RESUMEN
To investigate osteoclast formation in vivo and if leukotriene B4 (LTB4) loaded in microspheres (MS) could be used as a therapeutical strategy to promote a sustained delivery of the mediator and prevent osteoclast differentiation. Methods: In vivo, apical periodontitis was induced in mice to investigate osteoclast differentiation and signaling in absence of 5-lipoxygenase (5-LO). In vitro, LTB4-MS were prepared using an oil-in-water emulsion solvent extraction-evaporation process. Characterization and efficiency of LTB4 encapsulation were investigated. J774A.1 macrophages were cultured in the presence of monocyte colony-stimulating factor (M-CSF) and ligand for receptor activator of nuclear factor kappa B (RANKL) and then stimulated with LTB4-MS. Cytotoxicity, in vitro MS-LTB4 uptake, osteoclast formation and gene expression were measured. Results: We found that 5-LO negatively regulates osteoclastic formation in vivo during apical periodontitis development. In vitro, LTB4-MS were up-taken by macrophages and were not cytotoxic to the cells. LTB4-MS inhibited osteoclast formation and the synthesis of osteoclastogenic genes Acp5, Mmp9, Calcr and Ctsk. LTB4-MS inhibited differentiation of macrophages into an osteoclastic phenotype and cell activation under M-CSF and RANKL stimulus.
Asunto(s)
Leucotrieno B4 , Periodontitis Periapical , Ratones , Animales , Leucotrieno B4/metabolismo , Leucotrieno B4/farmacología , Osteoclastos/metabolismo , Araquidonato 5-Lipooxigenasa/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Factor Estimulante de Colonias de Macrófagos/metabolismo , Factor Estimulante de Colonias de Macrófagos/farmacología , Microesferas , Ligandos , Emulsiones/metabolismo , Diferenciación Celular/fisiología , Periodontitis Periapical/metabolismo , Solventes/metabolismo , AguaRESUMEN
BACKGROUND: Leukotriene B4 (LTB4) is a potent lipid mediator that stimulate the immune response. Because dental pulp inflammation and dentin repair are intrinsically related responses, the aim of this research was to investigate the potential of LTB4 in inducing differentiation of dental pulp stem cells. METHODS: Microspheres (MS) loaded with LTB4 were prepared using an oil emulsion solvent extraction evaporation process and sterility, characterization, efficiency of LTB4 encapsulation and in vitro LTB4 release assay were investigated. Mouse dental pulp stem cells (OD-21) were stimulated with soluble LTB4 or MS loaded with LTB4 (0.01 and 0.1 µM). Cytotoxicity and cell viability was determined by lactate dehydrogenase and methylthiazol tetrazolium assays. Gene expression were measured by quantitative reverse transcription polymerase chain reaction after 3, 6, 24, 48 and 72 h. Mineralized nodule formation was assessed after 28 days of OD-21 cell stimulation with LTB4 in mineralized media or not. Groups were compared using one-way ANOVA test followed by Dunnett's post-test (α = 0.05). RESULTS: Treatment with LTB4 or MS loaded with LTB4 (0.01 and 0.1 µm-µM) were not cytotoxic to OD-21 cells. Treatment with LTB4 modulated the expression of the Ibsp (integrin binding sialoprotein) and Runx2 (runt-related transcription factor 2) genes differently depending on the experimental period analyzed. Interestingly LTB4 loaded in microspheres (0.1 µM) allowed long term dental pulp cell differentiation and biomineralization. CONCLUSION: LTB4, soluble or loaded in MS, were not cytotoxic and modulated the expression of the Ibsp and Runx2 genes in cultured OD-21 cells. When LTB4 was incorporated into MS, odontoblast differentiation and mineralization was induced in long term culture.
Asunto(s)
Pulpa Dental , Leucotrieno B4 , Animales , Biomineralización , Diferenciación Celular , Células Cultivadas , Proteínas de la Matriz Extracelular/genética , Humanos , Leucotrieno B4/metabolismo , Leucotrieno B4/farmacología , Ratones , Microesferas , Odontoblastos/metabolismo , Células Madre/metabolismoRESUMEN
Serum levels of leukotriene-B4 (LTB4) are increased in type 1 diabetes (T1D) and it mediates systemic inflammation and macrophage reprogramming associated with this condition. Herein, we investigated the involvement of LTB4 in adiposity loss, hyperlipidemia, and changes in macrophage metabolism in a mouse model of streptozotocin-induced T1D. LTB4 receptor (BLT1) antagonist u75302 was employed to block LTB4 effects. As expected, hypoinsulinemia in T1D was associated with hyperglycemia, low levels of glucagon, hyperlipidemia, significant body fat loss, and increased white adipose tissue expression of Fgf21, a marker for lipolysis. With the exception of hyperglycemia and hypoglucagonemia, blockade of LTB4 signaling reverted these parameters in T1D mice. Along with hyperlipidemia, macrophages from T1D mice exhibited higher lipid uptake and accumulation. These cells also had enhanced glycolysis and oxidative metabolism and these parameters were dependent on the mitochondrial uncoupling respiration, as evidenced by elevated expression of oxidation markers carnitine palmitoyltransferase and uncoupling protein 1. Interestingly, all these parameters were at least partially reverted in T1D mice treated with u75302. Altogether, these findings suggest that in T1D mice LTB4/BLT1 is involved in the fat loss, hyperlipidemia, and increased macrophage lipid uptake and metabolism with an important involvement of mitochondrial uncoupling activity. These previously unrecognized LTB4/BLT1 functions may be explored in future to therapeutically alleviate severity of hyperlipidemia and systemic inflammation in T1D.
Asunto(s)
Adiposidad , Diabetes Mellitus Tipo 1/metabolismo , Leucotrieno B4/farmacología , Macrófagos Peritoneales/metabolismo , Adiposidad/efectos de los fármacos , Animales , Biomarcadores/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Ácidos Grasos/metabolismo , Glucólisis/efectos de los fármacos , Hiperlipidemias/metabolismo , Hiperlipidemias/patología , Lipogénesis/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Macrófagos Peritoneales/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxidación-Reducción/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Proteína Desacopladora 1/metabolismoRESUMEN
Tityus serrulatus sting causes thousands of deaths annually worldwide. T. serrulatus-envenomed victims exhibit local or systemic reaction that culminates in pulmonary oedema, potentially leading to death. However, the molecular mechanisms underlying T. serrulatus venom (TsV) activity remain unknown. Here we show that TsV triggers NLRP3 inflammasome activation via K(+) efflux. Mechanistically, TsV triggers lung-resident cells to release PGE2, which induces IL-1ß production via E prostanoid receptor 2/4-cAMP-PKA-NFκB-dependent mechanisms. IL-1ß/IL-1R actions account for oedema and neutrophil recruitment to the lungs, leading to TsV-induced mortality. Inflammasome activation triggers LTB4 production and further PGE2 via IL-1ß/IL-1R signalling. Activation of LTB4-BLT1/2 pathway decreases cAMP generation, controlling TsV-induced inflammation. Exogenous administration confirms LTB4 anti-inflammatory activity and abrogates TsV-induced mortality. These results suggest that the balance between LTB4 and PGE2 determines the amount of IL-1ß inflammasome-dependent release and the outcome of envenomation. We suggest COX1/2 inhibition as an effective therapeutic intervention for scorpion envenomation.
Asunto(s)
Proteínas Portadoras/genética , Dinoprostona/farmacología , Interleucina-1beta/efectos de los fármacos , Leucotrieno B4/farmacología , Macrófagos Peritoneales/efectos de los fármacos , Picaduras de Escorpión/inmunología , Venenos de Escorpión/farmacología , Animales , Araquidonato 5-Lipooxigenasa/genética , Western Blotting , Proteínas Portadoras/inmunología , Celecoxib/farmacología , AMP Cíclico/inmunología , Proteínas Quinasas Dependientes de AMP Cíclico/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/inmunología , Inhibidores de la Ciclooxigenasa/farmacología , Dinoprostona/inmunología , Técnicas In Vitro , Indoles/farmacología , Indometacina/farmacología , Inflamasomas/inmunología , Interleucina-1beta/inmunología , Leucotrieno B4/inmunología , Inhibidores de la Lipooxigenasa/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos Peritoneales/inmunología , Ratones , Ratones Noqueados , FN-kappa B/efectos de los fármacos , FN-kappa B/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR , Fosfoproteínas , Antagonistas de Prostaglandina/farmacología , Subtipo EP2 de Receptores de Prostaglandina E/efectos de los fármacos , Subtipo EP2 de Receptores de Prostaglandina E/inmunología , Subtipo EP4 de Receptores de Prostaglandina E/efectos de los fármacos , Subtipo EP4 de Receptores de Prostaglandina E/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Picaduras de Escorpión/mortalidad , Escorpiones , Xantonas/farmacologíaRESUMEN
Fish oil, a rich source of n-3 fatty acids, has been studied for its beneficial effects in many diseases. Recent studies have shown the robust anti-inflammatory activity of fish oil (FO), when administered orally to rats, in models of acute inflammation. Herein, we investigated if treatment with fish oil preparation (FOP) could interfere with the recruitment of leukocytes into the joint cavity of arthritic rats. We also evaluated the effect of treatment on rolling behavior and leukocyte adhesion in vivo and on leukocyte chemotaxis in vitro. Treatment with FOP (75, 150, and 300 mg/kg) initiated on the day of induction of arthritis (day 0) and maintained for 21 days reduced the total number of leukocytes recruited into the joint cavity, the number of rolling and adhered leukocytes in arthritic rats, and leukocyte migration in response to stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP) and leukotriene B4 (LTB4). Together, our data provide evidence that FOP plays an important inhibitory role in the recruitment of leukocytes into the joint cavity of arthritic rats.
Asunto(s)
Antiinflamatorios/farmacología , Artritis Experimental/tratamiento farmacológico , Quimiotaxis de Leucocito/efectos de los fármacos , Ácidos Grasos Omega-3/farmacología , Aceites de Pescado/farmacología , Animales , Adhesión Celular/efectos de los fármacos , Articulaciones/inmunología , Articulaciones/patología , Rodamiento de Leucocito/efectos de los fármacos , Leucocitos/inmunología , Leucotrieno B4/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , N-Formilmetionina Leucil-Fenilalanina/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
The aim of this study was to investigate the effect of anethole (AN) and eugenol (EUG) on leukocyte migration using in vitro chemotaxis and in situ microcirculation assays. BALB/c mice were used for the in vitro chemotaxis assay, and Wistar rats for the in situ microcirculation assay. We evaluated (a) the in vitro leukocyte migration in response to chemotactic factors (formyl-methionyl-leucyl-phenylalanine [fMLP] and leukotriene B4 [LTB4]) and (b) the rolling, adhesion, and migration of leukocytes induced by an injection of carrageenan (100 µg/cavity) into the scrotum of the animal. In the in vitro chemotaxis assay, AN and EUG at doses of 1, 3, 9, and 27 µg/ml significantly inhibited leukocyte migration when stimulated by the chemotactic agents fMLP and LTB4. In the in situ microcirculation assay, AN at doses of 125 and 250 mg/kg and EUG at a dose of 250 mg/kg significantly decreased the number of leukocytes that rolled, adhered, and migrated to perivascular tissue. The results indicate that AN and EUG exert inhibitory effects on leukocyte migration, highlighting their possible use to diminish excessive leukocyte migration in the inflammatory process.
Asunto(s)
Anisoles/farmacología , Quimiotaxis de Leucocito/efectos de los fármacos , Eugenol/farmacología , Derivados de Alilbenceno , Animales , Carragenina/farmacología , Humanos , Leucocitos/efectos de los fármacos , Leucocitos/fisiología , Leucotrieno B4/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , N-Formilmetionina Leucil-Fenilalanina/farmacología , Ratas , Ratas WistarRESUMEN
Rats were injected with rat recombinant (rr) IL3, rrSCF, rrIL-3+rrSCF, rrRANTES and LTB4. Six hours after subcutaneous injection of rrIL-3 or rrIL-3+rrSCF, there was an increase in mast cell numbers in the skin and spleen. Peritoneal mast cells were recruited following i.p. injection of rrIL-3, but with rrIL-3+rrSCF recruitment was delayed. Immunostaining with a mast cell specific antibody showed that immature orthochromatic mast cells were being recruited. rrIL-3 induced recruitment of mast cells to the peritoneal cavity was blocked by anti-integrin antibodies. Mast cell recruitment depended on the target tissue and the time of exposure to the chemoattractant.
Asunto(s)
Factores Quimiotácticos/inmunología , Mastocitos/inmunología , Animales , Movimiento Celular/inmunología , Quimiocina CCL5/farmacología , Factores Quimiotácticos/farmacología , Interleucina-3/farmacología , Leucotrieno B4/farmacología , Masculino , Mastocitos/efectos de los fármacos , Cavidad Peritoneal , Ratas , Ratas Wistar , Proteínas Recombinantes/farmacología , Piel/citología , Piel/inmunología , Bazo/citología , Bazo/inmunología , Factor de Células Madre/farmacologíaRESUMEN
The bioactive lipid mediator leukotriene B4 (LTB4) greatly enhances phagocyte antimicrobial functions against a myriad of pathogens. In murine histoplasmosis, inhibition of the LT-generating enzyme 5-lypoxigenase (5-LO) increases the susceptibility of the host to infection. In this study, we investigated whether murine resistance or susceptibility to Histoplasma capsulatum infection is associated with leukotriene production and an enhancement of in vivo and/or in vitro antimicrobial effector function. We show that susceptible C57BL/6 mice exhibit a higher fungal burden in the lung and spleen, increased mortality, lower expression levels of 5-LO and leukotriene B4 receptor 1 (BLT1) and decreased LTB4 production compared to the resistant 129/Sv mice. Moreover, we demonstrate that endogenous and exogenous LTs are required for the optimal phagocytosis of H. capsulatum by macrophages from both murine strains, although C57BL/6 macrophages are more sensitive to the effects of LTB4 than 129/Sv macrophages. Therefore, our results provide novel evidence that LTB4 production and BLT1 signaling are required for a histoplasmosis-resistant phenotype.
Asunto(s)
Histoplasma/inmunología , Histoplasmosis/veterinaria , Leucotrieno B4 , Receptores de Leucotrieno B4/inmunología , Enfermedades de los Roedores/inmunología , Animales , Araquidonato 5-Lipooxigenasa/genética , Araquidonato 5-Lipooxigenasa/inmunología , Susceptibilidad a Enfermedades , Inhibidores Enzimáticos/farmacología , Expresión Génica/inmunología , Histoplasma/patogenicidad , Histoplasmosis/genética , Histoplasmosis/inmunología , Histoplasmosis/metabolismo , Especificidad del Huésped , Interacciones Huésped-Patógeno , Leucotrieno B4/metabolismo , Leucotrieno B4/farmacología , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/microbiología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Fagocitosis/efectos de los fármacos , Receptores de Leucotrieno B4/genética , Enfermedades de los Roedores/genética , Enfermedades de los Roedores/metabolismo , Transducción de Señal , Bazo/efectos de los fármacos , Bazo/inmunología , Bazo/microbiologíaRESUMEN
Several emerging lines of evidence support an anti-inflammatory role for nicotinic acid (niacin); however, its role in the regulation of leukocyte migration in response to inflammatory stimuli has not been elucidated until now. Herein, we have examined the effect of nicotinic acid on neutrophil recruitment in experimentally induced inflammation. We demonstrated that nicotinic acid treatment inhibited interleukin (IL)-8-induced, leukotriene (LT)B4-induced, and carrageenan-induced neutrophil migration into the pleural cavity of BALB/c mice and reduced neutrophil rolling and adherence in a mouse cremaster muscle preparation. Surprisingly, nicotinic acid treatment increased the level of the neutrophil chemoattractant KC in response to carrageenan. These results suggest that nicotinic acid plays an important role in the regulation of inflammation due to its ability to inhibit the actions of the neutrophil chemoattractants IL-8 and LTB4. Further inhibition of chemoattractants leads to impairment of leukocyte rolling and adherence to the vascular endothelium in the microcirculation of inflamed tissues.
Asunto(s)
Antiinflamatorios/farmacología , Enfermedades del Sistema Inmune/prevención & control , Inflamación/tratamiento farmacológico , Trastornos Leucocíticos/prevención & control , Niacina/farmacología , Animales , Carragenina/farmacología , Adhesión Celular/efectos de los fármacos , Quimiocina CXCL1/metabolismo , Modelos Animales de Enfermedad , Inflamación/patología , Interleucina-8/farmacología , Rodamiento de Leucocito/efectos de los fármacos , Leucotrieno B4/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , Infiltración Neutrófila/efectos de los fármacos , Cavidad Pleural/efectos de los fármacos , Cavidad Pleural/metabolismoRESUMEN
The underlying mechanisms of itch are poorly understood. We have investigated a model involving the chemoattractant leukotriene B4 (LTB4) that is up-regulated in common skin diseases. Intradermal injection of LTB4 (0.1 nmol/site) into female CD1 mice induced significant scratching movements (used as an itch index) compared with vehicle-injected (0.1% bovine serum albumin-saline) mice. Intraperitoneal transient receptor potential (TRP) channel antagonist treatment significantly inhibited itch as follows: TRP vanilloid 1 (TRPV1) antagonist SB366791 (0.5 mg/kg, by 97%) and the TRP ankyrin 1 (TRPA1) antagonists TCS 5861528 (10 mg/kg; 82%) and HC-030031 (100 mg/kg; 76%). Leukotriene B4 receptor 2 antagonism by LY255283 (5 mg/kg i.p.; 62%) reduced itch. Neither TRPV1-knockout (TRPV1-KO) nor TRPA1-knockout (TRPA1-KO mice exhibited LTB4-induced itch compared with their wild-type counterparts. The reactive oxygen species scavengers N-acetylcysteine (NAC; 204 mg/kg i.p.; 86%) or superoxide dismutase (SOD; 10 mg/kg i.p.; 83%) also inhibited itch. LTB4-induced superoxide release was attenuated by TCS 5861528 (56%) and HC-030031 (66%), NAC (58%), SOD (50%), and LY255283 (59%) but not by the leukotriene B4 receptor 1 antagonist U-75302 (9 nmol/site) or SB366791. Itch, superoxide, and myeloperoxidase generation were inhibited by the leukocyte migration inhibitor fucoidan (10 mg/kg i.v.) by 80, 61, and 34%, respectively. Myeloperoxidase activity was also reduced by SB366791 (35%) and SOD (28%). TRPV1-KO mice showed impaired myeloperoxidase release, whereas TRPA1-KO mice exhibited diminished production of superoxide. This result provides novel evidence that TRPA1 and TRPV1 contribute to itch via distinct mechanisms.
Asunto(s)
Leucocitos/metabolismo , Leucotrieno B4/farmacología , Superóxidos/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Animales , Ancirinas/farmacología , Femenino , Leucocitos/efectos de los fármacos , Ratones , Ratones Noqueados , Prurito/tratamiento farmacológico , Prurito/metabolismo , Receptores de Leucotrieno B4/antagonistas & inhibidores , Canales Catiónicos TRPV/metabolismoRESUMEN
Leukotriene B(4), an arachidonic acid-derived lipid mediator, is a known proinflammatory agent that has a direct effect upon neutrophil physiology, inducing reactive oxygen species generation by the NADPH oxidase complex and impairing neutrophil spontaneous apoptosis, which in turn may corroborate to the onset of chronic inflammation. Despite those facts, a direct link between inhibition of neutrophil spontaneous apoptosis and NADPH oxidase activation by leukotriene B(4) has not been addressed so far. In this study, we aim to elucidate the putative role of NADPH oxidase-derived reactive oxygen species in leukotriene B(4)-induced anti-apoptotic effect. Our results indicate that NADPH oxidase-derived reactive oxygen species are critical to leukotriene B(4) pro-survival effect on neutrophils. This effect also relies on redox modulation of nuclear factor kappaB signaling pathway. We have also observed that LTB(4)-induced Bad degradation and mitochondrial stability require NADPH oxidase activity. All together, our results strongly suggest that LTB(4)-induced anti-apoptotic effect in neutrophils occurs in a reactive oxygen species-dependent manner. We do believe that a better knowledge of the molecular mechanisms underlying neutrophil spontaneous apoptosis may contribute to the development of more successful strategies to control chronic inflammatory conditions such as rheumatoid arthritis.
Asunto(s)
Apoptosis/efectos de los fármacos , Leucotrieno B4/farmacología , Mitocondrias/metabolismo , NADPH Oxidasas/metabolismo , FN-kappa B/metabolismo , Neutrófilos/citología , Neutrófilos/enzimología , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Oxidación-Reducción/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Several emerging lines of evidence support an anti-inflammatory role for nicotinamide and other vitamin B components. However, the mechanisms underlying their activity remain unclear. In the present study, we investigated the ability of nicotinamide to inhibit both neutrophil recruitment in IL-8-, LTB(4)- or carrageenan-induced pleurisy in mice and the rolling and adherence of neutrophils. Nicotinamide inhibited IL-8-, LTB(4)- and carrageenan-induced neutrophil migration, KC production and carrageenan-induced neutrophil rolling and adherence. We propose that the effects of nicotinamide in inhibiting neutrophil recruitment in carrageenan-induced pleurisy may be due to the ability of nicotinamide to inhibit the action of IL-8 and LTB(4), decrease KC production, and inhibit early events that regulate leukocyte migration from blood vessels into tissue.
Asunto(s)
Antiinflamatorios/farmacología , Infiltración Neutrófila/efectos de los fármacos , Niacinamida/farmacología , Pleuresia/tratamiento farmacológico , Animales , Carragenina/farmacología , Adhesión Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Interleucina-8/farmacología , Rodamiento de Leucocito/efectos de los fármacos , Leucotrieno B4/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , Pleuresia/inmunologíaRESUMEN
Maternal diabetes impairs fetoplacental metabolism and growth. Peroxisome proliferator-activated receptor α (PPARα) is a nuclear receptor capable of regulating lipid metabolism and inflammatory pathways. In this study, we analyzed whether placental and fetal PPARα activation regulates lipid metabolism and nitric oxide (NO) production in term placentas from diabetic rats. Diabetes was induced by neonatal streptozotocin administration. On day 21 of pregnancy, placentas from control and diabetic rats were cultured in the presence of PPARα agonists (clofibrate and leukotriene B(4) (LTB(4))) for further evaluation of levels, synthesis, and peroxidation of lipids as well as NO production. Besides, on days 19, 20, and 21 of gestation, fetuses were injected with LTB(4), and the placentas were explanted on day 21 of gestation for evaluation of placental weight and concentrations of placental lipids, lipoperoxides, and NO metabolites. We found that placentas from diabetic rats showed reduced PPARα concentrations. They presented no lipid overaccumulation but reduced lipid synthesis, parameters negatively regulated by PPARα activators. Lipid peroxidation and NO production, increased in placentas from diabetic rats, were negatively regulated by PPARα activators. Fetal PPARα activation in diabetic rats does not change placental lipid concentrations but reduced placental weight and NO production. In conclusion, PPARα activators regulate lipid metabolism and NO production in term placentas from diabetic rats, an activation that regulates placental growth and can partly be exerted by the developing fetus.
Asunto(s)
Clofibrato/farmacología , Diabetes Mellitus Experimental/fisiopatología , Leucotrieno B4/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , Óxido Nítrico/metabolismo , PPAR alfa/agonistas , Placenta/efectos de los fármacos , Animales , Colesterol/metabolismo , Diabetes Mellitus Experimental/metabolismo , Femenino , Feto/efectos de los fármacos , Técnicas In Vitro , Peroxidación de Lípido/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo II/metabolismo , Tamaño de los Órganos , PPAR alfa/metabolismo , Fosfolípidos/metabolismo , Placenta/metabolismo , Placenta/fisiopatología , Embarazo , Ratas , Ratas Wistar , Triglicéridos/metabolismoRESUMEN
BACKGROUND: peroxisome proliferator-activated receptor α (PPARα) is a crucial regulator of liver lipid metabolism. As maternal diabetes impairs foetal lipid metabolism and growth, we aimed to determine whether PPARα activation regulates lipid metabolism in the foetal liver from diabetic rats as well as foetal weight and foetal liver weight. METHODS: diabetes was induced by neonatal streptozotocin administration (90 mg/kg). For ex vivo studies, livers from 21-day-old foetuses from control and diabetic rats were explanted and incubated in the presence of PPARα agonists (clofibrate and leukotriene B(4) ) for further evaluation of lipid levels (by thin layer chromatography and densitometry), de novo lipid synthesis (by (14) C-acetate incorporation) and lipid peroxidation (by thiobarbituric reactive substances evaluation). For in vivo studies, foetuses were injected through the uterine wall with leukotriene B(4) on days 19, 20 and 21 of gestation. On day 21 of gestation, foetal liver concentrations of lipids and lipoperoxides were evaluated. RESULTS: foetuses from diabetic rats showed increased body weight and liver weight, as well as accumulation of triglycerides and cholesteryl esters, increased de novo lipid synthesis and lipid peroxidation in the liver when compared to controls. Ex vivo studies showed that PPARα ligands reduced both the concentrations and synthesis of the lipid species studied and lipid peroxidation in the foetal liver from diabetic rats. In vivo experiments showed that leukotriene B(4) reduced the concentrations of triglycerides, cholesteryl esters and phospholipids, as well as lipid peroxidation, foetal weight and foetal liver weight in diabetic rats. CONCLUSIONS: PPARα activation regulates the impaired foetal liver lipid metabolism, prevents hepatomegaly and reduces foetal overgrowth induced by maternal diabetes.
Asunto(s)
Diabetes Mellitus Experimental/patología , Feto/citología , Metabolismo de los Lípidos/fisiología , Hígado/metabolismo , PPAR alfa/metabolismo , Animales , Peso Corporal , Núcleo Celular/metabolismo , Diabetes Mellitus Experimental/metabolismo , Femenino , Leucotrieno B4/farmacología , Peroxidación de Lípido/efectos de los fármacos , Hígado/citología , Embarazo , Ratas , Ratas Wistar , Triglicéridos/metabolismoRESUMEN
OBJECTIVE AND DESIGN: Although proteinase-activated receptor (PAR)-4 has been implicated in inflammation, its role in regulating eosinophil recruitment in response to chemoattractants has not yet been demonstrated. To investigate the contribution of proteinases and PAR-4 activation to eosinophil migration in response to eotaxin-1 or leukotriene B(4) (LTB(4)), the effects of aprotinin or PAR-4 antagonist trans-cinnamoyl-YPGKF-NH(2) (tcY-NH(2)) on eosinophil migration induced by these chemoattractants were investigated. METHODS: BALB/c mice were pretreated with aprotinin or tcY-NH(2) (30 µg/mouse) prior to intrapleural injection of LTB(4) or eotaxin-1 and the number of infiltrating eosinophils was determined 48 h later. RESULTS: Aprotinin (1 mg/kg) inhibited eosinophil recruitment induced by eotaxin-1 (p < 0.01), but not that induced by LTB(4). Moreover, tcY-NH(2) treatment inhibited eosinophil recruitment in response to eotaxin-1 (p < 0.01 by ANOVA/Tukey post-test). CONCLUSION: These data suggest that aprotinin-inhibited proteinases participate in eosinophil migration induced by eotaxin-1 and that PAR-4 activation plays an important role in regulating this migration.
Asunto(s)
Aprotinina/farmacología , Quimiocina CCL11/farmacología , Eosinófilos/efectos de los fármacos , Receptores Proteinasa-Activados/metabolismo , Animales , Movimiento Celular , Cinamatos/farmacología , Eosinófilos/metabolismo , Leucotrieno B4/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , Oligopéptidos/farmacología , Ovalbúmina/inmunología , Cavidad Pleural/efectos de los fármacos , Cavidad Pleural/inmunología , Cavidad Pleural/metabolismo , Pleuresia/inmunologíaRESUMEN
Formaldehyde (FA) exposure induces upper airways irritation and respiratory abnormalities, but its mechanisms are not understood. Since mast cells are widely distributed in the airways, we hypothesized that FA might modify the airways reactivity by mechanism involving their activation. Tracheal rings of rats were incubated with Dulbecco's modified medium culture containing FA (0.1 ppm) in 96-well plastic microplates in a humid atmosphere. After 30 min, 6h, and 24-72 h, the rings were suspended in an organ bath and dose-response curve to methacholine (MCh) were determined. Incubation with FA caused a transient tracheal hyperresponsiveness to MCh that was independent from tracheal epithelium integrity. Connective tissue mast cell depletion caused by compound 48/80 or mast cell activation by the allergic reaction, before exposure of tracheal rings to FA prevented the increased responsiveness to MCh. LTB(4) concentrations were increased in the culture medium of tracheas incubated with FA for 48 h, whereas the LTB(4)-receptor antagonist MK886 (1 microM) added before FA exposure rendered the tracheal rings normoreactive to MCh. In addition, FA exposure did not cause hyperresponsiveness in tracheal segments incubated with l-arginine (1 microM). We suggest that airway connective tissue mast cells constitute the target and may provide the increased LTB(4) generation as well as an elevated consumption of NO leading to tracheal hyperresponsiveness to MCh.
Asunto(s)
Formaldehído/toxicidad , Leucotrieno B4/biosíntesis , Mastocitos/efectos de los fármacos , Contracción Muscular/efectos de los fármacos , Óxido Nítrico/biosíntesis , Tráquea/efectos de los fármacos , Animales , Arginina/farmacología , Células del Tejido Conectivo/inmunología , Técnicas In Vitro , Leucotrieno B4/antagonistas & inhibidores , Leucotrieno B4/farmacología , Masculino , Mastocitos/metabolismo , Cloruro de Metacolina/farmacología , Ovalbúmina/inmunología , Ratas , Ratas Wistar , Tráquea/fisiología , p-Metoxi-N-metilfenetilamina/farmacologíaRESUMEN
Chronic inflammation has long been associated with neoplastic progression. Our group had recently shown that the addition of a large number of apoptotic tumor cells to the tumor microenvironment induces a potent acute inflammatory reaction capable of promoting melanoma growth; however, primarily necrotizing cells do not cause such a reaction. Here, we show that potent inflammatory agents, such as lipopolysaccharide (LPS) and carrageenan, also promote growth of subtumorigenic doses of melanoma cells, having no effect on melanoma proliferation in vitro. Inhibition of 5-lipoxygenase (5-LOX) seems to have a pivotal role in this model because caffeic acid and MK886, a FLAP (5-LOX-activating protein) inhibitor, partially hindered tumor growth induced by apoptotic cells or LPS. Other enzymes of the arachidonic acid pathway, cyclooxygenase-1 and cyclooxygenase-2, seem to have no participation in this tumor promoter effect, as the inhibitor of both enzymes (indomethacin) did not alter melanoma growth. Leukotriene B4 (LTB4), the main product of the 5-LOX pathway, was able to induce growth of subtumorigenic inocula of melanoma cells, and a LTB4 receptor antagonist inhibited acute inflammation-associated tumor growth. Addition to the tumor inflammatory microenvironment of eicosapentaenoic acid, an omega3-polyunsaturated fatty acid with anti-inflammatory properties, or leukotriene B5, an eicosapentaenoic acid-derived leukotriene, significantly inhibited tumor development. These results give new insights to the mechanisms through which inflammation may contribute to tumor progression and suggest that LOX has an important role in tumor progression associated with an inflammatory state in the presence of apoptosis, which may be a consideration for apoptosis-inducing treatments, such as chemotherapy and radiotherapy.
Asunto(s)
Inflamación/patología , Leucotrieno B4/farmacología , Melanoma/patología , Animales , Apoptosis/fisiología , Araquidonato 5-Lipooxigenasa/metabolismo , Carragenina/farmacología , Procesos de Crecimiento Celular/efectos de los fármacos , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Progresión de la Enfermedad , Ácido Eicosapentaenoico/análogos & derivados , Ácido Eicosapentaenoico/metabolismo , Ácido Eicosapentaenoico/farmacología , Histocitoquímica , Indometacina/farmacología , Inflamación/inducido químicamente , Inflamación/metabolismo , Leucotrieno B4/análogos & derivados , Leucotrieno B4/metabolismo , Lipopolisacáridos/farmacología , Inhibidores de la Lipooxigenasa , Melanoma/metabolismo , Ratones , Prostaglandina-Endoperóxido Sintasas/metabolismoRESUMEN
Histoplasmosis is a pulmonary disease characterised by chronic granulomatous and suppurative inflammatory reactions caused by Histoplasma capsulatum. Regarding new therapies to control fungal infections, the aim of this study was to investigate whether pulmonary administration of leukotriene B(4) (LTB(4))-loaded microspheres (MS) could confer protection to 5-lipoxygenase knockout (5-LO(-/-)) mice infected by H. capsulatum. In this study, MS containing LTB(4) were administered intranasally to mice infected by H. capsulatum. On Day 14 after the infection, fungal recovery from the lungs and histology were evaluated and inflammatory cytokines were measured. Pulmonary administration of LTB(4)-loaded MS was able to reduce fungal recovery from infected lungs. Production of important inflammatory cytokines related to host defence was augmented following MS administration to the lungs. Lung histology also showed that infected mice presented a clear reduction in the fungal burden following the pulmonary release of LTB(4) from MS. Our study provides evidence that the proposed biodegradable microparticulate system, which can release LTB(4) to the lungs, can be employed as therapy, enhancing the antimicrobial activity of host cells during histoplasmosis.
Asunto(s)
Histoplasma/efectos de los fármacos , Histoplasmosis , Leucotrieno B4/farmacología , Microesferas , Administración Intranasal , Animales , Citocinas/inmunología , Citocinas/metabolismo , Glicolatos , Histoplasmosis/tratamiento farmacológico , Histoplasmosis/inmunología , Histoplasmosis/microbiología , Histoplasmosis/patología , Humanos , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Inflamación/microbiología , Ácido Láctico , Leucotrieno B4/administración & dosificación , Leucotrieno B4/inmunología , Lipooxigenasa/genética , Pulmón/inmunología , Pulmón/microbiología , Pulmón/patología , Enfermedades Pulmonares Fúngicas/tratamiento farmacológico , Enfermedades Pulmonares Fúngicas/inmunología , Enfermedades Pulmonares Fúngicas/microbiología , Enfermedades Pulmonares Fúngicas/patología , Masculino , Ratones , Ratones Noqueados , Ácido Poliglicólico , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Resultado del TratamientoRESUMEN
In alveolar macrophages, leukotriene (LT) B(4) and cysteinyl LTs (LTC(4), LTD(4) and LTE(4)) both enhance Fc gamma receptor (Fc gammaR)-mediated phagocytosis. In the present study we investigated the role of specific PKC isoforms (PKC-alpha and -delta), the MAP kinases p38 and ERK 1/2, and PI3K in mediating the potentiation of Fc gammaR-mediated phagocytosis induced by addition of leukotrienes to the AMs. It was found that exogenously added LTB(4) and LTD(4) both enhanced PKC-delta and -alpha phosphorylation during Fc gammaR engagement. Studies with isoform-selective inhibitors indicated that exogenous LTB(4) effects were dependent on both PKC-alpha and -delta, while LTD(4) effects were exclusively due to PKC-delta activation. Although both exogenous LTB(4) and LTD(4) enhanced p38 and ERK 1/2 activation, LTB(4) required only ERK 1/2, while LTD(4) required only p38 activation. Activation by both LTs was dependent on PI3K activation. Effects of endogenous LTs on kinase activation were also investigated using selective LT receptor antagonists. Endogenous LTB(4) contributed to Fc gammaR-mediated activation of PKC-alpha, ERK 1/2 and PI3K, while endogenous cysLTs contributes to activation of PKC-delta, p38 and PI3K. Taken together, our data show that the capacities of LTB(4) and LTD(4) to enhance Fc gammaR-mediated phagocytosis reflect their differential activation of specific kinase programs.
Asunto(s)
Leucotrieno B4/fisiología , Leucotrieno D4/fisiología , Macrófagos Alveolares/inmunología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Quinasa C/metabolismo , Receptores de IgG/fisiología , Animales , Células Cultivadas , Femenino , Leucotrieno B4/farmacología , Leucotrieno D4/farmacología , Macrófagos Alveolares/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Fagocitosis , Inhibidores de las Quinasa Fosfoinosítidos-3 , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Ratas , Ratas Wistar , Receptores de IgG/inmunologíaRESUMEN
Leukotriene B(4) (LTB(4)) is a potent inflammatory mediator and stimulates the immune response. In addition, LTB(4) promotes leukocyte functions such as phagocytosis, chemotaxis and chemokinesis of polymorphonuclear leukocytes, as well as modulates cytokine release. However, some physicochemical characteristics of leukotrienes, such as poor solubility in water and chemical instability, make them difficult to administer in vivo. The aim of this study was to develop LTB(4)-loaded microspheres (MS) that prolong and sustain the in vivo release of this mediator. An oil-in-water emulsion solvent extraction-evaporation method was chosen to prepare the lipid-loaded MS. We determined their diameters, evaluated the in vitro release of LTB(4), using enzyme immunoassay and evaluated in vitro MS uptake by peritoneal macrophages. To assess the preservation of neutrophil chemoattractant activity, LTB(4)-loaded MS were tested in vitro (in a modified Boyden microchamber) and in vivo, after intratracheal administration.