RESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Seed of Torreya nucifera (L.) Siebold & Zucc is used to treat several diseases in Asia. Reports document that T. nucifera has anti-cancer, anti-inflammatory, anti-oxidative activities. In spite of numerous findings on its pharmacological effects, the understanding of the molecular inhibitory mechanisms of the plant remains to be studied. Therefore, we aimed to explore in vitro anti-inflammatory mechanisms of ethyl acetate fraction (Tn-EE-BF) prepared from the seed of T. nucifera in LPS-stimulated macrophage inflammatory responses. MATERIALS AND METHODS: For this purpose, we measured nitric oxide (NO) and prostaglandin E2 (PGE2) in LPS-stimulated macrophages. Additionally, using RT-PCR, luciferase reporter gene assay, immunoblotting analysis, and kinase assay, the levels of inflammatory genes, transcription factors, and inflammatory signal-regulatory proteins were investigated. Finally, the constituent of Tn-EE-BF was identified using HPLC. RESULTS: Tn-EE-BF inhibits NO and PGE2 production and also blocks mRNA levels of inducible NO synthase (iNOS), tumor necrosis factor (TNF)-α, and cyclooxygenase (COX)-2 in a dose dependent manner. Tn-EE-BF reduces nuclear levels of the transcriptional factors NF-κB (p65) and AP-1 (c-Jun and FRA-1). Surprisingly, we found that Tn-EE-BF inhibits phosphorylation levels of Src and Syk in the NF-κB pathway, as well as, IRAK1 at the protein level, part of the AP-1 pathway. By kinase assay, we confirmed that Src, Syk, and IRAK1 are suppressed directly. HPLC analysis indicates that arctigenin, amentoflavone, and quercetin may be active components with anti-inflammatory activities. CONCLUSION: Tn-EE-BF exhibits anti-inflammatory activities by direct inhibition of Src/Syk/NF-κB and IRAK1/AP-1.
Asunto(s)
Antiinflamatorios/farmacología , Butanoles/química , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Lipopolisacáridos/farmacología , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Extractos Vegetales/farmacología , Solventes/química , Quinasa Syk/metabolismo , Taxaceae/química , Familia-src Quinasas/metabolismo , Animales , Antiinflamatorios/aislamiento & purificación , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Mediadores de Inflamación/metabolismo , Macrófagos/enzimología , Ratones , Óxido Nítrico , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fosforilación , Fitoterapia , Extractos Vegetales/aislamiento & purificación , Plantas Medicinales , Células RAW 264.7 , Transducción de Señal/efectos de los fármacos , Factor de Transcripción AP-1/metabolismo , Factor de Transcripción ReIA/metabolismo , Transfección , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Even though a lot of reports have suggested the anti-inflammatory activity of kaempferol (KF) in macrophages, little is known about its exact anti-inflammatory mode of action and its immunopharmacological target molecules. In this study, we explored anti-inflammatory activity of KF in LPS-treated macrophages. In particular, molecular targets for KF action were identified by using biochemical and molecular biological analyses. KF suppressed the release of nitric oxide (NO) and prostaglandin E2 (PGE2), downregulated the cellular adhesion of U937 cells to fibronectin (FN), neutralized the generation of radicals, and diminished mRNA expression levels of inflammatory genes encoding inducible NO synthase (iNOS), TNF-α, and cyclooxygenase- (COX-) 2 in lipopolysaccharide- (LPS-) and sodium nitroprusside- (SNP-) treated RAW264.7 cells and peritoneal macrophages. KF reduced NF-κB (p65 and p50) and AP-1 (c-Jun and c-Fos) levels in the nucleus and their transcriptional activity. Interestingly, it was found that Src, Syk, IRAK1, and IRAK4 responsible for NF-κB and AP-1 activation were identified as the direct molecular targets of KF by kinase enzyme assays and by measuring their phosphorylation patterns. KF was revealed to have in vitro and in vivo anti-inflammatory activity by the direct suppression of Src, Syk, IRAK1, and IRAK4, involved in the activation of NF-κB and AP-1.
Asunto(s)
Antiinflamatorios/química , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Quempferoles/química , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Animales , Núcleo Celular/metabolismo , Fibronectinas/metabolismo , Humanos , Inflamación/metabolismo , Lipopolisacáridos/química , Masculino , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fosforilación , Quinasa Syk , Factor de Transcripción AP-1/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Células U937RESUMEN
Kaempferol (KF) is the most abundant polyphenol in tea, fruits, vegetables, and beans. However, little is known about its in vivo anti-inflammatory efficacy and mechanisms of action. To study these, several acute mouse inflammatory and nociceptive models, including gastritis, pancreatitis, and abdominal pain were employed. Kaempferol was shown to attenuate the expansion of inflammatory lesions seen in ethanol (EtOH)/HCl- and aspirin-induced gastritis, LPS/caerulein (CA) triggered pancreatitis, and acetic acid-induced writhing.
Asunto(s)
Dolor Abdominal/tratamiento farmacológico , Gastritis/tratamiento farmacológico , Quempferoles/farmacología , Nocicepción/efectos de los fármacos , Pancreatitis/tratamiento farmacológico , Ácido Acético/toxicidad , Animales , Antiinflamatorios no Esteroideos/farmacología , Aspirina/efectos adversos , Ceruletida/efectos adversos , Modelos Animales de Enfermedad , Gastritis/inducido químicamente , Gastritis/patología , Ratones , Pancreatitis/inducido químicamenteRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Codariocalyx motorius (Houtt.) H. Ohashi (Fabaceae) is one of several ethnopharmacologically valuable South Asian species prescribed as an herbal medicine for various inflammatory diseases. Due to the lack of systematic studies on this plant, we aimed to explore the inhibitory activity of Codariocalyx motorius toward inflammatory responses using its ethanolic extract (Cm-EE). MATERIALS AND METHODS: Lipopolysaccharide (LPS)-treated macrophages and a HCl/EtOH-induced gastritis model were used for evaluation of the anti-inflammatory activity of Cm-EE. HPLC and spectroscopic analysis were employed to identify potential active components. Mechanistic approaches to determine target enzymes included kinase assays, reporter gene assays, and overexpression of target enzymes. RESULTS: Cm-EE strongly suppressed nitric oxide (NO) and prostaglandin E2 (PGE2) release. Cm-EE-mediated inhibition was observed at the transcriptional level in the form of suppression of NF-κB (p65) translocation and activation. This extract also lowered the levels of phosphorylation of Src and Syk, their kinase activity, and their formation of signalling complexes by binding to the downstream enzyme p85/PI3K. In accord with these findings, the phosphorylation of p85 induced by overexpression of Src or Syk was also diminished by Cm-EE. Orally administered Cm-EE clearly inhibited gastritic ulcer formation and the phosphorylation of IκBα and Src in HCl/EtOH-treated stomachs of mice. By phytochemical analysis, luteolin and its glycoside, apigenin-7-O-glucuronide, and scutellarein-6-O-glucuronide were identified as major components of Cm-EE. Among these, it was found that luteolin was able to strongly suppress NO and PGE2 production under the same conditions. CONCLUSION: Syk/Src-targeted inhibition of NF-κB by Cm-EE could be a major anti-inflammatory mechanism contributing to its ethno pharmacological role as an anti-inflammatory herbal medicine.
Asunto(s)
Antiinflamatorios/farmacología , Fabaceae/química , Inflamación/tratamiento farmacológico , Extractos Vegetales/farmacología , Animales , Antiinflamatorios/aislamiento & purificación , Dinoprostona/metabolismo , Modelos Animales de Enfermedad , Etanol/química , Gastritis/tratamiento farmacológico , Gastritis/patología , Humanos , Inflamación/patología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , FN-kappa B/metabolismo , Óxido Nítrico/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Quinasa Syk , Familia-src Quinasas/metabolismoRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Cerbera manghas L. (Apocynaceae), a semi-mangrove medicinal plant distributed throughout tropical and subtropical countries, is traditionally known to possess analgesic, anti-inflammatory, anti-convulsant, cardiotonic, and hypotensive activity. In vitro and in vivo anti-inflammatory activities of a methanol extract of the leaves of Cerbera manghas and the underlying molecular mechanisms were investigated to validate the ethnopharmacological use of this plant. MATERIALS AND METHODS: The effect of Cerbera manghas methanol extract (Cm-ME) on the production of inflammatory mediators and the induction of HCl/EtOH-treated gastritis was explored using macrophages, HEK293 cells, and ICR mice. The molecular targets of this extract and potential active components in Cm-ME were also investigated. RESULTS: Cm-ME inhibited the production of nitric oxide (NO) in lipopolysaccharide (LPS)-treated RAW264.7 cells and peritoneal macrophages in a dose-dependent manner. This extract also suppressed the expression of NO synthase (iNOS) and cyclooxygenase (COX)-2. NF-κB-mediated enhancement of luciferase activity, nuclear translocation of p50 and p65, and phosphorylation of IκBα were markedly reduced by Cm-ME treatment. Direct enzyme assays, reporter gene assays, and immunoprecipitation analysis of kinases revealed Syk and Src as immunopharmacological targets of Cm-ME. Moreover, this extract strongly ameliorated the gastric symptoms induced by HCl/EtOH treatment of mice. Finally, HPLC analysis and pharmacological tests identified kaempferol as an active component of the extract with Src/Syk inhibitory activities. CONCLUSION: Inhibition of Syk/Src and the NF-κB pathway by kaempferol could play a key role in the anti-inflammatory pharmacological action of Cerbera manghas.
Asunto(s)
Antiinflamatorios/farmacología , Apocynaceae , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Extractos Vegetales/farmacología , Proteínas Tirosina Quinasas/metabolismo , Familia-src Quinasas/metabolismo , Animales , Antiinflamatorios/uso terapéutico , Apocynaceae/química , Línea Celular , Células Cultivadas , Ciclooxigenasa 2/genética , Etanol , Gastritis/inducido químicamente , Gastritis/tratamiento farmacológico , Células HEK293 , Humanos , Ácido Clorhídrico , Quempferoles/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Metanol/química , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , FN-kappa B/metabolismo , Óxido Nítrico/farmacología , Óxido Nítrico Sintasa de Tipo II/genética , Extractos Vegetales/uso terapéutico , ARN Mensajero/metabolismo , Solventes/química , Quinasa SykRESUMEN
Chemical derivatization of resveratrol has been widely conducted in an effort to overcome its chemical instability and therapeutic potential. In the present study, we examined the anti-inflammatory effects of resveratrol derivatives containing an amide functionality using in vitro macrophage models that were stimulated by Toll-like receptor (TLR) ligands, and using several animal inflammatory disease models. Of the resveratrol derivatives tested, compound 8 (2,4-dihydroxy-N-(4-hydroxyphenyl)benzamide) most strongly inhibited the production of nitric oxide (NO), tumor necrosis factor (TNF)-α, and prostaglandin E2 (PGE2), as well as the mRNA expression of inducible NO synthase (iNOS), TNF-α, and cyclooxygenase (COX)-2 in lipopolysaccharide (LPS)-activated RAW264.7 cells, differentiated U937 cells, and peritoneal macrophages. The inhibitory activity of compound 8 was apparently mediated by suppressing the phosphorylation of signal transducer and activator of transcription (STAT)-1, STAT-3, STAT-5, and interferon regulatory factor (IRF)-3. The direct target of compound 8 was revealed to be Janus kinase 2 (JAK2) but not TANK-binding kinase (TBK) 1 using the direct kinase assay and analyses of complex formation with these molecules. Additionally, upstream kinase of TBK1 seems to be also inhibited by compound 8. This compound also strongly ameliorated mouse inflammatory symptoms seen in arachidonic acid-induced ear edema, dextran sodium sulfate (DSS)-treated colitis, EtOH/HCl-induced gastritis, collagen type II-triggered arthritis, and acetic acid-induced writhing. Therefore, of the resveratrol derivatives that we tested, compound 8 was determined to have the strongest anti-inflammatory activities in vitro and in vivo and may potentially be developed for use as a novel anti-inflammatory drug.
Asunto(s)
Antiinflamatorios/farmacología , Benzamidas/farmacología , Janus Quinasa 2/efectos de los fármacos , Estilbenos/administración & dosificación , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , Humanos , Mediadores de Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa , Resveratrol , Transducción de Señal , Factores de Transcripción/metabolismoRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Evodia lepta (Spreng.) Merr., in the Rutaceae family, is a medicinal plant traditionally used to treat inflammatory symptoms such as in meningitis and hepatitis. However, no study has systematically investigated its anti-inflammatory activities including its molecular mechanism. MATERIALS AND METHODS: The effects of a methanol extract from the roots Evodia lepta (El-ME) were evaluated using lipopolysaccharide (LPS)-treated RAW264.7 cells producing nitric oxide (NO) and prostaglandin E2 (PGE2), and an HCl/ethanol-induced mouse gastritis model. Target molecules were identified by analyzing the activation of transcription factors and their upstream kinases. RESULTS: El-ME reduced the production of NO and PGE2 from LPS-activated RAW264.7 cells in a dose-dependent manner. El-ME also ameliorated the gastritis symptoms of EtOH/HCl-treated mice. The extract suppressed production of mRNA for the inducible NO synthase (iNOS) and cyclooxygenase (COX)-2; the nuclear translocation of nuclear factor (NF)-κB; the phosphorylation of upstream kinases that activate NF-κB; and the kinase activities of Syk and Src. CONCLUSION: The anti-inflammatory effects of El-ME might be due to its suppression of Syk/Src and NF-κB. Considering the in vitro and in vivo efficacy of El-ME, Evodia lepta could be developed into an anti-inflammatory herbal remedy.
Asunto(s)
Antiinflamatorios/farmacología , Evodia , Extractos Vegetales/farmacología , Animales , Antiinflamatorios/uso terapéutico , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Dinoprostona/metabolismo , Etanol , Gastritis/inducido químicamente , Gastritis/tratamiento farmacológico , Gastritis/patología , Células HEK293 , Humanos , Ácido Clorhídrico , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Masculino , Metanol/química , Ratones , Ratones Endogámicos ICR , FN-kappa B/metabolismo , Óxido Nítrico/metabolismo , Extractos Vegetales/uso terapéutico , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Solventes/química , Estómago/patología , Quinasa SykRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Dipterocarpus tuberculatus Roxb. (Dipterocarpaceae) has been traditionally used to treat various inflammatory symptoms. However, no mechanistic studies on the anti-inflammatory actions of D. tuberculatus have been reported. This study is therefore aimed at exploring the anti-inflammatory effects of 95% ethanol extracts (Dt-EE) of this plant. MATERIALS AND METHODS: The regulatory activity of Dt-EE and its molecular mechanism on the release of nitric oxide (NO) and prostaglandin (PG)E2 in lipopolysaccharide (LPS)-treated macrophage-like RAW264.7 cells were elucidated by evaluating the activation of transcription factors and their upstream signals and by analyzing the kinase activities of target enzymes. Furthermore, to confirm its availability for oral use, an EtOH/HCl-induced acute gastritis model was tested with this extract. RESULTS: Dt-EE effectively suppressed LPS-mediated inflammatory responses such as the production of NO and PGE2 from macrophages in a dose-dependent manner. In particular, Dt-EE clearly blocked the activation of NF-κB by blocking the phosphorylation of its upstream enzymes IKK and Akt. Using a direct enzyme assay, Dt-EE was shown to block the enzyme activity of PDK1. Finally, this extract also remarkably ameliorated inflammatory lesions in the stomach induced by EtOH/HCl. CONCLUSION: Our data strongly suggest that Dt-EE can be considered as a novel anti-inflammatory remedy with PDK1/NF-κB inhibitory properties and can also be used to treat gastritis symptoms. In addition, our findings can serve as a basis for further phytochemical and pharmacological studies in the future.