Modulation of basophils' degranulation and allergy-related enzymes by monomeric and dimeric naphthoquinones

Abstract

Allergic disorders are characterized by an abnormal immune response towards non-infectious substances, being associated with life quality reduction and potential life-threatening reactions. The increasing prevalence of allergic disorders demands for new and effective anti-allergic treatments. Here we test the anti-allergic potential of monomeric (juglone, menadione, naphthazarin, plumbagin) and dimeric (diospyrin and diosquinone) naphthoquinones. Inhibition of RBL-2H3 rat basophils' degranulation by naphthoquinones was assessed using two complementary stimuli: IgE/antigen and calcium ionophore A23187. Additionally, we tested for the inhibition of leukotrienes production in IgE/antigen-stimulated cells, and studied hyaluronidase and lipoxidase inhibition by naphthoquinones in cell-free assays. Naphthazarin (0.1 µM) decreased degranulation induced by IgE/antigen but not A23187, suggesting a mechanism upstream of the calcium increase, unlike diospyrin (10 µM) that reduced degranulation in A23187-stimulated cells. Naphthoquinones were weak hyaluronidase inhibitors, but all inhibited soybean lipoxidase with the most lipophilic diospyrin, diosquinone and menadione being the most potent, thus suggesting a mechanism of competition with natural lipophilic substrates. Menadione was the only naphthoquinone reducing leukotriene C4 production, with a maximal effect at 5 µM. This work expands the current knowledge on the biological properties of naphthoquinones, highlighting naphthazarin, diospyrin and menadione as potential lead compounds for structural modification in the process of improving and developing novel anti-allergic drugs.

Figure 1. Chemical structures of monomeric and dimeric naphthoquinones.

Figure 2. Schematic representation of protocols of RBL-2H3 basophils' degranulation assays using IgE/antigen (A) or calcium ionophore (A23187) (B) as stimuli.

Figure 3. Solvent (DMSO) effect on RBL-2H3 basophils' degranulation assays.

Effects of DMSO on cell viability (i), β-hexosaminidase (ii) and histamine release (iii) in cells stimulated with IgE/antigen (A) or with calcium ionophore (A23187) (B), n = 3–4. *P<0.05, One way ANOVA with Bonferroni post-hoc (vs. control).

Figure 4. RBL-2H3 cells' degranulation inhibition by naphthoquinones.

Effect on cell viability and β-hexosaminidase and histamine release in (A) IgE-antigen- or in (B) calcium ionophore (A23187)-stimulated cells treated with quercetin (QCT) and naphthoquinones [diospyrin (DPR), diosquinone (DQN), juglone (JGL), menadione (MND), naphthazarin (NTZ) and plumbagin (PLB)], n = 3–10. *P<0.05, paired t test to respective control (100 ng/mL IgE/DNP + 0.1% DMSO or 1 µM A23187 + 0.5% DMSO).

Figure 5. Concentration-dependent hyaluronidase inhibition by sodium cromoglycate (black circles) menadione (grey circles) and naphthazarin (white circles).

n = 3–4.

Figure 6. Lipoxidase inhibition and leukotriene C₄ production.

Soybean lipoxidase inhibition by individual naphthoquinones (A) and by quercetin (B) in a cell-free assay and inhibition of leukotriene C₄ production in IgE/antigen-stimulated RBL-2H3 by menadione (C). Grey box represents LTC₄ production of stimulated (upper) and non-stimulated (lower) control cells. n = 3–5.

Figure 7. Simplified scheme of RBL-2H3 cells' degranulation pathways.

The DNP antigen activates multiple signal transduction pathways via the IgE anti-DNP/FcεRI receptor complex. DNP receptor binding activates the immunoreceptor tyrosine activation motifs (ITAM)-Spleen tyrosine kinase (SyK) pathway that can be inhibited by shikonin and probably by naphthazarin. Activated Syk catalyses protein phosphorylation of several proteins, leading indirectly to the activation of protein kinase C (PKC) that induces degranulation and the activation of phospholipase A2 (PLA2). PLA2 increases arachidonic acid (AA) bioavailability that can be converted in leukotrienes (LT) by 5-lipoxygenase (5LO; inhibited by menadione), or in oxidized lipids by means of ROS production. 5LO converts AA into 5-hydroperoxyeicosatetraenoic acid (5-HPETE), which is metabolised to an unstable epoxide, LTA₄, and finally in LTC₄, in RBL-2H3 cells. The increase in intracellular calcium by SyK pathway, as well as by A23187 promotes degranulation.