The interaction of Rubia cordifolia with iron redox status: a mechanistic aspect in free radical reactions.

The present publication investigates the antioxidant property and mechanistic aspect of alcoholic extract of R. cordifolia. The extract of R. cordifolia has shown significant inhibitory effect on FeSO4 induced lipid peroxidation. Study with iron redox status showed that R. cordifolia extract reduced or oxidixed; Fe3+ or Fe2+ respectively, in a dose dependent manner. Results with superoxide anion (O2-.) and hydroxyl radical (OH.), showed no radical scavenging activity. The alcoholic extract significantly maintains the reduced glutathione content both in time and dose dependent manner. It also reduced the rate of depletion of reduced glutathione (GSH) level in presence of ferrous sulphate (FeSO4) and cumene hydroperoxide (CHP). On the basis of these observations, it can be concluded that the antioxidant property of R. cordifolia is due to a direct interaction with iron.     

Reduced neuronal nitric oxide synthase is involved in ischemia-induced hippocampal neurogenesis by up-regulating inducible nitric oxide synthase expression

Nitric oxide (NO), a free radical with signaling functions in the CNS, is implicated in some developmental processes, including neuronal survival, precursor proliferation, and differentiation. However, neuronal nitric oxide synthase (nNOS) -derived NO and inducible nitric oxide synthase (iNOS) -derived NO play opposite role in regulating neurogenesis in the dentate gyrus after cerebral ischemia. In this study, we show that focal cerebral ischemia reduced nNOS expression and enzymatic activity in the hippocampus. Ischemia-induced cell proliferation in the dentate gyrus was augmented in the null mutant mice lacking nNOS gene (nNOS−/−) and in the rats receiving 7-nitroindazole, a selective nNOS inhibitor, after stroke. Inhibition of nNOS ameliorated ischemic injury, up-regulated iNOS expression, and enzymatic activity in the ischemic hippocampus. Inhibition of nNOS increased and iNOS inhibitor decreased cAMP response element-binding protein phosphorylation in the ipsilateral hippocampus in the late stage of stroke. Moreover, the effects of 7-nitroindazole on neurogenesis after ischemia disappeared in the null mutant mice lacking iNOS gene (iNOS−/−). These results suggest that reduced nNOS is involved in ischemia-induced hippocampal neurogenesis by up-regulating iNOS expression and cAMP response element-binding protein phosphorylation.     

Down-Regulation of Neuronal Nitric Oxide Synthase by Nitric Oxide After Oxygen-Glucose Deprivation in Rat Forebrain Slices

Abstract : The precise role that nitric oxide (NO) plays in the mechanisms of ischemic brain damage remains to be established. The expression of the inducible isoform (iNOS) of NO synthase (NOS) has been demonstrated not only in blood and glial cells using in vivo models of brain ischemia-reperfusion but also in neurons in rat forebrain slices exposed to oxygen-glucose deprivation (OGD). We have used this experimental model to study the effect of OGD on the neuronal isoform of NOS (nNOS) and iNOS. In OGD-exposed rat forebrain slices, a decrease in the calcium-dependent NOS activity was found 180 min after the OGD period, which was parallel to the increase during this period in calcium-independent NOS activity. Both dexamethasone and cycloheximide, which completely inhibited the induction of the calcium-independent NOS activity, caused a 40-70% recovery in calcium-dependent NOS activity when compared with slices collected immediately after OGD. The NO scavenger oxyhemoglobin produced complete recovery of calcium-dependent NOS activity, suggesting that NO formed after OGD is responsible for this down-regulation. Consistently, exposure to the NO donor ( Z )-1-[(2-aminoethyl)- N -(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NONOate) for 180 min caused a decrease in the calcium-dependent NOS activity present in control rat forebrain slices. Furthermore, OGD and DETA-NONOate caused a decrease in level of both nNOS mRNA and protein. In summary, our results indicate that iNOS expression down-regulates nNOS activity in rat brain slices exposed to OGD. These studies suggest important and complex interactions between NOS isoforms, the elucidation of which may provide further insights into the physiological and pathophysiological events that occur during and after cerebral ischemia.     

iNOS expression inhibits hypoxia-inducible factor-1 activity

Hypoxia-inducible factor-1 (HIF-1) activates genes important in vascular function such as vascular endothelial growth factor (VEGF), erythropoietin (EPO), and inducible nitric oxide synthase (iNOS). iNOS catalyzes the synthesis of nitric oxide (NO), a free radical gas that mediates a number of cellular processes, including regulation of gene expression, vasodilatation, and neurotransmission. Here we demonstrate that iNOS expression inhibits HIF-1 activity under hypoxia in C6 glioma cells transfected with an iNOS gene and a VEGF promoter-driven luciferase gene. HIF-1 induction of VEGF-luciferase activity in C6 cell is also inhibited by sodium nitroprusside (SNP). Furthermore, pretreatment of C6 cells with N-acetyl-l-cysteine (NAC), an antioxidant, nullified the inhibitory effect of iNOS on HIF-1 binding. These results demonstrate that NO generated by iNOS expression inhibits HIF-1 activity in hypoxic C6 cells and suggest a negative feedback loop in the HIF-1 --> iNOS cascade.     

Neuroprotective Effects of Cactus Polysaccharide on Oxygen and Glucose Deprivation Induced Damage in Rat Brain Slices

1. The neuroprotective effect of cactus polysaccharide (CP) on oxygen and glucose deprivation (OGD) and reoxygenation (REO)-induced damage in the cortical and hippocampal slices of rat brain was investigated. 2. Cell viability was evaluated by using the 2, 3, 5-triphenyl tetrazolium chloride (TTC) method. The fluorescence of propidium iodide (PI) staining was used for quantification of cellular survival, and lactate dehydrogenase (LDH) activity in incubation medium was assessed by LDH assay to evaluate the degree of injury. 3. The OGD ischemic condition significantly decreased cellular viability and increased LDH release in the incubation medium. CP (0.2 mg/l∼2 mg/l) protected brain slices from OGD injury in a dosage dependent manner as demonstrated by increased A 490 value of TTC, decreased PI intensity and LDH release. At the above concentration, CP also prevented the increase of nitric oxide (NO) content and inducible nitric oxide synthase (iNOS) activity induced by OGD. 4. CP can protect the brain slices (cortical and hippocampus) against injury induced by OGD. Its neuroprotective effect may be partly mediated by the NO/iNOS system induced by OGD insult. Xianju Huang and Qin Li have contributed equally to this article.     

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.     

Huperzine A protects C6 rat glioma cells against oxygen-glucose deprivation-induced injury

The protective effects of huperzine A against oxygen-glucose deprivation (OGD)-induced injury in C6 cells were investigated. OGD for 6h and reoxygenation for 6h enhanced phosphorylation and degradation of IkappaBalpha and nuclear translocation of nuclear factor-kappa B (NF-kappaB), triggered overexpression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and nitric oxide (NO) in C6 cells. Along with inhibiting acetylcholinesterase activity, treatment with 1 microM huperzine A inhibited activation of NF-kappaB, attenuated iNOS, COX-2 and NO overexpression, and promoted survival in C6 cells subjected to OGD insult. The protective effects of huperzine A were partly mediated by "cholinergic anti-inflammatory pathway" through alpha7 nicotinic acetylcholine receptor.     

姜黄素对脂多糖激活的小胶质细胞iNOS 表达的抑制及抗氧化作用

用姜黄素预处理小胶质细胞株BV,1 h后加用脂多糖(200 ng/ml)进行刺激,通过MTT检测细胞活性;硝酸还原酶法检测细胞上清液中一氧化氮(NO)的含量;Western 印迹、免疫细胞化学染色检测细胞活化后形态及诱导型一氧化氮合酶(iNOS)蛋白的表达;瞬时转染和荧光素酶报告基因鉴定iNOS和NF-κB基因表达活性;SOD和GSH-Px检测姜黄素的抗氧化能力.结果证明,脂多糖可促使小胶质细胞活化,使iNOS和NF-κB基因表达活性显著增强;iNOS蛋白表达明显上调,NO释放增多;细胞内SOD和GSH-Px活性明显下降.而姜黄素(10 μmol/L)可以显著抑制活化后小胶质细胞NO的产生、iNOS蛋白的表达及iNOS-Luc、NF-κB-Luc的表达活性,其机制可能通过NF-κB的信号转导途径抑制iNOS的表达.姜黄素可通过提高细胞内SOD和GSH-Px的活性发挥抗氧化作用.     

Leaf extracts of Carlowrightia cordifolia induce macrophage nitric oxide production

Carlowrightia cordifolia (Acanthaceae) is a medicinal plant used in northeastern Mexico as a traditional remedy against inflammation. As tissue release of nitric oxide (NO) has been correlated with both inflammatory and anti-inflammatory processes, the aim of this study was to determine the effect of C. cordifolia leaf extracts on macrophage NO production. Lipopolysaccharide (LPS)-stimulated and non-LPS-stimulated mouse peritoneal macrophages were incubated with aqueous, ethanol, methanol and hexane extracts of C. cordifolia leaves. All extracts inhibited NO release from LPS-stimulated macrophages, with methanol and hexane extracts showing the greatest inhibition. On the other hand, macrophage cultures treated with extracts without LPS-stimulation produced high releases of NO. These unexpected results suggest two different ways by which leaf extracts may act, depending on cell status. On the other hand, data on NO activity in relation to inflammatory/anti-inflammatory auto-regulatory feedback and high concentrations of NO release by non-stimulated macrophages agreed with the hypothesis that NO may have an inhibitory effect in vascular inflammation.     

Ischemia and reperfusion of liver induces eNOS and iNOS expression: effects of a NO donor and NOS inhibitor

The aim of this study was to investigate the role of nitric oxide (NO) in hepatic ischemia-reperfusion (I/R) injury in rats. Immunohistochemistry was used to examine the protein expression of endothelial and inducible nitric oxide synthases (eNOS, iNOS) and nitrotyrosine after I/R challenges to the liver, and blood levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactic dehydrogenase (LDH), hydroxyl radical and NO were measured before ischemia and after reperfusion. Ischemia was induced by occlusion of the common hepatic artery and portal vein for 40 min, followed by reperfusion for 90 min. Reperfusion of the liver induced a significant increase in the blood concentrations of AST, ALT, LDH (n = 8; P < 0.001), hydroxyl radical (n = 8; P < 0.001) and NO (n = 8; P < 0.01). The eNOS, iNOS, nitrotyrosine, SOD1 and SOD2 protein expression was also found to increase significantly after reperfusion (n = 3). Administration of the NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) (n = 8) had a protective effect on the I/R-related injury, but the NO donor L-arginine (L-Arg) (n = 8) potentiated the damage caused by I/R. These results suggest that reperfusion of the liver induces expression of NOS, which is related to the elevation of blood NO. The increase in hydroxyl radical concentration was accompanied by an increase in antioxidant enzyme expression (SOD1 and SOD2), and an increase in nitrotyrosine expression was also observed, reflecting the increased production of NO and oxygen radicals. We concluded from the protective effect of L-NAME and the potentiation by L-Arg that NOS expression and increases in NO and hydroxyl radical production have deleterious effects on the response to I/R in the liver.     

The redox pathway of S-nitrosoglutathione, glutathione and nitric oxide in cell to neuron communications

Recent results demonstrated that S-nitrosoglutathione (GSNO) and nitric oxide (*NO) protect brain dopamine neurons from hydroxyl radical (*OH)-induced oxidative stress in vivo because they are potent antioxidants. GSNO and *NO terminate oxidant stress in the brain by (i) inhibiting iron-stimulated hydroxyl radicals formation or the Fenton reaction, (ii) terminating lipid peroxidation, (iii) augmenting the antioxidative potency of glutathione (GSH), (iv) mediating neuroprotective action of brain-derived neurotrophin (BDNF), and (v) inhibiting cysteinyl proteases. In fact, GSNO--S-nitrosylated GSH--is approximately 100 times more potent than the classical antioxidant GSH. In addition, S-nitrosylation of cysteine residues by GSNO inactivates caspase-3 and HIV-1 protease, and prevents apoptosis and neurotoxicity. GSNO-induced antiplatelet aggregation is also mediated by S-nitrosylation of clotting factor XIII. Thus the elucidation of chemical reactions involved in this GSNO pathway (GSH GS* + *NO-->[GSNO]-->GSSG + *NO-->GSH) is necessary for understanding the biology of *NO, especially its beneficial antioxidative and neuroprotective effects in the CNS. GSNO is most likely generated in the endothelial and astroglial cells during oxidative stress because these cells contain mM GSH and nitric oxide synthase. Furthermore, the transfer of GSH and *NO to neurons via this GSNO pathway may facilitate cell to neuron communications, including not only the activation of guanylyl cyclase, but also the nitrosylation of iron complexes, iron containing enzymes, and cysteinyl proteases. GSNO annihilates free radicals and promotes neuroprotection via its c-GMP-independent nitrosylation actions. This putative pathway of GSNO/GSH/*NO may provide new molecular insights for the redox cycling of GSH and GSSG in the CNS.     

Differential biological effects of products of nitric oxide (NO) synthase: it is not enough to say NO

The radical gas nitric oxide (NO) is implicated in an enormous number of biological function both in physiological and pathological conditions. Often it is not clear if it plays a deleterious or beneficial role. Here briefly, are analyzed some of the reasons of this multitude of effects. Emphasis is given to factors influencing NO formation and to the type and quantity of radicals formed by nitric oxide synthase. In particular, a comparison between the biological effects of nitroxyl anion (HNO/NO(-)) and nitric oxide NO(.) is considered. These redox siblings often exhibit orthogonal behavior in physiological and pathological conditions. In the light of the multitude of effects of NO, the role of this gas, their siblings and their derivatives in cardiac ischemic preconditioning scenario is more extensively analyzed.     

Neuroprotective Potential of Fasudil Mesylate in Brain Ischemia-Reperfusion Injury of Rats

We previously reported that inhibition of Rho-kinase (ROCK) by hydroxyl fasudil improves cognitive deficit and neuronal damage in rats with chronic cerebral ischemia (Huang et al., Cell Mol Neurobiol 28:757–768, 2008). In this study, fasudil mesylate (FM) was investigated for its neuroprotective potential in rats with ischemia following middle cerebral artery occlusion (MCAO) and reperfusion. The effect of fasudil mesylate was also studied in rat brain cortical and hippocampal slices treated with oxygen-glucose deprivation (OGD) injury. Gross anatomy showed that cerebral infarct size, measured with 2,3,5-triphenyltetrazolium chloride (TTC) staining, was significantly smaller in the FM-treated than in the non-FM-treated ischemic rats. In the brain regions vulnerable to ischemia of ischemic rats, fasudil mesylate was also found to significantly restore the enzyme protein expression level of endothelial nitric oxide synthase (eNOS), which was decreased in ischemia. However, it remarkably reduced the protein synthesis of inducible nitric oxide synthase (iNOS) that was induced by ischemia and reperfusion. In rat brain slices treated with OGD injury, fasudil mesylate increased the neuronal cell viability by 40% for cortex and by 61% for hippocampus, respectively. Finally, in the presence of OGD and fasudil mesylate, superoxide dismutase (SOD) activity was increased by 50% for cortex and by 58% for hippocampus, compared to OGD only group. In conclusion, our in vivo study showed that fasudil mesylate not only decreased neurological deficit but also reduced cerebral infarct size, possibly and at least partially by augmenting eNOS protein expression and inhibiting iNOS protein expression after ischemia-reperfusion. Xian-Ju Huang contributed equally to this article.     

Inhibition of nitric oxide synthase inhibitors and lipopolysaccharide induced inducible NOS and cyclooxygenase-2 gene expressions by rutin, quercetin, and quercetin pentaacetate in RAW 264.7 macrophages

Several natural flavonoids have been demonstrated to perform some beneficial biological activities, however, higher-effective concentrations and poor-absorptive efficacy in body of flavonoids blocked their practical applications. In the present study, we provided evidences to demonstrate that flavonoids rutin, quercetin, and its acetylated product quercetin pentaacetate were able to be used with nitric oxide synthase (NOS) inhibitors (N-nitro-L-arginine (NLA) or N-nitro-L-arginine methyl ester (L-NAME)) in treatment of lipopolysaccharide (LPS) induced nitric oxide (NO) and prostaglandin E2 (PGE2) productions, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene expressions in a mouse macrophage cell line (RAW 264.7). The results showed that rutin, quercetin, and quercetin pentaacetate-inhibited LPS-induced NO production in a concentration-dependent manner without obvious cytotoxic effect on cells by MTT assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide as an indicator. Decrease of NO production by flavonoids was consistent with the inhibition on LPS-induced iNOS gene expression by western blotting. However, these compounds were unable to block iNOS enzyme activity by direct and indirect measurement on iNOS enzyme activity. Quercetin pentaacetate showed the obvious inhibition on LPS-induced PGE2 production and COX-2 gene expression and the inhibition was not result of suppression on COX-2 enzyme activity. Previous study demonstrated that decrease of NO production by L-arginine analogs effectively stimulated LPS-induced iNOS gene expression, and proposed that stimulatory effects on iNOS protein by NOS inhibitors might be harmful in treating sepsis. In this study, NLA or L-NAME treatment stimulated significantly on LPS-induced iNOS (but not COX-2) protein in RAW 264.7 cells which was inhibited by these three compounds. Quercetin pentaacetate, but not quercetin and rutin, showed the strong inhibitory activity on PGE2 production and COX-2 protein expression in NLA/LPS or L-NAME/LPS co-treated RAW 264.7 cells. These results indicated that combinatorial treatment of L-arginine analogs and flavonoid derivates, such as quercetin pentaacetate, effectively inhibited LPS-induced NO and PGE2 productions, at the same time, inhibited enhanced expressions of iNOS and COX-2 genes.     

Estradiol abolishes reduction in cell death by the opioid agonist Met5-enkephalin after oxygen glucose deprivation in isolated cardiomyocytes from both sexes

There is evidence for differences in the response to the treatment of cardiovascular disease in men and women. In addition, there are conflicting results regarding the effectiveness of pharmacologically induced protection or ischemic preconditioning in females. We investigated whether the ability of Met(5)-enkephalin (ME) to reduce cell death after oxygen-glucose deprivation (OGD) is influenced by the presence of 17beta-estradiol (E(2)) in a nitric oxide (NO)- and estrogen receptor-dependent manner. On postnatal day 7 to 8, murine cardiomyocytes from wild-type or inducible NO synthase (iNOS) knockout mice were separated by sex, isolated by collagenase digestion, cultured for 24 h, and subjected to 90 min OGD and 180 min reoxygenation at 37 degrees C (n = 4 to 5 replicates). Cell cultures were incubated in E(2) for 15 min or 24 h before OGD. ME was used to increase cell survival. Cell death was assessed by propidium iodide. More than 300 cells were examined for each treatment. Data are presented as means +/- SE. As a result, in both sexes, ME-induced cell survival was lost in the presence of E(2), and the ability of ME to improve cell survival was restored after treatment with the estrogen receptor antagonist ICI-182780. Furthermore, iNOS was necessary for ME to increase cell survival following OGD in vitro. We conclude that ME-induced reduction in cell death is abolished by E(2) in a sex-independent manner via activation of estrogen receptors, and this interaction is dependent on iNOS.