). The oxidation products of the fluorescently-labeled diselenide, Fmocselenocystine, have been characterised using UPLC and LCMS/MS. Oxidation by HOCl and H2O2 results in cleavage of the diselenide bond (with a loss of absorption at 300 nm) and formation of low molecular mass species in a dose- and time-dependent manner. The seleninic acid (RSeO2H) is a major oxidation product. Conclusions: Readily available and stable diselenides are rapidly oxidised by biologically-relevant oxidants, and may act as efficient oxidant scavengers in vivo, thereby ameliorating oxidative damage associated with inflammatory diseases.
doi: 10.1016/j.freeradbiomed.2016.10.245 205 Gambusia Punctata (Poeciliidae) as Alternative Biomodel in Oxidative Stress Evaluation for Ecotoxicological and Pharmaceutical Applications Frenkel Guisado Bourzac1, Juan Carlos Moreno Guisado1, Alina González Marañón1, Jesús Rafael Rodríguez Amado2, and Raimundo Nonato Picanço Souto2 1 University of Oriente, Santiago, Cuba, 2Universidade Federal do Amapá, Macapá, Brazil Background: Fish are sensitive organisms to changes caused by oxidative stress inducing agents, due to expression of biological changes based on the quality of the environment at cellular,
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individual, populations or community level. Objective: To evaluate Gambusia punctata as an alternative biomodel for evaluating physiological response facing oxidative stress and changes in cell REDOX potential. Method: Different criteria were analyzed: ubiquitous distribution, easy taxonomic identification, appropriate size, limited mobility, relatively long life cycle, trophic chain position, ecological relevance, tolerance to contamination and acceptable survival facing environmental variations, easy capture and handling for laboratory studies and proven correlation between environmental pollution and physiological response due to metals, inorganic and organic pollutants bioaccumulation, among others. Results: Molecular biomarkers response assessed for G. punctata is consistent for regular assessment of cell damage and antioxidant response determinations. Increase in multi-pollutants levels for the initial “in situ” evaluation, followed by Conductivity and Total Dissolved Solids (TDS), was positively correlated with molecular determinations (total dissolved proteins, NAD(P)H, H2O2, ascorbate, like-MDA reaction products) and physiological changes. Ionic strength increase in historic records is mainly based in freshwater bodies content of Na+, K+, Cl–, Mg2+, SO42–, HCO3–, NO3– , NO2–, NH4+, PO43–, and significant and persistent content in metals through water course such as Al, Ni and Mo due to anthropic effects. Biomarkers response reflects the cell damage for G. punctata and its antioxidant response. These initial results reveal this fish potentiality as alternative oxidative stress biomodel. Molecular criteria are useful for both, “in situ” evaluation for biomonitoring ecosystems health, “in vitro” early analytical stages of toxicological studies for drugs and chemical exposure and pharmaceutical studies. Conclusion: G. punctata fulfil basic standards for its use as alternative biomodel, allowing oxidative stress evaluation due to its adaptative response.
doi: 10.1016/j.freeradbiomed.2016.10.246 206 Chronic Ethanol Consumption Alters SOD2 Dynamics through Lysine Acetylation Mohammed Assiri1, Peter S Harris1, Hadi Ali1, Yongliang Liang2, Colin T Shearn1, James R Roede1, Donald S Backos1, and Kristofer S Fritz1 1 University of Colorado, Aurora, USA, 2Emory University, Atlanta, USA Chronic ethanol consumption is a significant source of morbidity and mortality worldwide and contributes to approximately 6% of global deaths. Understanding mechanisms of hepatic mitochondrial dysfunction resulting from ethanol metabolism remains a key area of interest. Our findings demonstrate that liver physiology is altered by ethanol-induced mitochondrial protein hyperacetylation. Since oxidative stress is a critical marker of earlystage ethanol toxicity in the liver, scrutiny is focused on the impact of ethanol toxicity on the acetylation status of mitochondrial antioxidant proteins and overall antioxidant status. We used a mouse model that employs a Lieber-DeCarli liquid diet to examine the impact of chronic ethanol consumption in a high-fat Western diet. Towards the goal of elucidating mechanisms of alcohol toxicity, we integrated a multi-omics approach utilizing techniques such as 1D- and 2D-SDS-PAGE followed by Western blot, immunohistochemistry (IHC), enzyme activity assays, quantitative acetylomics via HPLC-MS/MS, and computational modeling. These proteomic techniques were employed to assess changes in the acetylation status of individual lysine residues on mitochondrial antioxidant proteins from control and ethanol-fed mice. The results of our studies indicate that, as a result of chronic ethanol
SfRBM / SFRRI 2016
consumption, the global acetylation of SOD2 lysine residues was increased in liver by 270% and 400%, as quantified by Western blot and LC-MS/MS, respectively. Western blot analysis also showed an approximate 400% increase in SOD2 K68 acetylation, which is known to inhibit enzyme activity. IHC analysis revealed that ethanol-induced acetylation of SOD2-K68 is significantly increased in the zone 3 in liver, which is generally known to be hypoxic. Our findings also correlate with a decreased ratio of GSH/GSSG as a consequence of ethanol toxicity. These acetyl-related changes were then correlated with alterations in SOD2 enzymatic structure and activity. Interestingly, chronic ethanol consumption shifted the isoelectric point of SOD2 toward a more negative state as demonstrated by 2D- electrophoresis. Ethanol-induced acetylation in vivo also decreased SOD2 activity by 40%. Through the integration of enzyme assays, mass spectrometry, and computational biology, our goal is to further elucidate mechanisms of nutrient disruption and oxidative stress due to alcohol toxicity.
doi: 10.1016/j.freeradbiomed.2016.10.247 207 Redox Activity Induced by Metal Coordination with Prion Peptide Wakako Hiraoka1, Naoya Matsunaga1, Asuka Kato1, and Shinnosuke Kondo1 1 Meiji University, Tokyo, Japan Background: Physiological function of prion protein (PrP) is still unknown, however its redox and metal-reservoir potentials are viewed as meaningful. We also showed the conformational change of the human PrP binding to Cu2+ [1]. In this report, we have examined the redox ability and the stability of metals at binding site using octarepeat peptide, which is a primary site for Cu2+ uptake in the N-terminal region of PrP. Materials and Methods: An eight-residue peptide (PHGGGWGQ) of prion protein was used for the spectroscopic affinity assay for metal ions (Cu2+, Ni2+, Co2+, Zn2+, and Mn2+). For electron spin resonance (ESR), the peptide and Cu2+ were prepared as 0.1 mM in 10 mM HEPES buffer (pH 7.6) containing 30% glycerol. The ESR spectrum was acquired at 20 K. For circular dichroism (CD), the peptide and Cu2+ were prepared as 0.8 mM in 15 mM HEPES buffer. To verify competitive metal binding, octapeptide-Cu2+ was titrated with other divalent metal ions such as Ni2+, Co2+, Zn2+, and Mn2+. The reactivity of metal-containing peptide with ROS was analyzed by ESR spin trapping with CYPMPO. Results: The ESR at 20K showed two types of coordination modes for the binding of octapeptide-Cu2+. The CD spectrum of octapepide-Cu2+ indicated the characteristic positive peak of molecular ellipticity at visible wavelengths, which was also assigned to Cu2+ coordination complexes. The CD peak of octapeptideCu2+was reduced by titrating with other divalent metal ions, such as Ni2+, Co2+, Zn2+, and Mn2+. ESR spin trapping showed that the copper-peptide complex converted superoxide into hydroxyl radical. References: [1] Biochem. Biophys. Res. Commun., Vol. 366, 244, 2008. & Vol. 394, 522, 2010.
doi: 10.1016/j.freeradbiomed.2016.10.248
208 SOD1-Knockout Mice Are Resistant to Lethal Effects of Thioacetamide-Induced Hepatotoxicity Takujiro Homma1, Takaya Shirato1, Jaeyong Lee1, Toshihiro Kurahashi1,2, and Junichi Fujii1 1 Graduate School of Medical Science, Yamagata University, Japan, 2Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Japan We investigated the responses of mice that are defective for superoxide dismutase 1 (SOD1) to thioacetamide (TAA)-induced hepatotoxicity. When a lethal dose of TAA (500 mg/kg) was intraperitoneally injected, the wild-type (WT) mice all died within 36h, but all of the SOD1-knockout (KO) mice survived. To elucidate the mechanism responsible for this, we examined the acute effects of a sublethal dose of TAA (200 mg/kg) on the livers of WT and KO mice. The extent of TAA-induced liver damage was less in the KO mice. The levels of proteins modified with acetyl-lysine, a marker for TAA metabolism, were lower in the KO mice than the WT mice upon the TAA treatment. The KO mice, which were under oxidative stress per se, exhibited a lower CYP2E1 activity. Thus, we conclude that elevated TAA metabolites and ROS that are produced by CYP-mediated drug metabolism trigger lipogenesis as well as liver damage and determine the fate of the mice.
doi: 10.1016/j.freeradbiomed.2016.10.249 209 Luteolin Exerts Anti-Inflammatory Effects through Regulating Functional Diversity of Macrophage Phenotypes Hiroshi Ichikawa1, Mayuko Osada-Oka2, Keiko Kobayashi2, and Yukiko Minamiyama2 1 Doshisha University, Japan, 2Kyoto Prefectural University, Japan Objectives: Macrophages (Mφ) play major roles in innate immunity for host defense against infection with bacteria. Activated Mφ are classified into M1 and M2 phenotypes. Pro-inflammatory M1-Mφ have the function of phagocytosis and bactericidal activity. In contrast, M2-Mφ play an anti-inflammatory role and can decrease M1-derived immune reaction. However, an excessive or prolonged M1 polarization leads to tissue injury and chronic inflammation. Thus, it is an important to regulate M1/M2 balance. In this study, we investigated whether luteolin, including in many vegetables and fruit, affects M1 or M2 polarization, and this functional diversity. Materials & Methods: Polarization of mice bone marrow-derived Mφ (BMDMs) was induced by lipopolysaccharide (LPS) and interferon (IFN)-γ to M1-Mφ) or by interleukin (IL) -4 and IL-13 to M2-Mφ. Additionally, BMDMs were cultured with luteolin during activation to M1 or M2 phenotype. The expressions of hypoxia inducible factor-1α (HIF-1α), inducible nitric oxide synthase (iNOS), and tumor necrosis factor-α (TNF-α) for M1-marker, and mannose receptor (MR), arginase-1 and Ym-1 for M2-marker were determined by Western blot or quantitative real-time PCR. Nitrogen oxides (NOx) and phorbol 12-myristate 13-acetate-induced reactive oxygen species (ROS) production were measured by Griess reaction and chemiluminescence assay, respectively. Results: Mφ activated by LPS and IFN-γ increased the protein and mRNA levels for M1-marker and the productions of NOx and ROS. Mφ activated by IL-4 and IL-13 were up-regulated the expressions for M2-marker. Luteolin inhibited the increased in the expressions
SfRBM / SFRRI 2016
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