Exogenous GSH Research Articles

Reduced glutathione attenuates pediatric sepsis-associated encephalopathy by inhibiting inflammatory cytokine release and mitigating lipid peroxidation-induced brain injury.

Sepsis-associated encephalopathy (SAE) is a severe complication of sepsis. Reduced glutathione (GSH) has antioxidant properties and is used as a neuroprotective agent in some studies. However, research on the application of exogenous GSH in the treatment of SAE is limited. This study aimed to determine the effects of exogenous GSH in pediatric SAE patients and mice. We evaluated clinical parameters, inflammatory factors, and oxidative stress before and after GSH treatment. The clinical trials demonstrated that GSH treatment improved brain damage markers (S-100 beta protein, brain fatty acid-binding protein), increased neurological status scores (Glasgow coma scale), and reduced Pediatric Risk of Mortality III scores in children with SAE. GSH treatment also significantly reduced the levels of inflammatory factors (interleukin-6, tumor necrosis factor-α) and decreased lipid peroxidation (superoxide dismutase). Additionally, GSH reduced lipid peroxidation resulting from abnormal lipid metabolism, as indicated by the levels of acyl-CoA synthetase long-chain family member 4, lysophosphatidylcholine acyltransferase 3, and glutathione peroxidase 4. In-vivo experiments showed that the neuroprotective effect of GSH was dose-dependent, with better effects observed at medium and high doses. Furthermore, GSH alleviated brain damage, suppressed the release of inflammatory factors, and inhibited lipid peroxidation in SAE mice. The animal experiments also showed that GSH reduces lipid peroxidation through the 15-lipoxygenase/phosphatidylethanolamine binding protein 1/glutathione peroxidase 4 pathway. Our study suggests that exogenous GSH has neuroprotective effects in pediatric SAE. These findings provide a basis for the potential use of GSH as a therapeutic method for SAE.

Glutathione accelerates the cell cycle and cellular reprogramming in plant regeneration.

The plasticity of plant cells underlies their wide capacity to regenerate, with increasing evidence in plants and animals implicating cell cycle dynamics in cellular reprogramming. To investigate the cell cycle during cellular reprogramming, we developed a comprehensive set of cell cycle phase markers in the Arabidopsis root. Using single-cell RNA-seq profiles and live imaging during regeneration, we found that a subset of cells near an ablation injury dramatically increases division rate by truncating G1. Cells in G1 undergo a transient nuclear peak of glutathione (GSH) prior to coordinated entry into S phase followed by rapid divisions and cellular reprogramming. A symplastic block of the ground tissue impairs regeneration, which is rescued by exogenous GSH. We propose a model in which GSH from the outer tissues is released upon injury licensing an exit from G1 near the wound to induce rapid cell division and reprogramming.

Glutathione regulates CIA-activated splenic-lymphocytes via NF-κB/MMP-9 and MAPK/PCNA pathways manipulating immune response

Reduced glutathione (GSH) is an antioxidant involved in redox homeostasis, and recently regarded as an inducer of Reductive stress. Its immune-regulatory effects on lymphocytes have not been extensively studied. This study is based on the finding that much increased GSH level in collagen-induced arthritis (CIA) rat spleen, and aimed to investigate the effects of GSH (0, 1, 10, 100 mM) on normal and immune-stimulated spleen lymphocytes respectively. The elevated GSH level is associated with the increased levels of inflammatory factors; especially the increased DPP1 activity indicated immune-granulocytes activation in CIA rat spleen. Exogenous GSH had different influences on normal and CIA lymphocytes, affecting intracellular levels of GSH, Glutathione-S-transferases (GSTs) and Reactive oxygen species (ROS); as well as the expressions of NF-κB, MMP-9, Bcl-2, GST, P38, PCNA and TLR4. The increased extracellular GSH level disturbed redox homeostasis and induces reductive stress to spleen lymphocytes, which decreased intracellular GSH concentration and influenced the MAPK/PCNA and NF-κB/MMP-9 signaling pathways, as well as cell cycles respectively, leading to cell senescence/ferroptosis /apoptosis. This study also revealed the multiple faces of GSH in regulating spleen lymphocytes, which depends on its levels in tissue or in cells, and the activation status of lymphocytes. These findings indicate the immune-regulatory role of GSH on spleen-lymphocytes, and the high level GSH in CIA rat spleens may contribute to CIA development.

Nitric oxide signal is required for glutathione-induced enhancement of photosynthesis in salt-stressed Solanum lycopersicum L.

Reduced glutathione (γ-glutamyl-cysteinyl-glycine, GSH), the primary non-protein sulfhydryl group in organisms, plays a pivotal role in the plant salt stress response. This study aimed to explore the impact of GSH on the photosynthetic apparatus, and carbon assimilation in tomato plants under salt stress, and then investigate the role of nitric oxide (NO) in this process. The investigation involved foliar application of 5 mM GSH, 0.1% (w/v) hemoglobin (Hb, a nitric oxide scavenger), and GSH+Hb on the endogenous NO levels, rapid chlorophyll fluorescence, enzyme activities, and gene expression related to the Calvin cycle in tomato seedlings (Solanum lycopersicum L. cv. 'Zhongshu No. 4') subjected short-term salt stress (100 mM NaCl) for 24, 48 and 72 hours. GSH treatment notably boosted nitrate reductase (NR) and NO synthase (NOS) activities, elevating endogenous NO signaling in salt-stressed tomato seedling leaves. It also mitigated chlorophyll fluorescence (OJIP) curve distortion and damage to the oxygen-evolving complex (OEC) induced by salt stress. Furthermore, GSH improved photosystem II (PSII) electron transfer efficiency, reduced QA - accumulation, and countered salt stress effects on photosystem I (PSI) redox properties, enhancing the light energy absorption index (PIabs). Additionally, GSH enhanced key enzyme activities in the Calvin cycle and upregulated their genes. Exogenous GSH optimized PSII energy utilization via endogenous NO, safeguarded the photosynthetic reaction center, improved photochemical and energy efficiency, and boosted carbon assimilation, ultimately enhancing net photosynthetic efficiency (Pn) in salt-stressed tomato seedling leaves. Conversely, Hb hindered Pn reduction and NO signaling under salt stress and weakened the positive effects of GSH on NO levels, photosynthetic apparatus, and carbon assimilation in tomato plants. Thus, the positive regulation of photosynthesis in tomato seedlings under salt stress by GSH requires the involvement of NO.

Feasibility exploration of GSH in the treatment of acute hepatic encephalopathy from the aspects of pharmacokinetics, pharmacodynamics, and mechanism.

Our previous study highlighted the therapeutic potential of glutathione (GSH), an intracellular thiol tripeptide ubiquitous in mammalian tissues, in mitigating hepatic and cerebral damage. Building on this premise, we posited the hypothesis that GSH could be a promising candidate for treating acute hepatic encephalopathy (AHE). To verify this conjecture, we systematically investigated the feasibility of GSH as a therapeutic agent for AHE through comprehensive pharmacokinetic, pharmacodynamic, and mechanistic studies using a thioacetamide-induced AHE rat model. Our pharmacodynamic data demonstrated that oral GSH could significantly improve behavioral scores and reduce hepatic damage of AHE rats by regulating intrahepatic ALT, AST, inflammatory factors, and homeostasis of amino acids. Additionally, oral GSH demonstrated neuroprotective effects by alleviating the accumulation of intracerebral glutamine, down-regulating glutamine synthetase, and reducing taurine exposure. Pharmacokinetic studies suggested that AHE modeling led to significant decrease in hepatic and cerebral exposure of GSH and cysteine. However, oral GSH greatly enhanced the intrahepatic and intracortical GSH and CYS in AHE rats. Given the pivotal roles of CYS and GSH in maintaining redox homeostasis, we investigated the interplay between oxidative stress and pathogenesis/treatment of AHE. Our data revealed that GSH administration significantly relieved oxidative stress levels caused by AHE modeling via down-regulating the expression of NADPH oxidase 4 (NOX4) and NF-κB P65. Importantly, our findings further suggested that GSH administration significantly regulated the excessive endoplasmic reticulum (ER) stress caused by AHE modeling through the iNOS/ATF4/Ddit3 pathway. In summary, our study uncovered that exogenous GSH could stabilize intracerebral GSH and CYS levels to act on brain oxidative and ER stress, which have great significance for revealing the therapeutic effect of GSH on AHE and promoting its further development and clinical application.

Abstract 436: Extracellular glutathione catabolism as an alternative cyst(e)ine source in cancer

Abstract Glutathione (GSH) is the most abundant antioxidant in the human body and plays essential roles in physiology and disease. Unchecked oxidative stress is involved in tumor initiation and progression. Paradoxically, clinical and laboratory data show that ROS-scavenging molecules such as glutathione not only fail to impede tumor growth but, in many cases, accelerate it. Still, preliminary data from our lab show that genetically ablating tumors’ ability to synthesize GSH does not affect their growth. This finding suggests that the favorable influence of glutathione on tumor growth may arise from external sources. Yet, the mechanism cancer cells employ to take advantage of extracellular glutathione in this context is unknown. Here, we show that γ-glutamyl-transpeptidase 1 (GGT1), an enzyme that localizes on the outer side of the cell’s membrane, can break down extracellular GSH, yielding glutamate and the dipeptide cysteinylglycine. Cysteinylglycine can be further metabolized, generating cysteine and glycine to the cells. We found that the addition of exogenous GSH, as well as cysteinylglycine, can mitigate cell death, proliferation arrest, and oxidative stress caused by cystine depletion. The overexpression of human GGT1 in cancer cells significantly reduces the amount of glutathione needed to rescue cell fitness. Accordingly, pharmacologic inhibition of GGT1 activity prevented GSH's ability to rescue cell proliferation. Moreover, an unbiased pharmacological screening revealed that GGT1-mediated cysteine acquisition renders cancer cells resistant to inhibitors of cystine uptake and utilization, supporting the role of GGT1 as a source of cyst(e)ine. Our findings reveal that cancer cells can access cysteine, a vital yet scarce amino acid, through GGT1's ability to break down glutathione, which is present in remarkably high concentrations within the tumor microenvironment. Although the downstream proteins involved in the transport and utilization of GSH breakdown products are yet to be identified, we anticipate this pathway has the potential to uncover novel therapeutic avenues for cancer treatment by targeting alternative pathways of nutrient acquisition that are potentially implicated in cancer progression and therapy resistance. Citation Format: Fabio Hecht, Marco Zocchi, Emily Tuttle, Gloria Asantewaa, TashJaé Scales, Fatemeh Alimohammadi, Nathan Ward, Gina DeNicola, Isaac Harris. Extracellular glutathione catabolism as an alternative cyst(e)ine source in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 436.

Listeria monocytogenes utilizes glutathione and limited inorganic sulfur compounds as sources of essential cysteine.

Listeria monocytogenes (Lm) is a Gram-positive facultative intracellular pathogen that leads a biphasic lifecycle, transitioning its metabolism and selectively inducing virulence genes when it encounters mammalian hosts. Virulence gene expression is controlled by the master virulence regulator PrfA, which is allosterically activated by the host- and bacterially derived glutathione (GSH). The amino acid cysteine is the rate-limiting substrate for GSH synthesis in bacteria and is essential for bacterial growth. Unlike many bacteria, Lm is auxotrophic for cysteine and must import exogenous cysteine for growth and virulence. GSH is enriched in the host cytoplasm, and previous work suggests that Lm utilizes exogenous GSH for PrfA activation. Despite these observations, the import mechanism(s) for GSH remains elusive. Analysis of known GSH importers predicted a homologous importer in Lm comprised of the Ctp ABC transporter and the OppDF ATPases of the Opp oligopeptide importer. Here, we demonstrated that the Ctp complex is a high-affinity GSH/GSSG importer that is required for Lm growth at physiologically relevant concentrations. Furthermore, we demonstrated that OppDF is required for GSH/GSSG import in an Opp-independent manner. These data support a model where Ctp and OppDF form a unique complex for GSH/GSSG import that supports growth and pathogenesis. In addition, we show that Lm utilizes the inorganic sulfur sources thiosulfate and H2S for growth in a CysK-dependent manner in the absence of other cysteine sources. These findings suggest a pathoadaptive role for partial cysteine auxotrophy in Lm, where locally high GSH/GSSG or inorganic sulfur concentrations may signal arrival to distinct host niches.

Durian (Durio zibethinus L.) fruit: A superior dietary source of natural glutathione and γ-glutamylcysteine

Numerous age-related degenerative diseases have been linked to diminished intracellular levels of glutathione (GSH), a vital antioxidant in the human body. Unfortunately, exogenous GSH encounters various barriers that prevent its entry into most human cells. γ-Glutamylcysteine (γ-EC), an immediate precursor of GSH, is a potential therapeutic agent that restores cellular GSH levels. Additionally, both compounds can serve as kokumi taste substances and enhance basic taste sensations. Considering the growing interest among health-conscious consumers toward naturally derived diets, the incorporation of fresh fruits and vegetables abundant in GSH and γ-EC presents a promising alternative for supplementation. Screening of various plant samples, including fruits, vegetables, and mushrooms, revealed that ripe durian pulp exhibited the highest levels of GSH (∼2.6) and γ-EC (∼14) per milligram per gram of dry weight. Owing to the popularity of durian-related products, we extended our investigation to analyze GSH and γ-EC contents in processed products. Notably, durian chips and pastes displayed a significant decrease in GSH and γ-EC levels, suggesting their sensitivity to heat. This observation was further validated by the observed instability of GSH and γ-EC standards during heat treatment. Our findings underscore the potential of durian fruit as a rich dietary source of GSH and γ-EC.

Developing NIR xanthene-chalcone fluorophores with large Stokes shifts for fluorescence imaging.

A series of novel near-infrared (NIR) xanthene-chalcone fluorophores were constructed through a modular synthesis with the electron-donating xanthene moiety and the electron-withdrawing chalcone moiety. These fluorophores are convenient for fluorescence imaging in living cells, benefiting from their NIR emissions (650-710 nm), large Stokes shifts (>100 nm), moderate quantum yields and low cytotoxicity. The substituted hydroxyl group of the xanthene-chalcone fluorophore HCA-E facilitates the development of multifunctional fluorescent probes. As an example, a highly sensitive and selective probe N-HCA-E for glutathione (GSH) detection was developed based on the fluorophore HCA-E. A 4-nitrobenzenesulfonyl (4-Ns) group was introduced to cage the hydroxyl group of HCA-E, which was used as a selective recognition site for the thiol of GSH and an effective fluorescence quencher. Probe N-HCA-E revealed NIR "turn-on" fluorescence (709 nm) for endogenous and exogenous GSH detection in lysosomes with a large Stokes shift (129 nm) and high anti-interference ability.

Characterization of a Nicotiana tabacum phytochelatin synthase 1 and its response to cadmium stress.

Phytochelatin synthase (PCS) is a critical enzyme involved in heavy metal detoxification in organisms. In this study, we aim to comprehensively investigate the molecular and functional characteristics of the PCS1 gene from Nicotiana tabacum by examining its enzymatic activity, tissue-specific expression pattern, Cd-induced expression, as well as the impact on Cd tolerance and accumulation. The results demonstrated that the amino acid sequence of NtPCS1 shared a highsimilarity in its N-terminal region with PCS from other species. The enzymaticactivity of NtPCS1 was found to be enhanced in the order Ag2+ > Cd2+ > Cu2+ > Pb2+ > Hg2+ > Fe2+ > Zn2+. In addition, RT-PCR data indicated that NtPCS1 gene is constitutively expressed, with the highest expression observed in flowers, and that its transcript levels are up-regulated by CdCl2. When tobacco overexpressing NtPCS1 (PCS1 lines) were grown under CdCl2 stress, they produced more phytochelatins (PCs) than WT plants, but this did not result in increased Cd accumulation. However, in a root growth assay, the PCS1 lines exhibited hypersensitivity to Cd. The overexpression of NtPCS1 itself does not appear to be the primary cause of this heightened sensitivity to Cd, as the Arabidopsis thaliana Atpcs1 mutant overexpressing NtPCS1 actually exhibited enhanced tolerance to Cd. Furthermore, the addition of exogenous glutathione (GSH) progressively reduced the Cd hypersensitivity of the PCS1 lines, with the hypersensitivity even being completely eliminated. Surprisingly, the application of exogenous GSH led to a remarkably enhanced Cd accumulation in the PCS1 lines. This study enriches our understanding of the molecular function of the NtPCS1 gene and suggests a promising avenue for Cd tolerance through the heterologous expression of PCS genes in different species.

Exogenous Melatonin Promotes Glucoraphanin Biosynthesis by Mediating Glutathione in Hairy Roots of Broccoli (Brassica oleracea L. var. italica Planch).

To investigate the mechanism of melatonin (MT)-mediated glutathione (GSH) in promoting glucoraphanin (GRA) and sulforaphane (SF) synthesis, the gene expression pattern and protein content of hairy broccoli roots under MT treatment were analyzed by a combination of RNA-seq and tandem mass spectrometry tagging (TMT) techniques in this study. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that both proteins and mRNAs with the same expression trend were enriched in the "Glutathione metabolism (ko00480)" and "Proteasome (ko03050)" pathways, and most of the differentially expressed genes (DEGs) and differentially abundant proteins (DAPs) regulating the two pathways were downregulated. The results showed that endogenous GSH concentration and GR activity were increased in hairy roots after MT treatment. Exogenous GSH could promote the biosynthesis of GRA and SF, and both exogenous MT and GSH could upregulate the expression of the GSTF11 gene related to the sulfur transport gene, thus promoting the biosynthesis of GRA. Taken together, this study provides a new perspective to explore the complex molecular mechanisms of improving GRA and SF synthesis levels by MT and GSH regulation.

PLK1 regulating chemoradiotherapy sensitivity of esophageal squamous cell carcinoma through pentose phosphate pathway/ferroptosis

BackgroundEsophageal squamous cell carcinoma (ESCC) is the most common pathological type of esophageal cancer in China, accounting for more than 90 %. Most patients were diagnosed with advanced-stage ESCC, for whom new adjuvant therapy is recommended. Therefore, it is urgent to explore new therapeutic targets for ESCC. Ferroptosis, a newly discovered iron-dependent programmed cell death, has been shown to play an important role in carcinogenesis by many studies. This study explored the effect of Polo like kinase 1 (PLK1) on chemoradiotherapy sensitivity of ESCC through ferroptosis MethodsIn this study, we knocked out the expression of PLK1 (PLK1-KO) in ESCC cell lines (KYSE150 and ECA109) with CRISPR/CAS9. The effects of PLK1-knock out on G6PD, the rate-limiting enzyme of pentose phosphate pathway (PPP), and downstream NADPH and GSH were explored. The lipid peroxidation was observed by flow cytometry, and the changes in mitochondria were observed by transmission electron microscopy. Next, through the CCK-8 assay and clone formation assay, the sensitivity to cobalt 60 rays, paclitaxel, and cisplatin were assessed after PLK1-knock out, and the nude mouse tumorigenesis experiment further verified it. The regulation of transcription factor YY1 on PLK1 was evaluated by dual luciferase reporter assay. The expression and correlation of PLK1 and YY1, and their impact on prognosis were analyzed in more than 300 ESCC cases from the GEO database and our center. Finally, the above results were further proved by single-cell sequencing. ResultsAfter PLK1 knockout, the expression of G6PD dimer and the level of NADPH and GSH in KYSE150 and ECA109 cells significantly decreased. Accordingly, lipid peroxidation increased, mitochondria became smaller, membrane density increased, and ferroptosis was more likely to occur. However, with the stimulation of exogenous GSH (10 mM), there was no significant difference in lipid peroxidation and ferroptosis between the PLK1-KO group and the control group. After ionizing radiation, the PLK1-KO group had higher lipid peroxidation ratio, more cell death, and was more sensitive to radiation, while exogenous GSH (10 mM) could eliminate this difference. Similar results could also be observed when receiving paclitaxel combined with cisplatin and chemoradiotherapy. The expression of PLK1, G6PD dimer, and the level of NADPH and GSH in KYSE150, ECA109, and 293 T cells stably transfected with YY1-shRNAs significantly decreased, and the cells were more sensitive to radiotherapy and chemotherapy. ESCC patients from the GEO database and our center, YY1 and PLK1 expression were significantly positively-correlated, and the survival of patients with high expression of PLK1 was significantly shorter. Further analysis of single-cell sequencing specimens of ESCC in our center confirmed the above results. ConclusionIn ESCC, down-regulation of PLK1 can inhibit PPP, and reduce the level of NADPH and GSH, thereby promoting ferroptosis and improving their sensitivity to radiotherapy and chemotherapy. Transcription factor YY1 has a positive regulatory effect on PLK1, and their expressions were positively correlated. PLK1 may be a target for predicting and enhancing the chemoradiotherapy sensitivity of ESCC.

Synergistic effects of exogenous glutathione and calcium on ascorbate-glutathione cycle and glutathione-associated enzymes upregulation under lead stress in Brassica napus L.

Heavy metals (HMs) such as lead (Pb) pose a significant threat to global food security due to their adverse effect on the health of crop plants. Calcium (Ca) and Glutathione (GSH) are signaling molecules to scavenge free radicals in HM-stressed plants. In this study, GSH and Ca's role is examined in supporting canola seedlings against Pb toxicity. In a pot experiment, the administration of Glutathione (GSH, 0 and 100µM) and/or calcium (CaCl2, 0 and 500µM) in canola seedlings was examined under lead stress (0 and 100µM of Pb(NO3)2. Compared with the control samples, Pb treatment increased MDA and H2O2 values by 61 and 53%, respectively, indicative of oxidative burst. However, using a combination of GSH and Ca lowered oxidative stress in Pb-stressed plants by an approximately twofold reduction in MDA and H2O2 content. Total PC content increased by 78% in Pb-stressed plants, suggesting that these chelating peptides diminish the damaging effects of Pb. Interestingly, further boosts in total PC levels were recorded in Pb-stressed plants treated with GSH and Ca concurrently. The addition of exogenous GSH and Ca to Pb-stressed canola plants limited Pb uptake and translocation and improved ascorbate-glutathione cycle performance. Moreover, compared to their separate usage, the co-treatment of exogenous GSH and Ca strengthened the GSH pool by increasing the activities of enzymes involved in GSH metabolism. The findings demonstrate that exogenous GSH and Ca modulate GSH synthesis, metabolism, and redox homeostasis synergistically to enhance resistance to oxidative stress generated by Pb.

Promotion or Inhibition Effects of Exogenous Glutathione-Degraded Amino Acids on the Formation of 2,3-Butanedione and Pyrazines via Varied Pathways of Interaction with α-Dicarbonyl Compounds Derived from N-(1-Deoxy-d-xylulos-1-yl)-alanine.

The contribution of glutathione (GSH) and free amino acids degraded from GSH to the generation of pyrazines and 2,3-butanedione was illustrated during their interaction in the thermal treatment of the Amadori compound of alanine and xylose (ARP). GSH-degraded amino acids, glutamic acid (Glu), cysteine (Cys), and glycine (Gly), but not pyroglutamic acid (pGlu), could effectively capture α-dicarbonyls to facilitate the formation of pyrazines when ARP was heated with GSH. Deoxypentosones, the precursors of 2,3-butanedione, were largely consumed in the ARP-GSH model by the interaction with GSH and its degradative Cys compared with the ARP model. The addition of GSH and deoxypentosones inhibited the further degradation of deoxypentosones, resulting in less formation of 2,3-butanedione and other α-dicarbonyl compounds. Meanwhile, the reaction between GSH-degraded Cys and deoxypentosones to form sulfur-containing compounds such as thiols accelerated the consumption of deoxypentosones; thereby, the formation of 2,3-butanedione was severely interfered. However, this inhibition was compensated for by the GSH-degraded Gly through the addition between Gly and MGO and the subsequent deamination. The involvement of exogenous GSH could simultaneously boost the yields of 2,3-butanedione and pyrazines compared with those of ARP heated alone. As the degree of GSH degradation strengthened in the ARP-thermal-degraded GSH models, the yields of both pyrazines and 2,3-butanedione steadily increased.

The effect of glutathione on glucosinolate biosynthesis through the sulfur assimilation pathway in pakchoi associated with the growth conditions

Glucosinolates (GSLs) are a group of nitrogen- and sulfur-containing secondary metabolites, synthesized primarily in members of the Brassicaceae family, that play an important role in food flavor, plant antimicrobial activity, resistance to insect attack, stress tolerance, and human anti-cancer effects. As a sulfur-containing compound, glutathione has a strong connection with GSLs biosynthesis as a sulfur donor or redox system, and exists in reduced (glutathione; GSH) and oxidized (glutathione disulfide; GSSG) forms. However, the mechanism of GSH regulating GSLs biosynthesis remainds unclear. Hence, the exogenous therapy to pakchoi under normal growth condition and sulfur deficiency condition were conducted in this work to explore the relevant mechanism. The results showed that exogenous application of buthionine sulfoximine, an inhibitor of GSH synthesis, decreased the transcript levels of GSLs synthesis-related genes and transcription factors, as well as sulfur assimilation-related genes under the normal growth condition. Application of exogenous GSH inhibited the expression of GSLs synthesis- and sulfur assimilation-related genes under the normal condition, while the GSLs biosynthesis and the sulfur assimilation pathway were activated by exogenous application of GSH when the content of GSH in vivo of plants decreased owing to sulfur deficiency. Moreover, exogenous application of GSSG increased the transcript levels of GSLs synthesis- and sulfur assimilation-related genes under the normal growth condition and under sulfur deficiency. The present work provides new insights into the molecular mechanisms of GSLs biosynthesis underlying glutathione regulation.

Exogenous glutathione can alleviate chromium toxicity in kenaf by activating antioxidant system and regulating DNA methylation

Glutathione (GSH) participates in plant response to heavy metals (HMs) stress, however, the epigenetic regulating mechanisms of GSH in HMs detoxification remains unclear. In this study, to reveal the potential epigenetic regulating mechanisms, kenaf seedlings were treated with/without GSH under chromium (Cr) stress. A comprehensive physiological, genome-wide DNA methylation and gene functional analysis were performed. Results showed that external GSH obviously recovered Cr-induced growth inhibition, significantly decreased H2O2, O2.- and MDA accumulation, increased the activities of antioxidant enzymes (SOD, CAT, GR and APX) in kenaf exposed to Cr. In addition, the expression level of the main DNA methyltransferase (MET1, CMT3 and DRM1) and demethylase (ROS1, DEM, DML2, DML3 and DDM1) genes were investigated by qRT-PCR. The result indicated that Cr stress decreased DNA methyltransferase genes expression while increased demethylase genes expression; however, apply exogenous GSH led to the recovery trend. These indicating exogenous GSH alleviation Cr stress on kenaf seedlings by increasing DNA methylation level. At the same time, the MethylRAD-seq genome-wide DNA methylation analysis showed the DNA methylation level was significantly increased after GSH treatment compared with Cr treatment alone. The differentially methylated genes (DMGs) were uniquely enriched in DNA repair, flavin adenine dinucleotide binding and oxidoreductase activity. Furthermore, a ROS homeostasis-associated DMG, HcTrx, was selected for further functional analysis. Results showed that the knock-down of HcTrx kenaf seedlings displayed yellow-green phenotype and impaired antioxidant enzyme activity; in contrast, the overexpression lines of HcTrx improved chlorophyll levels and enhanced Cr tolerance in Arabidopsis. Taken together, our results illustrate the novel role of GSH-mediated Cr detoxification in kenaf by modulating the DNA methylation, and thus further affect the activation of antioxidant defense systems. The present characterized Cr tolerant gene resource could be further used for kenaf Cr tolerant breeding via genetic improvement.

Glutathione-mediated changes in productivity, photosynthetic efficiency, osmolytes, and antioxidant capacity of common beans (Phaseolus vulgaris) grown under water deficit.

Globally, salinity and drought are severe abiotic stresses that presently threaten vegetable production. This study investigates the potential exogenously-applied glutathione (GSH) to relieve water deficits on Phaseolus vulgaris plants cultivated in saline soil conditions (6.22 dS m-1) by evaluating agronomic, stability index of membrane, water satatus, osmolytes, and antioxidant capacity responses. During two open field growing seasons (2017 and 2018), foliar spraying of glutathione (GSH) at 0.5 (GSH1) or 1.0 (GSH1) mM and three irrigation rates (I100 = 100%, I80 = 80% and I60 = 60% of the crop evapotranspiration) were applied to common bean plants. Water deficits significantly decreased common bean growth, green pods yield, integrity of the membranes, plant water status, SPAD chlorophyll index, and photosynthetic capacity (Fv/Fm, PI), while not improving the irrigation use efficiency (IUE) compared to full irrigation. Foliar-applied GSH markedly lessened drought-induced damages to bean plants, by enhancing the above variables. The integrative I80 + GSH1 or GSH2 and I60 + GSH1 or GSH2 elevated the IUE and exceeded the full irrigation without GSH application (I100) treatment by 38% and 37%, and 33% and 28%, respectively. Drought stress increased proline and total soluble sugars content while decreased the total free amino acids content. However, GSH-supplemented drought-stressed plants mediated further increases in all analyzed osmolytes contents. Exogenous GSH enhanced the common bean antioxidative machinery, being promoted the glutathione and ascorbic acid content as well as up-regulated the activity of superoxide dismutase, catalase, ascorbate peroxidase, and glutathione peroxidase. These findings demonstrate the efficacy of exogenous GSH in alleviating water deficit in bean plants cultivated in salty soil.

Evaluation of erastin synergized cisplatin anti-nasopharyngeal carcinoma effect with a glutathione-activated near-infrared fluorescent probe

Nasopharyngeal carcinoma (NPC), a malignant tumor originating from the nasopharynx, is one of the common malignant tumors of the head and neck. There are significant geographical differences in the incidence of nasopharyngeal carcinoma, with a high incidence in China and Southeast Asian countries. Herein, we designed and synthesized a novel near-infrared fluorescent (NIRF) probe to detect glutathione (GSH) in cellular and tumor environments using semi-naphthofluorescein (SNAFL) as the fluorescent molecular backbone and 2-fluoro-4-nitrobenzenesulfonate as the recognition moiety. Upon reaction with GSH, SNAFL-GSH emitted a fluorescence signal, and its emission wavelength at 650 nm was remarkably enhanced. The results of selectivity experiments indicated that SNAFL-GSH was able to discriminate GSH from Cys, Hcy, and H2S. Moreover, SNAFL-GSH could image both endogenous and exogenous GSH and distinguish normal and cancer cells by fluorescence signal difference. At the cellular level, cisplatin (DDP)-induced ferroptosis and inhibition of proliferation of various NPC cell lines (CNE2, CNE1, 5–8F cells) by erastin combined with DDP were visualized with the help of SNAFL-GSH. In a mouse tumor xenograft model, we successfully employed SNAFL-GSH for the evaluation of the efficacy of erastin combined with DDP in the treatment of NPC. More importantly, the probe could image cancerous tissue sections from NPC patients with an imaging depth of approximately 80 µm. It was foreseen that SNAFL-GSH offered great potential for application in the diagnosis and evaluation of the therapeutic efficacy of NPC, and these results would also provide new ideas for the clinical treatment of NPC.

Ascorbate−glutathione cycle involving in response of Bangia fuscopurpurea (Bangiales, Rhodophyta) to hyposalinity

Bangia fuscopurpurea is a widespread intertidal seaweed that is commercially cultured in China. This seaweed is frequently exposed to hyposalinity stress, but little is known about the adaptation mechanisms. Ascorbate−glutathione (AsA−GSH) cycle plays important roles in many organisms under a variety of abiotic stress, including hyposaline stress. In this study, we investigated the response of key metabolites and enzymes involved in the AsA−GSH cycle of B. fuscopurpurea under hyposalinity, with the addition of exogenous GSH and Lbuthionine-sulfoximine (BSO). The quantification of BfAPX gene expression was assessed across varied treatment regimens. And the putative interaction proteins of BfAPX were screened by yeast two hybrid system. It was found that under hyposalinity (15 and/or 0 psu), the content of reduced glutathione (GSH), total glutathione (GSH+ oxidized glutathione, GSSG) and cysteine, the ratio of GSH/GSSG and ascorbic acid (AsA)/ dehydroascorbic acid (DHA), and the activity of ascorbic acid peroxidase (APX) and monodehydroascorbate reductase (MDHAR) was significantly up-regulated. The hyposality-promoted GSH/GSSG was weakened while the glutathione reductase (GR) activity was promoted by adding exogenous GSH and BSO. The hyposality-promoted AsA/DHA ratio was strengthened by exogenous GSH but weakened by BSO. The dehydroascorbate reductase (DHAR) activity had no significant changes either with or without exogenous GSH under all salinities, while DHAR activity together with DHA content was enhanced by BSO. The expression of APX gene markedly increased under hyposalinity+BSO treatment. Putative interacting proteins of APX, including glutamate dehydrogenase 1a and fructose diphosphate aldolase, were identified through screening. The results indicated that the AsA−GSH cycle was involved in response of B. fuscopurpurea to hyposalinity by means of increasing GSH/GSSG ratio (through promoting GSH biosynthesis pathway and GSH regeneration from GSSG by GR catalyzation) and AsA/DHA ratio (promoting AsA regeneration through MDHAR). These findings would contribute to improve the aquaculture of this promising economic species and unveil how intertidal seaweeds address the global climate challenges.

Exogenous GSH Supplementation to Raw Semen Alters Sperm Kinematic Parameters in Infertile Patients.

Glutathione is an important antioxidant found in all mammalian cells. Sperm motility is positively correlated with seminal reduced glutathione (GSH) levels, and infertile men are known to have lower GSH levels. Studies on GSH supplementation in improving sperm functions in infertility patients are limited. Here, we re-investigate the effect of exogenous GSH supplementation on human sperm motility and kinematic parameters. Residual semen samples from 71 infertility patients who came for routine semen analysis for infertility assessment were studied. Liquefied raw semen was supplemented with GSH (0-10 mM) for 1 h. The untreated sample was the blank control. Only a 5 mM concentration was tested in all 71 samples. After two washes, the sperm was incubated and then analyzed for sperm motility and kinematic parameters by computer-assisted semen analysis (CASA), followed by adenosine triphosphate (ATP), reactive oxygen species (ROS) levels, free thiols, and DNA damage analyses. At 2 hrs post-treatment, GSH supplementation significantly altered many of the kinematics, compared to the control. Straight line velocity (VSL) (p = 0.0459), curvilinear velocity (VCL) (p < 0.0001), average path velocity (VAP) (p < 0.0001), and lateral head amplitude (ALH) (p < 0.0001) were decreased, whereas straightness (STR) (p = 0.0003), linearity (LIN) (p = 0.0008), and beat cross frequency (BCF) (p = 0.0291) were increased in 5 mM group. Wobble (WOB) (p = 0.4917), motility (MOT) (p = 0.9574), and progressive motility (PROG) (p = 0.5657) were unchanged. ATP level was significantly increased in the 5 mM group (p < 0.05). It is concluded that exogenous GSH supplementation does alter sperm kinematics in humans. These altered kinematic parameters together with increased energy (ATP) may have a positive role in influencing the success rates of ART procedures.