RESUMO
Assessing the gut mucosa milieu is important to grade the inflammatory process in conditions such as food hypersensitivity, allergy, gut parasitosis, etc. However, the gastrointestinal tract comprises a challenging system to evaluate, due to its thin tubular structure and mucosa, which suffer fast autolysis after death. Irrespective of the preferred inflammatory score system, it is important to choose the technique that will render the best tissue analysis. Thus, our aim was to compare two of the most frequently used methods to collect, process and analyze gut segments, the Swiss Roll and the Intestinal Strips. Normal C57Bl/6 mice were randomly assigned to Rolls or Strips group. After an overdose of anesthetics, segments of the duodenum, jejunum and ileum were collected and prepared accordingly for histological processing and analysis. Our results show the villi in the Rolls tend to be shorter and wider than those in the Strips in the duodenum and jejunum but not the ileum. No significant differences were observed in intra-epithelial lymphocytes and goblet cells counts. Finally, we staged each segment using our histomorphometric classification system, which revealed that although all animals presented a normal intestinal mucosa, those assigned to the Rolls group had their mucosa staged in the Infiltrative Stage while the Strips group had their mucosa staged as Normal. In conclusion, Swiss Rolls might be desirable for a wider assessment of the intestine, as it allows large segments to be analyzed at once, while Strips are better suited when detailed evaluation of each villus is intended.
Assuntos
Duodeno/patologia , Trato Gastrointestinal/patologia , Íleo/patologia , Inflamação/patologia , Mucosa Intestinal/patologia , Jejuno/patologia , Manejo de Espécimes/métodos , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Manejo de Espécimes/classificaçãoRESUMO
Thrombosis related diseases are among the main causes of death and incapacity in the world. Despite the existence of antithrombotic agents available for therapy, they still present adverse effects like hemorrhagic risks which justify the search for new options. Recently, pachydictyol A, isopachydictyol A, and dichotomanol, three diterpenes isolated from Brazilian marine brown alga Dictyota menstrualis were identified as potent antithrombotic molecules through inhibition of thrombin, a key enzyme of coagulation cascade and a platelet agonist. Due to the biotechnological potential of these marine metabolites, in this work we evaluated their binding mode to thrombin in silico and identified structural features related to the activity in order to characterize their molecular mechanism. According to our theoretical studies including structure-activity relationship and molecular docking analysis, the highest dipole moment, polar surface area, and lowest electronic density of dichotomanol are probably involved in its higher inhibition percentage towards thrombin catalytic activity compared to pachydictyol A and isopachydictyol A. Interestingly, the molecular docking studies also revealed a good shape complementarity of pachydictyol A and isopachydictyol A and interactions with important residues and regions (e.g., H57, S195, W215, G216, and loop-60), which probably justify their thrombin inhibitor effects demonstrated in vitro. Finally, this study explored the structural features and binding mode of these three diterpenes in thrombin which reinforced their potential to be further explored and may help in the design of new antithrombotic agents.
Assuntos
Anticoagulantes/química , Anticoagulantes/farmacologia , Diterpenos/química , Diterpenos/farmacologia , Fibrinolíticos/química , Fibrinolíticos/farmacologia , Trombina/antagonistas & inibidores , Organismos Aquáticos/química , Coagulação Sanguínea/efeitos dos fármacos , Brasil , Modelos Moleculares , Simulação de Acoplamento Molecular/métodos , Phaeophyceae/química , Relação Estrutura-Atividade , Trombose/tratamento farmacológicoRESUMO
This report describes a modified, simple, low-cost and more sensitive method to determine bleeding patterns and haemoglobin concentration in a tail-bleeding assay using BALB/c mice and tail tip amputation. The cut tail was immersed in Drabkin's reagent to promote erythrocyte lysis and haemoglobin release, which was monitored over 30 min. The operator was blinded to individual conditions of the mice, which were treated with either saline (NaCl 0.15m), DMSO (0.5%) or clinical anti-thrombotic drugs. Our experimental protocols showed good reproducibility and repeatability of results when using Drabkin's reagent than water. Thus, the use of Drabkin's reagent offered a simple and low-cost method to observe and quantify the bleeding and rebleeding episodes. We also observed the bleeding pattern and total haemoglobin loss using untreated animals or those under anti-coagulant therapy in order to validate the new Drabkin method and thus confirm that it is a useful protocol to quantify haemoglobin concentrations in tail-bleeding assay. This modified method provided a more accurate results for bleeding patterns in mice and for identifying new anti-thrombotic drugs.
Assuntos
Fibrinolíticos/farmacologia , Hemorragia/tratamento farmacológico , Cauda/lesões , Animais , Modelos Animais de Doenças , Feminino , Proteínas Ligadas por GPI , Proteína da Hemocromatose , Masculino , Proteínas de Membrana/sangue , Camundongos Endogâmicos BALB C , Reprodutibilidade dos TestesRESUMO
Platelet aggregation is one of the main events involved in vascular thrombus formation. Recently, N'-substituted-phenylmethylene-3-methyl-1,6-diphenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazides were described as antiplatelet derivatives. In this work, we explore the properties of these antiplatelet agents through a series of pharmacological, biochemical and toxicological studies. The antiplatelet activity of each derivative was confirmed as 3a, 3b and 3 h significantly inhibited human platelet aggregation induced by arachidonic acid, with no detectable effect on clotting factors or healthy erythrocytes. Importantly, mice treated with derivative 3a showed a higher survival rate at an in vivo model of pulmonary thromboembolism with a lower bleeding risk in comparison to aspirin. The in silico studies pointed a series of structural parameters related to thromboxane synthase (TXS) inhibition by 3a, which was confirmed by tracking plasma levels of PGE2 and TXB2 through an in vitro enzyme immunoassay. Derivative 3a showed selective TXS inhibition allied with low bleeding risk and increased animal survival, revealing the derivative as a promising candidate for treatment of cardiovascular diseases.
Assuntos
Inibidores da Agregação Plaquetária/farmacologia , Pirazóis/farmacologia , Piridinas/farmacologia , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Inibidores da Agregação Plaquetária/química , Inibidores da Agregação Plaquetária/toxicidade , Pirazóis/química , Pirazóis/toxicidade , Piridinas/química , Piridinas/toxicidadeRESUMO
The incidence of hematological disorders has increased steadily in Western countries despite the advances in drug development. The high expression of the multi-resistance protein 4 in patients with transitory aspirin resistance, points to the importance of finding new molecules, including those that are not affected by these proteins. In this work, we describe the synthesis and biological evaluation of a series of N,N'-disubstituted thioureas derivatives using in vitro and in silico approaches. New designed compounds inhibit the arachidonic acid pathway in human platelets. The most active thioureas (compounds 3d, 3i, 3m and 3p) displayed IC50 values ranging from 29 to 84 µM with direct influence over in vitro PGE2 and TXA2 formation. In silico evaluation of these compounds suggests that direct blockage of the tyrosyl-radical at the COX-1 active site is achieved by strong hydrophobic contacts as well as electrostatic interactions. A low toxicity profile of this series was observed through hemolytic, genotoxic and mutagenic assays. The most active thioureas were able to reduce both PGE2 and TXB2 production in human platelets, suggesting a direct inhibition of COX-1. These results reinforce their promising profile as lead antiplatelet agents for further in vivo experimental investigations.
Assuntos
Ciclo-Oxigenase 1/química , Fibrinolíticos/síntese química , Fibrinolíticos/farmacologia , Inibidores da Agregação Plaquetária/síntese química , Inibidores da Agregação Plaquetária/farmacologia , Tioureia/análogos & derivados , Ácido Araquidônico/metabolismo , Domínio Catalítico/efeitos dos fármacos , Simulação por Computador , Ciclo-Oxigenase 1/efeitos dos fármacos , Ciclo-Oxigenase 1/metabolismo , Dinoprostona/metabolismo , Fibrinolíticos/química , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Inibidores da Agregação Plaquetária/química , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Tioureia/farmacologia , Tromboxano B2/metabolismoRESUMO
Platelets are cytoplasmatic fragments from bone marrow megakaryocytes present in blood. In this work, we review the basis of platelet mechanisms, their participation in syndromes and in arterial thrombosis, and their potential as a target for designing new antithrombotic agents. The option of new biotechnological sources is also explored.
Assuntos
Plaquetas/metabolismo , Transtornos Hemostáticos/patologia , Aspirina/farmacologia , Aspirina/uso terapêutico , Plaquetas/efeitos dos fármacos , Transtornos Hemostáticos/metabolismo , Humanos , Integrinas/genética , Integrinas/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Inibidores da Agregação Plaquetária/uso terapêutico , Deficiência do Pool Plaquetário/metabolismo , Deficiência do Pool Plaquetário/patologia , Trombose/tratamento farmacológico , Trombose/patologiaRESUMO
Thromboxane synthase (TXAS) is a P450 epoxygenase that synthesizes thromboxane A2 (TXA2), a potent mediator of platelet aggregation, vasoconstriction and bronchoconstriction. This enzyme plays an important role in several human diseases, including myocardial infarction, stroke, septic shock, asthma and cancer. Despite of the increasing interest on developing TXAS inhibitors, the structure and activity of TXAS are still not totally elucidated. In this study, we used a comparative molecular modeling approach to construct a reliable model of TXAS and analyze its interactions with Dazoxiben and Ozagrel, two competitive inhibitors. Our results were compatible with experimental published data, showing feasible cation-π interaction between the iron atom of the heme group of TXAS and the basic nitrogen atom of the imidazolyl group of those inhibitors. In the absence of the experimental structure of thromboxane synthase, this freely available model may be useful for designing new antiplatelet drugs for diseases related with TXA2.