Caracterização dos efeitos inflamatórios induzidos pela peçonha da vespa Parachartergus fraternus – estudos in vivo e in vitro

Abstract

Wasp venom has compounds that can trigger different reactions in victims, one of them is inflammation. On the other hand, these same compounds, when isolated can be useful as potential therapeutic tools. Among wasp species Parachartergus fraternus is endemic in Latin America. Recent studies have shown that venom of this wasp was able to induce edema in mice and that peptides isolated from the venom had antinociceptive activity. However, venom involvement in different aspects of inflammatory reaction, mechanisms and participation of isolated compounds have not been investigated. The aim of present work was to characterize in vivo and in vitro the inflammatory process induced by P. fraternus venom (Pfv), and the antimicrobial and cytotoxic potential of isolated peptides and derivatives of Pfv. For in vivo assays, male Swiss mice (n = 7/group, 18-25g) and Pfv at doses of 100, 200 and 400 μg/kg were used. In in vitro assays, mouse peritoneal macrophages and Pfv were used at concentrations of 2.5, 5 and 10 μg/mL. Peptides were evaluated at concentrations of 0.03 μM to 50 μM, according to each assay. The results were expressed as mean ± SEM; ANOVA; Bonferroni; p <0.05. In in vivo assays, it was observed that Pfv triggered paw edema in mice with a peak at the dose of 200 μg/kg, at 10 min. The treatments with the drugs dexamethasone, indomethacin, celecoxib, MK886, loratadine, cimetidine, cyproheptadine, captopril and L-NAME reduced this reaction. Pfv also increased vascular permeability at all doses and between 15 and 60 min. Pfv (200 μg/kg) induced mast cell degranulation in all periods, with peak at 15 min. This same dose induced leukocyte influx into peritoneal cavity of mice with a predominance of mononuclear cells in all periods. The increase in mRNA expression for COX-2, iNOS and IFN-γ occurred 1 and 6 hours after Pfv injection. An increase in IL-10 expression was also detected 48 hours after injection. Histopathological analysis showed a marked infiltration of macrophages and mast cells in the animals' skin region where the venom was injected. In in vitro assays, Pfv was not cytotoxic in the MTT assay and did not cause macrophage detachment in cell culture plates. However, it increased the adhesion of macrophages, spreading and phagocytosis, as well as induced the release of hydrogen peroxide and nitric oxide. In addition, circular dichroism and NMR assays performed with isolated peptides (agelaia-MPI and protonectin) and Pfv derivates (neuroVAL and protonectin-F) indicated the presence of a-helical structure using the solvent TFE 30%. All peptides showed antimicrobial and antitumor activity, with the exception of neuroVAL. Agelaia-MPI was the only hemolytic peptide. All peptides internalized normal and tumor cells, with protonectin and protonectin-F accumulating in both cell types. In conclusion, Pfv induced an inflammatory process causing vascular and cellular changes in vivo and activating macrophages in vitro. Still, the isolated peptides and derivates of Pfv have pharmacological potential, mainly neuroVAL, which can be used as a cell penetrating peptide, and protonectin and protonectin-F as antimicrobial and antitumor.