RESUMO
Based on the multiomics analysis, this study is aimed at investigating the underlying mechanism of didymin against acute liver injury (ALI). The mice were administrated with didymin for 2 weeks, followed by injection with lipopolysaccharide (LPS) plus D-galactosamine (D-Gal) to induce ALI. The pathological examination revealed that didymin significantly ameliorated LPS/D-Gal-induced hepatic damage. Also, it markedly reduced proinflammatory cytokines release by inhibiting the TLR4/NF-κB pathway activation, alleviating inflammatory injury. A transcriptome analysis proved 2680 differently expressed genes (DEGs) between the model and didymin groups and suggested that the PI3K/Akt and metabolic pathways might be the most relevant targets. Meanwhile, the metabolome analysis revealed 67 differently expressed metabolites (DEMs) between the didymin and model groups that were mainly clustered into the glycerophospholipid metabolism, which was consistent with the transcriptome study. Importantly, a comprehensive analysis of both the omics indicated a strong correlation between the DEGs and DEMs, and an in-depth study demonstrated that didymin alleviated metabolic disorder and hepatocyte injury likely by inhibiting the glycerophospholipid metabolism pathway through the regulation of PLA2G4B, LPCAT3, and CEPT1 expression. In conclusion, this study demonstrates that didymin can ameliorate LPS/D-Gal-induced ALI by inhibiting the glycerophospholipid metabolism and PI3K/Akt and TLR4/NF-κB pathways.
Assuntos
NF-kappa B , Proteínas Proto-Oncogênicas c-akt , Animais , Camundongos , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Lipopolissacarídeos/farmacologia , Transcriptoma , Receptor 4 Toll-Like/metabolismo , Fígado/metabolismo , Metaboloma , Glicerofosfolipídeos/metabolismo , Glicerofosfolipídeos/farmacologia , 1-Acilglicerofosfocolina O-Aciltransferase/genética , 1-Acilglicerofosfocolina O-Aciltransferase/metabolismo , 1-Acilglicerofosfocolina O-Aciltransferase/farmacologia , Fosfolipases A2 do Grupo IV/genética , Fosfolipases A2 do Grupo IV/metabolismo , Fosfolipases A2 do Grupo IV/farmacologiaRESUMO
PURPOSE: TMX-101 and TMX-202 are formulations of toll-like receptor 7 (TLR-7) agonists, under investigation for the treatment of urothelial carcinoma. Our goal was to evaluate the efficacy of intravesical instillations of TMX-101 or TMX-202 in an orthotopic bladder cancer rat model. METHODS: Four groups of 14 rats received an instillation with isogenic AY-27 tumor cells on day 0, starting tumor development. On day 2 and 5, the rats were treated with an intravesical instillation of TMX-101 0.1%, TMX-202 0.38%, vehicle solution or NaCl. On day 12 the rats were sacrificed and the bladders were evaluated histopathologically. RESULTS: No signs of toxicity were seen. The number of tumor-positive rats was 11 of 14 (79%) in the vehicle control group and in the NaCl control group, versus 9 of 14 (64%) in the TMX-101-treated group, and 8 of 14 (57%) in the TMX-20-treated group. The difference between tumor-bearing rats in the treated and control groups was not significant (p = 0.12). Bladder weight was significantly lower for TMX-202-treated rats compared to vehicle (p = 0.005). CONCLUSIONS: TMX-101 and TMX-202 are TLR-7 agonists with antitumor activity. Treatment with TMX-101 and TMX-202 resulted in less tumor-bearing rats compared to vehicle or saline control groups, although not statistically significant. In this aggressive bladder cancer model, a lower number of tumor-positive rats after treatment with TLR-7 agonists indicates activity for the treatment of non-muscle invasive bladder cancer.
Assuntos
Adenina/análogos & derivados , Carcinoma de Células de Transição/tratamento farmacológico , Glicerofosfolipídeos/farmacologia , Imiquimode/farmacologia , Fatores Imunológicos/farmacologia , Receptor 7 Toll-Like/agonistas , Neoplasias da Bexiga Urinária/tratamento farmacológico , Bexiga Urinária/efeitos dos fármacos , Adenina/farmacologia , Adjuvantes Imunológicos/farmacologia , Administração Intravesical , Animais , Carcinoma de Células de Transição/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Ratos , Ratos Endogâmicos F344 , Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVES: To evaluate and compare the pharmacokinetic and pharmacodynamic properties of 2 investigational Toll-like receptor 7 agonists, TMX-101, and TMX-202 after intravenous and intravesical administration in a rat model. TLR-7 agonists are successfully used as topical treatment for various (pre)malignant skin lesions and are now under investigation as intravesical therapy for non-muscle-invasive bladder cancer. METHODS: Rats received an intravesical instillation with TMX-101, TMX-202, or vehicle. Additionally 2 groups of rats received an intravenous injection with TMX-101 or TMX-202. Blood sampling was performed at different time points, including pre-exposure and postexposure to determine the plasma concentrations of study drugs for pharmacokinetic and pharmacodynamic analyses and to determine the plasma concentrations of cytokines (IL-2, IL-6, and TNF-α). RESULTS: We observed no signs of toxicity after intravesical or intravenous administration. There was a limited dose dependent systemic uptake of TMX-101 and TMX-202 after intravesical administration. The systemic uptake of TMX-202 after intravesical instillation was 25 times lower compared to TMX-101. CONCLUSIONS: This in vivo study confirms the safety of intravesical TMX-101 and TMX-202 administration, with TMX-202 showing lower systemic uptake. TMX-202 has a larger molecule-mass compared to TMX-101, and it may therefore have a favorable safety profile when treating patients with non-muscle-invasive bladder cancer intravesically.
Assuntos
Adenina/análogos & derivados , Aminoquinolinas/farmacologia , Aminoquinolinas/farmacocinética , Modelos Animais de Doenças , Glicerofosfolipídeos/farmacologia , Glicerofosfolipídeos/farmacocinética , Receptor 7 Toll-Like/agonistas , Neoplasias da Bexiga Urinária/tratamento farmacológico , Adenina/administração & dosagem , Adenina/farmacocinética , Adenina/farmacologia , Administração Intravenosa , Administração Intravesical , Aminoquinolinas/administração & dosagem , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Feminino , Glicerofosfolipídeos/administração & dosagem , Ratos , Ratos Endogâmicos F344 , Distribuição Tecidual , Neoplasias da Bexiga Urinária/patologiaRESUMO
Ceramide kinase (CerK) and ceramide-1-phosphate (C1P) are involved in various cellular functions, while regulation of the enzyme activity has not been well elucidated. We herein investigated the effects of several glycerophospholipids on human recombinant CerK activity with CaCl2 and MgCl2 by measuring the formation of fluorescent labeled C1P in vitro. CerK activities were 44.1±11.4 (pmol/µg/min) with vehicle, 137±29 with 2 mM CaCl2, and 144±32 with 2 mM MgCl2 in the glycerol/albumin buffer. The addition of glycerophospholipids such as phosphatidylcholine, phosphatidylinositol (PI), PI 4,5-bisphosphate (PI(4,5)P2), and phosphatidic acid had no effect on CerK activity with CaCl2, although PI(4,5)P2 and phosphatidic acid bound to CerK in the lipid-protein overlay assay. The addition of cardiolipin (diphosphatidylglycerol) at concentrations up to 0.1 µM increased, whereas those more than 1 µM decreased CerK activity with CaCl2/MgCl2. In the lipid-protein overlay assay, cardiolipin bound to CerK and CerK lacking pleckstrin homology (PH) domain, but not PH domain of CerK, in CaCl2-independent manner. Cardiolipin also bound to CerK in the multilamellar vesicle binding assay. A deviation from the normal range of cellular cardiolipin, both the decrease by phospholipase D6 expression and increase by an exogenous addition of the lipid, negatively regulated C1P formation in intact HepG2 cells. Our results revealed that cardiolipin bound to CerK and regulated the formation of C1P in vitro and in cells.
Assuntos
Cardiolipinas/metabolismo , Ceramidas/metabolismo , Glicerofosfolipídeos/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Cloreto de Cálcio/farmacologia , Ácido Edético/farmacologia , Ácido Egtázico/farmacologia , Células Hep G2 , Humanos , Cloreto de Magnésio/farmacologia , Fosfolipase D/genética , Fosfolipase D/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Plasmídeos , Proteínas Recombinantes/metabolismoRESUMO
OBJECTIVES: Monocytes are one of the major phagocytic cells that patrol for invading pathogens, and upon activation, differentiate into macrophages or antigen-presenting dendritic cells (DCs) capable of migrating to lymph nodes eliciting an adaptive immune response. The key role in regulating adaptive immune responses has drawn attention to modulate monocyte responses therapeutically within cancer, inflammation and infectious diseases. We present a technology for targeting of monocytes and delivery of a toll-like receptor (TLR) agonist in fresh blood using liposomes with a positively charged surface chemistry. METHODS: Liposomes were extruded at 100 nm, incubated with fresh blood, followed by leukocyte analyses by FACS. Liposomes with and without the TLR7 agonist TMX-202 were incubated with fresh blood, and monocyte activation measured by cytokine secretion by ELISA and CD14 and DC-SIGN expression. RESULTS: The liposomes target monocytes specifically over lymphocytes and granulocytes in human whole blood, and show association with 75 - 95% of the monocytes after 1 h incubation. Formulations of TMX-202 in cationic liposomes were potent in targeting and activation of monocytes, with strong induction of IL-6 and IL-12p40, and differentiation into CD14(+) and DC-SIGN+ DCs. CONCLUSION: Our present liposomes selectively target monocytes in fresh blood, enabling delivery of TLR7 agonists to the intracellular TLR7 receptor, with subsequent monocyte activation and boost in secretion of proinflammatory cytokines. We envision this technology as a promising tool in future cancer immunotherapy.
Assuntos
Adenina/análogos & derivados , Células Dendríticas/imunologia , Glicerofosfolipídeos/farmacologia , Monócitos/efeitos dos fármacos , Receptor 7 Toll-Like/agonistas , Adenina/administração & dosagem , Adenina/farmacologia , Moléculas de Adesão Celular/metabolismo , Diferenciação Celular/efeitos dos fármacos , Citocinas/metabolismo , Glicerofosfolipídeos/administração & dosagem , Humanos , Subunidade p40 da Interleucina-12/metabolismo , Lectinas Tipo C/metabolismo , Lipossomos , Macrófagos/metabolismo , Masculino , Monócitos/metabolismo , Receptores de Superfície Celular/metabolismoRESUMO
Membrane phospholipids can function as potent regulators of ion channel function. This study uncovers and investigates the effect of phosphatidic acid on Kv channel gating. Using the method of reconstitution into planar lipid bilayers, in which protein and lipid components are defined and controlled, we characterize two effects of phosphatidic acid. The first is a non-specific electrostatic influence on activation mediated by electric charge density on the extracellular and intracellular membrane surfaces. The second is specific to the presence of a primary phosphate group, acts only through the intracellular membrane leaflet and depends on the presence of a particular arginine residue in the voltage sensor. Intracellular phosphatidic acid accounts for a nearly 50 mV shift in the midpoint of the activation curve in a direction consistent with stabilization of the voltage sensor's closed conformation. These findings support a novel mechanism of voltage sensor regulation by the signaling lipid phosphatidic acid.
Assuntos
Ativação do Canal Iônico/efeitos dos fármacos , Ácidos Fosfatídicos/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacologia , Sequência de Aminoácidos , Animais , Glicerofosfolipídeos/farmacologia , Bicamadas Lipídicas/metabolismo , Dados de Sequência Molecular , Fosfatidilcolinas/farmacologia , Fosfolipase D/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Ratos , Alinhamento de SequênciaRESUMO
BACKGROUND: The Toll-like receptor 7 (TLR7) activator imiquimod (IMQ) is safe and effective in treating actinic keratosis; however, an intermittent treatment regimen is necessary because of excessive local reactions. OBJECTIVES: To evaluate in vitro potency, pharmacodynamics/pharmacokinetics, toxicity and efficacy in vivo of the newly developed TLR7 ligand-phospholipid conjugate, TMX-202, in a gel formulation. MATERIAL AND METHODS: The effects of TMX-202 were assessed both in vitro on a murine macrophage cell line and in primary bone marrow-derived dendritic cells and in vivo on mice (C57BL/6-wild type, Myd88(-/-) and Tlr7(-/-)). RESULTS: TMX-202 was more potent than IMQ in vitro using murine and human cells. In contrast, in vivo it showed less systemic pro-inflammatory activity and better safety than IMQ. Moreover, the TMX-202 gel formulation exhibited at least comparable efficacy to Aldara in a mouse model for skin proliferative diseases. CONCLUSION: TMX-202 is safe and efficacious without causing excessive adverse effects, suggesting that it may be an alternative to Aldara for the treatment of proliferative skin conditions.
Assuntos
Adenina/análogos & derivados , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Glicerofosfolipídeos/farmacologia , Glicerofosfolipídeos/uso terapêutico , Ceratose Actínica/tratamento farmacológico , Glicoproteínas de Membrana/genética , Receptor 7 Toll-Like/genética , Adenina/sangue , Adenina/farmacologia , Adenina/uso terapêutico , Aminoquinolinas/sangue , Aminoquinolinas/farmacologia , Animais , Antineoplásicos/sangue , Linhagem Celular , Fatores Quimiotáticos/sangue , Células Dendríticas/fisiologia , Géis/farmacologia , Géis/uso terapêutico , Glicerofosfolipídeos/sangue , Humanos , Imiquimode , Interferon gama/metabolismo , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Queratinócitos/fisiologia , Ceratose Actínica/genética , Leucócitos Mononucleares/efeitos dos fármacos , Macrófagos/fisiologia , Dose Máxima Tolerável , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fator 88 de Diferenciação Mieloide/genética , Proteínas Proto-Oncogênicas c-myc/genética , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND AND PURPOSE: The development of potent and selective inhibitors of the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) via DAG lipases (DAGL) α and ß is just starting to be considered as a novel and promising source of pharmaceuticals for the treatment of disorders that might benefit from a reduction in endocannabinoid tone, such as hyperphagia in obese subjects. EXPERIMENTAL APPROACH: Three new fluorophosphonate compounds O-7458, O-7459 and O-7460 were synthesized and characterized in various enzymatic assays. The effects of O-7460 on high-fat diet intake were tested in mice. KEY RESULTS: Of the new compounds, O-7460 exhibited the highest potency (IC50 = 690 nM) against the human recombinant DAGLα, and selectivity (IC50 > 10 µM) towards COS-7 cell and human monoacylglycerol lipase (MAGL), and rat brain fatty acid amide hydrolase. Competitive activity-based protein profiling confirmed that O-7460 inhibits mouse brain MAGL only at concentrations ≥ 10 µM, and showed that this compound has only one major 'off-target', that is, the serine hydrolase KIAA1363. O-7460 did not exhibit measurable affinity for human recombinant CB1 or CB2 cannabinoid receptors (Ki > 10 µM). In mouse neuroblastoma N18TG2 cells stimulated with ionomycin, O-7460 (10 µM) reduced 2-AG levels. When administered to mice, O-7460 dose-dependently (0-12 mg·kg⻹, i.p.) inhibited the intake of a high-fat diet over a 14 h observation period, and, subsequently, slightly but significantly reduced body weight. CONCLUSIONS AND IMPLICATIONS: O-7460 might be considered a useful pharmacological tool to investigate further the role played by 2-AG both in vitro and in vivo under physiological as well as pathological conditions.
Assuntos
Fármacos Antiobesidade/uso terapêutico , Ácidos Araquidônicos/antagonistas & inibidores , Endocanabinoides/antagonistas & inibidores , Inibidores Enzimáticos/uso terapêutico , Glicerídeos/antagonistas & inibidores , Glicerofosfolipídeos/uso terapêutico , Lipase Lipoproteica/antagonistas & inibidores , Obesidade/tratamento farmacológico , Ácidos Oleicos/uso terapêutico , Organofosfonatos/uso terapêutico , Animais , Fármacos Antiobesidade/administração & dosagem , Fármacos Antiobesidade/farmacologia , Ácidos Araquidônicos/metabolismo , Comportamento Animal/efeitos dos fármacos , Linhagem Celular , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Endocanabinoides/metabolismo , Ingestão de Energia/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Glicerídeos/metabolismo , Glicerofosfolipídeos/administração & dosagem , Glicerofosfolipídeos/farmacologia , Humanos , Hipotálamo/efeitos dos fármacos , Hipotálamo/enzimologia , Hipotálamo/metabolismo , Lipase Lipoproteica/genética , Lipase Lipoproteica/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Neurônios/metabolismo , Obesidade/enzimologia , Obesidade/metabolismo , Ácidos Oleicos/administração & dosagem , Ácidos Oleicos/farmacologia , Organofosfonatos/administração & dosagem , Organofosfonatos/farmacologia , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Esterol Esterase/antagonistas & inibidores , Esterol Esterase/metabolismoRESUMO
In this paper, using high-performance liquid chromatography methods were quantitatively and qualitatively identified most biological important high fatty acids, contained in Rkatsiteli grape seed oil of 2010 years crop in Signakhi region of Georgia. The chromatography investigation showed, that the grape seed oil contained 61% linolic acid, 19% oleic, 8% palmitic, 4.5% stearic, 1.4% linolenic and 0.6% arachidonuic acids. In standard diet grape seed oil was added as a food additive (5.0 gr and 10.0 gr on 1.0 kg food). After 15 days fatty acids are carried out from mouse liver (120 inbred mouse), fractioned and using high-performance liquid chromatography the retention values of individual fatty asids are identiced. The investigation showed different sensitivity of components contained in grape seed oil.
Assuntos
Ácidos Graxos Insaturados/análise , Ácidos Graxos Insaturados/farmacologia , Aditivos Alimentares/química , Aditivos Alimentares/farmacologia , Extrato de Sementes de Uva/química , Óleos de Plantas/química , Animais , Membrana Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Glicerofosfolipídeos/análise , Glicerofosfolipídeos/farmacologia , Humanos , Ácido Linoleico/análise , Ácido Linoleico/farmacologia , Fígado/efeitos dos fármacos , Camundongos , Ácido Oleico/análise , Ácido Oleico/farmacologia , Triglicerídeos/análise , Triglicerídeos/farmacologiaRESUMO
This paper describes the inhibitory activities of diacylglyceride phospholipids, such as phosphatidylcholine (lecithin), phosphatidylethanolamine (cephalin), phosphatidylserine, phosphatidylglycerol, bisphosphatidylglycerol (cardiolipin), phosphatidylinositol, and phosphatidic acid (phosphatidate) (compounds 1 - 7, respectively) against DNA polymerase (pol), DNA topoisomerase (topo), and human cancer cell growth. Among the compounds tested, compounds 3 - 7 were revealed to be potent inhibitors of animal pols: compound 4 was the strongest inhibitor, with IC(50) values for different pols of 1.7 - 15 mM. Compounds 4 - 7 also inhibited the activity of human topo II: compound 7 was the strongest inhibitor, with an IC50 value of 20 mM. The glycerophospholipids had no effect on the activities of plant (cauliflower) pol a, prokaryotic pols, or other DNA metabolic enzymes, such as calf primase of pol a, T7 RNA polymerase, T4 polynucleotide kinase, and bovine deoxyribonuclease I. These results suggest that compounds 3 - 7 are selective inhibitors of animal pols and human topos. Compounds 4 and 7 also suppressed the growth of a human colon carcinoma cell line that lacked p53 (HCT116 p53(-/-)); their LD(50) values were 63.6 and 51.1 mM, respectively, suggesting that cell growth inhibition by these compounds leads to the inhibition of pols and/or topos. From these findings, diacylglyceride phospholipids, which are present in various foods, might be effective nutrients for promoting human anti-cancer health promotion.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , DNA Topoisomerases Tipo II/metabolismo , DNA Topoisomerases Tipo I/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Glicerofosfolipídeos/farmacologia , Inibidores da Topoisomerase/farmacologia , Animais , Afidicolina/farmacologia , Bovinos , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Células HCT116 , Humanos , Concentração Inibidora 50 , Inibidores da Síntese de Ácido Nucleico , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
An imbalance in (n-6)/(n-3) PUFA has been reported in cystic fibrosis (CF) patients. Glycerophospholipids enriched in docosahexaenoic acid (GPL-DHA) have been shown to regulate the (n-6)/(n-3) fatty acid ratio in the elderly. Here, we tested the effect of GPL-DHA supplementation on PUFA status in F508del homozygous CF mice. GPL-DHA liposomes were administrated by gavage (60 mg DHA/kg daily, i.e. at maximum 1.4 mg DHA/d) to 1.5-mo-old CF mice (CF+DHA) and their corresponding wild-type (WT) homozygous littermates (WT+DHA) for 6 wk. The PUFA status of different tissues was determined by GC and compared with control groups (CF and WT). There was an alteration in the (n-6) PUFA pathway in several CF-target organs in CF compared with WT mice, as evidenced by a higher level of arachidonic acid (AA) in membrane phospholipids or whole tissue (21 and 39% in duodenum-jejunum, 32 and 38% in ileum, and 19 and 43% in pancreas). Elevated AA levels were associated with lower linoleic acid (LA) and higher dihomo-gamma-linolenic acid levels. No DHA deficiency was observed. GPL-DHA treatment resulted in different PUFA composition changes depending on the tissue (increase in LA, decrease in elevated AA, DHA increase, increase in (n-6)/(n-3) fatty acid ratio). However, the DHA/AA ratio consistently increased in all tissues in CF+DHA and WT+DHA mice. Our study demonstrates the effectiveness of an original oral DHA formulation in counter-balancing the abnormal (n-6) fatty acid metabolism in organs of CF mice when administrated at a low dose and highlights the potential of the use of GPL-DHA as nutritherapy for CF patients.
Assuntos
Ácido Araquidônico/metabolismo , Fibrose Cística/metabolismo , Suplementos Nutricionais , Ácidos Docosa-Hexaenoicos/farmacologia , Glicerofosfolipídeos/farmacologia , Ácido Linoleico/metabolismo , Animais , Membrana Celular/metabolismo , Fibrose Cística/genética , Modelos Animais de Doenças , Ácidos Graxos/análise , Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Ômega-6/metabolismo , Glicerofosfolipídeos/química , Humanos , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Lipídeos/química , Lipídeos/isolamento & purificação , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Fosfolipídeos/metabolismo , Deleção de SequênciaRESUMO
BACKGROUND: Previous epidemiological studies have shown that light to moderate alcohol consumption has protective effects against coronary heart disease but the mechanisms of the beneficial effect of alcohol are not known. Ethanol may increase high density lipoprotein (HDL) cholesterol concentration, augment the reverse cholesterol transport, or regulate growth factors or adhesion molecules. To study whether qualitative changes in HDL phospholipids mediate part of the beneficial effects of alcohol on atherosclerosis by HDL receptor, we investigated whether phosphatidylethanol (PEth) in HDL particles affects the secretion of vascular endothelial growth factor (VEGF) by a human scavenger receptor CD36 and LIMPII analog-I (CLA-1)-mediated pathway. METHODS: Human EA.hy 926 endothelial cells were incubated in the presence of native HDL or PEth-HDL. VEGF concentration and CLA-1 protein expression were measured. Human CLA-1 receptor-mediated mechanisms in endothelial cells were studied using CLA-1 blocking antibody and protein kinase inhibitors. RESULTS: Phosphatidylethanol-containing HDL particles caused a 6-fold increase in the expression of CLA-1 in endothelial cells compared with the effect of native HDL. That emergent effect was mediated mainly through protein kinase C and p44/42 mitogen-activated protein kinase pathways. PEth increased the secretion of VEGF and that increase could be abolished by a CLA-1 blocking antibody. CONCLUSIONS: High density lipoprotein particles containing PEth bind to CLA-1 receptor and thereby increase the secretion of VEGF from endothelial cells. Ethanol-induced protective effects against coronary heart disease may be explained, at least partly, by the effects of PEth-modified HDL particles on VEGF via CLA-1-mediated mechanisms in endothelial cells.
Assuntos
Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Glicerofosfolipídeos/farmacologia , Lipoproteínas HDL/efeitos dos fármacos , Receptores de Lipoproteínas/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Western Blotting , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Carbazóis/farmacologia , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Flavonoides/farmacologia , Glicerofosfolipídeos/antagonistas & inibidores , Humanos , Indóis , Lipoproteínas/biossíntese , Lipoproteínas/genética , Maleimidas , Proteína Quinase 1 Ativada por Mitógeno/biossíntese , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/fisiologiaRESUMO
BACKGROUND: Lactobacilli represent a major component of the human microbiota. In this study we investigated whether and how Lactobacillus fermentum inhibits the proinflammatory responses of human epithelial cells on Yersinia enterocolitica infection. METHODS: Human epithelial cells were exposed to Y. enterocolitica pYV(-) or L. fermentum or to both strains, combinations of heat-killed L. fermentum or supernatant of L. fermentum cultures and Y. enterocolitica. The modulation of Y. enterocolitica induced IL-8 levels in the culture supernatants was determined and activation of Rac, p38, and NF-kappaB was investigated. RESULTS: Exposure of human epithelial cells to L. fermentum does not induce NF-kappaB activation and subsequent IL-8 secretion in HeLa cells, whereas Y. enterocolitica induces NF-kappaB activation and high levels of IL-8. Viable L. fermentum, supernatant of L. fermentum cultures, but not heat-killed L. fermentum, inhibited IL-8 secretion of HeLa cells triggered by Y. enterocolitica. Lactobacillus fermentum-exposed HeLa cells showed decreased Rac, p38, and NF-kappaB activation after Y. enterocolitica infection. Treatment of L. fermentum supernatants with phospholipase C abolished the inhibitory effect, indicating that a secreted phospholipid mediates the antiinflammatory properties of L. fermentum. Adhesion to or invasion of Y. enterocolitica into epithelial cells was not altered by coincubation with L. fermentum. CONCLUSION: Our results lead to the conclusion that L. fermentum inhibits the Y. enterocolitica-induced IL-8 production by a possibly secreted phospholipid of <10 kDa molecular weight. These data suggest that L. fermentum may have probiotic properties modulating intestinal inflammatory responses and might offer new therapeutic strategies in the treatment of intestinal inflammatory diseases.
Assuntos
Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Mediadores da Inflamação/metabolismo , Interleucina-8/metabolismo , Limosilactobacillus fermentum/fisiologia , Yersiniose/metabolismo , Yersinia enterocolitica/patogenicidade , Aderência Bacteriana , Células Cultivadas , Meios de Cultivo Condicionados , Endopeptidase K/farmacologia , Glicerofosfolipídeos/química , Glicerofosfolipídeos/farmacologia , Células HeLa , Humanos , Inflamação , Compostos de Ferro/farmacologia , NF-kappa B/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
A new erythroblastic leukemia cell line (EEB) was established from a patient with early erythroblastic leukemia. The cells had features of immature erythroblasts, including an agranular basophilic cytoplasm and CD36, CD71, CD175s (sialyl-Tn) and CD235a (glycophorin A) expression without CD41 expression, myeloperoxidase activity and platelet-peroxidase activity. The cells were confirmed to be of the erythroid lineage based on expression of the gamma-globin message. They were induced to differentiate into benzidine-positive cells by hemin and delta-amino levulinic acid (delta-ALA). An analysis of cell membrane lipids showed that EEB cells contain a type of glycerolipid, phosphatidylglucose (PhGlc), but not unbranched type 2 chains, i antigens. GL-7 which is a recombinant Fab fragment of GL-2 and binds to PhGlc, induced production of hemoglobin F (HbF) associated with accumulation of the gamma-globin (gamma-globin) message in EEB cells. The GL-7-mediated erythroid differentiation was associated with apoptosis. These results suggest that direct signaling to PhGlc mediates erythroid differentiation and apoptosis in EEB cells.
Assuntos
Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Células Eritroides/efeitos dos fármacos , Glicerofosfolipídeos/farmacologia , Fragmentos Fab das Imunoglobulinas/farmacologia , Leucemia Eritroblástica Aguda/tratamento farmacológico , Leucemia Eritroblástica Aguda/metabolismo , Diferenciação Celular/efeitos dos fármacos , Análise Citogenética , Ensaios de Seleção de Medicamentos Antitumorais , Células Eritroides/citologia , Globinas/efeitos dos fármacos , Globinas/genética , Glicerofosfolipídeos/análise , Humanos , Imunofenotipagem , Leucemia Eritroblástica Aguda/genética , Masculino , Pessoa de Meia-Idade , Fosforilação/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sensibilidade e Especificidade , Relação Estrutura-Atividade , Tirosina/efeitos dos fármacos , Tirosina/metabolismoRESUMO
Two new analogues derived from the platelet activating factor (PAF), containing glucosamine instead of the acetyl group, were synthesized, and their effect on the human keratinocyte cell line HaCaT was evaluated with respect to cytotoxicity, proliferation, adhesion, and migration. Starting with (R)-1,2-isopropylideneglycerol (3), the glycosylation acceptor 1-O-octadecyl-3-O-tert-butyldimethylsilyl-sn-glycerol (6) was synthesized in three steps. Glycosylation of 6 with the already known O-(3,4,6-tri-O-acetyl-2-deoxy-2-dimethylmaleimido-beta-D-glycopyranosyl)trichloracetimidate gave 1-O-octadecyl-2-O-(3',4',6'-tri-O-acetyl-2'-deoxy-2'-dimethylmaleimido-beta-D-glucopyranosyl)-3-O-tert-butyldimethylsilyl-sn-glycerol (7). After removing the (tert-butyldimethyl)silyl (TBDMS) group with FeCl3x6H2O, phosphoryl choline was introduced, yielding [1-O-octadecyl-2-O-(2'-deoxy-2'-dimethylmaleimido-beta-D-glucopyranosyl)-sn-glycero(3)]phosphorylcholine (2) (glucosimide-PAF). pH controlled cleavage of the amino protection group gave [1-O-octadecyl-2-O-(2'-deoxy-2'-amino-beta-D-glucopyranosyl)-sn-glycero(3)]phosphorylcholine hydrochloride (1) (glucosamine-PAF). 2 inhibited proliferation of HaCaT cells by 26% at nontoxic concentrations, while 1 increased the proliferation rate by 30% at low concentrations. At higher concentrations, both compounds showed cytotoxic properties with LD50 = 30 micromol/L (1) and LD50 = 5-6 micromol/L (2). Both 1 and 2 were potent promoters of cell adhesion and migration of HaCaT cells.
Assuntos
Antineoplásicos/síntese química , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Glucosamina/análogos & derivados , Glucosamina/síntese química , Glicerofosfolipídeos/síntese química , Glicolipídeos/síntese química , Antineoplásicos/farmacologia , Antineoplásicos/toxicidade , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Glucosamina/farmacologia , Glucosamina/toxicidade , Glicerofosfolipídeos/farmacologia , Glicerofosfolipídeos/toxicidade , Glicolipídeos/farmacologia , Glicolipídeos/toxicidade , Humanos , Relação Estrutura-AtividadeAssuntos
Ativação Linfocitária/efeitos dos fármacos , Microdomínios da Membrana/efeitos dos fármacos , Fosfatidiletanolaminas/farmacologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Animais , Glicerofosfolipídeos/farmacologia , Humanos , Microdomínios da Membrana/fisiologia , Camundongos , Transdução de Sinais , Linfócitos T Citotóxicos/fisiologia , Células Tumorais CultivadasRESUMO
In human neutrophils, the activation of phospholipase D and the Tyr phosphorylation of proteins are early signaling events upon cell stimulation. We found that the pretreatment of neutrophils with ethanol (0.8%) or 1-butanol (0.3%), which results in the accumulation of phosphatidylalcohol at the expense of phosphatidic acid (PA), decreased the phorbol myristate acetate-stimulated Tyr phosphorylation of endogenous proteins (42, 115 kDa). When neutrophil cytosol was incubated in the presence or absence of PA, these and other endogenous proteins became Tyr-phosphorylated in a PA-dependent manner. In contrast, phosphatidylalcohols exhibited only 25% (phosphatidylethanol) or 5% (phosphatidylbutanol) of the ability of PA to stimulate Tyr phosphorylation in the cell-free assay. Similarly, other phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, polyphosphoinositides, and sphingosine 1-phosphate) showed little ability to stimulate Tyr phosphorylation. These data suggest that PA can function as an intracellular regulator of Tyr phosphorylating activity. Gel filtration chromatography of leukocyte cytosol revealed a peak of PA-dependent Tyr phosphorylating activity distinct from a previously described PA-dependent phosphorylating activity (Waite, K. A., Wallin, R., Qualliotine-Mann, D., and McPhail, L. C. (1997) J. Biol. Chem. 272, 15569-15578). Among the protein Tyr kinases expressed in neutrophils, only Fgr eluted exclusively in the peak of PA-dependent Tyr phosphorylating activity. Importantly, Fgr isolated from unstimulated neutrophil lysates showed increased activity in the presence of PA but not phosphatidylbutanol. Moreover, the pretreatment of neutrophils with 1-butanol decreased Fgr activity in cells stimulated with formyl-methionyl-leucyl phenylalanine plus dihydrocytochalasin B. Together, these results suggest a new second messenger role for PA in the regulation of Tyr phosphorylation.
Assuntos
Neutrófilos/metabolismo , Ácidos Fosfatídicos/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , 1-Butanol/farmacologia , Células Cultivadas , Etanol/farmacologia , Glicerofosfolipídeos/farmacologia , Humanos , Ácidos Fosfatídicos/farmacologia , Fosfolipase D/metabolismo , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Quinases da Família srcRESUMO
The synthesis of inflammation mediators produced from arachidonic acid is regulated primarily by the cellular concentration of free arachidonic acid. Since intracellular arachidonic acid is almost totally present as phospholipid esters, the concentration of intracellular arachidonic acid is primarily dependent on the balance between the release of arachidonic acid from membrane phospholipids and the uptake of arachidonic acid into membrane phospholipids. Cytosolic phospholipase A(2) is a calciumdependent enzyme that catalyzes the stimulus-coupled hydrolysis of arachidonic acid from membrane phospholipids. Following exposure of macrophages to various foreign or endogenous stimulants, cytosolic phospholipase A(2) is activated. Treatment with these compounds may also stimulate phospholipase D activity, and, in the presence of ethanol, phospholipase D catalyzes the synthesis of phosphatidylethanol. A cell-free system was used to evaluate the effect of phosphatidylethanol on cytosolic phospholipase A(2) activity. Phosphatidylethanol (0.5 microM) added to 1-stearoyl-2-[(3)H]-arachidonoyl-sn-glycero-3-phosphocholine vesicles stimulated cytosolic phospholipase A(2) activity. However, high concentrations (20-100 microM) of phosphatidylethanol inhibited cytosolic phospholipase A(2) activity. Phosphatidic acid, the normal phospholipase D product, also stimulated cytosolic phospholipase A(2) activity at 0.5 microM, but had an inhibitory effect on cytosolic phospholipase A(2) activity at concentrations of 50 and 100 microM. Ethanol (20-200 mM), the precursor of phosphatidylethanol, added directly to the assay did not alter cytosolic phospholipase A(2) activity. These results suggest that phosphatidylethanol alters the physical properties of the substrate, and at lower concentrations of anionic phospholipids the substrate is more susceptible to hydrolysis. However, at high concentrations, phosphatidylethanol either reverses the alterations in physical properties of the substrate or phosphatidylethanol may be competing as the substrate. Both interactions may result in lower cytosolic phospholipase A(2) activity.
Assuntos
Cálcio/metabolismo , Citosol/enzimologia , Glicerofosfolipídeos/farmacologia , Macrófagos/enzimologia , Fosfolipases A/metabolismo , Animais , Ácido Araquidônico/metabolismo , Extratos Celulares , Quelantes/farmacologia , Citosol/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Etanol/farmacologia , Hidrólise/efeitos dos fármacos , Cinética , Lipossomos/química , Lipossomos/efeitos dos fármacos , Lipossomos/metabolismo , Macrófagos/citologia , Camundongos , Ácidos Fosfatídicos/farmacologia , Fosfatidilcolinas/metabolismo , Células Tumorais CultivadasRESUMO
The lipid A (endotoxin) moiety of lipopolysaccharide (LPS) elicits rapid cellular responses from many cell types, including macrophages, lymphocytes, and monocytes. In CD14 transfected 70Z/3 pre-B lymphocyte tumor cells, these responses include activation of the MAP kinase homolog, p38, activation of NF-kappaB, and transcription of kappa light chains, leading to the assembly of surface IgM. In this work, we explored the specificity of the response with regard to lipid structure, and the requirement for p38 kinase activity prior to NF-kappaB activation in control and CD14 transfected 70Z/3 (CD14-70Z/3) cells. A p38-specific inhibitor, SB203580, was used to block p38 kinase activity in cells. CD14-70Z/3 cells were incubated with 1-50 microM SB203580, and then stimulated with LPS. Nuclear extracts were prepared, and NF-kappaB activation was measured using an electrophoretic mobility shift assay. SB203580 did not inhibit LPS induced NF-kappaB activation. In addition, LPS failed to activate p38 tyrosine phosphorylation in 70Z/3 cells lacking CD14, in spite of rapid NF-kappaB activation and robust surface IgM production with appropriate higher doses of LPS. LPS stimulation of p38 phosphorylation, NF-kappaB activation, and surface IgM expression were all blocked completely by lipid A-like endotoxin antagonists whether or not CD14 was present. Acidic glycerophospholipids and ceramides did not mimic lipid A-like molecules either as agonists or antagonists in this system. Our data support the hypothesis that lipid A-mediated activation of cells requires stimulation of a putative lipid A sensor that is downstream of CD14, but upstream of p38 and NF-kappaB.
Assuntos
Lipídeo A/farmacologia , Proteínas Quinases Ativadas por Mitógeno , Transdução de Sinais/efeitos dos fármacos , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Ceramidas/farmacologia , Inibidores Enzimáticos/farmacologia , Glicerofosfolipídeos/farmacologia , Imidazóis/farmacologia , Lipídeo A/antagonistas & inibidores , Receptores de Lipopolissacarídeos/genética , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Camundongos , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Piridinas/farmacologia , Transfecção , Células Tumorais Cultivadas , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
We studied the influence of membrane lipids, MgCl2, and ATP on the ability of a soluble diacylglycerol kinase to bind to 100-nm lipid vesicles. The enzyme did not bind detectably to vesicles that contained phosphatidylcholine alone or to vesicles that contained 50 mol % phosphatidylcholine + 50 mol % phosphatidylethanolamine. But it did bind to vesicles that contained anionic phosphoglycerides, and maximal binding occurred (in the presence of MgCl2) when the vesicles contained anionic phosphoglycerides alone. When increasing amounts of phosphatidylcholine were included in phosphatidylserine-containing vesicles, enzyme binding to the vesicles decreased by as much as 1000-fold. However, when increasing amounts of phosphatidylethanolamine were included in phosphatidylserine-containing vesicles, little change in binding occurred until the concentration of phosphatidylserine was reduced to below 25 mol %. These results and results obtained with vesicles that contained various mixtures of anionic phosphoglycerides, phosphatidylcholine, phosphatidylethanolamine, and unesterified cholesterol provided evidence that anionic phosphoglycerides were positive effectors of binding, phosphatidylcholine was a negative effector, and phosphatidylethanolamine and unesterified cholesterol were essentially neutral diluents. Other experiments showed that diacylglycerol and some of its structural analogues also were important, positive effectors of enzyme binding and that addition of ATP to the medium increased their effects. The combined results of the study suggest that the enzyme may bind to vesicles via at least two types of binding sites: one type that requires anionic phospholipids and is enhanced by Mg2+ but inhibited by phosphatidylcholine, and one type that requires diacylglycerol and is enhanced by ATP.