Hypoxia-Induced Generation of Nitric Oxide Free Radicals in Cerebral Cortex of Newborn Guinea Pigs

Previous studies have shown that brain tissue hypoxia results in increased N-methyl-D-aspartate (NMDA) receptor activation and receptor-mediated increase in intracellular calcium which may activate Ca⁺⁺-dependent nitric oxide synthase (NOS). The present study tested the hypothesis that tissue hypoxia will induce generation of nitric oxide (NO) free radicals in cerebral cortex of newborn guinea pigs. Nitric oxide free radical generation was assayed by electron spin resonance (ESR) spectroscopy. Ten newborn guinea pigs were assigned to either normoxic (FiO₂ = 21%, n = 5) or hypoxic (FiO₂ = 7%, n = 5) groups. Prior to exposure, animals were injected subcutaneously with the spin trapping agents diethyldithiocarbamate (DETC, 400 mg/kg), FeSO₄.7H₂O (40 mg/kg) and sodium citrate (200mg/kg). Pretreated animals were exposed to either 21% or 7% oxygen for 60 min. Cortical tissue was obtained, homogenized and the spin adducts extracted. The difference of spectra between 2.047 and 2.027 gauss represents production of NO free radical. In hypoxic animals, there was a difference (16.75 ± 1.70 mm/g dry brain tissue) between the spectra of NO spin adducts identifying a significant increase in NO free radical production. In the normoxic animals, however, there was no difference between the two spectra. We conclude that hypoxia results in Ca²⁺- dependent NOS mediated increase in NO free radical production in the cerebral cortex of newborn guinea pigs. Since NO free radicals produce peroxynitrite in presence of superoxide radicals that are abundant in the hypoxic tissue, we speculate that hypoxia-induced generation of NO free radical will lead to nitration of a number of cerebral proteins including the NMDA receptor, a potential mechanism of hypoxia-induced modification of the NMDA receptor resulting in neuronal injury.     

Immunochemical detection of nitric oxide and nitrogen dioxide trapping of the tyrosyl radical and the resulting nitrotyrosine in sperm whale myoglobin

We demonstrate herein that nitric oxide (*NO) and nitrogen dioxide (*NO2) both react with the tyrosyl radical formed in sperm whale myoglobin (swMb) by reaction with hydrogen peroxide. The tyrosyl radical was detected by Western blotting using a novel anti-5,5-dimethyl-1-pyrroline N-oxide (DMPO) polyclonal antiserum that specifically recognizes protein radical-derived DMPO nitrone adducts. In the presence of DMPO, hydrogen peroxide reacts with swMb to form the DMPO tyrosyl radical as is known from both electron spin resonance and immuno-spin trapping investigations. Both *NO and NO2- significantly suppressed DMPO-Mb formation under the physiological oxygen tension of 30 mm Hg. If this inhibition of DMPO trapping of the tyrosyl radical is due, at least in part, to the reaction of the tyrosyl radical with *NO and *NO2, then nitrotyrosine should be formed. In line with this expectation, swMb treated with low concentrations of *NO or NO2- formed nitrotyrosine when hydrogen peroxide was added under 30 mm Hg oxygen tension as detected by Western blotting. The amount of nitrotyrosine generated with *NO was higher than with NO2-, implying that there are two different peroxynitrite-independent nitrotyrosine formation mechanisms and that *NO is not just a source of *NO2.     

Formation of protein tyrosine ortho-semiquinone radical and nitrotyrosine from cytochrome c-derived tyrosyl radical

Oxidative alteration of mitochondrial cytochrome c (cyt c) has been linked to disease pathophysiology and is one of the causative factors for pro-apoptotic events. Hydrogen peroxide induces a short-lived cyt c-derived tyrosyl radical as detected by the electron spin resonance (ESR) spin-trapping technique. This investigation was undertaken to characterize the fate and consequences of the cyt c-derived tyrosyl radical. The direct ESR spectrum from the reaction of cyt c with H(2)O(2) revealed a single-line signal with a line width of approximately 10 G. The detected ESR signal could be prevented by pretreatment of cyt c with iodination, implying that the tyrosine residue of cyt c was involved. The ESR signal can be enhanced and stabilized by a divalent metal ion such as Zn(2+), indicating the formation of the protein tyrosine ortho-semiquinone radical (ToQ.). The production of cyt c-derived ToQ. is inhibited by the spin trap, 2-methyl-2-nitrosopropane (MNP), suggesting the participation of tyrosyl radical in the formation of the ortho-semiquinone radical. The endothelium relaxant factor nitric oxide is well known to mediate mitochondrial respiration and apoptosis. The consumption of NO by cyt c was enhanced by addition of H(2)O(2) as verified by inhibition electrochemical detection using an NO electrode. The rate of NO consumption in the system containing cyt c/NO/H(2)O(2) was decreased by the spin traps 5,5-dimethyl pyrroline N-oxide and MNP, suggesting NO trapping of the cyt c-derived tyrosyl radical. The above result was further confirmed by NO quenching of the ESR signal of the MNP adduct of cyt c tyrosyl radical. Immunoblotting analysis of cyt c after exposure to NO in the presence of H(2)O(2) revealed the formation of 3-nitrotyrosine. The addition of superoxide dismutase did not change the cyt c nitration, indicating that it is peroxynitrite-independent. The results of this study may provide useful information in understanding the interconnection among cyt c, H(2)O(2), NO, and apoptosis.     

The production of reactive oxygen species by dietary flavonols

Flavonols are a group of naturally occurring compounds which are widely distributed in nature where they are found glycosylated primarily in vegetables and fruits. A number of studies have found both anti- and prooxidant effects for many of these compounds. The most widely studied because of their ubiquitous nature have been quercetin, a B-dihydroxylated and myricetin, a B-trihydroxylated flavonol. Some of their prooxidant properties have been attributed to the fact that they can undergo autooxidation when dissolved in aqueous buffer. Studying a number of factors affecting autooxidation, we found the rate of autooxidation for both quercetin and myricetin to be highly pH dependent with no autooxidation detected for quercetin at physiologic pH. Both the addition of iron for the two flavonols and the addition of iron followed by SOD for quercetin at physiologic pH. Both the addistantially. Neither kaempferol, a monohydroxylated flavonol nor rutin, a glycosylated quercetin showed any ability to autooxidize. The results with rutin differ from what we expected based on the B-ring structural similarity to quercetin. The autooxidation of quercetin and myricetin was further studied by electron spin resonance spectroscopy (ESR). Whereas quercetin produced a characteristic DMPO-OH radical, it was not detected below a pH of 9. However, the addition of iron allowed the signal to be detected at a pH as low as 8.0. On the other hand, myricetin autooxidation yielded a semiquinone signal which upon the addition of iron, converted to a DMPO-OH signal detected at a pH of 7.5. In a microsome-NADPH system, quercetin produced an increase in oxygen utilization and with ESR, an ethanol-derived radical signal which could be completely suppressed by catalase indicating the dependence of the signal on hydrogen peroxide. These studies demonstrate that the extracellular production of active oxygen species by dietary flavonols is not likely to occur in vivo but the potential for intracellular redox cycling may have toxicologic significance.     

Nitric oxide generation in aqueous solutions of cigarette smoke and approaches to its origin.

By using the ESR spin trapping technique with the N-methyl-D-glucamine dithiocarbamate (MGD)2-Fe(II) complex, the generation of nitric oxide (NO), a gaseous free radical, was observed in NO spin trapping solution bubbled with the filtered main-stream of cigarette smoke. The ESR signal with a three-line spectrum characteristic of an NO radical, which was not observed immediately after bubbling of smoke, started rapidly increasing with time up to around 25 min after the last addition of ferrous ions Fe(II), and then slowly approached a peak value dependent on the burned cigarette mass and on the smoking speed. The production of NO was, however, much affected by air oxidation and enhanced by the addition of ascorbic acid. A certain concentration of sodium nitrite (NaNO2) solution, in which nitrite NO2- is assumed as the main origin of the NO, mimicked closely the time course of NO generation resulting from the smoke of one cigarette. The cigarette smoke that was passed through alkaline pyrogallol solution as a deoxidizer; however, it exhibited an unchanged intensity of NO signal throughout the measurement. These results strongly suggest that NO would be gradually reproduced from NO2- in the reductive aqueous solution containing excess Fe(II) through NO2, which is initially formed and is concomitantly oxidized from NO in cigarette smoke.     

Antihypertensive effect of quercetin in rats fed with a high-fat high-sucrose diet

The effects of different levels of quercetin on the blood pressure were studied in 6-week-old male Sprague-Dawley rats. The rats were fed with a control diet or a high-fat high-sucrose (HFS) diet containing 0, 0.02, 0.07, 0.2, or 0.5% quercetin for 4 weeks. The systolic blood pressure and the lipid peroxides in the plasma were both higher in the rats fed with the HFS diet without quercetin than in the rats fed with the control diet. The nitric oxide synthase (NOS) activity in the vascular tissues and nitric oxide (NO) metabolites in the plasma and urine were both lower in these rats. A distinct depression of the increase in blood pressure was found in the rats fed with the HFS diets containing quercetin. Each level of quercetin examined was effective, the 0.5% level being much more effective than other levels. Dietary quercetin decreased lipid peroxidation in the plasma of the rats fed with the HFS diets. Quercetin also suppressed the decrease in NO metabolites in the plasma and urine, and the NOS activity in the vascular tissues of these rats. These results suggest that the increased NO availability caused by the elevated NOS activity, and the antioxidative activity in these rats fed with quercetin may be sources of the antihypertensive effect of quercetin.     

Inhibition of lipoxygenase-dependent lipid peroxidation by quercetin: Mechanism of antioxidative function

Quercetin inhibited soybean lipoxygenase-1-dependent linoleic acid peroxidation. Two to three μM quercetin was required for 50% inhibition. During the inhibition, quercetin was oxidized. The oxidation was observed as an absorbance decrease at about 380 nm and an absorbance increase at about 335 nm. Inhibition of linoleic acid peroxidation by quercetin seems to be due to reduction by the reagent of the linoleic acid radical formed as an intermediate during lipoxygenation. Quercetin oxidation was suppressed by ascorbate under conditions when ascorbate did not affect lipoxygenase-dependent linoleic acid peroxidation. The results suggest that ascorbate can reduce the quercetin oxidized by the linoleic acid radical back to quercetin. Based on the results, the significance of a redox reaction between oxidized quercetin and ascorbate is discussed.     

Electron paramagnetic resonance (EPR) spin trapping of biological nitric oxide

Nitric oxide (NO) is a free radical species with multiple physiological functions. Because of low concentrations and short half-life of NO, its direct measurement in living tissues remains a difficult task. Electron paramagnetic resonance (EPR) spin trapping is probably one of the best suitable platforms for development of new methods for quantification of biological NO. The most reliable EPR-based approaches developed so far are based on the reaction of NO with various iron complexes, both intrinsic and exogenously applied. This review is focused on the current state and perspectives of EPR spin trapping for experimental and clinical NO biology.     

Early specific free radical-related cytotoxicity of gas phase cigarette smoke and its paradoxical temporary inhibition by tar: An electron paramagnetic resonance study with the spin trap DEPMPO

Electron paramagnetic resonance (EPR) spin trapping studies demonstrated aqueous tar particulate matter (TPM) and gas phase cigarette smoke (GPCS) to behave as different sources of free radicals in cigarette smoke (CS) but their cytotoxic implications have been only assessed in CS due to its relevance to the natural smoking process. Using a sensitive spin trapping detection with 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), this study compared the respective roles of CS- and GPCS-derived free radicals on smoke-induced cytotoxicity and lipid peroxidation of filtered and unfiltered, machine-smoked experimental and reference cigarettes yielding a wide range of TPM yields. In buffer bubbled with CS the DEPMPO/superoxide spin adduct was the major detected nitroxide. Use of appropriate control experiments with nitric oxide radical (NO*) or carbonyl sulfide, and a computer analysis of spin adduct diastereoisomery showed that the hydroxyl radical (HO*) adduct of DEPMPO seen in GPCS-bubbled was rather related to metal-catalyzed nucleophilic synthesis than to direct HO* trapping. Unexpectedly a protective effect of TPM on murine 3T3 fibroblasts was observed in early (<3h) free radical-, GPCS-induced cell death, and carbon filtering decreased free radical formation, toxicity and lipid peroxidation in three cell lines (including human epithelial lung cells) challenged with GPCS. These results highlight an acute, free radical-dependent, harmful mechanism specific to the GPCS phase, possibly involving NO* chemistry, whose physical or chemical control may be of great interest with the aim of reducing the toxicity of smoke.     

Antimutagenic and antioxidant/prooxidant activity of quercetin

The present study has been performed to evaluate the antimutagenic activity of quercetin, ascorbic acid and their combination against an oxidative mutagen. An effort was also made to correlate this activity to the in vitro antioxidant activity of these agents. Antimutagenicity testing was done in Ames Salmonella Assay system using Salmonella typhimurium TA102 against t-butylhydroperoxide as an oxidative mutagen. In vitro antioxidant scavenging activity was tested for DPPH free radical, superoxide anion, hydrogen peroxide and hydroxyl radical in their specific test systems. Quercetin (0.5-8 nmole/plate) and ascorbic acid (0.1-100 micromole/plate) showed significant effect. Quercetin (4 and 8 nmole/plate) when combined with ascorbic acid (500 nmole/plate) showed an increase in the antimutagenic activity. In vitro antioxidant activity of quercetin was better than ascorbic acid in all the test systems used. The study indicated that the antimutagenic activity of quercetin was not solely accountable by its antioxidant nature. However, in vitro free radical scavenging activity of quercetin correlated well with the antimutagenic activity.     

Investigation of the existence and biological role of L-arginine/nitric oxide pathway in human platelets by spin-trapping/EPR studies.

The aim of the present study was to apply spin trapping/EPR spectroscopy to investigate the existence and biological role of the L-arginine/nitric oxide pathway in human platelet aggregation. Three different spin traps were used: two nitroso, 3,5-dibromo-4-nitrosobenzenesulfonate (DBNBS) and 2-methyl-2-nitrosopropane (MNP), and a nitrone, 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The effect of spin-trap concentration on the collagen-induced human platelet aggregation was compared to the anti-aggregatory effect caused by L-arginine. The results show that the nitroso spin traps (DBNBS and MNP) are more effective than L-arginine in preventing platelet aggregation. DMPO has virtually no effect on the collagen-induced aggregation except at a high concentration (300 mM). Furthermore, activation of platelets with a low concentration of collagen (17 micrograms/ml) and in the presence of DBNBS or MNP yields several EPR-detectable spin adducts. Some of the observed spin adducts do not correspond to those originating from the interaction of a free radical, nitric oxide (NO.) gas, with the spin traps [Arroyo, C.M. & Kohno, M. (1991) Free Radical Res. Commun. 14, 145-155]. Only one adduct of DBNBS, with a relative intensity of 0.1, observed in the washed-platelet experiment and in the presence of superoxide dismutase, is similar to the EPR spectrum obtained following a reaction of pure NO. gas with DBNBS. This suggests that the EPR spectrum of the DBNBS adduct consisting of a triplet may originate from the production of NO. by these cells. Additional DBNBS and MNP spin adducts were generated during platelet activation in the presence of Ca2+ and of a cytosol-depleted L-arginine preparation from washed platelets to which L-arginine was subsequently added. The formation of these DBNBS and MNP spin adducts were inhibited by N omega-methyl-L-arginine (MeArg, 100 microM), suggesting that these originated from a product of NO synthase. Furthermore, the formation of DBNBS and MNP spin adducts in platelet suspensions was enhanced by the presence of superoxide dismutase; however, their formation was prevented by the endothelial-derived relaxing factor (EDRF) inhibitors methylene blue and hemoglobin. The results from the MeArg and EDRF inhibitor experiments support the existence of the L-arginine/NO pathway in platelets. In addition, the prevention of spin-adduct formation by EDRF inhibitors, suggests that the mechanisms of EDRF formation and the L-arginine/NO pathway in endothelial cells and platelets are similar.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interactions of peroxynitrite,tetrahydrobiopterin, ascorbic acid,and thiols: implications for uncoupling endothelial nitric-oxide synthase

Tetrahydrobiopterin (BH4) serves as a critical co-factor for the endothelial nitric-oxide synthase (eNOS). A deficiency of BH4 results in eNOS uncoupling, which is associated with increased superoxide and decreased NO* production. BH4 has been suggested to be a target for oxidation by peroxynitrite (ONOO-), and ascorbate has been shown to preserve BH4 levels and enhance endothelial NO* production; however, the mechanisms underlying these processes remain poorly defined. To gain further insight into these interactions, the reaction of ONOO- with BH4 was studied using electron spin resonance and the spin probe 1-hydroxy-3-carboxy-2,2,5-tetramethyl-pyrrolidine. ONOO- reacted with BH4 6-10 times faster than with ascorbate or thiols. The immediate product of the reaction between ONOO- and BH4 was the trihydrobiopterin radical (BH3.), which was reduced back to BH4 by ascorbate, whereas thiols were not efficient in recycling of BH4. Uncoupling of eNOS caused by peroxynitrite was investigated in cultured bovine aortic endothelial cells (BAECs) by measuring superoxide and NO* using spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine and the NO*-spin trap iron-diethyldithiocarbamate. Bolus ONOO-, the ONOO- donor 3-morpholinosydnonimine, and an inhibitor of BH4 synthesis (2,4-diamino-6-hydroxypyrimidine) uncoupled eNOS, increasing superoxide and decreasing NO* production. Exogenous BH4 supplementation restored endothelial NO* production. Treatment of BAECs with both BH4 and ascorbate prior to ONOO- prevented uncoupling of eNOS by ONOO-. This study demonstrates that endothelial BH4 is a crucial target for oxidation by ONOO- and that the BH4 reaction rate constant exceeds those of thiols or ascorbate. We confirmed that ONOO- uncouples eNOS by oxidation of tetrahydrobiopterin and that ascorbate does not fully protect BH4 from oxidation but recycles BH3. radical back to BH4.     

Modification of membranes by quercetin, a naturally occurring flavonoid, via its incorporation in the polar head group

Quercetin is a naturally occurring flavonoid that has a lot of beneficial properties to human health. In this report, using the spin label technique, the influence of quercetin on the fluidity of multilamellar DPPC liposomes was studied. The polarity of the environment preferred by quercetin was also examined by determining the dependence of the position of electronic absorption maxima on dielectric properties of different environments. Autofluorescence of quercetin was also used to examine its distribution in cells. An additional aim of the study was to find how quercetin presence affects human skin fibroblasts. The results showed that incorporation of quercetin at physiological pH into DPPC liposomes caused changes in the partition coefficient of the Tempo spin label between water and polar head group phases. By determining the electronic absorption maxima, we observed that the chromophore of quercetin is localized in the polar head region. Fluorescence microscopy of HSF cells showed quercetin presence in the membrane, cytoplasm and inside the nucleus. Ultrastructural observation revealed some changes, especially in membranous structures, after flavonol treatment. From the results we have concluded that quercetin present in the membrane and other structures can cause changes within cells crucial for its pharmacological activity.     

Modification of membranes by quercetin, a naturally occurring flavonoid, via its incorporation in the polar head group

Quercetin is a naturally occurring flavonoid that has a lot of beneficial properties to human health. In this report, using the spin label technique, the influence of quercetin on the fluidity of multilamellar DPPC liposomes was studied. The polarity of the environment preferred by quercetin was also examined by determining the dependence of the position of electronic absorption maxima on dielectric properties of different environments. Autofluorescence of quercetin was also used to examine its distribution in cells. An additional aim of the study was to find how quercetin presence affects human skin fibroblasts. The results showed that incorporation of quercetin at physiological pH into DPPC liposomes caused changes in the partition coefficient of the Tempo spin label between water and polar head group phases. By determining the electronic absorption maxima, we observed that the chromophore of quercetin is localized in the polar head region. Fluorescence microscopy of HSF cells showed quercetin presence in the membrane, cytoplasm and inside the nucleus. Ultrastructural observation revealed some changes, especially in membranous structures, after flavonol treatment. From the results we have concluded that quercetin present in the membrane and other structures can cause changes within cells crucial for its pharmacological activity.     

Regeneration of phenolic antioxidants from phenoxyl radicals: an ESR and electrochemical study of antioxidant hierarchy

Radicals from the flavonoids quercetin, (+)-catechin, (+/-)-taxifolin and luteolin, and from all-rac-alpha-tocopherol have been generated electrochemically by one-electron oxidation in deaerated dimethylformamide (DMF), and characterised by electron spin resonance spectroscopy (ESR) after spin-trapping by 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Simulations of the ESR spectrum based on estimated coupling constants of the spin-trapped quercetin radical, confirmed that this antioxidant radical is oxygen-centered. The complex mixture of radicals, quinoid intermediates and stable two-electron oxidation products, were for each antioxidant allowed to react with each of the four other antioxidants, and the progression of reaction followed by ESR after addition of DMPO, and the product solution further analysed by HPLC. All-rac-alpha-tocopherol was found to be most efficient in regenerating each of the other antioxidants from their oxidation products with a regeneration index (defined as moles regenerated of the oxidised phenolic antioxidant divided with moles of all-rac-alpha-tocopherol consumed) of 0.90+/-0.16 for quercetin, 0.48+/-0.11 for (+)-catechin, 0.48+/-0.06 for (+/-)-taxifolin and 0.50+/-0.10 for luteolin in equimolar 1.00 mM solution. Quercetin was found to have the highest regeneration index among the flavonoids: 0.88+/-0.13 for (+/-)-catechin, 0.41+/-0.03 for (+/-)-taxifolin and 0.41+/-0.02 for luteolin. The antioxidant hierarchy based on the reduction potentials determined by cyclic voltammetry under similar conditions (0.93 V for all-rac-alpha-tocopherol, 1.07 V for quercetin, 1.15 V for luteolin, 1.16V for (+)-catechin and 1.20 V for (+/-)-taxifolin) is compared with the observed over-all regeneration (34% for quercetin, 34% for (+)-catechin, 52% for (+/-)-taxifolin and 43% for luteolin by all-rac-alpha-tocopherol).     

Quercetin Attenuates Inflammatory Responses in BV-2 Microglial Cells: Role of MAPKs on the Nrf2 Pathway and Induction of Heme Oxygenase-1

A large group of flavonoids found in fruits and vegetables have been suggested to elicit health benefits due mainly to their anti-oxidative and anti-inflammatory properties. Recent studies with immune cells have demonstrated inhibition of these inflammatory responses through down-regulation of the pro-inflammatory pathway involving NF-κB and up-regulation of the anti-oxidative pathway involving Nrf2. In the present study, the murine BV-2 microglial cells were used to compare anti-inflammatory activity of quercetin and cyanidin, two flavonoids differing by their alpha, beta keto carbonyl group. Quercetin was 10 folds more potent than cyanidin in inhibition of lipopolysaccharide (LPS)-induced NO production as well as stimulation of Nrf2-induced heme-oxygenase-1 (HO-1) protein expression. In addition, quercetin demonstrated enhanced ability to stimulate HO-1 protein expression when cells were treated with LPS. In an attempt to unveil mechanism(s) for quercetin to enhance Nrf2/HO-1 activity under endotoxic stress, results pointed to an increase in phospho-p38MAPK expression upon addition of quercetin to LPS. In addition, pharmacological inhibitors for phospho-p38MAPK and MEK1/2 for ERK1/2 further showed that these MAPKs target different sites of the Nrf2 pathway that regulates HO-1 expression. However, inhibition of LPS-induced NO by quercetin was not fully reversed by TinPPIX, a specific inhibitor for HO-1 activity. Taken together, results suggest an important role of quercetin to regulate inflammatory responses in microglial cells and its ability to upregulate HO-1 against endotoxic stress through involvement of MAPKs.     

巨噬细胞产生NO.和O_2~-自由基的分子机理

建立了用顺磁共振（ＥＳＲ）和化学发光技术测定巨噬细胞产生ＮＯ和氧自由基的方法．捕捉到了巨噬细胞受佛波酯刺激产生的ＮＯ．和Ｏ－２自由基．测定了在不同浓度Ｌ－精氨酸存在时佛波酯刺激后巨噬细胞产生的ＮＯ自由基．研究了巨噬细胞产生的ＮＯ和氧自由基的分子机理．结果表明巨噬细胞不仅产生氧自由基而且产生ＮＯ自由基．ＮＡＤＰＨ氧化酶产生氧自由基的部位位于巨噬细胞膜的外侧．ＮＯ合成酶活化产生ＮＯ自由基比ＮＡＤＰＨ氧化酶活化产生氧自由基晚几分钟．     

The interaction between nitrogen oxides and hemoglobin and endothelium-derived relaxing factor

Among nitrogen oxides, NO and NO₂ are free radicals and show a variety of biological effects. NO₂ is a strongly oxidizing toxicant, although NO, not oxidizing as NO₂, is toxic in that it interacts with hemoglobin to form nitrosyl-and methemoglobin. Nitrosylhemoglobin shows a characteristic electron spin resonance (ESR) signal due to an odd electron localized on the nitrogen atom of NO and reacts with oxygen to yield nitrate and methemoglobin, which is rapidly reduced by methemoglobin reductase in red cells. NO was found to inhibit the reductase activity. Part of NO inhaled in the body is oxidized by oxygen to NO₂, which easily dissolves in water and converts to nitrite. The nitrite oxyhemoglobin autocatalytically after a lag. The mechanism of the oxidation, particularly the involvement of superoxide, was controversial. The stoichiometry of the reaction has now been established using nitrate ion electrode and a methemoglobin free radical was detected by ESR during the oxidation. Complete inhibition of the autocatalysis by aniline or aminopyrine suggests that the radical catalyzes conversion of nitrite to NO₂, which oxidizes oxyhemoglobin. Recently NO was shown to be one of endothelium- derived relaxing factors and the relaxation induced by the factor was inhibited by hemoglobin and potentiated by superoxide dismutase.     

Quercetin inhibits LPS-induced macrophage migration by suppressing the iNOS/FAK/paxillin pathway and modulating the cytoskeleton

The natural flavonoid quercetin has antioxidant, anti-inflammatory, and anticancer effects. We investigated the effect of quercetin on lipopolysaccharide (LPS)-induced macrophage migration. Quercetin significantly attenuated LPS-induced inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO) production in RAW264.7 cells without affecting their viability. Additionally, quercetin altered the cell size and induced an elongated morphology and enlarged the vacuoles and concentrated nuclei. Quercetin significantly disrupted the F-actin cytoskeleton structure. Furthermore, quercetin strongly inhibited LPS-induced macrophage adhesion and migration in a dose-dependent manner. Moreover, quercetin inhibited the LPS-induced expression of p-FAK, p-paxillin, FAK, and paxillin as well as the cytoskeletal adapter proteins vinculin and Tensin-2. Therefore, quercetin suppresses LPS-induced migration by inhibiting NO production, disrupting the F-actin cytoskeleton, and suppressing the FAK–paxillin pathway. Quercetin may thus have potential as a therapeutic agent for chronic inflammatory diseases.