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Rami B. Kassab, Ondrej Vasicek, Milan Ciz, Antonin Lojek and Tomas Perecko

Abstract

The health benefits of berberine have been recognized for years. Even so, its effects on human neutrophils, the first line of immune defense, have not been reported. The purpose of this study was to investigate the effects of berberine on the human neutrophil oxidative burst. Reactive oxygen species production was analyzed by luminol-enhanced chemiluminescence. The analysis was performed in spontaneous and stimulated (phorbol myristate acetate (PMA) or opsonized zymosan particles (OZP)) whole blood and isolated neutrophils in the presence or absence of berberine. The effects of berberine on oxidant production in cell-free assays were evaluated using luminescence (H2O2-peroxidase-luminol) and fluorescence (Oxygen Radical Absorbance Capacity – ORAC) techniques. Berberine decreased the production of reactive oxygen species in human whole blood and isolated neutrophils stimulated with either PMA or OZP with a different efficiency (EC50 was 69 μM and 197 μM for PMA and OZP, respectively). The effect was more pronounced in isolated neutrophils. Cell-free assays showed the antioxidant activity of berberine against peroxyl radicals and hydrogen peroxide. Based on our results, we suggest that the effects of berberine on reactive oxygen species production in human neutrophils are due to its antioxidant activity.

Open access

Jana Králová, Michaela Pekarová, Katarína Drábiková, Viera Jančinová, Radomír Nosál, Milan Číž and Antonín Lojek

The effects of dithiaden on nitric oxide production by RAW 264.7 cells

Asreported in our previous studies, dithiaden (an antagonist of histamine H1-receptor, used clinically as an anti-allergic or anti-emetic drug) in a concentration range of 5×10-5-10-4 M decreased the production of reactive oxygen species by phagocytes. In this study we investigated the influence of dithiaden on nitric oxide (NO) production by LPS-stimulated macrophages.

The cell viability in the presence of 10-4-5×10-5 M dithiaden was evaluated by an ATP-test. RAW 264.7 cells (2.5×106/well) were preincubated with dithiaden for 60 mins and subsequently stimulated with 0.1 μg/ml of bacterial lipopolysaccharide. After incubating for 24 hours the NO production was determined spectrophotometrically using Griess reaction as a concentration of nitrites (the end product of NO metabolism) accumulated in the cell supernatants. The expression of inducible nitric oxide synthase (iNOS) in cell-lysates was evaluated using Western blot analysis. Scavenging properties of dithiaden against NO were evaluated amperometrically. Our data demonstrate that dithiaden in the concentration of 5×10-5 M (approved by ATP test as non toxic) caused a significant decrease in the accumulation of nitrites, and in addition, this decline was followed by a marked reduction of iNOS protein expression. Amperometrical analysis did not show any scavenging properties of dithiaden against NO.

From this data it can be suggested that the inhibition effect of dithiaden on macrophage NO production is caused exclusively by the suppression of iNOS protein expression.

Open access

Antonin Lojek, Milan Číž, Michaela Pekarová, Gabriela Ambrožová, Ondřej Vašíček, Jana Moravcová, Lukáš Kubala, Katarína Drábiková, Viera Jančinová, Tomáš Perečko, Jana Pečivová, Tatiana Mačičková and Radomír Nosáľ

Modulation of metabolic activity of phagocytes by antihistamines

The purpose of the study was to investigate the effects of H1-antihistamines of the 1st generation (antazoline, bromadryl, brompheniramine, dithiaden, cyclizine, chlorcyclizine, chlorpheniramine, clemastine) and the 2nd generation (acrivastine, ketotifen, and loratadine) on the respiratory burst of phagocytes. Reactive oxygen species generation in neutrophils isolated from rat blood was measured using luminol-enhanced chemiluminescence. Changes in nitrite formation and iNOS protein expression by RAW 264.7 macrophages were analysed using Griess reaction and Western blotting. The antioxidative properties of drugs in cell-free systems were detected spectrophotometrically, luminometrically, fluorimetrically, and amperometrically. The majority of the H1-antihistamines tested (bromadryl, brompheniramine, chlorcyclizine, chlorpheniramine, clemastine, dithiaden, and ketotifen) exhibited a significant inhibitory effect on the chemiluminescence activity of phagocytes. H1-antihistamines did not show significant scavenging properties against superoxide anion and hydroxyl radical, thus this could not contribute to the inhibition of chemiluminescence. H1-antihistamines had a different ability to modulate nitric oxide production by LPS-stimulated macrophages. Bromadryl, clemastine, and dithiaden were the most effective since they inhibited iNOS expression, which was followed by a significant reduction in nitrite levels. H1-antihistamines had no scavenging activity against nitric oxide. It can be concluded that the effects observed in the H1-antihistamines tested are not mediated exclusively via H1-receptor pathway or by direct antioxidative properties. Based on our results, antihistamines not interfering with the microbicidal mechanisms of leukocytes (antazoline, acrivastine and cyclizine) could be used preferentially in infections. Other antihistamines should be used, under pathological conditions accompanied by the overproduction of reactive oxygen species.