Glutathione Primes T Cell Metabolism for Inflammation.

Activated T cells produce reactive oxygen species (ROS), which trigger the antioxidative glutathione (GSH) response necessary to buffer rising ROS and prevent cellular damage. We report that GSH is essential for T cell effector functions through its regulation of metabolic activity. Conditional gene targeting of the catalytic subunit of glutamate cysteine ligase (Gclc) blocked GSH production specifically in murine T cells. Gclc-deficient T cells initially underwent normal activation but could not meet their increased energy and biosynthetic requirements. GSH deficiency compromised the activation of mammalian target of rapamycin-1 (mTOR) and expression of NFAT and Myc transcription factors, abrogating the energy utilization and Myc-dependent metabolic reprogramming that allows activated T cells to switch to glycolysis and glutaminolysis. In vivo, T-cell-specific ablation of murine Gclc prevented autoimmune disease but blocked antiviral defense. The antioxidative GSH pathway thus plays an unexpected role in metabolic integration and reprogramming during inflammatory T cell responses.

Targeting PAX8 sensitizes ovarian cancer cells to ferroptosis by inhibiting glutathione synthesis.

Ovarian cancer is a malignant tumor originating from the ovary, characterized by its high mortality rate and propensity for recurrence. In some patients, especially those with recurrent cancer, conventional treatments such as surgical resection or standard chemotherapy yield suboptimal results. Consequently, there is an urgent need for novel anti-cancer therapeutic strategies. Ferroptosis is a distinct form of cell death separate from apoptosis. Ferroptosis inducers have demonstrated promising potential in the treatment of ovarian cancer, with evidence indicating their ability to enhance ovarian cancer cell sensitivity to cisplatin. However, resistance of cancer cells to ferroptosis still remains an inevitable challenge. Here, we analyzed genome-scale CRISPR-Cas9 loss-of function screens and identified PAX8 as a ferroptosis resistance protein in ovarian cancer. We identified PAX8 as a susceptibility gene in GPX4-dependent ovarian cancer. Depletion of PAX8 rendered GPX4-dependent ovarian cancer cells significantly more sensitive to GPX4 inhibitors. Additionally, we found that PAX8 inhibited ferroptosis in ovarian cancer cells. Combined treatment with a PAX8 inhibitor and RSL3 suppressed ovarian cancer cell growth, induced ferroptosis, and was validated in a xenograft mouse model. Further exploration of the molecular mechanisms underlying PAX8 inhibition of ferroptosis mutations revealed upregulation of glutamate-cysteine ligase catalytic subunit (GCLC) expression. GCLC mediated the ferroptosis resistance induced by PAX8 in ovarian cancer. In conclusion, our study underscores the pivotal role of PAX8 as a therapeutic target in GPX4-dependent ovarian cancer. The combination of PAX8 inhibitors such as losartan and captopril with ferroptosis inducers represents a promising new approach for ovarian cancer therapy.

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

BACKGROUND: Ferroptosis 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. METHODS: This 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. RESULTS: Our 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. CONCLUSIONS: Together, 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.

NaAsO2 decreases GSH synthesis by inhibiting GCLC and induces apoptosis through Hela cell mitochondrial damage, mediating the activation of the NF-κB/miR-21 signaling pathway.

BACKGROUND: Cervical cancer is the fourth most common cancer in women worldwide, and arsenic has a certain effect in solid tumor chemotherapy. As the rate-limiting enzyme subunit of GSH synthesis, GCLC may be an important target for arsenic to induce apoptosis through mitochondrial apoptosis pathway to exert anti-tumor effect. NF-κB plays an important role in the occurrence and development of cervical cancer and can regulate the expression of GCLC. miR-21 is a potential biomarker of cervical cancer, which can induce apoptosis through ROS regulated the mitochondrial pathway of cells. However, the role of miR-21 in the mitochondrial pathway of cervical cancer cells induced by NaAsO2 through NF-κB/GCLC and GSH synthesis regulated oxidative stress is rarely reported. Therefore, the purpose of this study was to investigate whether NaAsO2 might induce mitochondrial damage and apoptosis of cervical cancer cells through NF-κB/ miR-21 /GCLC induced oxidative stress, and play the anti-tumor role of arsenic as a potential drug for the treatment of cervical cancer. METHODS: Hela cells were treated with different concentrations of NaAsO2, D, L-Buthionine-(SR)-sulfoximine (BSO), IκBα inhibitor (BAY 11-7082) and miR-21 Inhibitor. CCK-8 assay, Western Blot, qRT PCR, immunofluorescence, transmission electron microscopy, mitochondrial Membrane Potential Assay Kit with JC-1,2',7'-Dichlorofluorescin diacetate fluorescent probe and Annexin V-FITC were used to measure cell activity, GSH and ROS, mitochondrial morphology and membrane potential (ΔΨm), protein and mRNA expression of GCLC, GCLM, p65, IκBα, p-P65, p-I κBα, Bcl-2, BAX, Caspase3, cleaved-caspase3 and miR-21. RESULTS: Compared with the control group, with the gradual increasing dose of NaAsO2, cell viability was considerable reduced, and increased rate of apoptosis, intracellular GSH level was decreased significantly, ROS was increased, mitochondrial structure was damaged, mitochondrial membrane potential ΔΨm and Bcl2/BAX lowered, the expression of Caspase3 and cleaved-caspase3 were significantly increased, resulting in mitochondrial apoptosis. When Hela cells were treated with 15, 20, and 25 µmol/L NaAsO2, the mRNA and protein levels of GCLC and GCLM were reduced, the expression of p65 in the nucleus was increased, the expression of p-p65/p65, p-IκBα/IκBα and miR-21 were significantly increased. When BSO increased the inhibitory effect of NaAsO2 on GCLC, Compared with NaAsO2 group, the ΔΨm and protein of Bcl-2/BAX, caspase3 and cleaved-capsase3 were increased. When BAY 11-7082 combined with NaAsO2 co-treated, compared with the NaAsO2 group, the protein and mRNA expression of GCLC was increased, NaAsO2-increased expression level of miR-21 was suppressed, and the ΔΨm and cell viability were higher. In addition, compared with the combination of NaAsO2 and miR-21NC, the protein expression of GCLC was increased, the ΔΨm and cell viability reduction were alleviated by miR-21 Inhibitor combined with NaAsO2. CONCLUSION: NaAsO2 may lead to ROS accumulation in Hela cells and trigger mitochondrial apoptosis. The mechanism may be related to the activation of NF-κB signaling pathway and the promotion of miR-21 expression which leads to the inhibition of GCLC expression and the significant decrease of intracellular reductive GSH synthesis.

Sulodexide Increases Glutathione Synthesis and Causes Pro-Reducing Shift in Glutathione-Redox State in HUVECs Exposed to Oxygen-Glucose Deprivation: Implication for Protection of Endothelium against Ischemic Injury.

Sulodexide (SDX), a purified glycosaminoglycan mixture used to treat vascular diseases, has been reported to exert endothelial protective effects against ischemic injury. However, the mechanisms underlying these effects remain to be fully elucidated. The emerging evidence indicated that a relatively high intracellular concentration of reduced glutathione (GSH) and a maintenance of the redox environment participate in the endothelial cell survival during ischemia. Therefore, the aim of the present study was to examine the hypothesis that SDX alleviates oxygen-glucose deprivation (OGD)-induced human umbilical endothelial cells' (HUVECs) injury, which serves as the in vitro model of ischemia, by affecting the redox state of the GSH: glutathione disulfide (GSSG) pool. The cellular GSH, GSSG and total glutathione (tGSH) concentrations were measured by colorimetric method and the redox potential (ΔEh) of the GSSG/2GSH couple was calculated, using the Nernst equation. Furthermore, the levels of the glutamate-cysteine ligase catalytic subunit (GCLc) and the glutathione synthetase (GSS) proteins, a key enzyme for de novo GSH synthesis, were determined using enzyme-linked immunoassay (ELISA). We demonstrated that the SDX treatment in OGD conditions significantly elevated the intracellular GSH, enhanced the GSH:GSSG ratio, shifting the redox potential to a more pro-reducing status. Furthermore, SDX increased the levels of both GCLc and GSS. The results show that SDX protects the human endothelial cells against ischemic stress by affecting the GSH levels and cellular redox state. These changes suggest that the reduction in the ischemia-induced vascular endothelial cell injury through repressing apoptosis and oxidative stress associated with SDX treatment may be due to an increase in GSH synthesis and modulation of the GSH redox system.

Carnosic Acid Encapsulated in Albumin Nanoparticles Induces Apoptosis in Breast and Colorectal Cancer Cells.

Carnosic acid (CA) is a natural phenolic compound with several biomedical actions. This work was performed to study the use of CA-loaded polymeric nanoparticles to improve the antitumor activity of breast cancer cells (MCF-7) and colon cancer cells (Caco-2). CA was encapsulated in bovine serum albumin (BSA), chitosan (CH), and cellulose (CL) nanoparticles. The CA-loaded BSA nanoparticles (CA-BSA-NPs) revealed the most promising formula as it showed good loading capacity and the best release rate profile as the drug reached 80% after 10 h. The physicochemical characterization of the CA-BSA-NPs and empty carrier (BSA-NPs) was performed by the particle size distribution analysis, transmission electron microscopy (TEM), and zeta potential. The antitumor activity of the CA-BSA-NPs was evaluated by measuring cell viability, apoptosis rate, and gene expression of GCLC, COX-2, and BCL-2 in MCF-7 and Caco-2. The cytotoxicity assay (MTT) showed elevated antitumor activity of CA-BSA-NPs against MCF-7 and Caco-2 compared to free CA and BSA-NPs. Moreover, apoptosis test data showed an arrest of the Caco-2 cells at G2/M (10.84%) and the MCF-7 cells at G2/M (4.73%) in the CA-BSA-NPs treatment. RT-PCR-based gene expression analysis showed an upregulation of the GCLC gene and downregulation of the BCL-2 and COX-2 genes in cells treated with CA-BSA-NPs compared to untreated cells. In conclusion, CA-BSA-NPs has been introduced as a promising formula for treating breast and colorectal cancer.

Quercetin modulates NRF2 and NF-κB/TLR-4 pathways to protect against isoniazid- and rifampicin-induced hepatotoxicity in vivo.

Isoniazid and rifampicin are crucial for treating tuberculosis (TB); however, they can cause severe hepatotoxicity leading to liver failure. Therapeutic options are limited and ineffective. We hypothesized that prophylaxis with quercetin attenuates isoniazid- and rifampicin-induced liver injury. We randomly divided Wistar rats into seven groups (n = 6). The animals received isoniazid and rifampicin or were co-treated with quercetin or silymarin for 28 days. The protective effect of quercetin was assessed using liver function tests and liver histology. Nuclear factor erythroid 2-related factor 2 (NRF2) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways were explored to elucidate the mechanism of action. Quercetin co-administration prevented the elevation of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and bilirubin compared with isoniazid and rifampicin treatment alone. In the histological analysis, we observed that quercetin prophylaxis lessened the severity of hepatic necrosis and inflammation compared with the anti-TB drug-treated group. Quercetin attenuated anti-TB drug-induced oxidative stress by increasing NRF2 activation and expression, boosting endogenous antioxidant levels. Additionally, quercetin blocked inflammatory mediators high mobility group box-1 (HMGB-1) and interferon γ (IFN-γ), inhibiting activation of the NF-κB/ toll like receptor 4 (TLR-4) axis. Quercetin protects against anti-TB liver injury by activating NRF2 and blocking NF-κB/TLR-4.

Neuroprotective effect of targeted regulatory Nrf2 gene on rats with acute brain injury induced by carbon monoxide poisoning.

Oxidative stress has been considered as an important cause of neurocyte damage induced by carbon monoxide (CO) poisoning; however, the precise mechanisms are not fully understood. The study aimed to elucidate the molecular mechanism and the neuroprotective effect of targeted regulatory nuclear factor erythroid2-related factor 2 (Nrf2) gene on acute brain injury in CO poisoning rats. An acute CO poisoning rat model was established by CO inhalation in hyperbaric oxygen chamber and followed by the administration of Nrf2 gene-loaded lentivirus. Mitochondrial membrane potential (ΔΨM), the levels of Nrf2, glutamate-cysteine ligase catalytic subunit (GCLC), catalase (CAT) and glutathione peroxidase (GSH-Px), and cell apoptosis were determined in brain tissue in rats. We found that CO poisoning could decrease ΔΨm of cells, slightly increase the expressions of Nrf2 and GCLC at mRNA and protein levels, reduce CAT and GSH-Px, and thus initiate apoptosis process. The Nrf2 gene treatment could obviously enhance the expressions of Nrf2 at mRNA and protein levels, and increase the concentrations of CAT and GSH-Px, maintain the ΔΨm of cells in brain tissue, significantly inhibit cell apoptosis as compared with the CO poisoning group (p < .05). These findings suggest that CO poisoning could induce oxidative stress and impair mitochondrial function of cells in brain tissue. The administration of Nrf2 gene could notably strengthen the antioxidant capacity of cells through regulating the downstream genes of Nrf2/antioxidant responsive element signal pathway, and positively protect cells against brain injury induced by acute severe CO poisoning.

Genetic susceptibility analysis of GCLC rs17883901 polymorphism to preeclampsia in Chinese Han women.

Preeclampsia (PE) is a specific obstetric disorder that may result in maternal and neonatal morbidity and mortality. Increasing evidence has been indicated that some candidate genes related to oxidative stress, such as glutamate-cysteine ligase, catalytic subunit (GCLC), glutamate-cysteine ligase, modifier subunit (GCLM), involve in the pathogenesis of PE. After the genetic contribution of GCLC rs17883901 polymorphism was analyzed by TaqMan allelic discrimination real-time PCR in 1001 PE patients and 1182 normal pregnant women, a case-control association analysis was performed. Although no statistical difference was found in genetic distribution of rs17883901 in GCLC between PE and control group (χ2 = 2.201, p = .333 by genotypic, χ2 = 0.524, p = .469, OR = 0.932, 95%CI = 0.771-1.128 by allelic), significant differences in the genotypic frequencies were investigated between mild PE group (χ2 = 6.999, p = .030) or late-onset PE group (χ2 = 6.197, p = .045) and control group. Furthermore, when dividing the mild PE patients, the late-onset PE patients and the controls into TT/CT + CC, TT + CT/CC, and TT/CC subgroups, we found statistical differences between mild PE and controls (TT/CT + CC:χ2 = 5.132, p = .023, OR = 2.948, 95%CI = 1.107-7.854; TT/CC:χ2 = 4.564, p = .033, OR = 2.793, 95%CI = 1.046-7.460) as well as late-onset PE and controls (TT/CT + CC:χ2 = 4.043, p = .044, OR = 2.248, 95%CI = 1.000-5.055). This is the first study to indicate GCLC rs17883901 polymorphism may be associated with a risk of mild PE and late-onset PE in Chinese Han women. However, additional well-designed studies with multi-ethnic and large-scale samples should be performed to validate our results.

Crocin attenuates cigarette smoke-induced lung injury and cardiac dysfunction by anti-oxidative effects: the role of Nrf2 antioxidant system in preventing oxidative stress.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) has been emerging as a great health problem in world. Cigarette smoke is known to cause oxidative stress and deplete glutathione (GSH) levels. Nuclear erythroid-related factor 2 (Nrf2) is involved in transcriptional regulation of glutamate-cysteine ligase catalytic subunit (GCLc). Antioxidant compounds may be of therapeutic value in monitoring disease progression. Crocin demonstrates antioxidant and anti-inflammatory functions. The aim of this study was to investigate the protective role of crocin against CSE-mediated oxidative stress, inflammatory process, Nrf2 modifications and impairment of cardiac function in rats with COPD. METHODS: Eighty rats were divided into four groups: Control, Cigarette smoke exposure (CSE), Crocin, Crocin+CS. Each group was divided into the two parts: 1) to evaluate lung inflammatory and oxidative process, 2) to evaluate the effect of Cigarette smoke induced-lung injuries on cardiac electrocardiogram (such as heart rate and QRS complex) and hemodynamic parameters (such as perfusion pressure and left ventricular developed pressure). RESULTS: CSE rats showed a significant increase in cotinine concentration (17.24 ng/ml), and inflammatory parameters and a decrease in PO2 (75.87 mmHg) and expression of PKC (0.86 fold), PI3K (0.79 fold), MAPK (0.87 fold), Nrf2 (0.8 fold) and GCLc (0.75 fold) genes, antioxidant activity, and finally cardiac abnormalities in electrocardiogram and hemodynamic parameters. Co-treatment whit crocin could restore all these values to normal levels. CONCLUSIONS: CS induced-COPD in rat model provides evidence that chronic CS exposure leads to lung injury and mediated cardiac dysfunction. Crocin co-treatment by modulating of Nrf2 pathway protected lung injury caused by COPD and its related cardiac dysfunction. In this study, we showed the importance of Nrf2 activators as a therapeutic target for the development of novel therapy for lung oxidative injuries.

Effects of CeO2 nanoparticles on the HO-1, NQO1, and GCLC expression in the testes of diabetic rats.

CeO2 nanoparticles (CNPs) as effective ROS scavengers exhibit potent antioxidant activity. In this study the effect of CNPs investigated was on HO-1, NQO1, and GCLC expression in the streptozotocin (STZ)-induced diabetic rats. Twenty-four male Wistar rats were divided into 4 groups: controls did not receive any treatment; diabetic rats received STZ (60 mg/kg daily); CNPs group received CNPs 30 mg/kg daily for 2 weeks; and rats in STZ + CNPs group received CNPs 30 mg/kg daily for 2 weeks following STZ injection. Oxidative stress was evaluated by measurement of total antioxidant capacity (TAC) and total oxidative status (TOS levels). HO-1, NQO1, and GCLC expression was measured using quantitative real-time PCR. Following STZ injection, significant lower levels of TAC and higher levels of TOS were observed. CNPs could alleviate deleterious effects of diabetes through the enhancement of TAC levels and a significant decline in TOS levels. HO-1, NQO1, and GCLC expression in the diabetic rats were lower than controls. HO-1, NQO1, and GCLC was upregulated in the diabetic rats treated with CNPs. There were significant correlations between NQO1 and GCLC, NQO1 and HO-1, and between HO-1 and GCLC expression. Moreover, Nrf2 was associated with NQO1, GCLC, and HO-1 expression. CNPs as Nrf2 upregulator confer protection against oxidative stress in the testes of STZ-induced diabetic rats by upregulating HO-1, GCLC, and NQO1 cytoprotective genes.

Clinical and molecular characterization of 6 children with glutamate-cysteine ligase deficiency causing hemolytic anemia.

Glutathione (gamma-glutamylcysteinylglycine) has diverse functions including free radicals scavenging and modulating many critical cellular processes. Glutathione is synthesized by the consecutive action of the enzymes glutamate-cysteine ligase (GCL) and glutathione synthetase. GCL is composed of a catalytic subunit encoded by the GCLC gene and a regulatory subunit encoded by the GCLM gene. GCL deficiency due to homozygous mutations in GCLC has been reported in 6 individuals from 4 independent families. All presented with hemolytic anemia and 4 had additional neurological manifestations including cognitive impairment, neuropathy, ataxia, and myopathy. In this report, we present additional 6 children from 2 independent consanguineous families with GCL deficiency. All the children presented with neonatal hemolytic anemia. Beyond the neonatal period, they did not have jaundice or hemolysis, but continued to have mild anemia. They all had normal development and neurological examination. The affected children from the first family had the homozygous mutation c.1772G>A (p.S591N) and the second family had the homozygous mutation c.514T>A (p.S172T) in GCLC. GCL deficiency can have a mild non-neurological phenotype or a more severe phenotype with neurological manifestations. GCL deficiency can be an underdiagnosed cause of hemolytic anemia, thus awareness may aid in early diagnosis, appropriate genetic counseling, and management.

Overexpression of the regulatory subunit of glutamate-cysteine ligase enhances monoclonal antibody production in CHO cells.

For decades, Chinese hamster ovary (CHO) cells have been the preferred host for therapeutic monoclonal antibody (mAb) production; however, increasing mAb titer by rational engineering remains a challenge. Our previous proteomic analysis in CHO cells suggested that a higher content of glutathione (GSH) might be related to higher productivity. GSH is an important antioxidant, cell detoxifier, and is required to ensure the formation of native disulfide bonds in proteins. To investigate the involvement of GSH in mAb production, we generated stable CHO cell lines overexpressing genes involved in the first step of GSH synthesis; namely the glutamate-cysteine ligase catalytic subunit (Gclc) and the glutamate-cysteine ligase modifier subunit (Gclm). The two genes were reconstructed from our RNA-Seq de novo assembly and then were functionally annotated. Once the sequences of the genes were confirmed using proteogenomics, a transiently expressed mAb was introduced into cell lines overexpressing either Gclc or Gclm. The new cell lines were compared for mAb production to the parental cell line and changes at the proteome level were measured using SWATH. As per our previous proteomics observations, overexpressing Gclm improved productivity, titer, and the frequency of high producer clones by 70%. In contrast, overexpressing Gclc, which produced a higher amount of GSH, did not increase mAb production. We show that GSH cannot be linked to higher productivity and that Gclm may be controlling other cellular processes involved in mAb production yet to be elucidated. Biotechnol. Bioeng. 2017;114: 1825-1836. © 2017 Wiley Periodicals, Inc.

Novel antioxidants' synthesis and their anti-oxidative activity through activating Nrf2 signaling pathway.

Novel structure compounds (WS) containing 3,4,5-trimethoxyphenyl and acyl pyrazole were designed and synthesized based combination principles. Among them, WS13 was screened out to possess desirable anti-oxidative activity in vitro. Cell survival assay and apoptosis experiment in H2O2 induced PC12 cells injury model all showed that its cytoprotection exhibited a concentration-effect manner. WS13 at 10µM could remove ROS with equal effiency to edaravone. Further, it clearly activated Nrf2 nuclear translocation and upregulated GCLC mRNA transcription and protein expression in dose-dependent manner, and its cytoprotection was reversed by GCLC protein inhibitor. In total, WS13 with further promotion can serve as Nrf2-GCLC activator in anti-oxidative therapy.

Exercise induced upregulation of glutamate-cysteine ligase catalytic subunit and glutamate-cysteine ligase modifier subunit gene expression in Thoroughbred horses.

OBJECTIVE: This study was performed to reveal the molecular structure and expression patterns of horse glutamate-cysteine ligase catalytic subunit (GCLC) and glutamate-cysteine ligase modifier subunit (GCLM) genes whose products form glutamate cysteine ligase, which were identified as differentially expressed genes in the previous study. METHODS: We performed bioinformatics analyses, and gene expression assay with quantitative polymerase chain reaction (qPCR) for horse GCLC and GCLM genes in muscle and blood leukocytes of Thoroughbred horses. RESULTS: Expression of GCLC showed the same pattern in both blood and muscle tissues after exercise. Expression of GCLC increased in the muscle and blood of Thoroughbreds, suggesting a tissue-specific regulatory mechanism for the expression of GCLC. In addition, expression of the GCLM gene increased after exercise in both the blood and muscle of Thoroughbreds. CONCLUSION: We established the expression patterns of GCLC and GCLM in the skeletal muscle and blood of Thoroughbred horses in response to exercise. Further study is now warranted to uncover the functional importance of these genes in exercise and recovery in racehorses.

HNF1ß drives glutathione (GSH) synthesis underlying intrinsic carboplatin resistance of ovarian clear cell carcinoma (OCCC).

Chemoresistance to platinum-based antineoplastic agents is a consistent feature among ovarian carcinomas; however, whereas high-grade serous carcinoma (OSC) acquires resistance during chemotherapy, ovarian clear cell carcinoma (OCCC) is intrinsically resistant. The main objective of this study was to explore, in vitro and in vivo, if hepatocyte nuclear factor 1ß (HNF1ß) and glutaminolysis contribute for the resistance of OCCC to carboplatin through the intrinsically increased GSH bioavailability. To disclose the role of HNF1ß, experiments were also performed in an OSC cell line, which does not express HNF1ß. Metabolic profiles, GSH quantification, HNF1ß, and γ-glutamylcysteine ligase catalytic subunit (GCLC) and modifier subunit (GCLM) expression, cell cycle, and death were assessed in ES2 cell line (OCCC) and OVCAR3 cell line (OSC); HNF1ß knockdown was performed in ES2 and murine model of subcutaneous and peritoneal OCCC tumors was established to test buthionine sulphoxamine (BSO), as a sensitizer to carboplatin. Glutaminolysis is activated in ES2 and OVCAR3, though ES2 exclusively synthesizes amino acids and GSH. ES2 cells are more resistant to carboplatin than OVCAR3 and the abrogation of GSH production by BSO sensitizes ES2 to carboplatin. HNF1ß regulates the expression of GCLC, but not GCLM, and consequently GSH production in ES2. In vivo, BSO prior to carboplatin reduces dramatically subcutaneous tumor size and GSH levels, as well as peritoneal dissemination. Our study discloses HNF1ß as the mediator of intrinsic OCCC chemoresistance and sheds a light to re-explore a cancer adjuvant therapeutic approach using BSO to overcome the lack of efficient therapy in OCCC.

Protective effects of naringenin in cardiorenal syndrome.

BACKGROUND: Cardiorenal syndrome is a complicated and bidirectional interrelationship between the heart and kidneys. Naringenin (NG) is a naturally occurring flavonoid possessing various biological and pharmacological properties. MATERIALS AND METHODS: We tested whether NG could improve cardiac and renal function in a rat model of cardiorenal syndrome. RESULTS: The results showed that NG-attenuated cardiac remodeling and cardiac dysfunction in rats with cardiorenal syndrome, as evidenced by decrease of left ventricle weight (LVW), increase of body weight (BW), decrease of LVW/BW, decrease of concentrations of serum creatinine, blood urea nitrogen, type-B natriuretic peptide, aldosterone, angiotensin (Ang) II, C-reactive protein, and urine protein, increase of left ventricular systolic pressure and falling rates of left ventricular pressure (dp/dtmax), and decrease of left ventricular diastolic pressure, left ventricular end-diastolic pressure, and -dp/dtmax. NG significantly inhibited the increase of lipid profiles including low-density lipoprotein, TC, and TG in rats. In addition, NG significantly inhibited the increase of cardiac expression of IL-1ß, IL-6, and interferon γ. Moreover, NG decreased malonaldehyde level, increased superoxide dismutase activity and glutathione content in rats, and increased the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and catalytic subunit of γ-glutamylcysteine ligase (GCLc) in rats and Ang II-treated cardiac fibroblasts. Inhibition of Nrf2 and glutathione synthesis significantly suppressed NG-induced decrease of ROS level. Inhibition of Nrf2 markedly suppressed NG-induced increase of GCLc expression in Ang II-treated cardiac fibroblasts. CONCLUSIONS: The data provide novel options for therapy of patients and new insights into the cardioprotective effects of NG in cardiorenal syndrome.

Tetramethylpyrazine inhibits CoCl2 -induced neurotoxicity through enhancement of Nrf2/GCLc/GSH and suppression of HIF1α/NOX2/ROS pathways.

Hypoxia-mediated neurotoxicity contributes to various neurodegenerative disorders, including Alzheimer's disease and multiple sclerosis. Tetramethylpyrazine (TMP), a major bioactive component purified from Ligusticum wallichii Franchat, exhibited potent neuroprotective effect. However, the mechanism of TMP-exerted neuroprotective effect against hypoxia was not clear. In the study, we investigated the mechanism of the neuroprotective effect of TMP against hypoxia induced by CoCl2 in vitro and in vivo. The results showed that TMP could protect against CoCl2 -induced neurotoxicity in PC12 cells and in rats, as evidenced by enhancement of cell viability in PC12 cells and improvement of learning and memory ability in rats treated with CoCl2 . TMP could inhibit mitochondrial dysfunction, mitochondrial apoptotic molecular events, and thus apoptosis induced by CoCl2 . TMP inhibited CoCl2 -increased reactive oxygen species (ROS) level, which may contribute to hypoxia-related neurotoxicity induced by CoCl2 . The antioxidant and neuroprotective activities of TMP involved two pathways: one was the enhancement of nuclear factor erythroid 2-related factor 2 (Nrf2)/catalytic subunit of γ-glutamylcysteine ligase-mediated regulation of GSH and the other was the inhibition of hypoxia-inducible factor 1 α/NADPH oxidase 2 (NOX2)-mediated ROS generation. These two pathways contributed to improvement of oxidative stress and thus the amelioration of apoptosis under hypoxic conditions. These results have appointed a new path toward the understanding of pathogenesis and TMP-related therapy of hypoxia-related neurodegenerative diseases. We proposed two cascades for tetramethylpyrazine-exhibited protective effects against CoCl2 -induced neurotoxicity: One is enhancement of nuclear factor erythroid 2-related factor 2-catalytic subunit of γ-glutamylcysteine ligase-mediated regulation of glutathone and the other was the inhibition of hypoxia-inducible factor 1 α-NADPH oxidase-2-mediated ROS generation. We think these findings should provide a new understanding of pathogenesis and tetramethylpyrazine-related therapy of hypoxia-related neurodegenerative diseases.

Inhibition of the glutamate-cysteine ligase catalytic subunit with buthionine sulfoximine enhances the cytotoxic effect of doxorubicin and cyclophosphamide in Burkitt lymphoma cells.

Burkitt lymphoma (BL) is a highly aggressive lymphoma that mainly affects children and young adults. Chemotherapy is effective in young BL patients but the outcome in adults is less satisfactory. Therefore, there is a need to enhance the cytotoxic effect of drugs used in BL treatment. Glutathione (GSH) is an important antioxidant involved in processes such as regulation of oxidative stress and drug detoxification. Elevated GSH levels have been observed in many cancers and were associated with chemoresistance. We previously identified GCLC, encoding an enzyme involved in GSH biosynthesis, as an essential gene in BL. We now confirm that knockout of GCLC decreases viability of BL cells and that the GCLC protein is overexpressed in BL tissues. Moreover, we demonstrate that buthionine sulfoximine (BSO), a known inhibitor of GCLC, decreases growth of BL cells but does not affect control B cells. Furthermore, we show for the first time that BSO enhances the cytotoxicity of compounds commonly used in BL treatment, doxorubicin, and cyclophosphamide. Given the fact that BSO itself was not toxic to control cells and well-tolerated in clinical trials, combination of chemotherapy with BSO may allow reduction of the doses of cytotoxic drugs required to obtain effective responses in BL patients.

Gclc as a Marker for Injured Distal Nephron in Ischemia-Reperfusion Induced Acute Kidney Injury.

Purpose: The distal nephron of kidney plays a pivotal role in advancing acute kidney injury (AKI). Understanding the role of distal nephrons in AKI and identifying markers of injured distal nephrons are critical to comprehending the mechanism of renal injury and identifying novel therapeutic targets. Methods: We analyzed single-cell RNA sequencing (scRNA-seq) data from mice with AKI induced by ischemia-reperfusion (IR), unilateral ureteral obstruction (UUO), cisplatin (CP), sodium oxalate (SO) and lipopolysaccharide (LPS). Additionally, we analyzed renal transcriptomics samples for AKI. Subsequently, we validated the effectiveness of targeting the biomarker Gclc in vitro and in vivo through metabolomics and immunofluorescence. Results: The LOH-Inj and DCT-Inj subtypes were identified through scRNA-seq. Compared to normal distal nephrons, the injured distal nephrons exhibited higher levels of ferroptosis, pro-inflammation, and fibrosis. The expression of ferroptosis-related gene Gclc were high in various AKI models. Furthermore, Gclc was exclusively expressed in the distal nephron and upregulated in the injury subtype. To confirm our findings, we suppressed GCLC expression in the kidneys, resulting to aggravated IR-induced AKI. Inhibition of Gclc promoted damage to primarily renal tubular epithelial cells by promoting inflammatory infiltration, inhibiting glutathione metabolism and exacerbating oxidative stress. Conclusion: Our research findings suggest that Gclc is a potential marker for injured distal nephron.