Effects of venoms on neutrophil respiratory burst: a major inflammatory function

Neutrophils play a pivotal role in innate immunity and in the inflammatory response. Neutrophils are very motile cells that are rapidly recruited to the inflammatory site as the body first line of defense. Their bactericidal activity is due to the release into the phagocytic vacuole, called phagosome, of several toxic molecules directed against microbes. Neutrophil stimulation induces release of this arsenal into the phagosome and induces the assembly at the membrane of subunits of the NAPDH oxidase, the enzyme responsible for the production of superoxide anion that gives rise to other reactive oxygen species (ROS), a process called respiratory burst. Altogether, they are responsible for the bactericidal activity of the neutrophils. Excessive activation of neutrophils can lead to extensive release of these toxic agents, inducing tissue injury and the inflammatory reaction. Envenomation, caused by different animal species (bees, wasps, scorpions, snakes etc.), is well known to induce a local and acute inflammatory reaction, characterized by recruitment and activation of leukocytes and the release of several inflammatory mediators, including prostaglandins and cytokines. Venoms contain several molecules such as enzymes (phospholipase A2, L-amino acid oxidase and proteases, among others) and peptides (disintegrins, mastoporan, parabutoporin etc.). These molecules are able to stimulate or inhibit ROS production by neutrophils. The present review article gives a general overview of the main neutrophil functions focusing on ROS production and summarizes how venoms and venom molecules can affect this function.(AU)

O fator de crescimento dos vasos (VEGF) como mediador neuroprotetor na quebra da barreira hematoencefálica no envenenamento pela aranha "Phoneutria nigriventer" em ratos: análise das vias de sinalização e da neuroquímica cerebral por espectroscopia no infravermelho com transformada de Fourier / Vascular endothelial growth factor (VEGF) as a neuroprotector mediator in the blood-brain barrier breakdown caused by "Phoneutria nigriventer" spider envenomation in rats: analysis of signaling pathways and cerebral neurochemistry by Fourier transform infrared spectroscopy

Resumo: O veneno da aranha Phoneutria nigriventer (PNV) contém neuropeptídeos que afetam canais iônicos e a neurotransmissão, induzindo a quebra da barreira hematoencefálica (BHE) no hipocampo de ratos, o que ocorre paralelamente ao aumento do fator de crescimento endotelial vascular (VEGF). Sabe-se que a resposta biológica do VEGF é desencadeada através da regulação transcricional promovida pelo domínio tirosina-quinase de receptores transmembranares do VEGF, dos quais o VEGFR-2 (Flk-1) é considerado o principal mediador e ativador de várias vias de sinalização. O trabalho propõe investigar o possível papel neuroprotetor do VEGF após inibir sua ligação ao receptor Flk-1 pelo itraconazol (ITZ). Para isso, examinamos o status bioquímico do hipocampo por espectroscopia no Infravermelho com Transformada de Fourier (FT-IR), bem como avaliamos as proteínas envolvidas nas rotas paracelular e transcelular da BHE e quais vias de sinalização, relacionadas à neuroproteção do VEGF, foram ativadas. Os ratos receberam PNV ou foram pré-tratados com ITZ (30 min) seguido de PNV pela veia da cauda e depois sacrificados em 1 e 2 h (intervalos com maiores sinais de intoxicação), 5 h (intervalo com sinais incipientes de recuperação) e 24 h (intervalo sem sinal visual detectável de envenenamento), sendo comparados aos controles, salina e ITZ. O pré-tratamento com o antifúngico agravou os efeitos do veneno e aumentou danos à BHE. Os espectros FT-IR do veneno, hipocampo dos controles, PNV e ITZ-PNV mostraram as bandas de 1400 cm-1 (carboxilato) e de 1467 cm-1 (flexão de CH2: principalmente lipídios), que foram considerados bandas biomarcadora e referência, respectivamente. A inibição da ligação VEGF/Flk-1 produziu mudanças marcantes na estabilidade lipídios/proteínas em 1-2 h. As maiores diferenças ocorreram nas regiões espectrais atribuídas à lípides simétricos (2852 cm-1) e assimétricos (2924 e 2968 cm-1). As análises quantitativas mostraram maiores aumentos na razão 1400 cm-1/1467 cm-1 no período de intoxicação grave (1 h), e referem-se à região espectral de 3106 cm-1 a 687 cm-1. Ademais, a desativação da ligação VEGF/Flk-1 pelo itraconazol (ITZ) aumentou o fator indutor de hipóxia (H1F1-?), VEGF, Flk-1, Flt-1, Neu-N e caspase-3 às 5 horas após a injeção do PNV. No mesmo intervalo, a permeabilidade transcelular da BHE aumentou (caveolina-1?, dinamina-2 e família Src de não receptores tirosina-quinase (SKFs)), enquanto laminina e a via paracelular (occludina, ?-catenina) foram reforçadas e a proteína de efluxo glicoproteína-P (P-gp) aumentou. Ao mesmo tempo (5 h), ocorreu auto-fosforilação da via pró-proliferação celular (p38-fosforilada). Às 24 h, apesar da ausência de sinais de intoxicação, a via pró-sobrevivência celular (Akt-fosforilada) diminuiu nos animais pré-tratados com ITZ, enquanto aumentou nos tratados com PNV apenas. Os dados indicam ativação de mecanismos de neuroproteção relacionados ao VEGF envolvendo o receptor Flk-1 e principalmente à serina-treonina-quinase Akt, provavelmente via PI3K. ERK-fosforilada (2 h) e p38-fosforilada (5 h) sugerem interação entre as vias de sinalização com o objetivo de restabelecer a homeostase do hipocampo. O intervalo de 5 h parece ser o ponto de virada orquestrando respostas biológicas variadas. Os dados permitem concluir sobre o papel neuroprotetor do VEGF e que o mesmo pode ser explorado como possível alvo terapêutico no envenenamento por P. nigriventer.(AU)

Abstract: Phoneutria nigriventer spider venom (PNV) contains ion channels-acting neuropeptides that affect neurotransmission and induces transitory blood-brain barrier (BBB) breakdown in rat¿s hippocampus, which run in parallel with (vascular endothelial growth factor) VEGF upregulation. It is known that VEGF biological response is triggered through transcriptional regulation promoted by transmembrane tyrosine kinase receptors, being VEGFR-2 (Flk-1) considered the major mediator of VEGF effect through activation of a number of signaling pathways. The purpose of this work is to investigate a putative neuroprotective role of VEGF by inhibiting its binding to receptor Flk-1 by itraconazole (ITZ). To do this, we examined the biochemical status of the hippocampus by Infrared Spectroscopy and Fourier Transform (FT-IR), as well as evaluated the proteins involved in the BBB paracellular and transcellular routes and which signaling pathways related to VEGF neuroprotection were activated. Rats were administered PNV alone or were pre-treated with ITZ (30 min) followed by PNV through the tail vein, and then euthanized at 1 and 2 h (intervals with greatest signs of intoxication), 5 h (interval with incipient signs of animals¿ recovery) and 24 h (interval with no visually detectable envenomation sign) and compared to saline and ITZ controls. The antifungal pre-treatment aggravated PNV toxic effects and increased BBB damage. FT-IR spectra of venom and from hippocampi of controls, PNV and ITZ-PNV showed a 1400 cm-1 band linked to symmetric stretch of carboxylate and 1467 cm-1 band (CH2 bending: mainly lipids), which were considered biomarker and reference bands, respectively. Inhibition of VEGF/Flk-1 binding produced marked changes in lipid/protein stability at 1-2 h. The largest differences were observed in spectra regions assigned to lipids, both symmetric (2852 cm-1) and asymmetric (2924 and 2968 cm-1). Quantitative analyses showed greatest increases in the 1400 cm-1/1467 cm-1 ratio also at 1 h. Such changes at period of rats¿ severe intoxication referred to wavenumber region from 3106 cm-1 to 687 cm-1. Furthermore, the deactivation of Flk-1 receptor by VEGF through itraconazole (ITZ) showed increased hypoxia inducible factor (H1F-1?), VEGF, Flk-1, Flt-1, Neu-N and caspase-3 at 5 h after PNV injection. At same interval, BBB transcellular permeability increased (caveolin-1?, dynamin-2 and Src family of non-receptor tyrosine kinases (SKFs)), while laminin and paracellular route (occludin, ?-catenin) were reinforced and P-glycoprotein (P-gp) efflux protein was increased. Such effects were timely followed by upregulation of auto-phosphorylation of the pro-proliferation (phosphorylated-p38) pathway. At 24 h, despite absence of intoxication signs, the pro-survival (p-Akt) pathway was downregulated in animals underwent inhibition of VEGF-Flk-1 binding, whereas it was upregulated in PNV rats non-treated with ITZ. The data indicate triggering of VEGF-related mechanisms involving Flk-1 receptor and serine-threonine kinase Akt, probably via PI3K, as the main mechanism of neuroprotection. Phosphorylated ERK (2 h) and p-p38 (5 h) indicates interplay among transduction pathways likely aiming at re-establishment of hippocampal homeostasis. The findings suggest 5 h interval as the turning point that orchestrates varied biological responses. Taking together the data of the present study allow concluding that VEGF expression exerts neuroprotective role and can be explored as a possible therapeutic target in P. nigriventer envenomation.(AU)

Snake venom L-amino acid oxidases: an overview on their antitumor effects

The L-amino acid oxidases (LAAOs) constitute a major component of snake venoms and have been widely studied due to their widespread presence and various effects, such as apoptosis induction, cytotoxicity, induction and/or inhibition of platelet aggregation, hemorrhage, hemolysis, edema, as well as antimicrobial, antiparasitic and anti-HIV activities. The isolated and characterized snake venom LAAOs have become important research targets due to their potential biotechnological applications in pursuit for new drugs of interest in the scientific and medical fields. The current study discusses the antitumor effects of snake venom LAAOs described in the literature to date, highlighting the mechanisms of apoptosis induction proposed for this class of proteins.

Snake venom L-amino acid oxidases: an overview on their antitumor effects

The L-amino acid oxidases (LAAOs) constitute a major component of snake venoms and have been widely studied due to their widespread presence and various effects, such as apoptosis induction, cytotoxicity, induction and/or inhibition of platelet aggregation, hemorrhage, hemolysis, edema, as well as antimicrobial, antiparasitic and anti-HIV activities. The isolated and characterized snake venom LAAOs have become important research targets due to their potential biotechnological applications in pursuit for new drugs of interest in the scientific and medical fields. The current study discusses the antitumor effects of snake venom LAAOs described in the literature to date, highlighting the mechanisms of apoptosis induction proposed for this class of proteins.