Gamma-glutamylcysteine Synthetase Research Articles

A Th17 cell-intrinsic glutathione/mitochondrial-IL-22 axis protects against intestinal inflammation.

The intestinal tract generates significant reactive oxygen species (ROS), but the role of Tcell antioxidant mechanisms in maintaining intestinal homeostasis is poorly understood. We used Tcell-specific ablation of the catalytic subunit of glutamate cysteine ligase (Gclc), which impaired glutathione (GSH) production, crucially reducing IL-22 production by Th17 cells in the lamina propria, which is critical for gut protection. Under steady-state conditions, Gclc deficiency did not alter cytokine secretion; however, C.rodentium infection induced increased ROS and disrupted mitochondrial function and TFAM-driven mitochondrial gene expression, resulting in decreased cellular ATP. These changes impaired the PI3K/AKT/mTOR pathway, reducing phosphorylation of 4E-BP1 and consequently limiting IL-22 translation. The resultant low IL-22 levels led to poor bacterial clearance, severe intestinal damage, and high mortality. Our findings highlight a previously unrecognized, essential role of Th17 cell-intrinsic GSH in promoting mitochondrial function and cellular signaling for IL-22 protein synthesis, which is critical for intestinal integrity and defense against gastrointestinal infections.

A Th17 cell-intrinsic glutathione/mitochondrial-IL-22 axis protects against intestinal inflammation.

The intestinal tract generates significant reactive oxygen species (ROS), but the role of Tcell antioxidant mechanisms in maintaining intestinal homeostasis is poorly understood. We used Tcell-specific ablation of the catalytic subunit of glutamate cysteine ligase (Gclc), which impaired glutathione (GSH) production, crucially reducing IL-22 production by Th17 cells in the lamina propria, which is critical for gut protection. Under steady-state conditions, Gclc deficiency did not alter cytokine secretion; however, C.rodentium infection induced increased ROS and disrupted mitochondrial function and TFAM-driven mitochondrial gene expression, resulting in decreased cellular ATP. These changes impaired the PI3K/AKT/mTOR pathway, reducing phosphorylation of 4E-BP1 and consequently limiting IL-22 translation. The resultant low IL-22 levels led to poor bacterial clearance, severe intestinal damage, and high mortality. Our findings highlight a previously unrecognized, essential role of Th17 cell-intrinsic GSH in promoting mitochondrial function and cellular signaling for IL-22 protein synthesis, which is critical for intestinal integrity and defense against gastrointestinal infections.

Evolution of the pathogenic mold Aspergillus fumigatus on high copper levels identifies novel resistance genes.

Aspergillus fumigatus is the leading cause of severe mold infections in immunocompromised patients. This common fungus possesses innate attributes that allow it to evade the immune system, including its ability to survive the high copper (Cu) levels in phagosomes. Our previous work has revealed that under high Cu levels, the A. fumigatus transcription factor AceA is activated, inducing the expression of the copper exporter CrpA to expel excess Cu. To identify additional elements in Cu resistance, we evolved A. fumigatus wild-type and mutant ΔaceA or ΔcrpA strains under increasing Cu concentrations. Sequencing of the resultant resistant strains identified both shared and unique evolutionary pathways to resistance. Reintroduction of three of the most common mutations in genes encoding Pma1 (plasma membrane H+-ATPase), Gcs1 (glutamate cysteine-ligase), and Cpa1 (carbamoyl-phosphate synthetase), alone and in combination, into wild-type A. fumigatus confirmed their additive role in conferring Cu resistance. Detailed analysis indicated that the pma1 mutation L424I preserves Pma1 H+-ATPase activity under high Cu concentrations and that the cpa1 mutation A37V confers a survival advantage to conidia in the presence of Cu. Interestingly, simultaneous mutations of all three genes did not alter virulence in infected mice. Our work has identified novel Cu-resistance pathways and provides an evolutionary approach for dissecting the molecular basis of A. fumigatus adaptation to diverse environmental challenges.IMPORTANCEAspergillus fumigatus is the most common mold infecting patients with weakened immunity. Infection is caused by the inhalation of mold spores into the lungs and is often fatal. In healthy individuals, spores are engulfed by lung immune cells and destroyed by a combination of enzymes, oxidants, and high levels of copper. However, the mold can protect itself by pumping out excess copper with specific transporters. Here, we evolved A. fumigatus under high copper levels and identified new genetic mutations that help it resist the toxic effects of copper. We studied how these mutations affect the mold's ability to resist copper and how they impact its ability to cause disease. This is the first such study in a pathogenic mold, and it gives us a better understanding of how it manages to bypass our body's defenses during an infection.

Pro-Oxidant Auranofin and Glutathione-Depleting Combination Unveils Synergistic Lethality in Glioblastoma Cells with Aberrant Epidermal Growth Factor Receptor Expression.

Glioblastoma (GBM) is the most prevalent and advanced malignant primary brain tumor in adults. GBM frequently harbors epidermal growth factor receptor (EGFR) wild-type (EGFRwt) gene amplification and/or EGFRvIII activating mutation. EGFR-driven GBM relies on the thioredoxin (Trx) and/or glutathione (GSH) antioxidant systems to withstand the excessive production of reactive oxygen species (ROS). The impact of EGFRwt or EGFRvIII overexpression on the response to a Trx/GSH co-targeting strategy is unknown. In this study, we investigated Trx/GSH co-targeting in the context of EGFR overexpression in GBM. Auranofin is a thioredoxin reductase (TrxR) inhibitor, FDA-approved for rheumatoid arthritis. L-buthionine-sulfoximine (L-BSO) inhibits GSH synthesis by targeting the glutamate-cysteine ligase catalytic (GCLC) enzyme subunit. We analyzed the mechanisms of cytotoxicity of auranofin and the interaction between auranofin and L-BSO in U87MG, U87/EGFRwt, and U87/EGFRvIII GBM isogenic GBM cell lines. ROS-dependent effects were assessed using the antioxidant N-acetylsteine. We show that auranofin decreased TrxR1 activity and increased ROS. Auranofin decreased cell vitality and colony formation and increased protein polyubiquitination through ROS-dependent mechanisms, suggesting the role of ROS in auranofin-induced cytotoxicity in the three cell lines. ROS-dependent PARP-1 cleavage was associated with EGFRvIII downregulation in U87/EGFRvIII cells. Remarkably, the auranofin and L-BSO combination induced the significant depletion of intracellular GSH and synergistic cytotoxicity regardless of EGFR overexpression. Nevertheless, molecular mechanisms associated with cytotoxicity were modulated to a different extent among the three cell lines. U87/EGFRvIII exhibited the most prominent ROS increase, P-AKT(Ser-473), and AKT decrease along with drastic EGFRvIII downregulation. U87/EGFRwt and U87/EGFRvIII displayed lower basal intracellular GSH levels and synergistic ROS-dependent DNA damage compared to U87MG cells. Our study provides evidence for ROS-dependent synergistic cytotoxicity of auranofin and L-BSO combination in GBM in vitro. Unraveling the sensitivity of EGFR-overexpressing cells to auranofin alone, and synergistic auranofin and L-BSO combination, supports the rationale to repurpose this promising pro-oxidant treatment strategy in GBM.

Effect of N-acetyl-L-cysteine administration in drinking water of broilers exposed to heat stress on performances, intestinal integrity, and oxidative status

Supplementing the diet of broiler with N-acetyl-L-cysteine (NAC) has been shown to support small intestinal integrity, and as a precursor of L-cysteine, it may also support antioxidant functions. As heat-stressed chickens suffer from intestinal disturbances caused by oxidative stress and at the same time increase water consumption, it was hypothesised that NAC supplemented via the drinking water could reinforce finisher broiler productivity under heat stress. Hence, three hundred 15-day-old male Ross 308 broilers were assigned to three treatments until 39 days of age: thermoneutral (TN), heat-stressed (HS), or HS + NAC. NAC was administered to the drinking water at 1000 mg/L starting on day 20. At 22 days, a chronic cyclic heat stress model was initiated in the HS and HS + NAC groups. On day 39, birds were euthanized and sampled for physiological measurements. Heat stress significantly decreased weight gain and feed intake from 22 to 39 days but did not affect feed efficiency. Rectal temperature and respiration rate significantly increased in heat-stressed broilers on days 23, 30, and 38. Also, ileal mucosal malondialdehyde (MDA) and plasma MDA levels were significantly higher in the HS group and the HS + NAC group, respectively, as compared to the TN group, whereas MDA levels in lung tissue were lower in both HS groups. Small intestinal villus height was significantly lower and serum FITC-d levels were significantly higher in the HS group compared to the TN group, without the effect of NAC supplementation. Furthermore, hepatic gene transcript of glutamate-cysteine ligase catalytic subunit was upregulated under heat stress conditions which may reflect higher glutathione synthesis, whereas in HS + NAC birds cystathionine-beta synthase-like (CBSL) mRNA level was dramatically reduced, indicating decreased transsulfuration activity. In conclusion, the administration of 1000 mg/L NAC in drinking water did not alleviate the reduced performances of heat-stressed finisher broilers. Nevertheless, it resulted in the downregulation of CBSL expression, suggesting a potential methionine-sparing effect.

Liver epigenomic signature associated with chronic oxidative stress in a mouse model of glutathione deficiency

Oxidative stress is intimately involved in the pathogenesis of fatty liver disease (FLD). A major factor contributing to oxidative stress is the depletion of the ubiquitous antioxidant glutathione (GSH). Unexpectedly, chronic GSH deficiency renders glutamate-cysteine ligase modifier subunit (Gclm)-null mice protected from fatty liver injuries. Epigenetic regulation serves as an important cellular mechanism in modulating gene expression and disease outcome in FLD, although it is not well understood how systemic redox imbalance modifies the liver epigenome. In the current study, utilizing the Gclm-null mouse model, we aimed to elucidate redox-associated epigenomic changes and their implications in liver stress response. We performed high-throughput array-based DNA methylation profiling (MeDIP array) in 22,327 gene promoter regions (from −1300 bp to +500 bp of the Transcription Start Sites) in the liver and peripheral blood cells. Results from the MeDIP array demonstrate that, although global methylation enrichment in gene promoters did not change, low GSH resulted in prevalent demethylation at the individual promoter level. Such an effect likely attributed to a declined availability of the methyl donor S-adenosyl methionine (SAM) in Gclm-null liver. Functional enrichment analysis of liver target genes is suggestive of a potential role of epigenetic mechanisms in promoting cellular survival and lipid homeostasis in Gclm-null liver. In comparison with the liver tissue, MeDIP array in peripheral blood cells revealed a panel of 19 gene promoters that are candidate circulating biomarkers for hepatic epigenomic changes associated with chronic GSH deficiency. Collectively, our results provided new insights into the in vivo interplay between liver redox state and DNA methylation status. The current study laid the groundwork for future epigenetic/epigenomic investigations in experimental settings or human populations under conditions of liver oxidative stress induced by environmental or dietary challenges.

Neuronal glutathione depletion elevates the Aβ42/Aβ40 ratio and tau aggregation in Alzheimer's disease mice.

Alzheimer's disease (AD) involves reduced glutathione levels, causing oxidative stress and contributing to neuronal cell death. Our prior research identified diminished glutamate-cysteine ligase catalytic subunit (GCLC) as linked to cell death. However, the effect of GCLC on AD features such as amyloid and tau pathology remained unclear. To address this, we investigated amyloid pathology and tau pathology in mice by combining neuron-specific conditional GCLC knockout mice with amyloid precursor protein (App) knockin (KI) or microtubule-associated protein tau (MAPT) KI mice. Intriguingly, GCLC knockout resulted in an increased Aβ42/40 ratio. Additionally, GCLC deficiency in MAPT KI mice accelerated the oligomerization of tau through intermolecular disulfide bonds. These findings suggest that the decline in glutathione levels, due to aging or AD pathology, may contribute to the progression of AD.

IRE1α determines ferroptosis sensitivity through regulation of glutathione synthesis

Cellular sensitivity to ferroptosis is primarily regulated by mechanisms mediating lipid hydroperoxide detoxification. We show that inositol-requiring enzyme 1 (IRE1α), an endoplasmic reticulum (ER) resident protein critical for the unfolded protein response (UPR), also determines cellular sensitivity to ferroptosis. Cancer and normal cells depleted of IRE1α gain resistance to ferroptosis, while enhanced IRE1α expression promotes sensitivity to ferroptosis. Mechanistically, IRE1α’s endoribonuclease activity cleaves and down-regulates the mRNA of key glutathione biosynthesis regulators glutamate-cysteine ligase catalytic subunit (GCLC) and solute carrier family 7 member 11 (SLC7A11). This activity of IRE1α is independent of its role in regulating the UPR and is evolutionarily conserved. Genetic deficiency and pharmacological inhibition of IRE1α have similar effects in inhibiting ferroptosis and reducing renal ischemia–reperfusion injury in mice. Our findings reveal a previously unidentified role of IRE1α to regulate ferroptosis and suggests inhibition of IRE1α as a promising therapeutic strategy to mitigate ferroptosis-associated pathological conditions.

Implications of GCLC in prognosis and immunity of lung adenocarcinoma and multi-omics regulation mechanisms

BackgroundFerroptosis is an iron-dependent type of regulated cell death, and has been implicated in lung adenocarcinoma (LUAD). Evidence has proved the key role of glutamate-cysteine ligase catalytic subunit (GCLC) in ferroptosis, but its role in LUAD remains unclear. Herein, we explored the implications of GCLC and relevant genes in LUAD prognosis and immunity as well as underlying molecular mechanisms.MethodsThis work gathered mRNA, miRNA, DNA methylation, somatic mutation and copy-number variation data from TCGA-LUAD. WGCNA was utilized for selecting GCLC-relevant genes, and a GCLC-relevant prognostic signature was built by uni- and multivariate-cox regression analyses. Immune compositions were estimated via CIBERSORT, and two immunotherapy cohorts of solid tumors were analyzed. Multi-omics regulatory mechanisms were finally assessed.ResultsOur results showed that GCLC was overexpressed in LUAD, and potentially resulted in undesirable survival. A prognostic model was generated, which owned accurate and independent performance in prognostication. GCLC, and relevant genes were notably connected with immune compositions and immune checkpoints. High GCLC expression was linked with better responses to anti-PD-L1 and anti-CTLA-4 treatment. Their possible DNA methylation sites were inferred, e.g., hypomethylation in cg19740353 might contribute to GCLC up-regulation. Frequent genetic mutations also affected their expression. Upstream transcription factors (E2F1/3/4, etc.), post-transcriptional regulation of miRNAs (hsa-mir-30c-1, etc.), lncRNAs (C8orf34-AS1, etc.), and IGF2BP1-mediated m6A modification were identified. It was also found NOP58-mediated SUMOylation post-translational modification.ConclusionsTogether, we show that GCLC and relevant genes exert crucial roles in LUAD prognosis and immunity, and their expression can be controlled by complex multi-omics mechanisms.

Nuclear factor erythroid 2-related factor 2 promotes radioresistance by regulating glutamate-cysteine ligase modifier subunit and its unique immunoinvasive pattern.

The enzyme glutamate-cysteine ligase modifier subunit (GCLM) serves as the initial rate-limiting factor in glutathione (GSH) synthesis. GSH is the preferred substrate for glutathione peroxidase 4 (GPX4), directly impacting its activity and stability. This study aims to elucidate the expression of GCLM and its correlation with the nuclear factor erythroid 2-related factor 2 (NFE2L2), commonly referred to as NRF2, in esophageal squamous cell carcinoma (ESCC) and further investigate the potential signaling axis of radiotherapy resistance caused by NRF2-mediated regulation of ferroptosis in ESCC. The expression of NRF2, GCLM, and GPX4 in ESCC was analyzed by bioinformatics, and their relationship with ferroptosis was verified through cell function experiments. Their role in radioresistance was then investigated through multiple validation steps. Bioinformatics analysis was employed to determine the immune infiltration pattern of NRF2 in ESCC. Furthermore, the effect of NRF2-mediated massive macrophage M2 infiltration on radiotherapy and ferroptosis was validated through in vivo experiments. In vitro assays demonstrated that overactivated NRF2 promotes radioresistance by directly binding to the promoter region of GCLM. The Tumor Immune Estimation Resource (TIMER) and quanTIseq analyses revealed NRF2 enrichment in M2 macrophages with a positive correlation. Co-culturing KYSE450 cells with M2 macrophages demonstrated that a significant infiltration of macrophages M2 can render ESCC cells resistant to radiotherapy but restore their sensitivity to ferroptosis in the process. Our study elucidates a link between the NRF2-GCLM-GSH-GPX4 signaling axis in ESCC, highlighting its potential as a therapeutic target for antagonistic biomarkers of resistance in the future. Additionally, it provides a novel treatment avenue for ESCC metastasis and radioresistance.

TMT-Based Quantitative Proteomics Reveal the Metabolic Changes Underlying Growth Superiority in a Novel Gymnocypris Hybrid, Gymnocypris przewalskii ♀ × G. eckloni ♂

Hybrid breeding is an effective approach to generate better varieties and prevent variety degradation. The present study investigated the metabolic changes underlying growth superiority in the novel Gymnocypris hybrid (GH), Gymnocypris przewalskii ♀ (GP) × G. eckloni ♂ (GE). The ranking of survival rate was GH > GE > GP, whereas the ranking of growth rate was GE > GH > GP. A proteomic analysis of G. hybrid and its parents was conducted to elucidate the metabolic changes underlying growth superiority. Identified pathways were primarily associated with amino acid, carbohydrate, energy, lipid, and nucleotide metabolism. These metabolic pathways, which are closely associated with growth, are controlled through regulation of the expression of numerous proteins, including adenosylhomocysteinase, hydroxypyruvate reductase, glutamate-cysteine ligase, L-lactate dehydrogenase, creatine kinase, GDP-L-fucose synthase, pyruvate kinase, fructose-bisphosphate aldolase, carbonic anhydrase, phosphopyruvate hydratase, phosphoglycerate kinase, S-(hydroxymethyl) glutathione dehydrogenase, and AMP deaminase. Real-time PCR assays showed that the level of mRNA expression of differentially expressed genes was positively correlated with growth. Proteins that were differentially expressed in GH exhibited fewer differences from GP and more differences from GE. These data are the first to reveal the molecular mechanism whereby growth is regulated in G. hybrid and its parents at the protein level. The study thus provides important information for genetic breeding and improvement of G. hybrid for aquaculture production.

Med1 inhibits ferroptosis and alleviates liver injury in acute liver failure via Nrf2 activation

BackgroundExtensive hepatocyte mortality and the absence of specific medical therapy significantly contribute to the unfavorable prognosis of acute liver failure (ALF). Ferroptosis is a crucial form of cell death involved in ALF. In this study, we aimed to determine the impact of Mediator complex subunit 1 (Med1) on ferroptosis and its potential hepatoprotective effects in ALF.ResultsMed1 expression is diminished in the liver of lipopolysaccharide (LPS)/D-galactosamine (D-GalN)-induced ALF mice, as well as in hepatocytes damaged by H2O2 or TNF-α/D-GalN in vitro. Med1 overexpression mitigates liver injury and decreases the mortality rate of ALF mice by ferroptosis inhibition. The mechanism by which Med1 inhibits erastin-induced ferroptosis in hepatocytes involves the upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream antioxidant genes heme oxygenase-1 (HO-1), glutamate cysteine ligase catalytic (GCLC), and NAD(P)H quinone oxidoreductase 1 (NQO1). Furthermore, Med1 overexpression suppresses the transcription of proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the liver of mice with LPS/D-GalN-induced ALF.ConclusionOverall, our research findings indicate that Med1 suppresses ferroptosis and alleviates liver injury in LPS/D-GalN-induced ALF through the activation of Nrf2. These findings substantiate the therapeutic viability of targeting the Med1-Nrf2 axis as a means of treating individuals afflicted with ALF.Graphical

Solid state fermentation of Diaphragma juglandis fructus with Aspergillus niger alleviates hydrogen peroxide-induced oxidative stress in HepG2 cells via Nrf2 signaling pathway

Diaphragma juglandis fructus (DJF) is a traditional medicine, which is rich in bioactive polyphenols and has been proven to exhibit better antioxidant activity. However, the potential effects of fermentation of DJF on oxidative stress remains unclear. Herein, this study aimed to investigate the impact of solid-state fermentation (SSF) with Aspergillus niger (A. niger) on the polyphenol content and biological activity in DJF. Furthermore, compared with unfermented Diaphragma juglandis fructus (UF-DJF) extracts, ellagic acid (EA), sinapic acid (SA) and (+)-catechin (CA) displayed the highest increase in soluble phenolics (SPs) of fermented Diaphragma juglandis fructus (F-DJF) extracts. Thus, we explored the protective effect of EA, SA, CA and co-existing of EA, SA, CA against hydrogen peroxide (H2O2)-induced oxidative stress in HepG2 cells. Our results demonstrated that the insoluble-bound phenolics (IBPs) could be released, thereby increasing the SPs content and in vitro antioxidant activity after SSF. In addition, we also found that F-DJF extracts had the best inhibitory effect on three digestive enzymes among UF-DJF, before fermented Diaphragma juglandis fructus (BF-DJF) and F-DJF extracts. Moreover, our results revealed that pre-treatment with EA, SA, CA and co-present of EA, SA, CA could protect HepG2 cells from oxidative damage through activating the Nrf2 signaling pathway and up-regulating the expression of heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase quinone1 (NQO1), glutamate-cysteine ligase catalytic subunit (GCLC), cytoplasmic Cu-Zn-SOD (SOD1), mitochondrial Mn-SOD (SOD2) and catalase (CAT). Remarkably, the co-presence of EA, SA, CA showed the best protective effect. In conclusion, our research highlights SSF is an effective method for enhancing polyphenol content and biological activity in DJF. Moreover, these findings provide novel insights into the role of F-DJF extracts in prevention chronic diseases associated with oxidative stress and the possibility of their application in functional foods. In addition, fermentation may be a reliable strategy to enhance the medicinal value of DJF.

Pharmacological elevation of glutathione inhibits status epilepticus-induced neuroinflammation and oxidative injury

Glutathione (GSH) is a major endogenous antioxidant, and its depletion has been observed in several brain diseases including epilepsy. Previous studies in our laboratory have shown that dimercaprol (DMP) can elevate GSH via post-translational activation of glutamate cysteine ligase (GCL), the rate limiting GSH biosynthetic enzyme and inhibit neuroinflammation in vitro. Here we determined 1) the role of cysteamine as a new mechanism by which DMP increases GSH biosynthesis and 2) its ability to inhibit neuroinflammation and neuronal injury in the rat kainate model of epilepsy. DMP depleted cysteamine in a time- and concentration-dependent manner in a cell free system. To guide the in vivo administration of DMP, its pharmacokinetic profile was determined in the plasma, liver, and brain. The results confirmed DMP's ability to cross the blood-brain-barrier. Treatment of rats with DMP (30 mg/kg) depleted cysteamine in the liver and hippocampus that was associated with increased GCL activity in these tissues. GSH levels were significantly increased (20 %) in the hippocampus 1 h after 30 mg/kg DMP administration. Following DMP (30 mg/kg) administration once daily, a marked attenuation of GSH depletion was seen in the SE model. SE-induced inflammatory markers including cytokine release, microglial activation, and neuronal death were significantly attenuated in the hippocampus with DMP treatment. Taken together, these results highlight the importance of restoring redox status with rescue of GSH depletion by DMP in post epileptogenic insults.

Binge alcohol induces NRF2-related antioxidant response in the skeletal muscle of female mice

BackgroundChronic alcohol enhances oxidative stress, but the temporal response of antioxidant genes in skeletal muscle following a binge drinking episode remains unknown. MethodsExperiment 1: C57BL/6Hsd female mice received an IP injection of saline (CON; n = 39) or ethanol (ETOH; n = 39) (5 g/kg). Gastrocnemius muscles were collected from baseline (untreated; n = 3), CON (n = 3), and ETOH (n = 3) mice every 4 h for 48 h. Experiment 2: Gastrocnemius muscles were collected from control-fed (CON-FED; n = 17), control-fasted (CON-FAST; n = 18), or alcohol-fed (ETOH-FED; n = 18) mice every 4hrs for 20hrs after saline or ethanol (5 g/kg). ResultsEtOH enhanced Superoxide dismutase 1 (Sod1) and NADPH Oxidase 4 (Nox4) from 24 to 48hr after the binge, while Sod2 and Nox2 were suppressed. Nuclear factor erythroid-derived 2-like 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1) increased 12hrs after intoxication. Cytochrome P450 oxidoreductase (Por), Heme oxygenase 1 (Ho1), Peroxiredoxin 6 (Prdx6), Glutamate-cysteine ligase catalytic subunit (Gclc), Glutamate-cysteine ligase modifier subunit (Gclm), and Glutathione-disulfide reductase (Gsr) were increased by ETOH starting 12–16hrs post-binge. Fasting had similar effects on Nrf2 compared to alcohol, but downstream targets of NRF2, including Por, Ho1, Gclc, and Gclm, were differentially altered with fasting and EtOH. ConclusionThese data suggest that acute alcohol intoxication induced markers of oxidative stress and antioxidant signaling through the NRF2 pathway and that there were effects of alcohol independent of a possible decrease in food intake caused by binge intoxication.

Glutamate rescues heat stress-induced apoptosis of Sertoli cells by enhancing the activity of antioxidant enzymes and activating the Trx1-Akt pathway in vitro

Heat stress reduces the number of Sertoli cells, which is closely related to an imbalanced redox status. Glutamate functions to maintain the equilibrium of redox homeostasis. However, the role of glutamate in heat treated Sertoli cells remains unclear. Herein, Sertoli cells from 3-week-old piglets were treated at 44 °C for 30 min (heat stress). Glutamate levels increased significantly following heat stress treatment, followed by a gradual decrease during recovery, while glutathione (GSH) showed a gradual increase. The addition of exogenous glutamate (700 μM) to Sertoli cells before heat stress significantly reduced the heat stress-induced apoptosis rate, mediated by enhanced levels of antioxidant substances (superoxide dismutase (SOD), total antioxidant capacity (TAC), and GSH) and reduced levels of oxidative substances (reactive oxygen species (ROS) and malondialdehyde (MDA)). Glutamate addition to Sertoli cells before heat stress upregulated the levels of glutamate-cysteine ligase, modifier subunit (Gclm), glutathione synthetase (Gss), thioredoxin (Trx1) and B-cell leukemia/lymphoma 2 (Bcl-2), and the ratio of phosphorylated Akt (protein kinase B)/total Akt. However, it decreased the levels of Bcl2-associated X protein (Bax) and cleaved-caspase 3. Addition of the inhibitor of glutaminase (Gls1), Bptes (Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide, 30 μM)to Sertoli cells before heat stress reversed these effects. These results inferred that glutamate rescued heat stress-induced apoptosis in Sertoli cells by enhancing activity of antioxidant enzymes and activating the Trx1-Akt pathway. Thus, glutamate supplementation might represent a novel strategy to alleviate the negative effect of heat stress.

Preparation and functional properties of rice bran globulin-chitooligosaccharide-quercetin-resveratrol covalent complex.

As the major protein (approximately 36%) in rice bran, globulin exhibits excellent foaming and emulsifying properties, endowing its useful application as a foaming and emulsifying agent in the food industry. However, the low water solubility restricts its commercial potential in industrial applications. The present study aimed to improve this protein's processing and functional properties. A novel covalent complex was fabricated by a combination of the Maillard reaction and alkaline oxidation using rice bran globulin (RBG), chitooligosaccharide (C), quercetin (Que) and resveratrol (Res). The Maillard reaction improved the solubility, emulsifying and foaming properties of RBG. The resultant glycosylated protein was covalently bonded with quercetin and resveratrol to form a (RBG-C)-Que-Res complex. (RBG-C)-Que-Res exhibited higher thermal stability and antioxidant ability than the native protein, binary globulin-chitooligosaccharide or ternary globulin-chitooligosaccharide-polyphenol (only containing quercetin or resveratrol) conjugates. (RBG-C)-Que-Res exerted better cytoprotection against the generation of malondialdehyde and reactive oxygen species in HepG2 cells, which was associated with increased activities of antioxidative enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) through upregulated genes SOD1, CAT, GPX1 (i.e. gene for glutathione peroxidase-1), GCLM (i.e. gene for glutamate cysteine ligase modifier subunit), SLC1A11 (i.e. gene for solute carrier family 7, member 11) and SRXN1 (i.e. gene for sulfiredoxin-1). The anti-apoptotic effect of (RBG-C)-Que-Res was confirmed by the downregulation of caspase-3 and p53 and the upregulation of B-cell lymphoma-2 gene expression. The present study highlights the potential of (RBG-C)-Que-Res conjugates as functional ingredients in healthy foods. © 2024 Society of Chemical Industry.

Towards zero excess sludge discharge with built-in ozonation for wastewater biological treatment

In this study, a biological treatment process, which used a built-in ozonation bypass to achieve sludge reduction, was built to treat the industrial antifreeze production wastewater (mainly composed of ethylene glycol). The results indicated there is a positive correlation between ozone dosage and sludge reduction. At the laboratory level, the MLSS in the system can be stably controlled at around 3400 mg MLSS L−1 under the dosage of 0.18 g O3 g−1 MLSS. Ozonation can increase the compactness of sludge flocs (fractal dimension increased from 1.89 to 1.92). Ozone destroys microbial cell membranes and alters the structure of sludge flocs through direct oxidation through electrophilic reactions. It leads to the release of intracellular polysaccharides, proteins, and other biological macromolecules in microorganisms, thereby promoting the implicit growth of microbial populations. Some bacteria such as g_Pseudomonas, g_Gemmobacter, etc. have strong ethylene glycol degradation ability and tolerance to ozonation. The removal of ethylene glycol includes the glyoxylate cycle, glycine serine carbon cycle, and the glutamate-cysteine ligase pathway of assimilation. Gene KatG and gpx may be key factors in improving microbial tolerance to ozonation. The comprehensive evaluation from the perspectives of cost and carbon emission shows that choosing ozone cracking-implicit growth in wastewater treatment systems has significant cost advantages and application value.

Inhibition of glycogen synthase kinase-3 enhances NRF2 protein stability, nuclear localisation and target gene transcription in pancreatic beta cells

Accumulation of reactive oxygen species (i.e., oxidative stress) is a leading cause of beta cell dysfunction and apoptosis in diabetes. NRF2 (NF-E2 p45-related factor-2) regulates the adaptation to oxidative stress, and its activity is negatively regulated by the redox-sensitive CUL3 (cullin-3) ubiquitin ligase substrate adaptor KEAP1 (Kelch-like ECH-associated protein-1). Additionally, NRF2 is repressed by the insulin-regulated Glycogen Synthase Kinase-3 (GSK3). We have demonstrated that phosphorylation of NRF2 by GSK3 enhances β-TrCP (beta-transducin repeat-containing protein) binding and ubiquitylation by CUL1 (cullin-1), resulting in increased proteasomal degradation of NRF2. Thus, we hypothesise that inhibition of GSK3 activity or β-TrCP binding upregulates NRF2 and so protects beta cells against oxidative stress. We have found that treating the pancreatic beta cell line INS-1 832/13 with the KEAP1 inhibitor TBE31 significantly enhanced NRF2 protein levels. The presence of the GSK3 inhibitor CT99021 or the β-TrCP-NRF2 protein-protein interaction inhibitor PHAR, along with TBE31, resulted in prolonged NRF2 stability and enhanced nuclear localisation (P < 0.05). TBE31-mediated induction of NRF2-target genes encoding NAD(P)H quinone oxidoreductase 1 (Nqo1), glutamate-cysteine ligase modifier (Gclm) subunit and heme oxygenase (Hmox1) was significantly enhanced by the presence of CT99021 or PHAR (P < 0.05) in both INS-1 832/13 and in isolated mouse islets. Identical results were obtained using structurally distinct GSK3 inhibitors and inhibition of KEAP1 with sulforaphane. In summary, we demonstrate that GSK3 and β-TrCP/CUL1 regulate the proteasomal degradation of NRF2, enhancing the impact of KEAP1 regulation, and so contributes to the redox status of pancreatic beta cells. Inhibition of GSK3, or β-TrCP/CUL1 binding to NRF2 may represent a strategy to protect beta cells from oxidative stress.

Protective Mechanisms of Juncus effusus and Carbonized Juncus effusus against D-Galactosamine-Induced Acute Liver Injury in Mice.

This study investigated the hepatoprotective effects of Juncus effusus (J. effusus) and Carbonized J. effusus against liver injury caused by D-galactosamine (D-GalN) in mice. J. effusus and Carbonized J. effusus were administered by gavage once daily starting seven days before the D-GalN treatment. The results of the study indicated that J. effusus and Carbonized J. effusus suppressed the D-GalN-induced generation of serum alanine transaminase (ALT), aspartate aminotransferase (AST), hepatic malondialdehyde (MDA) and tumor necrosis factor-alpha (TNF-α) was observed. The values of superoxide dismutase (SOD) exhibited an increase. In addition, J. effusus and Carbonized J. effusus promoted the protein expression of nuclear factor erythroid 2-related factor 2 (Nrf2), NADPH quinone oxidoreductase-1 (NQO-1), heme oxygenase-1 (HO-1) as well as the mRNA expression of Nrf2, HO-1, NQO-1 and Glutamate cysteine ligase catalytic subunit (GCLC). The compressed Carbonized J. effusus demonstrated the optimum impact. These results suggest that J. effusus and Carbonized J. effusus protect against D-GalN-induced acute liver injury through the activation of the Nrf2 pathway.