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.
Table 1.
IC50 values (mean±SEM) for soybean lipoxidase inhibition by naphthoquinones and quercetin, using a cell-free assay.
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 [35] 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.
Table 2.
Maximal inhibition (mean±SEM) of the main studied targets and respective naphthoquinones' concentration.