RESUMEN
Synthesis and anti-inflammatory effects of certain furo[3',2':3,4]naphtho[1,2-d]imidazole derivatives 12-18 were studied. These compounds were synthesized from naphtho[1,2-b]furan-4,5-dione (10) which in turn was prepared from the known 2-hydroxy-1,4-naphthoquinone (7) in a one pot reaction. Furo[3',2':3,4]naphtho[1,2-d]imidazole (12) was inactive (IC(50) value of >30 microM) while its 5-phenyl derivative 13, with an IC(50) value of 16.3 and 11.4 microM against lysozyme and beta-glucuronidase release, respectively, was comparable to the positive trifluoperazine. The same potency was observed for 5-furan derivative 16 with an IC(50) value of 19.5 and 11.3 microM against lysozyme and beta-glucuronidase release, respectively. An electron-withdrawing NO(2) substituted on 5-phenyl or 5-furanyl group led to the devoid of activity as in the cases of 14 and 17. Among them, compound 15 exhibited significant inhibitory effects, with an IC(50) value of 7.4 and 5.0 microM against lysozyme and beta-glucuronidase release, respectively. For the LPS-induced NO production, the phenyl derivatives 12-15 were inactive while the nitrofuran counterparts 17 and 18 suppress LPS-induced NO production significantly, with an IC(50) value of 1.5 and 1.3 microM, respectively, which are more active than that of the positive 1400 W. Compounds 16-18 were capable of inhibiting LPS-induced iNOS protein expression at a dose-dependent manner in which compound 18, with an IC(50) of 0.52 microM in the inhibition of iNOS expression, is approximately fivefold more potent than that of the positive 1400 W. In the CLP rat animal model, compound 18 was found to be more active than the positive hydrocortisone in the inhibition of the iNOS mRNA expression in rat lung tissue. The sepsis-induced PGE2 production in rat serum decreased 150% by the pretreatment of 18 in a dose of 10 mg/kg.
Asunto(s)
Antiinflamatorios/química , Antiinflamatorios/farmacología , Imidazoles/química , Imidazoles/farmacología , Sepsis/tratamiento farmacológico , Animales , Antiinflamatorios/uso terapéutico , Línea Celular , Furanos/química , Expresión Génica/efectos de los fármacos , Imidazoles/uso terapéutico , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Prostaglandinas E/antagonistas & inhibidores , Prostaglandinas E/metabolismo , Ratas , Ratas Sprague-Dawley , Superóxidos/metabolismoRESUMEN
In the present study, a novel synthetic compound 4-(2-(cyclohex-2-enylidene)hydrazinyl)quinolin-2(1H)-one (CYL-4d) was found to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) production without affecting cell viability or enzyme activity of expressed inducible NO synthase (iNOS) in RAW 264.7 macrophages. CYL-4d exhibited parallel inhibition of LPS-induced expression of iNOS protein, iNOS mRNA and iNOS promoter activity in the same concentration range. LPS-induced activator protein-1 (AP-1) DNA binding, AP-1-dependent reporter gene activity and c-Jun nuclear translocation were all markedly inhibited by CYL-4d with similar efficacy, whereas CYL-4d produced a weak inhibition of nuclear factor-kappaB (NF-kappaB) DNA binding, NF-kappaB-dependent reporter gene activity and p65 nuclear translocation without affecting inhibitory factor-kappa B alpha (I kappa B alpha) degradation. CYL-4d had no effect on the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK) and its upstream activator MAPK kinase (MEK) 3, whereas it significantly attenuated the phosphorylation of c-Jun, c-Jun NH(2)-terminal kinase (JNK) and its upstream activator MEK4 in a parallel concentration-dependent manner. Other Toll-like receptors (TLRs) ligands (peptidoglycans, double-stranded RNA, and oligonucleotide containing unmethylated CpG motifs)-induced iNOS protein expression were also inhibited by CYL-4d. Furthermore, the NO production from BV-2 microglial cells as well as rat alveolar macrophages in response to LPS was diminished by CYL-4d. These results indicate that the blockade of NO production by CYL-4d in LPS-stimulated RAW 264.7 cells is attributed mainly to interference in the MEK4-JNK-AP-1 signaling pathway. CYL-4d inhibition of NO production is not restricted to TLR4 activation and immortalized macrophage-like cells.
Asunto(s)
Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , MAP Quinasa Quinasa 4/metabolismo , Óxido Nítrico/metabolismo , Quinolonas/farmacología , Factor de Transcripción AP-1/metabolismo , Animales , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/metabolismo , Microglía/efectos de los fármacos , Microglía/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fosforilación/efectos de los fármacos , Quinolonas/síntesis química , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
5-[4-Acridin-9-ylamino]phenyl]-5-methyl-3-methylenedihydrofuran-2-one (CYL-26z) inhibited the polymorphonuclear leukocyte (PMNL) infiltration and protein leakage into the lungs in lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice as determined on the basis of PMNL and protein contents in bronchoalveolar lavage (BAL) fluid and myeloperoxidase (MPO) content in whole lung extracts. CYL-26z also attenuated the formyl-Met-Leu-Phe (fMLP)-induced neutrophil chemotaxis and respiratory burst in vitro (IC(50) 8.4+/-0.9microM and 2.0+/-0.6microM, respectively). CYL-26z had no effect on superoxide anion (O(2)(-)) generation during dihydroxyfumaric acid autoxidation or on the NADPH oxidase activity in two cell-free systems (the arachidonic acid-induced assembly of NADPH oxidase and the preassembled oxidase caused by phorbol ester treatment), whereas it inhibited NaF-induced respiratory burst. Inhibition of respiratory burst by CYL-26z was readily reversible by washing. Only slight, but significant, inhibition of extracellular signal regulated kinase (ERK) phosphorylation and p38 mitogen-activated protein kinase (MAPK) activation in response to fMLP by CYL-26z up to 30microM was obtained. CYL-26z effectively blocked the formation of phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) as determined by immunofluorescence microscopy and flow cytometry assays and the dual phosphorylation of protein kinase B (PKB/Akt) on S473 and T308 residues in fMLP-stimulated neutrophils. The membrane recruitment of p110gamma and Ras, the Ras activation, and the association between p110gamma and Ras were also attenuated by CYL-26z. These results indicate that the blockade of Ras activation by CYL-26z attenuated the downstream phosphoinositide 3-kinase (PI3K) gamma signaling, which is involved in chemoattractant-induced neutrophil chemotaxis and respiratory burst, and may have a beneficial anti-inflammatory effect on ALI.
Asunto(s)
Acridinas/uso terapéutico , Furanos/uso terapéutico , Activación Neutrófila/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Síndrome de Dificultad Respiratoria/tratamiento farmacológico , Acridinas/farmacología , Animales , Fosfatidilinositol 3-Quinasa Clase I , Furanos/farmacología , Ratones , Activación Neutrófila/fisiología , Neutrófilos/fisiología , Transducción de SeñalRESUMEN
Artocarpol A (ART), a natural product isolated from Artocarpus rigida, stimulated superoxide anion (O2*-) generation, which was inhibited by 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294002), a phosphoinositide 3-kinase (PI3K) inhibitor, in rat neutrophils. ART stimulated phosphorylation of protein kinase B (PKB/Akt) on both T308 and S473 residues, and LY 294002 inhibited these effects. Rat neutrophils expressed both class IA PI3K subunits (p85, p110alpha, p110beta, and p110delta) and a class IB PI3K subunit (p110gamma) as assessed by a combination of Western blotting and reverse transcription-polymerase chain reaction (RT-PCR) approaches. Stimulation of neutrophils with ART evoked phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) formation, which reached a maximal level at 2 min and was attenuated by LY 294002, as evidenced by immunofluorescence microscopy and by flow cytometry. Detectable membrane-association of class IA PI3Ks, class IB PI3K and Ras was seen as early as 1.5, 0.5 and 1.5 min, respectively, after stimulation with ART. The kinetics of ART-induced Ras activation paralleled the kinetics of class IA PI3Ks recruitment to membrane caused by ART, and the p85 and p110gamma immunoprecipitates contain Ras. ART stimulated Src family kinase activation, which was detectable within 1.5 min of incubation with ART. Both Src kinase activity and PtdIns(3,4,5)P3 formation in ART-stimulated neutrophils were inhibited by 4-amino-1-tert-butyl-3-(1'-naphthyl)pyrazolo[3,4-d]pyrimidine (PP1 analog). PP1 analog also attenuated the ART-stimulated O2*- generation in rat neutrophils. These results indicate that the stimulation of respiratory burst by ART in neutrophils implicates PI3K signaling.
Asunto(s)
Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Neutrófilos/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Estallido Respiratorio , Familia-src Quinasas/metabolismo , Animales , Activación Enzimática , Inmunoprecipitación , Microscopía Fluorescente , Neutrófilos/enzimología , Neutrófilos/metabolismo , Fosfatos de Fosfatidilinositol/biosíntesis , Fosforilación , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Superóxidos/metabolismoRESUMEN
We demonstrated that 5-amino-3-(3,4-dichlorophenyl)1,2,3,4-oxatriazolium (GEA3162), a nitric oxide (NO)-releasing agent, stimulated [Ca2+]i rise in rat neutrophils. This Ca2+ response was prevented by the thiol reducing agents, 2-mercaptoethanol, N-acetyl-L-cysteine, dithiothreitol, 2,3-dimercaptopropane-1-sulfonic acid (DMPS) and tris-(2-carboxyethyl)phosphine (TCEP), but slightly reduced by the antioxidant, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox). GEA3162 also increased the formation of cellular reactive oxygen intermediates and decreased the cellular content of low molecular thiols. These responses were greatly reduced by Trolox, dithiothreitol and N-acetyl-L-cysteine. GEA3162 stimulated the protein tyrosine phosphorylation in neutrophils. The [Ca2+]i rise caused by formyl-Met-Leu-Phe (fMLP) and cyclopiazonic acid (CPA) was suppressed by GEA3162. TCEP prevented the inhibition of fMLP-induced [Ca2+]i rise by GEA3162. In the absence of external Ca2+, GEA3162 inhibited the CPA-induced [Ca2+]i rise, whereas it only slightly affected the fMLP-induced mobilization of the Ca2+ store. Application of GEA3162 after the stimulation with fMLP or CPA suppressed the robust Ca2+ entry followed by the readdition of Ca2+ into medium. Moreover, the Ca2+ entry was more susceptible to inhibition by treatment with GEA3162 prior to than after the fMLP stimulation. GEA3162 had no effect on the mitochondrial membrane potential. GEA3162 induced actin reorganization and condensed filament network at the cell periphery. These results indicate that GEA3162 exerted both the stimulation of Ca2+ entry and the inhibition of the store-operated Ca2+ entry in rat neutrophils. The dual effects of GEA3162 on the regulation of the external Ca2+ entry are mainly through the thiol modification of target protein(s) residing on the outside of the plasma membrane.
Asunto(s)
Calcio/metabolismo , Neutrófilos/efectos de los fármacos , Donantes de Óxido Nítrico/farmacología , Compuestos de Sulfhidrilo/metabolismo , Triazoles/farmacología , Acetilcisteína/farmacología , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Animales , Señalización del Calcio/efectos de los fármacos , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Quelantes/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Indoles/farmacología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Mercaptoetanol/farmacología , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/fisiología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/metabolismo , Oxidación-Reducción/efectos de los fármacos , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Factores de Tiempo , Tirosina/metabolismo , Unitiol/farmacologíaRESUMEN
1 Artocarpol A (ART), a natural phenolic compound isolated from Artocarpus rigida, stimulated a slow onset and long-lasting superoxide anion generation in rat neutrophils, whereas only slightly activated the NADPH oxidase in a cell-free system. 2 Pretreatment of neutrophils with pertussis toxin (1 microg ml(-1)), 50 microM 2'-amino-3'-methoxyflavone (PD 98059), or 1 microM 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126) had no effect on ART-stimulated superoxide anion generation. ART (30 microM) did not induce extracellular signal-regulated kinase (ERK) phosphorylation. 3 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB 203580) markedly attenuated the ART-stimulated superoxide anion generation (IC50 value of 4.3+/-0.3 microM). Moreover, ART induced p38 mitogen-activated PK (MAPK) phosphorylation and activation. 4 The superoxide anion generation in response to ART was also substantially inhibited in a Ca2+-free medium, and by pretreatment with 1 microM 1-[6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U-73122) and 100 microM 2-aminoethyldiphenyl borate (2-APB). ART (30 microM) stimulated the [Ca2+]i elevation in the presence or absence of external Ca2+, and also increased the D-myo-inositol 1,4,5-trisphosphate formation. 5 2-[1-(3-Dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF 109203X) greatly inhibited the ART-stimulated superoxide anion generation (IC50 value of 7.8+/-1.0 nM). ART increased the recruitment of PKC-alpha, -betaI, and -betaII to the plasma membrane of neutrophils, and stimulated Ca2+-dependent PKC activation in the cytosol preparation. 6 ART induced the phosphorylation of p47phox, which was attenuated by GF 109203X. Moreover, ART evoked the membrane association of p47(phox), which was inhibited by GF 109203X and SB 203580. 7 These results indicate that the ART stimulation of superoxide anion generation involved the activation of p38 MAPK, PLC/Ca2+, and PKC signaling pathways in rat neutrophils.
Asunto(s)
Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Neutrófilos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Superóxidos/metabolismo , Animales , Butadienos/farmacología , Calcio/metabolismo , Sistema Libre de Células/efectos de los fármacos , Sistema Libre de Células/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Estrenos/farmacología , Flavonoides/farmacología , Imidazoles/farmacología , Immunoblotting , Indoles/farmacología , Maleimidas/farmacología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/enzimología , Neutrófilos/metabolismo , Nitrilos/farmacología , Fosforilación/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Piridinas/farmacología , Pirrolidinonas/farmacología , Ratas , Ratas Sprague-Dawley , Transducción de Señal/fisiología , Acetato de Tetradecanoilforbol/farmacología , Fosfolipasas de Tipo C/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The large amount of nitric oxide (NO) produced by inducible NO synthase (iNOS) contributes to cellular injury in inflammatory disease. In the present study, a novel synthetic compound (3E)-4-(2-hydroxyphenyl)but-3-en-2-one (HPB) was found to inhibit lipopolysaccharide (LPS)-induced NO generation, but not through the inhibition of iNOS activity, in RAW 264.7 macrophages. Administration of HPB into mice also inhibited the LPS-induced increase in serum nitrite/nitrate levels. To evaluate the underlying mechanisms of HPB inhibition of NO generation, the expression of the iNOS gene in RAW 264.7 macrophages was examined. HPB abolished the LPS-induced expression of iNOS protein, iNOS mRNA and iNOS promoter activity in a similar concentration-dependent manner. LPS-induced nuclear factor-kappaB (NF-kappaB) DNA binding and NF-kappaB-dependent reporter gene activity were both significantly inhibited by HPB. This effect was mediated through the inhibition of inhibitory factor-kappaBalpha (IkappaBalpha) phosphorylation and degradation, and of p65 nuclear translocation. HPB had no effect on the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinases (MAPK), and c-Jun NH(2)-terminal kinase (JNK). However, HPB suppressed the LPS-induced intracellular reactive oxygen species (ROS) production. These results indicate that HPB down-regulates iNOS gene expression probably through the inhibition of LPS-induced intracellular ROS production, which has been implicated in the activation of NF-kappaB.
Asunto(s)
Butanonas/farmacología , Inhibidores Enzimáticos/farmacología , Lipopolisacáridos/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Fenoles/farmacología , Animales , Secuencia de Bases , Línea Celular , ADN/metabolismo , Cartilla de ADN , Ensayo de Cambio de Movilidad Electroforética , Activación Enzimática , Ratones , Ratones Endogámicos BALB C , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Especies Reactivas de OxígenoRESUMEN
5-[4-Acridin-9-ylamino]phenyl]-5-methyl-3-methylenedihydrofuran-2-one (CYL-26z) inhibited the formyl-Met-Leu-Phe (fMLP)-stimulated phospholipase D (PLD) activity, which was assessed by the production of phosphatidylethanol (PEt) in the presence of ethanol, in rat neutrophils (IC50 1.2+/-0.2 microM). CYL-26z caused a slight but significant attenuation of the global protein tyrosine phosphorylation stimulated by fMLP only at concentrations of CYL-26z up to 30 microM. CYL-26z blocked the membrane recruitment of protein kinase C-alpha (PKC-alpha) at concentrations of CYL-26z > or =3 microM, but failed to affect the membrane association of PKC-betaI and -betaII. The translocation of RhoA to the membrane was attenuated by CYL-26z (IC50 3.8+/-0.8 microM) in fMLP-stimulated neutrophils, whereas CYL-26z caused no significant inhibition of the membrane recruitment of ADP-ribosylation factor (Arf). CYL-26z inhibited the activation of RhoA and dissociation of the RhoA-Rho guanine nucleotide dissociation inhibitor (GDI) complex in fMLP-stimulated neutrophils (IC50 1.8+/-1.0 microM and 1.8+/-0.9 microM, respectively). In a cell-free system, CYL-26z effectively attenuated the membrane association of RhoA in response to GTPgammaS (IC50 1.3+/-0.5 microM). In contrast, the GTPgammaS-stimulated translocation of Arf to membrane was suppressed only at concentrations of CYL-26z up to 30 microM. CYL-26z inhibited the fMLP-stimulated membrane expression of CD11b, CD45 and CD63, and the release of lysozyme and beta-glucuronidase. These results indicate that CYL-26z inhibited the fMLP-stimulated PLD activity, mainly through the blockade of RhoA activation, and degranulation in rat neutrophils.
Asunto(s)
Acridinas/farmacología , Inhibidores Enzimáticos/farmacología , Furanos/farmacología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Fosfolipasa D/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , Animales , Degranulación de la Célula/efectos de los fármacos , Activación Enzimática , Inmunoprecipitación , Neutrófilos/enzimología , Fosfolipasa D/metabolismo , Fosforilación , Ratas , Ratas Sprague-Dawley , Tirosina/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
In this study, we demonstrate that N-ethylmaleimide (NEM), a cell permeable thiol-alkylating agent, enhanced the [Ca2+]i rise caused by stimulation with cyclopiazonic acid (CPA), a sarcoplasmic-endoplasmic reticulum Ca2+-ATPase inhibitor, in rat neutrophils. In addition, NEM attenuated the formyl-Met-Leu-Phe (fMLP)-induced [Ca2+]i rise whether NEM was added to cells prior to or after fMLP stimulation. Moreover, application of NEM after fMLP activation in the absence of external Ca2+ inhibited the Ca2+ signal upon addition of Ca2+ to the medium. Similar patterns were also obtained by using 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), a cell impermeable dithiol-oxidizing agent, which replaced NEM in the CPA- and fMLP-induced [Ca2+]i rise experiments. Treatment with dithiothreitol (DTT), a cell permeable dithiol-reducing agent, N-acetyl-l-cysteine (NAC), a cell permeable monothiol-reducing agent, and tris-(2-carboxyethyl)phosphine (TCEP), a cell impermeable reductant without a thiol group, all rescued the fMLP-induced Ca2+ signal from NEM. Rat neutrophils express the mRNA encoding for transient receptor potential (TRP) C6, inositol trisphosphate receptor (IP3R) 2 and IP3R3. NEM had no effect on the mitochondrial membrane potential. NEM could restore the polarization and F-actin accumulation of fMLP-treated cells to those of the control. In the absence of external Ca2+, NEM rendered the CPA-induced [Ca2+]i elevation persistently but inhibited the fMLP-induced Ca2+ spike, which was reversed by tris-(2-cyanoethyl)phosphine (TCP), a cell permeable reductant without a thiol group. DTNB did not affect the Ca2+ spike caused by fMLP. These results indicate that through protein thiol oxidation, NEM affects the receptor-activated and the store depletion-derived Ca2+ signals in an opposing manner.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Etilmaleimida/farmacología , Indoles/farmacología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Actinas/química , Animales , Canales de Calcio/análisis , Ácido Ditionitrobenzoico/farmacología , Receptores de Inositol 1,4,5-Trifosfato , Masculino , Potenciales de la Membrana/efectos de los fármacos , Fosfinas/farmacología , ARN Mensajero/análisis , Ratas , Receptores Citoplasmáticos y Nucleares/análisis , Canales Catiónicos TRPC/genéticaRESUMEN
In an effort to develop potent anti-inflammatory and cancer chemopreventive agents, a series of chalcones were prepared by Claisen-Schmidt condensation of appropriate acetophenones with suitable aromatic aldehyde or prepared with appropriate dihydrochalcone reacted with appropriate alkyl bromide or prepared in one-pot procedure involving acetophenone and convenient aromatic aldehyde using ultrasonic agitation on basic alumina. The synthesized products were tested for their inhibitory effects on the activation of mast cells, neutrophils, macrophages, and microglial cells. The potent inhibitors of NO production in macrophages and microglial cells were further evaluated for their in vitro cytotoxic effects against several human cancer cell lines. 2'-Hydroxychalcones 1-3, and 2',5'-dihydroxychalcone 7 exhibited potent inhibitory effects on the release of beta-glucuronidase or lysozyme from rat neutrophils stimulated with formyl-Met-Leu-Phe (fMLP)/cytochalasin B (CB). Two 2'-hydroxychalcones (1 and 3) showed potent inhibitory effects on superoxide anion generation in rat neutrophils in response to fMLP/CB. The previously reported chalcone, 5, 6, and 12, exhibited potent inhibitory effect on NO production in lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma)-activated N9 microglial cells or in LPS-activated RAW 264.7 macrophage-like cells. The potent inhibitors 5, 6, and 12 of NO production in macrophages or microglial cells revealed significant or marginal cytotoxic effects against several human cancer lines. Compound 12 manifested potent selective cytotoxicity against human MCF-7 cells and caused cell death by apoptosis. The present results demonstrated that 1-3, and 7 have anti-inflammatory effects and 5, 6, and 12 are potential anti-inflammatory and cancer chemopreventive agents.
Asunto(s)
Antiinflamatorios/síntesis química , Anticarcinógenos/síntesis química , Chalconas/síntesis química , Animales , Antiinflamatorios/farmacología , Anticarcinógenos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Chalconas/farmacología , Quimioprevención , Humanos , Lipopolisacáridos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Mastocitos/efectos de los fármacos , Ratones , Microglía/efectos de los fármacos , Microglía/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Óxido Nítrico/biosíntesis , Ratas , Relación Estructura-ActividadRESUMEN
Mast cells, neutrophils, and macrophages are important inflammatory cells that have been implicated in the pathogenesis of acute and chronic inflammatory diseases. To explore a novel antiinflammatory agent, we have synthesized two types of acridines, 9-anilinoacridine and 9-phenoxyacridine derivatives, for evaluation on the grounds that acridine is a versatile heterocycle possessing a wide variety of biological properties. The title compounds were synthesized by reaction of 9-chloroacridine with appropriate Ar-NH(2) and Ar-OH, and their antiinflammatory activities on inhibitory effects on the activation of mast cells, neutrophils, and macrophages were studied. Three acridine derivatives 4, 10, and 11 were proved to be more potent than the reference inhibitor mepacrine for the inhibition of rat peritoneal mast cell degranulation with similar IC(50) values (16-21 microM). Compound 3 also showed potent inhibitory activity (IC(50) = 8.2 and 4.4 microM, respectively) for the secretion of lysosomal enzyme and beta-glucuronidase from neutrophils. Moreover, compounds 5 and 9 were shown to be efficacious inhibitors of TNF-alpha production in macrophage-like cell lines RAW 264.7. Compounds 2 and 12 were the potent inhibitors of TNF-alpha production in murine microglial cell lines N9. To further explore the cytotoxic properties of these acridine derivatives, (E)-12 was selected for NCI's in vitro disease-oriented tumor cells screen. The results indicated that this compound had no significant cytotoxicity with a mean GI(50) of 58.0 microM. These results indicated that the antiinflammatory effects of acridine derivatives were mediated, at least in part, through the suppression of chemical mediators released from mast cells, neutrophils, and macrophages and that these compounds have the potential to be novel antiinflammatory agents with no significant cytotoxicity.
Asunto(s)
Acridinas/síntesis química , Amsacrina/análogos & derivados , Antiinflamatorios/síntesis química , Antineoplásicos/síntesis química , Acridinas/química , Acridinas/farmacología , Amsacrina/química , Compuestos de Anilina/química , Animales , Antiinflamatorios/química , Antiinflamatorios/farmacología , Antineoplásicos/química , Antineoplásicos/farmacología , Degranulación de la Célula/efectos de los fármacos , Células Cultivadas , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Mastocitos/efectos de los fármacos , Mastocitos/ultraestructura , Ratones , Neutrófilos/efectos de los fármacos , Neutrófilos/ultraestructura , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
In activated macrophages, large amounts of nitric oxide (NO) are generated by inducible nitric oxide synthase (iNOS). This is an important mechanism in macrophage-induced cytotoxicity and inflammation. In the present study, a synthetic carbazole compound, 9-(2-chlorobenzyl)-9H-carbazole-3-carbaldehyde (LCY-2-CHO), was found to have an inhibitory effect on lipopolysaccharide (LPS)-stimulated NO generation in RAW 264.7 macrophages (IC50 value of 1.3+/-0.4 microM). LCY-2-CHO did not induce cytotoxicity and had a negligible effect on iNOS activity. To explore the mechanism of inhibition of NO generation by LCY-2-CHO, the expression of the iNOS gene was examined. LCY-2-CHO abolished the LPS-induced expression of both iNOS protein and mRNA in a parallel concentration-dependent manner with IC50 values similar to those required for inhibition of NO generation. LCY-2-CHO did not enhance the degradation of iNOS mRNA. In cells transiently transfected with an iNOS promoter-chloramphenicol acetyltransferase (CAT) reporter construct, LCY-2-CHO attenuated the LPS-induced iNOS promoter activity. However, LCY-2-CHO had no effect on the degradation of IkappaB-alpha or IkappaB-beta, DNA binding activity, or transcriptional activity of nuclear factor-kappaB (NF-kappaB). These results indicate that LCY-2-CHO inhibits NO generation via a decrease in the transcription of iNOS mRNA through a signaling pathway that does not involve NF-kappaB activation.
Asunto(s)
Proteínas I-kappa B , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Óxido Nítrico/metabolismo , Animales , ADN/efectos de los fármacos , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Interacciones Farmacológicas , Macrófagos/metabolismo , Ratones , Inhibidor NF-kappaB alfa , FN-kappa B/efectos de los fármacos , FN-kappa B/metabolismo , Óxido Nítrico Sintasa/efectos de los fármacos , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Regiones Promotoras Genéticas/efectos de los fármacos , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismoRESUMEN
We have demonstrated that magnolol suppressed thromboxane B2 (TXB2) and leukotriene B4 (LTB4) formation in A23187-stimulated rat neutrophils. Maximum inhibition was obtained with about 10 microM magnolol. Magnolol was more effective in the inhibition of cyclooxygenase (COX) activity than in the inhibition of 5-lipoxygenase (5-LO) activity as assessed by means of enzyme activity determination in vitro and COX and 5-LO metabolic capacity analyses in vivo. Magnolol alone stimulated cytosolic phospholipase A2 (cPLA2) phosphorylation and the translocation of 5-LO and cPLA2 to the membrane, and evoked arachidonic acid (AA) release. Recruitment of both 5-LO and cPLA2 to the membranes was suppressed by EGTA. Arachidonyl trifluoromethyl ketone (AACOCF3), a PLA2 inhibitor, bromoenol lactone (BEL), a Ca2+-independent PLA2 (iPLA2) inhibitor, and EGTA suppressed the magnolol-induced AA release. However, none of the follows affected magnolol-induced AA-release: 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580), a p38 mitogen-activated protein kinase (MAPK) inhibitor, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126), a MAPK kinase (MEK) inhibitor, or 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF109203X), a protein kinase C (PKC) inhibitor. In addition, magnolol at 30 microM did not stimulate the p38 MAPK and extracellular signal-regulated kinase 2 (ERK2) enzyme activities. These results indicated that magnolol inhibits the formation of prostaglandins and leukotrienes in A23187-stimulated rat neutrophils, probably through a direct blockade of COX and 5-LO activities. The stimulatory effects of magnolol at high concentration on the membrane association of 5-LO and cPLA2 are attributable to the elevation of [Ca2+]i, and on the AA release is likely via activation of cPLA2 and iPLA2.
Asunto(s)
Ácido Araquidónico/metabolismo , Compuestos de Bifenilo/farmacología , Lignanos , Neutrófilos/efectos de los fármacos , Fosfolipasas A/metabolismo , Animales , Araquidonato 5-Lipooxigenasa/metabolismo , Transporte Biológico/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Ácido Egtácico/metabolismo , Eicosanoides/metabolismo , Activación Enzimática , Técnicas In Vitro , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neutrófilos/enzimología , Neutrófilos/metabolismo , Fosfolipasas A2 , Prostaglandina-Endoperóxido Sintasas/metabolismo , RatasRESUMEN
2-Benzyloxybenzaldehyde (CCY1a) inhibited the formyl-Met-Leu-Phe (fMLP)-induced elevation of cytosolic [Ca(2+)]() ([Ca(2+)](i)) in rat neutrophils. The late plateau phase, but not the initial Ca(2+) spike, of the fMLP-induced [Ca(2+)](i) change was inhibited by CCY1a. In the absence of external Ca(2+), CCY1a had no appreciable effect on either the fMLP- or cyclopiazonic acid (CPA)-induced [Ca(2+)](i) elevation. CCY1a failed to inhibit [Ca(2+)](i) changes induced by N-ethylmaleimide, GEA3162, ionomycin or sphingosine, but slightly inhibited the Ca(2+) signals elicited by ATP or interleukin-8 (IL-8). In a classical Ca(2+) readdition protocol, addition of CCY1a after cell activation strongly inhibited the [Ca(2+)](i) response to fMLP, whilst that to CPA was only slightly reduced. CCY1a nearly abrogated the fMLP-stimulated Mn(2+) influx but was less effective on the CPA-induced response. CCY1a attenuated the levels of tyrosine-phosphorylated bands in the 70-85 kDa molecular mass range. CCY1a had no effect on the basal [Ca(2+)](i) level, the pharmacologically isolated plasma membrane Ca(2+)-ATPase activity or on the mitochondrial membrane potential. Thus, CCY1a blocks fMLP-induced Ca(2+) entry into neutrophils probably by blocking the relevant Ca(2+) channel directly or, alternatively, indirectly through the attenuation of tyrosine phosphorylation of some cellular proteins.
Asunto(s)
Benzaldehídos/farmacología , Calcio/metabolismo , Líquido Intracelular/efectos de los fármacos , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Animales , Calcio/antagonistas & inhibidores , Líquido Intracelular/metabolismo , N-Formilmetionina Leucil-Fenilalanina/antagonistas & inhibidores , Neutrófilos/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Excessive nitric oxide (NO) production by activated microglia plays a critical role in neurodegenerative disorders. In this study, we found that 9-(2-chlorobenyl)-9H-carbazole-3-carbaldehyde (LCY-2-CHO) suppressed the NO production in lipopolysaccharide (LPS)/interferon-gamma (IFNgamma)-stimulated murine microglial N9 and BV-2 cells and in LPS-stimulated N9 cells and rat primary microglia. LCY-2-CHO had no cytotoxic effect on microglia. In activated N9 cells, LCY-2-CHO abolished the expression of inducible nitric oxide synthase (iNOS) protein and mRNA but failed to alter the stability of expressed iNOS mRNA and the enzymatic activity of expressed iNOS protein. LCY-2-CHO did not block DNA-binding activity of nuclear factor-kappaB (NF-kappaB) or cyclic AMP response element-binding protein (CREB), but abolished that of activator protein-1 (AP-1), CCAAT/enhancer-binding protein (C/EBP) and nuclear factor IL6 (NF-IL6). LCY-2-CHO attenuated the nuclear levels of c-Jun and C/EBPbeta, but not those of p65, p50, C/EBPdelta, signal transducer and activator of transcription-1 (STAT-1) or the nuclear expression of IFN regulatory factor-1 (IRF-1). LCY-2-CHO had no effect on the phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), MAPK-activated protein kinase-2 (MAPKAPK-2), STAT-1, CREB or c-Jun in LPS/IFNgamma-stimulated N9 cells, whereas it attenuated the phosphorylation of C/EBPbeta at Ser105 and Thr235 residues, which occurred concomitantly with LCY-2-CHO inhibition of C/EBPbeta expression and phosphorylation. Taken together, these results suggest that LCY-2-CHO inhibits NO production in microglia through the blockade of AP-1 and C/EBP activation.
Asunto(s)
Antiinflamatorios/farmacología , Proteínas Potenciadoras de Unión a CCAAT/antagonistas & inhibidores , Carbazoles/farmacología , Microglía/metabolismo , Óxido Nítrico/antagonistas & inhibidores , Factor de Transcripción AP-1/antagonistas & inhibidores , Animales , Células Cultivadas , Interferón gamma/efectos adversos , Lipopolisacáridos/efectos adversos , Ratones , Microglía/efectos de los fármacos , Microglía/patología , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa de Tipo II/genética , Proteínas Nucleares/efectos de los fármacos , ARN Mensajero/efectos de los fármacos , RatasRESUMEN
Crotafuran B, a natural pterocarpanoid isolated from Crotalaria pallida, inhibited the lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production (IC50 16.4+/-0.7 microM) and inducible nitric oxide synthase (iNOS) protein and mRNA expression (IC50 11.5+/-0.6 microM and 11.8+/-2.2 microM, respectively), but not via its cytotoxicity or the inhibition of iNOS enzyme activity, in RAW 264.7 macrophages. Crotafuran B also reduced the iNOS promoter activity (IC50 13.4+/-0.1 microM) in piNOS-LUC-transfected cells. Crotafuran B treatment inhibited the p65 nuclear translocation and the nuclear factor-kappaB (NF-kappaB) DNA binding activity in LPS-activated macrophages. Crotafuran B also reduced the NF-kappaB transcriptional activity in pNF-kappaB-LUC-transfected cells. Crotafuran B had no effect on the LPS-induced phosphorylation of inhibitory kappaBalpha (IkappaBalpha), but enhanced the cellular level of IkappaBalpha that rebounded to the basal levels and increased the IkappaBalpha mRNA expression. These results indicate that the crotafuran B inhibition of NO production involves a decrease in the iNOS gene expression via the inhibition of NF-kappaB activation through the increase in IkappaBalpha synthesis.
Asunto(s)
Crotalaria/química , Furanos/farmacología , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Proteínas I-kappa B/biosíntesis , Lipopolisacáridos , Macrófagos/efectos de los fármacos , FN-kappa B/antagonistas & inhibidores , Óxido Nítrico/biosíntesis , Animales , Northern Blotting , Western Blotting , Línea Celular , Relación Dosis-Respuesta a Droga , Ensayo de Cambio de Movilidad Electroforética , Luciferasas/genética , Macrófagos/metabolismo , Ratones , Inhibidor NF-kappaB alfa , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Óxido Nítrico Sintasa de Tipo II/genética , TransfecciónRESUMEN
One new diterpene, 8(14),15-sandaracopimaradiene-2alpha,3beta,18-triol (1), two new phenylpropane derivatives, i.e., (E)-methyl 2-(3,4-methylene-dioxyphenyl)-3-methoxypropenoate (2) and (E)-2-(3,4-methylene-dioxyphenyl)-3-methoxypropenoic acid (3), and two known diterpenes, ent-8(14),15-sandaracopimaradiene-2alpha,18-diol (4) and 8(14),15-sandaracopimaradiene-2alpha,18,19-triol (5), were isolated from the heartwoods and barks of Amentotaxus formosana, respectively. The anti-inflammatory activity of the diterpenes 1, 4, and 5 was assessed in vitro by determining their inhibitory effects on the chemical mediators released from mast cells, neutrophils, macrophages, and microglial cells. Compounds 1, 4, and 5 showed significant concentration-dependent inhibitory effects on the release of beta-glucuronidase from rat neutrophils in response to formyl-Met-Leu-Phe/cytochalasin B (fMLP/CB) with IC50 values of 5.5 +/- 1.8, 8.4 +/- 2.9 and 19.2 +/- 3.3 microM, respectively. Compounds 1 and 5 also showed significant concentration-dependent inhibitory effects on superoxide anion generation in rat neutrophils stimulated with fMLP/CB and phorbol 12-myristate 13-acetate (PMA) with IC50 values of 12.6 +/- 1.2 and 9.4 +/- 1.7, and 10.7 +/- 3.3 and 12.9 +/- 0.9 microM, respectively.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Fitoterapia , Extractos Vegetales/farmacología , Taxaceae , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Antiinflamatorios no Esteroideos/uso terapéutico , Diterpenos/administración & dosificación , Diterpenos/farmacología , Diterpenos/uso terapéutico , Relación Dosis-Respuesta a Droga , Hidrocarburos Aromáticos/administración & dosificación , Hidrocarburos Aromáticos/farmacología , Hidrocarburos Aromáticos/uso terapéutico , Concentración 50 Inhibidora , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Ratones , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Corteza de la Planta , Extractos Vegetales/administración & dosificación , Extractos Vegetales/uso terapéutico , Ratas , Ratas Sprague-Dawley , MaderaRESUMEN
Exposure of macrophages to lipopolysaccharide (LPS) induces release of tumor necrosis factor-alpha (TNF-alpha), which is initially synthesized as a 26-kDa pro-TNF-alpha followed by proteolytic processing to a 17-kDa secreted form. In this study, justicidin A, an arylnaphthalide lignan isolated from Justicia procumbens, was found to inhibit LPS-stimulated TNF-alpha release from RAW 264.7 macrophages in a concentration- and time-dependent manner, and the underlying mechanism was investigated. In the presence of justicidin A, challenge with LPS increased the steady-state level of the 26-kDa membrane-bound form of TNF-alpha protein, whereas justicidin A had little effect on the expression of TNF-alpha mRNA and on the synthesis of pro-TNF-alpha protein. Results of the pulse-chase experiment, revealed that the conversion of pro-TNF-alpha to mature TNF-alpha was inhibited by justicidin A. Moreover, justicidin A suppressed the transport of TNF-alpha to cell surface as analyzed by flow cytometry. The immunofluorescence analysis demonstrated that large amounts of LPS-induced TNF-alpha accumulated primarily within Golgi complex. These results indicate that justicidin A inhibits TNF-alpha release at the step of transport of pro-TNF-alpha to cell surface, and this leads to the accumulation of TNF-alpha in Golgi complex in RAW 264.7 macrophages.
Asunto(s)
Dioxolanos/farmacología , Expresión Génica/efectos de los fármacos , Lignanos , Macrófagos/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Línea Celular , Interacciones Farmacológicas , Interleucina-6/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Ratones , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Three novel phloroglucinol derivatives, garcinielliptones F (1), H (3), and I (4), and two novel terpenoids, garcinielliptones G (2) and J (5), with a new skeleton have been isolated from the seeds of Garcinia subelliptica. Their structures, including relative configurations, were elucidated by spectroscopic methods and computer-generated molecular modeling. Compound 1 showed potent inhibitory effects on the release of beta-glucuronidase and lysozyme from rat neutrophils that had been stimulated with formyl-Met-Leu-Phe (fMLP)/cytochalasin B (CB). This effect was concentration-dependent with IC(50) values of 26.9+/-2.6 and 20.0+/-1.3 microM, respectively. Compound 1 also showed a potent concentration-dependent inhibitory effect on superoxide anion generation in rat neutrophils stimulated with fMLP/CB, with an IC(50) value of 17.0+/-0.9 microM. Compound 4 showed a potent inhibitory effect on NO production in culture media of N9 cells in response to lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma) in a concentration-dependent manner with an IC(50) value of 7.4+/-0.2 microM.
Asunto(s)
Antiinflamatorios no Esteroideos/química , Garcinia/química , Terpenos/química , Animales , Antiinflamatorios no Esteroideos/aislamiento & purificación , Antiinflamatorios no Esteroideos/farmacología , Modelos Químicos , Estructura Molecular , Neutrófilos/efectos de los fármacos , Ratas , Terpenos/aislamiento & purificación , Terpenos/farmacologíaRESUMEN
Four novel phloroglucinol derivatives, garcinielliptones A (1), B (2), C (3), D (4), a novel triterpenoid, garcinielliptone E (5), and three known compounds were isolated from the seeds of Garcinia subelliptica. The structures, including relative configurations, were elucidated by means of spectroscopic data. Known compounds garsubellin A (6) and garcinielliptin oxide (7) showed potent inhibitory effects on the release of beta-glucuronidase, and beta-glucuronidase and histamine, respectively, from peritoneal mast cells stimulated with compound 48/80 in a concentration-dependent manner with IC(50) values of 15.6+/-2.5, and 18.2+/-3.6 and 20.0+/-2.7 microM, respectively. Compound 7 showed potent inhibitory effects on the release of beta-glucuronidase and lysozyme from neutrophils stimulated with formyl-Met-Leu-Phe(fMLP)/cytochalasin B (CB) in a concentration-dependent manner with IC(50) values of 15.7+/-3.0 and 23.9+/-3.2 microM, respectively. Compound 7 also showed potent inhibitory effect on superoxide formation from neutrophils stimulated with fMLP/CB also in a concentration-dependent manner with an IC(50) value of 17.9+/-1.5 microM.