Glutamate Cysteine Ligase Expression Research Articles

Efficacy of oltipraz in preventing acetaminophen-induced liver injury in mice.

Oltipraz (OPZ) is a synthetic dithiolethione with potential as a cancer chemopreventive agent, which can work by inducing detoxification enzymes. OPZ is an activator of nuclear factor erythroid 2-related factor 2 (Nrf2), suggesting its involvement in enzyme induction and possible protection against drug-induced liver injury. In this study, we present OPZ-mediated protection of mice against acetaminophen (APAP)-induced liver injury and discuss its possible contributing factors. Overnight-fasted male CD-1 mice were administered APAP intraperitoneally, and some mice were administered OPZ 16h before APAP. Hepatotoxicity was assessed by measuring serum alanine aminotransferase leakage and histopathological evaluation. The hepatic mRNA expressions of CYP2E1, glutamate cysteine ligase (GCL), and NAD(P)H:quinone oxidoreductase (NQO1) were measured by real-time reverse-transcription polymerase chain reaction. OPZ protected mice from APAP-induced liver injury in a dose-dependent manner, but did not alter hepatic glutathione (GSH) content or GCL expression in control mice, indicating that its hepatoprotective effect is not due to changes in basal GSH levels. OPZ did not affect CYP2E1 expression or APAP-induced early GSH depletion, suggesting it does not inhibit the metabolic activation of APAP to produce N-acetyl-p-benzoquinone imine. In contrast, after GSH depletion, OPZ accelerated hepatic GSH recovery. APAP significantly increased GCL expression during liver injury, but OPZ treatment only led to additional NQO1 expression. This suggests that NQO1 is responsible for the enhanced GSH recovery and protection against APAP-induced liver injury seen in OPZ-treated mice. In summary, OPZ protects against APAP-induced liver injury by inducing NQO1 expression and resulting in improved GSH recovery.

Elemene induces cell apoptosis via inhibiting glutathione synthesis in lung adenocarcinoma.

The rhizome of Curcuma wenyujin Y.H. Chen & C. Ling, also known as Wen-E-Zhu, has been used for cancer treatment since ancient times, with roots dating back to the Song Dynasty. Elemene (EE), a sesquiterpene extract with potent anticancer properties, is extracted from Wen-E-Zhu, with β-elemene (BE) being its main active compound, along with trace amounts of β-caryophyllene (BC), γ-elemene and δ-elemene isomers. EE has demonstrated broad-spectrum anti-cancer effects and is commonly used in clinical treatments for various types of malignant cancers, including lung cancer. Studies have shown that EE can arrest the cell cycle, inhibit cancer cell proliferation, and induce apoptosis and autophagy. However, the exact mechanism of its anti-lung cancer activity remains unclear and requires further research and investigation. In this study, the possible mechanism of EE and its main active components, BE and BC, against lung adenocarcinoma was investigated by using A549 and PC9 cell lines. The subcutaneous tumor model of nude mice was constructed to evaluate the efficacy of EE in vivo, then the in vitro half-inhibitory concentration (IC50) of EE and its main active components, BE and BC, on A549 and PC9 cells at different concentrations were determined by CCK-8. Flow cytometry was used to detect the apoptosis and cycle of A549 and PC9 cells treated with different concentrations of BE and BC for 24h. Non-targeted metabolomics analysis was performed on A549cells to explore potential target pathways, which were subsequently verified through kit detection and western blot analysis. Injection of EE in A549 tumor-bearing mice effectively suppressed cancer growth in vivo. The IC50 of EE and its main active components, BE and BC, was around 60μg/mL. Flow cytometry analysis showed that BE and BC blocked the G2/M and S phases of lung adenocarcinoma cells and induced apoptosis, leading to a significant reduction in mitochondrial membrane potential (MMP). Results from non-targeted metabolomics analysis indicated that the glutathione metabolism pathway in A549cells was altered after treatment with the active components. Kit detection revealed a decrease in glutathione (GSH) levels and an increase in the levels of oxidized glutathione (GSSG) and reactive oxygen (ROS). Supplementation of GSH reduced the inhibitory activity of the active components on lung cancer and also decreased the ROS content of cells. Analysis of glutathione synthesis-related proteins showed a decrease in the expression of glutaminase, cystine/glutamate reverse transporter (SLC7A11), and glutathione synthase (GS), while the expression of glutamate cysteine ligase modified subunit (GCLM) was increased. In the apoptosis-related pathway, Bax protein and cleaved caspase-9/caspase-9 ratio were up-regulated and Bcl-2 protein was down-regulated. EE, BE, and BC showed significant inhibitory effects on the growth of lung adenocarcinoma cells, and the mechanism of action was linked to the glutathione system. By down-regulating the expression of proteins related to GSH synthesis, EE and its main active components BE and BC disrupted the cellular redox system and thereby promoted cell apoptosis.

Nrf2 Regulation by Curcumin: Molecular Aspects for Therapeutic Prospects.

Nuclear factor erythroid 2 p45-related factor (2Nrf2) is an essential leucine zipper protein (bZIP) that is primarily located in the cytoplasm under physiological conditions. Nrf2 principally modulates endogenous defense in response to oxidative stress in the brain.In this regard, Nrf2 translocates into the nucleus and heterodimerizes with the tiny Maf or Jun proteins. It then attaches to certain DNA locations in the nucleus, such as electrophile response elements (EpRE) or antioxidant response elements (ARE), to start the transcription of cytoprotective genes. Many neoplasms have been shown to have over activated Nrf2, strongly suggesting that it is responsible for tumors with a poor prognosis. Exactly like curcumin, Zinc–curcumin Zn (II)–curc compound has been shown to induce Nrf2 activation. In the cancer cell lines analyzed, Zinc–curcumin Zn (II)–curc compound can also display anticancer effects via diverse molecular mechanisms, including markedly increasing heme oxygenase-1 (HO-1) p62/SQSTM1 and the Nrf2 protein levels along with its targets. It also strikingly decreases the levels of Nrf2 inhibitor, Kelch-like ECH-associated protein 1 (Keap1) protein.As a result, the crosstalk between p62/SQSTM1 and Nrf2 could be used to improve cancer patient response to treatments. The interconnected anti-inflammatory and antioxidative properties of curcumin resulted from its modulatory effects on Nrf2 signaling pathway have been shown to improve insulin resistance. Curcumin exerts its anti-inflammatory impact through suppressing metabolic reactions and proteins such as Keap1 that provoke inflammation and oxidation. A rational amount of curcumin-activated antioxidant Nrf2 HO-1 and Nrf2-Keap1 pathways and upregulated the modifier subunit of glutamate-cysteine ligase involved in the production of the intracellular antioxidant glutathione. Enhanced expression of glutamate-cysteine ligase, a modifier subunit (GLCM), inhibited transcription of glutamate-cysteine ligase, a catalytic subunit (GCLC). A variety of in vivo, in vitro and clinical studies has been done so far to confirm the protective role of curcumin via Nrf2 regulation. This manuscript is designed to provide a comprehensive review on the molecular aspects of curcumin and its derivatives/analogs via regulation of Nrf2 regulation.

Epalrestat suppresses cadmium-induced cytotoxicity through Nrf2 in endothelial cells.

Cadmium (Cd) is an industrial and environmental pollutant that targets the vascular endothelium. The vascular system is critically affected by Cd toxicity. Recent studies have indicated an association between Cd and vascular diseases, although the mechanisms of Cd implications in vascular diseases are not clear. The purpose of the present study was to determine whether epalrestat (EPS), which is used for the treatment of diabetic neuropathy, protects against Cd-induced cytotoxicity in bovine aortic endothelial cells (BAECs). In the present study, the effects of EPS at near-plasma concentration were examined on Cd-induced cytotoxicity in BAECs. Cd-induced cytotoxicity was suppressed by pretreatment with EPS. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor that serves a role in regulating the expression of glutamate cysteine ligase, the rate-limiting enzyme in glutathione (GSH) synthesis. In a previous study, EPS was demonstrated to increase GSH levels in BAECs in association with the Nrf2 pathway. In the present study, EPS increased GSH levels in BAECs exposed to Cd. The protective ability of EPS against the Cd-induced cytotoxicity disappeared following Nrf2 small interfering RNA transfection. In addition, EPS affected the intracellular levels of Cd, Cd transporter ZIP8 and metallothionein. To the best of our knowledge, the current study demonstrated, for the first time, that EPS suppresses Cd-induced cytotoxicity in BAECs. The upregulation of GSH may be associated with the suppression of Cd-induced cytotoxicity by EPS. From these findings, it may be proposed that the regulation of GSH, ZIP8 and metallothionein by EPS is a promising therapeutic approach to prevent Cd-induced toxicity.

Whey protein boosts the antioxidant profile of rats by enhancing the activities of crucial antioxidant enzymes in a tissue-specific manner

Whey protein, a by-product of cheese industry, is harmful for the environment (i.e., surface and subterranean waters, soil) and, therefore, for humans due to its high polluting burden. Concomitantly, it has been reported that it is a mixture with potent antioxidant action since it is rich in cysteine residues, which are necessary for glutathione synthesis in vivo. On this basis, this study intended to examine the role of whey protein on the intensification of tissue antioxidant arsenal. To this end, a dose of sheep/goat whey protein equal to 1 g/kg of body weight/day dissolved in drinking water was administered to rats for 28 consecutive days. According to our findings, whey protein improved the antioxidant profile of liver, small intestine, lung and muscle whereas it did not affect the redox state of kidney. Our results were based on the alterations found in the protein expression of glutamate cysteine ligase, catalase and superoxide dismutase-1 measured in all tissues and the activity of glutathione S-transferase evaluated in muscle. Although tissue-specific, it is obvious that the action of whey protein is biologically beneficial and could serve as a biofunctional constituent for foods able to improve redox profile when administered against redox-related diseases.

The Sulphur Response in Wheat Grain and Its Implications for Acrylamide Formation and Food Safety.

Free (soluble, non-protein) asparagine concentration can increase many-fold in wheat grain in response to sulphur deficiency. This exacerbates a major food safety and regulatory compliance problem for the food industry because free asparagine may be converted to the carcinogenic contaminant, acrylamide, during baking and processing. Here, we describe the predominant route for the conversion of asparagine to acrylamide in the Maillard reaction. The effect of sulphur deficiency and its interaction with nitrogen availability is reviewed, and we reiterate our advice that sulphur should be applied to wheat being grown for human consumption at a rate of 20 kg per hectare. We describe the genetic control of free asparagine accumulation, including genes that encode metabolic enzymes (asparagine synthetase, glutamine synthetase, glutamate synthetase, and asparaginase), regulatory protein kinases (sucrose nonfermenting-1 (SNF1)-related protein kinase-1 (SnRK1) and general control nonderepressible-2 (GCN2)), and basic leucine zipper (bZIP) transcription factors, and how this genetic control responds to sulphur, highlighting the importance of asparagine synthetase-2 (ASN2) expression in the embryo. We show that expression of glutamate-cysteine ligase is reduced in response to sulphur deficiency, probably compromising glutathione synthesis. Finally, we describe unexpected effects of sulphur deficiency on carbon metabolism in the endosperm, with large increases in expression of sucrose synthase-2 (SuSy2) and starch synthases.

Inhibition of MEK/ERK upregulates GSH production and increases RANKL-induced osteoclast differentiation in RAW 264.7 cells

Osteoclasts (OCs) are multinucleated cells that are phylogenetically evolved from monocyte–macrophage lineage and are essential for skeletal coupling processes. During bone development, bone formation by osteoblasts and bone resorption by OCs are tightly coupled and are involved in bone homeostasis. Therefore, it is essential to understand the mechanisms that regulate OC differentiation in order to develop effective therapeutics for the treatment of OC-associated diseases. This study aimed to determine the molecular mechanisms regulating OC differentiation. The mitogen-activated protein kinases and extracellular signal-regulated kinase (ERK) are recognised to be crucial factors regulating OC differentiation and activation. RAW 264.7 cells were differentiated into OCs in the presence of RANKL and were treated with inhibitors of several signal pathways. Although PD98059 is an ERK inhibitor, it inhibited the phosphorylation of ERK, JNK, Akt, and Src kinase. PD98059 increased OC differentiation and expression of OC markers, such as TRAP, calcitonin receptor, and cathepsin K, and increased the expression of NFATc1. Moreover, it also increased the expression of glutamate-cysteine ligase and production of glutathione (GSH). Thus, we examined the involvement of GSH in OC differentiation and observed that GSH treatment alone increased the OC numbers and cotreatment with PD98059 further enhanced OC differentiation. Our results suggested that inhibition of the ERK pathway may promote OC differentiation via upregulation of GSH. These findings reveal that ERK and GSH modulate the signal pathway necessary for OC differentiation, and this may form the basis of a new therapeutic strategy for treating OC-related diseases.

5-Caffeoylquinic acid ameliorates oxidative stress-mediated cell death via Nrf2 activation in hepatocytes

Context 5-Caffeoylquinic acid (5-CQA) is one of the most abundant compounds found in natural foods including coffee. Objective We investigated whether 5-CQA had a cytoprotective effect through the NF-E2-related factor 2 (Nrf2)-antioxidant response element (ARE) signalling pathway. Materials and methods Nrf2 activation in response to 5-CQA treatment at the concentration of 10–100 μM is evaluated by Western blotting of Nrf2 and ARE reporter gene assay as well as its target gene expression in HepG2 cells. Intracellular reactive oxygen species (ROS) and glutathione (GSH) levels were measured in the tert-butyl hydroperoxide-induced hepatocytes to examined cytoprotective effect of 5-CQA (10–100 μM). The specific role of 5-CQA on Nrf2 activation was examined using Nrf2 knockout cells or Nrf2 specific inhibitor, ML-385. Results Nuclear translocation of Nrf2 is increased by 5-CQA in HepG2 cells which peaked at 6 h. Consequently, 5-CQA significantly increases the ARE reporter gene activity and downstream antioxidant proteins, including glutamate cysteine ligase (GCL), hemeoxygenase-1 (HO-1), NAD(P)H quinone oxidoreductase 1, and Sestrin2. Nrf2 deficiency or inhibition completely antagonized ability of 5-CQA to induce HO-1 and GCL expression. Cells pre-treated with 5-CQA were rescued from tert-butyl hydroperoxide-induced ROS production and GSH depletion. Nrf2 activation by 5-CQA was due to increased phosphorylation of MAPKs, AMPK and PKCδ. Discussion and conclusions Taken together, our results demonstrate that as a novel Nrf2 activator, 5-CQA, may be a promising candidate against oxidative stress-mediated liver injury. Additional efforts are needed to assess 5-CQA, as a potential therapeutic in liver diseases in vivo and in humans.

The Association of Cigarette Smoke Exposure with Lung Cellular Toxicity and Oxidative Stress: the Protective Role of Crocin.

Cigarette smoke (CS) contains many free radicals and toxic chemicals. Nuclear erythroid-related factor-2 (Nrf2) is a transcriptional regulator of several phase II antioxidant genes, including glutamate-cysteine ligase (GCL). In this study, it was hypothesized that Crocin may mediate antioxidant signaling pathway to protect human lung epithelial cells against CS-mediated toxicity and oxidative stress via inducing glutathione (GSH) biosynthesis and activation of Nrf2 pathway. Alveolar epithelial cells (A549) were exposed to 1, 2.5 and 5% cigarette smoke extracts (CSE) with or without Crocin (500μM). After 48h exposure, the cytotoxicity, oxidant/antioxidant parameters and the Nrf2 pathway modification were assayed. Treatment of A549 cells with all concentrations of CSE dose dependently decreased cell viability, antioxidant levels, GCL and Nrf2 gene expression, which was associated with increased production of reactive oxygen species. Crocin not only restored CSE-depleted GSH levels by enhancing GCL expression via activation of Nrf2 but also quenched the CSE-generation and release of reactive oxygen species. Crocin attenuated CSE-mediated Nrf2 modifications, thereby inducing its nuclear accumulation associated with GCL gene transcription leading to enhanced GSH levels. By inducing GSH synthesis, Crocin attenuates CSE-mediated GSH depletion and protects cells against CSE-induced oxidative stress via Nrf2 pathway. These results may have implications in dietary modulation of natural antioxidants in treatment of pulmonary diseases.

Curcumin attenuates oxidative stress in RAW264.7 cells by increasing the activity of antioxidant enzymes and activating the Nrf2-Keap1 pathway.

Large-scale breeding environments often lead to oxidative stress. Macrophages play an important role in the immune system and are vulnerable to reactive oxygen species (ROS), which result in macrophage death. Curcumin is the main active component of turmeric and exerts antioxidant effects. Here, we measured the activity of some antioxidant enzymes and chose the Nrf2-Keap1 signaling pathway to study the protective effects of curcumin on macrophages under oxidative stress in vitro. We used RAW264.7 cells as a research model, and oxidative damage was induced by hydrogen peroxide (H2O2). Cell viability was measured by an MTT assay. Flow cytometry was used to measure cellular ROS and apoptosis. The effect of curcumin on Nrf2-Keap1 signaling pathway-related genes was analyzed by qRT-PCR. Furthermore, the translocation of Nrf2 protein was also investigated by Western blot analysis of total and nuclear proteins. All curcumin-treated groups exhibited increased activity of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX). Low- and middle-dose curcumin decreased malondialdehyde (MDA) and ROS levels, but high-dose curcumin increased MDA and ROS production. We found that low-dose curcumin protected cells from apoptosis, while apoptosis in the middle- and high-dose curcumin-treated groups were stagnant in the early stage. Furthermore, middle-dose curcumin upregulated Nrf2 expression after H2O2 treatment for 4 h. Low- and middle-dose curcumin could activate Nrf2 and promote it to migrate into nuclei. The translocation of Nrf2 to the nucleus to upregulate the expression of haemoxygenase-1 (HO-1) was promoted in the low- and middle-dose curcumin-treated groups. The middle-dose curcumin-treated group also exhibited enhanced expression of glutamate-cysteine ligase, a modifier subunit (GLCM), but inhibited transcription of glutamate-cysteine ligase, a catalytic subunit (GCLC). Curcumin resisted oxidants by increasing the activity of antioxidant enzymes and activating the Nrf2-Keap1 pathway, which could potentially promote cell survival.

Alterations in oocyte mitochondrial number and function are related to spindle defects and occur with maternal aging in mice and humans†

The objective of this work was to determine the role of mitochondria in the loss of oocyte quality with maternal aging. Our results show that mitochondrial DNA (mtDNA) copy number and function are reduced in eggs from aged mice after both in vivo and in vitro maturation. Higher incidences of spindle abnormalities were observed in old eggs. However, no correlation with egg ATP content was found. In vitro matured eggs from aged mice did not have a normal cortical distribution of active mitochondria and were subject to increased oxidative stress due to higher levels of reactive oxygen species and lower expression of glutamate-cysteine ligase, catalytic subunit (Gclc). Supplementation of antioxidants during in vitro maturation of old eggs mitigated this affect, resulting in increased mtDNA copy number and mitochondrial function, a mitochondria distribution pattern similar to young eggs, and improved chromosomal alignment. Eggs from women of advanced maternal age (AMA) had lower mitochondrial function than eggs from young women, although both age groups displayed a cortical distribution pattern of active mitochondria. In contrast to the mouse, human eggs from AMA women had higher mtDNA copy number than eggs from young women following in vitro maturation. In summary, oocytes of older females are more susceptible to perturbations in mitochondrial number and function, which are associated with increased spindle abnormalities and oxidative stress during in vitro maturation. These results demonstrate that oocyte mitochondria play a critical role in age-related infertility.

Depletion of thiols leads to redox deregulation, production of 4-hydroxinonenal and sperm senescence: a possible role for GSH regulation in spermatozoa†.

We hypothesized that thiols and particularly glutathione (GSH) are essential for the regulation of stallion sperm functionality. To test this hypothesis, we initially investigated the relationship between sperm function and GSH content, revealing highly significant correlations between GSH, sperm viability, motility, and velocity parameters (P<0.001). Furthermore, the deleterious effects of GSH depletion using menadione and 1,3 dimethoxy 1,4, naphtoquinone (DMNQ) were able to be prevented by the addition of cysteine, but no other antioxidant. Pre-incubation with cysteine prevented menadione and DMNQ induced damage to sperm membranes after 1 h (P<0.001; P<0.05) and after 3 h of incubation (P<0.001, P<0.05). Pre-incubation with cysteine ameliorated both the menadione- and DMNQ-induced increase in 4-hydroxynonenal (P<0.001). As cysteine is a precursor of GSH, we hypothesized that stallion spermatozoa are able to synthesize this tripeptide using exogenous cysteine. To test this hypothesis, we investigated the presence of two enzymes required to synthesize GSH (GSH and GCLC) and using western blotting and immunocytochemistry we detected both enzymes in stallion spermatozoa. The inhibition of GCLC reduced the recovery of GSH by addition of cysteine after depletion, suggesting that stallion spermatozoa may use exogenous cysteine to regulate GSH. Other findings supporting this hypothesis were changes in sperm functionality after BSO treatment and changes in GSH and GSSG validated using HPLC-MS, showing that BSO prevented the increase in GSH in the presence of cysteine, although important stallion to stallion variability occurred and suggested differences in expression of glutamate cysteine ligase. Mean concentration of GSH in stallion spermatozoa was 8.2±2.1 μM/109 spermatozoa, well above the nanomolar ranges per billion spermatozoa reported for other mammals.

Retinol but not retinoic acid can enhance the glutathione level, in a manner similar to β-carotene, in a murine cultured macrophage cell line.

ScopeWe evaluated the potential of retinol and retinoic acid (RA) to enhance intracellular glutathione (GSH) levels in a murine cultured macrophage cell line, RAW264, to investigate whether the RA signaling pathway is involved in the β‐carotene‐induced GSH enhancement.Methods and resultsWe examined GSH levels in RAW264 cells cultured in media supplemented with β‐carotene and various inhibitors (ER50891 for RA receptor (RAR)α, CD2665 for RARβ/γ, or HX531 for all subtypes of retinoid X receptor (RXR)), to verify each inhibitor's activity against β‐carotene, as well as in media supplemented with various stimulants (AM80 for RARα, CD2314 for RARβ, CD437 for RARγ, or SR11237 for RXR), to compare their activity with that of β‐carotene. We also examined the GSH level and glutamate‐cysteine‐ligase (GCL) expression in RAW264 cells cultured in all‐trans RA‐ or retinol‐supplemented media. Enhanced GSH production was not inhibited by any tested antagonist, and, apart from β‐carotene, no agonist induced GSH production. Retinol, but not all‐trans RA, enhanced GSH synthesis and increased GCL expression, similar to that observed with β‐carotene.ConclusionThe RA signaling pathway may not be involved in the β‐carotene‐induced enhancement of GSH levels in RAW264 cells, whereas, like β‐carotene, retinol can enhance the GSH level and GCL expression.

Effect of a Sulforaphane and Maitake Mushroom Extract Combination on the Expression of Glutamate Cysteine Ligase

Oxidative stress contributes to numerous health issues including neurodegenerative diseases and cancer. Reduced glutathione (GSH) is a major antioxidant that neutralizes reactive oxygen species (ROS) occurring both endogenously through respiration and exogenously during exposure to environmental stressors. Increasing levels of GSH to combat ROS is critical to reducing oxidative stress. The rate‐limiting step of glutathione synthesis is catalyzed by the enzyme glutamate cysteine ligase, a heterodimer consisting of a Glutamate‐Cysteine Ligase Catalytic Subunit (GCLC) and a Glutamate‐Cysteine Ligase Modifier Subunit (GCLM). In this study, we focus on the effect the combination of sulforaphane and maitake mushroom extract has in elevating the production of GSH as a means of preventing cellular damage caused by oxidative stress. HepG2 liver cancer cells were treated over a time course of 24h with maitake mushroom extract (250 μg/mL) and a physiologically achievable level of sulforaphane (0.5 μM). The combination induced gene expression of both GCLC and GCLM an average of 2‐fold as early as 6h. Additionally, we examined the levels of GSH in HepG2 cells treated with the combination.Previously, our laboratory demonstrated that the combination induced the expression of Phase 2 detoxifying enzymes NAD(P)H quinone dehydrogenase 1(NQO‐1), UDP glucuronosyltransferase 1 family, polypeptide A1 (UGT1A1), and heme oxygenase 1 (HMOX‐1/HSP32) as well as reduced the expression of pro‐inflammatory markers interleukin‐6 (IL‐6) and cyclooxygenase‐2 (COX‐2). These results show that the sulforaphane and maitake mushroom extract combination possesses anti‐oxidative properties that may promote cellular health.Support or Funding InformationResearch supported by Nutramax LaboratoriesThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Modulated Gene Expression of Toxoplasma gondii Infected Retinal Pigment Epithelial Cell Line (ARPE-19) via PI3K/Akt or mTOR Signal Pathway.

Due to the critical location and physiological activities of the retinal pigment epithelial (RPE) cell, it is constantly subjected to contact with various infectious agents and inflammatory mediators. However, little is known about the signaling events in RPE involved in Toxoplasma gondii infection and development. The aim of the study is to screen the host mRNA transcriptional change of 3 inflammation-related gene categories, PI3K/Akt pathway regulatory components, blood vessel development factors and ROS regulators, to prove that PI3K/Akt or mTOR signaling pathway play an essential role in regulating the selected inflammation-related genes. The selected genes include PH domain and leucine- rich-repeat protein phosphatases (PHLPP), casein kinase2 (CK2), vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), glutamate-cysteine ligase (GCL), glutathione S-transferase (GST), and NAD(P)H: quinone oxidoreductase (NQO1). Using reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), we found that T. gondii up-regulates PHLPP2, CK2β, VEGF, GCL, GST, and NQO1 gene expression levels, but down-regulates PHLPP1 and PEDF mRNA transcription levels. PI3K inhibition and mTOR inhibition by specific inhibitors showed that most of these host gene expression patterns were due to activation of PI3K/Akt or mTOR pathways with some exceptional cases. Taken together, our results reveal a new molecular mechanism of these gene expression change dependent on PI3K/Akt or mTOR pathways and highlight more systematical insight of how an intracellular T. gondii can manipulate host genes to avoid host defense.

T24. REDOX REGULATORS AND OXIDATIVE STRESS IN SCHIZOPHRENIA

BackgroundElevated levels of oxidative stress have been reported in schizophrenia and may play a role in the underlying psychopathology. The antioxidant defense system may be disrupted in schizophrenia. We have earlier shown how levels of membrane polyunsaturated fatty acids (PUFA) change during the course of schizophrenia, increasing from a low level in a subgroup, remaining stable in another. Here, we aimed at comparing levels of redox regulators, oxidative stress, and related genotypes in schizophrenia patients and healthy controls, and at identifying how these biomarkers are related to membrane fatty acids and clinical characteristics in acute and chronic phase of schizophrenia.MethodsPatients with schizophrenia spectrum disorders (n=55) examined during an acute phase and five years later during a chronic phase, and healthy controls (n=51) were included. We assessed blood levels of redox regulators [(alpha-tocopherol, bilirubin, uric acid, glutathione (GSH), glutathione peroxidase (GPx)], glutathione reductase (GR), markers of oxidative stress [F2-isoprostane, reactive oxygen metabolites (D-ROMs)] and PUFA. We examined genotypes and gene expression related to glutamate cysteine ligase (GCL), the rate-limiting enzyme of GSH synthesis, and its catalytic (GCLC) and modulator (GCLM) subunits. Links between redox measures and Positive and Negative Syndrome Scale (PANSS) were studied.ResultsIn the chronic phase, levels of alpha-tocopherol (p=0.03) and bilirubin (p<0.001) were lower, while uric acid (p=0.02) was higher in patients than in controls. In patients, the levels of alpha-tocopherol were higher in the acute phase than in the chronic phase (p=0.001). However, the changes depended on the PUFA levels in the acute phase. In the low PUFA group, alpha-tocopherol levels remained stable, whereas in the high PUFA group, they decreased to those of the low PUFA group. Levels of D-ROMs were linked to PUFA (r=0.39, p=0.007) and long-chain PUFA (r=0.42, p=0.003) in controls but not in patients.There was no significant difference in the distribution of GCLC genotypes between patients and controls. Compared to other GAG trinucleotide-repeat (TNR) genotypes, 7/9 GAG genotype was linked to higher gene expression of GCL (mRNA GCLC, p=0.049; mRNA GCLM, p=0.02), higher levels of GSH in blood (p=0.02) and higher GR activity in blood (p=0.007). Only when combined with C-129T high risk genotype, the 7/7 GAG genotype induced a strong reduction of CGLC expression (p=0.045, bootstrapping 10000 samples). Gene expression related to glutathione synthesis was non-significantly (p=0.10–0.19) higher among patients. Levels of long-chain PUFA were significantly positively linked to gene expression of glutathione related enzymes.DiscussionThe findings of abnormal levels of alpha-tocopherol, bilirubin, uric acid and glutathione synthesis in the chronic phase of schizophrenia indicate oxidative stress. Dysregulation of antioxidant defenses may be involved in the pathophysiology of schizophrenia and warrants further experimental studies.

Huangqi Decoction Alleviates Alpha-Naphthylisothiocyanate Induced Intrahepatic Cholestasis by Reversing Disordered Bile Acid and Glutathione Homeostasis in Mice.

Intrahepatic cholestasis is a serious symptom of liver disorders with limited therapies. In this study, we investigated the efficacy of Huangqi decoction (HQD), a two-herb classic traditional Chinese medicine (TCM), in the treatment of alpha-naphthylisothiocyanate (ANIT)-induced intrahepatic cholestasis in mice. HQD treatment ameliorated impaired hepatic function and tissue damage. A metabolomics study revealed that the endogenous metabolites significantly affected by HQD were related to bile acid (BA) biosynthesis and glutathione metabolism pathways. HQD treatment decreased the intrahepatic accumulation of cytotoxic BAs, normalized serum BA levels, and increased biliary and urinary BA excretion. Additionally, HQD restored the hepatic glutathione content and suppressed reactive oxygen species (ROS) in cholestatic mice. Protein and gene analysis revealed that HQD increased the expression of the hepatic metabolizing enzymes cytochrome P450 (CYP) 2B10 and UDP glucuronosyltransferase family 1 member A1 (UGT1A1), as well as multidrug resistance-associated protein 2 (Mrp2), Mrp3, and Mrp4, which play crucial roles in BA homeostasis. Further, HQD increased the protein expression of glutamate-cysteine ligase, which is involved in the synthesis of glutathione. Importantly, HQD increased the nuclear expression of nuclear factor-E2-related factor-2 (Nrf2). In conclusion, HQD protects against intrahepatic cholestasis by reversing the disordered homeostasis of BAs and glutathione.

Glutathione homeostasis is significantly altered by quercetin via the Keap1/Nrf2 and MAPK signaling pathways in rats

Previously, we showed that 0.5% quercetin simultaneously decreased serum homocysteine and glutathione (GSH) levels in rats. The aim of the present study was to investigate the effects of 0.5% quercetin on GSH metabolism, related enzymes and signal pathways in rats. Rats were fed the control diet and 0.5% quercetin-supplemented diet for 6 weeks. The results showed that quercetin reduced serum and hepatic content of GSH and the ratio of GSH and oxidized glutathione (GSSG), enhanced hepatic activity and mRNA expression of glutathione S-transferase (GST), inhibited hepatic activity and mRNA expression of glutamate cysteine ligase (GCL), and decreased hepatic glutathione reductase (GR) mRNA expression. Levels of phosphorylated p38 and extracellular signal-regulated kinase (ERK) 1/2 mitogen-activated protein kinases (MAPKs) increased, while that of nuclear factor E2-like 2 (Nrf2) protein decreased after quercetin treatment. However, no significant hepatotoxicity was noted. We concluded that quercetin treatment altered hepatic GSH metabolism by modulating GSH metabolic enzyme activities and mRNA expression in rats, and p38, ERK1/2 MAPKs, and Nrf2 were involved in modulating GSH metabolism-related enzymes.

Effect of biomechanical stress on endogenous antioxidant networks in bovine articular cartilage

Mechanosensitve pathways in chondrocytes are essential for maintaining articular cartilage homeostasis. Traumatic loading increases cartilage oxidation and causes cell death and osteoarthritis. However, sub-lethal doses of the pro-oxidant molecule tert-Butyl hydroperoxide (tBHP) protects against loading-induced chondrocyte death. We hypothesized that compressive cyclic loading at moderate strains (<20%) causes sub-lethal cartilage oxidation that induces an adaptive increase in the endogenous antioxidant defense network. We tested this hypothesis by subjecting healthy bovine articular cartilage explants to in vitro static or cyclic (1 Hz) compressive loading at 50 kPa (15% strain, "physiologic") versus 300 kPa (40% strain, "hyper-physiologic") for 12 h per day for 2 days. We also treated unloaded explants with 100 μM tBHP for 12 h per day for 2 days to differentiate between biomechanical and chemical pro-oxidant stimulation. All loading conditions induced glutathione oxidation relative to unloaded controls, but only the 50 kPa cyclic loading condition increased total glutathione content (twofold). This increase was associated with a greater expression of glutamate-cysteine ligase, the rate-limiting step in glutathione synthesis, compared to 300 kPa cyclic loading. 50 kPa cyclic loading also increased the expression of superoxide dismutase-1 and peroxiredoxin-3. Like 50 kPa loading, tBHP treatment also increased total glutathione content. However, tBHP treatment and 50 kPa cyclic loading differed in their effect on the expression of genes regulating antioxidant defense and cartilage matrix synthesis and degradation. These findings suggest that glutathione metabolism is a mechanosensitive antioxidant defense pathway in chondrocytes and that intermittent pro-oxidant treatment alone is insufficient to account for all changes in mediators of cartilage homeostasis associated with cyclic loading. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:760-769, 2018.

Temporal changes in glutathione biosynthesis during the lipopolysaccharide-induced inflammatory response of THP-1 macrophages

How macrophages maintain redox homeostasis in the inflammatory process, in which a large amount of oxidants are produced, remains elusive. In this study, we investigated the temporal changes in the intracellular glutathione (GSH), the master antioxidant, and the expression of glutamate cysteine ligase (GCL), the rate-limiting enzyme for GSH biosynthesis, in the inflammatory response of human macrophages (THP1 cells) to lipopolysaccharide. Intracellular GSH concentration was decreased significantly in the early phase (~6h) of LPS exposure, and then gradually went back to the basal level in the late phase (9–24h). The expression level of the catalytic subunit of GCL (GCLC) followed a similar pattern of change as GSH: its mRNA and protein levels were reduced in the early phase and then back to basal level in the late phase. In contrast, the expression of the modifier subunit of GCL (GCLM) was significantly increased in the phase of LPS exposure. Activation Nrf2, the transcription factor involved in the induction of both GCLC and GCLM, occurred at as early as 3h after LPS exposure; whereas the activation of NF-κB occurred at as early as 30min. Inhibition of NF-κB signaling with SN50 prevented the decrease of GCLC and inhibited Nrf2 activation in response to LPS. These data demonstrate time-dependent changes in the expression of GCL and Nrf2 signaling during the inflammatory response, and that the regulation of GCLC and GCLM might be through different pathways in this process.