RESUMO
It has been described that the metalloprotease BmooMP-alpha-I purified from Bothrops moojeni snake venom is able to hydrolyze the TNF molecule. However, this observation has been based mainly on in vitro investigation, in addition to molecular modeling and docking approaches. Considering that there is no in vivo study to demonstrate the biological effects of this enzyme, the major aim to the present work was to investigate whether the BmooMP-alpha-I has any anti-inflammatory efficacy by setting up a murine experimental design of colitis induced by dextran sulfate sodium (DSS). For this purpose, C57BL/6 mice were divided into six groups, as follows: (i) animals without intestinal inflammation, (ii) animals without intestinal inflammation treated with BmooMP-alpha-I (50 µg/animal/day), and (iii) animals with intestinal inflammation induced by 3% of DSS, (iv) mice with intestinal inflammation induced by DSS and treated with BmooMP-alpha-I enzyme at the 50, 25, or 12.5 µg/animal/day dosages by intraperitoneal route. Clinical signs of colitis were observed daily for calculating the morbidity scores, cytokine measurements, and histological features. We observed that the animals treated with different doses of the enzyme presented a remarkable improvement of colitis signs, as confirmed by a significant increase of the intestine length in comparison to the DSS group. Also, no difference was observed between the groups treated with the enzyme or vehicle, as the colon length of these animals was slightly lower than that of the group of healthy animals, without induction of intestinal inflammation. The cytokine quantification in supernatants of intestinal tissue homogenates showed a significant reduction of 38% in IFN-gamma levels, when the animals were treated with 50 µg of the BmooMP-alpha-I compared to the animals receiving DSS only. A significant reduction of 39% in TNF levels was also observed in all doses of treatment with BmooMP-alpha-I, in addition to a significant reduction of 35% in the amount of IL-12p40. Histological examinations revealed that the BmooMP-alpha-I 50 µg treated group preserved colon architecture and goblet cells and reduced the ulcer area, when compared with DSS mice, which showed typical inflammatory changes in tissue architecture, such as ulceration, crypt dilation, loss of tissue architecture, and goblet cell depletion, accompanied by a significant cell infiltration. In conclusion, our results suggest that the improvement of clinical scores and histological findings related to BmooMP-alpha-I treatment in this experimental model could be attributed to the metalloprotease ability to modulate cytokine production locally at the inflamed intestine. These findings highlight the potential anti-inflammatory role and effectiveness of this enzyme as a therapeutic alternative in this type of immunopathological condition.
Assuntos
Colite/induzido quimicamente , Colite/tratamento farmacológico , Sulfato de Dextrana/toxicidade , Metaloendopeptidases/uso terapêutico , Animais , Bothrops , Colite/metabolismo , Citocinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Malignant melanoma is a less common but highly dangerous form of skin cancer; it starts in the melanocytes cells found in the outer layer of the skin. Jararhagin toxin, a metalloproteinase isolated from Bothrops jararaca snake venom acts upon several biological processes, as inflammation, pain, platelet aggregation, proliferation and apoptosis, though not yet approved for use, may one day be employed to treat tumors. METHODS: B16F10 murine melanoma cells were treated with jararhagin (jara), a disintegrin-like metalloproteinase isolated from Bothrops jararaca snake venom, and jari (catalytic domain inactivated with 1,10-phenanthroline). Viability and adhesion cells were evaluated by MTT assay. The expression of caspase-3 active, phases of the cell cycle and apoptosis were assessed by flow cytometry. We analyze in vivo the effects of jararhagin on melanoma growth, apoptosis and metastasis. RESULTS: The tumor cells acquired round shapes, lost cytoplasmic expansions, formed clusters in suspension and decreased viability. Jari was almost 20 times more potent toxin than jara based on IC50 values and on morphological changes of the cells, also observed by scanning electron microscopy. Flow cytometry analysis showed 48.3% decrease in the proliferation rate of cells and 47.2% increase in apoptosis (jara) and necrosis (jari), following 1.2 µM jara and 0.1 µM jari treatments. Caspase-3 activity was increased whereas G0/G1 cell cycle phase was on the decline. Proliferative rate was assessed by staining with 5,6-carboxyfluoresceindiacetate succinimidyl ester, showing a significant decrease in proliferation at all concentrations of both toxins. CONCLUSIONS: In vivo treatment of the toxins was observed reduction in the incidence of nodules, and metastasis and antiproliferative inhibition capacity. This data strengthens the potential use jararhagin as an anti-neoplastic drug.
Assuntos
Antineoplásicos/uso terapêutico , Bothrops , Venenos de Crotalídeos/uso terapêutico , Melanoma/tratamento farmacológico , Metaloendopeptidases/uso terapêutico , Neoplasias Cutâneas/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Adesão Celular/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Venenos de Crotalídeos/isolamento & purificação , Venenos de Crotalídeos/farmacologia , Melanoma/metabolismo , Metaloendopeptidases/isolamento & purificação , Metaloendopeptidases/farmacologia , Metaloproteases/farmacologia , Metaloproteases/uso terapêutico , Camundongos , Agregação Plaquetária , Inibidores da Agregação Plaquetária/farmacologia , Inibidores da Agregação Plaquetária/uso terapêutico , Neoplasias Cutâneas/metabolismo , Veneno de Bothrops jararaca , Melanoma Maligno CutâneoAssuntos
Humanos , Aterosclerose/tratamento farmacológico , Aterosclerose/fisiopatologia , Aterosclerose/terapia , Lipoproteínas HDL , Óxido Nítrico , Fumar/efeitos adversos , Trombofilia/complicações , Ácido Ascórbico/uso terapêutico , Aspirina/uso terapêutico , Fatores Quimiotáticos/sangue , Chlamydophila pneumoniae/patogenicidade , Heparina de Baixo Peso Molecular/uso terapêutico , Macrófagos/química , Metaloendopeptidases/uso terapêutico , Proteína C-Reativa , Selectinas/uso terapêutico , TiclopidinaAssuntos
Humanos , Aterosclerose/fisiopatologia , Aterosclerose/tratamento farmacológico , Aterosclerose/terapia , Trombofilia/complicações , Tabagismo/efeitos adversos , Óxido Nítrico , Lipoproteínas HDL , Aspirina/uso terapêutico , Ácido Ascórbico/uso terapêutico , Metaloendopeptidases/uso terapêutico , Heparina de Baixo Peso Molecular/uso terapêutico , Ticlopidina , Fatores Quimiotáticos/sangue , Macrófagos/química , Proteína C-Reativa/diagnóstico , Chlamydophila pneumoniae/patogenicidade , Selectinas/uso terapêuticoRESUMO
Metalloproteinases and disintegrins are important components of most viperid and crotalid venoms. Large metalloproteinases referred to as MDC enzymes are composed of an N-terminal Metalloproteinase domain, a Disintegrin-like domain and a Cys-rich C-terminus. In contrast, disintegrins are small non-enzymatic RGD-containing cysteine-rich polypeptides. However, the disintegrin region of MDC enzymes bears a high degree of structural homology to that of the disintegrins, although it lacks the RGD motif. Despite these differences, both components share the property of being able to recognize integrin cell surface receptors and thereby to inhibit integrin-dependent cell reactions. Recently, several membrane-bound MDC enzymes, closely related to soluble venom MDC enzymes, have been described in mammalian cells. This group of membrane-anchored mammalian enzymes is also called the ADAM family of proteins due to the structure revealing A Disintegrin And Metalloproteinase domains. ADAMs are involved in the shedding of molecules from the cell surface, a property which is also shared by some venom MDC enzymes.