RESUMEN
Cr-LAAO, an L-amino acid oxidase isolated from Calloselasma rhodosthoma snake venom, has been demonstrated as a potent stimulus for neutrophil activation and inflammatory mediator production. However, the mechanisms involved in Cr-LAAO induced neutrophil activation has not been well characterized. Here we investigated the mechanisms involved in Cr-LAAO-induced lipid body (also known as lipid droplet) biogenesis and eicosanoid formation in human neutrophils. Using microarray analysis, we show for the first time that Cr-LAAO plays a role in the up-regulation of the expression of genes involved in lipid signalling and metabolism. Those include different members of phospholipase A2, mostly cytosolic phospholipase A2-α (cPLA2-α); and enzymes involved in prostaglandin synthesis including cyclooxygenases 2 (COX-2), and prostaglandin E synthase (PTGES). In addition, genes involved in lipid droplet formation, including perilipin 2 and 3 (PLIN 2 and 3) and diacylglycerol acyltransferase 1 (DGAT1), were also upregulated. Furthermore, increased phosphorylation of cPLA2-α, lipid droplet biogenesis and PGE2 synthesis were observed in human neutrophils stimulated with Cr-LAAO. Treatment with cPLA2-α inhibitor (CAY10650) or DGAT-1 inhibitor (A922500) suppressed lipid droplets formation and PGE2 secretion. In conclusion, we demonstrate for the first time the effects of Cr-LAAO to regulate neutrophil lipid metabolism and signalling.
Asunto(s)
Venenos de Crotálidos/enzimología , Dinoprostona/metabolismo , Fosfolipasas A2 Grupo IV/metabolismo , L-Aminoácido Oxidasa/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Adolescente , Adulto , Animales , Venenos de Crotálidos/farmacología , Crotalinae/metabolismo , Citosol/metabolismo , Humanos , Técnicas In Vitro , Gotas Lipídicas/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Metabolismo de los Lípidos/genética , Modelos Biológicos , Activación Neutrófila/efectos de los fármacos , Activación Neutrófila/genética , Activación Neutrófila/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Regulación hacia Arriba/efectos de los fármacos , Adulto JovenRESUMEN
INTRODUCTION: Crotalus envenomations cause serious complications and can be fatal without appropriate treatment. Venom isoforms present and inter/intraspecific variations in the venom composition can result in different symptoms presented by bites by snakes from the same species but from different geographical regions. We comparatively evaluated the local and systemic effects caused by Crotalus durissus terrificus (Cdt), C.d. collilineatus (Cdcolli), and C.d. cascavella (Cdcasc) envenomation. METHODS: Venom chromatography was performed. Proteolytic, phospholipase, and LAAO activities were analyzed. Edema, myotoxicity, hepatotoxicity, nephrotoxicity, and coagulation alterations were evaluated. RESULTS: The venom SDS-PAGE analyses found the presence of convulxin, gyroxin, crotoxin, and crotamine in Cdt and Cdcolli venoms. Crotamine was not present in the Cdcasc venom. Cdt, Cdcollli, and Cdcasc venoms had no proteolytic activity. Only Cdcasc and Cdt venoms had phospholipase activity. LAAO activity was observed in Cdcolli and Cdcasc venoms. Cdcolli and Cdcasc venoms caused 36.7% and 13.3% edema increases, respectively. Cdt venom caused a 10% edema induction compared to those by other venoms. All venoms increased TOTAL-CK, MB-CK, and LDH levels (indicating muscle injury) and ALT, AST, GGT, and ALP levels (markers of liver damage) and were able to induce a neuromuscular blockade. Urea and creatinine levels were also altered in both plasma and urine, indicating kidney damage. Only Cdcolli and Cdcasc venoms increased TAPP and TAP. CONCLUSIONS: Together, these results allow us to draw a distinction between local and systemic effects caused by Crotalus subspecies, highlighting the clinical and biochemical effects produced by their respective venoms.
Asunto(s)
Venenos de Crotálidos/toxicidad , Crotalus/clasificación , Edema/inducido químicamente , Riñón/efectos de los fármacos , Hígado/efectos de los fármacos , Fosfatasa Alcalina/sangre , Fosfatasa Alcalina/efectos de los fármacos , Animales , Creatina Quinasa/sangre , Creatina Quinasa/efectos de los fármacos , Creatinina/sangre , Edema/patología , Electroforesis en Gel de Poliacrilamida , Riñón/patología , L-Lactato Deshidrogenasa/sangre , L-Lactato Deshidrogenasa/efectos de los fármacos , Hígado/patología , Ratones , Modelos Animales , Transaminasas/sangre , Transaminasas/efectos de los fármacos , Urea/sangreRESUMEN
Abstract INTRODUCTION: Crotalus envenomations cause serious complications and can be fatal without appropriate treatment. Venom isoforms present and inter/intraspecific variations in the venom composition can result in different symptoms presented by bites by snakes from the same species but from different geographical regions. We comparatively evaluated the local and systemic effects caused by Crotalus durissus terrificus (Cdt), C.d. collilineatus (Cdcolli), and C.d. cascavella (Cdcasc) envenomation. METHODS: Venom chromatography was performed. Proteolytic, phospholipase, and LAAO activities were analyzed. Edema, myotoxicity, hepatotoxicity, nephrotoxicity, and coagulation alterations were evaluated. RESULTS: The venom SDS-PAGE analyses found the presence of convulxin, gyroxin, crotoxin, and crotamine in Cdt and Cdcolli venoms. Crotamine was not present in the Cdcasc venom. Cdt, Cdcollli, and Cdcasc venoms had no proteolytic activity. Only Cdcasc and Cdt venoms had phospholipase activity. LAAO activity was observed in Cdcolli and Cdcasc venoms. Cdcolli and Cdcasc venoms caused 36.7% and 13.3% edema increases, respectively. Cdt venom caused a 10% edema induction compared to those by other venoms. All venoms increased TOTAL-CK, MB-CK, and LDH levels (indicating muscle injury) and ALT, AST, GGT, and ALP levels (markers of liver damage) and were able to induce a neuromuscular blockade. Urea and creatinine levels were also altered in both plasma and urine, indicating kidney damage. Only Cdcolli and Cdcasc venoms increased TAPP and TAP. CONCLUSIONS: Together, these results allow us to draw a distinction between local and systemic effects caused by Crotalus subspecies, highlighting the clinical and biochemical effects produced by their respective venoms.
Asunto(s)
Animales , Crotalus/clasificación , Venenos de Crotálidos/toxicidad , Edema/inducido químicamente , Riñón/efectos de los fármacos , Hígado/efectos de los fármacos , Urea/sangre , Creatina Quinasa/efectos de los fármacos , Creatina Quinasa/sangre , Creatinina/sangre , Modelos Animales , Edema/patología , Electroforesis en Gel de Poliacrilamida , Fosfatasa Alcalina/efectos de los fármacos , Fosfatasa Alcalina/sangre , Transaminasas/efectos de los fármacos , Transaminasas/sangre , Riñón/patología , L-Lactato Deshidrogenasa/efectos de los fármacos , L-Lactato Deshidrogenasa/sangre , Hígado/patología , RatonesRESUMEN
The action of Cr-LAAO, an l-amino acid oxidase isolated from Calloselasma rhodosthoma snake venom, on NADPH oxidase activation in isolated human neutrophil function was investigated. This enzyme has an intrinsic activity of hydrogen peroxide production. Cr-LAAO, in its native form, induces the ROS production in neutrophil and migration of cytosolic NADPH oxidase components p40phox, p47phox and p67phox to the membrane, and Rac, a GTPase protein member, with the involvement of intracellular signaling mediated by phospho PKC-α. In its inactive form, iCr-LAAO does not induce NADPH oxidase activation in neutrophil showing that the intrinsic enzymatic activity does not have a role in this process, suggesting that its primary structure is essential for the cell's stimulation. Accordingly, the data showed for the first time that the Cr-LAAO has a role in NADPH oxidase complex activation triggering relevant proinflammatory events in human neutrophils.
Asunto(s)
Venenos de Crotálidos/farmacología , L-Aminoácido Oxidasa/farmacología , NADPH Oxidasas/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Adolescente , Adulto , Animales , Venenos de Crotálidos/enzimología , Crotalinae , Activación Enzimática , Humanos , L-Aminoácido Oxidasa/aislamiento & purificación , Neutrófilos/enzimología , FosfoproteínasRESUMEN
BjcuL is a C-type lectin with specificity for the binding of ß-d-galactose units isolated from Bothrops jararacussu venom. It triggers cellular infiltration in post capillary venules, increases edema and vascular permeability in murine models, contributes to in vitro neutrophil activation and modulates macrophage functional activation towards an M1 state. The purpose of this study was to investigate the effect of BjcuL on human peripheral blood mononuclear cells (PBMCs) activation with a focus on PBMCs proliferation and inflammatory mediators release. Results showed that BjcuL is not toxic to PBMCs, that BjcuL inhibits PBMCs proliferation and that it stimulates PBMCs to produce superoxide anion and hydrogen peroxide, primarily via lymphocyte stimulation, but does not stimulate the production of nitric oxide and PGE2. These results demonstrate that BjcuL has an immunomodulatory effect on PBMCs. Further studies are needed to confirm the immunomodulatory effect of BjcuL, to elucidate the molecular mechanisms of action responsible for its effects and to determine its potential application as an immunopharmacological and biotechnological tool.
Asunto(s)
Venenos de Crotálidos/toxicidad , Factores Inmunológicos/toxicidad , Leucocitos Mononucleares/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Ciclooxigenasa 1/genética , Ciclooxigenasa 2/genética , Dinoprostona/metabolismo , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Expresión Génica/efectos de los fármacos , Hemaglutininas/metabolismo , Humanos , Lectinas Tipo C , Leucocitos Mononucleares/metabolismo , Óxido Nítrico/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The aim of the study was to evaluate the in vitro effects of Bothrops bilineata crude venom (BbV) on isolated human neutrophil function. We proved that BbV isn't toxic towards human neutrophils. During an incubation of human neutrophils with BbV hydrogen peroxide was produced. Moreover, BbV was able to stimulate neutrophil release of proinflammatory mediators such as IL-8 and IL-6 as well as PGE2 and NETs liberation. There is no data in the literature showing the effect of BbV on the production of IL-6 and IL-8 or NETs liberation by isolated human neutrophils. Taken together our results testify that BbV triggers relevant proinflammatory events in human neutrophils.