Inhibición de las actividades proteolítica y fosfolipasa A2 del veneno de Bothrops asper por el extracto etanólico de Neurolaena lobata (L) Cass / Inhibition of proteolytic and phospholipase A2 activities of Bothrops asper venom by the ethanolic extract of Neurolaena lobata (L) Cass

Neurolaena lobata es utilizada tradicionalmente en Centroamérica para tratar la mordedura de serpiente, pero su efectividad para contrarrestar el envenenamiento producido por Bothrops asper ha sido poco estudiada. Se evaluó la capacidad del extracto etanólico de sus hojas para inhibir las actividades proteolítica, fosfolipasa A2 (PLA2; evaluada como hemólisis indirecta) y coagulante del veneno in vitro. El material vegetal fue colectado en Izabal, Guatemala, secado, se hicieron extracciones con etanol y se evaluó la presencia de actividades proteolítica, PLA2 y coagulante in-trínsecas en ensayos de concentración-actividad. Los efectos inhibitorios de la actividad proteolítica y PLA2 del veneno se evaluaron después de pre-incubar concentraciones variables del extracto con concentraciones fijas de veneno. La inhibición de la actividad coagulante del veneno no fue evaluada porque el extracto presentó actividad anticoagulante intrínseca dependiente de la concentración. El extracto inhibió completamente las actividades proteolítica (CE50 = 15.7 µg/µl) y PLA2 (CE50 = 32.5 µg/µl) del veneno. El análisis fitoquímico utilizando ensayos macro y semimicrométricos de cromatografía en capa fina, demostró la presencia de flavonoides, cumarinas, saponinas, taninos, sesquiterpenlactonas y aceites esenciales en el extracto. Su efecto sobre las proteínas del veneno se evaluó por electroforesis SDS-PAGE, mostrando cambios en el patrón electroforético atribuidos a la formación de complejos moleculares con los metabo-litos del extracto. Los resultados indican que el extracto podría inhibir los efectos tóxicos del veneno inducidos por las metaloproteinasas dependientes de zinc (SVMPs) y PLA2s, pero podría afectar las alteraciones en la coagulación, coadyuvando en la desfibrinogenación inducida por el veneno.

Neurolaena lobata has been used by traditional healers in Central America to treat snakebite, but its ability to neutralize Bothrops asper envenomations needs to be proved. This study evaluated the inhibitory potential of the ethanolic extract of the leaves of N. lobata against proteolytic, phospholipase A2 (PLA2) and coagulant activities of the venom in vitro. Leaves were collected in Izabal, Guatemala, dried, extracted with ethanol and concentration-response assays were conducted to detect intrinsic proteolytic, PLA2 (evaluated as indirect hemolysis) and coagulant activities. Assays for anti-proteolytic and anti-PLA2 activities were performed after pre-incubation of several amounts of extract with a fixed concentration of venom. Inhibition assay for the coagulant effect of the venom was not tested because pre-incubation of thrombin with the extract prolonged the clotting time of plasma in a concentration-dependent manner. Proteolytic (EC50 = 15.7 µg/µl) and PLA2 (EC50 = 32.5 µg/µl) activities of the venom resulted completely inhibited by the extract. Phytochemical profiles, determined by micrometric assays and semi microanalysis by thin layer chro-matography, showed the presence of flavonoids, coumarins, saponins, tannins, sesquiterpene lactones and essential oils in the extract. SDS-PAGE was used to assess the action of the extract on the venom proteins. Results showed changes in the electrophoretic profile, probably due to the formation of insoluble complexes with plant specialized metabolites. These findings demonstrated that the extract could be able to inhibit toxic effects triggered by zinc-dependent snake venom metalloproteinases (SVMPs) y PLA2s but might aggravate the alterations induced by the venom in coagulation.

Use of protein kinase C and phospholipase A2 inhibitors in bovine endometrial cells treated with estradiol and calcium ionophore / Uso de inibidores de proteína quinase C e fosfolipase A2 em células endometriais bovinas tratadas com estradiol e ionóforo de cálcio

The release of endometrial prostaglandin-F2α (PGF2α) in bovine females can be induced in vivo by estradiol (E2). However, its role in this mechanism has not been clarified. We hypothesized that E2 stimulates the activity and abundance of protein kinase C (PKC) and phospholipase A2 (PLA2). Our objective in this study was to analyze the effects of PKC and PLA2 inhibitors on PGF2α synthesis induced by E2 and calcium ionophore (CI) in bovine endometrial cells (BEND cells; Experiment 1). Additionally, we evaluated the abundance of PKC and PLA2 in endometrial explants of cows treated or not with E2 17 days after estrus (D17, D0 = estrus; Experiment 2). In Experiment 1, BEND cells were submitted to a PKC inhibitor (10 µM of C25H24N4O2; bisindolylmaleimide I, or BIS I), a PLA2 inhibitor (20 µM of arachydoniltrifluoromethane or AACOCF3), or none. The BEND cells were subsequently treated with E2 and CI, and PGF2α concentrations were measured in the culture medium through radioimmunoassay. For DIF-12 (PGF2α concentration 12 h after treatment subtracted from PGF2α concentration at hour 0), no PKC inhibitor effect was observed (P= 0.2709). However, DIF-12 was lower (P < 0.05) for groups treated with the PLA2 inhibitor and PLA2 inhibitor + CI + E2 groups than the control and CI + E2 groups. Thus, AACOCF3 was an efficient PLA2 inhibitor in the BEND cells culture system, and E2 did not stimulate the synthesis of PKC and PLA2. In Experiment 2, cyclic Nellore heifers received none (n = 5) or 3 mg (n = 6) of 17ß-E2 on D17 and were slaughtered 2 h after administration. The abundance of PKC and PLA2 in the endometrial tissue was evaluated using Western blotting analysis. No E2 effect was observed on PKC (P = 0.08) and PLA2 (P = 0.56). We concluded that E2 did not stimulate the activity and abundance of PKC and PLA2.(AU)

A liberação endometrial de prostaglandina-F2α (PGF2α) em fêmeas bovinas pode ser induzida in vivo pelo estradiol (E2). Entretanto o seu mecanismo de ação ainda não foi bem esclarecido. Nossa hipótese é que o E2 estimula a atividade e a abundância da proteína quinase C (PKC) e da fosfolipase A2 (PLA2). Nosso objetivo com este estudo foi analizar os efeitos de inibidores de PKC e PLA2 na síntese de PGF2α induzida por E2 e ionóforo de cálcio (CI) em células endometriais bovinas (células BEND; Experimento 1). Adicionalmente, nós avaliamos a abundância de PKC e PLA2 em explantes endometriais de vacas tratadas com ou sem E2 17 dias após o estro (D17, D0 = estro; Experimento 2). No Experimento 1, células BEND foram submetidas ao inibidor de PKC (10 µM de C25H24N4O2; bisindolylmaleimide I, ou BIS I), e ao inibidor de PLA2 (20 µM de arachydoniltrifluoromethane ou AACOCF3) ou a nenhum inibidor. As células BEND foram subsequentemente tratadas com E2 e CI e concentrações de PGF2α foram mensuradas no meio de cultura por radioimunoenssaio. Para DIF-12 (concentração de PGF2α 12 horas depois do tratamento, subtraída da concentração de PGF2α na hora 0), não foi observado efeito do inibidor de PKC (P = 0.2709). Entretanto DIF-12 foi menor (P < 0.05) nos grupos tratados com inibidor de PLA2 e inibidor de PLA2 + CI + E2 quando comparados com o grupo controle e o grupo CI + E2. O AACOCF3 foi um eficiente inibidor de PLA2 em sistema de cultura de células BEND e o E2 não estimulou a síntese de PKC e PLA2. No Experimento 2, novilhas Nelore cíclicas receberam 3 mg de 17ß-E2 (n = 6) ou nenhum tratamento (n = 5) no D17 e foram abatidas duas horas depois da administração dos tratamentos. A quantidade de PKC and PLA2 no tecido endometrial foi avaliada pela técnica de Western Blotting. Não foi observado efeito do E2 sobre a PKC (P= 0.08) e nem sobre a PLA2 (P= 0.56). Conclui-se que o E2 não estimulou a atividade e abundância de PKC e PLA2.(AU)

Identification and characterization of the first endogenous phospholipase A2 inhibitor from a non-venomous tropical snake, Boa constrictor (Serpentes: Boidae)

Abstract Background: Endogenous phospholipase A2 inhibitors from snake blood (sbPLIs) have been isolated from several species around the world, with the primary function of self-protection against the action of toxic phospholipases A2. In American snakes, sbPLIs were solely described in pit vipers, in which the natural protection role is justified. In this study, we described a sbPLI in Boa constrictor (popularly known as jiboia), a non-venomous snake species from America. Methods: PLA2 inhibitory activity was tested in the blood plasma of B. constrictor using C. d. terrificus venom as the enzyme source. Antibodies developed against CNF, a sbγPLI from Crotalus durissus terrificus, were used to investigate the presence of homologues in the blood plasma of B. constrictor. A CNF-like molecule with a PLA2 inhibitory activity was purified by column chromatography. The encoding gene for the inhibitor was cloned from B. constrictor liver tissue. The DNA fragment was cloned, purified and sequenced. The deduced primary sequence of interest was aligned with known sbγPLIs from the literature. Results: The blood plasma of B. constrictor displayed PLA2 inhibitory activity. A CNF-like molecule (named BcNF) was identified and purified from the blood plasma of B. constrictor. Basic properties such as molecular mass, composing amino acids, and pI were comparable, but BcNF displayed reduced specific activity in PLA2 inhibition. BcNF showed highest identity scores (ISs) with sbγPLIs from pit vipers from Latin America (90-100%), followed by gamma inhibitors from Asian viperid (80-90%). ISs below 70% were obtained for BcNF and non-venomous species from Asia. Conclusion: A functional sbγPLI (BcNF) was described in the blood plasma of B. constrictor. BcNF displayed higher primary identity with sbγPLIs from Viperidae than to sbγPLIs from non-venomous species from Asia. The physiological role played by sbγPLIs in non-venomous snake species remains to be understood. Further investigation is needed.(AU)

Effect of phospholipase A2 inhibitors during infection caused by Leishmania (Leishmania) amazonensis

Lipid metabolites play an important role in parasite differentiation and virulence. Studies have revealed that Leishmania sp. uses prostaglandins to evade innate barriers, thus enabling the parasites to survive inside immune cells. Despite the role of the enzyme Phospholipase A2 (PLA2) in prostaglandins production, few studies have investigated the role of parasite PLA2 during the interaction between L. (L.) amazonensis and the host (in vitro and in vivo) immune cells. Methods: In the present work, the leishmanicidal effect of PLA2 inhibitors, methyl arachidonyl fluorophosphonate (MAFP), bromoenol lactone (BEL) and aristolochic acid (AA) were investigated in vitro (promastigote and intracellular amastigote forms of L. (L.) amazonensis) and during in vivo infection using BALB/c mice. Results: The aforementioned inhibitors were deleterious to promastigote and amastigote forms of the L. (L.) amazonensis and were non-toxic to peritoneal macrophages from BALB/c mice. L. (L.) amazonensis-infected BALB/c mice treated with the inhibitor BEL presented decreased lesion size and skin parasitism; however, BEL treatment induced hepatotoxicity in BALB/c mice. Conclusions: Results presented herein suggested that PLA2 inhibitors altered L. (L.) amazonensis viability. In spite of liver toxicity, treatment with BEL was the most selective compound in vitro, as well in vivo, resulting in lower skin parasitism in the infected mice. These findings corroborate the role of PLA2 in parasite virulence and maintenance in vertebrate hosts, and suggest that molecules structurally related to BEL should be considered when planning compounds against Leishmania sp.(AU)

Preparation of monoclonal antibodies against gamma-type phospholipase A2 inhibitors and immunodetection of these proteins in snake blood

Background: Paracoccidioidomycosis (PCM) is a neglected systemic mycosis caused by a dimorphic fungus of the Paracoccidioides genus. The standard diagnosis is based on isolation of the fungi in culture, and by microscopic visualization of characteristic multiple budding yeast cells in biological samples. However, in some situations, access to the site of injury prevents the collection of biological material. A variety of immuno-serological techniques has proven useful for allowing inferring diagnosis with a certain degree of certainty, thus optimizing time. The aim of this study was to standardize and validate the Dot-ELISA (DE) assay, comparing it with the serological standard, double immunodiffusion (DI). Methods: In order to standardize the DE assay, 143 serum samples were used. Out of those, 23 were from apparently healthy patients, 77 were from patients with confirmed PCM and 43 were from patients with other lung infections (tuberculosis, aspergillosis and histoplasmosis). To validate the DE technique, 300 serum samples from patients with PCM clinical suspicion (probable and possible cases) were employed, and these results were compared with those of DI. Results: The DE assay showed sensitivity of 91%, specificity of 95.4%, positive predictive value of 96%, negative predictive value of 98.2%, accuracy of 93%, and great precision (k = 0.93). In addition, the nitrocellulose membranes have proved to be viable for using at least 90 days after P. brasiliensis B-339 antigen sensitization. Conclusion: Dot-ELISA method was found to be an extremely promising tool as serologic screening technique, because of its high sensitivity. Furthermore, Dot-ELISA shows the prospect of being transferred to laboratories of mycoserology including those with fewer resources or even to be used directly in the field. It has an excellent shelf life ­ membranes coated with antigen can be used for testing without changes in the pattern of reactivity among laboratories ­ and presents reliable values of sensitivity, specificity, predictive values, accuracy and a high correlation with the serological standard methodology. Based on the present findings, it possible to state that this technique constitutes a remarkable option to be used in routine diagnosis for public health centers.(AU)

Alpha-type phospholipase A2 inhibitors from snake blood

It is of popular and scientific knowledge that toxins from snake venom (among them the PLA2 and myotoxins) are neutralized by various compounds, such as antibodies and proteins purified from animal blood. Venomous and nonvenomous snakes have PLA2 inhibitory proteins, called PLIs, in their blood serum. One hypothesis that could explain the presence of these PLIs in the serum of venomous snakes would be self-protection against the enzymes of their own venom, which eventually could reach the circulatory system. However, the presence of PLIs in non-venomous snakes suggests that their physiological role might not be restricted to protection against PLA2 toxins, but could be extended to other functions, as in the innate immune system and local regulation of PLA2s. The present study aimed to review the currently available literature on PLA2 and myotoxin alpha inhibitors present in snake plasma, thus helping to improve the research on these molecules. Furthermore, this review includes current information regarding the mechanism of action of these inhibitors in an attempt to better understand their application, and proposes the use of these molecules as new models in snakebite therapy. These molecules may help in the neutralization of different types of phospholipases A2 and myotoxins, complementing the conventional serum therapy.(AU)

Darapladib, a Lipoprotein-Associated Phospholipase A2 Inhibitor, Reduces Rho Kinase Activity in Atherosclerosis

PURPOSE: Increased lipoprotein-associated phospholipase A2 (Lp-PLA2) activity and Rho kinase activity may be associated with atherosclerosis. The principal aim of this study was to examine whether darapladib (a selective Lp-PLA2 inhibitor) could reduce the elevated Lp-PLA2 and Rho kinase activity in atherosclerosis. MATERIALS AND METHODS: Studies were performed in male Sprague-Dawley rats. The atherosclerosis rats were prepared by feeding them with a high-cholesterol diet for 10 weeks. Low-dose darapladib (25 mg.kg-1.d-1) and high-dose darapladib (50 mg.kg-1.d-1) interventions were then administered over the course of 2 weeks. RESULTS: The serum levels of triglycerides, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), high-sensitivity C-reactive protein (hs-CRP), and Lp-PLA2, significantly increased in atherosclerosis model groups, as did Rho kinase activity and cardiomyocyte apoptosis (p0.05). CONCLUSION: Darapladib, a Lp-PLA2 inhibitor, leads to cardiovascular protection that might be mediated by its inhibition of both Rho kinase and Lp-PLA2 in atherosclerosis.

Endogenous phospholipase A2 inhibitors in snakes: a brief overview

The blood plasma of numerous snake species naturally comprises endogenous phospholipase A2 inhibitors, which primarily neutralize toxic phospholipases A2 that may eventually reach their circulation. This inhibitor type is generally known as snake blood phospholipase A2 inhibitors (sbPLIs). Most, if not all sbPLIs are oligomeric glycosylated proteins, although the carbohydrate moiety may not be essential for PLA2 inhibition in every case. The presently known sbPLIs belong to one of three structural classes - namely sbαPLI, sbβPLI or sbγPLI - depending on the presence of characteristic C-type lectin-like domains, leucine-rich repeats or three-finger motifs, respectively. Currently, the most numerous inhibitors described in the literature are sbαPLIs and sbγPLIs, whereas sbβPLIs are rare. When the target PLA2 is a Lys49 homolog or an Asp49 myotoxin, the sbPLI is denominated a myotoxin inhibitor protein (MIP). In this brief overview, the most relevant data on sbPLIs will be presented. Representative examples of sbαPLIs and sbγPLIs from two Old World - Gloydius brevicaudus and Malayopython reticulatus - and two New World - Bothrops alternatus and Crotalus durissus terrificus - snake species will be emphasized.(AU)

Lipoprotein associated phospholipase A2 activity & its correlation with oxidized LDL & glycaemic status in early stages of type-2 diabetes mellitus.

Background & objectives: Lipoprotein associated phospholipase A2 (Lp-PLA2) is an important risk predictor of coronary artery disease (CAD). This study was aimed to evaluate Lp-PLA2 activity and oxidized low density lipoprotein (oxLDL) in newly diagnosed patients of type 2 diabetes mellitus and to determine the correlation of Lp-PLA2 activity with oxLDL and plasma glucose levels. Methods: Blood samples were collected in patients with newly diagnosed type 2 diabetes (n=40) before any treatment was started and healthy controls (n=40). These were processed for estimating plasma glucose: fasting and post prandial, ox LDL, and Lp-PLA2 activity. The parameters in the two groups were compared. Correlation between different parameters was calculated by Pearson correlation analysis in both groups. Results: Lp-PLA2 activity (24.48 ± 4.91 vs 18.63 ± 5.29 nmol/min/ml, P<0.001) and oxLDL levels (52.46 ± 40.19 vs 33.26 ± 12.54 μmol/l, P<0.01) were significantly higher in patients as compared to those in controls. Lp-PLA2 activity correlated positively with oxLDL in both controls (r=0.414, P<0.01), as well in patients (r=0.542, P<0.01). A positive correlation between Lp-PLA2 activity and fasting plasma glucose levels was observed only in patients (r=0.348, P<0.05). Interpretation & conclusions: Result of this study implies that higher risk of CAD in patients with diabetes may be due to increase in Lp-PLA2 activity during the early course of the disease. A positive correlation between enzyme activity and fasting plasma glucose indicates an association between hyperglycaemia and increased activity of Lp-PLA2. This may explain a higher occurrence of CAD in patients with diabetes. A positive correlation between oxLDL and Lp-PLA2 activity suggests that Lp-PLA2 activity may be affected by oxLDL also.

Controlling arachidonic acid metabolic network: from single- to multi-target inhibitors of key enzymes / 药学学报

Inflammatory diseases are common medical conditions seen in disorders of human immune system. There is a great demand for anti-inflammatory drugs. There are major inflammatory mediators in arachidonic acid metabolic network. Several enzymes in this network have been used as key targets for the development of anti-inflammatory drugs. However, specific single-target inhibitors can not sufficiently control the network balance and may cause side effects at the same time. Most inflammation induced diseases come from the complicated coupling of inflammatory cascades involving multiple targets. In order to treat these complicated diseases, drugs that can intervene multi-targets at the same time attracted much attention. The goal of this review is mainly focused on the key enzymes in arachidonic acid metabolic network, such as phospholipase A2, cyclooxygenase, 5-lipoxygenase and eukotriene A4 hydrolase. Advance in single target and multi-targe inhibitors is summarized.