Equol Effectively Inhibits Toxic Activity of Human Neutrophils without Influencing Their Viability.

Equol (7,4'-dihydroxy-isoflavan, or 4',7-isoflavandiol) is a chroman derivative produced by intestinal bacteria in response to soy isoflavone intake in some, but not in all, humans. Equol shows strong anti-oxidant, anti-estrogenic, anti-cancerous and anti-inflammatory properties. The antioxidative capacity of equol has recently received considerable attention, and it has been used for preventing and treating several diseases. We investigated the effect of equol on human neutrophils, extra- and intracellular formation of oxidants, the phosphorylation of protein regulating NADPH oxidase and its effect on apoptosis. Neutrophils, isolated from blood from healthy subjects, were tested upon activation with various stimulants, proper for reactive oxygen species (ROS) production and treated by equol. Equol has the ability to reduce the toxic action of neutrophils. With increasing concentrations, equol decreased the amount of oxidants produced by neutrophils both extra- and intracellularly. The phosphorylation of p40(phox) (a component of NADPH oxidase, responsible for the assembly of functional oxidase in intracellular membranes) was reduced in the presence of equol. The experiments showed that equol did not change the number of viable, apoptotic or dead neutrophils significantly in all concentrations used. These results indicate the promising effect of equol in the operation of ROS in different mechanisms in the model of inflammation.

Molecular pharmacology of antihistamines in inhibition of oxidative burst of professional phagocytes.

Antihistamines of the H1and H3/H4groups interfere with oxidative burst of human professional phagocytes in vitro. In the concentration of 10 µM, H1antihistamines of the 1st and 2nd generation inhibited oxidative burst of human neutrophils in the rank order of potency: dithiaden > loratadine > brompheniramine > chlorpheniramine > pheniramine. Of the H1antihistamines, the most effective was dithiaden in suppressing oxidative burst of whole human blood and dose-dependently the chemiluminescence of isolated neutrophils at extra- and intracellular level. Inhibition of free oxygen radical generation in isolated neutrophils by dithiaden resulted from the inhibition of protein kinase C activation. The potentiation of recombinant caspase-3 by dithiaden is supportive of the antiinflammatory effect of dithiaden and suggestive of increasing the apoptosis of professional phagocytes. Of the H3/H4antihistamines, the most effective was JNJ7777120 in decreasing chemiluminescence in whole blood and also at extra- and intracellular sites of isolated neutrophils. JNJ 10191584 and thioperamide were less effective and the latter significantly potentiated free oxygen radical generation intracellularly. The results demonstrated that, compared with the H3/H4antihistamines investigated, H1antihistamines were much more potent in inhibiting free oxygen radical generation in human professional phagocytes. This finding should be taken into account therapeutically.

The natural stilbenoid pinosylvin and activated neutrophils: effects on oxidative burst, protein kinase C, apoptosis and efficiency in adjuvant arthritis.

AIM: To investigate the effects of the naturally occurring stilbenoid pinosylvin on neutrophil activity in vitro and in experimental arthritis, and to examine whether protein kinase C (PKC) activation served as an assumed target of pinosylvin action. METHODS: Fresh human blood neutrophils were isolated. The oxidative burst of neutrophils was evaluated on the basis of enhanced chemiluminescence. Neutrophil viability was evaluated with flow cytometry, and PKC phosphorylation was assessed by Western blotting analysis. Adjuvant arthritis was induced in Lewis rats with heat-killed Mycobacterium butyricum, and the animals were administered with pinosylvin (30 mg/kg, po) daily for 21 d after arthritis induction. RESULTS: In isolated human neutrophils, pinosylvin (10 and 100 µmol/L) significantly decreased the formation of oxidants, both extra- and intracellularly, and effectively inhibited PKC activation stimulated by phorbol myristate acetate (0.05 µmol/L). The inhibition was not due to neutrophil damage or increased apoptosis. In arthritic rats, the number of neutrophils in blood was dramatically increased, and whole blood chemiluminescence (spontaneous and PMA-stimulated) was markedly enhanced. Pinosylvin administration decreased the number of neutrophils (from 69 671 ± 5588/µL to 51 293 ± 3947/µL, P=0.0198) and significantly reduced the amount of reactive oxygen species in blood. CONCLUSION: Pinosylvin is an effective inhibitor of neutrophil activity, and is potentially useful as a complementary medicine in states associated with persistent inflammation.

Suppression of oxidative burst in human neutrophils with the naturally occurring serotonin derivative isomer from Leuzea carthamoides.

OBJECTIVE: Neutrophil leukocytes and macrophages represent professional phagocytic cells. When appropriately stimulated, they undergo dramatic physiological and biochemical changes resulting in phagocytosis, chemotaxis and degranulation with the activation of reactive oxygen species (ROS) production known as the respiratory burst. DESIGN: In this study we analysed the effect of a crystalline complex fraction of four N-feruloyl-serotonin isomers isolated from the seeds of Leuzea carthamoides on the mechanism of oxidative burst of human neutrophils in vitro. RESULTS: N-feruloyl-serotonin (N-f-5HT) inhibited dose-dependently oxidative burst of human whole blood and isolated neutrophils in vitro stimulated with phorbol-myristate-acetate (PMA) as measured by luminol/isoluminol enhanced chemiluminescence.In isolated neutrophils stimulated with PMA, N-f-5HT was effective against extracellular as well as intracellular reactive oxygen species. Western blot analysis documented that N-f-5HT in concentrations of 10 and 100 µM significantly decreased PMA-induced phosphorylation of protein kinase C alpha/beta II. CONCLUSION: The results suggest that N-f-5HT represents an effective naturally occurring substance with potent effect on the oxidative burst of human neutrophils and could be further investigated for its pharmacological activity against oxidative stress in ischaemia-reperfusion, inflammation and other pathological conditions.

In vivo effect of pinosylvin and pterostilbene in the animal model of adjuvant arthritis.

OBJECTIVE: The aim of this study was to evaluate the effects of pinosylvin (PIN) and pterostilbene (PTE), natural substances from the stilbenoid group, on the development of adjuvant arthritis in rats. METHODS: Adjuvant arthritis (AA) was induced by a single intradermal injection of Mycobacterium butyricum in incomplete Freund's adjuvant in male Lewis rats. Our experiments included healthy intact animals as reference controls, arthritic animals without any drug administration, and arthritic animals with administration of PIN and PTE in the oral daily dose of 30 mg/kg b.w. The treatment involved administration of the substances tested from day 0, i.e. the day of immunization, to the experimental day 28. The following parameters were monitored: change of the hind paw volume (HPV) on day 14, 21 and 28, luminol-enhanced chemiluminescence (CL) of the joint and myeloperoxidase (MPO) activity in hind paw joint homogenates (day 28). RESULTS: Arthritic animals treated with PIN showed a decrease in HPV, significantly on days 14 and 28. PIN decreased CL of the joint as well as MPO activity of the joint homogenate, in comparison with untreated animals. PTE had no effect on HPV and MPO activity in hind paw joint homogenates and exerted only a partial effect on luminol-enhanced CL. CONCLUSIONS: On the basis of our results we conclude that the effect of PTE on CL was only partial. PIN, on the other hand, had a beneficial anti-inflammatory and antioxidant effect on oxidative stress induced biochemical changes occurring in AA, as determined by all three functional parameters.

Molecular targets of the natural antioxidant pterostilbene: effect on protein kinase C, caspase-3 and apoptosis in human neutrophils in vitro.

OBJECTIVE: Pterostilbene, a naturally occurring phenolic derivative, exhibits various pharmacological effects, e.g. anti-cancerous, antioxidant, anti-inflammatory and anti-diabetic. Based on our previous study, we assessed the cellular and molecular effects of pterostilbene on human neutrophils and in cell free systems. Experimental and theoretical molecular descriptors of stilbene derivatives were also determined. METHODS: We assessed the antioxidant properties of pterostilbene using cell free system and computational methods. The effect of pterostilbene on protein kinase C activation/phosphorylation was detected by special anti-phospho protein kinase C antibodies. Membrane associated changes determining the life span of neutrophils and human recombinant caspase-3 assay were examined. RESULTS: Pterostilbene possessed comparable antioxidant properties as resveratrol in cell free system. Computational methods were used to establish the molecular characteristics of stilbene derivatives. The values of electronic parameters suggest a slight enhancement of electron donor properties of pterostilbene compared to resveratrol. Phosphorylation and thus activation of protein kinase C alpha/beta II in activated neutrophils was not decreased by pterostilbene. Pterostilbene in concentrations of 10-100 µM was found to inhibit the activity of human caspase-3 purified enzyme and did not influence cell viability significantly. CONCLUSION: Pterostilbene, an analog of resveratrol, was identified as a good natural antioxidant compound. However, reducing the oxidative burst of human neutrophils during their activation in vitro with pterostilbene does not include protein kinase C phosphorylation pathway. Pterostilbene showed dose dependent activation/inhibition of caspase-3 enzyme activity.

Different effect of two synthetic coumarin-stilbene hybrid compounds on phagocyte activity.

OBJECTIVE: Activated phagocytes, generating a variety of powerful inflammatory mediators, such as oxygen and nitrogen species, may participate in oxidative stress-mediated inflammation and organ toxicity. At present, great attention is devoted to the important class of phenolic compounds - coumarins - due to their antiinflammatory/antioxidant activities. We compared two synthetic phenylcoumarins: 7-hydroxy-3-(4´-hydroxyphenyl) coumarin (HHC; 0.01-100 µmol/l) and its hydrogenated analogue: 7-hydroxy-3-(4´-hydroxyphenyl)-3,4-dihydrocoumarin (HHDC; 0.01-100 µmol/l) as their ability to inhibit reactive oxygen species (ROS) generation in human neutrophils and nitric oxide (NO) production by RAW 264.7 macrophages in vitro, with respect to some of their physicochemical characteristics. METHODS: ROS production was measured with luminol-enhanced chemiluminescence (CL) in the microplate luminometer Immunotech LM-01T, nitrite formation was determined by the Griess reaction - spectrophotometrically. The radical scavenging assays were employed to assess the antiradical activity values. The relevant physico-chemical parameters of the compounds tested, electronic and hydrophobic, were determined experimentally as well as by suitable computational programmes. RESULTS: Both HHC and HHDC were found to decrease significantly (p<0.01) CL of whole blood stimulated with phorbol myristate acetate (PMA) from the concentration of 1 µmol/l. While HHC significantly inhibited CL stimulated by A23187 and opsonized zymosan (OpZ), HHDC was ineffective. Unlike HHDC, HHC in the concentrations of 10 and 100 µmol/l significantly (p<0.01) reduced NO formation in lipopolysaccharide (LPS) -stimulated murine macrophages RAW 264.7. HHC possessed the higher free radical reducing efficacy in accordance with its more favourable values of electronic parameters in comparison with HHDC. CONCLUSIONS: Our results show the different inhibitory effects of HHC and HHDC on phagocytic activity that might be the result of their diverse free radical scavenging properties and lipophilicity features.

Glucomannan reduces neutrophil free radical production in vitro and in rats with adjuvant arthritis.

The effect of glucomannan (GM), a natural polysaccharide isolated from the yeast Candida utilis, on reactive oxygen species (ROS) generation in human neutrophils in vitro and in rats with Mycobacterium butyricum induced adjuvant arthritis (AA) was tested by the luminol/isoluminol-enhanced chemiluminescence (CL) method. In vitro, GM (500 microg/ml) significantly decreased spontaneous CL of human whole blood, while PMA (4beta-phorbol-12beta-myristate-alpha13acetate)-stimulated CL was decreased by GM in the concentrations of 100 and 500 microg/ml. To specify the site of action of GM, its effect on extra- and intracellular ROS generation in isolated neutrophils was evaluated. GM significantly decreased spontaneous and PMA-stimulated CL and it was more effective extracellularly than intracellularly. In vivo experiments included healthy animals as controls, arthritic animals without any drug administration, and arthritic animals with GM administration (once daily in the oral dose of 15 mg/kg, over a period of 28 days). On day 28, CL in whole blood, spleen and joint was monitored. Arthritic animals treated with GM showed decrease in spontaneous and PMA-stimulated CL of whole blood as well as CL of the joint, in comparison with untreated animals. The obtained findings demonstrated an antioxidant effect of GM in vitro and in rats with AA, which may be due to its free radical scavenger activity and to interaction with different receptors and/or modulation of postreceptor intracellular signalling pathways. The specific physicochemical parameters, such as structure of GM, its low molecular weight and good water solubility, play an important role in the above effects.

H1-antihistamines and oxidative burst of professional phagocytes.

OBJECTIVES: We analysed and compared the effect of five H1-antihistamines on stimulated oxidative burst at extra- and intracellular level of isolated and stimulated human polymorphonuclear leukocytes. DESIGN: Oxidative burst of isolated human neutrophils was studied by means of luminol and isoluminol enhanced chemiluminescence. RESULTS: The following rank order of potency for H1-antihistamines to decrease chemiluminescence was evaluated extracellularly: dithiaden> loratadine> chlorpheniramine> brompheniramine> pheniramine and at intracellular site: loratadine> dithiaden. CONCLUSION: H1-antihistamines differ substantially according to their chemical structure in suppressing oxidative burst both at extra- and intracellular site of isolated stimulated human neutrophils.

Novel aspects of the activation of NADPH oxidase in neutrophils of rheumatic patients on biological therapy.

The relationship between inflammation and formation of reactive oxygen species (ROS) is still not completely understood and excessive inflammatory reaction is attributed to increased yet also to reduced ROS formation. To compare ROS formation in severe and low inflammation, neutrophil oxidative burst was analyzed in rheumatic patients before and during therapy with TNFα- or interleukin-6 receptor-neutralizing antibodies. Intracellular and extracellular ROS productions were evaluated on the basis of luminol- and isoluminol-enhanced chemiluminescence in isolated peripheral neutrophils. Disease activity score DAS28 and platelet to lymphocyte ratio were used as markers of arthritis activity and the intensity of systemic inflammation. Biological therapy effectively reduced the intensity of inflammation. Of the twenty-six patients studied eighteen achieved remission or low disease activity. Highly active arthritis persisted only in one patient, though prior to the therapy it was evident in all subjects tested. In patients receiving biological therapy, intracellular chemiluminescence was significantly higher than in patients before this therapy; ROS produced by neutrophils extracellularly were not affected. The increased ROS formation associated with reduced inflammation supports the need to revise the view of the role of ROS in inflammation - from toxic agents promoting inflammation towards a more complex view of ROS as regulators of immune pathways with inflammation-limiting capacity. From this perspective, the interference with neutrophil-derived oxidants may represent a new mechanism involved in the anti-inflammatory activity of biological therapy.

Structure-efficiency relationship in derivatives of stilbene. Comparison of resveratrol, pinosylvin and pterostilbene.

OBJECTIVES: Oxidative stress is related to a number of autoimmune diseases, e.g. rheumatoid arthritis, cancer, etc. The main source of pathologically working reactive oxygen species (ROS) are activated polymorphonuclear leukocytes (PMNL). OBJECTIVE: There are some papers comparing structure - pharmacological efficiency relationship of vegetal substances from the stilbenoid group. We compared the effect of trans-resveratrol, which is well-known by its antioxidative activity, with the effect of pinosylvin and pterostilbene. METHODS: Luminol-enhanced chemiluminescence (CL) was used to study the antioxidative action. The effect was observed in whole blood and in isolated PMNL. The concentrations of substances tested were 0.01-100 microM. Due to the different abilities of luminol and isoluminol to pass through the cell membrane, we studied the effect of the substances tested on intracellular and extracellular ROS. To stimulate the production of ROS we used phorbol-myristate-acetate (PMA), which activates PMNL via protein kinase C. RESULTS: Resveratrol, pinosylvin and pterostilbene inhibited significantly the CL of whole blood and extra- and intracellular CL of isolated PMNL in a dosedependent manner. Depending on different functional groups of the stilbene molecule, resveratrol inhibited CL of whole blood and isolated PMNL, whereas pinosylvin influenced mainly intracellular CL and pterostilbene extracellular CL. CONCLUSION: The presence of different functional groups in the molecules of stilbenoids influence their antioxidative effect. Modification of these functional groups may result in derivatives with required antioxidative properties, targeting mainly extracellular ROS which are responsible for tissue damage during chronic inflammation.

Mechanism of anti-inflammatory action of liquorice extract and glycyrrhizin.

The antiradical activity, protective effect against lipid peroxidation of liposomal membrane, and inhibitory effect on whole blood reactive oxygen species (ROS) liberation of Glycyrrhiza glabra crude extract and glycyrrhizin, its major compound, were assessed. The liquorice extract showed significant activity in all the three assay systems used in a dose dependent manner. It displayed remarkable reactivity with free stable 1,1'-diphenyl-2-picrylhydrazyl (DPPH) radical, inhibitory efficacy in peroxidatively damaged unilamellar dioleoyl phosphatidylcholine (DOPC) liposomes, and inhibition of ROS chemiluminescence, generated by whole blood, induced by both receptor-bypassing stimuli (PMA) and receptor operating stimuli (Opz) in the ranking order of stimuli PMA> Opz. These activities may be attributed to phenolic antioxidants involving isoflavan derivatives, coumarins and chalcones. Nonetheless, triterpene saponin glycyrrhizin exhibited no efficacy in the system of DPPH reaction and peroxidation of liposomal membrane, and negligible inhibition of chemiluminescence generated by inflammatory cells. These results indicate that the mechanism of anti-inflammatory effect of glycyrrhizin most probably does not involve ROS and this major constituent is not responsible for the inhibition effects of liquorice extract on neutrophil functions.

Pharmacological intervention with oxidative burst in human neutrophils.

In this study we investigated the effect of five therapeutically used drugs and four natural polyphenolic compounds on the mechanism of oxidative burst of human neutrophils concerning their participation in the generation of reactive oxygen species (ROS). The compounds investigated decreased the oxidative burst of whole blood in the rank order of potency: N-feruloylserotonin > quercetin > curcumin > arbutin > dithiaden > carvedilol. The generation of intracellular reactive oxygen species in isolated neutrophils decreased in the same rank order, while carvedilol was ineffective. Scavenging of extracellular oxygen radicals followed the rank order of potency: N-feruloylserotonin > curcumin > quercetin > dithiaden. Arbutin and carvedilol had no effect. All compounds tested increased the activity of caspase-3 in cell-free system indicating a positive effect on apoptosis of neutrophils. Activation of protein kinase C was significantly decreased by dithiaden, curcumin, quercetin and N-feruloylserotonin. Carvedilol, dithiaden, quercetin and arbutin reduced activated neutrophil myeloperoxidase release more significantly compared with their less pronounced effect on superoxide generation The presented results are indicative of pharmacological intervention with neutrophils in pathological processes. Of particular interest was the effect of natural compounds. Intracellular inhibition of oxidative burst in isolated neutrophils by the drugs tested and natural antioxidants has to be further analysed since ROS play an important role in immunological responses of neutrophils.

Extra- and intracellular formation of reactive oxygen species by human neutrophils in the presence of pheniramine, chlorpheniramine and brompheniramine.

BACKGROUND: Allergic inflammation was found to be accompanied by activation of neutrophils. Since this resulted in increased formation of reactive oxygen species, the antioxidative activity of antiallergic drugs was considered to decrease the risk of tissue damage. However, if the drug-induced inhibition of radical formation occurred intracellularly, it could disturb regulation of neutrophil functions and decrease bactericidal activity of these cells. Separate analysis of extra- and intracellular activity of antioxidative drugs is thus of particular importance. OBJECTIVE: To differentiate the effects of three antiallergic H1-antihistamine drugs (pheniramine, chlor- and brompheniramine) on radical formation outside and inside human neutrophils. METHODS: Formation of reactive oxygen species was determined in vitro using the chemiluminescence method. The chemiluminescence signal, initiated by phorbol-12-myristate-13-acetate, was enhanced either with isoluminol (extracellular) or with luminol in the presence of extracellular scavengers, superoxide dismutase and catalase (intracellular). RESULTS: The antihistamines tested displayed dual activity - they inhibited the extracellular and potentiated the intracellular chemiluminescence. Chlor- and brompheniramine were found to be more effective than pheniramine. CONCLUSION: Compared to other H1-antihistamines (such as dithiaden or loratadine, active both extra- and intracellularly), the observed inhibition caused by the pheniramines tested was unique since it occurred selectively outside neutrophils. This might indicate the ability of these drugs to minimise toxic effects of extracellular radicals without affecting intracellular oxidant production involved in regulation of neutrophil functions and in microbial killing.

On the antioxidant activity of carvedilol in human polymorphonuclear leukocytes in vitro and ex vivo.

OBJECTIVES: In the present study we investigated whether the beta-adrenoceptor antagonist carvedilol (CARV) [Dilatrend] prevented reactive oxygen metabolite (ROM) production in human polymorphonuclear leukocytes (PMNL) or interfered with ROM already generated. To specify the site of action of CARV, we evaluated its effect on extra- and intracellular ROM generation. In addition, we studied the effect of CARV therapy on ROM production in whole blood obtained from patients with congestive heart failure (CHF) combined with type II diabetes mellitus. METHODS: ROM generation in whole blood and isolated PMNL was determined after phorbol 12-myristate-13-acetate (PMA-0.05 micromol/l) stimulation by luminol/isoluminol-enhanced chemiluminescence (CL) in the microplate luminometer Immunotech LM-01T. RESULTS: CARV significantly decreased CL of human whole blood in the concentrations of 10, 20, 50 and 100 micromol/l, both when applied simultaneously and after stimulation. In isolated PMNL, CARV significantly decreased extracellular CL in the concentrations of 50 and 100 micromol/l, intracellular CL was decreased in the concentrations of 20, 50 and 100 micromol/l. The nonstimulated and PMA stimulated CL of whole blood was increased in patients before therapy in comparison with healthy controls. After CARV therapy, 25 mg/day and 50 mg/day, there was a trend to reduce CL as compared to values before therapy. CONCLUSIONS: In human PMNL, CARV interfered in vitro and ex vivo with ROM generation as well as with already generated ROM, suggestive of its both "preventive" and "therapeutic" effect.

On the pharmacology and toxicology of neutrophils.

OBJECTIVES: To study the effect of H1-antihistamines dithiaden (Dit) and loratadine (Lor) and compare it with that of histamine (His) on phorbolmyristate acetate (PMA) stimulated chemiluminescence (CL) of whole blood, isolated neutrophils, release of myeloperoxidase (MPO), and on superoxide (SO) generation. METHODS: Luminol- and isoluminol-enhanced CL was applied for measuring the oxidative burst, spectrophotometry was used for determination of MPO (o-dianisidine) and SO generation (superoxide dismutase inhibition of cytochrome c). RESULTS: Dit and Lor dose-dependently inhibited CL of whole blood and significantly decreased oxidative burst both at the extra- and intracellular sites of neutrophils. Release of MPO was decreased with both drugs tested in 10-times lower concentrations than was SO inhibition. Histamine (His) was much less effective in the inhibition of the parameters investigated. CONCLUSIONS: Histaminergic drugs of the 1st generation inhibited oxidative burst of human phagocytes in whole blood and in isolated neutrophils. The rank order of potency to inhibit CL, MPO release and SO generation in PMA stimulated phagocytes was: Dit>Lor>His.

Ischaemia/reperfusion-induced organ injury in low dose streptozotocin diabetes.

OBJECTIVES: The influence of low dose streptozotocin diabetes on intestinal and vascular injury induced by mesenteric ischaemia/reperfusion (I/R) was studied in rats. The role of reactive oxygen species (ROS) in the exacerbation of ischaemic/postischaemic damage in diabetes was addressed. METHODS: Diabetes was induced by i.p. injection of 3 x 30 mg/kg streptozotocin and after 5 weeks male Wistar rats underwent 60 min ischaemia followed by 30 min reperfusion of the superior mesenteric artery (SMA). The extent of intestinal haemorrhagic injury was assessed macroscopically. Relaxation to acetylcholine of precontracted SMA rings was tested. Chemiluminescence (CL) enhanced by luminol of tissue samples excised from the ileum and SMA was measured. RESULTS: In diabetic rats I/R-induced intestinal injury was significantly more pronounced compared to non-diabetic rats (63.6% potentiation). Decreased endothelial-dependent relaxation of diabetic SMA was not further influenced by I/R. Diabetes itself did not change the CL response of SMA and there was a similar CL increase in the diabetic group with I/R as in the controls. In the intestinal samples CL response was suppressed and I/R only mildly increased CL in the diabetic group. CONCLUSIONS: Diabetes renders the intestinal tissue more vulnerable to the effects of I/R. Endothelial-dependent relaxation of diabetic SMA was not further worsened by I/R. CL responses showed a different involvement of ROS in diabetic intestinal versus vascular tissue.

Participation of reactive oxygen species in diabetes-induced endothelial dysfunction.

OBJECTIVES: In the present study, the relationship between diabetes-induced hyperglycemia, reactive oxygen species production and endothelium-mediated arterial function was examined. The effect of antioxidant on the reactive oxygen species induced damage was tested. METHODS: Diabetes was induced by streptozotocin (STZ), 3 x 30 mg/kg i.p., administered on three consecutive days. After 10 weeks of diabetes, the functional state of the endothelium of the aorta was tested, endothelemia evaluation was performed and systolic blood pressure was measured. Reactive oxygen species (ROS) formation in blood and the aorta was measured using luminol-enhanced chemiluminescence (CL). Levels of reduced glutathione (GSH) were determined in the aorta, kidney, and plasma. To study the involvement of hyperglycemia in functional impairment of the endothelium, aortal rings incubated in solution with high glucose concentration were tested in in vitro experiments. RESULTS: After 10 weeks of diabetes, endothelial injury was observed, exhibited by diminished endothelium-dependent relaxation of the aorta, increased endothelemia and by elevated systolic blood pressure. Using luminol-enhanced CL, a significant increase of ROS production was found in arterial tissue and blood. GSH levels were significantly increased in the kidney, while there were no GSH changes in plasma and the aorta. Incubation of aortic rings in solution with high glucose concentration led to impairment of endothelium-dependent relaxation. The synthetic antioxidant SMe1EC2 was able to restore reduced endothelium-mediated relaxation. CONCLUSIONS: Our results suggest an important role of hyperglycemia-induced ROS production in mediating endothelial dysfunction in experimental diabetes, confirmed by CL and the protective effect of the antioxidant SMe1EC2.

Water Extract of Mentha x villosa: Phenolic Fingerprint and Effect on Ischemia-Reperfusion Injury.

Qualitative analysis of the water extract of Mentha x villosa Huds. leaves was performed by liquid chromatography mass spectrometry (LC-MS/MS) and quantitative analysis was made by reverse-phase liquid chromatography coupled with photodiode array detection (LC-DAD). Sixteen phenolic compounds were identified and quantified consisting of 8 phenolic acids/derivatives and 8 flavonoid glycosides (quinic acid, chlorogenic acid, coumaroyl-hexoside, caffeic acid, coumaroylquinic acid, lithospermic acid, rosmarinic acid, salvianolic acid A, luteolin-7-O-glucuronide, luteolin-7-O-glucoside, luteolin-7-O-rutinoside, eriodictyol-7-O-rutinoside, apigenin-7-O-glucuronide, kaempferol-3-O-glucuronide, chrysoeriol-7-O-rutinoside, and hesperetin-7-O-rutinoside). Luteolin-7- O-rutinoside (25.6 ± 0.7 mg/g dry extract) and rosmarinic acid (17.9 ± 0.4 mg/g dry extract) were the most abundant. High antioxidant activity of this phenolic-rich water extract was confirmed in vitro by DPPH and ABTS tests and ex vivo in the ischemia-reperfusion injured rat superior mesenteric artery. Thus, the water extract of M. x villosa leaves seems to be a promising agent in prevention of tissue injury caused by oxidative stress.

Selective inhibition of extracellular oxidants liberated from human neutrophils--A new mechanism potentially involved in the anti-inflammatory activity of hydroxychloroquine.

Hydroxychloroquine is used in the therapy of rheumatoid arthritis or lupus erythematosus. Although these diseases are often accompanied by activation of neutrophils, there are still few data relating to the impact of hydroxychloroquine on these cells. We investigated the effect of orally administered hydroxychloroquine on neutrophil oxidative burst in rats with adjuvant arthritis. In human neutrophils, extra- and intracellular formation of oxidants, mobilisation of intracellular calcium and the phosphorylation of proteins regulating NADPH oxidase assembly were analysed. Administration of hydroxychloroquine decreased the concentration of oxidants in blood of arthritic rats. The inhibition was comparable with the reference drug methotrexate, yet it was not accompanied by a reduction in neutrophil count. When both drugs were co-applied, the effect became more pronounced. In isolated human neutrophils, treatment with hydroxychloroquine resulted in reduced mobilisation of intracellular calcium, diminished concentration of external oxidants and in decreased phosphorylation of Ca(2+)-dependent protein kinase C isoforms PKCα and PKCßII, which regulate activation of NADPH oxidase on plasma membrane. On the other hand, no reduction was observed in intracellular oxidants or in the phosphorylation of p40(phox) and PKCδ, two proteins directing the oxidase assembly to intracellular membranes. Hydroxychloroquine reduced neutrophil-derived oxidants potentially involved in tissue damage and protected those capable to suppress inflammation. The observed effects may represent a new mechanism involved in the anti-inflammatory activity of this drug.