Katarína Drábiková

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.

Decreased activity of neutrophils in the presence of diferuloylmethane (curcumin) involves protein kinase C inhibition.

Diferuloylmethane (curcumin) has been shown to act beneficially in arthritis, particularly through downregulated expression of proinflammatory cytokines and collagenase as well as through the modulated activities of T lymphocytes and macrophages. In this study its impact on activated neutrophils was investigated both in vitro and in experimental arthritis. Formation of reactive oxygen species in neutrophils was recorded on the basis of luminol- or isoluminol-enhanced chemiluminescence. Phosphorylation of neutrophil protein kinases C alpha and beta II was assessed by Western blotting, using phosphospecific antibodies. Adjuvant arthritis was induced in Lewis rats by heat-killed Mycobacterium butyricum. Diferuloylmethane or methotrexate was administered over a period of 28 days after arthritis induction. Under in vitro conditions, diferuloylmethane (1-100 microM) reduced dose-dependently oxidant formation both at extra- and intracellular level and it effectively reduced protein kinase C activation. Adjuvant arthritis was accompanied by an increased number of neutrophils in blood and by a more pronounced spontaneous as well as PMA (phorbol myristate acetate) stimulated chemiluminescence. Whereas the arthritis-related alterations in neutrophil count and in spontaneous chemiluminescence were not modified by diferuloylmethane, the increased reactivity of neutrophils to PMA was less evident in diferuloylmethane-treated animals. The effects of diferuloylmethane were comparable with those of methotrexate. Diferuloylmethane was found to be a potent inhibitor of neutrophil functions both in vitro and in experimental arthritis. As neutrophils are considered to be cells with the greatest capacity to inflict damage within diseased joints, the observed effects could represent a further mechanism involved in the antirheumatic activity of diferuloylmethane.

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.

The combined luminol/isoluminol chemiluminescence method for differentiating between extracellular and intracellular oxidant production by neutrophils

Abstract To address the question why isoluminol, but not luminol, failed to detect oxidants produced intracellularly, differences between these luminophores were investigated with respect to physicochemical parameters and the character of chemiluminescence signal. Our results showed the isoluminol molecule to be more polar, more hydrophilic and possessing lower ability to form intramolecular bonds than the luminol molecule. Therefore, isoluminol: (i) only slightly pervaded biological membranes; (ii) depended essentially on extracellular peroxidase; (iii) did not produce chemiluminescence in the presence of extracellular scavengers; and (iv) it could be considered a specific detector of extracellular radicals. On the other hand, the physicochemical parameters of luminol and partial resistance of its chemiluminescence to the effect of extracellular inhibitors proved the lipo/hydrophilic character of this luminophore and thus its ability to interact with radicals both outside and inside of cells. The luminol chemiluminescence measured in the presence of extracellular scavengers and the isoluminol chemiluminescence were used with the intention to differentiate the effects of two antihistamine drugs on intra- and extracellular radical formation. In activated human neutrophils, brompheniramine inhibited the extracellular and potentiated the intracellular part of chemiluminescence signal, whereas a reducing effect of loratadine was observed in both compartments.

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.

Reactive oxygen metabolite production is inhibited by histamine and H1-antagonist dithiaden in human PMN leukocytes.

The study evaluated the distinction between extracellular and intracellular production of reactive oxygen metabolites (ROM) in isolated polymorphonuclear leukocytes (PMNL) stimulated with opsonised zymosan (OZ) and investigated its modulation by the endogenous mediator histamine (0.1-100 w mol/l) and by the H 1 -antagonist dithiaden (1-100 w mol/l). For this observation, a modified luminol and an isoluminol amplified chemiluminescence (CL) technique were used. Our results showed that PMNL activated with OZ responded with a respiratory burst accompanied by both extra- and intracellular generation of ROM. Histamine and dithiaden significantly decreased both the extra- and intracellular component of chemilumiescence stimulated with OZ. While dithiaden decreased both the extra- and intracellular part of CL with the same potency, histamine decreased preferentially the extracellular part of CL. The fact that histamine as well as the H 1 -antagonist dithiaden decreased the respiratory burst indicates that not only histamine receptors but also non-receptor mechanisms could be involved in the reduction of CL. Interaction with enzymes (NADPH-oxidase, myeloperoxidase, phospholipase A 2 ) or interference with PMNL membrane structure may well result in reduction of the chemiluminescence signal.

Evidence for intracellular histamine liberation in isolated rat mast cells

The beta-adrenoceptor blocking drug exaprolol liberated histamine from isolated rat mast cells in a dose- and time-dependent way. Histamine was liberated within seconds and was not followed by a parallel granule liberation. The inhibition of histamine liberation was induced with low temperature, low pH, high concentration of Ca2+, TTD, suramin and EDTA. Subcellular distribution of3H-exaprolol demonstrated a quantitative relationship between histamine depletion against exaprolol uptake in isolated rat mast cell granules. A nonspecific mechanism of action in the effect of exaprolol on mast cells is discussed. It is proposed that the drug acts on mast cells due to the direct and indirect ion exchange mechanism resulted in disproportion between histamine and granule liberation.

Effect of chloroquine on isolated mast cells

Chloroquine liberated a relatively low amount of histamine from isolated rat mast cells. In a dose-dependent way, this drug inhibited histamine liberation from mast cells stimulated with compound 48/80, A23187, concanavalin A plus phosphatidylserine (Con A+PS) and abolished histamine liberation induced by exaprolol. The degranulation was decreased in cells stimulated with 48/80, Con A+PS and exaprolol. Chloroquine significantly inhibited the formation of thromboxane B2 in mast cells stimulated with 48/80, Con A+PS and A23187. We assume that chloroquine interferes with mast cells at a plasmic membrane site as well as intracellularly.

Protection of the vascular endothelium in experimental situations.

Protection of the vascular endothelium in experimental situations One of the factors proposed as mediators of vascular dysfunction observed in diabetes is the increased generation of reactive oxygen species (ROS). This provides support for the use of antioxidants as early and appropriate pharmacological intervention in the development of late diabetic complications. In streptozotocin (STZ)-induced diabetes in rats we observed endothelial dysfuction manifested by reduced endothelium-dependent response to acetylcholine of the superior mesenteric artery (SMA) and aorta, as well as by increased endothelaemia. Changes in endothelium-dependent relaxation of SMA were induced by injury of the nitric oxide radical (·NO)-signalling pathway since the endothelium-derived hyperpolarising factor (EDHF)-component of relaxation was not impaired by diabetes. The endothelial dysfunction was accompanied by decreased ·NO bioavailabity as a consequence of reduced activity of eNOS rather than its reduced expression. The results obtained using the chemiluminiscence method (CL) argue for increased oxidative stress and increased ROS production. The enzyme NAD(P)H-oxidase problably participates in ROS production in the later phases of diabetes. Oxidative stress was also connected with decreased levels of reduced glutathione (GSH) in the early phase of diabetes. After 10 weeks of diabetes, adaptational mechanisms probably took place because GSH levels were not changed compared to controls. Antioxidant properties of SMe1EC2 found in vitro were partly confirmed in vivo. Administration of SMe1EC2 protected endothelial function. It significantly decreased endothelaemia of diabetic rats and improved endothelium-dependent relaxation of arteries, slightly decreased ROS-production and increased bioavailability of ·NO in the aorta. Further studies with higher doses of SMe1EC2 may clarify the mechanism of its endothelium-protective effect in vivo.

Cooperation of chloroquine and blood platelets in inhibition of polymorphonuclear leukocyte chemiluminescence.

Effect of activated blood platelets and chloroquine on concentration of reactive oxygen species produced by polymorphonuclear leukocytes (PMNL) stimulated with Ca(2+)-ionophore A23187 was investigated. Oxygen metabolites localized outside PMNL were visualized by isoluminol enhanced chemiluminescence, whereas chemiluminescence, enhanced with luminol and measured in the presence of the extracellular scavengers superoxide dismutase and catalase, was used for the detection of radicals originated intracellularly. Significant reduction of chemiluminescence was observed in the presence of platelets (added to PMNL in the physiological cell ratio 50:1) and of chloroquine (10 and 100 micromol/L). Although chloroquine decreased effectively both the extra- as well as the intracellular part of the chemiluminescence signal, the activity of platelets occurred largely outside PMNL. Serotonin liberated from platelets by A23187 appeared to be involved in inhibition of chemiluminescence; its concentrations achieved in platelet supernatants were found to be sufficient for elimination of PMNL-derived oxygen metabolites. The presented results indicated that chloroquine and blood platelets cooperate in inhibition of chemiluminescence because their common effect was found to be much more extensive than reduction induced by these inhibitors separately. Therefore, for accurate prediction of drug effect in the whole organism, the use of multicellular test systems seems to be pertinent.