RESUMO
Despite the fact that Esenbeckia leiocarpa, a Brazilian plant, possesses potential anti-inflammatory properties, its effect in neutrophils, key players in inflammation, has never been investigated. In this study, a crude hydroalcoholic extract (CHE) was used to evaluate the potential toxic or agonistic effect of E. leiocarpa in human neutrophils. At a noncytotoxic concentration of 500 µg/mL, CHE increased actin polymerization and cell signaling events, especially p38 MAPK. Its modulatory activity on neutrophil cell apoptosis was investigated by cytology and by flow cytometry and, although CHE increased the apoptotic rate (by cytology) and increased annexin-V binding, it did not, unexpectedly, increase CD16 shedding. CHE increased the degradation of the cytoskeletal proteins gelsolin and paxillin but, surprisingly, not of vimentin. The proapoptotic activity of CHE was reversed by a pan-caspase inhibitor but not by a p38 inhibitor. We conclude that CHE is a novel human neutrophil agonist that induces apoptosis by a caspase-dependent and p38-independent mechanism in an atypical fashion based on its lack of effect on CD16 shedding and vimentin degradation. Since the resolution of inflammation occurs by elimination of apoptotic neutrophils, the ability of CHE to induce neutrophil apoptosis correlates well with its anti-inflammatory properties, as previously reported.
Assuntos
Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Extratos Vegetais/farmacologia , Rutaceae/química , Actinas/metabolismo , Células Cultivadas , Humanos , Extratos Vegetais/químicaRESUMO
UNLABELLED: This study was conducted to investigate the anti-inflammatory efficacy of Esenbeckia leiocarpa against the inflammation caused by the carrageenan using a murine air pouch model. MATERIAL AND METHODS: The effects of the crude hydroalcoholic extract (CHE), fractions (n-hexane (Hex) and ethyl acetate (AcOEt)), subfractions (polar (Pol) and nonpolar (Nonpol)), or isolated compounds (dihydrocorynantheol (DHC) and beta-sitosterol (ß-Sit)) isolated from CHE upon leukocytes, exudate, myeloperoxidase (MPO) adenosine-deaminase (ADA), nitrate/nitrite (NO(x)), interleukin-1 beta (IL-1ß), tumor necrosis factor-alpha (TNF-α), and inhibitory kappa-B-alpha (IκB-α) degradation were evaluated. The CHE, Alk, Pol, Nonpol, DHC and ß-Sit, inhibited leukocytes, exudate, MPO and ADA, NO(x), IL-1ß, and TNF-α (P<0.05). The Hex and AcOEt fractions inhibited all of the proinflammatory parameters, except the exudate. The compound DHC prevented the IκB-α degradation. CONCLUSION: E. leiocarpa possesses important anti-inflammatory properties. These inhibitory effects occurred along with the downregulation of nitric oxide, IL-1ß and TNF-α levels. The isolated compounds DHC and ß-Sit may be partially responsible for these anti-inflammatory effects.
Assuntos
Anti-Inflamatórios/uso terapêutico , Inflamação/tratamento farmacológico , Magnoliopsida/química , Casca de Planta/química , Extratos Vegetais/uso terapêutico , Adenosina Desaminase , Alcaloides/análise , Animais , Carragenina/farmacologia , Citocinas/biossíntese , Citocinas/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Inflamação/induzido quimicamente , Leucócitos/efeitos dos fármacos , Camundongos , Sitosteroides/análise , Resultado do TratamentoRESUMO
The role of the anti-cancer agent Viscum album agglutinin-I (VAA-I) in leukaemia PLB-985 cells differentiated toward a neutrophil-like phenotype by dimethylsulphoxide (PLB-985D) has never been studied. This study investigated whether or not VAA-I can induce cytoskeletal breakdown in PLB-985D cells, as previously observed in undifferentiated PLB-985 cells. VAA-I was found to induce apoptosis in PLB-985D cells, as assessed by cytology and by degradation of gelsolin, an event known to occur via caspase-3 activation. VAA-I induced cytoskeletal breakdown based on the disruption of the F-actin network and cleavage of paxillin, vimentin and lamin B(1). In addition, we demonstrated, for the first time, that non-muscle myosin heavy chain IIA (NMHC-IIA) was cleaved by VAA-I treatment. Degradation of NMHC-IIA was reversed by the pan caspase inhibitor z-VAD-fmk in PLB-985D cells and neutrophils. However, unlike lamin B(1), no NMHC-IIA was detected on the cell surface of apoptotic neutrophils. In conclusion, PLB-985D cells responded in a similar manner to neutrophils regarding the degradation of the tested cytoskeletal. Therefore, PLB-985D cells may provide a suitable substitute for neutrophils in screening experiments, preventing extensive neutrophil cell isolation.
Assuntos
Antineoplásicos/uso terapêutico , Caspases/metabolismo , Leucemia/tratamento farmacológico , Neutrófilos/imunologia , Miosina não Muscular Tipo IIA/metabolismo , Preparações de Plantas/uso terapêutico , Proteínas de Plantas/uso terapêutico , Toxinas Biológicas/uso terapêutico , Apoptose/efeitos dos fármacos , Caspases/genética , Diferenciação Celular , Linhagem Celular Tumoral , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Citometria de Fluxo , Humanos , Lamina Tipo B/análise , Lamina Tipo B/metabolismo , Leucemia/metabolismo , Miosina não Muscular Tipo IIA/análise , Paxilina/análise , Paxilina/metabolismo , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Inativadoras de Ribossomos , Proteínas Inativadoras de Ribossomos Tipo 2 , Vimentina/análise , Vimentina/metabolismoRESUMO
Viscum album agglutinin-I (VAA-I) is a potent inducer of cell apoptosis and possesses anti-tumoral activity. Using PLB-985 and chronic granulomatous disease (X-CGD) cells, which lack expression of gp91(phox), VAA-I was found to induce apoptosis in both cell lines as assessed by cytology, DNA laddering and degradation of the cytoskeletal protein gelsolin. Both cell lines expressed caspase-3 and -8 and VAA-I activated these caspases. We demonstrated that lamin B(1) is a novel target to VAA-I and its degradation was reversed by a pan-caspase inhibitor and by a caspase-6, but not a caspase-8, inhibitor.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas do Citoesqueleto/efeitos dos fármacos , Leucemia/patologia , Lectinas de Plantas/farmacologia , Preparações de Plantas/farmacologia , Proteínas de Plantas/farmacologia , Toxinas Biológicas/farmacologia , Caspase 3 , Caspase 8 , Caspases/efeitos dos fármacos , Caspases/metabolismo , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/metabolismo , Gelsolina/antagonistas & inibidores , Humanos , Lamina Tipo B/antagonistas & inibidores , Leucemia/tratamento farmacológico , Proteínas Inativadoras de Ribossomos , Proteínas Inativadoras de Ribossomos Tipo 2 , Viscum album/químicaRESUMO
Although there are several agents that induce neutrophil apoptosis, few are known as inducers of eosinophil apoptosis. As eosinophils are potent effector cells contributing to allergic inflammation and asthma, we investigated whether the pro-apoptotic agent Viscum album agglutinin-I (VAA-I) could induce eosinophil apoptosis. VAA-I was found to induce apoptosis in eosinophilic AML14.3D10 (3D10) cells and that these cells expressed caspases-1, -2, -3, -4, -7, -8, -9 and -10. VAA-I-induced gelsolin degradation was reversed by the pan-caspase inhibitor N-benzyloxycarbonyl-V-A-D-O-methylfluoromethyl ketone (z-VAD). Also, paxillin, vimentin and lamin B1 were cleaved by caspases in VAA-I-induced 3D10 cells. VAA-I activated caspase-3 and -8 in 3D10 cells but, unlike z-VAD, treatment with a caspase-8 inhibitor slightly reversed apoptosis. Treatment of purified human eosinophils with VAA-I was found to induce apoptosis, degradation of gelsolin and lamin B1, but unlike 3D10 cells, cleavage of lamin B1 and cell apoptosis was not reversed by z-VAD. We conclude that VAA-I is a potent inducer of eosinophil apoptosis and that proteases other than those inhibited by z-VAD in 3D10 cells are involved in VAA-I-induced peripheral blood eosinophil apoptosis and lamin B1 cleavage. Thus, VAA-I represents a potential candidate for the reduction of the number of eosinophils in diseases where they play important roles.
Assuntos
Caspases/metabolismo , Proteínas do Citoesqueleto/metabolismo , Leucemia Mieloide/metabolismo , Preparações de Plantas/farmacologia , Proteínas de Plantas/farmacologia , Toxinas Biológicas/farmacologia , Doença Aguda , Apoptose , Caspase 3 , Caspase 8 , Caspases/análise , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/análise , Proteínas do Citoesqueleto/efeitos dos fármacos , Fragmentação do DNA , Eosinófilos/metabolismo , Gelsolina/análise , Gelsolina/metabolismo , Humanos , Laminina/metabolismo , Paxilina , Fosfoproteínas/análise , Fosfoproteínas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Inativadoras de Ribossomos , Proteínas Inativadoras de Ribossomos Tipo 2 , Vimentina/análise , Vimentina/metabolismoRESUMO
Chemicals of environmental concern are known to alter the immune system. Recent data indicate that some contaminants possess proinflammatory properties by activating neutrophils, an area of research that is still poorly investigated. We have previously documented that toxaphene activates human neutrophils to produce reactive oxygen species (ROS) and accelerates apoptosis by a yet unknown mechanism. In this study, we found that toxaphene induces another neutrophil function, chemotaxis. Furthermore, we found that toxaphene induces both chemotaxis and apoptosis via a ROS-dependent mechanism, since these responses were blocked by the addition of catalase to the culture. In addition, toxaphene was found to induce the degradation of the cytoskeletal proteins gelsolin, paxillin, and vimentin during apoptosis, and this was reversed by the addition of z-VAD-FMK (caspase inhibitor) or catalase, demonstrating the importance of caspases and ROS in this process. In contrast to toxaphene, we found that beryllium does not induce superoxide production, and, this correlates with its inability to induce chemotaxis and apoptosis. We conclude that toxaphene induces chemotaxis and apoptosis via ROS and that caspases and ROS are involved in the degradation of cytoskeletal proteins.
Assuntos
Apoptose , Berílio/farmacologia , Inibidores de Caspase , Quimiotaxia de Leucócito/fisiologia , Proteínas do Citoesqueleto/metabolismo , Neutrófilos/efeitos dos fármacos , Preparações de Plantas , Proteínas de Plantas , Espécies Reativas de Oxigênio/metabolismo , Toxafeno/farmacologia , Clorometilcetonas de Aminoácidos/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Radicais Livres , Gelsolina/metabolismo , Humanos , Neutrófilos/metabolismo , Neutrófilos/fisiologia , Paxilina , Fosfoproteínas/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 2 , Superóxidos/metabolismo , Toxinas Biológicas/farmacologia , Vimentina/metabolismoRESUMO
Viscum album agglutinin-I (VAA-I) is a plant lectin that possesses interesting potential therapeutic properties and immunomodulatory activities. We have recently found that VAA-I is a potent inducer of human neutrophil apoptosis, but the mechanism(s) involved require further elucidation. In this study, we found that VAA-I alters mitochondrial transmembrane potential and increases intracellular levels of reactive oxygen species (ROS). Despite these observations, treatment with the mitochondrial stabilizer, bongkrekic acid, or with catalase, known to degrade H(2)O(2), fails to reverse VAA-I-induced apoptosis. Moreover, VAA-I was found to induce apoptosis in PLB-985 cells deficient in gp91(phox), indicating that the lectin acts via an ROS-independent mechanism. Pretreatment of neutrophils with brefeldin A, an inhibitor of vesicular transport, was found to reverse VAA-I-induced apoptosis. Protein expression of Mcl-1 was decreased by VAA-I. The role of caspases in the degradation of cytoskeletal proteins during both spontaneous and VAA-I-induced neutrophil apoptosis was also investigated. Paxillin and vimentin were markedly degraded by VAA-I when compared with neutrophils that undergo spontaneous apoptosis, but not vinculin or alpha- and beta-tubulin. Caspases were involved in cytoskeletal protein degradation because preincubation with the pan-caspase inhibitor N-benzyloxycarbonyl-V-A-D-O-methylfluoromethyl ketone was found to reverse protein cleavage. We conclude that VAA-I needs to be internalized to mediate apoptosis and that its activity is not dependent on a cell surface receptor-mediated pathway. Also, we conclude that VAA-I induces apoptosis by ROS-independent and Mcl-1-dependent mechanisms and that caspases are involved in cytoskeletal protein degradation in both spontaneous and VAA-I-induced neutrophil apoptosis.