RESUMO
Fisetin (3,7,3',4'-tetrahydroxyflavone), a natural flavonoid, is a therapeutic agent for respiratory inflammatory diseases such as chronic obstructive pulmonary disease (COPD). However, detailed molecular mechanisms regarding the target protein of fisetin remain unknown. Fisetin significantly reduces tumour necrosis factor alpha (TNF-α)-induced interleukin (IL)-8 levels by inhibiting both nuclear factor kappa B (NF-κB) transcriptional activity and the phosphorylation of its upstream effectors. We show that fisetin prevents interactions between protein kinase C (PKC)δ and TNF receptor-associated factor 2 (TRAF2), thereby inhibiting the inhibitor of kappa B kinase (IKK)/NF-κB downstream signalling cascade. Furthermore, we found that fisetin directly binds to PKCδ in vitro. Our findings provide evidence that fisetin inhibits the TNF-α-activated IKK/NF-κB cascade by targeting PKCδ, thereby mediating inflammatory diseases such as COPD. These data suggest that fisetin is a good therapeutic drug for the treatment of inflammatory lung diseases, such as COPD, by inhibiting the TNF-α/NF-κB signalling pathway.
Assuntos
Células Epiteliais/efeitos dos fármacos , Flavonoides/farmacologia , Interleucina-8/metabolismo , NF-kappa B/antagonistas & inibidores , Proteína Quinase C-delta/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Células Epiteliais/enzimologia , Flavonóis , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , NF-kappa B/metabolismo , Fosforilação , Ligação Proteica , Fator 2 Associado a Receptor de TNF/metabolismoRESUMO
Pseudolysimachion rotundum var. subintegrum is utilized as a traditional herbal remedy to treat cough, bronchitis, and asthma in Korea, Russia, China, and Europe. Here, we show that 3-methoxy-catalposide, a novel iridoide glycoside isolated from P. rotundum var. subintegrum has the anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated macrophages. The chemical structure of 3-methoxy-catalposide was determined by NMR, optical rotation and HRESIMS. In in vitro experiment, RAW264.7 cells were treated with 3-methoxy-catalposide for 2h before exposure to LPS for different times. Inflammatory gene and protein expressions were assayed using RT-PCR and ELISA. Activities of signal proteins were examined using western analysis. Our results demonstrated that 3-methoxy-catalposide significantly inhibits the expression of cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) in RAW264.7 cells stimulated by LPS, thereby suppressing the release of prostaglandin E2 (PGE2) and nitric oxide (NO). Moreover, 3-methoxy-catalposide markedly reduced the LPS-induced expression of pro-inflammatory genes, such as interleukin (IL)-6, IL-1ß, and TNF-α. Further, 3-methoxy-catalposide inhibited both LPS-induced activation of three MAP kinases (ERK 1/2, JNK, and p38) and the nuclear translocation of NF-κB and AP-1. These results support that 3-methoxy-catalposide may be a promising candidate for inflammation treatment.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/imunologia , Glucosídeos Iridoides/farmacologia , Lipopolissacarídeos/toxicidade , Macrófagos/imunologia , Monocinas/imunologia , Animais , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/imunologia , Inflamação/patologia , Macrófagos/patologia , Camundongos , NF-kappa B/imunologia , Células RAW 264.7 , Fator de Transcrição AP-1/imunologiaRESUMO
As part of an ongoing search for new natural products from medicinal plants to treat respiratory disease, six new compounds, a dihydroflavonol (1) and five C-geranylated flavanones (3, 6, 8, 13, and 14), and 13 known compounds were isolated from mature fruits of Paulownia tomentosa. The structures of the new compounds were determined via interpretation of their spectroscopic data (1D and 2D NMR, UV, IR, ECD, and MS). In biological activity assays with human alveolar basal epithelial cells, the expression of TNF-α-induced proinflammatory cytokines (IL-8 and IL-6) was reduced significantly by the EtOAc fraction of a P. tomentosa extract as well as by the new compounds isolated from this fraction. Furthermore, the majority of the isolates (1-19 except 5-7) were found to inhibit human neutrophil elastase (HNE) activity, with IC50 values ranging from 2.4 ± 1.0 to 74.7 ± 8.5 µM. In kinetic enzymatic assays with the HNE substrate MeOSuc-AAPV-pNA, compound 17 exhibited the highest inhibitory activity (Ki = 3.2 µM) via noncompetitive inhibition. These findings suggest that the flavanone constituents of P. tomentosa fruits may be valuable for the development of new drug candidates to treat airway inflammation.
Assuntos
Anti-Inflamatórios não Esteroides/isolamento & purificação , Anti-Inflamatórios não Esteroides/farmacologia , Flavanonas/isolamento & purificação , Flavanonas/farmacologia , Frutas/química , Magnoliopsida/química , Proteínas Secretadas Inibidoras de Proteinases/isolamento & purificação , Proteínas Secretadas Inibidoras de Proteinases/farmacologia , Anti-Inflamatórios não Esteroides/química , Flavanonas/química , Humanos , Interleucina-6/análise , Interleucina-8/análise , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Proteínas Secretadas Inibidoras de Proteinases/química , República da Coreia , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Precise control of transgene expression is desirable in biological and clinical studies. However, because the binary feature of currently employed gene switches requires the transfer of two therapeutic expression units concurrently into a single cell, the practical application of the system for gene therapy is limited. To simplify the transgene expression system, we generated a gene switch designated as pEUI(+) encompassing a complete set of transgene expression modules in a single vector. Comprising of the GAL4 DNA-binding domain and modified EcR (GvEcR), a minimal VP16 activation domain fused with a GAL4 DNA-binding domain, as well as a modified Drosophila ecdysone receptor (EcR), the newly developed singular gene switch is highly responsive to the administration of a chemical inducer in a time- and dosage-dependent manner. The pEUI(+) vector is a potentially powerful tool for improving the control of transgene expression in both biological research and pre-clinical studies. Here, we present a detailed protocol for modulation of a transient and stable transgene expression using pEUI(+) vector by the treatment of tebufenozide (Teb). Additionally, we share important guidelines for the use of Teb as a chemical inducer.
Assuntos
Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Transgenes , Animais , Células HEK293 , Humanos , TransfecçãoRESUMO
BACKGROUND: Piscroside C, isolated from Pseudolysimachion rotundum var. subintegrum, is a novel iridoid glycoside with therapeutic efficacy in a mouse model of chronic obstructive pulmonary disease (COPD). Piscroside C has been reported as a constituent of YPL-001 (under Phase 2a study, ClinicalTrials.gov identifier NCT02272634). PURPOSE: To investigate the mechanisms behind piscroside C therapeutic effects on COPD in human airway epithelial NCI-H292 cells. METHODS: We tested if piscroside C effectively suppresses MUC5AC gene expression and TNF-RSC/IKK/NF-κB cascades in TNF-α-stimulated NCI-H292 cells by employing, reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, luciferase reporter assays, chromatin immunoprecipitation assays and immunoprecipitation. RESULTS: Piscroside C markedly suppressed the expression of TNF-α-induced MUC5AC mucus protein by inhibiting the transcriptional activity of NF-κB in NCI-H292 cells. Indeed, piscroside C negatively regulated the function of TNF receptor 1 signaling complex (TNF-RSC, an upstream regulator of the NF-κB pathway) without affecting its extracellular interaction with the TNF-α ligand. This inhibitory effect by piscroside C is mediated by the inactivation of protein kinase C (PKC), an essential regulator of TNF-RSC. PKC inactivation by piscroside C results in decreased PKCδ binding to a TRAF2 subunit of TNF-RSC and subsequent reduced IKK phosphorylation, resulting in NF-κB inactivation. CONCLUSION: We propose that piscroside C is a promising therapeutic constituent of YPL-001 through its inhibition of PKCδ activity in the TNF-RSC/IKK/NF-κB/MUC5AC signaling cascade.
Assuntos
Glicosídeos Iridoides/farmacologia , NF-kappa B/metabolismo , Proteína Quinase C-delta/antagonistas & inibidores , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Fator de Necrose Tumoral alfa/metabolismo , Brônquios/citologia , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Humanos , Mucina-5AC/genética , Mucina-5AC/metabolismo , Complexos Multiproteicos , Fosforilação/efeitos dos fármacos , Proteína Quinase C-delta/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator 2 Associado a Receptor de TNF/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The extract tea of Dendropanax morbifera is popular beverages in Korea, and their preventive and therapeutic roles in metabolic disorders have been reported. However, the molecular mechanism has not been studied despite the known efficacy of D. morbifera. Eleven fractions (fr.1-fr.11) were divided by MPLC to find the active compound. Among them, Fr.5 was superior to others in that the inhibitory efficacy of de novo triglyceride (TG) biosynthesis. NMR analysis revealed that Fr.5 is composed 98% or more (9Z,16S)-16-hydroxy-9,17-octadecadiene-12,14-diynoic acid (HOD). Treatment of HOD diminished oleic acid (OA)-induced TG accumulation in HepG2 hepatocytes and differentiation of 3T3-L1 preadipocytes by activating LKB1/AMPK. In addition, we determined the effect of the oral administration of the extract of D. morbifera on obesity and hepatic steatosis in high-fat diet (HFD)-induced obese mice. This study proved that D. morbifera containing HOD, the active substance, can show preventive or therapeutic efficacy on obesity and hepatic steatosis through the targeting LKB1/AMPK axis.
RESUMO
The flower buds of Magnolia fargesii, known traditionally as Xinyi, exert anti-inflammatory effects against inflammatory lung diseases such as COPD. Lignans isolated from Xinyi are an important group of plant-derived anti-inflammatory compounds. However, the mechanisms of action underlying their protective effects against COPD are not yet fully understood. Here, we showed that seven lignans (lignans 1-7) obtained from a CHCl3 fraction of Xinyi effectively suppress the inflammatory response in CSC-stimulated airway epithelial cells (in vitro) and in a mouse model of COPD established by exposure to CS and LPS. The CHCl3 fraction was found to inhibit CSC-induced IL-6 expression in human airway epithelial cells and to suppress the infiltration of inflammatory cells (neutrophils and macrophages) and secretion of inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the mouse model. Similarly, each of the seven lignans isolated from the CHCl3 fraction also suppressed the infiltration of inflammatory cells (neutrophils and macrophages) and secretion of inflammatory mediators such as reactive oxygen species (ROS), TNF-α, and IL-6 in vivo. Notably, all lignan compounds significantly suppressed both extracellular signal-related kinase (ERK) and Akt phosphorylation levels in CSC-stimulated human lung mucoepidermoid carcinoma (NCI-H292) cells. Of these, lignan 1 (dimethylpinoresinol) inhibited the expression of CSC-induced inflammatory cytokines (IL-1ß, -6, and -8) in vitro in a dose-dependent manner by suppressing the activation of epidermal growth factor receptor (EGFR) and its downstream effectors, including ERK and Akt, in NCI-H292 cells. Our results show that the lignans isolated from Xinyi may prevent airway inflammatory diseases through the suppression of EGFR and its downstream effectors.
RESUMO
Controlled gene expression is an indispensable technique in biomedical research. Here, we report a convenient, straightforward, and reliable way to induce expression of a gene of interest with negligible background expression compared to the most widely used tetracycline (Tet)-regulated system. Exploiting a Drosophila ecdysone receptor (EcR)-based gene regulatory system, we generated nonviral and adenoviral singular vectors designated as pEUI(+) and pENTR-EUI, respectively, which contain all the required elements to guarantee regulated transgene expression (GAL4-miniVP16-EcR, termed GvEcR hereafter, and 10 tandem repeats of an upstream activation sequence promoter followed by a multiple cloning site). Through the transient and stable transfection of mammalian cell lines with reporter genes, we validated that tebufenozide, an ecdysone agonist, reversibly induced gene expression, in a dose- and time-dependent manner, with negligible background expression. In addition, we created an adenovirus derived from the pENTR-EUI vector that readily infected not only cultured cells but also rodent tissues and was sensitive to tebufenozide treatment for regulated transgene expression. These results suggest that EcR-based singular gene regulatory switches would be convenient tools for the induction of gene expression in cells and tissues in a tightly controlled fashion.