RESUMO
The formation of a thrombus is a key event in thromboembolic disorders, that contribute to high mortality and morbidity in affected patients. In the present study, we synthesized a library of novel substituted 3,3-dibutyl-8-methoxy-2,3-dihydrobenzo [b] [1,4] thiazepin-4(5H)-one derivatives which were tested for their platelet aggregation and thrombin inhibitory activity. Among the tested compounds, 3,3-dibutyl-7-(2-chlorophenyl)-8-methoxy-2,3-dihydrobenzo[b] [1,4]thiazepin-4(5H)-one (DCT) displayed the maximum thrombin inhibition with an IC50 value of 3.85⯵M and thus DCT was chosen for further studies. Next, the effect of DCT on primary hemostasis was evaluated using agonist-induced platelet aggregation model. The lead compound inhibited the collagen- or ADP- or thrombin-induced platelet aggregation in a dose-dependent manner. Furthermore, DCT prolonged the process of clot formation when evaluating plasma re-calcification time (320⯱â¯11â¯sec at 5⯵g DCT), activated partial thromboplastin time (58.0⯱â¯0.01â¯sec at 2⯵g), and prothrombin time (14.7⯱â¯0.01â¯sec at 5⯵g). Molecular docking studies suggested that the benzothiazepinones evaluated here consistently display hydrogen bonding with Ser214 of thrombin, which is similar to that of the co-crystallized ligand (1-(2R)-2-amino-3-phenyl-propanoyl-N-(2,5dichlorophenyl)methylpyrrolidine-2-carboxamide). DCT displayed additional hydrogen bonding to Ser195 and π-π interactions between its methoxyphenyl groups and Trp60, thereby providing a structural rationale for the observed biological effect.
Assuntos
Inibidores de Serina Proteinase/farmacologia , Tiazepinas/farmacologia , Trombina/antagonistas & inibidores , Relação Dose-Resposta a Droga , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Inibidores de Serina Proteinase/síntese química , Inibidores de Serina Proteinase/química , Relação Estrutura-Atividade , Tiazepinas/síntese química , Tiazepinas/química , Trombina/metabolismoRESUMO
PURPOSE: The dietary sesamol is one of the important constituent of sesame seed that has been mainly claimed to combat cardiovascular disease and diabetes, which are the major secondary complications of arthritis. Thus, the present study was designed to evaluate the anti-arthritic, anti-inflammatory and anti-stress potentials of sesamol. METHODS: Arthritis was induced using Freund's complete adjuvant to hind paw of experimental rats. The physical and biochemical alterations and its recovery by sesamol were assessed by measuring enzymatic and non-enzymatic mediators. Arthritis-induced inflammation, oxidative stress and their protective by sesamol were measured by determining the levels of pro-inflammatory cytokines and oxidative stress markers. RESULTS: In the present study, sesamol was demonstrated to alleviate arthritis-induced cartilage degeneration by mitigating augmented serum levels of hyaluronidase and matrix metalloproteinases (MMP-13, MMP-3 and MMP-9). It also protected bone resorption by reducing the elevated levels of bone joint exoglycosidases, cathepsin D and tartarate-resistant acid phosphatases. Sesamol also abrogated the non-enzymatic inflammatory markers (TNF, IL-1ß, IL-6, COX-2, PGE2, ROS, and H2O2,) effectively. In addition, sesamol neutralizes arthritis-induced oxidative stress by restoring the levels of reactive oxygen species, lipid and hydro peroxides and sustained antioxidant homeostasis by re-establishing altered activities of superoxide dismutase, catalase and glutathione-s-transferase. CONCLUSION: Taken together, the study demonstrated the anti-arthritic, anti-inflammatory, anti-oxidative stress and chondro-protective potentials of sesamol in vivo. Thus, sesamol could be a single bullet that can fight arthritis as well as the secondary complications of arthritis such as cardio vascular disorders and diabetes.
Assuntos
Anti-Inflamatórios/administração & dosagem , Antioxidantes/administração & dosagem , Artrite Experimental/tratamento farmacológico , Benzodioxóis/administração & dosagem , Matriz Extracelular/metabolismo , Mediadores da Inflamação/metabolismo , Fenóis/administração & dosagem , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Catalase/metabolismo , Ciclo-Oxigenase 2/metabolismo , Glutationa Transferase/metabolismo , Peróxido de Hidrogênio/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Spider venom is a complex mixture of protein and peptide toxins. Hyaluronidase a 'spreading factor' has not been studied extensively in spider venom. In this paper, we describe the purification and characterization of a hyaluronidase from Hippasa partita venom gland extract. Hyaluronidase (HPHyal) has been purified by the successive chromatography on a Sephadex G-100 and on CM-Sephadex C-25 columns. HPHyal has been purified to an extent of about approximately 20-folds. The molecular mass was found to be 42.26 kDa by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. HPHyal was optimally active at pH 5.8 at 37 degrees C and in the presence of 300 mM NaCl in the reaction mixture. HPHyal showed absolute specificity for hyaluronan and belongs to neutral active group of enzymes. HPHyal revealed single-precipitin line, while venom gland extract revealed multiple bands in Western blotting with the antiserum prepared against venom gland extract. HPHyal indirectly potentiates the myotoxicity of VRV-PL-VIII myotoxin and also the hemorrhagic potency of hemorrhagic complex-I. Cations, Na(+) and K(+) enhanced the activity and chloride ions do not have any effect while, divalent cations, inhibited the enzyme activity.
Assuntos
Hialuronoglucosaminidase/química , Hialuronoglucosaminidase/metabolismo , Venenos de Aranha/enzimologia , Aranhas/enzimologia , Animais , Edema/induzido quimicamente , Feminino , Camundongos , Coelhos , Venenos de Aranha/química , Especificidade por SubstratoRESUMO
Indian Echis carinatus bite causes sustained tissue destruction at the bite site. Neutrophils, the major leukocytes in the early defence process, accumulate at the bite site. Here we show that E. carinatus venom induces neutrophil extracellular trap (NET) formation. The NETs block the blood vessels and entrap the venom toxins at the injection site, promoting tissue destruction. The stability of NETs is attributed to the lack of NETs-degrading DNase activity in E. carinatus venom. In a mouse tail model, mice co-injected with venom and DNase 1, and neutropenic mice injected with the venom, do not develop NETs, venom accumulation and tissue destruction at the injected site. Strikingly, venom-induced mice tail tissue destruction is also prevented by the subsequent injection of DNase 1. Thus, our study suggests that DNase 1 treatment may have a therapeutic potential for preventing the tissue destruction caused by snake venom.
Assuntos
Antídotos/farmacologia , Desoxirribonuclease I/farmacologia , Armadilhas Extracelulares/efeitos dos fármacos , Necrose/prevenção & controle , Neutropenia/imunologia , Venenos de Víboras/antagonistas & inibidores , Animais , Antídotos/metabolismo , Desoxirribonuclease I/metabolismo , Armadilhas Extracelulares/imunologia , Feminino , Injeções Intralesionais , Masculino , Camundongos , Necrose/induzido quimicamente , Necrose/patologia , Neutrófilos/citologia , Neutrófilos/imunologia , Fatores de Proteção , Cauda/efeitos dos fármacos , Cauda/imunologia , Cauda/patologia , Venenos de Víboras/administração & dosagem , Venenos de Víboras/toxicidade , Viperidae/fisiologiaRESUMO
Medicinal plants are employed in the treatment of human ailments from time immemorial. Several studies have validated the use of medicinal plant products in arthritis treatment. Arthritis is a joint disorder affecting subchondral bone and cartilage. Degradation of cartilage is principally mediated by enzymes like matrix metalloproteinases (MMPs), hyaluronidases (HAase), aggrecanases and exoglycosidases. These enzymes act upon collagen, hyaluronan and aggrecan of cartilage respectively, which would in turn activate bone deteriorating enzymes like cathepsins and tartrate resistant acid phosphatases (TRAP). Besides, the incessant action of reactive oxygen species and the inflammatory mediators is reported to cause further damage by immunological activation. The present study demonstrated the anti-arthritic efficacy of tamarind seed extract (TSE). TSE exhibited cartilage and bone protecting nature by inhibiting the elevated activities of MMPs, HAase, exoglycosidases, cathepsins and TRAP. It also mitigated the augmented levels of inflammatory mediators like interleukin (IL)-1ß, tumor necrosis factor-α, IL-6, IL-23 and cyclooxygenase-2. Further, TSE administration alleviated increased levels of ROS and hydroperoxides and sustained the endogenous antioxidant homeostasis by balancing altered levels of endogenous antioxidant markers. Overall, TSE was observed as a potent agent abrogating arthritis-mediated cartilage/bone degradation, inflammation and associated stress in vivo demanding further attention.
Assuntos
Artrite Experimental/tratamento farmacológico , Osso e Ossos/efeitos dos fármacos , Cartilagem/efeitos dos fármacos , Inflamação/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/uso terapêutico , Sementes/química , Tamarindus/embriologia , Animais , Osso e Ossos/patologia , Cartilagem/patologia , Mediadores da Inflamação/metabolismo , Extratos Vegetais/farmacologia , Ratos , Ratos WistarRESUMO
Thrombocytopenia in methotrexate (MTX)-treated cancer and rheumatoid arthritis (RA) patients connotes the interference of MTX with platelets. Hence, it seemed appealing to appraise the effect of MTX on platelets. Thereby, the mechanism of action of MTX on platelets was dissected. MTX (10 µM) induced activation of pro-apoptotic proteins Bid, Bax and Bad through JNK phosphorylation leading to ΔΨm dissipation, cytochrome c release and caspase activation, culminating in apoptosis. The use of specific inhibitor for JNK abrogates the MTX-induced activation of pro-apoptotic proteins and downstream events confirming JNK phosphorylation by MTX as a key event. We also demonstrate that platelet mitochondria as prime sources of ROS which plays a central role in MTX-induced apoptosis. Further, MTX induces oxidative stress by altering the levels of ROS and glutathione cycle. In parallel, the clinically approved thiol antioxidant N-acetylcysteine (NAC) and its derivative N-acetylcysteine amide (NACA) proficiently alleviate MTX-induced platelet apoptosis and oxidative damage. These findings underpin the dearth of research on interference of therapeutic drugs with platelets, despite their importance in human health and disease. Therefore, the use of antioxidants as supplementary therapy seems to be a safe bet in pathologies associated with altered platelet functions.
Assuntos
Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Antimetabólitos Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , MAP Quinase Quinase 4/genética , Metotrexato/farmacologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Regulação da Expressão Gênica , Humanos , MAP Quinase Quinase 4/metabolismo , Potencial da Membrana Mitocondrial , Metotrexato/antagonistas & inibidores , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Oxirredução , Estresse Oxidativo , Fosforilação , Cultura Primária de Células , Transdução de Sinais , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína de Morte Celular Associada a bcl/genética , Proteína de Morte Celular Associada a bcl/metabolismoRESUMO
Although arthritis is primarily a joint disorder that mainly targets the articular cartilage and subchondral bone, several recent investigations have reported oxidative burst and vital organ damage that are being considered as secondary complications of arthritis. The continuous generation of free radicals like reactive oxygen and nitrogen species is considered as a key culprit in the initiation and propagation of oxidative damage. In addition, activation of T and B cells, macrophages, inflammatory mediators such as TNF-α, IL-1ß and IL-6 aggravates the oxidative damage of the vital organs, particularly the liver. The current piece of work demonstrates oxidative stress in the liver of arthritic rats and its amelioration by the procyanidin-rich tamarind seed extract (TSE). The arthritic liver homogenate, mitochondrial and cytosolic fractions were found with increased levels of oxidative stress markers including free radicals. As a consequence, depletion in the levels of glutathione, total thiols, glutathione peroxidase and reductase was evident. Furthermore, the activities of endogenous antioxidant enzymes like superoxide dismutase, catalase and glutathione-S-transferase were found to be significantly altered. The increased and decreased activity of transaminases respectively in serum and liver, along with histological observations, further confirms the liver damage. Unfortunately, the commonly used drugs like NSAIDs and DMARDs have failed to prevent oxidative damage, rather they were found to be the inducers themselves. Interestingly, TSE supplementation was found to significantly inhibit oxidative burst in the liver and maintain homeostasis. Thus, the study clearly demonstrates the protective efficacy of TSE against arthritis-associated oxidative liver damage, including mitochondrial oxidative burst and its associated secondary complications.
Assuntos
Antioxidantes/administração & dosagem , Artrite/tratamento farmacológico , Fígado/efeitos dos fármacos , Extratos Vegetais/administração & dosagem , Sementes/química , Tamarindus/química , Animais , Artrite/metabolismo , Feminino , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Fígado/metabolismo , Masculino , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The current study intended to explore the effect of fusaric acid on blood coagulation including plasma coagulation and platelet aggregation. Fusaric acid exhibited biphasic effects on citrated human plasma recalcification time. At concentrations below 50 ng, fusaric acid decreased the clotting time of plasma dose-dependently from 130 ± 3s control value to 32 ± 3s; however, above 50 ng, fusaric acid increased the clotting time from 32 ± 3s and reached a maximum of 152 s at 100 ng and remained unaltered thereafter for the increased dose of fusaric acid. Fusaric acid without damaging red blood cells and platelets, inhibited agonists such as collagen, ADP, thrombin, and epinephrine-induced aggregation of both platelet-rich plasma (PRP) and washed platelets preparations of human. Interestingly, fusaric acid showed biphasic effects only in thrombin-induced platelet aggregation of washed platelets, and at lower concentration (below 900 ng) it activated platelet aggregation; however, in increased concentration (above 900 ng) it inhibited the platelet aggregation of washed platelets. In addition, fusaric acid also inhibited the agonist ADP-induced platelet aggregation of washed platelet suspension but did not show biphasic effect. Further, fusaric acid did not induce the platelets to generate reactive oxygen species (ROS) that clearly suggests that the induction of platelet function could be the result of the fusaric acid-mediated receptor interaction but not through the morphological shape change.