Osteoblastic MG-63 cell differentiation, contraction, and mRNA expression in stress-relaxed 3D collagen I gels

The present study was designed to evaluate the apoptotic efficacy of selenium (Se) under glutathione-deprived conditions. Testicular cells were used as a model to assess the above. For the study, cells were maintained for 4 h under various treatments; control (media only), selenium (0.5 microM and 1.5 microM), BSO (20 nM), selenium + BSO (0.5 microM Se + 20 nM BSO and 1.5 microM Se + 20 nM BSO). The treated cells were harvested for various estimations viz. viability, GSH, GSSG, redox ratio, ROS generation and integrity of DNA. mRNA was extracted for RT-PCR analysis of JNK, p38, caspase 3 and Bcl-2. It was observed that the cell viability decreased concomitant with the decrease in GSH levels, increase in GSSG levels and increase in the generation of ROS in the combined treatment group in comparison to control and individual treatments. Also, there was an increase in the mRNA expression of JNK and p38 MAPK along with an increase in caspase 3 expression and decrease in Bcl-2 expression. The integrity of DNA was also found to be altered in the combined treatment. Thus, the results presented in this work agree with those earlier reports in a notion that sodium selenite causes apoptosis and the toxicity of selenite is mediated by increase of intracellular ROS. Also, reduction in endogenous GSH along with selenite treatment is associated with increased apoptosis, increased expression of p38 and JNK MAPK, decreased Bcl-2 expression, and increase in caspase-3 expression. Our data indicates that GSH participates in apoptosis in testicular cells and that depletion of this molecule may be critical in predisposing these cells to apoptotic cell death.     

Protective mechanisms of N‐acetyl‐cysteine against pyrrolizidine alkaloid clivorine‐induced hepatotoxicity

Pyrrolizidine alkaloid (PA) clivorine, isolated from traditional Chinese medicinal plant Ligularia hodgsonii Hook, has been shown to induce apoptosis in hepatocytes via mitochondrial‐mediated apoptotic pathway in our previous research. The present study was designed to observe the protection of N‐acetyl‐cysteine (NAC) on clivorine‐induced hepatocytes apoptosis. Our results showed that 5 mM NAC significantly reversed clivorine‐induced cytotoxicity via MTT and Trypan Blue staining assay. DNA apoptotic fragmentation analysis and Western‐blot results showed that NAC decreased clivorine‐induced apoptotic DNA ladder and caspase‐3 activation. Further results showed that NAC inhibited clivorine‐induced Bcl‐xL decrease, mitochondrial cytochrome c release and caspase‐9 activation. Intracellular glutathione (GSH) is an important ubiquitous redox‐active reducing sulfhydryl (? SH) tripeptide, and our results showed that clivorine (50 µM) decreased cellular GSH amounts and the ratio of GSH/GSSG in the time‐dependent manner, while 5 mM NAC obviously reversed this depletion. Further results showed that GSH synthesis inhibitor BSO augmented clivorine‐induced cytotoxicity, while exogenous GSH reversed its cytotoxicity on hepatocytes. Clivorine (50 µM) significantly induced cellular reactive oxygen species (ROS) generation. Further results showed that 50 µM Clivorine decreased glutathione peroxidase (GPx) activity and increased glutathione S transferase (GST) activity, which are both GSH‐related antioxidant enzymes. Thioredoxin‐1 (Trx) is also a ubiquitous redox‐active reducing (? SH) protein, and clivorine (50 µM) decreased cellular expression of Trx in a time‐dependent manner, while 5 mM NAC reversed this decrease. Taken together, our results demonstrate that the protection of NAC is major via maintaining cellular reduced environment and thus prevents clivorine‐induced mitochondrial‐mediated hepatocytes apoptosis. J. Cell. Biochem. 108: 424–432, 2009. © 2009 Wiley‐Liss, Inc.     

Glutathione reductase plays an anti-apoptotic role against oxidative stress in human hepatoma cells

The cellular roles of glutathione reductase (GR) in the reactive oxygen species (ROS)-induced apoptosis were studied using the HepG2 cells transfected with GR. The overexpression of GR caused a marked enhancement in reduced and oxidized glutathione (GSH/GSSG) ratio, and significantly decreased ROS levels in the stable transfectants. Hydrogen peroxide (H₂O₂), under the optimal condition for apoptosis, significantly decreased cellular viability and total GSH content, and rather increased ROS level, apoptotic percentage and caspase-3 activity in the mock-transfected cells. However, hydrogen peroxide could not largely generate these apoptotic changes in cellular viability, ROS level, apoptotic percentage, caspase-3 activity and total GSH content in the cells overexpressing GR. Taken together, GR may play a protective role against oxidative stress.     

Enhancement of catalase activity by repetitive low-grade H2O2 exposures protects fibroblasts from subsequent stress-induced apoptosis

Exposure of Chinese hamster V79 fibroblasts to mild and repetitive H2O2 doses in culture for 15 weeks produced no change in lipid peroxidation status, GSH/GSSG ratio and glutathione peroxidase activity of these cells (VST cells). In contrast, in VST cells catalase levels underwent a prominent increase which could be significantly inhibited and brought down to control levels after treatment with the catalase inhibitor 3-aminotriazole (3-AT). When control (VC) cells were exposed to UV radiation (UVC 5 J/m2) or H2O2 (7.5mM, 15 min), intracellular reactive oxygen species (ROS) levels rose prominently with significant activation of caspase-3. Marked nuclear fragmentation and lower cell viability were also noted in these cells. In contrast, VST cells demonstrated a significantly lower ROS level, an absence of nuclear fragmentation and an unchanged caspase-3 activity after exposure to UVC or H2O2. Cell viability was also significantly better preserved in VST cells than VC cells after UV or H2O2 exposures. Following 3-AT treatment of VST cells, UVC radiation or H2O2 brought about significantly higher elevations in intracellular ROS, increases in caspase-3 activity, significantly lowered cell viability and marked nuclear fragmentation, indicating the involvement of high catalase levels in the cytoprotective effects of repetitive stress. Therefore, upregulation of the antioxidant defense after repetitive oxidative stress imparted a superior ability to cope with subsequent acute stress and escape apoptotic death and loss of viability.     

Accumulation of glutathione disulfide mediates NF-kappaB activation during immune stimulation with CpG DNA

Innate immune cells recognize pathogens by detecting molecular patterns that are distinct from those of the host. One such pattern is unmethylated CpG dinucleotides, which are common in bacterial DNA but not in vertebrate genomes. Macrophages respond to such CpG motifs in bacterial DNA or synthetic oligodeoxynucleotides (ODN) by inducing NF-kappaB and secreting proinflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), but the mechanisms regulating this have been unclear. CpG ODN-stimulated cells produce reactive oxygen species (ROS) and have a decreased ratio of intracellular glutathione/glutathione disulfide (GSH/GSSG), indicating a shift to a more oxidized intracellular redox state. To determine whether this may play a role in mediating the CpG-induced macrophage activation, the GSH/GSSG redox state was manipulated in the murine macrophagelike cell line RAW264.7. Treatment of cells with BCNU to inhibit glutathione reductase (GR) enhanced the CpG-induced intracellular oxidation and decreased the GSH/GSSG, with increased activation of NF-kappaB and a doubling in the CpG-induced production of IL-6 and TNF-alpha. Experimental manipulation of the intracellular GSSG concentration during inhibition of cellular prooxidant production demonstrated that increased intracellular GSSG is a primary signal that is directly or indirectly required for CpG-induced NF-kappaB activation but is not in itself sufficient to trigger this in the absence of CpG ODN. These data suggest the existence of a second CpG-induced intracellular signal, independent of GSSG, mediating the activation of innate immunity by bacterial DNA.     

Protective role of a coumarin-derived schiff base scaffold against tertiary butyl hydroperoxide (TBHP)-induced oxidative impairment and cell death via MAPKs, NF-κB and mitochondria-dependent pathways

The present study investigated the antioxidant signalling mechanism of a coumarin-derived schiff base (CSB) scaffold against tert-butylhydroperoxide (TBHP) induced oxidative insult in murine hepatocytes. CSB possesses DPPH and other free radical scavenging activities. TBHP reduced cell viability and intracellular antioxidant status accompanied by an increase in intracellular ROS production in hepatocytes. TBHP also activated phospho-ERK1/2, phospho-p38 and NF-κB, altered the Bcl-2/Bad ratio, reduced mitochondrial membrane potential, released cytochrome C and activated caspase 3, suggesting that TBHP induced oxidative stress responsive cell death via apoptotic pathway. FACS analysis and DNA fragmentation studies also confirmed the apoptotic cell death in TBHP exposed hepatocytes. Treatment with CSB effectively reduced these adverse effects by preventing the oxidative insult, alteration in the redox-sensitive signalling cascades and mitochondrial events. Combining, results suggest that antioxidant property of CSB make the molecule to be a potential protective measure against oxidative insult, cytotoxicity and cell death.     

Role of oxidative stress in vinorelbine-induced vascular endothelial cell injury

Vinorelbine (VNR), a vinca alkaloid anticancer drug, often causes vascular injury such as venous irritation, vascular pain, phlebitis, and necrotizing vasculitis. The purpose of this study was to identify the mechanisms that mediate the cell injury induced by VNR in porcine aorta endothelial cells (PAECs). PAECs were exposed to VNR for 10 min followed by further incubation in serum-free medium without VNR. The exposure to VNR (0.3–30 μM) decreased the cell viability concentration and time dependently. The incidence of apoptotic cells significantly increased at 12 h after transient exposure to VNR. At the same time, VNR increased the activity of caspases. Interestingly, VNR rapidly depleted intracellular glutathione (GSH) and increased intracellular reactive oxygen species (ROS) production. Moreover, VNR depolarized the mitochondrial membrane potential and decreased cellular ATP levels. These VNR-induced cell abnormalities were almost completely inhibited by GSH and N-acetylcysteine. On the other hand, l-buthionine-(S,R)-sulfoximine, a specific inhibitor of GSH synthesis, aggravated the VNR-induced loss of cell viability. These results clearly demonstrate that VNR induces oxidative stress by depleting intracellular GSH and increasing ROS production in PAECs, and oxidative stress plays an important role in the VNR-induced cell injury.     

Allicin inhibits cell growth and induces apoptosis through the mitochondrial pathway in HL60 and U937 cells

In this article, the effects of allicin, a biological active compound of garlic, on HL60 and U937 cell lines were examined. Allicin induced growth inhibition and elicited apoptotic events such as blebbing, mitochondrial membrane depolarization, cytochrome c release into the cytosol, activation of caspase 9 and caspase 3 and DNA fragmentation. Pretreatment of HL60 cells with cyclosporine A, an inhibitor of the mitochondrial permeability transition pore (mPTP), inhibited allicin-treated cell death. HL60 cell survival after 1 h pretreatment with cyclosporine A, followed by 16 h in presence of allicin (5 microM) was approximately 80% compared to allicin treatment alone (approximately 50%). Also N-acetyl cysteine, a reduced glutathione (GSH) precursor, prevented cell death. The effects of cyclosporine A and N-acetyl cysteine suggest the involvement of mPTP and intracellular GSH level in the cytotoxicity. Indeed, allicin depleted GSH in the cytosol and mitochondria, and buthionine sulfoximine, a specific inhibitor of GSH synthesis, significantly augmented allicin-induced apoptosis. In HL60 cells treated with allicin (5 microM, 30 min) the redox state for 2GSH/oxidized glutathione shifted from EGSH -240 to -170 mV. The same shift was observed in U937 cells treated with allicin at a higher concentration for a longer period of incubation (20 microM, 2 h). The apoptotic events induced by various concentrations of allicin correlate to intracellular GSH levels in the two cell types tested (HL60: 3.7 nmol/10(6) cells; U937: 7.7 nmol/10(6) cells). The emerging mechanistic basis for the antiproliferative function of allicin, therefore, involves the activation of the mitochondrial apoptotic pathway by GSH depletion and by changes in the intracellular redox status.     

Protective role of a coumarin-derived schiff base scaffold against tertiary butyl hydroperoxide (TBHP)-induced oxidative impairment and cell death via MAPKs,NF-κB and mitochondria-dependent pathways

Abstract

The present study investigated the antioxidant signalling mechanism of a coumarin-derived schiff base (CSB) scaffold against tert-butylhydroperoxide (TBHP) induced oxidative insult in murine hepatocytes. CSB possesses DPPH and other free radical scavenging activities. TBHP reduced cell viability and intracellular antioxidant status accompanied by an increase in intracellular ROS production in hepatocytes. TBHP also activated phospho-ERK1/2, phospho-p38 and NF-κB, altered the Bcl-2/Bad ratio, reduced mitochondrial membrane potential, released cytochrome C and activated caspase 3, suggesting that TBHP induced oxidative stress responsive cell death via apoptotic pathway. FACS analysis and DNA fragmentation studies also confirmed the apoptotic cell death in TBHP exposed hepatocytes. Treatment with CSB effectively reduced these adverse effects by preventing the oxidative insult, alteration in the redox-sensitive signalling cascades and mitochondrial events. Combining, results suggest that antioxidant property of CSB make the molecule to be a potential protective measure against oxidative insult, cytotoxicity and cell death.     

Zinc stimulates the production of toxic reactive oxygen species (ROS) and inhibits glutathione reductase in astrocytes

The release of zinc (Zn) from glutamatergic synapses contributes to the neuropathology of ischemia, traumatic brain injury, and stroke. Astrocytes surround glutamatergic synapses and are vulnerable to the toxicity of Zn, which impairs their antioxidative glutathione (GSH) system and elevates the production of reactive oxygen species (ROS). It is not known whether one or both of these actions are the primary cause of Zn-induced cell death in astrocytes. Using primary rat astrocyte cultures we have examined whether Zn-mediated impairment of GSH redox cycling is the main source of its toxicity. Zn acetate at concentrations of 100 microM or greater were found to inactivate glutathione reductase (GR) via an NADPH-dependent mechanism, while concentrations of 150 microM and above caused substantial cell death. Furthermore, Zn increased the ratio of GSSG:GSH in astrocytes, increased their export of GSSG, slowed their clearance of exogenous H2O2, and promoted the intracellular production of ROS. In contrast, the GR inhibitor, carmustine, did not induce cell death, cause the production of ROS, or alter the GSH thiol redox balance. Taken together these results indicate that Zn toxicity in astrocytes is primarily associated with the generation of intracellular ROS, rather than the inhibition of GR.     

Evidence of prooxidant and antioxidant action of melatonin on human liver cell line HepG2

The aim of this study was to evaluate melatonin cytotoxicity by measuring its effects on various cellular targets. Cell viability, intracellular reduced glutathione (GSH) level, and reactive oxygen species (ROS) production were assessed in the human liver cell line (HepG2), after incubation with increasing melatonin concentrations (0.1-10,000 microM). The incubation times tested were 24, 72, and 96 h for cell viability and intracellular GSH level, and 15 and 45 minutes for ROS production. Cellular target evaluations were possible in living cells by means of a new microplate cytofluorimeter. This technology was suitable for the assessment of cell viability, GSH level, and ROS overproduction with, respectively, neutral red, monochlorobimane (mBCl), and 2',7'-dichlorofluorescin diacetate (DCFH-DA) fluorescent probes. At the lowest melatonin concentrations (0.1-10 microM) and for a relatively short incubation time (24 h), the antioxidant effect of melatonin was revealed by an increased intracellular GSH level, associated to cell viability improvement. In contrast, after longer incubation (96 h), cell viability significantly decreased with these lowest melatonin concentrations (0.1-10 microM). Moreover, high melatonin concentrations (1,000-10,000 microM) induced GSH depletion. This oxidative stress is associated with ROS overproduction from 10 microM after only 15 minutes of incubation. This dual effect is strong evidence that, in vitro, melatonin can be both antioxidant and prooxidant on the human liver cell line, depending on the concentration and incubation time.     

Regulation of ionizing radiation-induced apoptosis by a manganese porphyrin complex

Ionizing radiation induces the production of reactive oxygen species, which play an important causative role in apoptotic cell death. Therefore, compounds that scavenge reactive oxygen species may confer regulatory effects on apoptosis. Superoxide dismutase (SOD) mimetics have been shown to be protective against cell injury caused by reactive oxygen species. We investigated the effects of the manganese (III) tetrakis(N-methyl-2-pyridyl)porphyrin (MnTMPyP), a cell-permeable SOD mimetic, on ionizing radiation-induced apoptosis. Upon exposure to 2 Gy of gamma-irradiation, there was a distinct difference between the control cells and the cells pre-treated with 5 microM MnTMPyP for 2 h with regard to apoptotic parameters, cellular redox status, mitochondria function, and oxidative damage to cells. MnTMPyP effectively suppressed morphological evidence of apoptosis and DNA fragmentation in U937 cells exposed to ionizing radiation. The [GSSG]/[GSH+GSSG] ratio and the generation of intracellular reactive oxygen species were higher and the [NADPH]/[NADP(+)+NADPH] ratio was lower in control cells compared to MnTMPyP-treated cells. The ionizing radiation-induced mitochondrial damage reflected by the altered mitochondrial permeability transition, the increase in the accumulation of reactive oxygen species, and the reduction of ATP production were significantly higher in control cells compared to MnTMPyP-treated cells. MnTMPyP pre-treated cells showed significant inhibition of apoptotic features such as activation of caspase-3, up-regulation of Bax and p53, and down-regulation of Bcl-2 compared to control cells upon exposure to ionizing radiation. This study indicates that MnTMPyP may play an important role in regulating the apoptosis induced by ionizing radiation presumably through scavenging of reactive oxygen species.     

Mechanisms of Trazodone‐Induced Cytotoxicity and the Protective Effects of Melatonin and/or Taurine toward Freshly Isolated Rat Hepatocytes

It has been reported that the bioactive intermediate metabolites of trazodone might cause hepatotoxicity. This study was designed to investigate the exact mechanism of hepatocellular injury induced by trazodone as well as the protective effects of taurine and/or melatonin against this toxicity. Freshly isolated rat hepatocytes were used. Trazodone was cytotoxic and caused cell death with LC₅₀ of 300 µm within 2 h. Trazodone caused an increase in reactive oxygen species (ROS) formation, malondialdehyde accumulation, depletion of intracellular reduced glutathione (GSH), rise of oxidized glutathione disulfide (GSSG), and a decrease in mitochondrial membrane potential, which confirms the role of oxidative stress in trazodone‐induced cytotoxicity. Preincubation of hepatocytes with taurine prevented ROS formation, lipid peroxidation, depletion of intracellular reduced GSH, and increase of oxidized GSSG. Taurine could also protect mitochondria against trazodone‐induced toxicity. Administration of melatonin reduced the toxic effects of trazodone in isolated rat hepatocytes. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:457‐462, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21509     

6-formylpterin, a xanthine oxidase inhibitor, intracellularly generates reactive oxygen species involved in apoptosis and cell proliferation

The chemical property of 6-formylpterin and its biological functions were examined. Polarographic studies revealed that 6-formylpterin reacted with NAD(P)H and consumed oxygen. In contrast, other conjugated pterins, such as biopterin and neopterin, showed no consumption of oxygen. The production analysis using high-performance liquid chromatography documented that 6-formylpterin catalyzes the conversion from NADH to NAD. Electroparamagnetic resonance spin trapping experiments demonstrated that this reaction is accompanied with the generation of reactive oxygen species (ROS), superoxide anion and hydrogen peroxide. When 6-formylpterin was administered to HL-60 cells, intracellular ROS generation was observed and apoptosis was induced. In contrast, other conjugated pterins induced neither intracellular ROS generation nor apoptosis in HL-60 cells. The intracellular ROS generation by 6-formylpterin was observed in other cells, such as PanC-1 cells and Jurkat cells. 6-formylpterin suppressed cell proliferation in PanC-1 cells and inhibited Fas-mediated apoptosis in Jurkat cells. These findings indicate that, among conjugated pterins, 6-formylpterin has the unique property to transfer electron from NAD(P)H to oxygen and that the property brings about intracellular ROS generation, which exerts various biological functions such as induction of apoptosis, suppression of cell proliferation, and inhibition of Fas-mediated apoptosis.     

Hepatocyte susceptibility to glyoxal is dependent on cell thiamin content

Glyoxal, a reactive dicarbonyl, is detoxified primarily by the glyoxalase system utilizing glutathione (GSH) and by the aldo-keto reductase enzymes which utilizes NAD[P]H as the co-factor. Thiamin (Vitamin B(1)) is an essential coenzyme for transketolase (TK) that is part of the pentose phosphate pathway which helps maintain cellular NADPH levels. NADPH plays an intracellular role in regenerating glutathione (GSH) from oxidized GSH (GSSG), thereby increasing the antioxidant defenses of the cell. In this study we have focused on the prevention of glyoxal toxicity by supplementation with thiamin (3mM). Thiamin was cytoprotective and restored NADPH levels, glyoxal detoxification and mitochondrial membrane potential. Hepatocyte reactive oxygen species (ROS) formation, lipid peroxidation and GSH oxidation were decreased. Furthermore, hepatocytes were made thiamin deficient with oxythiamin (3mM) as measured by the decreased hepatocyte TK activity. Under thiamin deficient conditions a non-toxic dose of glyoxal (2mM) became cytotoxic and glyoxal metabolism decreased; while ROS formation, lipid peroxidation and GSH oxidation was increased.     

黄连素预处理对6-羟基多巴胺所致PC12细胞损伤的影响

目的:观察黄连素(Berberine,BBR)预处理对6-羟基多巴胺(6-hydroxydopamine,6-OHDA)诱导的PC12细胞的影响,并探讨二型超氧化物歧化酶(Mn-SOD,SOD2)是否介导了BBR的保护作用。方法:将PC12细胞分为5组,分别为正常培养的对照组(Control)、25μM的6-OHDA损伤组、1μM的BBR预处理24 h组(BBR+6-OHDA)、SOD2-siRNA干扰组(SOD2-siRNA+BBR+6-OHDA)和乱序siRNA处理组(SC-siRNA+BBR+6-OHDA),孵育24 h后,采用噻唑蓝法(Methylthiazolyldiphenyl-tetrazolium bromide,MTT)检测细胞活力,试剂盒检测培养基乳酸脱氢酶(Lactic Dehydrogenase,LDH)、细胞内活性氧(Reactive Oxygen Species,ROS)、还原型谷胱甘肽(Glutathione,GSH)和过氧化氢酶(Catalase,CAT)的含量,使用流式细胞仪检测凋亡率,Western blot检测SOD2和凋亡蛋白Cleaved caspase-3的表达。结果:与Control组相比,6-OHDA诱导PC12细胞24 h后,细胞活力显著降低,SOD2表达、GSH和CAT的含量明显减少,培养基上清液LDH活力、细胞凋亡率、Cleaved caspase-3表达和ROS水平显著增加(P<0.05),而BBR预处理可显著恢复6-OHDA诱导的PC12细胞活力、SOD2表达、GSH和CAT水平,并降低细胞凋亡率、凋亡蛋白表达和细胞ROS水平(P<0.05),而SOD2-siRNA显著逆转了BBR预处理产生的上述保护作用(P<0.05),SC-siRNA则未对BBR预处理产生的上述作用造成明显影响(P>0.05)。结论:黄连素预处理可减轻6-OHDA诱导的PC12细胞损伤,而SOD2分子介导了BBR预处理对暴露于6-OHDA的PC12细胞的保护作用。     

Progressive iron accumulation induces a biphasic change in the glutathione content of neuroblastoma cells

Glutathione (GSH) constitutes the single most important antioxidant in neurons, whereas iron causes oxidative stress that leads to cell damage and death. Although GSH and iron produce opposite effects on redox cell status, no mechanistic relationships between iron and GSH metabolism are known. In this work, we evaluated in SH-SY5Y neuroblastoma cells the effects of iron accumulation on intracellular GSH metabolism. After 2 d exposure to increasing concentrations of iron, cells underwent concentration-dependent iron accumulation and a biphasic change in intracellular GSH levels. Increasing iron from 1 to 5 microM resulted in a marked increase in intracellular oxidative stress and increased GSH levels. Increased GSH levels were due to increased synthesis. Further increases in iron concentration led to significant reduction in both reduced (GSH) and total (GSH + (2 x GSSG)) glutathione. Cell exposure to high iron concentrations (20-80 microM) was associated with a marked decrease in the GSH/GSSG molar ratio and the GSH half-cell reduction potential. Moreover, increasing iron from 40 to 80 microM resulted in loss of cell viability. Iron loading did not change GSH reductase activity but induced significant increases in GSH peroxidase and GSH transferase activities. The changes in GSH homeostasis reported here recapitulate several of those observed in Parkinson's disease substantia nigra. These results support a model by which progressive iron accumulation leads to a progressive decrease in GSH content and cell reduction potential, which finally results in impaired cell integrity.     

京尼平保护高糖诱导损伤小鼠胰岛MIN6细胞的机制

京尼平(genipin,Gen)是一种重要的抗氧化物,在细胞内抵抗氧化应激损伤过程中发挥重要的作用.为了探讨京尼平对高糖诱导损伤的小鼠胰岛MIN6细胞的影响,采用CCK-8法检测细胞存活率.高糖损伤组细胞活力下降(P＜0.05),京尼平作用高糖损伤的细胞后,细胞活力增加(P＜0.05);小鼠胰岛素(insulin)检测...     

Glutathione production in copper-deficient isolated rat hepatocytes.

Dietary copper deficiency has been shown to reduce copper-dependent superoxide dismutase (SOD) activity and to increase lipid peroxidation in rats. Circulating reduced glutathione (GSH) concentrations are elevated in copper-deficient (CuD) rats, which suggests an increased GSH synthesis or decreased degradation, perhaps as an adaptation to the oxidative stress of copper deficiency. GSH synthesis was examined in isolated hepatocytes from CuD rats. Isolated hepatocytes were prepared by collagenase perfusion and incubated in Krebs-Henseleit bicarbonate buffer, pH 7.4, 10 mM glucose, 2.5 mM Ca2+ in the presence and absence of 1.0 mM buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis. Cell viability was assessed by trypan blue exclusion. GSH and oxidized glutathione (GSSG) were measured by the glutathione reductase recycling assay. Copper deficiency depressed hepatocyte Cu by greater than 90% and increased intracellular GSH by 41-117% over the 3-h incubation, with a two- to threefold increase in the rate of intracellular GSH synthesis. Intracellular GSSG values were minimally influenced by CuD, with a constant mol% GSSG. Extracellular total glutathione (GSH + 2GSSG) synthesis was increased by approximately 33%. Both intracellular GSH and extracellular total glutathione synthesis were inhibited by BSO. The pattern of food consumption in CuD rats, meal fed versus ad libitum fed, had no effect on glutathione synthesis. The results indicate an increased hepatic GSH synthesis as a response to dietary copper deficiency and suggest an interrelationship between the essential nutrients involved in oxyradical metabolism.