RESUMO
Infection with the protozoan Trypanosoma cruzi causes Chagas disease and consequently leads to severe inflammatory heart condition; however, the mechanisms driving this inflammatory response have not been completely elucidated. Nitric oxide (NO) is a key mediator of parasite killing in T. cruzi-infected mice, and previous studies have suggested that leukotrienes (LTs) essentially regulate the NO activity in the heart. We used infected 5-lipoxygenase-deficient mice (5-LO-/-) to explore the participation of nitric oxide synthase isoforms, inducible (iNOS) and constitutive (cNOS), in heart injury, cytokine profile, and oxidative stress during the early stage of T. cruzi infection. Our evidence suggests that the cNOS of the host is involved in the resistance of 5-LO-/- mice during T. cruzi infection. iNOS inhibition generated a remarkable increase in T. cruzi infection in the blood and heart of mice, whereas cNOS inhibition reduced cardiac parasitism (amastigote nests). Furthermore, this inhibition associates with a higher IFN-γ production and lower lipid peroxidation status. These data provide a better understanding about the influence of NO-interfering therapies for the inflammatory response toward T. cruzi infection.
Assuntos
Araquidonato 5-Lipoxigenase/sangue , Doença de Chagas/sangue , Doença de Chagas/enzimologia , Animais , Antioxidantes/metabolismo , Citocinas/sangue , Camundongos , Camundongos Knockout , Óxido Nítrico/sangue , Óxido Nítrico Sintase Tipo II/sangue , Trypanosoma cruzi/patogenicidadeRESUMO
Objective: Monosodium urate-induced inflammation plays a vital role in acute gout (AG). Inflammation is a multi-stage process involved in the acute release of arachidonic acid and its metabolites. However, the function of the metabolism of arachidonic acid and other polyunsaturated fatty acids in AG is not well understood. This study aimed to investigate the modification of polyunsaturated fatty acid metabolism by AG. Methods: Plasma samples from patients with an AG attack (n = 26) and gender-matched healthy controls (n = 26) were analysed by metabolic profiling of polyunsaturated fatty acids. The findings were further validated with a second cohort (n = 20 each group). The associated mechanisms were investigated in whole blood cells from the second cohort and neutrophils in vitro. Results: Plasma metabolic profiling revealed a significant increase in leukotriene B4 (LTB4) for AG patients in both cohorts. The increase in plasma LTB4 was accounted for by the dynamic balance between the activation of 5-lipoxygenase and CYP4F3, the former mediating the biosynthesis of LTB4 and the latter mediating its metabolism. This was supported by significantly increased transcriptional levels of 5-lipoxygenase and CYP4F3 in whole blood cells from AG patients compared with those of controls, and the uric acid-caused dose-relevant and time-dependent activation of 5-lipoxygenase and CYP4F3 at the transcriptional and molecular levels in vitro. Conclusion: Increased LTB4 in AG patients is mainly due to activation of 5-lipoxygenase. 5-Lipoxygenase inhibition may be of therapeutic value clinically.
Assuntos
Araquidonato 5-Lipoxigenase/sangue , Artrite Gotosa/enzimologia , Doença Aguda , Adolescente , Adulto , Idoso , Artrite Gotosa/sangue , Estudos de Casos e Controles , Células Cultivadas , Família 4 do Citocromo P450/sangue , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Ácidos Graxos Insaturados/sangue , Feminino , Humanos , Leucotrieno B4/sangue , Masculino , Metaboloma/fisiologia , Pessoa de Meia-Idade , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Ácido Úrico/administração & dosagem , Ácido Úrico/farmacologia , Adulto JovemRESUMO
The inflammatory process plays a key role in neurodegenerative disorder. The inflammatory molecule, 5-lipooxygenase (5-LOX), protein is involved in the pathologic phenotype of Alzheimer's disease (AD) which includes Aß amyloid deposition and tau hyperphosphorylation. This study determined the level of 5-LOX in serum of AD patients, mild cognitive impairment (MCI) patients, and the normal elderly, and the rescue effect by YWCS, a peptide inhibitor of 5-LOX on neurotoxicity by Aß amyloid25-35 (Aß25-35) in neuroblastoma cells. The concentration of serum 5-LOX was estimated by surface plasmon resonance and western blot. The neuroprotective effect of 5-LOX peptide inhibitor YWCS in Aß25-35-induced neurotoxicity was analyzed by MTT assay and western blotting. We found significant upregulated serum 5-LOX in AD patients and also in MCI patients compared to the normal control group. The peptide inhibitor of 5-LOX, YWCS, prevented the neurotoxic effect of Aß25-35 by reducing the expression of γ-secretase as well as p-Tau181 in SH-SY5Y cells. However, YWCS was nontoxic towards normal HEK cells. The differential expression of serum 5-LOX among the study groups suggests it can be one of potential serum protein marker and a therapeutic regimen for AD and MCI. The negative correlation with neuropsychological parameters, i.e., MoCA and HMSE, increases its importance and makes it useful during the clinical setup which is very needful in developing countries. Peptide YWCS can serve as a new platform as a 5-LOX inhibitor which can prevent neurotoxicity developed in AD.
Assuntos
Doença de Alzheimer/sangue , Doença de Alzheimer/enzimologia , Araquidonato 5-Lipoxigenase/sangue , Inibidores de Lipoxigenase/uso terapêutico , Síndromes Neurotóxicas/sangue , Síndromes Neurotóxicas/tratamento farmacológico , Peptídeos/uso terapêutico , Idoso , Secretases da Proteína Precursora do Amiloide/metabolismo , Biomarcadores , Estudos de Casos e Controles , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Inibidores de Lipoxigenase/farmacologia , Masculino , Peptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Curva ROC , Proteínas tau/metabolismoRESUMO
The traditional Chinese medicine Yunnan Baiyao (YNB) has been reported to possess antiinflammatory properties, however its mechanism of action remains unclear. It was previously reported that YNB ameliorated depression of arachidonic acid (AA) levels in a rat model of collagen-induced arthritis. In the current study, the capacity of YNB to ameliorate inflammation was compared in carrageenaninduced and AAinduced acute inflammation of the rat paw with celecoxib and mizolastine, respectively (n=24 per group). The capacity of YNB to affect the phospholipase A2 (PLA2)/AA pathway (using reverse transcriptionquantitative polymerase chain reaction) and release of inflammatory lipid mediators (by ELISA) were investigated. Celecoxib ameliorated carrageenaninduced paw edema, and mizolastine ameliorated AAinduced rat paw edema. YNB alleviated paw edema and inhibited inflammatory cell infiltration in the two models. YNB inhibited production of 5LOX AA metabolite leukotriene B4 (LTB4), and suppressed expression of 5LOX, cytosolic PLA2 (cPLA2), 5LOXactivating protein, and LTB4 receptor mRNA in the AAinduced inflammation model (P<0.05). YNB Inhibited the production of the COX2 AA metabolite prostaglandin E2 (PGE2) and suppressed expression of COX2, cPLA2, PGE2 mRNA in the carrageenaninduced inflammation mode (P<0.05). Taken together, the data suggest that modulation of COX and LOX pathways in AA metabolism represent a novel anti-inflammatory mechanism of YNB.
Assuntos
Anti-Inflamatórios/farmacologia , Ácido Araquidônico/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Inflamação/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Fosfolipases A2/metabolismo , Doença Aguda , Animais , Araquidonato 5-Lipoxigenase/sangue , Biomarcadores , Biópsia , Carragenina/efeitos adversos , Ciclo-Oxigenase 2/sangue , Ciclo-Oxigenase 2/metabolismo , Modelos Animais de Doenças , Expressão Gênica , Inflamação/tratamento farmacológico , Inflamação/etiologia , Inflamação/patologia , Mediadores da Inflamação , Lipídeos/sangue , Masculino , RatosRESUMO
OBJECTIVE: The goal of the present study was to investigate the effects of 5-lipoxygenase (5-LOX) inhibition, alone and with cyclooxygenase (COX) inhibitors, on inflammatory parameters and apoptosis in ischemia/reperfusion (I/R)-induced myocardial damage in rats. For this purpose, zileuton, a selective and potent inhibitor of 5-LOX, resulting in suppression leukotriene production, was used. METHODS: Male Wistar rats (200-250 g; n=12 per group) were used in the study. I/R was performed by occluding the left coronary artery for 30 minutes and 2 hours of reperfusion of the heart. Experimental groups were I/R group, sham I/R group, zileuton (5 mg/kg orally, twice daily)+I/R group, zileuton+indomethacin (5 mg/kg intraperitoneally)+I/R group, zileuton+ketorolac (10 mg/kg subcutaneously)+I/R group, and zileuton+nimesulide (5 mg/kg subcutaneously)+I/R group. Following I/R, blood samples were collected to measure tumor necrosis factor alpha (TNF-α), and left ventricles were excised for evaluation of microscopic damage; malondialdehyde (MDA), glutathione, nuclear factor (NF)-κB assays; and evaluation of apoptosis. RESULTS: Left ventricle MDA in I/R group was higher compared to sham group; however, it did not show significant change with zileuton. Although tissue injury in I/R group was less severe in all treatment groups, it was not statistically significant. NF-κB H-score and apoptotic index, which were higher in I/R group compared to sham I/R, were decreased with application of zileuton (H-score: p<0.01; apoptotic index: p<0.001). Zileuton had no significant effect on increased serum TNF-α levels in I/R group. CONCLUSION: 5-LOX inhibition in rat myocardial infarction model attenuated increased left ventricle NF-κB expression and apoptosis and these actions were not modulated by COX inhibitors.
Assuntos
Araquidonato 5-Lipoxigenase/efeitos dos fármacos , Hidroxiureia/análogos & derivados , Antagonistas de Leucotrienos/uso terapêutico , Infarto do Miocárdio/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Animais , Araquidonato 5-Lipoxigenase/sangue , Modelos Animais de Doenças , Hidroxiureia/farmacologia , Hidroxiureia/uso terapêutico , Antagonistas de Leucotrienos/farmacologia , Masculino , Infarto do Miocárdio/sangue , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/sangueRESUMO
BACKGROUND AND AIM: Eosinophils are markers of the eosinophilic esophagitis (EoE) disease, and this work aimed to assess whether activation of eosinophils could be a noninvasive test to contribute for EoE diagnosis. METHODS: The activation state of peripheral blood eosinophils in EoE patients and control subjects was assessed based on the morphological aspects of the eosinophil after adherence to slide. Cyclooxygenase-2 and 5-lipoxygenase expressions were evaluated by means of immunofluorescence microscopy to verify if and which eicosanoid pathway is triggered in eosinophils in blood in EoE. RESULTS: The eosinophils of patients with EoE were significantly more activated than those of control individuals. The lowest percentage of normal eosinophils for control subjects was 40%, while the highest percentage of eosinophils of normal aspect for patients with EoE was 32%. Considering 36% as a cutoff for normal eosinophils, this value differentiated all individuals with EoE from individuals without the disease with a sensitivity of 100%, considering the diagnosis of EoE as currently defined. Eosinophils of EoE patients showed higher expression of cyclooxygenase-2 than those of control subjects. CONCLUSIONS: The quantification of morphological changes in eosinophils is a feasible, easy, and reliable manner to identify EoE patients. Therefore, patients with symptoms of esophageal dysfunction showing higher than 36% activated eosinophils in peripheral blood could be a useful way to help definition and diagnostic criterion for EoE.
Assuntos
Esofagite Eosinofílica/diagnóstico , Eosinófilos/imunologia , Adulto , Araquidonato 5-Lipoxigenase/sangue , Biomarcadores/sangue , Estudos Transversais , Ciclo-Oxigenase 2/sangue , Esofagite Eosinofílica/imunologia , Eosinófilos/enzimologia , Eosinófilos/patologia , Feminino , Humanos , Masculino , Adulto JovemRESUMO
Lipoxygenase (LOX) pathway has emerged to have a role in carcinogenesis. There is an evidence that both 12-LOX and 5-LOX have procarcinogenic role. We have previously reported the elevated level of serum 12-LOX in breast cancer patients. This study evaluated the serum level of 5-LOX in breast cancer patients and its in vitro inhibition assessment with peptide inhibitor YWCS. The level of 5-LOX was determined by surface plasmon resonance (SPR). The peptide inhibitor of 5-LOX was designed by molecular modeling and kinetic assay was performed by spectrophotometry. The siRNA mediated 5-LOX gene silencing was performed to investigate the effect on proliferation of MDA-MB-231, breast cancer cell line. The serum 5-LOX level in breast cancer (5.69±1.97ng/µl) was almost 2-fold elevated compared to control (3.53±1.0ng/µl) (P < 0.0001). The peptide YWCS had shown competitive inhibitory effects with IC50, 2.2 µM and dissociation constant (K D), 4.92×10(-8) M. The siRNA mediated knockdown of 5-LOX, resulted in the decreased gene expression for 5-LOX and increased cell death in MDA-MB-231 cell line and thereby play a key role in reducing tumor proliferation. Thus, it can be concluded that 5-LOX is one of the potential serum protein marker for breast cancer and a promising therapeutic target for the same.
Assuntos
Araquidonato 5-Lipoxigenase/sangue , Biomarcadores Tumorais/sangue , Neoplasias da Mama/enzimologia , Adulto , Araquidonato 5-Lipoxigenase/fisiologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Inibidores de Lipoxigenase/farmacologia , Inibidores de Lipoxigenase/uso terapêutico , Pessoa de Meia-Idade , Simulação de Acoplamento Molecular , EspectrofotometriaRESUMO
Jasminum lanceolarium Roxb is an important traditional Chinese medicine. Its stems and roots have been used for the treatment of rheumatism and fever while the leaves are used as an anti-inflammatory agent to relieve pain. In order to support its traditional Chinese medicinal uses, five animal models were designed and the anti-inflammatory and analgesic properties of the 70% EtOH-H2O extracts of J. lanceolarium (EJL) were investigated. Meanwhile, biochemical parameters such as cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX) in blood serum of rats exposed to acute (carrageenan) inflammation model were evaluated. At doses of 400 mg/kg, EJL exhibited higher anti-inflammation effect than that of indomethacin and better analgesic activity than that of aspirin (P<0.001). Furthermore, eleven isolated compounds including six lignanoids (1, 2, 6, 7, 8, and 11) and five iridoids (3, 4, 5, 9, and 10) were isolated from the active extracts and showed significant anti-inflammatory activities with the IC50 values of 1.76-5.22 mg/mL, respectively, when testing their inhibitory effects on phospholipase A2 in vitro. The results demonstrated that the possible anti-inflammatory mechanisms might be attributed to inhibit the hydrolysis of membrane phospholipids, production on both COX-2 and 5-LOX, and then finally inhibit the release of prostaglandins (PGs), which suggested that EJL had a non-selective inhibitory effect on the release or actions of these mediators, and might be a dual LOX-COX inhibitor for the treatment of inflammation from the natural resource. The studies on the animals and the inflammatory mediators, along with the bioactive compounds presumed that the existences of iridoids and lignanoids could be response for their bioactivities of the whole plants.
Assuntos
Analgésicos/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Edema/tratamento farmacológico , Jasminum , Dor/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Ácido Acético , Analgésicos/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Araquidonato 5-Lipoxigenase/sangue , Permeabilidade Capilar/efeitos dos fármacos , Carragenina , Ciclo-Oxigenase 2/sangue , Edema/sangue , Edema/induzido quimicamente , Feminino , Temperatura Alta , Masculino , Camundongos , Dor/etiologia , Fosfolipases A2/metabolismo , Fitoterapia , Extratos Vegetais/farmacologia , Raízes de Plantas , Caules de Planta , Prostaglandinas/metabolismo , Ratos Wistar , XilenosRESUMO
Benzothiazine and pyrazole derivatives possess anti-inflammatory properties. Previously, synergism of both heterocyclic moieties into a single nucleus has shown to produce biologically active N'-arylmethylidene-2-(3,4-dimethyl-5,5-dioxidopyrazolo(4,3-c)(1,2)benzothiazin-2(4H)yl)acetohydrazides) compound. Present study investigates the anti-arthritic effect and possible mechanism of 2,4-dihydroxyphenyl derivative (DHP) in Freund's complete adjuvant-induced arthritic rat model. Ankle joint histopathology was performed with Hematoxylin & Eosin staining, while serum C-reactive protein (CRP) levels were measured by agglutination method. mRNA expression levels and protein levels of proinflammatory markers were measured by real time reverse transcription polymerase chain reaction and Enzyme linked immunosorbent assay (ELISA), respectively. in vitro Concanavalin A (ConA)-stimulated splenocyte proliferation was also measured by ELISA reader. DHP treatment reduced the macroscopic arthritic score, CRP levels, synovial inflammation, bone erosion and pannus formation. Levels of cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), Prostaglandin-E2 (PGE2), and 5-lipoxygenase (5-LOX), were significantly attenuated by DHP. It also significantly decreased the levels of toll-like receptor 2, nuclear factor-kappaB (NF-ĸB), and tissue necrosis factor-α (TNF-α) and non-significantly elevated interleukin-4 (IL-4) levels. Piroxicam, used as reference drug, significantly reduced the levels of COX-1, COX-2, PGE2, NF-ĸB, and TNF-α, but did not show reduction in 5-LOX and toll-like receptor 2 levels. However piroxicam significantly enhanced the levels of IL-4. Both DHP and piroxicam suppressed ConA-stimulated splenocyte proliferation. DHP normalized all altered hematological markers and did not show any sign of hepatotoxicity or nephrotoxicity as determined by alanine transaminase, aspartate aminotransferase, urea, and creatinine levels. Results showed that DHP possesses significant anti-arthritic activity which may be attributed to its immunomodulatory and anti-inflammatory effects.
Assuntos
Anti-Inflamatórios/farmacologia , Artrite Experimental/tratamento farmacológico , Hidrazinas/farmacologia , Pirazóis/farmacologia , Tiazinas/farmacologia , Animais , Anti-Inflamatórios/uso terapêutico , Araquidonato 5-Lipoxigenase/sangue , Artrite Experimental/imunologia , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Proteína C-Reativa/metabolismo , Proliferação de Células/efeitos dos fármacos , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 2/genética , Dinoprostona/sangue , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hidrazinas/uso terapêutico , Imunomodulação/efeitos dos fármacos , Masculino , NF-kappa B/genética , Pirazóis/uso terapêutico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Baço/citologia , Baço/efeitos dos fármacos , Tiazinas/uso terapêutico , Receptor 2 Toll-Like/genética , Fator de Necrose Tumoral alfa/genéticaRESUMO
Although substantial variation exists in individual responses to omega-3 (ω-3) (n-3) fatty acid supplementation, the causes for differences in response are largely unknown. Here we investigated the associations between the efficacy of ω-3 fatty acid supplementation and a broad range of nutritional and clinical factors collected during a double-blind, placebo-controlled trial in participants of African ancestry, randomly assigned to receive either 2 g eicosapentaenoic acid (EPA) + 1 g docosahexaenoic acid (n = 41) or corn/soybean oil placebo (n = 42) supplements for 6 wk. Food-frequency questionnaires were administered, and changes in erythrocyte lipids, lipoproteins, and monocyte 5-lipoxygenase-dependent metabolism were measured before and after supplementation. Mixed-mode linear regression modeling identified high (n = 28) and low (n = 13) ω-3 fatty acid response groups on the basis of changes in erythrocyte EPA abundance (P < 0.001). Compliance was equivalent (â¼88%), whereas decreases in plasma triglycerides and VLDL particle sizes and reductions in stimulated monocyte leukotriene B4 production were larger in the high-response group. Although total diet quality scores were similar, the low-response group showed lower estimated 2005 Healthy Eating Index subscores for dark-green and orange vegetables and legumes (P = 0.01) and a lower intake of vegetables (P = 0.02), particularly dark-green vegetables (P = 0.002). Because the findings reported here are associative in nature, prospective studies are needed to determine if dietary dark-green vegetables or nutrients contained in these foods can enhance the efficacy of ω-3 fatty acid supplements. This trial was registered at clinicaltrials.gov as NCT00536185.
Assuntos
Negro ou Afro-Americano , Doenças Cardiovasculares/sangue , Dieta , Suplementos Nutricionais , Ácidos Graxos Ômega-3/farmacologia , Comportamento Alimentar , Verduras , Adulto , Araquidonato 5-Lipoxigenase/sangue , Doenças Cardiovasculares/etnologia , Doenças Cardiovasculares/prevenção & controle , VLDL-Colesterol/sangue , Dieta/normas , Ácidos Docosa-Hexaenoicos/farmacologia , Método Duplo-Cego , Ácido Eicosapentaenoico/sangue , Ácido Eicosapentaenoico/farmacologia , Ingestão de Energia , Eritrócitos/metabolismo , Ácidos Graxos Ômega-3/sangue , Feminino , Humanos , Leucotrieno B4/biossíntese , Modelos Lineares , Lipoproteínas/sangue , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismo , Cooperação do Paciente , Inquéritos e Questionários , Triglicerídeos/sangueRESUMO
Human 5-lipoxygenase (5-LOX) is one of the key anti-inflammatory drug targets due to its key role in leukotrienes biosynthesis. We have built a model for the active conformation of human 5-LOX using comparative modeling, docking of known inhibitors, and molecular dynamics simulation. Using this model, novel 5-LOX inhibitors were identified by virtual screen. Of the 105 compounds tested in a cell-free assay, 30 have IC(50) values less than 100 µM and 11 less than 10 µM with the strongest inhibition of 620 nM. Compounds 4, 7, and 11 showed strong inhibition activity in the human whole blood (HWB) assay with IC(50) values of 8.6, 9.7, 8.1 µM, respectively. Moreover, compounds 4 and 7 were also found to inhibit microsomal prostaglandin E synthase (mPGES)-1 with micromolar IC(50) values, similar to licofelone, a dual functional inhibitor of 5-LOX/mPGES-1. The compounds reported here provide new scaffolds for anti-inflammatory drug design.
Assuntos
Anti-Inflamatórios não Esteroides/química , Araquidonato 5-Lipoxigenase/química , Inibidores de Lipoxigenase/química , Simulação de Dinâmica Molecular , Anti-Inflamatórios não Esteroides/farmacologia , Araquidonato 5-Lipoxigenase/sangue , Sistema Livre de Células , Bases de Dados Factuais , Dinoprostona/sangue , Humanos , Oxirredutases Intramoleculares/antagonistas & inibidores , Oxirredutases Intramoleculares/sangue , Leucotrieno B4/sangue , Inibidores de Lipoxigenase/farmacologia , Microssomos/enzimologia , Prostaglandina-E Sintases , Ligação Proteica , Conformação Proteica , Pirróis/química , Pirróis/farmacologia , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Relação Estrutura-AtividadeRESUMO
BACKGROUND AND PURPOSE Arachidonic acid derivatives play a central role in inflammation processes. Arachidonic acid is metabolized by several enzymes, particularly cyclooxygenases (COX), 5-lipoxygenase (5-LOX) and microsomal prostaglandin E-synthase-1 (mPGES-1) to pro-inflammatory mediators. EXPERIMENTAL APPROACH We determined the effect of LP105, a pirinixic acid derivative which acts as inhibitor of 5-LOX, COX and mPGES-1, on aortic aneurysm development in mice and on 5-LOX activity in murine monocytes. KEY RESULTS In a monocyte cell line (RAW264.7), LP105 inhibited 5-LOX in whole cells (IC(50) : 1-3 µM) and in supernatants (IC(50) : â¼10 µM). Oral administration of LP105 to mice resulted in therapeutic tissue and plasma levels. Aortic aneurysms were induced in ApoE(-/-) mice by angiotensin II (AngII) and LP105 (5 mg·day(-1) per animal) was co-administered to a subgroup. Compared with animals receiving AngII alone, the LP105+AngII group showed a lower heart rate, a trend towards reduced heart to body weight ratio but similar hypertensive responses. AngII alone significantly increased aortic weight and diameter but co-treatment with LP105+AngII prevented these changes. LC/MS-MS studies revealed increased 15-hydroxytetraenoic acid (15-HETE) and 14,15-epoxyeicosatrienoic acid (14,15-EET) plasma levels in LP105-treated animals. In the murine kidney, mRNAs of EET-generating or metabolizing enzymes and of 5-LOX and 15-LOX were unaffected by LP105. LP105 also did not inhibit the EET-metabolizing soluble epoxide hydrolase. CONCLUSIONS AND IMPLICATIONS LP105 was a potent inhibitor of monocyte 5-LOX and reduced AngII-induced vascular remodelling in mice. A shift of arachidonic acid metabolism to the protective EET pathway may contribute to the beneficial effects of LP105.
Assuntos
Aneurisma Aórtico/patologia , Araquidonato 5-Lipoxigenase/metabolismo , Cardiotônicos/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Oxirredutases Intramoleculares/antagonistas & inibidores , Inibidores de Lipoxigenase/farmacologia , Pirimidinas/farmacologia , Administração Oral , Angiotensina II/administração & dosagem , Angiotensina II/toxicidade , Animais , Aorta/patologia , Aneurisma Aórtico/metabolismo , Araquidonato 5-Lipoxigenase/sangue , Cardiotônicos/farmacocinética , Cardiotônicos/uso terapêutico , Sistema Cardiovascular/efeitos dos fármacos , Linhagem Celular , Inibidores de Ciclo-Oxigenase/uso terapêutico , Modelos Animais de Doenças , Epóxido Hidrolases/sangue , Epóxido Hidrolases/metabolismo , Injeções Subcutâneas , Inibidores de Lipoxigenase/uso terapêutico , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Prostaglandina-E Sintases , Pirimidinas/metabolismo , Pirimidinas/uso terapêuticoRESUMO
OBJECTIVE: To investigate the expressions of 15- and 5-lipoxygenases in leukocytes and the changes of the levels of blood lipoxin A4 (LXA4) and leukotriene C4 (LTC4) in children with asthma. METHODS: The mRNA levels of 15- and 5-lipoxygenases in leukocytes were assessed by RT-PCR, and the levels of blood LXA4 and LTC4 were determined by ELISA, in 106 children with mild, moderate and severe asthma. Forty healthy children served as the controls. RESULTS: In children with mild, moderate and severe asthma, the relative mRNA levels of 15-lipoxygenase in leukocytes were 1.78 ± 0.56, 1.28 ± 0.45 and 0.58 ± 0.22 (F = 16.72, P < 0.01), respectively, and all were higher than that of the controls (0.26 ± 0.12, P < 0.05). The levels of blood LXA4 were (5.52 ± 1.97), (1.86 ± 0.72) and (0.81 ± 0.36) µg/L (F = 22.59, P < 0.01), respectively, decreasing with the severity of asthma, and all were higher than that of the controls [(0.04 ± 0.01) µg/L, P < 0.05]. There was a positive correlation between PEF, FEV(1) and blood LXA4. The relative levels of 5-lipoxygenase mRNA in leukocytes were 0.26 ± 0.12, 0.79 ± 0.34 and 1.21 ± 0.52, respectively in children with asthma of mild, moderate and severe degree (F = 18.64, P < 0.01), which showed an increase with the severity of the disease, and all of which were higher than that of the controls (0.12 ± 0.05, P < 0.05). The levels of blood LTC4 were (22.4 ± 8.2), (54.6 ± 28.4) and (118.7 ± 41.1) ng/L (F = 25.91, P < 0.01), respectively, also showing an increase with the severity of asthma, and were higher than that of the controls [(6.8 ± 2.5) ng/L, P < 0.05]. There was a negative correlation between PEF, FEV1 and blood LTC4. CONCLUSION: The reversed changes of 15-lipoxygenase product LXA4 and 5-lipoxygenase product LTC4 in children with asthma of mild, moderate and severe degree suggests that insufficiency of LXA4, an physiological antagonist to leukotrienes, and an overproduction of LTC4, may be involved in the pathogenesis of worsening of asthma in children.
Assuntos
Araquidonato 15-Lipoxigenase/sangue , Araquidonato 5-Lipoxigenase/sangue , Asma/sangue , Lipoxinas/sangue , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Leucócitos/metabolismo , MasculinoRESUMO
Recently, we showed that leukotrienes (LTs) regulate ovarian cell function in vitro. The aim of this study was to examine the role of LTs in corpus luteum (CL) function during both the estrous cycle and early pregnancy in vivo. mRNA expression of LT receptors (BLT for LTB(4) and CYSLT for LTC(4)), and 5-lipoxygenase (5-LO) in CL tissue and their localization in the ovary were studied during the estrous cycle and early pregnancy. Moreover, concentrations of LTs (LTB(4) and C(4)) in the CL tissue and blood were measured. 5-LO and BLT mRNA expression increased on days 16-18 of the cycle, whereas CYSLT mRNA expression increased on days 16-18 of the pregnancy. The level of LTB(4) was evaluated during pregnancy compared with the level of LTC(4), which increased during CL regression. LT antagonists influenced the duration of the estrous cycle: the LTC(4) antagonist (azelastine) prolonged the luteal phase, whereas the LTB(4) antagonist (dapsone) caused earlier luteolysis in vivo. Dapsone decreased progesterone (P(4)) secretion and azelastine increased P(4) secretion during the estrous cycle. In summary, LT action in the bovine reproductive tract is dependent on LT type: LTB(4) is luteotropic during the estrous cycle and supports early pregnancy, whereas LTC(4) is luteolytic, regarded as undesirable in early pregnancy. LTs are produced/secreted in the CL tissue, influence prostaglandin function, and serve as important factors during the estrous cycle and early pregnancy in cattle.
Assuntos
Araquidonato 5-Lipoxigenase/fisiologia , Bovinos/fisiologia , Corpo Lúteo/fisiologia , Ciclo Estral/fisiologia , Leucotrieno B4/fisiologia , Leucotrieno C4/fisiologia , Animais , Araquidonato 5-Lipoxigenase/sangue , Dapsona/farmacologia , Feminino , Imuno-Histoquímica/veterinária , Leucotrieno B4/antagonistas & inibidores , Leucotrieno B4/sangue , Leucotrieno B4/genética , Leucotrieno C4/antagonistas & inibidores , Leucotrieno C4/sangue , Leucotrieno C4/genética , Inibidores de Lipoxigenase/farmacologia , Ftalazinas/farmacologia , Gravidez , RNA Mensageiro/química , RNA Mensageiro/genética , Receptores de Leucotrienos/fisiologia , Receptores do Leucotrieno B4/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterináriaRESUMO
An inhibition test was used to study mechanisms responsible for L-arginine metabolic disturbances in the blood monocytes of patients with diabetic foot syndrome (DFS). It showed enhanced baseline iNOS activity and inhibition of the arginase pathway with lower nitrite production in response to the administration of lipopolysaccharide in the monocytes of patients with DFS. Impaired L-arginine metabolism was related to the higher activities of protein kinase C (PKC), phosphodiesterase (PDE), and 5-lipoxygenase (5-LO) along with decreased cyclooxygenase activity and drastic protein kinase A (PKA) inhibition. Within the first week, no changes in the wound process were associated with persistent metabolic disturbances of arachidonic acid and serine-threonine kinases with the higher sensitivity of AT1 receptors. In patients with DFS, the condition for wound process termination was decreased baseline iNOS activity and enhanced arginase-1 activity during PKA stimulation with the lower activity of 5-LO, PDE, and PKS. However, impaired mechanisms in the regulation of monocytic L-arginine metabolism persisted even a month later, which predetermines skin remodeling disturbance and the likelihood of recurrent DFS
Assuntos
Arginina/sangue , Pé Diabético/sangue , Monócitos/metabolismo , Adulto , Idoso , Araquidonato 5-Lipoxigenase/sangue , Ácido Araquidônico/sangue , Arginase/sangue , Pé Diabético/patologia , Feminino , Humanos , Lipopolissacarídeos/farmacologia , Masculino , Pessoa de Meia-Idade , Óxido Nítrico Sintase Tipo II/sangue , Diester Fosfórico Hidrolases/sangue , Prostaglandina-Endoperóxido Sintases/sangue , Proteínas Serina-Treonina Quinases/sangue , Receptor Tipo 1 de Angiotensina/sangueRESUMO
Recent studies have shown a correlation between 5-lipoxygenase (5-LO) pathway up-regulation and cardiovascular risk. Despite the existence of several assays for products of the 5-LO pathway, a reliable method for clinical determination of 5-LO activity remains to be established. In the present communication, we report conditions that allow measurement of 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B(4) (LTB(4)) in peripheral blood mononuclear cells (PBMCs) isolated from the blood of atherosclerosis patients before and after stimulation by the calcium ionophore, A23187. LTB(4), a potent mediator of inflammation-linked cardiovascular disease, was measured using an existing competitive enzyme immunoassay (EIA) kit after making specific methodological improvements that allowed PBMCs to be used in this format for the first time. LTB(4) was also measured by LC/MS/MS along with 5-HETE, a direct by-product of the action of 5-LO on arachidonic acid and a molecule for which no commercial EIA kit exists. The LC/MS/MS assay was validated over a range of 0.025-25ng/mL for LTB(4) and 0.1-25ng/mL for 5-HETE. The EIA method has a validated range covering 0.025-4ng/mL. When both assays were applied to analyze LTB(4) from stimulated PBMCs isolated from 25 subjects with various degrees of atherosclerosis, a high correlation was obtained (r=0.9426, Pearson's correlation coefficient). A high correlation was also observed between the levels of LTB(4) and 5-HETE measured by LC/MS/MS after ionophore stimulation (r=0.9159). Details are presented for optimized sample collection, processing, storage, and analysis in accordance with the logistical demands of clinical analysis.
Assuntos
Araquidonato 5-Lipoxigenase/sangue , Araquidonato 5-Lipoxigenase/metabolismo , Técnicas Imunoenzimáticas/métodos , Leucócitos Mononucleares/enzimologia , Araquidonato 5-Lipoxigenase/genética , Calcimicina/metabolismo , Calcimicina/farmacologia , Cromatografia Líquida , Humanos , Ácidos Hidroxieicosatetraenoicos/genética , Ácidos Hidroxieicosatetraenoicos/metabolismo , Ionóforos/metabolismo , Ionóforos/farmacologia , Leucotrieno B4/análise , Leucotrieno B4/genética , Leucotrieno B4/metabolismo , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem , Temperatura , Fatores de TempoRESUMO
The 5-lipoxygenase (5-LOX) pathway has been associated with a variety of inflammatory diseases including asthma, atherosclerosis, rheumatoid arthritis, pain, cancer and liver fibrosis. Several classes of 5-LOX inhibitors have been identified, but only one drug, zileuton, a redox inhibitor of 5-LOX, has been approved for clinical use. To better evaluate the efficacy of 5-LOX inhibitors for pharmacological intervention, a rat model was modified to test the in vivo efficacy of 5-LOX inhibitors. Inflammation was produced by adding carrageenan into a newly formed air pouch and prostaglandins produced. While macrophages and neutrophils are present in the inflamed pouch, little 5-LOX products are formed. Cellular 5-LOX activation was obtained by adding calcium ionophore (A23187) into the pouch thus providing a novel model to evaluate the efficacy and selectivity of 5-LOX inhibitors. Also, we described modifications to the in vitro 5-LOX enzyme and cell assays. These assays included a newly developed fluorescence-based enzyme assay, a 5-LOX redox assay, an ex vivo human whole blood assay and an IgE-stimulated rat mast cell assay, all designed for maximal production of leukotrienes. Zileuton and CJ-13,610, a competitive, non-redox inhibitor of 5-LOX, were evaluated for their pharmacological properties using these assays. Although both compounds achieved dose-dependent inhibition of 5-LOX enzyme activity, CJ-13,610 was 3-4 fold more potent than zileuton in all-assays. Evaluation of 5-LOX metabolites-by LC/MS/MS and ELISA confirmed that both compounds selectively inhibited all products downstream of 5-hydroperoxy eicosatetraenoic acid (5-HPETE), including 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxoETE), without inhibition of 12-lipoxygenase (12-LOX), 15-lipoxygenase (15-LOX), or cyclooxygenase (COX) products. In the rat air pouch model, oral dosing of CJ-13,610 and zileuton resulted in selective inhibition 5-LOX activity from pouch exudate and ex vivo rat whole blood with similar potency to in vitro assay. These data show that the rat air pouch model is a reliable and useful tool for evaluating in vivo efficacy of 5-LOX inhibitors and may aid in the development of the next generation of 5-LOX inhibitors, such as the non-redox inhibitors similar to CJ-13,610.
Assuntos
Hidroxiureia/análogos & derivados , Imidazóis/farmacologia , Inflamação/enzimologia , Leucotrienos/metabolismo , Inibidores de Lipoxigenase/farmacologia , Mastócitos/efeitos dos fármacos , Sulfetos/farmacologia , Ar , Animais , Araquidonato 5-Lipoxigenase/sangue , Araquidonato 5-Lipoxigenase/metabolismo , Bioensaio/métodos , Calcimicina/farmacologia , Carragenina , Linhagem Celular Tumoral , Cromatografia Líquida , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ativação Enzimática , Ativadores de Enzimas/farmacologia , Ensaio de Imunoadsorção Enzimática , Humanos , Hidroxiureia/farmacologia , Imunoglobulina E/imunologia , Inflamação/induzido quimicamente , Ionóforos/farmacologia , Leucotrienos/sangue , Masculino , Mastócitos/enzimologia , Mastócitos/imunologia , Oxirredução , Ratos , Ratos Endogâmicos Lew , Reprodutibilidade dos Testes , Espectrometria de Massas em TandemRESUMO
Diacylglycerides (DAGs) such as 1-oleoyl-2-acetyl-sn-glycerol (OAG) stimulate 5-lipoxygenase (5-LO) enzyme activity and function as agonists for human polymorphonuclear leukocytes (PMNL) to induce 5-LO product synthesis. Here, we addressed the role of endogenous DAG generation in agonist-induced 5-LO activation in human PMNL. Preincubation of PMNL with the phospholipase D (PLD) inhibitor 1-butanol potently suppressed 5-LO product synthesis induced by the Ca(2)(+) ionophore A23187 or thapsigargin (TG) and blocked A23187-evoked translocation of 5-LO from the cytosol to the nuclear membrane, analyzed by subcellular fractionation as well as by indirect immunofluorescence microscopy. Tertiary-butanol, a rather poor inhibitor of PLD, caused only moderate suppression of 5-LO and hardly inhibited 5-LO translocation. Interestingly, 1-butanol failed to inhibit 5-LO product formation when PMNL were stimulated with OAG (30 microM). Moreover, coincubation of A23187- or TG-stimulated PMNL with OAG reversed inhibition of 5-LO product formation by 1-butanol in a concentration-dependent manner (EC(50), approximately 1 muM) and also restored 5-LO translocation. In addition, inhibition of phosphatidic acid phosphatase (PA-P) by propranolol or bromoenol lactone caused suppression of 5-LO product formation and of translocation, which could be reversed by addition of exogenous OAG. Together, our data suggest that in agonist-stimulated PMNL, the endogenous formation of DAGs via the PLD/PA-P pathway determines 5-LO activation.
Assuntos
Araquidonato 5-Lipoxigenase/sangue , Diglicerídeos/metabolismo , Neutrófilos/enzimologia , Fosfatidato Fosfatase/sangue , Fosfolipase D/sangue , 1-Butanol/farmacologia , Adulto , Calcimicina/farmacologia , Ativação Enzimática , Humanos , Inibidores de Lipoxigenase , Microscopia de Fluorescência , Valores de Referência , Tapsigargina/farmacologiaRESUMO
5-Lipoxygenase (5-LOX) is the key enzyme responsible for the synthesis of the biologically active leukotrienes. Its presence has been reported in cells of the myeloid lineage and B lymphocytes but has not been formally defined in T lymphocytes. In this study, we provide evidence for 5-LOX expression on both transcriptional and translational levels in highly purified peripheral blood T cells as well as in human T lymphoblastoid cell lines (MOLT4 and Jurkat). Messenger RNA (mRNA) of 5-LOX was amplified by conventional reverse transcription-polymerase chain reaction (RT-PCR; MOLT4 and Jurkat cells) and by in situ RT-PCR (T lymphocytes). 5-LOX protein expression was confirmed by Western blot and immunofluorescence studies. 5-LOX was present primarily in the cytoplasm with some nuclear localization and was translocated to the nuclear periphery after culture in a mitosis-supporting medium. Fluorescence-activated cell sorter analysis of different T-lymphocyte populations, including CD4, CD8, CD45RO, CD45RA, T helper type 2, and T-cell receptor-alphabeta and -gammadelta expressing cells, did not identify a differential distribution of the enzyme. Purified peripheral blood T lymphocytes were incapable of synthesizing leukotrienes in the absence of exogenous arachidonic acid. Jurkat cells produced leukotriene C(4) and a small amount of leukotriene B(4) in response to CD3-CD28 cross-linking. This synthesis was abolished by two inhibitors of leukotriene synthesis, MK-886 and AA-861. The presence of 5-LOX in T lymphocytes but the absence of endogenous lipoxygenase metabolite production compared to Jurkat cells may constitute a fundamental difference between resting peripheral lymphocytes and leukaemic cells.
Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Linfócitos T/enzimologia , Araquidonato 5-Lipoxigenase/sangue , Araquidonato 5-Lipoxigenase/genética , Western Blotting/métodos , Técnica Indireta de Fluorescência para Anticorpo/métodos , Humanos , Células Jurkat , RNA Mensageiro/genética , RNA Neoplásico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Células Tumorais CultivadasRESUMO
The beneficial influence of dietary curcumin, capsaicin and their combination on the susceptibility of low-density lipoprotein (LDL) to oxidation was examined in an animal study. Individually, both dietary curcumin and capsaicin significantly inhibited the in vivo iron-induced LDL oxidation, as well as copper-induced oxidation of LDL in vitro. The protective effect of the combination of curcumin and capsaicin on LDL oxidation was greater than that of individual compounds. This protective influence of spice principles was also indicated by the relative anodic electrophoretic mobility of oxidized LDL on agarose gel. In another study, rats injected with iron showed hepatic toxicity as measured by an increase in lipid peroxides and elevated serum enzymes, alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase. Dietary curcumin, capsaicin and their combination reduced the activities of these enzymes, and lowered the liver lipid peroxide level, indicating amelioration of the severity of iron-induced hepatotoxicity. In yet another study, a comparison of the extent of carrageenan-induced paw inflammation showed that both dietary curcumin and capsaicin moderately lowered inflammation, while the spice principles in combination were more effective. Dietary curcumin and capsaicin significantly decreased the activity of 5'-lipoxygenase activity in the polymorphonuclear lymphocytes in carrageenan-injected rats, the decrease being even higher in the case of combination of these two spice principles. Results suggest that dietary curcumin and capsaicin individually are protective to LDL oxidation both in vivo and in vitro, to iron-induced hepatotoxicity and to carrageenan-induced inflammation. This beneficial effect was higher when the two compounds were fed in combination.