GPX1 mRNA Research Articles

The presence of the ovary prevents hepatic mitochondrial oxidative stress in young and aged female mice through glutathione peroxidase 1

BackgroundFor unknown reasons a woman's risk for developing Metabolic Syndrome (MetS) increases dramatically with age and/or loss of ovarian function. The MetS is characterized by hepatic insulin resistance (IR), which is strongly associated with intrahepatic lipid (IHL) accumulation, mitochondrial dysfunction, and oxidative stress. Although circumstantial evidence suggests that the endocrine function of the ovary can directly impact hepatic mitochondrial function, this hypothesis remains untested. Thus, the purpose of this study was to assess the influence of age and secretory function of the ovary on mechanisms that regulate hepatic mitochondrial function. MethodsAdult (10week-old) and aged (88week-old) female C57BL/6 mice were separated into two groups to undergo bilateral ovariectomy (OVX) or control surgery (SHAM). Eight weeks after surgery hepatic tissue was removed for measurements of total IHL and fatty acid species within hepatic triglycerides, mitochondrial function, and reactive oxygen species (ROS) production. ResultsHepatic IHL content was not affected by OVX, but was increased by age. OVX had no effect on mitochondrial respiration, however, hepatic mitochondria from aged mice had lower O2 consumption, lower complex IV and higher complex I content. Mitochondrial H2O2 production was highest in OVX groups and exacerbated by age, while mitochondrial lipid peroxidation was highest in the aged mice and exacerbated by OVX. Regardless of age, OVX resulted in lower mitochondrial content of antioxidant glutathione peroxidase 1 (Gpx1). Isolated liver tissue from a sub-set of animals were acutely treated with conditioned ovarian media which increased Gpx1 mRNA expression compared to vehicle treated liver tissue. ConclusionOvarian secretory function is necessary for the maintenance of hepatic ROS buffering capacity in the mitochondria, while age significantly influences mitochondrial respiration. These data suggest that when age is coupled with loss of ovarian function there is an increased risk for developing hepatic mitochondrial dysfunction, which may influence the onset of metabolic disease. Thus, in females there is critical organ cross-talk occurring between hepatic tissue and the ovary that impacts hepatic mitochondrial function.

The Protective Effects of Different Sources of Maternal Selenium on Oxidative Stressed Chick Embryo Liver.

The experiment was conducted to investigate the protective effects of different sources of maternal selenium (Se) on oxidative stressed chick embryo. A total of 270 Lingnan Yellow broiler breeders were randomly allocated into three treatments with five replicates for 18 birds each. Breeders were fed with basal diet (BD) including 0.04mg/kg Se or BD supplemented with sodium selenite (SS) or selenomethionine (SM) at a level of 0.15mg Se/kg. The rearing experiment lasted for 8weeks after an 8-week pre-test. Twenty eggs were collected from each replicate during the last 10-day, then incubated in a commercial incubator. On embryonic 17th, fertile eggs were transferred into 39.5°C temperature stimulation for 6h. Afterward, five eggs were randomly selected from each replicate for collecting chick embryo sample. The results showed that Se supplementation in the diet of breeders resulted in lower reactive oxygen species (ROS), heat shock protein 70 (HSP70), malondialdehyde (MDA), carbonyl and 8-hydroxydeoxyguanosine (8-OHdG) concentrations and higher glutathione peroxidase (GPx), total superoxide dismutase (T-SOD), and catalase (CAT) activities in heat stress treated chick embryo (P < 0.05), and ROS, MDA, carbonyl, 8-OHdG concentrations in SM treatment were lower than those in SS treatment (P < 0.05). Se supplementation elevated cellular glutathione peroxidase (GPx1) mRNA level and activity, cytoplasmic thioredoxin reductase (TrxR1) activity and selenoprotein P (SelP) mRNA and protein level (P < 0.05), and maternal SM showed a higher value than maternal SS in upregulating GPx1, TrxR1, and SelP mRNA levels as well as GPx1 and TrxR1 activities or SelP protein level (P < 0.05). This study indicated that maternal Se can enhance antioxidative capacity and reduce ROS concentration and oxidative damage by upregulating the expression of antioxidative selenoprotein, and maternal SM is superior to SS in heat stress treated chick embryo.

Expression and DNA methylation alterations of seven cancer-associated 3p genes and their predicted regulator miRNAs (miR-129-2, miR-9-1) in breast and ovarian cancers

The methylation of promoter CpG islands and interactions between microRNAs (miRNAs) and messenger RNAs (mRNAs) of target genes are considered two crucial epigenetic mechanisms for inducing gene and pathway deregulation in tumors. Here, the expression levels of seven cancer-associated 3p genes (RASSF1(isoform A), RARB(isoform 2), SEMA3B, RHOA, GPX1, NKIRAS1, and CHL1) and their predicted regulator miRNAs (miR-129-2, miR-9-1) were analyzed in breast (BC, 40 samples) and ovarian (OC, 14 samples) cancers using RT-PCR and qPCR. We first revealed a negative correlation between the level of the miR-129-2 precursor and RASSF1(A) and GPX1 mRNA levels in BC (Spearman's correlation coefficient (rs) was −0.26 in both cases). Similar results were observed for the miR-129-2 precursor and the RASSF1(A), GPX1, RARB(2), and CHL1 genes in OC (rs was in the range−0.48 to −0.54). Using methylation-specific PCR, a significant correlation was shown between promoter hypermethylation and the down-regulation of the RASSF1(A), GPX1, RARB(2), SEMA3B, MIR-129-2, and MIR-9-1 genes in BC (rs=0.41 to 0.75) and of the RASSF1(A) gene in OC (rs=0.67). We first demonstrated a high hypermethylation frequency of MIR-129-2 and SEMA3B (up to 45 to 48%) in both BC (69 samples) and OC (41 samples). Moreover, we observed a positive correlation between the hypermethylation of MIR-129-2 and the up-regulation of the RASSF1(A) and GPX1 genes in BC (rs=0.38 and 0.42, respectively). QPCR analysis of the expression of RASSF1(A) and mature miR-129-2 in additional BC sample set (24 samples) revealed a negative correlation between them (rs=−0.41) that strengthened the results obtained during the analysis of miR-129-2 precursor level. In summary, the obtained data indicate the involvement of methylation in the down-regulation of the studied coding and miRNA genes and suggest the involvement of miR-129-2 in the deregulation of RASSF1(A) via a direct interaction or/and mediators in common pathways (according to KEGG, Gene Ontology (FDR<0.01), and GeneCards data) in the examined gynecological tumors.

Characterization and expression of a new cytoplasmic glutathione peroxidase 1 gene in the Antarctic fish Trematomus bernacchii

Glutathione peroxidases are a family of antioxidant enzymes catalyzing the reduction of H2O2 or organic hydroperoxides. In the present study, we report the molecular characterization and gene expression analysis of a new GPx-1 from the Antarctic fish Trematomus bernacchii. To expand our knowledge on the GPx-1 s evolution within the group of Antarctic fish, in this work, we also presented the cDNA sequencing of this enzyme in other three species, belonging to two families—Nototheniidae (Trematomus eulepidotus, T. lepidorhinus) and Bathydraconidae (Cygnodraco mawsoni). The deduced amino acid sequences were compared with GPx-1 s of other vertebrates by multiple alignment, in order to evaluate the conservation of amino acids involved in the enzyme activity. The results of phylogenetic analyses indicated that fish GPx-1 s possibly originated from independent duplication events, and Antarctic GPx-1 s evolved according to the molecular and morphological phylogeny of Antarctic fish. Basal GPx-1 mRNA expression analyses in various tissues of T. bernacchii specimens indicated that liver and heart displayed the highest mRNA accumulation; probably a protection of these organs against lipid peroxidation is needed.

Cloning of catalase and expression patterns of catalase and selenium-dependent glutathione peroxidase from Exopalaemon carinicauda in response to low salinity stress

Catalase (CAT) and selenium-dependent glutathione peroxidase (Se-GPx) play a vital role in protecting organisms against various oxidative stresses by eliminating H2O2. The objective of this paper is to evaluate the roles of these antioxidant molecules in the ridgetail white prawn Exopalaemon carinicauda in response to low salinity stress. A complementary DNA (cDNA) containing the complete coding sequence of CAT was cloned from the hepatopancreas using reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends. The full-length cDNA of CAT (2 649 bp) contains a 5′-untranslated region (UTR) of 78 bp, a 3′-UTR of 1 017 bp, with a poly (A) tail, and an open reading frame of 1 554 bp encoding a 517-amino-acid polypeptide with predicted molecular mass of 58.46 kDa and estimated isoelectric point of 6.64. This CAT sequence contained the proximal active site signature (60FDRERIPERVVHAKGAG76), proximal heme-ligand signature sequence (350RLFSYPDTH358) and three catalytic amino acid residues (His71, Asn144 and Tyr354). Sequence comparison showed that the CAT deduced amino acid sequence of E. carinicauda shared 68%-92% of identities with those of other species. Quantitative real-time PCR analysis revealed that CAT mRNA was widely expressed in the hepatopancreas (highest), hemocyte, eyestalk, heart, gill, muscle, ovary and stomach. Under low salinity stress, CAT and GPx mRNA expression levels both in the gill and hepatopancreas increased significantly at the first 48 h and 6 h respectively, indicating a tissue- and time-dependent antioxidant response in E. carinicauda. All these results indicate that E. carinicauda CAT is a member of the CAT family and might be involved in the acute response against low salinity stress.

Effect of tamoxifen and fulvestrant long-term treatments on ROS production and (pro/anti)-oxidant enzymes mRNA levels in a MCF-7-derived breast cancer cell line.

Reactive oxygen species (ROS) are key players in the apoptotic effects induced by short-term tamoxifen treatment of breast cancer cells, but also in acquired resistance following long-term treatment. Whereas the use of the selective estrogen receptor down-regulator fulvestrant is promising, especially in patients who develop tamoxifen resistance, only few studies addressed its implication in the modulation of cellular redox status. The regulation of (pro/anti)-oxidant players were first investigated at the mRNA level in a MCF-7-derived cell line after short-term (24h) estradiol treatment. Long-term anti-estrogen treated MCF-7 derived cell lines were also developed: 3months of 4-hydroxytamoxifen alone (MCF7L-OHTLT) or followed by 3months of fulvestrant (MCF7L-ICILT). Growth properties, hormone sensitivity, receptor content, ROS production and relative mRNA expression of pro or antioxidant enzymes were evaluated in these long-term treated cell lines. Short-term estradiol treatment showed a hormone sensitivity of Nox2, GPx1, GPx2 and SOD1 mRNA levels. The long-term fulvestrant treatment (3months) of MCF7L-OHTLT led to a reduced level of ROS production accompanied with a drastic drop of the accessory protein p22(phox) mRNA. This ROS reduction, although not clearly related to antioxidant enzymes level, seems to be involved in fulvestrant sensitivity of long-term anti-estrogen treated cells, as suggested by the effects of antiradical tempol treatment. When compared to long-term 4-hydroxytamoxifen-treated breast cancer cells, addition of fulvestrant treatment was able to diminish ROS production and p22(phox) mRNA level, and made cells more sensitive to growth inhibition induced by tempol. These effects may be a valuable asset of the fulvestrant treatment.

Cloning, Sequencing, and Expression of Selenoprotein Transcripts in the Turkey (Meleagris gallopavo).

The minimum Se requirement for male turkey poults is 0.3 μg Se/g – three times higher than requirements found in rodents – based on liver and gizzard glutathione peroxidase-4 (GPX4) and GPX1 activities. In addition, turkey liver GPX4 activity is 10-fold higher and GPX1 activity is 10-fold lower than in rats, and both GPX1 and GPX4 mRNA levels are dramatically down-regulated by Se deficiency. Currently, the sequences of all annotated turkey selenoprotein transcripts and proteins in the NCBI database are only “predicted.” Thus we initiated cloning and sequencing of the full turkey selenoprotein transcriptome to demonstrate expression of selenoprotein transcripts in the turkey, and to develop tools to investigate Se regulation of the full selenoproteome. Total RNA was isolated from six tissues of Se-adequate adult tom turkeys, and used to prepare reverse-transcription cDNA libraries. PCR primers were designed, based initially on chicken, rodent, porcine, bovine and human sequences and later on turkey shotgun cloning sequences. We report here the cloning of full transcript sequences for 9 selenoproteins, and 3'UTR portions for 15 additional selenoproteins, which include SECIS elements in 22 3'UTRs, and in-frame Sec (UGA) codons within coding regions of 19 selenoproteins, including 12 Sec codons in SEPP1. In addition, we sequenced the gap between two contigs from the shotgun cloning of the turkey genome, and found the missing sequence for the turkey Sec-tRNA. RTPCR was used to determine the relative transcript expression in 6 tissues. GPX3 expression was high in all tissues except kidney, GPX1 expression was high in kidney, SEPW1 expression was high in heart, gizzard and muscle, and SELU expression was high in liver. SEPP2, a selenoprotein not found in mammals, was highly expressed in liver but not in other tissues. In summary, transcripts for 24 selenoproteins are expressed in the turkey, not just predicted.

Anti-oxidative responses of zebrafish (Danio rerio) gill, liver and brain tissues upon acute cold shock

The present study seeks to detect oxidative damage and to compare anti-oxidative responses among liver, gills and brain of adult zebrafish that were cooled from 28°C (control) to 12°C (treatment) for 0–24h. The lipid peroxidation of liver, gill and brain tissues significantly increased at 1h after transfer, but reactive oxygen species in the treatment group increased significantly after 24h as compared to the control. The fish were found to develop a cascading anti-oxidative mechanism beginning with an increase in Cu/Zn-SOD levels, followed by increased CAT and GPx mRNA expressions in the three tissue types. Both smtB and mt2 mRNAs increased in the hepatic and brain tissues following 1h of cold stress, but only smtB exhibited a significant increase in the gills at 1h and 6h after transfer to 12°C. Furthermore, cellular apoptosis in the brain was not evident after cold shock, but liver and gills showed cellular apoptosis at 1–3h, with another peak in the liver at 6h after cold shock. The results suggest that the cold shock induced oxidative stress, and the enzymatic (SOD, GPx and CAT) and non-enzymatic (mt-2 and smt-B) mRNA expressions all play a role in the resulting anti-oxidation within 1–6h of cold shock. A functional comparison showed that the brain had the most powerful antioxidant defense system of the three tissue types since it had the highest smtB mRNA expression and a lower level of cell apoptosis than the liver and gills after exposure to cold stress.

Selenium Status Biomarkers and Selenium Requirements in the Turkey (M. gallopavo)

Glutathione peroxidase (Gpx) activity has been used extensively to determine selenium (Se) status and requirements. The identification of the full selenoproteome and the use of transcriptomics offer additional biomarkers for assessment of Se status. We recently reported the Se requirement for the turkey based on Gpx1 and Gpx4 activity was 0.3 mg Se/g diet (vs. 0.2 mg/g NRC 1994), and that both Gpx mRNAs were reduced by Se deficiency (Exp Biol Mol 235:23). To determine Se status in today's rapidly growing commercial turkeys using both traditional and mRNA biomarkers, we fed day‐old male poults (n=50) a Se‐deficient diet (0.005 mg/g) supplemented with 10 graded levels of Se (0‐1.0 mg/g) for 28 d. There was no significant effect of Se on growth, although deficient (–Se) poults averaged 533±134 g vs. 769±202 g in 0.4 mg/g poults. Se response curves and plateau breakpoints (BP) were used to determine the impact of deficiency and minimal Se requirements. Plasma Gpx3 activity (n=5/trt) in –Se poults fell to &lt;2% of Se‐adequate levels (+Se), showing our birds were Se deficient. The BP for plasma Gpx3 activity was 0.22 mg/g. Gpx1 in red blood cells fell to 34% of +Se and reached a BP at 0.25 mg/g. Initial analysis (n=3/trt) showed liver Gpx1 and Gpx4 activity in –Se poults fell to 4 and 6% of +Se, respectively, and reached BP at 0.28 and 0.14 mg/g, respectively. qRT‐PCR of total RNA (n=3/trt) showed –Se liver Gpx1 and Gpx4 mRNA levels fell to 32 and 17%, respectively, of +Se, with both BPs at 0.05 mg/g. Turkey Se requirements based on Gpx activity are ~3x higher than in rats but Gpx1 mRNA‐based requirements are the same as in rats. This study shows Gpx4 liver mRNA and activity are highly regulated by Se status in turkeys, in contrast to rodents, and that the turkey minimal Se requirement is 0.3 mg Se/g. Support: WIS 1829 and UW Se Nutrition Research Fund

Effect of exercise training on liver antioxidant enzymes in STZ-diabetic rats.

Abstract Aims Although exercise is shown to improve the antioxidant defense in many organs and pathologies, little research has focused on the effect of chronic exercise training on diabetes-induced liver oxidative stress. We evaluated the effect of chronic treadmill exercise on the hepatic antioxidant enzymes and redox status in STZ-diabetic rats. Main methods Male Wistar rats were randomly assigned into control, trained, diabetic, and diabetic trained groups. Type 1 diabetes mellitus was induced via intraperitoneal injection of STZ. The animals were trained by running on a non-graded motorized treadmill 5 days weekly for a period of 9 weeks. The activity and mRNA levels of antioxidant enzymes, hydrogen peroxide generation, thiol content, and GSH/GSSG ratio were evaluated. Key findings Diabetes negatively affected the hepatic antioxidant defense as indicated by reduced SOD activity, downregulation of SOD1 and GPX1 mRNA levels, increased catalase activity, increased H2O2 production, reduced thiol content and GSH/GSSG ratio. Nine weeks of treadmill exercise training either prevented (SOD1 mRNA, GPX1 mRNA, and thiol content) or ameliorated (catalase activity, SOD activity, H2O2 generation, GSH/GSSG ratio) these diabetes hepatic effects. Significance Our data suggest that exercise training attenuates the diabetes-induced alterations on antioxidant enzymes in the liver of STZ-diabetic rats by improving the hepatic ROS scavenging and redox status. This result strongly implicates a positive effect of exercise training on diabetes-induced liver oxidative stress.

Epigallocatechin-3-gallate enhances the therapeutic effects of leptomycin B on human lung cancer a549 cells.

Our previous studies have shown Leptomycin B (LMB) is a promising antilung cancer drug. Epigallocatechin-3-gallate (EGCG) has antitumor properties but a debatable clinical application. The objective of this study is to evaluate the combination therapeutic effect of LMB and EGCG and its molecular mechanisms in human lung cancer A549 cells. Increased cytotoxicity was observed in LMB+EGCG-treated cells compared to LMB-treated cells. Elevated ROS was maximized 2 h after treatment, and LMB+EGCG-treated cells had higher ROS levels compared to LMB. N-Acetyl-L-cysteine (NAC) studies confirmed the oxidative role of LMB and/or EGCG treatment. In comparison to the control, CYP3A4, SOD, GPX1, and p21 mRNA expression levels were increased 7.1-, 2.0-, 4.6-, and 13.1-fold in LMB-treated cells, respectively, while survivin was decreased 42.6-fold. Additionally, these increases of CYP3A4, SOD, and GPX1 were significantly reduced, while p21 was significantly increased in LMB+EGCG-treated cells compared to LMB-treated cells. The qRT-PCR results for p21 and survivin were further confirmed by Western blot. Our study first shows that LMB produces ROS and is possibly metabolized by CYP3A4, GPX1, and SOD in A549 cells, and combination treatment of LMB and EGCG augments LMB-induced cytotoxicity through enhanced ROS production and the modulation of drug metabolism and p21/survivin pathways.

Estimation of dietary selenium requirement for Chinese egg-laying ducks

The purpose of this study was to estimate the selenium (Se) requirement of egg-laying ducks based on daily egg production and the selenoprotein glutathione peroxidase (Gpx). Five-hundred and forty laying ducks were divided into six treatments, each containing six replicates of 15 ducks. The birds were caged individually and received a Se-deficient basal diet (0.04 mg/kg) or diets supplemented with 0.08, 0.16, 0.24, 0.32, 0.40 mg/kg Se (as sodium selenite) for 6 months. The experiment consisted of two periods: an early-laying period of 2 months and the peak-laying period of 4 months. Egg production and feed intake were recorded daily. At the end of the experiment, blood samples were drawn for determination of Gpx activity in plasma (Gpx3) and in erythrocytes (Gpx1). Hepatic Gpx1 activity and relative expression of Gpx1 mRNA were also determined. Eggs (n = 6) were sampled for quality determination and Se content at the end of the experiment. The activities of plasma Gpx3, erythrocyte Gpx1 and liver Gpx1 increased in a quadratic manner (P &lt; 0.001) with increasing supplemental Se. The mRNA abundance of hepatic Gpx1 increased linearly (P &lt; 0.001) with dietary Se supplementation. Egg shell thickness was significantly reduced in the ducks fed 0.44 mg Se/kg (P &lt; 0.05), indicating that higher dietary Se tends to compromise egg shell quality. Yolk and albumen contents of Se increased linearly (P &lt; 0.0001) with dietary Se supplementation. Using quadratic broken line models, the Se requirement for daily egg production was 0.18 mg/kg for early-laying ducks and 0.24 mg/kg for peak-laying ducks; for optimal function of Gpx (peak-laying ducks), it was 0.37 mg Se/kg.

192 EXPRESSION OF SELECTED ANTIOXIDANT ENZYMES IN BOVINE OVIDUCT EPITHELIAL CELL (BOEC) IN RESPONSE TO ELEVATED TEMPERATURES IN VITRO

Elevated temperatures have a negative impact on bovine reproduction. One of its effects is an increased concentration of reactive oxygen species (ROS) which may lead to female infertility. Oxidative stress impairs oocyte maturation, fertilization, and embryo development, and it also influences the reproductive tract. One of the defence mechanisms against the increase of ROS is the synthesis of antioxidants. Thus, the aim of this study was to analyse the expression of antioxidant enzymes (superoxide dismutase 1, SOD1; catalase, CAT; and glutathione peroxidase 1, GPX1) in bovine oviduct epithelial cells (BOEC) cultured with or without embryos at elevated temperatures. Ovaries and oviducts were collected from a slaughterhouse. BOECs were mechanically isolated from the oviducts. The oocytes were isolated from ovaries and then maturated and fertilized in vitro. BOEC, after formation of aggregates, were cultured (variant I) in 40-µL droplets of cultured medium (TCM199 25 mM HEPES medium supplemented with 10% FBS, 10 µg mL–1 gentamicin, and 50 µg mL–1 streptomycin) overlaid with mineral oil. Twenty aggregates per droplet were cultured at control (38.5°C) and elevated (41°C) temperatures for 168 h in 5% CO2 in air. Analogously, in variant II, BOEC aggregates were co-cultured with 15 bovine embryos per droplet. Subsequently, the SOD1, CAT, and GPX1 mRNA levels were analysed in BOEC by real-time RT–PCR (Light Cycler, Roche Diagnostics, Warsaw, Poland) and normalized to S18/H2A gene expression. Relative quantification was determined with LightCycler software version 3.5 (Roche Diagnostics) by the second derivative maximum method. Statistical analyses were performed by Portable Statgraphics 5.0 Centurion (Statpoint Technologies Inc., Warrenton, VA). Mean values of SOD1, CAT, and GPX1 expression in BOEC in RT-qPCR analysis were compared using Tukey's HSD test (a = 0.01). Elevated temperature leads to an up-regulation of SOD1 in BOEC cultured (38°C: 0.76 ± 0.12 a.u., n = 44; 41°C: 1.07 ± 0.21 a.u., n = 48) and co-cultured with bovine embryos (38°C: 0.71 ± 0.11 a.u., n = 36; 41°C: 1.04 ± 0.2 a.u., n = 36) and the difference was statistically significant (P &lt; 0.01). The CAT gene expression in BOEC was constant in variant I (38°C: 0.56 ± 0.22 a.u., n = 56; 41°C: 0.58 ± 0.27 a.u., n = 56) and variant II (38°C: 0.48 ± 0.27 a.u., n = 32; 41°C: 0.59 ± 0.29 a.u., n = 24). Also, GPX1 gene expression in BOEC was constant in variant I (38°C: 0.66 ± 0.23 a.u., n = 60; 41°C: 0.61 ± 0.19 a.u., n = 56) and in variant II (38°C: 0.59 ± 0.19 a.u., n = 36; 41°C: 0.64 ± 0.22 a.u., n = 36). In conclusion, elevated temperature leads to an activation of the BOEC's defence mechanisms which are based on SOD1 expression, and which may protect cells against oxidative stress. Elevated temperature doesn't affect the cat and GPX1 expression in BOEC. The presence of embryos does not affect the expression of antioxidant enzymes in BOEC. Research was supported by COST DPN/DWM/MZ/5670/08/09.

Effects of Nano-Selenium on Kindey Selenium Contents, Glutathione Peroxidase Activities and GPx-1 mRNA Expression in Mice

The experiment was conducted to evaluate kindey selenium status, glutathione peroxidase (GPx) activity and GPx-1 expression in mice fed with nanoselenium. Sixty KM mice, female and male in half, were randomly divided into control, sodium selenite and nanoselenium groups. 0.5 milliliter of water, sodium selenite (2 μg Se/mL) and nanoselenium (2 μg Se/mL) were respectively supplemented to the three groups in oral (ig) every day. Whole experiment lasted for 28 days. Kindey selenium contents, GPx activities and GPx-1 mRNA expression were analyzed at experiment trrmination. The results showed that kindey selenium contents and GPx activities in nanoselenium group and sodium selenite group were very significantly higher than those in control group (P&lt;0.01); kindey GPx activities in nanoselenium group were significantly higher than that in sodium selenite group (P&lt;0.05). Kindey mRNA expression of GPx-1 was approx 166% higher in nanoselenium group and approx 157% higher in sodium selenite group than that in control group. Kindey mRNA expression of GPx-1 was approx 3.50 % higher in nanoselenium group than that in sodium selenite group. The results indicated that nanoselenium supplementation could significantly enhance kindey selenium contents, GPx activities and GPx-1 mRNA expression in mice, nanoselenium was more available than sodium selenite in increasing kindey selenium contents, GPx activities and GPx-1 mRNA expression in mice.

Methylation of promoter and expression silencing of GPX3 gene in hepatocellular carcinoma tissue

Accumulating evidence has identified transcriptional silencing by aberrant methylation of CpG islands as a potential mechanism for the inactivation of tumor suppressor genes. The role of aberrant methylation of the GPX3 promoter in hepatocellular carcinoma (HCC) is not yet clear. We investigated the association of the status of GPX3 promoter methylation and GPX3 protein expression with the clinicopathological progression of HCC. Sixty HCC tumor and matched non-cancerous tissues were included in this study, and methylation was examined using MSP. GPX3 mRNA and protein levels were evaluated using RT-PCR and western blot analysis, respectively. Clinicopathological data were compiled for correlation analyses. Among the 60 HCC cases, 76.7% (46/60) showed at elevated DNA methylation and displayed significantly lower levels of GPX3 mRNA and protein expression. Low or undetectable GPX3 protein expression was observed in 10 of 60 tumors. GPX3 promoter methylation was detected in 46 of 60 (76.7%) tumors, while no GPX3 gene promoter methylation was observed in the matched non-cancerous specimens. There was a negative correlation between promoter methylation and GPX3 mRNA levels (P<0.05). Analysis of clinicopathological data revealed that both mRNA and protein were significantly associated with portal tumor thrombosis, metastasis and differentiation. In additional, GPX3 methylation showed a relationship with portal tumor thrombosis, metastasis and differentiation. Our results suggest that promoter methylation may be a mechanism for inactivation of GPX3, possibly leading to subsequent carcinogenesis and progression of HCC.

Pigment epithelium–derived factor exerts antioxidative effects in granulosa cells

To determine whether supplementing granulosa cells cultures with pigment epithelium-derived factor (PEDF) can protect them from oxidative stress. We used either granulosa cell line or human primary granulosa cell culture from women undergoing invitro fertilization (IVF) treatments. University research facilities. Imprinting control region female mice. Recombinant PEDF (rPEDF) was added to cultures of either primary granulosa cell culture or granulosa cell line in the present or absence of H2O2 triggering. We followed cell viability with the use of methylthiazolyl tetrazolium assay and tracked PEDF mechanism of action with the use of Western blot analysis, measuring the level of SOD-1 and GPX-1 mRNA, protein level of BAX, and phosphorylation of AKT. We found that granulosa cell viability and the level of PEDF mRNA were both significantly reduced, in a dose-dependent manner, after exposure to H2O2. The rate of H2O2-induced apoptosis was significantly attenuated in granulosa cells treated with rPEDF. We showed that granulosa cells, of both humans and rodents, express the PEDF receptor, PNPLA2; once stimulated by rPEDF, the cells exhibited phosphorylation of AKT. Finally, we showed that PEDF exerts its antioxidative activity through the AKT signaling pathway. This study demonstrates that PEDF represents a novel intrinsic antioxidant of granulosa cells.

APOA1 mRNA expression in ovarian serous carcinoma effusions is a marker of longer survival.

We previously described the overexpression of APOA1 and GPX3 in ovarian/peritoneal serous carcinoma compared with breast carcinoma effusions using gene expression array analysis. The objective of the present study was to validate this finding and to analyze the association between these genes and clinicopathologic parameters, including survival, in advanced-stage ovarian serous carcinoma. APOA1 and GPX3 mRNA expression using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was analyzed in 121 effusions (101 ovarian, 20 breast carcinomas) and 85 solid ovarian carcinoma specimens (43 primary carcinomas, 42 metastases). APOA1 and GPX3 transcript levels were significantly higher in ovarian carcinoma at all anatomic sites compared with breast carcinoma effusions (P < .001). GPX3 mRNA levels were significantly higher in primary carcinomas and solid metastases from patients who received neoadjuvant chemotherapy compared with chemo-naïve tumors (P = .016). APOA1 and GPX3 mRNA levels in the entire effusion series were unrelated to clinicopathologic parameters. However, higher APOA1 mRNA levels in primary diagnosis pre-chemotherapy effusions were significantly related to better overall survival (P = .045), a finding that retained its significance in Cox multivariate analysis (P = .016). APOA1 and GPX3 mRNA levels on qRT-PCR effectively differentiate ovarian from breast carcinoma. APOA1 may be a novel prognostic marker in metastatic serous carcinoma.

The oxido‐reductase enzyme glutathione peroxidase 4 (GPX4) governsSalmonella Typhimurium‐induced neutrophil transepithelial migration

SummaryNeutrophil (polymorphonuclear leucocytes; PMN) transmigration across mucosal surfaces contributes to dysfunction of epithelial barrier properties, a characteristic underlying many mucosal inflammatory diseases. Using Salmonella enterica serovar Typhimurium (S. Typhimurium) as a prototypic proinflammatory insult, we have previously reported that the eicosanoid hepoxilin A3 (HXA3), an endogenous product of 12-lipoxygenase (12-LOX) activity, is secreted from the apical surface of the intestinal epithelium to establish a chemotactic gradient that guides PMN across the epithelial surface. Since little is known regarding the molecular mechanisms that regulate 12-LOX during S. Typhimurium infection, we investigated this pathway. We found that expression of phospholipid glutathione peroxidase (GPX4), which is known to have an inhibitory effect on 12-LOX activity, is significantly decreased at both the mRNA and protein level during infection with S. Typhimurium. Moreover, employing intestinal epithelial cell monolayers expressing siRNA against GPX4 mRNA, S. Typhimurium-induced PMN migration was significantly increased compared with the non-specific siRNA control cells. Conversely, in cells engineered to overexpress GPX4, S. Typhimurium-induced PMN migration was significantly decreased, which is consistent with the finding that partial depletion of GPX4 by RNAi resulted in a significant increase in HXA3 secretion during S. Typhimurium infection. Mechanistically, although we found Salmonella entry not to be required for the induced decrease in GPX4, the secreted effector, SipA, which is known to induce epithelial responses leading to stimulation of HXA3, governed the decrease in GPX4 in a process that does not lead to an overall increase in the levels of ROS. Taken together, these results suggest that S. Typhimurium induces apical secretion of HXA3 by decreasing the expression of phospholipid GPX, which in turn leads to an increase in 12-LOX activity, and hence HXA3 synthesis.

Panax ginseng extract modulates oxidative stress, DNA fragmentation and up-regulate gene expression in rats sub chronically treated with aflatoxin B1 and fumonisin B 1.

Aflatoxins and fumonisins are important food-borne mycotoxins implicated in human health and have cytotoxic effects. The aims of the current study were to evaluate the protective role of Panax ginseng extract (PGE) against the synergistic effect of subchronic administration of aflatoxin B1 (AFB1) and fumonisin B1 (FB1) on DNA and gene expression in rat. Female Sprague-Dawley rats were divided into eight groups (ten rats/group) and treated for 12weeks including the control group, the group having received AFB1 (80µg/kg bw), the group having received FB1 (100µg/kg bw), the group having received AFB1 plus FB1 and the groups having received PGE (20mg/kg bw) alone or with AFB1 and/or FB1. At the end of experiment, liver and kidney were collected for the determination of DNA fragmentation, lipid peroxidation (LP), glutathione (GSH) contents and alterations in gene expression. The results indicated that these mycotoxins increased DNA fragmentation, LP and decreased GSH content in liver and kidney and down-regulated gene expression of antioxidants enzymes. The combined treatments with AFB1 and/or FB1 plus PGE suppressed DNA fragmentation only in the liver, normalized LP and increased GSH in the liver and kidney as well as up-regulated the expression of GPx, SOD1 and CAT mRNA. It could be concluded that AFB1 and FB1 have synergistic genotoxic effects. PGE induced protective effects against their oxidative stress and genotoxicity through its antioxidant properties.

Effects of deer velvet extract from Formosan sika deer on the embryonic development and anti-oxidative enzymes mRNA expression in mouse embryos

Ethnopharmacrological relevanceThe deer velvet or its extracts has been widely used in clinic. It has been used in promoting reproductive performances and treating of oxidation and aging process. The aim of this study is to investigate the effects of velvet extract from Formosan sika deer (Formosan sika deer; Cervus nippon taiouanus, FSD) velvet on mouse embryonic development and anti-oxidant ability in vitro. Materials and methodsMouse 4-cells embryos were divided into 16 groups for 72h in vitro incubation. The embryonic development stages and morphology were evaluated every 12h in experimental period. The quantitative real time PCR was used to measure the CuZn-SOD, GPx and CAT mRNA expression of the blastocysts. ResultsThe 4-cells embryos of hydrogen peroxide (HP) groups did not continue developing after oxidant stress challenged. The blastocyst developmental rate (90.0–90.4%, P>0.05) and normal morphological rate (84.4–85.1%, P>0.05) of the 1% and 2% DV extract groups were similar to those in the control group (90.7% and 88.8%, respectively). The embryos challenged by HP (5, 10 and 25μM) and subsequently incubated in mHTF medium with 1% and 2% of deer velvet (DV) extracts were able to continue development; the blastocyst developmental rate of these groups were similar to that in the control group. The relative mRNA expression of the focused anti-oxidative enzymes in the mouse embryos did not significantly differ among the designed DV treatment groups (P>0.05). ConclusionThe FSD velvet extract in adequate concentration could promote anti-oxidative enzymes mRNA expression followed the challenge of hydrogen peroxide, relieve the mouse embryo under oxidative stress, and maintain the blastocyst developmental ability in vitro.