Puerarin protects rat pancreatic islets from damage by hydrogen peroxide

Periodate-oxidized 2′,3′-dialdehyde ATP (oxidized ATP) has been used extensively as a selective antagonist at P2X₇ receptors, although P2X₇-independent actions on pro-inflammatory cytokine release have also been reported. Because P2X₇ receptors in astrocytes have been suggested as potential targets of anti-inflammatory drug therapy, we examined the effect of oxidized ATP on β-actin expression and superoxide production of RBA-2 type-2 astrocytes known to possess P2X₇ receptors. Oxidized ATP per se decreased β-actin expression time and dose dependently. Treatment with oxidized ATP for 8 h caused an approximately 50% decrease in β-actin expression whereas other P2 receptor antagonists, brilliant blue G (BBG), suramin and pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), were not effective. In addition, oxidized ATP per se decreased the intracellular superoxide concentration, whereas ATP and the P2X₇ receptor-selective agonist 3′-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate (BzATP) stimulated intracellular superoxide production, an effect inhibited by oxidized ATP. In addition, oxidized ATP neither affected cellular viability nor affected interleukin-1β, converting enzyme (ICE)-like protease activity in these astrocytes. To further elucidate the mechanism, the effects of oxidized ATP on intracellular superoxide concentration and β-actin expression were examined in a P2X₇ receptor-negative astrocyte cell line, IA-1g1. Oxidized ATP-induced a time-dependent decrease in intracellular superoxide concentration whereas oxidized ATP had no effect on β-actin expression. Nevertheless, oxidized ATP altered f-actin cytoskeleton arrangement in IA-1g1 astrocytes. Taken together, these results indicate that oxidized ATP per se caused a cell specific decrease in β-actin expression in RBA-2 type-2 astrocytes. In addition, oxidized ATP decreased intracellular superoxide concentrations and altered f-actin cytoskeleton arrangement in both P2X₇ receptor-positive and -negative astrocytes. Thus, we conclude from these results that the effects of oxidized ATP on actin and superoxide are mediated through mechanisms that are at least in part, independent of P2X₇ receptors.     

Iron chlorin e6 scavenges hydroxyl radical and protects human endothelial cells against hydrogen peroxide toxicity

Iron chlorin e6 (FeCe6) has recently been proposed to be potentially antimutagenic and antioxidative. However, the antioxidant property of FeCe6 has not been elucidated in detail. In this study, we investigated the ability of FeCe6 to scavenge hydroxyl radical and to protect biomolecules and mammalian cells from oxidative stress-mediated damage. In electron spin resonance (ESR) experiments, FeCe6 showed excellent hydroxyl radical scavenging activity, whereas its iron-deficient molecule, chlorin e6 (Ce6) showed little effect. FeCe6 also significantly reduced hydroxyl radical-induced thiobarbituric acid reactive substance (TBARS) formation and benzoate hydroxylation in a dose-dependent manner. The rate constant for reaction between FeCe6 and hydroxyl radical was measured as 8.5 x 10(10) M(-1) s(-1) by deoxyribose degradation method, and this value was much higher than that of most hydroxyl radical scavengers. Superoxide dismutase (SOD) activity of FeCe6 was also confirmed by ESR study and cytochrome c reduction assay, but its in vitro activity appeared to be less efficient in comparison with other well-known SOD mimics. In addition, FeCe6 appreciably diminished hydroxyl radical-induced DNA single-strand breakage and protein degradation in Fe-catalyzed and Cu-catalyzed Fenton systems, and it significantly protected human endothelial cells against hydrogen peroxide (H2O2) toxicity. These results suggest that FeCe6 is a novel hydroxyl radical scavenger and may be useful for preventing oxidative injury in biological systems.     

TMPDP, a tetramethylpyrazine derivative, protects vascular endothelial cells from oxidation damage by hydrogen peroxide

A novel ligustrazine derivative, tetramethylpyrazine diphenylmethyl piperazidine (TMPDP), prepared by hybridization and bioisosteric replacement of the molecular structure of TMP, was studied for its protective effects on oxidative damage of human umbilical vein endothelial cells (HUVECs) in response to hydrogen peroxide (H2O2). The antioxidative effect of TMPDP was assessed by the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) test. Cell viability was measured using a 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The activity of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and glutathione peroxidase (GSH) and the content of malondialdehyde (MDA) in cells were determined by commercial kits. The intracellular formation of reactive oxygen species (ROS) and the concentration of free intracellular calcium ([Ca2+]i) were determined using DCFH-DA assay and with fura-2/AM fluorimetry, respectively. Results showed that TMPDP had a moderate antioxidative effect against DPPH. Cell viability was decreased markedly by exposure to H2O2. Introduction of TMPDP, however, significantly increased cell viability, markedly reduced LDH release from cells and decreased lipid peroxidation in response to H2O2 treatment. These effects of TMPDP were accompanied by increased activity of the endogenous antioxidant enzymes, SOD and GSH, reduced production of ROS and reduced intracellular concentration of Ca2+. These results suggest that TMPDP protects HUVECs against oxidative damage by scavenging ROS and regulates intracellular calcium concentration. This might have important implications for the development of new agents for the effective treatment of vascular disease.     

A comparative study of plant and animal mitochondria exposed to paraquat reveals that hydrogen peroxide is not related to the observed toxicity

Rat liver mitochondria are much more susceptible to protein oxidation induced by paraquat than plant mitochondria. The unsaturated index and the peroxidizability index are higher in rat than in potato tuber. The levels of superoxide dismutase and glutathione reductase are concurrent with the different sensitivities to paraquat, with higher activities in plant mitochondria. However, glutathione peroxidase and catalase activities are higher in rat mitochondria. Paraquat (10 mM) inhibited all the enzymatic activities; excluding catalase all the other activities were inhibited to a similar degree. The differential sensitivities of plant and animal mitochondria to paraquat correlate with fatty acid composition of mitochondrial lipids and a similar correlation was also established for some antioxidant enzymes. At the mitochondrial level, H₂O₂ is not a major factor of paraquat toxicity since rat liver mitochondria which exhibit higher activities of glutathione peroxidase and catalase are however more susceptible to paraquat.     

Iron protects astrocytes from 6-hydroxydopamine toxicity

The role of iron in 6-hydroxydopamine (6-OHDA) toxicity towards astrocytes was investigated in vitro using rat primary astrocytes, rat astrocytoma cell line C6, and human astrocytoma cell line U251. The assessment of mitochondrial respiration or lactate dehydrogenase release has shown a dose-dependent decrease in the viability of astrocytes treated with 6-OHDA, which coincided with DNA fragmentation and the changes in cellular morphology. This was a consequence of the oxidative stress mediated by 6-OHDA autoxidation products hydrogen peroxide, superoxide anion, and hydroxyl radical. Both FeSO(4) and FeCl(3) markedly alleviated detrimental effects of 6-OHDA treatment, while MgSO(4) was without effect. The protective action of iron was neutralized by a membrane-permeable iron chelator o-phenanthroline, which also augmented astrocyte killing in the absence of exogenous iron. The mechanisms responsible for iron-mediated protection of astrocytes did not involve interference with either 6-OHDA autoxidation, hydrogen peroxide toxicity, or 6-OHDA-induced activation of extracellular signal-regulated kinase. Finally, the addition of iron potentiated and its chelation blocked 6-OHDA toxicity towards neuronal PC12 cells, suggesting the opposite roles for this transition metal in regulating the survival of astrocytes and dopaminergic neurons.     

Apotransferrin protects cortical neurons from hemoglobin toxicity

The protective effect of iron chelators in experimental models of intracerebral hemorrhage suggests that nonheme iron may contribute to injury to perihematomal cells. Therapy with high affinity iron chelators is limited by their toxicity, which may be due in part to sequestration of metals in an inaccessible complex. Transferrin is unique in chelating iron with very high affinity while delivering it to cells as needed via receptor-mediated endocytosis. However, its efficacy against iron-mediated neuronal injury has never been described, and was therefore evaluated in this study using an established cell culture model of hemoglobin neurotoxicity. At concentrations similar to that of CSF transferrin (50-100 micrograms/ml), both iron-saturated holotransferrin and apotransferrin were nontoxic per se. Overnight exposure to 3 μM purified human hemoglobin in serum-free culture medium resulted in death, as measured by lactate dehydrogenase release assay, of about three-quarters of neurons. Significant increases in culture iron, malondialdehyde, protein carbonyls, ferritin and heme oxygenase-1 were also observed. Holotransferrin had no effect on these parameters, but all were attenuated by 50-100 micrograms/ml apotransferrin. The effect of apotransferrin was very similar to that of deferoxamine at a concentration that provided equivalent iron binding capacity, and was not antagonized by concomitant treatment with holotransferrin. Transferrin receptor-1 expression was localized to neurons and was not altered by hemoglobin or transferrin treatment. These results suggest that apotransferrin may mitigate the neurotoxicity of hemoglobin after intracerebral hemorrhage. Increasing its concentration in perihematomal tissue may be beneficial.     

Aripiprazole protects cortical neurons from glutamate toxicity

Neurodegeneration is thought to be a component of schizophrenia pathology, and some antipsychotics appear to slow degenerative changes in patients. Aripiprazole, the first partial dopamine D(2) receptor agonist approved for the treatment of schizophrenia, is suggested to be neuroprotective based on non-clinical studies using transformed cell lines and in vivo stress and lesion paradigms. However, aripiprazole-induced neuroprotection has not been studied in a neuronal glutamate toxicity assay, which may model aspects of neurodegeneration occurring in schizophrenia. This study examined whether therapeutically relevant concentrations of aripiprazole protect rat embryonic cortical neurons from glutamate toxicity in biochemical and high-content imaging assays. Aripiprazole inhibited glutamate-induced neurotoxicity by 40% in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay, in contrast to risperidone and olanzapine, which had little neuroprotective activity. This neuroprotective effect of aripiprazole was not mediated by the activation of serotonin 5-HT(1A) or dopamine D(2) receptors, Akt or glycogen-synthase kinase-3β signaling (GSK-3β), or through the inhibition of poly-ADP ribose polymerase (PARP). Further experiments are required to determine the biochemical nature of aripiprazole-induced neuroprotection and whether any such activity might have clinical relevance.     

Mercurial-induced hydrogen peroxide generation in mouse brain mitochondria: protective effects of quercetin

Plants of the genus Polygala have been shown to possess protective effects against neuronal death and cognitive impairments in neurodegenerative disorders related to excitotoxicity. Moreover, previous reports from our group have shown the neuroprotective effects of the plant Polygala paniculata against methylmercury (MeHg)-induced neurotoxicity. In this work, we have examined the potential protective effects of three compounds (7-prenyloxy-6-methoxycoumarin, quercetin, and 1,5-dihidroxi-2,3-dimethoxy xanthone) from Polygala species against MeHg- and mercuric chloride (HgCl2)-induced disruption of mitochondrial function under in vitro conditions using mitochondrial-enriched fractions from mouse brain. MeHg and HgCl2 (10-100 microM) significantly decreased mitochondrial viability; this phenomenon was positively correlated to mercurial-induced glutathione oxidation. Among the isolated compounds, only quercetin (100-300 microM) prevented mercurial-induced disruption of mitochondrial viability. Moreover, quercetin, which did not display any chelating effect on MeHg or HgCl2, prevented mercurial-induced glutathione oxidation. The present results suggest that the protective effects of quercetin against mercurial-induced mitochondrial dysfunction is related to the removal of oxidant species generated in the presence of either MeHg or HgCl2. Reinforcing this hypothesis, MeHg and HgCl2 increased the production of hydrogen peroxide in the brain mitochondria, as well as the levels of malondialdehyde. These oxidative phenomena were prevented by co-incubation with quercetin or catalase. These results are the first to show the involvement of hydrogen peroxide as a crucial molecule related to the toxic effects of both organic and inorganic mercurials in brain mitochondria. In addition, the study is the first to show the protective effect of quercetin against mercurial-induced toxicity, pointing to its capability to counteract mercurial-dependent hydrogen peroxide generation as a potential molecular mechanism of protection. Taken together, these data render quercetin a promising molecule for pharmacological studies with respects to mercurials' poisoning.     

Carnitine protects mitochondria and removes toxic acyls from xenobiotics

The carnitine system is altered by several xenobiotics (drugs and chemicals). These alterations are responsible for most toxic effects, which can be reverted or minimized by L-carnitine administration. Formation of nonmetabolizable acyl coenzyme A (CoA) is a typical step in the biotransformation of pivaloyl antibiotics, valproate and ifosfamide. The elevated levels of acylcarnitine occurring in human urine due to impaired metabolism of specific acyl CoA support the role of L-carnitine as an acceptor of specific, nonmetabolizable acyl CoA. The consequence of this process is a secondary carnitine deficiency. The formation of stable complexes with an essential component of mitochondrial membrane, cardiolipin, and the inhibition of myocardial specific isoform of carnitine-palmitoyl transferase are presumably the basis of adriamycin cardiotoxicity. L-carnitine interacts with cardiolipin, modifying membrane permeability and protecting the functions of the mitochondria. This mechanism can be proposed to explain the protective effects of L-carnitine against adriamycin cardiotoxicity, ammonium acetate and zidovudine-induced mitochondrial ultrastructural and functional alterations. Cisplatin, cephalosporin and carbapenem antibiotics inhibit carnitine reabsorption in renal tubules and cause proximal tubular damage. The study of peroxisomal producing agents belonging to largely different chemical classes showed that these agents caused carnitine system perturbations which may have the potential to be highly relevant biomarkers of exposure to the nongenotoxic peroxisomal proliferating agent class of hepatic tumorigens.     

Effect of ursodeoxycholic acid on hydrogen peroxide induced lipid peroxidation in sheep liver mitochondria

The effect of various concentrations of ursodeoxycholic acid (UDCA), a potent hepatoprotective agent on hydrogen peroxide-induced mitochondrial swelling was evaluated in vitro to find out the mechanism of action of the drug. Aliquots of sheep liver mitochondria were pre-incubated with various concentrations of UDCA [0-600 micrograms] and swelling was induced by hydrogen peroxide [1 mM]. Swelling was assessed at various time intervals and lipid peroxide, reduced glutathione status were also evaluated simultaneously. UDCA minimized hydrogen peroxide-induced swelling in a dose-dependent manner. Time-dependent elevation in the level of lipid peroxides was noted in mitochondria treated with hydrogen peroxide and this elevation was minimized in UDCA pre-treatment. UDCA also maintains the reduced glutathione level in mitochondria. UDCA acts against the oxidative stress imposed in liver mitochondria. It reduces lipid peroxidation-induced abnormalities such as swelling and thiol group depletion and the anti lipid peroxidative efficacy of the drug may be related to its hydrophilic nature which might protect the hydrophobic regions of the mitochondrial membranes which are prone for free radical-mediated reactions.     

Tanshinone IIA protects rat primary hepatocytes against carbon tetrachloride toxicity via inhibiting mitochondria permeability transition

Tanshinone IIA (Tan IIA), one of the key components of Salvia milthorrhiza Bunge (Lamiaceae), is used to treat liver disease. The present study was carried out to investigate the possible mechanisms involved in the hepatoprotective effects of Tan IIA on carbon tetrachloride (CCl₄)-induced hepatocyte toxicity. In cultures treated with 1 or 2 μM CCl₄, Tan IIA (10–75 μM) significantly increased hepatocyte survival rates. However, only at a concentration of 75 μM could Tan IIA partially reverse the CCl₄ (3 μM)-induced decrease of survival rate (34?±?3% vs. 18?±?3%, n?=?8, p?<?0.01). In isolated mitochondria energized with succinate, Tan IIA could inhibit the large swelling effect induced by CCl₄ (1 and 2 μM). Base on these results, Tan IIA could protect rat primary cultured hepatocytes from CCl₄-induced toxicity partially by the inhibitory effect on the opening of mitochondrial permeability transition (MPT).     

Caffeic acid protects hydrogen peroxide induced cell damage in WI-38 human lung fibroblast cells

Cytoprotective effect of caffeic acid (3,4-dihydroxy cinnamic acid) on human lung fibroblast (WI-38) cells against hydrogen peroxide induced damage was investigated. Caffeic acid was found to scavenge intracellular reactive oxygen species, and 1,1-diphenyl-2-picrylhydrazyl radical, and thus prevented lipid peroxidation. The caffeic acid protected cell damage of WI-38 cells exposed to hydrogen peroxide (H(2)O(2)), via the activation of extracellular signal regulated kinase protein. Caffeic acid increased the activity of catalase and its protein expression. Hence, from the present study, it is suggestive that caffeic acid protects WI-38 cells against H2O2 damage by enhancing the cellular antioxidant activity.     

过氧化氢对大鼠气管上皮细胞氧化性损伤研究

应用无血清培养方法，建立大鼠气管上皮细胞体外试验模型，研究过氧化氢对大鼠气管上皮细胞的氧化性损伤作用。试验结果表明，大鼠气管上皮细胞与５ｍｍｏｌ过氧化氢共同培养４小时，可引起细胞膜脂质过氧化，导致细胞膜通透性增加，从而对细胞产生损伤作用。无血清培养可排除血清中一些抗氧化物对试验结果的影响。     

Melatonin protects against clomiphene citrate-induced generation of hydrogen peroxide and morphological apoptotic changes in rat eggs

The present study was aimed to determine whether clomiphene citrate-induces generation of hydrogen peroxide in ovary, if so, whether melatonin could scavenge hydrogen peroxide and protect against clomiphene citrate-induced morphological apoptotic changes in rat eggs. For this purpose, forty five sexually immature female rats were given single intramuscular injection of 10 IU pregnant mare's serum gonadotropin for 48 h followed by single injections of 10 IU human chorionic gonadotropin and clomiphene citrate (10 mg/kg bw) with or without melatonin (20 mg/kg bw) for 16 h. The histology of ovary, ovulation rate, hydrogen peroxide concentration and catalase activity in ovary and morphological changes in ovulated eggs were analyzed. Co-administration of clomiphene citrate along with human chorionic gonadotropin significantly increased hydrogen peroxide concentration and inhibited catalase activity in ovary, inhibited ovulation rate and induced egg apoptosis. Supplementation of melatonin reduced hydrogen peroxide concentration and increased catalase activity in the ovary, delayed meiotic cell cycle progression in follicular oocytes as well as in ovulated eggs since extrusion of first polar body was still in progress even after ovulation and protected against clomiphene citrate-induced egg apoptosis. These results clearly suggest that the melatonin reduces oxidative stress by scavenging hydrogen peroxide produced in the ovary after clomiphene citrate treatment, slows down meiotic cell cycle progression in eggs and protects against clomiphene citrate-induced apoptosis in rat eggs.     

Butein protects human dental pulp cells from hydrogen peroxide-induced oxidative toxicity via Nrf2 pathway-dependent heme oxygenase-1 expressions

Rhus verniciflua Stokes is a plant that is native to East Asian countries, such as Korea, China, and Japan. Butein, a plant polyphenol, is one of the major active components of R. verniciflua. Reactive oxygen species (ROS), produced via dental adhesive bleaching agents and pulpal disease, can cause oxidative stress. Here, we found that butein possesses cytoprotective effects on hydrogen peroxide (H₂O₂)-induced dental cell death. H₂O₂ is a representative ROS and causes cell death through necrosis in human dental pulp (HDP) cells. H₂O₂-induced cytotoxicity and production of ROS were blocked in the presence of butein, and these effects were dose dependent. Butein also increased heme oxygenase-1 (HO-1) protein expression and HO activity. In addition, butein-dependent HO-1 expression was required for the inhibition of H₂O₂-induced cell death and ROS generation. Furthermore, butein treatment caused nuclear accumulation of nuclear factor-E2-related factor 2 (Nrf2) and increased the promoter activity of antioxidant response elements (AREs). Treatment of HDP cells with a c-Jun NH2-terminal kinase (JNK) inhibitor also reduced butein-induced HO-1 expression, and butein treatment led to increased JNK phosphorylation. These results indicate that butein may be used to prevent functional dental cell death and thus may be useful as a pulpal disease agent.     

羊栖菜硫酸多糖通过PI3K/AKT通路抑制H2O2诱导的MRC-5细胞氧化损伤

目的 为阐明羊栖菜硫酸多糖(SFPS Ⅲ)对过氧化氢(H2O2)诱导的正常人胚肺细胞(MRC-5)氧化应激损伤的保护作用及潜在分子机制。方法 试验通过噻唑蓝(MTT)法检测MRC-5细胞暴露于H2O2或SFPS Ⅲ后的活力,Hoechst染色法检测细胞凋亡形态,流式细胞仪测定细胞凋亡率,酶联免疫吸附试验测定超氧化物歧化酶(SOD)和谷胱甘肽过氧化酶(GSH-Px)活性、一氧化氮(NO)和丙二醛(MDA)含量。通过免疫印迹Western blot检测相关蛋白水平。结果 H2O2处理使MRC-5细胞活力显著降低(P < 0.05),而SFPS Ⅲ(200～600 μg.mL-1)干预可明显增强MRC-5细胞活力。酶联免疫法结果表明,SFPS Ⅲ干预抑制H2O2诱导的细胞内NO和MDA的过度分泌,并增加了H2O2诱导的细胞内SOD和GSH-Px的活性。免疫印迹结果表明,120 μmol.L-1 H2O2处理的细胞中p-AKT和p-mTOR相对蛋白表达水平下调,而p-AKT和p-mTOR相对蛋白表达水平在SFPS Ⅲ处理的MRC-5细胞中被逆转。结论 SFPS Ⅲ对H2O2诱导的MRC-5细胞氧化应激损伤具有保护作用,其机制可能是激活PI3K/AKT/mTOR通路发挥其抗凋亡作用。研究结果为羊栖菜高附加值产品开发及天然、安全抗氧化功能因子的筛选提供了一定的理论依据。     

Effect of glutathione depletion on sites and topology of superoxide and hydrogen peroxide production in mitochondria

In this work, the topology of mitochondrial O2(-)(radical) and H2O2 generation and their interplay with matrix GSH in isolated heart mitochondria were examined. We observed that complex I releases O2(-)(radical) into the matrix (where it is converted to H2O2 by Mn-SOD) but not into the intermembrane space. No free radical generation was observed from complex II, but succinate treatment caused H2O2 generation from the matrix through a reverse electron flow to complex I. Complex III was found to release O2(-)(radical) into the matrix and into the intermembrane space. Antimycin, which increases steady-state levels of UQO>- (ubisemiquinone at the Qo site) in complex III, enhanced both H2O2 generation from the matrix and O2(-)(radical) production from the intermembrane space. On the other hand, myxothiazol, which inhibits UQO>- formation, completely inhibited antimycin induced O2(-)(radical) toward the intermembrane space and inhibited H2O2 generation from the matrix by 70%. However, myxothiazol alone enhanced H2O2 production from complex III, suggesting that other components of complex III besides the UQO- can cause O2(-)(radical) generation toward the matrix. As expected, mitochondrial GSH was found to modulate H2O2 production from the matrix but not O2- generation from the intermembrane space. Low levels of GSH depletion (from 0-40%, depending on the rate of H2O2 production) had no effect on H2O2 diffusion from mitochondria. Once this GSH depletion threshold was reached, GSH loss corresponded to a linear increase in H2O2 production by mitochondria. The impact of 50% mitochondrial GSH depletion, as seen in certain pathological conditions in vivo, on H2O2 production by mitochondria depends on the metabolic state of mitochondria, which governs its rate of H2O2 production. The greater the rate of H2O2 generation the greater the effect 50% GSH depletion had on enhancing H2O2 production.