GPx1 Protein Expression Research Articles

A263 IRONING OUT THE DETAILS: CROHN’S DISEASE PATIENT DERIVED MACROPHAGES ARE MORE SUSCEPTIBLE TO FERROPTOSIS

Abstract Background Death of intestinal epithelial cells is ubiquitous in Crohn’s disease (CD) but the death of immune cells such as macrophages is less explored. We have found that monocyte derived macrophages from patients with active CD are more susceptible to H2O2-induced cytotoxicity but the form of regulated cell death these cells undergo remains to be elucidated. Selenium (Se)—a common micronutrient deficiency in patients with CD—is used in the synthesis of selenoproteins that have antioxidant properties (e.g., glutathione peroxidases (GPx)) and are highly expressed in macrophages. Whether Se deficiency plays a role in the increased cytotoxicity also remains to be elucidated. Aims To determine the form of cell death of macrophages in response to H2O2-induced cytotoxicity and whether selenium deficiency plays a role in the increased susceptibility to H2O2-induced cytotoxicity. Methods Blood collected from healthy volunteers and patients with active CD was analyzed for serum GPx activity. Monocytes isolated by plastic adherence were treated with M-CSF (10ng/ml, 7d) to derive macrophages. Macrophages were treated with H2O2 (500μm, 2h) and lactate dehydrogenase release was measured. GPX1, SLC7A11 and PTGS2 mRNA expression were determined by qPCR. The presence of cleaved caspase 3 was determined by immunoblotting and IL-1β by ELISA. Lipid peroxidation a marker of ferroptosis was assessed by staining macrophages with Bodipy C-11 (± liproxstatin-1, an inhibitor of ferroptosis). Results CD macrophages were two times more susceptible to H2O2-evoked cell death. In response to H2O2 macrophages did not express cleaved caspase 3 or IL-1β, but there was an induction of ferroptosis markers (SLC7A11 and PTGS2). Moreover, lipid peroxidation was induced and could be blocked with liproxstatin-1. Dietary Se intake did not differ between groups but serum GPx activity was greater in patients with CD compared to control. In contrast, GPX1 mRNA expression and GPx1 protein expression were decreased in CD macrophages compared to healthy controls. Conclusions Macrophages derived from patients with CD are inherently more sensitive to ferroptosis potentially through reduced GPx1 expression. Future studies warrant testing if GPx1 prevents ferroptotic macrophage cell death and if it holds therapeutic relevance in CD pathophysiology. Funding Agencies Helmsley Charitable Trust, Crohn's and Colitis Foundation of America

Hypothermia Treatment after Hypoxia-Ischemia in Glutathione Peroxidase-1 Overexpressing Mice

The developing brain is uniquely susceptible to oxidative stress, and endogenous antioxidant mechanisms are not sufficient to prevent injury from a hypoxic-ischemic challenge. Glutathione peroxidase (GPX1) activity reduces hypoxic-ischemic injury. Therapeutic hypothermia (HT) also reduces hypoxic-ischemic injury, in the rodent and the human brain, but the benefit is limited. Here, we combined GPX1 overexpression with HT in a P9 mouse model of hypoxia-ischemia (HI) to test the effectiveness of both treatments together. Histological analysis showed that wild-type (WT) mice with HT were less injured than WT with normothermia. In the GPX1-tg mice, however, despite a lower median score in the HT-treated mice, there was no significant difference between HT and normothermia. GPX1 protein expression was higher in the cortex of all transgenic groups at 30 min and 24 h, as well as in WT 30 min after HI, with and without HT. GPX1 was higher in the hippocampus of all transgenic groups and WT with HI and normothermia, at 24 h, but not at 30 min. Spectrin 150 was higher in all groups with HI, while spectrin 120 was higher in HI groups only at 24 h. There was reduced ERK1/2 activation in both WT and GPX1-tg HI at 30 min. Thus, with a relatively moderate insult, we see a benefit with cooling in the WT but not the GPX1-tg mouse brain. The fact that we see no benefit with increased GPx1 here in the P9 model (unlike in the P7 model) may indicate that oxidative stress in these older mice is elevated to an extent that increased GPx1 is insufficient for reducing injury. The lack of benefit of overexpressing GPX1 in conjunction with HT after HI indicates that pathways triggered by GPX1 overexpression may interfere with the neuroprotective mechanisms provided by HT.

Immunohistochemical Expression of Glutathione Peroxidase 1 (Gpx-1) as an Independent Prognostic Factor in Colon Adenocarcinoma Patients.

Several studies revealed that expression levels of glutathione peroxidase 1 (Gpx-1) can be associated with cancer development, mainly through its role in hydroperoxide scavenging by regulating intracellular reactive oxygen species (ROS) levels. Therefore, our aim was to investigate the expression of Gpx-1 protein in a population of Polish patients with colon adenocarcinoma in the absence of any therapy prior to radical surgery. The study was carried out using colon tissue from patients with adenocarcinoma of the colon confirmed by histopathological examination. Gpx-1 antibody was used to determine the immunohistochemical expression of Gpx-1. The Chi2test or Chi2Yatesa test were used to analyse the associations between the immunohistochemical expression of Gpx-1 and clinical parameters. The relationship between Gpx-1 expression, and 5-year patient survival was examined using Kaplan-Meier analysis and the log-rank test. Intracellular localisation of Gpx-1 was detected by the use of transmission electron microscopy (TEM). Western blot analysis was used for the evaluation of Gpx-1 protein expression levels in cancer cell lines in vitro. Immunohistochemical study revealed that the high expression of Gpx-1 was associated with the tumour's histological grade, proliferating cell nuclear antigen (PCNA) immunohistochemical expression, depth of invasion, and angioinvasion (all p < 0.001) (4). The high immunohistochemical expression of Gpx-1 is correlated with poor prognosis of colon adenocarcinoma patients.

Obesity-induced oxidative stress and mitochondrial dysfunction negatively affect sperm quality.

Obesity is a systemic metabolic disease that can induce male infertility or subfertility through oxidative stress. The aim of this study was to determine how obesity impairs sperm mitochondrial structural integrity and function, and reduces sperm quality in both overweight/obese men and mice on a high-fat diet (HFD). Mice fed the HFD demonstrated higher body weight and increased abdominal fat content than those fed the control diet. Such effects accompanied the decline in antioxidant enzymes, such as glutathione peroxidase (GPX) and catalase and superoxide dismutase (SOD) in testicular and epidydimal tissues. Moreover, malondialdehyde (MDA) content significantly increased in sera. Mature sperm in HFD mice demonstrated higher oxidative stress, including increased mitochondrial reactive oxygen species (ROS) levels and decreased protein expression of GPX1, which may impair mitochondrial structural integrity and reduce mitochondrial membrane potential (MMP) and ATP production. Moreover, cyclic AMPK phosphorylation status increased, whereas sperm motility declined in the HFD mice. Clinical studies demonstrated that being overweight/obese reduced SOD enzyme activity in the seminal plasma and increased ROS in sperm, accompanied by lower MMP and low-quality sperm. Furthermore, ATP content in the sperm was negatively correlated with increases in the BMI of all clinical subjects. In conclusion, our results suggest that excessive fat intake had similar disruptive effects on sperm mitochondrial structure and function, as well as oxidative stress levels in humans and mice, which in turn induced lower sperm motility. This agreement strengthens the notion that fat-induced increases in ROS and impaired mitochondrial function contribute to male subfertility.

Green rooibos extract attenuates high glucose induced oxidative stress in a human derived (HepG2) liver cell line

• Oxidative stress biomarkers are attenuated by REE in a glucotoxic HepG2 cell model. • REE enhances antioxidant enzyme profiles of HepG2 cells exposed to high glucose. • REE activates NRF2 antioxidant pathway in HepG2 cells exposed to high glucose. The exposure of hepatocytes to high concentrations of glucose results in a glucotoxic environment and ensuing hepatocellular injury, often due to oxidative stress. The popularly consumed herbal tea, Rooibos ( Aspalathus linearis ), demonstrates pleiotropic pharmacological and antioxidant activities. Our study investigated the molecular events regulating the antioxidant effects of a Rooibos ethanolic extract (REE) in a glucotoxic HepG2 liver cell model. It is hypothesized that REE enhances antioxidant enzyme profiles of HepG2 cells exposed to high glucose to attenuate glucotoxic damage. Glucotoxicity was induced by treating the cells with 25 mM glucose. For this study, colorimetric, luminometric and western blot analyses were used to determine the potential of the REE to reduce biomarkers associated with oxidative damage and its potential to enhance the intracellular antioxidant defense system. The REE was found to be hepatoprotective as evidenced by reduced levels of oxidative damage biomarkers (MDA and protein carbonylation) and enhanced antioxidant capacity under glucotoxic conditions as evidenced by elevated GSH (reduced form). We also observed decreased intracellular hydrogen peroxide levels and caspase activity in the REE treated cells and further showed that the REE enhanced SOD2, CAT, and GPx1 protein expression. These effects were dependent on the transcriptional activity of NRF2. Collectively, our results show that the REE enhances the cellular antioxidant defense capacity and may provide a relevant supportive therapeutic option for treating diabetic complications associated with oxidative stress.

Follicular fluid exosomes regulate oxidative stress resistance, proliferation, and steroid synthesis in porcine theca cells

Theca cells (TCs) are regulated by various factors during ovarian development. However, the role of follicular fluid exosomes in ovarian TCs has not yet been reported. In the present study, we explored the effects of follicular fluid exosomes on porcine ovarian TCs. TCs were treated with follicular fluid exosomes in vitro, and the differential gene expression profiles of TCs in the exosome and control groups were obtained via transcriptome sequencing. Differentially expressed genes were identified and found to be associated with antioxidative stress, proliferation, and steroid hormone synthesis of TCs. In addition, exosomes were found to increase antioxidative stress, proliferation, and steroid synthesis, as revealed by a higher mRNA and protein expression of GPX1, CCND1, PCNA, CYP11A1, and HSD3B1 and lower mRNA and protein expression of TNFR1 and BAX. In conclusion, we demonstrated that exosomes are essential components in regulating the physiological function of TCs.

A Novel Diselenide-Probucol-Analogue Protects Against Methylmercury-Induced Toxicity in HT22 Cells by Upregulating Peroxide Detoxification Systems: a Comparison with Diphenyl Diselenide.

Methylmercury (MeHg) is a ubiquitous environmental neurotoxicant whose mechanisms of action involve oxidation of endogenous nucleophilic groups (mainly thiols and selenols), depletion of antioxidant defenses, and disruption of neurotransmitter homeostasis. Diphenyl diselenide-(PhSe)2-a model diaryl diselenide, has been reported to display significant protective effects against MeHg-induced neurotoxicity under both in vitro and in vivo experimental conditions. In this study, we compared the protective effects of (PhSe)2 with those of RC513 (4,4'-diselanediylbis(2,6-di-tert-butylphenol), a novel diselenide-probucol-analog) against MeHg-induced toxicity in the neuronal (hippocampal) cell line HT22. Although both (PhSe)2 and RC513 significantly mitigated MeHg- and tert-butylhydroperoxide (t-BuOOH)-cytotoxicity, the probucol analog exhibited superior protective effects, which were observed earlier and at lower concentrations compared to (PhSe)2. RC513 treatment (at either 0.5µM or 2µM) significantly increased glutathione peroxidase (GPx) activity, which has been reported to counteract MeHg-toxicity. (PhSe)2 was also able to increase GPx activity, but only at 2µM. Although both compounds increased the Gpx1 transcripts at 6h after treatments, only RC513 was able to increase mRNA levels of Prx2, Prx3, Prx5, and Txn2, which are also involved in peroxide detoxification. RC513 (at 2µM) significantly increased GPx-1 protein expression in HT22 cells, although (PhSe)2 displayed a minor (nonsignificant) effect in this parameter. In agreement, RC513 induced a faster and superior capability to cope with exogenously-added peroxide (t-BuOOH). In summary, when compared to the prototypical organic diaryl diselenide [(PhSe)2], RC513 displayed superior protective properties against MeHg-toxicity in vitro; this was paralleled by a more pronounced upregulation of defenses related to detoxification of peroxides, which are well-known MeHg-derived intermediate oxidant species.

Selenogenome and AMPK signal insight into the protective effect of dietary selenium on chronic heat stress-induced hepatic metabolic disorder in growing pigs

BackgroundChronic heat stress (CHS) disrupts hepatic metabolic homeostasis and jeopardizes product quality of pigs. Selenium (Se) may regulate the metabolic state through affect selenoprotein. Thus, we investigate the protective effect of dietary hydroxy-4-methylselenobutanoic acid (HMSeBA) on CHS induced hepatic metabolic disorder in growing pigs, and the corresponding response of selenoprotein.MethodsForty crossbreed growing pigs were randomly assigned to five groups: control group raised in the thermoneutral environment (22 ± 2 °C) with basal diet; four CHS groups raised in hyperthermal condition (33 ± 2 °C) with basal diet and supplied with 0.0, 0.2, 0.4, and 0.6 mg Se/kg HMSeBA, respectively. The trial lasted 28 d. The serum biochemical, hepatic metabolism related enzyme, protein and gene expression and 25 selenoproteins in liver tissue were determined by real-time PCR, ELISA and western blot.ResultsCHS significantly increased the rectal temperature, respiration rate, serum aspartate aminotransferase (AST) and low-density lipoprotein cholesterol (LDL-C) of pigs, up-regulated hepatic heat shock protein 70 (HSP70) and induced lower liver weight, glycogen content, hepatic glucokinase and glutathione peroxidase (GSH-Px). The CHS-induced liver metabolic disorder was associated with the aberrant expression of 6 metabolism-related gene and 11 selenoprotein encoding genes, and decreased the protein abundance of GCK, GPX4 and SELENOS. HMSeBA improved anti-oxidative capacity of liver. 0.4 or 0.6 mg Se/kg HMSeBA supplementation recovered the liver weight, glycogen content and rescue of mRNA abundance of genes related to metabolism and protein levels of GCK. HMSeBA supplementation changed expressions of 15 selenoprotein encoding genes, and enhanced protein expression of GPX1, GPX4 and SELENOS in the liver affected by CHS. CHS alone showed no impact while HMSeBA supplementation increased protein levels of p-AMPKα in the liver.ConclusionsIn summary, HMSeBA supplementation beyond nutrient requirement mitigates CHS-induced hepatic metabolic disorder, recovered the liver glycogen content and the processes that are associated with the activation of AMPK signal and regulation of selenoproteins in the liver of growing pigs.

Dietary Cacao Powder Supplementation in D-galactose Induced Aging Rat Model Improves Antioxidant Activities

Abstract Objectives Healthy aging is one of the most attentive topics since senescence may lead to retrogression of antioxidative ability, inflammatory regulation and neurodegeneration. This study was performed to determine the antioxidative effects of cacao powder supplementation in d-galactose induced aging rat model. Methods After 3 weeks of adaptation period, 12-week-old SD rats were randomly divided into four groups (n = 8 each): Control group (C), D-galactose induced aging group (G), D-galactose injection with 10% cacao powder group (LC), D-galactose injection with 16% cacao powder group (HC) (10%, 16% is % per total food weight). G, LC, HC groups were intraperitoneally injected with D-galactose for 8 weeks and C group was treated with saline as a substitute. Results After 8 weeks of cacao powder supplementation, serum malondialdehyde (MDA), advanced glycation end (AGE) levels, and Liver malondialdehyde (MDA) level significantly decreased in LC, HC group compared to G group (P &amp;lt; 0.05). Protein expression of SOD2 and CAT in the brain showed significant increase in LC and HC group compared to G group (P &amp;lt; 0.05). Protein expression of Gpx1 in the brain tends to increase in LC and HC group compared to G group but did not show statistical significance. Conclusions This study demonstrates that dietary cacao powder supplementation can alleviate oxidative stress by regulating oxidative stress markers like MDA and AGE or antioxidant enzymes such as SOD2 and CAT in d-galactose induced aging rat model. Funding Sources This study was funded by Nutritional Immunology Lab of Sookmyung Women's University.

Effects of selenium on selenoprotein synthesis and antioxidant parameters of bovine mammary epithelial cells

This study aimed to investigate the effects of selenium (Se) on the selenoproteins synthesis and antioxidant parameters of bovine mammary epithelial cells (BMECs). The experiment was conducted as a single factor completely randomized design to explore the effect of different levels of Se supplementation (0, 10, 20, 50, and 100 nmol/l) on selenoproteins synthesis and antioxidant parameters of BMECs, and to screen the appropriate dose of Se supplementation ensuring a better antioxidant function. Se supplementation increased cell proliferation, the activities of glutathione peroxidase (GPx) and superoxide dismutase, total antioxidant capacity and seleoprotein P (SelP) content, and decreased reactive oxygen species and malondialdehyde levels in a dose-dependent manner. Se supplementation of 50–100 nmol/l had a better effect. Se supplementation also increased thioredoxin reductase (TrxR) activity in a dose-dependent manner, and Se supplementation of 20–50 nmol/l had a better promoting effect. The dose-dependent response between Se supplementation and mRNA and protein expression of GPx1 and TrxR1, as well as SelP mRNA expression was also observed in this experiment. The mRNA and protein expression of GPx1 was up-regulated with the addition of 50–100 nmol/l Se, and the mRNA expression of TrxR1 and SelP was up-regulated with the addition of 20–100 nmol/l Se. Results indicated that Se supplementation of 50 nmol/l had a better promoting effect on the selenoproteins synthesis and antioxidant parameters of BMECs.

Fumonisin B1 induces oxidative stress in oesophageal (SNO) cancer cells

Fumonisin B1 (FB1) is a ubiquitous contaminant of maize that is epidemiologically linked to oesophageal cancer (OC) in South Africa. FB1-induced oxidative stress mediates toxicity in animals and human cell lines, but the effects relating to OC are limited. Given the species-specific effects of FB1, this study investigated FB1-mediated toxicity and oxidative stress in spindle-shaped N-cadherin (+) CD45 (−) osteoblastic (SNO) cells. Following exposure to FB1 (0–20 μM) for 48 h, mitochondrial membrane potential and intracellular reactive oxygen species (iROS) were measured (flow cytometry). Malondialdehyde concentration (lipid peroxidation) was determined spectrophotometrically. ATP and reduced glutathione (GSH) concentrations were quantified using luminometry, gene expression of SOD2 by qPCR and protein expression of SOD2, GPx1, Nrf2 and HSP70 by western blotting. Mitochondrial depolarization increased at 10 μM and 20 μM FB1, with a concomitant reduction in ATP, iROS and GSH at both concentrations. Lipid peroxidation increased at 10 μM FB1 exposure. While transcript levels of SOD2 were significantly increased, protein levels decreased. Protein expression of GPx1, Nrf2 and HSP70 increased. In contrast to the 10 μM and 20 μM FB1 treatment, mitochondrial depolarization decreased at 1.25 μM FB1. Intracellular ROS and ATP were decreased and lipid peroxidation increased. Decreased GSH was accompanied by a decrease in GPx1 protein levels, and increased HSP70 and Nrf2. SOD2 expression and protein levels were significantly increased. Overall these results indicate that FB1 caused increased ROS that were counteracted by engaging the antioxidant defense. Furthermore, the peculiar response at 1.25 μM FB1 is noteworthy, as compared to the other two concentrations tested.

Expression of hepatic antioxidant genes in l-thyroxine-induced hyperthyroid rats: Regulation by vitamin E and curcumin

Earlier we have demonstrated that oral supplementation of vitamin E and curcumin alleviates hyperthyroidism-induced oxidative stress and distorted histoarchitecture in rat liver [5]. To delineate the underlying mechanism of protection, the present study was undertaken to investigate the regulatory role of vitamin E and curcumin on antioxidant gene (AOG) expression in hyperthyroid rat liver. Adult male rats were rendered hyperthyroid by administration of 0.0012% l-thyroxine in their drinking water, while vitamin E (200 mg/kg body weight) and curcumin (30 mg/kg body weight) were supplemented orally for 30 days. l-Thyroxine-induced hyperthyroidism decreased the transcript levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx1) and glutathione reductase (GR) in liver. Alleviated message levels of SOD and CAT were noticed following simultaneous administration of curcumin and vitamin E to hyperthyroid rats. Moreover vitamin E or curcumin treatment ameliorated GPx1 and GR mRNA levels. Translated products of AOGs showed differential expression in the liver of hyperthyroid rats, where Cu/Zn SOD (SOD1), CAT and GR were decreased in contrast to Mn SOD (SOD2) and GPx1. Vitamin E administration was able to alleviate SOD1, CAT and GR translated products while only CAT protein was restored to normal level by curcumin. Co-administration of both antioxidants normalized GPx1 protein expression. Interestingly decreased activities of cytosolic CAT and GPx1 were alleviated following vitamin E and curcumin administration. Increased mitochondrial SOD1 and decreased GR activities were also normalized by antioxidant treatment. Above findings suggest that administration of vitamin E and curcumin may alleviate the hepatic AOG expression in hyperthyroid rats.

Effects of selenium supplementation on expression of glutathione peroxidase isoforms in cultured human lung adenocarcinoma cell lines

Selenium is an essential nutrient, a component of several anti-oxidant enzymes, and a possible factor in cancer risk, including lung cancer. We determined the subtoxic range of selenium concentration (as sodium selenite) required to increase and maintain the expression of anti-oxidant selenoproteins gluthathione peroxidases GPX1 and GPX4 at a constant level in cultures of human lung adenocarcinoma cell lines (H460, H1703 and H1944) and in HPL1D, a non-transformed lung epithelial cell line. Selenium dose-dependently increased GPX1 protein expression 1.8-fold in HPL1D cells and approximately 40-fold in H460 and H1944 cancer cells, with maximum effects at 20-40 nM. GPX4 protein was also increased, but more so in HPL1D (five-fold) than in H460 or H1944 cells (two- to three-fold). GPX1 mRNA showed similar patterns but differences of lesser magnitude. GPX1 protein and activity level was not consistently detectable in H1703 cells, with or without Se supplementation; its mRNA was present but very low. GPX4 protein level was also low in H1703 cells, but was markedly increased by selenium supplementation (48-fold). These results confirm a role for selenium in risk of lung cancer and the independent regulation of GPX1 and GPX4. Characterization of individual tumors with regard to GPX1 and GPX4 levels and regulation might be useful for interpretation of clinical studies on effects of selenium in lung cancer risk.

Homocysteine Down-regulates Cellular Glutathione Peroxidase (GPx1) by Decreasing Translation

Hyperhomocysteinemia contributes to vascular dysfunction and an increase in the risk of cardiovascular disease. An elevated level of homocysteine in vivo and in cell culture systems results in a decrease in the activity of cellular glutathione peroxidase (GPx1), an intracellular antioxidant enzyme that reduces hydrogen peroxide and lipid peroxides. In this study, we show that homocysteine interferes with GPx1 protein expression without affecting transcript levels. Expression of the selenocysteine (SEC)-containing GPx1 protein requires special translational cofactors to "read-through" a UGA-stop codon that specifies SEC incorporation at the active site of the enzyme. These factors include a selenocysteine incorporation sequence (SECIS) in the 3'-untranslated region of the GPx1 mRNA and cofactors involved in the biosynthesis and translational insertion of SEC. To monitor SEC incorporation, we used a reporter gene system that has a UGA codon within the protein-coding region of the luciferase mRNA. Addition of either the GPx1 or GPx3 SECIS element in the 3'-untranslated region of the luciferase gene stimulated read-through by 6-11-fold in selenium-replete cells; absence of selenium prevented translation. To alter cellular homocysteine production, we used methionine in the presence of aminopterin, a folate antagonist, co-administered with hypoxanthine and thymidine (HAT/Met). This treatment increased homocysteine levels in the media by 30% (p < 0.01) and decreased GPx1 enzyme activity by 45% (p = 0.0028). HAT/Met treatment decreased selenium-mediated read-through significantly (p < 0.001) in luciferase constructs containing the GPx1 or GPx3 SECIS element; most importantly, the suppression of selenium-dependent read-through was similar whether an SV40 promoter or the GPx1 promoter was used to drive transcription of the SECIS-containing constructs. Furthermore, HAT/Met had no effect on steady-state GPx1 mRNA levels but decreased GPx1 protein levels, suggesting that this effect is not transcriptionally mediated. These data support the conclusion that homocysteine decreases GPx1 activity by altering the translational mechanism essential for the synthesis of this selenocysteine-containing protein.