RESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Snakebite is a severe problem in many parts of the world, specifically in tropical and subtropical regions. A range of medicinal plant extracts are administered for treating snake bite. Of the many common plants, extracts of Citrus species have been documented to be used for treating snake bite and have been shown to decrease the snake venom toxicity. AIM: The aim of the current work is to evaluate the utility of citrus peel extracts (Citrus aurantium L. and Citrus reticulate Blanco) in the management of Indian cobra envenomation. MATERIALS AND METHODS: Peels of citrus species were evaluated for their phospholipase A2, protease and haemolytic inhibition properties. The phytochemicals present in the extract were inferred using GC-MS. In-vivo studies, using mice model, were done to confirm the inhibitory effect of the extracts. Molecular docking was used to understand the possible binding modes of selected phytochemicals to snake venom phospholipase. RESULTS: Citrus peel extracts are rich in polyphenols, flavonoids and tannins. The methanolic extract of Citrus aurantium L. and Citrus reticulate Blanco inhibits phospholipase (75%), protease (71%) and hemolysis (80%) activity of the venom. GC-MS analyses indicate the presence of ß-sitosterol, n-hexadecanoic acid, eicosanoic acid, and flavone in both the extracts. In addition, C. reticulate extract contains α-tocopherol and squalene. Molecular docking revealed that α-tocopherol, spiro [androst-5-ene-17,1'-cyclobutan]-2'-one,3-hydroxy-(3ß,17ß)- and ß-sitosterol acetate bind with moderate affinity to the catalytic site of phospholipase A2. CONCLUSION: The present study provides new molecular insight and scientific evidence on the utility of the methanolic extracts of citrus peels to neutralize the venom toxins of Naja naja.
Assuntos
Citrus , Venenos Elapídicos/enzimologia , Naja naja , Inibidores de Fosfolipase A2/farmacologia , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/farmacologia , Inibidores de Proteases/farmacologia , Proteínas de Répteis/antagonistas & inibidores , Animais , Hemólise/efeitos dos fármacos , Masculino , Camundongos , Peptídeo Hidrolases/metabolismo , Fosfolipases A2/metabolismo , Compostos Fitoquímicos/análise , Extratos Vegetais/química , Proteínas de Répteis/metabolismoRESUMO
Present study was carried out regarding enzymatic assay for 5'-nucleotidase enzymes present in snake venom Naja naja karachiensis and to evaluate twenty eight medicinal plants as their antidotes. Elevated enzymatic activities i.e., 119, 183, 262 and 335 U/mL were observed in 10, 20, 30 and 40 µg of crude venom, respectively, in dose dependent manner. Among various plant extracts only two (Bauhinia vaiiegate L. and Citms linion (L.) Burm. f.) were found 94% effective at 160 µg to neutralize 112 U/mL activities (p 0.5) while reference standard was proved 93.2% useful at 80 pg to halt 111 U/mL activities. Cedrus deodara G. Don, Enicostemna hyssopifolium (Willd.) Verdoom, Terminalia arjuma Wight & Am. and Zingiber officinalis Rosc. (at 160 µg) were found ≥90% effective (0.5 ≥ p ≥ 0.1) while Citrulus colocynthis, Fogonia cretica L., Rhazya stticta Dcne and Stenolobiun stans (L.) D. Don (at 320 µg) were proved 90% effective (0.05 ≥ p ≥ 0.02). The remaining plant extracts were observed abortive (p ≥ 0.001) in neutralization of 5'-nucleotidases enzymatic actions. This study emphasizes further characterization of active plant extracts to further explore the antivenom influences of these herbal remedies against deleterious effects produced by 5'-nucleotidase enzymes after snake bite envenomation.
Assuntos
5'-Nucleotidase/análise , 5'-Nucleotidase/toxicidade , Venenos Elapídicos/enzimologia , Elapidae , Plantas Medicinais/química , 5'-Nucleotidase/antagonistas & inibidores , Animais , Antivenenos/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Extratos Vegetais/farmacologia , Mordeduras de Serpentes/tratamento farmacológicoRESUMO
Context Withania somnifera (L.) Dunal is traditionally used for treating various ailments, but lacks scientific evaluation. Objective This study evaluates Withania somnifera (WS) for its effect on platelet activity and inflammatory enzymes. Materials and methods Aqueous and ethanolic (1:1) leaf extracts were subjected to in vitro indirect haemolytic activity using Naja naja venom, human platelet aggregation was quantified for lipid peroxidation using arachidonic acid (AA) as agonist and 5-lipoxygenase (5-LOX) levels were determined using standard spectrometric assays. Further, molecular docking was performed by the ligand fit method using molegro software package (Molegro ApS, Aarhus, Denmark). Results The study found that aqueous and ethanol extracts have very negligible effect (15%) with an IC50 value of 13.8 mg/mL on PLA2 from Naja naja venom. Further, extracts of WS also had very little effect (18%) with an IC50 value of 16.6 mg/mL on malondialdehyde (MDA) formation. However, a 65% inhibition of 5-LOX with an IC50 value of 0.92 mg/mL was observed in 1:1 ethanol extracts. The same was evident from SAR model with the active ingredient withaferin A binding predominantly on Phe 77, Tyr 98, Arg 99, Asp 164, Leu 168, Ser 382, Arg 395, Tyr 396 and Tyr 614 with an atomic contact energy value of -128.96 compared to standard phenidone (-103.61). Thus, the current study validates the application of WS for inflammatory diseases. Conclusion This study reveals the inhibitory potential of W. somnifera on inflammatory enzymes and platelet aggregation. Thus, WS can serve as a newer, safer and affordable medicine for inflammatory diseases.
Assuntos
Anti-Inflamatórios/farmacologia , Plaquetas/efeitos dos fármacos , Simulação de Acoplamento Molecular , Extratos Vegetais/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Withania , Anti-Inflamatórios/isolamento & purificação , Plaquetas/enzimologia , Inibidores de Ciclo-Oxigenase/isolamento & purificação , Inibidores de Ciclo-Oxigenase/farmacologia , Venenos Elapídicos/enzimologia , Etanol/química , Hemólise/efeitos dos fármacos , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Inibidores de Lipoxigenase/isolamento & purificação , Inibidores de Lipoxigenase/farmacologia , Estrutura Molecular , Inibidores de Fosfolipase A2/isolamento & purificação , Inibidores de Fosfolipase A2/farmacologia , Fosfolipases A2 Secretórias/metabolismo , Fitoterapia , Extratos Vegetais/isolamento & purificação , Folhas de Planta , Plantas Medicinais , Inibidores da Agregação Plaquetária/isolamento & purificação , Solventes/química , Relação Estrutura-Atividade , Withania/química , Vitanolídeos/isolamento & purificação , Vitanolídeos/farmacologiaRESUMO
Phospholipases A2 (PLA2) are the most lethal and noxious component of Naja naja karachiensis venom. They are engaged to induce severe toxicities after their penetration in victims. Present study was designed to highlight hydrolytic actions of PLA. in an egg yolk mixture and to encounter their deleterious effects via medicinal plants of Pakistan. PLA2 were found to produce free fatty acids in a dose dependent manner. Venom at concentration of 0.1 mg was found to liberate 26.6 pmoles of fatty acids with a decline in pH1 of 0.2 owing to the presence of PLA2 (133 Unit/mg). When quantity of venom was increased up to 8 mg, it caused to release 133 pmoles of free fatty acids with a decrease in 1.0 pH due to abundance in PLA, (665 Unit/mg). The rest of other doses of venom (0.3-4.0 mg) was found to liberate fatty acids between these two upper and lower limits. Twenty eight medicinal plants (0.1-0.6 mg) were tried to abort PLA, hydrolytic action, however, all were found useful (50-100%) against PLA,. Bauhinia variegate L., Citrus limon (L.). Burm.f. Enicostemnma hyssopifolium (Willd.) Verdoorn, Ocimum sanctum. Psoralea corylifolia L. and Stenolobium stans (L.) D. Don were found excellent in switching off 100% phospholipases A, at their lowest concentration (0.1 mg). Three plants extract were found useful only at lower concentration (0.1 mg), however, their higher doses were seemed to aggravate venom response. Eight medicinal plants failed to neutralize PLA, rather their higher doses were found effective. Standard antidote and rest of other plants extract were able to show maximum of 50% efficiencies. Therefore, it is necessary to identify and isolate bioactive constituent(s) from above cited six medicinal plants to eradicate the problem of snake bite in the future.
Assuntos
Venenos Elapídicos/enzimologia , Elapidae , Fosfolipases A2/toxicidade , Extratos Vegetais/farmacologia , Plantas Medicinais , Animais , PaquistãoRESUMO
To elucidate the anti-venom mechanism of persimmon tannin, the interaction between a polymeric persimmon proanthocyanidin fraction (PT40) and phospholipase A2 (PLA2) or bovine serum albumin (BSA) were studied using a competitive binding assay and spectroscopic methods including Fourier transform infrared spectroscopy (FT-IR), circular dichroism (CD), and resonance light scattering (RLS) spectroscopy. The results revealed that PT40 has a higher affinity for PLA2 than for BSA at physiological pH and induced greater conformational changes in PLA2 than in BSA. PT40 covalently bound to PLA2 in a reaction probably involving Lys residues. We propose that the high affinity of PT40 for PLA2 and the covalent modification of PLA2 by PT40 may be responsible for the ability of the tannin to irreversibly inhibit PLA2 catalytic activity, to prevent edema, and to neutralize the lethality of Chinese cobra PLA2 in vivo.
Assuntos
Diospyros/química , Venenos Elapídicos/enzimologia , Fosfolipases A2/química , Proantocianidinas/química , Soroalbumina Bovina/química , Animais , Ligação Competitiva , Bovinos , Dicroísmo Circular/métodos , Elapidae/fisiologia , Luz , Inibidores de Fosfolipase A2 , Extratos Vegetais/química , Espalhamento de Radiação , Espectroscopia de Infravermelho com Transformada de Fourier/métodosRESUMO
Luffa egyptiaca and Nicotiana rustica are used in traditional medicine to treat snakebites and were evaluated for inhibitory activities on Naja nigricolis venom protease. The aqueous and ethanolic extracts of L. egyptiaca significantly reduced the maximum velocity (Vmax) and the computed index of physiological efficiency (Kcat) of the enzyme in a dose dependent fashion. The protease activity was non-competitively inhibited by the aqueous extract of N. rustica with the Vmax significantly decreased and the K(M) remained unchanged. However, the N. rustica ethanol extract completely inhibited the protease activity. Ethyl acetate fractions partitioned from ethanol extracts of both plants were also found to completely inhibit the N. nigricolis venom protease activity at 0.1 and 0.05%. The use of these plants could be important in the treatment of snakebites.
Assuntos
Venenos Elapídicos/enzimologia , Elapidae , Luffa/química , Nicotiana/química , Extratos Vegetais/farmacologia , Inibidores de Proteases/farmacologia , Animais , Peptídeo Hidrolases/metabolismo , Extratos Vegetais/isolamento & purificação , Inibidores de Proteases/isolamento & purificaçãoRESUMO
AIM OF THE STUDY: Evaluations of the anti-snake venom efficacy of Mimosa pudica tannin isolate (MPT) obtained from root of the plant. MATERIALS AND METHOD: MPT was investigated in vitro and in vivo for its efficacy against the venom of Naja kaouthia snake. RESULTS: In vitro: (1) mice injected i.p. with MPT pre-incubated with Naja kaouthia venom at concentrations as low as 0.625 mg/ml showed 100% survival after a 24-h observation period. (2) In the proteomics study, mice injected with MPT pre-incubated with the Naja kaouthia venom showed down-regulation of five serum proteins. (3) In the protein-dye-binding study, the percentage of Bradford dye-protein binding showed a reduction relative to the decrease in MPT concentration used to incubate with the venom. In vivo: the results from the animal studies showed that MPT had no in vivo protection against the Naja kaouthia venom (0.875 mg/kg) in four different rescue modes and in an oral pre-treatment experiment. CONCLUSION: The study indicated the promising ability of MPT to neutralize the Naja kaouthia venom in in vitro experiments but fell short in its in vivo potential. As such, the use of Mimosa pudica (Mimosaceae) as therapeutics for snake bites is questionable as all the possible in vivo rescue studies and pre-treatment of the active constituents showed no protection against the affected mice.
Assuntos
Antivenenos/farmacologia , Venenos Elapídicos/toxicidade , Elapidae , Mimosa , Mordeduras de Serpentes/tratamento farmacológico , Taninos/farmacologia , Animais , Antivenenos/isolamento & purificação , Antivenenos/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Venenos Elapídicos/enzimologia , Masculino , Camundongos , Mimosa/química , Raízes de Plantas , Ligação Proteica , Proteômica/métodos , Mordeduras de Serpentes/sangue , Taninos/isolamento & purificação , Taninos/metabolismoRESUMO
Venom hyaluronidases help in rapid spreading of the toxins by destroying the integrity of the extra-cellular matrix of the tissues in the victims. A hyaluronidase inhibitor (WSG) is purified from a folk medicinal plant, Withania somnifera. The glycoprotein inhibited the hyaluronidase activity of cobra (Naja naja) and viper (Daboia russelii) venoms, which was demonstrated by zymogram assay and staining of the skin tissues for differential activity. WSG completely inhibited the activity of the enzyme at a concentration of 1:1 w/w of venom to WSG. Thus we are able to demonstrate that the glycoprotein inhibits hyaluronidase activity of the venoms. External application of the plant extract as an antidote in rural parts of India to snakebite victims appears to have a scientific basis.
Assuntos
Antivenenos/farmacologia , Daboia , Venenos Elapídicos/enzimologia , Elapidae , Glicoproteínas/farmacologia , Hialuronoglucosaminidase/antagonistas & inibidores , Venenos de Víboras/enzimologia , Withania/química , Animais , Antivenenos/isolamento & purificação , Venenos Elapídicos/toxicidade , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Glicoproteínas/isolamento & purificação , Humanos , Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/metabolismo , Técnicas In Vitro , Plantas Medicinais , Pele/efeitos dos fármacos , Pele/metabolismo , Venenos de Víboras/toxicidadeRESUMO
A post-synaptic neurotoxic phospholipase A(2) (PLA(2)) has been purified from Indian cobra Naja naja venom. It was associated with a peptide in the venom. The association was disrupted using 8 M urea. It is denoted to be a basic protein by its behavior on both ion exchange chromatography and electrophoresis. It is toxic to mice, LD(50) 1.9 mg/kg body weight (ip). It is proved to be post-synaptic PLA(2) by chymographic experiment using frog nerve-muscle preparation. A glycoprotein, (WSG) was isolated from a folk medicinal plant Withania somnifera. The WSG inhibited the phospholipase A(2) activity of NN-XIa-PLA(2,) isolated from the cobra venom, completely at a mole-to-mole ratio of 1:2 (NN-XIa-PLA(2): WSG) but failed to neutralize the toxicity of the molecule. However, it reduced the toxicity as well as prolonged the death time of the experimental mice approximately 10 times when compared to venom alone. The WSG also inhibited several other PLA(2) isoforms from the venom to varying extent. The interaction of the WSG with the PLA(2) is confirmed by fluorescence quenching and gel-permeation chromatography. Chemical modification of the active histidine residue of PLA(2) using p-brophenacyl bromide resulted in the loss of both catalytic activity as well as neurotoxicity of the molecule. These findings suggest that the venom PLA(2) has multiple sites on it; perhaps some of them are overlapping. Application of the plant extract on snakebite wound confirms the medicinal value associated with the plant.
Assuntos
Proteínas Neurotóxicas de Elapídeos/antagonistas & inibidores , Venenos Elapídicos/enzimologia , Glicoproteínas/farmacologia , Fosfolipases A/antagonistas & inibidores , Fosfolipases A/isolamento & purificação , Withania/química , Animais , Anuros , Sobrevivência Celular/efeitos dos fármacos , Proteínas Neurotóxicas de Elapídeos/isolamento & purificação , Relação Dose-Resposta a Droga , Elapidae , Eletroforese em Gel de Ágar , Feminino , Glicoproteínas/isolamento & purificação , Técnicas In Vitro , Masculino , Camundongos , Músculo Esquelético , Fosfolipases A2 , Fitoterapia , Raízes de Plantas/química , Espectrometria de FluorescênciaRESUMO
Trimeresurus malabaricus is an endemic snake found in the Southern region of Western Ghats section of India along with the more widely distributed species like Naja naja and Daboia russelii. T. malabaricus venom is not lethal when injected (i.p.) up to 20 mg/kg body weight in mice, but causes extensive local tissue degeneration. N. naja and D. russelii are highly toxic (i.p.) with minimum local tissue damage in experimental mice. In this study a comparative analysis of local tissue damage of T. malabaricus venom is made with N. naja and D. russelii snake venoms of the Southern regions of Western Ghats. T. malabaricus venom exhibits caseinolytic activity 16 and 24 times more than N. naja and D. russelii venom. Inhibition studies with specific protease inhibitors reveal that the major proteases belong to metalloproteases. T. malabaricus venom hydrolyses gelatin and induces strong hemorrhagic activity in mice. Both N. naja and D. russelii fail to hydrolyze gelatin even at very high concentration and did not induce any hemorrhagic activity. With D. russelii venom small hemorrhagic spot was observed at the site of injection. The hemorrhagic activity of T. malabaricus venom is completely neutralized by metalloprotease inhibitors and not by serine protease inhibitor. The i.m. injection of T. malabaricus venom causes extensive degradation of muscle tissue within 24 h. The light microscopic observation of muscle tissue showed congestion of blood vessels and hemorrhage at the early stage followed by extensive necrosis of muscle fibers. The elevated levels of serum CK and LDH activity further supported the muscle degeneration. Such pathological symptoms were not seen with N. naja and D. russelii snake venom. The hemorrhagic and the muscle necrosis was completely neutralized by metalloprotease inhibitors and not by serine protease inhibitor strongly suggests that the major toxin component in the T. malabaricus venom is metalloprotease and its activity can be easily neutralized using chelating agents and its use in the first aid as chelation therapy is beneficial.
Assuntos
Daboia , Elapidae , Metaloproteases/metabolismo , Músculo Esquelético/patologia , Trimeresurus , Venenos de Víboras/toxicidade , Animais , Venenos de Crotalídeos/química , Venenos de Crotalídeos/enzimologia , Venenos de Crotalídeos/toxicidade , Venenos Elapídicos/química , Venenos Elapídicos/enzimologia , Venenos Elapídicos/toxicidade , Gelatina/química , Hemorragia/induzido quimicamente , Hemorragia/patologia , Hidrólise , Metaloproteases/antagonistas & inibidores , Camundongos , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/efeitos dos fármacos , Necrose , Inibidores de Proteases/farmacologia , Dermatopatias/induzido quimicamente , Dermatopatias/patologia , Venenos de Víboras/química , Venenos de Víboras/enzimologiaRESUMO
The inhibitory effect of several bioactive compounds on the activity of hyaluronidase enzyme purified from Naja naja venom was investigated in vitro. Compounds were found to inhibit the hyaluronidase activity dose dependently. Among glycosaminoglycans, heparin, heparan sulfate, and dermatan sulfate showed maximum inhibition compared to chondroitin sulfates. Different molecular forms of chitosan inhibit the enzyme, and inhibition appears to depend on the chain length. In addition, plant-derived bioactive compounds also inhibited the activity of hyaluronidase dose dependently. Among those tested, aristolochic acid, indomethacin, quercetin, curcumin, tannic acid, and flavone exhibited inhibition, with aristolochic acid and quercetin completely inhibiting the enzyme activity. It is concluded that the inhibitors of hyaluronidase could be used as potent first aid agents in snakebite therapy. Furthermore, these inhibitors not only reduce the local tissue damage but also retard the easy diffusion of systemic toxins and hence increase survival time.
Assuntos
Venenos Elapídicos/enzimologia , Elapidae , Hialuronoglucosaminidase/antagonistas & inibidores , Preparações de Plantas/farmacologia , Polissacarídeos/farmacologia , Alcaloides/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Quitosana/farmacologia , Curcumina/farmacologia , Dexametasona/farmacologia , Relação Dose-Resposta a Droga , Flavonoides/farmacologia , Glicosaminoglicanos/farmacologia , Hialuronoglucosaminidase/metabolismo , Indometacina/farmacologia , Preparações de Plantas/química , Quercetina/farmacologia , Reserpina/farmacologiaRESUMO
Enzyme-linked immunosorbent assay (ELISA) has been modified for screening plants with antagonistic activity to Naja naja siamensis cobra venom. Aqueous extracts from plants were investigated for their inhibitory effects on the binding of anti-cobra venom antibody to antigen, cobra venom, fixed onto 96-well microtiter plates. Ingredients in extracts were allowed to react with immobilized venom before the subsequent addition of antivenom antibody. Venom components affected by exposure to the extracts, unable to interact with their specific antibody, were predicted to be unable to bind to their native destinations or natural receptors. Curcuma cf. zedoaria, an old Thai medicinal plant, showed clear inhibitory activity in the ELISA test. Neurotoxin and protein degradative enzymes, major components in venom, were identified as targets of this extract in Western immunoblotting analysis. Ingredients in the extract showed high affinity to the toxin in competition assay by immunoprecipitation. The extract attenuated toxin activity by extending contraction time of diaphragm muscle after envenomation and had a potency to protect cellular proteins from venom degradative enzymes. Curcuma parviflora, with less activity in ELISA, exhibited acceptable results in two experiments but negative results in two experiments, whereas Curcuma longa, having low activity in the ELISA test, never showed any favorable results. Screening of 36 samples could classify plants into an inhibition range of 0 to 86%. This modified ELISA is recommended as a preliminary screening method for inhibitors with a large number of samples.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Venenos Elapídicos/antagonistas & inibidores , Ensaio de Imunoadsorção Enzimática/métodos , Extratos Vegetais/farmacologia , Animais , Antivenenos/imunologia , Proteínas Neurotóxicas de Elapídeos/análise , Proteínas Neurotóxicas de Elapídeos/antagonistas & inibidores , Curcuma/química , Venenos Elapídicos/enzimologia , Venenos Elapídicos/imunologia , Imunoprecipitação , Nervo Frênico/efeitos dos fármacos , Extratos Vegetais/química , RatosRESUMO
Aqueous and alcoholic extracts of dried roots of Mimosa pudica were tested for their inhibitory activity on lethality, myotoxicity and toxic enzymes of Naja kaouthia venom. The aqueous extract, particularly the normal water extract, displayed a significant inhibitory effect on the lethality, myotoxicity and tested enzyme activities of venom compared with alcoholic extracts. The present finding suggests that aqueous extracts of M. pudica root possess compound(s), which inhibit the activity of cobra venom.
Assuntos
Venenos Elapídicos/enzimologia , Fosfolipases A/antagonistas & inibidores , Extratos Vegetais/farmacologia , Raízes de Plantas/química , Rosales/química , Animais , Creatina Quinase/metabolismo , Elapidae , Dose Letal Mediana , Camundongos , Fosfolipases A/toxicidadeRESUMO
We constructed a plasmid containing a chimeric gene composed of the gene encoding acetylcholinesterase (AChE) from Bungarus fasciatus venom and a gene encoding a single chain antibody fragment (scFv) directed against one of the two subunits of a presynaptic neurotoxin from rattlesnake. Large quantities of the fusion protein were produced in the culture medium of transfected COS cells. Fusion to AChE did not affect the ability of the scFv to recognise its antigen. Similarly, the AChE activity was not impaired in the fusion. The fusion protein was purified from the culture medium in a single step by affinity chromatography. The immunoconjugate obtained consisted of a soluble monomeric form of AChE fused to scFv. It was monovalent and had a molecular weight of 94 kDa. The properties of this scFv-AChE fusion show that the simple, reproducible preparation of various recombinant monovalent immunoenzymatic tracers with low molecular weight is possible. In addition, in the construct presented, the scFv domain can be easily changed to another one taking advantage of the SfiI-NotI restriction sites surrounding this domain.
Assuntos
Acetilcolinesterase/genética , Venenos Elapídicos/enzimologia , Região Variável de Imunoglobulina/genética , Recombinação Genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bungarus , Células COS , Clonagem Molecular , DNA Complementar , Eletroforese em Gel de Poliacrilamida , Técnicas Imunoenzimáticas , Região Variável de Imunoglobulina/isolamento & purificação , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
Secretory nonpancreatic phospholipase A2 (group II sPLA2) is induced in inflammation and present in atherosclerotic lesions. In an accompanying publication we demonstrate that transgenic mice expressing group II sPLA2 developed severe atherosclerosis. The current study was undertaken to determine whether 1 mechanism by which group II sPLA2 might contribute to the progression of inflammation and atherosclerosis is by increasing the formation of biologically active oxidized phospholipids. In vivo measurements of bioactive lipids were performed, and in vitro studies tested the hypothesis that sPLA2 can increase the accumulation of bioactive phospholipids. We have shown previously that 3 oxidized phospholipids derived from the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC) stimulated endothelial cells to bind monocytes, a process that is known to be an important step in atherogenesis. We now show that these 3 biologically active phospholipids are significantly increased in livers of sPLA2 transgenic mice fed a high-fat diet as compared with nontransgenic littermates. We present in vitro evidence for several mechanisms by which these phospholipids may be increased in sPLA2 transgenics. These studies demonstrated that polyunsaturated free fatty acids, which are liberated by sPLA2, increased the formation of bioactive phospholipids in LDL, resulting in increased ability to stimulate monocyte-endothelial interactions. Moreover, sPLA2-treated LDL was oxidized by cocultures of human aortic endothelial cells and smooth muscle cells more efficiently than untreated LDL. Analysis by electrospray ionization-mass spectrometry revealed that the bioactive phospholipids, compared with unoxidized PAPC, were less susceptible to hydrolysis by human recombinant group II sPLA2. In addition, HDL from the transgenic mice and human HDL treated with recombinant sPLA2 in vitro failed, in the coculture system, to protect against the formation of biologically active phospholipids in LDL. This lack of protection may in part relate to the decreased levels of paraoxonase seen in the HDL isolated from the transgenic animals. Taken together, these studies show that levels of biologically active oxidized phospholipids are increased in sPLA2 transgenic mice; they also suggest that this increase may be mediated by effects of sPLA2 on both LDL and HDL.