Effect of Rubia cordifolia, Fagonia cretica linn, and Tinospora cordifolia on free radical generation and lipid peroxidation during oxygen-glucose deprivation in rat hippocampal slices

The major damaging factor during and after the ischemic/hypoxic insult is the generation of free radicals, which leads to apoptosis, necrosis, and ultimately cell death. Rubia cordifolia (RC), Fagonia cretica linn (FC), and Tinospora cordifolia (TC) have been reported to contain a wide variety of antioxidants and have been in use in the eastern system of medicine for various disorders. Hippocampal slices were subjected to oxygen-glucose deprivation (OGD) and divided into three groups, control, OGD, and OGD+drug treated. Cytosolic reduced glutathione (GSH), nitric oxide [NO, measured as nitrite (NO2)]. EPR was used to establish the antioxidant effect of RC, FC, and TC with respect to superoxide anion (O*2-), hydroxyl radicals (*OH), nitric oxide (NO) radical, and peroxynitrite anion (ONOO-) generated from pyrogallol, menadione, DETA-NO, and Sin-1, respectively. RT-PCR was performed for the three herbs to assess their effect on the expression of gamma-glutamylcysteine ligase (GCLC), iNOS, and GAPDH gene expression. All the three herbs were effective in elevating the GSH levels and expression of the GCLC. The herbs also exhibited strong free radical scavenging properties against reactive oxygen and nitrogen species as revealed by electron paramagnetic resonance spectroscopy, diminishing the expression of iNOS gene. RC, FC, and TC therefore attenuate oxidative stress mediated cell injury during OGD and exert the above effects at both the cytosolic as well as at gene expression levels and may be effective therapeutic tool against ischemic brain damage.     

Molecular mechanism of glutathione-mediated protection from oxidized low-density lipoprotein-induced cell injury in human macrophages: role of glutathione reductase and glutaredoxin

Macrophage death is a hallmark of advanced atherosclerotic plaque, and oxidized low-density lipoprotein (OxLDL) found in these lesions is believed to contribute to macrophage injury. However, the underlying mechanisms of this phenomenon are only poorly understood. Here we show that in human monocyte-derived macrophages, OxLDL depleted intracellular glutathione (GSH) and inhibited glutathione reductase, resulting in a marked diminution of the glutathione/glutathione disulfide ratio. In the absence of OxLDL, an 80% depletion of intracellular GSH levels did not affect cell viability, but glutathione depletion dramatically increased OxLDL-induced cell death. Conversely, supplementation of intracellular GSH stores with glutathione diethyl ester substantially diminished OxLDL toxicity. OxLDL also promoted protein-S-glutathionylation, which was increased in macrophages pretreated with the glutathione reductase inhibitor BCNU. Knockdown experiments with siRNA directed against glutathione reductase and glutaredoxin showed that both enzymes are essential for the protection of macrophages against OxLDL. Finally, the peroxyl-radical scavenger Trolox did not prevent GSH depletion but completely blocked OxLDL-induced protein-S-glutathionylation and cell death. These data suggest that OxLDL promotes ROS formation and protein-S-glutathionylation by a mechanism independent from its effect on GSH depletion. Neither mechanism was sufficient to induce macrophage injury, but when stimulated concurrently, these pathways promoted the accumulation of protein-glutathione mixed disulfides and cell death.     

A role of vitamin E in protection against cell injury. Maintenance of intracellular glutathione precursors and biosynthesis

The depletion of cell calcium from isolated rat hepatocytes results in stimulated lipid peroxidation, loss of intracellular and mitochondrial GSH (reduced glutathione), and enhancement of both efflux and oxidation of GSH. These events are followed by cell injury and enhance the susceptibility of the cells to toxic chemicals. It is shown herein that an initial event in the generation of such injury is the depletion of cellular alpha-tocopherol. alpha-Tocopheryl succinate addition (25 microM) to the calcium-depleted cells markedly elevated the alpha-tocopherol content of the cells, inhibited the associated lipid peroxidation, and maintained intracellular GSH levels without affecting its efflux or redox status. This resulted in an enhanced formation of total glutathione after a 5-h incubation, which correlated with the alpha-tocopherol content of the cells, and was greater than that expected by a direct sparing action of vitamin E. Inhibition of hepatocyte glutathione biosynthesis by buthionine sulfoximine (0.5 mM) eliminated the enhancement of GSH formation by vitamin E. Analysis of endogenous and 35S-labelled precursors of glutathione biosynthesis by high-performance liquid chromatography demonstrated that the depletion of cellular alpha-tocopherol resulted in the efflux of glutathione precursors. It is concluded that cell injury associated with alpha-tocopherol depletion is partly the result of the efflux of glutathione precursors, and hence diminished biosynthesis and intracellular levels of GSH. These losses and resultant cell injury are preventable by maintenance of cellular alpha-tocopherol levels.     

15-Deoxy-Delta12,14-prostaglandin J(2) protects against nitrosative PC12 cell death through up-regulation of intracellular glutathione synthesis

Nitrosative stress with subsequent inflammatory cell death has been associated with many neurodegenerative disorders. Expression of inducible nitric-oxide synthase and production of nitric oxide (NO) have been frequently elevated in many inflammatory disorders. NO can rapidly react with superoxide anion, producing more reactive peroxynitrite. In the present study, exposure of rat pheochromocytoma (PC12) cells to the peroxynitrite donor 3-morpholinosydnonimine hydrochloride (SIN-1) induced apoptosis, which accompanied depletion of intracellular glutathione (GSH), c-Jun N-terminal kinase activation, mitochondrial membrane depolarization, the cleavage of poly(ADP-ribose)polymerase, and DNA fragmentation. During SIN-1-induced apoptotic cell death, expression of inducible cyclooxygenase (COX-2), and peroxisome proliferator-activated receptor-gamma (PPARgamma) was elevated. SIN-1 treatment resulted in elevated production of 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), an endogenous PPARgamma activator. Preincubation with 15d-PGJ(2) rendered PC12 cells resistant to nitrosative stress induced by SIN-1. 15d-PGJ(2) fortified an intracellular GSH pool through up-regulation of glutamylcysteine ligase, thereby preventing cells from SIN-1-induced GSH depletion. The above findings suggest that 15d-PGJ(2) may act as a survival mediator capable of augmenting cellular thiol antioxidant capacity through up-regulation of the intracellular GSH synthesis in response to the nitrosative insult.     

Adipose-derived mesenchymal stem cells stereotactic transplantation alleviate brain edema from intracerebral hemorrhage

Adipose-derived mesenchymal stromal cells (ADSCs) exhibited high potential in tissue repair and regeneration, and it has been proved that ADSCs could protect brain cells from apoptosis and maintaining blood-brain barrier stability after cerebral vascular disease. In this study, we evaluated the therapeutic potential and mechanism of ADSCs stereotactic transplantation in intracerebral hemorrhage (ICH) mice model and hemin-treated astrocytes. Mice were divided into three groups: sham group, ICH + PBS group, and ICH + ADSC group. Mice in ICH + ADSC group received ADSCs cell suspension stereotactic transplantation into the area beside the bleeding region. Astrocytes were divided into three groups: control group, hemin group, and hemin + ADSC group. Astrocytes in hemin + ADSC group were cultured in ADSCs-astrocyte no-contact coculture system and treated with 30 μM hemin solution. The results showed that ADSCs stereotactic transplantation improved functional outcomes and reduced cell apoptosis after ICH. Moreover, ADSCs stereotactic transplantation could alleviate brain edema and inflammation and AQP4 protein expression contributed to the alleviation of brain edema. In addition, mitogen-activated protein kinase (MAPK) pathways, including p38/MAPK pathway and c-Jun N-terminal kinase pathway, were involved in AQP4 modulation by ADSCs transplantation in ICH. In conclusion, ADSCs transplantation could alleviate the nervous tissue injury, reduce cell apoptosis, and relieve brain edema in ICH. And the edema regulation effect of ADSCs transplantation is associated with inhibition of inflammation and AQP4 protein expression.     

Heme oxygenase-1 induction by endogenous nitric oxide: influence of intracellular glutathione

To investigate the influence of glutathione (GSH) on cellular effects of nitric oxide (NO) formation, human colon adenocarcinoma cells were transfected with a vector allowing controlled expression of inducible nitric oxide synthase (iNOS). Protein levels of oxidative stress-sensitive heme oxygenase-1 (HO-1) were analyzed in the presence or absence of GSH depletion using L-buthionine-[S,R]-sulfoximine and iNOS induction. While no effect was observed in the presence of iNOS activity alone, a synergistic effect on HO-1 expression was observed in the presence of iNOS expression and GSH depletion. This effect was prevented by addition of N-methyl-L-arginine. Therefore, targeting of endogenous NO may be modulated by intracellular GSH.     

Neuroprotective effects of valproic acid against hemin toxicity: Possible involvement of the down-regulation of heme oxygenase-1 by regulating ubiquitin–proteasomal pathway

During hemorrhagic stroke induced by intracerebral hemorrhage (ICH), brain injury occurs from the deleterious actions of hemoglobin byproducts; induction of heme oxygenase-1 (HO-1) also plays a critical role in the neurotoxicity in ICH. Valproic acid (VPA), which is a commonly used drug in the treatment of epilepsy, has been reported to have neuroprotective effects against various neuronal insults including ischemic stroke. We investigated the effect of VPA on HO-1-mediated neurotoxicity in an experimental model of ICH. We investigated the effects of VPA on HO-1 protein in primary cortical neurons: (1) the expression levels of HO-1 mRNA and protein measured by RT-PCR and Western blotting; (2) the cell viability and ROS generation by MTT reduction assay and ROS measurement; (3) the signal pathway regulated by VPA using IP-Western blotting; (4) the effects of VPA on hemin-induced cell death by hemin microinjection and immunohistochemistry in vivo. VPA treatment partially blocked cell death induced by hemin, which is released from hemoglobin during ICH, both in rat primary cortical neurons and rat brain. Treatment of VPA significantly decreased the expression of HO-1 protein both in vitro and in vivo. Hemin treatment induced HO-1 protein expression and this was partially blocked by pretreatment with VPA, which might be mediated by increased ubiquitination and degradation of HO-1 via ERK1/2 and JNK activation in primary cortical neurons. Our results indicate that VPA inhibits hemin toxicity by downregulating HO-1 protein expression, and provide a therapeutic strategy to attenuate intracerebral hemorrhagic injury.     

Synergistic depletion of astrocytic glutathione by glucose deprivation and peroxynitrite: correlation with mitochondrial dysfunction and subsequent cell death

Previously we reported that immunostimulated astrocytes were highly vulnerable to glucose deprivation. The augmented death was mimicked by the peroxynitrite (ONOO )-producing reagent 3-morpholinosydnonimine (SIN-1). Here we show that glucose deprivation and ONOO- synergistically deplete intracellular reduced glutathione (GSH) and augment the death of astrocytes via formation of cyclosporin A-sensitive mitochondrial permeability transition (MPT) pore. Astrocytic GSH levels were only slightly decreased by glucose deprivation or SIN-1 (200 microM) alone. In contrast, a rapid and large depletion of GSH was observed in glucose-deprived/ SIN-1-treated astrocytes. The depletion of GSH occurred before a significant release of lactate dehydrogenase (a marker of cell death). Superoxide dismutase and ONOO-scavengers completely blocked the augmented death, indicating that the reaction of nitric oxide with superoxide to form ONOO was implicated. Furthermore, nitrotyrosine immunoreactivity (a marker of ONOO-) was markedly enhanced in glucose-deprived/SIN-1 -treated astrocytes. Mitochondrial transmembrane potential (MTP) was synergistically decreased in glucose-deprived/SIN-1-treated astrocytes. The glutathione synthase inhibitor L-buthionine-(S,R)-sulfoximine markedly decreased the MTP and increased lactate dehydrogenase (LDH) releases in SIN-1-treated astrocytes. Cyclosporin A, an MPT pore blocker, completely prevented the MTP depolarization as well as the enhanced LDH releases in glucose-deprived/SIN-1-treated astrocytes.     

Glutathione depletion inhibits IL-1 beta-stimulated nitric oxide production by reducing inducible nitric oxide synthase gene expression

L-buthionine-S,R-sulfoximine (BSO), an inhibitor of GSH synthesis, decreased IL-1 beta-induced nitrite release in rat islets and purified rat beta cells, nitrite formation and iNOS gene promoter activity in insulinoma cells, and iNOS mRNA expression in rat islets. The thiol depletor diethyl maleate (DEM) and an inhibitor of glutathione reductase 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) reduced IL-1 beta-stimulated nitrite release in islets. We conclude that GSH regulates IL-1 beta-induced NO production in islets, purified beta cells and insulinoma cells by modulation of iNOS gene expression.     

Increased efflux of glutathione conjugate in acutely diabetic cardiomyocytes

In diabetes, cell death and resultant cardiomyopathy have been linked to oxidative stress and depletion of antioxidants like glutathione (GSH). Although the de novo synthesis and recycling of GSH have been extensively studied in the chronically diabetic heart, their contribution in modulating cardiac oxidative stress in acute diabetes has been largely ignored. Additionally, the possible contribution of cellular efflux in regulating GSH levels during diabetes is unknown. We used streptozotocin to make Wistar rats acutely diabetic and after 4 days examined the different processes that regulate cardiac GSH. Reduction in myocyte GSH in diabetic rats was accompanied by increased oxidative stress, excessive reactive oxygen species, and an elevated apoptotic cell death. The effect on GSH was not associated with any change in either synthesis or recycling, as both gamma-glutamylcysteine synthetase gene expression (responsible for bio syn thesis) and glutathione reductase activity (involved with GSH recycling) remained unchanged. However, gene expression of multidrug resistance protein 1, a transporter implicated in effluxing GSH during oxidative stress, was elevated. GSH conjugate efflux mediated by multidrug resistance protein 1 also increased in diabetic cardiomyocytes, an effect that was blocked using MK-571, a specific inhibitor of this transporter. As MK-571 also decreased oxidative stress in diabetic cardiomyocytes, an important role can be proposed for this transporter in GSH and reactive oxygen species homeostasis in the acutely diabetic heart.     

Chronic iron overload enhances inducible nitric oxide synthase expression in rat liver.

Iron is an essential micronutrient promoting oxidative stress in the liver of overloaded animals and human, which may trigger the expression of redox-sensitive genes. We have tested the hypothesis that chronic iron overload (CIO) enhances inducible nitric oxide synthase (iNOS) expression in rat liver by extracellular signal-regulated kinase (ERK1/2) and NF-kappaB activation. CIO (diet enriched with 3%(wt/wt) carbonyl-iron for 12 weeks) increased liver protein carbonylation and decreased reduced glutathione (GSH) content and the GSH/GSSG ratio after 6 weeks, parameters that are normalized after 8-12 weeks of treatment. These changes are paralleled by higher phosphorylated-ERK1/2 to non-phosphorylated-ERK1/2 ratios at 6 and 8 weeks, increased NF-kappaB DNA binding to the iNOS gene promoter at 8-12 weeks, and higher iNOS mRNA expression and activity at 8 and 12 weeks. It is concluded that CIO triggers liver oxidative stress at early times, with upregulation of iNOS expression involving the ERK/NF-kappaB pathway at later times, a finding that may represent a hepatoprotective mechanism against CIO toxicity in addition to the recovery of GSH homeostasis.     

Glutamine potentiates TNF-alpha-induced tumor cytotoxicity

L-glutamine (Gln) sensitizes tumor cells to tumor necrosis factor (TNF)-alpha-induced cytotoxicity. The type and mechanism of cell death induced by TNF-alpha was studied in Ehrlich ascites tumor (EAT)-bearing mice fed a Gln-enriched diet (GED; where 30% of the total dietary nitrogen was from Gln). A high rate of Gln oxidation promotes a selective depletion of mitochondrial glutathione (mtGSH) content to approximately 58% of the level found in tumor mitochondria of mice fed a nutritionally complete elemental diet (standard diet, SD). The mechanism of mtGSH depletion involves a glutamate-induced inhibition of GSH transport from the cytosol into mitochondria. The increase in reactive oxygen intermediates (ROIs) production induced by TNF-alpha further depletes mtGSH to approximately 35% of control values, which associates with a decrease in the mitochondrial transmembrane potential (MMP), and elicits mitochondrial membrane permeabilization and release of cytochrome c. Mitochondrial membrane permeabilization was also found in intact tumor cells cultured with a Gln-enriched medium under conditions of buthionine sulfoximine (BSO)-induced selective GSH synthesis inhibition. Enforced expression of the bcl-2 gene in tumor cells could not avoid the glutamine- and TNF-alpha-induced cell death under conditions of mtGSH depletion. However, addition of GSH ester, which delivers free intracellular GSH and increases mtGSH levels, preserved cell viability. These findings show that glutamine oxidation and TNF-alpha, by causing a change in the glutathione redox status within tumor mitochondria, activates the molecular mechanism of apoptotic cell death.     

Induction of PR-1 accumulation accompanied by runaway cell death in the lsd1 mutant of Arabidopsis is dependent on glutathione levels but independent of the redox state of glutathione

The lesions simulating disease (lsd) mutants of Arabidopsis spontaneously develop hypersensitive-response-like lesions in the absence of pathogens. To address the function of the redox regulator glutathione in disease resistance, we examined the relationship between endogenous glutathione and PR-1 accumulation using one of these mutants, lsd1, as a disease resistance model. Lesion formation on lsd1 was suppressed by weak light and initiated by the subsequent transition to normal light. The application of buthionine sulfoximine, a specific inhibitor of glutathione biosynthesis, suppressed conditionally induced runaway cell death and expression of the PR-1 gene, suggesting that glutathione regulates the conditional cell death and PR-1 gene expression. The application of reduced (GSH) or oxidized (GSSG) glutathione to lsd1 upregulated the level of total glutathione ([GSH]+[GSSG]) accompanied by hastened accumulation of PR-1, and the basal level of total glutathione in lsd1 was higher than that in wild-type plants. The glutathione redox state defined as [GSH]/([GSH]+[GSSG]) decreased following the conditional transition, but the suppression of this decrease by the application of GSH did not inhibit the accumulation of PR-1. Taken together, conditional PR-1 accumulation in lsd1 is regulated not by the redox state but by the endogenous level of glutathione.     

Schisandrin B enhances the glutathione redox cycling and protects against oxidant injury in different types of cultured cells

Tert-butylhydroperoxide (tBHP) challenge caused an initial depletion of cellular reduced glutathione (GSH), which was followed by a gradual restoration of cellular GSH in AML12, H9c2, and differentiated PC12 cells. The time-dependent changes in cellular GSH induced by tBHP were monitored as a measure of GSH recovery capacity (GRC), of which glutathione reductase (GR)-mediated glutathione redox cycling and γ-glutamate cysteine ligase (GCL)-mediated GSH synthesis were found to play an essential role. While glutathione redox cycling sustained the GSH level during the initial tBHP-induced depletion, GSH synthesis restores the GSH level thereafter. The effects of (-)schisandrin B [(-)Sch B] and its analogs (Sch A and Sch C) on GRC were also examined in the cells. (-)Sch B and Sch C, but not Sch A, ameliorated the extent of tBHP-induced GSH depletion, indicative of enhanced glutathione redox cycling. However, the degree of restoration of GSH post-tBHP challenge was not affected or even decreased. Pretreatment with (-)Sch B and Sch C, but not Sch A, protected against oxidant injury in the cells. The (-)Sch B afforded cytoprotection was abolished by N,N'-bis(chloroethyl)-N-nitrosourea pretreatment suggesting the enhancement of glutathione redox cycling is crucially involved in the cytoprotection afforded by (-)Sch B against oxidative stress-induced cell injury.     

Ethylene and salicylic acid control glutathione biosynthesis in ozone‐exposed Arabidopsis thaliana

Ozone produces reactive oxygen species and induces the synthesis of phytohormones, including ethylene and salicylic acid. These phytohormones act as signal molecules that enhance cell death in response to ozone exposure. However, some studies have shown that ethylene and salicylic acid can instead decrease the magnitude of ozone‐induced cell death. Therefore, we studied the defensive roles of ethylene and salicylic acid against ozone. Unlike the wild‐type, Col‐0, Arabidopsis mutants deficient in ethylene signaling (ein2) or salicylic acid biosynthesis (sid2) generated high levels of superoxide and exhibited visible leaf injury, indicating that ethylene and salicylic acid can reduce ozone damage. Macroarray analysis suggested that the ethylene and salicylic acid defects influenced glutathione (GSH) metabolism. Increases in the reduced form of GSH occurred in Col‐0 6 h after ozone exposure, but little GSH was detected in ein2 and sid2 mutants, suggesting that GSH levels were affected by ethylene or salicylic acid signaling. We performed gene expression analysis by real‐time polymerase chain reaction using genes involved in GSH metabolism. Induction of γ‐glutamylcysteine synthetase (GSH1), glutathione synthetase (GSH2), and glutathione reductase 1 (GR1) expression occurred normally in Col‐0, but at much lower levels in ein2 and sid2. Enzymatic activities of GSH1 and GSH2 in ein2 and sid2 were significantly lower than in Col‐0. Moreover, ozone‐induced leaf damage observed in ein2 and sid2 was mitigated by artificial elevation of GSH content. Our results suggest that ethylene and salicylic acid protect against ozone‐induced leaf injury by increasing de novo biosynthesis of GSH.     

Biological Characterization of Gene Response to Insulin-Induced Hypoglycemia in Mouse Retina

Glucose is the most important metabolic substrate of the retina and maintenance of normoglycemia is an essential challenge for diabetic patients. Chronic, exaggerated, glycemic excursions could lead to cardiovascular diseases, nephropathy, neuropathy and retinopathy. We recently showed that hypoglycemia induced retinal cell death in mouse via caspase 3 activation and glutathione (GSH) decrease. Ex vivo experiments in 661W photoreceptor cells confirmed the low-glucose induction of death via superoxide production and activation of caspase 3, which was concomitant with a decrease of GSH content. We evaluate herein retinal gene expression 4 h and 48 h after insulin-induced hypoglycemia. Microarray analysis demonstrated clusters of genes whose expression was modified by hypoglycemia and we discuss the potential implication of those genes in retinal cell death. In addition, we identify by gene set enrichment analysis, three important pathways, including lysosomal function, GSH metabolism and apoptotic pathways. Then we tested the effect of recurrent hypoglycemia (three successive 4h periods of hypoglycemia spaced by 48 h recovery) on retinal cell death. Interestingly, exposure to multiple hypoglycemic events prevented GSH decrease and retinal cell death, or adapted the retina to external stress by restoring GSH level comparable to control situation. We hypothesize that scavenger GSH is a key compound in this apoptotic process, and maintaining “normal” GSH level, as well as a strict glycemic control, represents a therapeutic challenge in order to avoid side effects of diabetes, especially diabetic retinopathy.     

Neurotoxicity from glutathione depletion is mediated by Cu-dependent p53 activation

Loss of intracellular neuronal glutathione (GSH) is an important feature of neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. The consequences of GSH depletion include increased oxidative damage to proteins, lipids, and DNA and subsequent cytotoxic effects. GSH is also an important modulator of cellular copper (Cu) homeostasis and altered Cu metabolism is central to the pathology of several neurodegenerative diseases. The cytotoxic effects of Cu in cells depleted of GSH are not well understood. We have previously reported that depletion of neuronal GSH levels results in cell death from trace levels of extracellular Cu due to elevated Cu(I)-mediated free radical production. In this study we further examined the molecular pathway of trace Cu toxicity in neurons and fibroblasts depleted of GSH. Treatment of primary cortical neurons or 3T3 fibroblasts with the glutathione synthetase inhibitor buthionine sulfoximine resulted in substantial loss of intracellular GSH and increased cytotoxicity. We found that both neurons and fibroblasts revealed increased expression and activation of p53 after depletion of GSH. The increased p53 activity was induced by extracellular trace Cu. Furthermore, we showed that in GSH-depleted cells, Cu induced an increase in oxidative stress resulting in DNA damage and activation of p53-dependent cell death. These findings may have important implications for neurodegenerative disorders that involve GSH depletion and aberrant Cu metabolism.