云芝多糖对巨噬细胞GPx基因表达的影响

细胞内存在两种谷胱甘肽过氧化物酶;硒谷胱甘肽过氧化物酶和非硒谷肽甘肽过氧化物酶,它们在保护细胞免受氧化损伤等过程中起重要作用。为揭示云芝多糖作用与细胞抗氧化酶的关系,采用酶活性测定,斑点杂交等方法,探讨云芝多糖对小鼠腹腔巨噬细胞过氧化物酶表达的影响。结果显示,腹腔注射云芝多糖可以提高小鼠腹腔巨噬细胞的两种过氧化物酶活性,并使其ｍＲＮＡ含量增加,应用阻断剂的研究发现,云芝多糖对巨噬细胞ＳｅＧＰｘ及Ｇ     

Molecular characterization and mRNA expression of glutathione peroxidase and glutathione S-transferase during osmotic stress in olive flounder (Paralichthys olivaceus)

Glutathione peroxidase (GPX) and glutathione S-transferase (GST) are key enzymes of cellular detoxification systems that defend cells against reactive oxygen species (ROS). In this study, we isolated the GPX and GST full-length cDNA and investigated the expression of these mRNAs from livers of olive flounder during salinity changes (35, 17.5, 8.75, 4 and 0 psu) by quantitative PCR (QPCR). GPX cDNA consists of 429 base pairs (bp) and encodes a protein of 142 amino acids. GST cDNA consists of 663 bp and encodes a protein of 220 amino acids. Both of GPX and GST mRNA expressions were the highest in 4 psu and then decreased in 0 psu. Also, the levels of Na(+) and Cl(-) decreased, and aspartate aminotransferase (AST) and alanine aminotransferase (ALT) increased during the experimental period. These findings provide molecular characterization of GPX and GST in olive flounder and suggest that GPX and GST play important roles in detoxification of ROS, thereby these maybe indicators of oxidative stress responses by salinity changes in olive flounder.     

The responses of HT22 cells to the blockade of mitochondrial complexes and potential protective effect of selenium supplementation

Mitochondria are the major reactive oxygen species (ROS)--generating sites in mammalian cells. Blockade of complexes in the electron transport chain (ETC) increases the leakage of single electrons to O(2) and therefore increases ROS levels. Complexes I and III have been reported to be the major ROS-generating sites in mitochondria. In this study, using mouse hippocampal HT22 cells as in vitro model, we monitored the change of intracellular ROS level in response to the blockade of ETC at different complex, and measured changes of gene expression of antioxidant enzymes and phase II enzymes, also evaluated potential protective effect of selenium (Se) supplementation to the cells under this oxidative stress. In summary, our results showed that complex I was the major ROS-generating site in HT22 cells. Complex I blockade upregulated the mRNA levels of glutamylcysteine synthetase heavy and light chains, glutathione-S-transferases omega1 and alpha 2, hemoxygenase 1, thioredoxin reductase 1, and selenoprotein H. Unexpectedly, the expression of the enzymes that directly scavenge ROS decreased, including superoxide dismutases 1 and 2, glutathione peroxidase 1, and catalase. Se supplementation increased glutathione levels and glutathione peroxidase activity, indicating a potential protective role in oxidative stress caused by ETC blockade.     

Analysis of reactive oxygen species and antioxidant defenses in complex I deficient patients revealed a specific increase in superoxide dismutase activity

The mechanism of free radical production by complex I deficiency is ill-defined, although it is of significant contemporary interest. This study studied the ROS production and antioxidant defenses in children with mitochondrial NADH dehydrogenase deficiency. ROS production has remained significantly elevated in patients compared to controls. The expression of all antioxidant enzymes significantly increased at mRNA level. However, the enzyme activities did not correlate with high mRNA or protein expression. Only the activity of superoxide dismutase (SOD) was found to correlate with higher mRNA expression in patient derived cell lines. The activities of the enzymes such as glutathione peroxidase (GPx), Catalase (CAT) and glutathione-S-transferase (GST) were significantly reduced in patients (p<0.05 or p<0.01). Glutathione reductase (GR) activity and intracellular glutathione (GSH) levels were not changed. Decreased enzyme activities could be due to post-translational or oxidative modification of ROS scavenging enzymes. The information on the status of ROS and marking the alteration of ROS scavenging enzymes in peripheral lymphocytes or lymphoblast cell lines will provide a better way to design antioxidant therapies for such disorders.     

Expression and regulation of antioxidant enzymes in the developing limb support a function of ROS in interdigital cell death

Vertebrate limb development is a well-studied model of apoptosis; however, little is known about the intracellular molecules involved in activating the cell death machinery. We have shown that high levels of reactive oxygen species (ROS) are present in the interdigital 'necrotic' tissue of mouse autopod, and that antioxidants can reduce cell death. Here, we determined the expression pattern of several antioxidant enzymes in order to establish their role in defining the areas with high ROS levels. We found that the genes encoding the superoxide dismutases and catalase are expressed in autopod, but they are downregulated in the interdigital regions at the time ROS levels increased and cell death was first detected. The possible role of superoxide and/or peroxide in activating cell death is supported by the protective effect of a superoxide dismutase/catalase mimetic. Interestingly, we found that peroxidase activity and glutathione peroxidase-4 gene (Gpx4) expression were restricted to the non-apoptotic tissue (e.g., digits) of the developing autopod. Induction of cell death with retinoic acid caused an increase in ROS and decrease in peroxidase activity. Even more inhibition of glutathione peroxidase activity leads to cell death in the digits, suggesting that a decrease in antioxidant activity, likely due to Gpx4, caused an increase in ROS levels, thus triggering apoptosis.     

Endophytic bacterium Bacillus subtilis (BERA 71) improves salt tolerance in chickpea plants by regulating the plant defense mechanisms

Plant growth-promoting endophytic bacteria can stimulate the growth, nutrient acquisition, symbiotic performance and stress tolerance of chickpea plants under saline soil conditions. The aim of this study was to investigate the stress-adaptive mechanisms of chickpea plants mediated by Bacillus subtilis (BERA 71) under saline conditions. Inoculation with BERA 71 enhanced plant biomass and the synthesis of photosynthetic pigments and reduced the levels of reactive oxygen species (ROS) and lipid peroxidation in plants under conditions of stress. Furthermore, the activities of ROS-scavenging antioxidant enzymes (superoxide dismutase, peroxidase, catalase and glutathione reductase), the levels of non-enzymatic antioxidants (ascorbic acid and glutathione) and the total phenol content were increased in stressed plants during bacterial association. The bacteria decreased sodium accumulation and enhanced the nitrogen, potassium, calcium and magnesium content in the plants. The suppression of ROS generation and of lipid peroxidation and the accumulation of proline in BERA-71-inoculated plants enhanced the membrane stability under salinity stress and non-stress conditions.     

Analysis of reactive oxygen species and antioxidant defenses in complex I deficient patients revealed a specific increase in superoxide dismutase activity

The mechanism of free radical production by complex I deficiency is ill-defined, although it is of significant contemporary interest. This study studied the ROS production and antioxidant defenses in children with mitochondrial NADH dehydrogenase deficiency. ROS production has remained significantly elevated in patients compared to controls. The expression of all antioxidant enzymes significantly increased at mRNA level. However, the enzyme activities did not correlate with high mRNA or protein expression. Only the activity of superoxide dismutase (SOD) was found to correlate with higher mRNA expression in patient derived cell lines. The activities of the enzymes such as glutathione peroxidase (GPx), Catalase (CAT) and glutathione-S-transferase (GST) were significantly reduced in patients (p<0.05 or p<0.01). Glutathione reductase (GR) activity and intracellular glutathione (GSH) levels were not changed. Decreased enzyme activities could be due to post-translational or oxidative modification of ROS scavenging enzymes. The information on the status of ROS and marking the alteration of ROS scavenging enzymes in peripheral lymphocytes or lymphoblast cell lines will provide a better way to design antioxidant therapies for such disorders.     

Expression of antioxidant enzymes in rat kidney during ischemia-reperfusion injury

The effect of ischemia-reperfusion on activity, protein and m-RNA levels of catalase, copper-zinc and manganese containing superoxide dismutases and glutathione peroxidase, the enzymes that are involved in free radical detoxification was studied in rat kidney. Ischemia alone did not alter either the activities or protein levels of superoxide dismutase and glutathione peroxidase. However, catalase activity was found to be inhibited to 82% of control. The inhibition of catalase was due to the inactivation of the enzyme as there was no significant change in enzyme protein level. Reperfusion following ischemia, however, led to a significant decrease in both the activities as well as the protein levels of all the antioxidant enzymes. The observed overall decrease in total superoxide dismutase activity was the net effect of a decrease in copper-zinc superoxide dismutase while manganese superoxide dismutase activity was found to be increased following reperfusion. This observed increased manganese superoxide dismutase activity was the result of its increased protein level. The mRNA levels for catalase, superoxide dismutases, and glutathione peroxidase were observed to be increased (100–145% of controls) following ischemia; reperfusion of ischemic kidneys, however, resulted in a significant decrease in the levels of mRNAs coding for all the enzymes except manganese superoxide dismutase which remained high. These results suggest that in tissue, the down regulation of the antioxidant enzyme system could be responsible for the pathophysiology of ischemia-reperfusion injury.     

Skin Cornification Proteins Provide Global Link between ROS Detoxification and Cell Migration during Wound Healing

Wound healing is a complex dynamic process characterised by a uniform flow of events in nearly all types of tissue damage, from a small skin scratch to myocardial infarction. Reactive oxygen species (ROS) are essential during the healing process at multiple stages, ranging from the initial signal that instigates the immune response, to the triggering of intracellular redox-dependent signalling pathways and the defence against invading bacteria. Excessive ROS in the wound milieu nevertheless impedes new tissue formation. Here we identify small proline-rich (SPRR) proteins as essential players in this latter process, as they directly link ROS detoxification with cell migration. A literature-based meta-analysis revealed their up-regulation in various forms of tissue injury, ranging from heart infarction and commensal-induced gut responses to nerve regeneration and burn injury. Apparently, SPRR proteins have a far more widespread role in wound healing and tissue remodelling than their established function in skin cornification. It is inferred that SPRR proteins provide injured tissue with an efficient, finely tuneable antioxidant barrier specifically adapted to the tissue involved and the damage inflicted. Their recognition as novel cell protective proteins combining ROS detoxification with cell migration will provide new venues to study and manage tissue repair and wound healing at a molecular level.     

Global regulation of reactive oxygen species scavenging genes in alfalfa root and shoot under gradual drought stress and recovery

Reactive oxygen species (ROS) production and scavenging in plants under drought stress have been studied intensively in recent years. Here we report a global analysis of gene expression for the major ROS generating and scavenging proteins in alfalfa root and shoot under gradual drought stress followed by one-day recovery. Data from two alfalfa varieties, one drought tolerant and one drought sensitive, were compared and no qualitative differences in ROS gene regulation between the two were found. Conserved, tissue-specific patterns of gene expression in response to drought were observed for several ROS-scavenging gene families, including ascorbate peroxidase, monodehydroascorbate reductase, and peroxiredoxin. In addition, differential gene expression within families was observed. Genes for the ROS-generating enzyme, NADPH oxidase were generally induced under drought, while those for glycolate oxidase were repressed. Among the ROS-scavenging protein genes, Ferritin, Cu/Zn superoxide dismutase (SOD), and the majority of the glutathione peroxidase family members were induced under drought in both roots and shoots of both alfalfa varieties. In contrast, Fe-SOD, CC-type glutaredoxins, and thoiredoxins were downregulated.     

Lack of transketolase-like (TKTL) 1 aggravates murine experimental colitis

Transketolase-like (TKTL) 1 indirectly replenishes NADPH preventing damage induced by reactive oxygen species (ROS) formed upon intestinal inflammation. We investigated the function of TKTL1 during murine colitis and ROS detoxification for prevention of tissue damage. Mucosal damage in TKTL1(-/-) and wild-type (WT) mice was assessed by miniendoscopy and histology during dextran sodium sulfate (DSS) colitis. mRNA levels of interferon (IFN)-γ, inducible nitric oxide synthase (iNOS), interleukin (IL)-6, tumor necrosis factor (TNF), transketolase (TKT), and TKTL2 were determined by PCR and/or Western blotting. To assess oxidative and nitrosative stress nitrosylation, carbonylation and antioxidative enzymes catalase (Cat), superoxide dismutase 1 and 2, as well as glutathione (GSH) were determined. Myeloperoxidase (MPO) was determined for assessment of tissue neutrophils. TKTL1 knockout or DSS treatment did not influence TKT and TKTL2 mRNA or protein expression. Mucosal damage was significantly increased in TKTL1(-/-) mice indicated by miniendoscopy as well as a significantly shorter colon and more severe histological scores compared with WT mice during DSS colitis. This was associated with higher mRNA levels of IFN-γ, iNOS, IL-6, and TNF. In addition, iNOS protein expression was significantly enhanced in TKTL1(-/-) mice as well as MPO activity. Protein modification by nitric oxide (nitrotyrosine) was induced in TKTL1(-/-) mice. However, introduction of carbonyl groups by ROS was not induced in these mice. The expression of SOD1, SOD2, Cat, as well as GSH content was not significantly changed in TKTL1(-/-) mice. We conclude that induced colitis in TKTL1(-/-) mice was more severe compared with WT. This indicates a role of TKTL1 during mucosal repair and restoration.     

L929 cell conditioned medium protects RAW264.7 cells from oxidative injury through inducing antioxidant enzymes

We have previously found that L929 cell conditioned medium (L929-CM) could protect mouse peritoneal macrophages from oxidative injury. To uncover the mechanism further, we investigated the effect of L929-CM on the oxidative injury caused by tbOOH to RAW264.7 cell lines. The results showed that L929-CM could protect RAW264.7 cells from oxidative injury (presented by cell morphology and cell survival rate), and L929-CM could also improve total superoxide dismutase (SOD), selenium-dependent and non-selenium-dependent glutathione peroxidase (SeGPx and non-SeGPx) activities in RAW264.7 cells. RT-PCR analysis showed that, L929-CM could induce plasma glutathione peroxidase (PLGPx) mRNA expression, while there was no inducing effect of L929-CM on phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA expression in RAW264.7 cells. 5 microg/ml actinomycin D, 30 microg/ml cycloheximide (de novo protein synthesis inhibitor) and 50 microg/ml acetovanilone (intracellular superoxide anion production inhibitor) had no effects in attenuating the induction of PLGPx expression by L929-CM.     

The Fas death signaling pathway connecting reactive oxygen species generation and FLICE inhibitory protein down-regulation

Fas-mediated apoptosis plays an important role in normal tissue homeostasis, and disruption of this death pathway contributes to many human diseases. Induction of apoptosis via Fas activation has been associated with reactive oxygen species (ROS) generation and down-regulation of FLICE inhibitory protein (FLIP); however, the relationship between these two events and their role in Fas-mediated apoptosis are unclear. We show herein that ROS are required for FLIP down-regulation and apoptosis induction by Fas ligand (FasL) in primary lung epithelial cells. ROS mediate the down-regulation of FLIP by ubiquitination and subsequent degradation by proteasome. Inhibition of ROS by antioxidants or by ectopic expression of ROS-scavenging enzymes glutathione peroxidase and superoxide dismutase effectively inhibited FLIP down-regulation and apoptosis induction by FasL. Hydrogen peroxide is a primary oxidative species responsible for FLIP down-regulation, whereas superoxide serves as a source of peroxide and a scavenger of NO, which positively regulates FLIP via S-nitrosylation. NADPH oxidase is a key source of ROS generation induced by FasL, and its inhibition by dominant-negative Rac1 expression or by chemical inhibitor decreased the cell death response to FasL. Taken together, our results indicate a novel pathway of FLIP regulation by an interactive network of reactive oxygen and nitrogen species that provides a key mechanism of apoptosis regulation in Fas-induced cell death and related apoptosis disorders.     

ROS signaling by NOX4 drives fibroblast-to-myofibroblast differentiation in the diseased prostatic stroma

Stromal remodeling, in particular fibroblast-to-myofibroblast differentiation, is a hallmark of benign prostatic hyperplasia (BPH) and solid tumors, including prostate cancer (PCa). Increased local production of TGFβ1 is considered the inducing stimulus. Given that stromal remodeling actively promotes BPH/PCa development, there is considerable interest in developing stromal-targeted therapies. Microarray and quantitative PCR analysis of primary human prostatic stromal cells induced to undergo fibroblast-to-myofibroblast differentiation with TGFβ1 revealed up-regulation of the reactive oxygen species (ROS) producer reduced nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) and down-regulation of the selenium-containing ROS-scavenging enzymes glutathione peroxidase 3, thioredoxin reductase 1 (TXNRD1), and the selenium transporter selenoprotein P plasma 1. Consistently, NOX4 expression correlated specifically with the myofibroblast phenotype in vivo, and loss of selenoprotein P plasma 1 was observed in tumor-associated stroma of human PCa biopsies. Using lentiviral NOX4 short hairpin RNA-mediated knockdown, pharmacological inhibitors, antioxidants, and selenium, we demonstrate that TGFβ1 induction of NOX4-derived ROS is required for TGFβ1-mediated phosphorylation of c-jun N-terminal kinase, which in turn is essential for subsequent downstream cytoskeletal remodeling. Significantly, selenium supplementation inhibited differentiation by increasing ROS-scavenging selenoenzyme biosynthesis because glutathione peroxidase 3 and TXNRD1 expression and TXNRD1 enzyme activity were restored. Consistently, selenium depleted ROS levels downstream of NOX4 induction. Collectively, this work demonstrates that dysregulated redox homeostasis driven by elevated NOX4-derived ROS signaling underlies fibroblast-to-myofibroblast differentiation in the diseased prostatic stroma. Further, these data indicate the potential clinical value of selenium and/or NOX4 inhibitors in preventing the functional pathogenic changes of stromal cells in BPH and PCa.     

The effect of finger millet feeding on the early responses during the process of wound healing in diabetic rats

In the present study, the role of finger millet feeding on skin antioxidant status, nerve growth factor (NGF) production and wound healing parameters in healing impaired early diabetic rats is reported. Hyperglycemic rats received food containing 50 g/100 g finger millet (FM). Non-diabetic controls and diabetic controls received balanced nutritive diet. Full-thickness excision skin wounds were made after 2 weeks prior feeding of finger millet diet. The rate of wound contraction, and the levels of collagen, hexosamine and uronic acid in the granulation tissue were determined. The skin antioxidant status and lipid peroxide concentration were also monitored during the study. In hyperglycemic rats fed with finger millet diet, the healing process was hastened with an increased rate of wound contraction. Skin levels of glutathione (GSH), ascorbic acid and alpha-tocopherol in alloxan-induced diabetic rat were lower as compared to non-diabetics. Altered activities of superoxide dismutase (SOD) and catalase (CAT) were also recorded in diabetics. Interestingly, thiobarbituric acid reactive substances (TBARS) were elevated in the wound tissues of all the groups, when compared to normal (unwounded) skin tissues. However, in diabetic rats the TBARS levels of both normal and wounded skin tissues were significantly elevated (P < 0.001) when compared with control (non-diabetic) and diabetics fed with FM. Impaired production of NGF, determined by ELISA, in diabetic rats was improved upon FM feeding and further confirmed by immunocytochemical observations reflects the increased expression of NGF in hyperglycemic rats supplemented with FM-enriched diet. Histological and electron microscopical evaluations revealed the epithelialization, increased synthesis of collagen, activation of fibroblasts and mast cells in FM-fed animals. Thus, increased levels of oxidative stress markers accompanied by decreased levels of antioxidants play a vital role in delaying wound healing in diabetic rats. However, FM feeding to the diabetic animals, for 4 weeks, controlled the glucose levels and improved the antioxidant status, which hastened the dermal wound healing process.     

Improved repair of dermal wounds in mice lacking microRNA‐155

Wound healing is a well-regulated but complex process that involves haemostasis, inflammation, proliferation and maturation. Recent reports suggest that microRNAs (miRs) play important roles in dermal wound healing. In fact, miR deregulation has been linked with impaired wound repair. miR-155 has been shown to be induced by inflammatory mediators and plays a central regulatory role in immune responses. We have investigated the potential role of miR-155 in wound healing. By creating punch wounds in the skin of mice, we found an increased expression of miR-155 in wound tissue when compared with healthy skin. Interestingly, analysis of wounds of mice lacking the expression of miR-155 (miR-155^−/−) revealed an increased wound closure when compared with wild-type animals. Also, the accelerated wound closing correlated with elevated numbers of macrophages in wounded tissue. Gene expression analysis of wounds tissue and macrophages isolated from miR-155^−/− mice that were treated with interleukin-4 demonstrated an increased expression of miR-155 targets (BCL6, RhoA and SHIP1) as well as, the finding in inflammatory zone-1 (FIZZ1) gene, when compared with WT mice. Moreover, the up-regulated levels of FIZZ1 in the wound tissue of miR-155^−/− mice correlated with an increased deposition of type-1 collagens, a phenomenon known to be beneficial in wound closure. Our data indicate that the absence of miR-155 has beneficial effects in the wound healing process.