7-Hydroxycoumarin modulates the oxidative metabolism, degranulation and microbial killing of human neutrophils
Introduction
Neutrophils are professional killers of invading microorganisms and also contribute to the regulation of inflammatory processes. These cells secrete types of chemokines, cytokines, lipid mediators, granule proteins, and reactive oxygen species (ROS) that serve as signaling molecules for subsequent recruitment of inflammatory cells. These molecules help regulate the initiation of specific T and B cell immunity and the resolution phase of inflammation [1], [2], [3]. Thus, modulating the effector functions of neutrophils is a promising therapeutic strategy to manage inflammation.
Coumarin and its derivatives have arisen as promising natural products with potent anti-inflammatory effect and low toxicity to humans [4], [5], [6], [7], [8]. They constitute a large class of phenolic compounds produced by plants of different botanical families, primarily in Angiospermae [9]. The coumarin (1,2-benzopyrone; 5,6-benzo-α-pyrone) has been clinically used to treat cancer, lymphoedema, venous insufficiency and chronic infections [5], [6], [7], [8]. Coumarin alone is ineffective in treating some diseases, but it can increase the beneficial effects of other compounds in combination therapy [8]. However, it has a short half-life in humans due to hepatic metabolism (70–90%) to 7-hydroxycoumarin (Fig. 1) and its glucuronide. Coumarin is considered a prodrug and 7-hydroxycoumarin is the bioactive molecule [6], [8].
7-Hydroxycoumarin, also known as umbelliferone, occurs in plants and edible fruits and roots, such as golden apple, bitter orange, carrot, coriander, garden angelica, and mouse-ear hawkweed [10], [11]. This coumarin displays a wide range of beneficial bioactivities: it reduces lymphoedema in humans [6] and exerts anti-hyperglycemic [12], bronchodilating [10], antinociceptive [13], and antiedematogenic [4], [14], [15] effects in rats. The in vitro and in vivo antioxidant, anti-inflammatory and immunomodulatory effects of 7-hydroxycoumarin have been recently reported [4], [5], [11], [16], [17], [18], [19], [20], [21], [22]. This compound inhibits migration of neutrophils and eosinophils [10], degranulation of mast cells [15], release of NO by macrophages [19], [20], [21], release of cytokines [10], [13], biosynthesis of prostaglandin, and activity of cycloxygenase-2 and 5-lipoxygenase [4], [5], [14]. In addition, 7-hydroxycoumarin activates the protective immune responses of mice against influenza virus [19] and Salmonella typhimurium[22].
However, there are few reports on the action of 7-hydroxycoumarin on neutrophils. 7-Hydroxycoumarin and 7-hydroxy-4-methylcoumarin inhibit the generation of by zymosan-stimulated rabbit neutrophils [23], phorbol-12-myristate-13-acetate (PMA)-stimulated human neutrophils, and n-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated rat leukocytes [5]. 7-Hydroxycoumarin also inhibits the migration of neutrophils in mouse models of inflammation [10], [13]. Therefore, the high potential of 7-hydroxycoumarin for the development of an effective anti-inflammatory drug and the fundamental contribution of neutrophils to the regulation of inflammatory processes have motivated us to evaluate the modulator effect of 7-hydroxycoumarin, 7-hydroxy-4-methylcoumarin, and their acetylated analogs (Fig. 1) on some effector functions of human neutrophils. In particular, we investigated their effects on the oxidative metabolism, degranulation, microbial killing ability, and myeloperoxidase and elastase activity of neutrophils. We also evaluated their physicochemical properties, antioxidant activity, and HOCl scavenging potential in cell-free systems.
Section snippets
Chemicals
l-Ascorbic acid (Asc), cytochalasin B, 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), N,N-dimethylformamide, 5-dimethyl-1-pyrroline-N-oxide (DMPO), α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN), horseradish peroxidase (HRP, type VI-A, EC.1.11.1.7), luminol (5-amine-2,3-dihydro-1,4-phtalazinedione), lucigenin (N,N’-dimethyl-9,9′-biacridinium dinitrate), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), phorbol-12-myristate-13-acetate (PMA), superoxide dismutase (SOD),
Modulation of the ROS generation by neutrophils
We evaluated the modulator effect of the four coumarins (1–4) on the production of and total ROS by SOZ- or PMA-stimulated human neutrophils using the lucigenin-(CL-luc) and luminol-(CL-lum) enhanced chemiluminescence assays, respectively.
All the tested coumarins inhibited the PMA- and SOZ-stimulated neutrophil CL-luc in a concentration-dependent manner (Fig. 2A–D). They inhibited the SOZ-stimulated CL-luc twice as effectively as they inhibited the PMA-stimulated CL-luc. The hydroxylated
Discussion
We assessed whether 7-hydroxycoumarin (1) and its derivatives (2–4) modulate the oxidative metabolism, elastase and myeloperoxidase activity, degranulation, and microbial killing ability of human neutrophils. We also evaluated the physicochemical properties and antioxidant effect of the coumarins, as well as their potential to scavenge HOCl in cell-free systems.
We evaluated the modulator effect of the tested coumarins on the oxidative metabolism of SOZ- or PMA-stimulated human neutrophils by CL
Conclusion
7-Hydroxycoumarin (1), 7-hydroxy-4-methylcoumarin (2), and their acetylated analogs (3 and 4, respectively) modulate the effector functions of human neutrophils studied: oxidative metabolism, degranulation and microbial killing. They act through different mechanisms, by interfering in intracellular signaling pathways, scavenging ROS, interacting with MPO and generating oxidizing species. In general, the pro- and antioxidant effect of 7-hydroxycoumarin in cellular and cell-free systems, as well
Acknowledgements
The authors thank Mr. Alcides S. Pereira and Mrs. Nadir Mazzucato for technical assistance, and Prof. Dr. Gilberto U.L. Braga (FCFRP-USP, Brazil) for providing the C. albicans strain used in the microbial killing assay. This study was supported by the Brazilian agencies: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Grants 2000/06233-7, 2000/10641-3, 2002/09518-8, 2005/60596-8, 2007/02487-3, 2007/00840-8), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES),
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2018, Free Radical Biology and MedicineCitation Excerpt :The MPO inhibitors HX1 and ABAH interact with the same amino acid residues [35]. Thus, the direct interaction of the 3-PDs with the catalytic site of MPO, associated with their intrinsic free radical scavenger effect [21] explain, at least in part, why the 3-PDs 19 and 24 inhibit the neutrophil CL-lum more strongly than the other compounds – this assay measures the overall neutrophil ROS generation, which is highly dependent on MPO activity [4]. It is worth to note that the catalytic cycle of MPO also involves the formation of free radical intermediates in the porphyrin ring, mainly compound I and compound II, as well as the transient conversion of the substrate to its free radical form [37].
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2015, BiomaterialsCitation Excerpt :After 20 days the average length of MWCNT 2 decreased from 451 nm to 324 nm with only 9% of nanotubes shorter than 150 nm. From this result we hypothesize that the 7-hydroxyl group on the benzopyran ring of the coumarin is important to enhance the activity of HRP [48]. Indeed, for the derivative covalently bound to MWCNT 1 the hydroxyl group remained free, while for the second type of nanotubes, the hydroxyl function was used to insert the linker for the coupling to the tubes.
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Present address: Unidade de Biotecnologia, Universidade de Ribeirão Preto (UNAERP), Avenida Costábile Romano 2201, Bairro Ribeirânea, 14096-380 Ribeirão Preto, SP, Brazil.
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Present address: Universidade Nove de Julho (UNINOVE), Avenida Dr. Adolpho Pinto 109, Barra Funda, São Paulo, SP 01156-050, Brazil.