RESUMEN
BACKGROUND: The aim of this study was to evaluate the inhibitory effect of tamarixetin on the production of inflammatory mediators in IgE/antigen-induced mouse bone marrow-derived mast cells (BMMCs). MATERIALS AND METHODS: The effects of tamarixetin on mast cell activation were investigated with regard to degranulation, eicosanoid generation, Ca2+ influx, and immunoblotting of various signaling molecules. RESULTS: Tamarixetin effectively decreased degranulation and the eicosanoid generation such as leukotriene C4 and prostaglandin D2 in BMMCs. To elucidate the mechanism involved, we investigated the effect of tamarixetin on the phosphorylation of signal molecules. Tamarixetin inhibited the phosphorylation of Akt and its downstream signal molecules including IKK and nuclear factor κB. In addition, tamarixetin downregulated the phosphorylation of cytosolic phospholipase A2 (cPLA2) and p38 mitogen-activated protein kinase. CONCLUSIONS: Taken together, this study suggests that tamarixetin inhibits degranulation and eicosanoid generation through the PLCγ1 as well as Akt pathways in BMMCs, which would be potential for the prevention of allergic inflammatory diseases.
Asunto(s)
Degranulación de la Célula/efectos de los fármacos , Disacáridos/farmacología , Eicosanoides/biosíntesis , Mediadores de Inflamación/metabolismo , Inula/química , Mastocitos/efectos de los fármacos , Quercetina/análogos & derivados , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Calcio/metabolismo , Leucotrieno C4/biosíntesis , Mastocitos/metabolismo , Mastocitos/fisiología , Ratones , Ratones Endogámicos BALB C , FN-kappa B/metabolismo , Fosfolipasa C gamma/metabolismo , Fosfolipasas A2/metabolismo , Fosforilación/efectos de los fármacos , Prostaglandina D2/biosíntesis , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Quercetina/farmacología , beta-N-Acetilhexosaminidasas/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Immunoglobulin G (IgG) is currently the most studied immunoglobin class and is frequently used in antibody therapeutics in which its beneficial effector functions are exploited. IgG is composed of two heavy chains and two light chains, forming the basic antibody monomeric unit. In contrast, immunoglobulin A (IgA) and immunoglobulin M (IgM) are usually assembled into dimers or pentamers with the contribution of joining (J)-chains, which bind to the secretory component (SC) of the polymeric Ig receptor (pIgR) and are transported to the mucosal surface. IgA and IgM play a pivotal role in various immune responses, especially in mucosal immunity. Due to their structural complexity, 3D structural study of these molecules at atomic scale has been slow. With the emergence of cryo-EM and X-ray crystallographic techniques and the growing interest in the structure-function relationships of IgA and IgM, atomic-scale structural information on IgA-Fc and IgM-Fc has been accumulating. Here, we examine the 3D structures of IgA and IgM, including the J-chain and SC. Disulfide bridging and N-glycosylation on these molecules are also summarized. With the increasing information of structure-function relationships, IgA- and IgM-based monoclonal antibodies will be an effective option in the therapeutic field.
Asunto(s)
Inmunoglobulina A/química , Fragmentos Fc de Inmunoglobulinas/química , Cadenas J de Inmunoglobulina/química , Inmunoglobulina M/química , Receptores de Inmunoglobulina Polimérica/química , Animales , Glicosilación , HumanosRESUMEN
Alisol B 23-acetate (AB23A), a natural triterpenoid, has been reported to exert hepatoprotective and antitumor activities. Aiming to investigate the anti-inflammatory activity, this study examined the effect of AB23A on mast cells and allergic reaction. AB23A inhibited the degranulation of mast cells stimulated by immunoglobulin E/antigen (IgE/Ag), and also decreased the synthesis of leukotriene C4 (LTC4), production of interlukin-6 (IL-6), and expression of cyclooxygenase-2 (COX-2) in a concentration-dependent manner with no significant cytotoxicity in bone marrow-derived mast cells (BMMCs). AB23A inhibited spleen tyrosine kinase (Syk) and the downstream signaling molecules including phospholipase Cγ (PLCγ), serine-threonine protein kinase/inhibitor of nuclear factor kappa-B kinase/nuclear factor kappa-B (Akt/IKK/NF-κB), and mitogen-activated protein kinases/cytosolic phospholipase A2 (MAPK/cPLA2). Furthermore, AB23A blocked mobilization of Ca2+. Similar results were obtained in other mast cell lines Rat basophilic leukemia (RBL)-2H3 cells and a human mast cell line (HMC-1). In addition, AB23A attenuated allergic responses in an acute allergy animal model, passive cutaneous anaphylaxis (PCA). Taken together, this study suggests that AB23A inhibits the activation of mast cells and ameliorates allergic reaction, and may become a lead compound for the treatment of mast cell-mediated allergic diseases.
Asunto(s)
Antialérgicos/farmacología , Colestenonas/farmacología , Hipersensibilidad/tratamiento farmacológico , Mastocitos/efectos de los fármacos , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/inmunología , Degranulación de la Célula/efectos de los fármacos , Humanos , Hipersensibilidad/inmunología , Hipersensibilidad/patología , Inmunoglobulina E/inmunología , Leucotrieno C4/biosíntesis , Mastocitos/inmunología , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Fosfolipasa C gamma/genética , Proteínas Serina-Treonina Quinasas/genética , Ratas , Bazo/efectos de los fármacos , Bazo/enzimología , Quinasa SykRESUMEN
BACKGROUND: Immunoglobulin A (IgA) plays a pivotal role in various immune responses, especially that of mucosal immunity. IgA is usually assembled into dimers with the contribution of J-chains. There are two N-glycosylation sites in human IgA1-Fc and one in the J-chain. There is no consensus as yet on the functional role of the N-glycosylation. METHODS: To gain a better understanding of their role, we designed a series of IgA1-Fc mutants, which were expressed in the absence or presence of the J-chain. RESULTS: IgA1-Fc without the J-chain, was predominantly expressed as a monomer, and in its presence dimers and some polymers appeared. N263 (Fc Cα2), N459 (Fc tailpiece) and N49 (J-chain) were shown to be site-specifically modified with N-glycans by mass spectrometry analysis. Mutant IgA1-Fc N459Q failed to form a proper dimer in the presence of the J-chain, instead higher-order aggregates appeared. Fluorescence experiments suggest that the N459-glycans cover a hydrophobic surface at the Fc tailpiece that prevents other Fc molecules from approaching the dimeric IgA. A thermofluor assay revealed that the N-glycans at N263 (Fc) and N49 (J-chain) both contribute in different ways to the thermal stability of the Fc-J-chain complex. NMR analysis of 13C-labeled Fc suggests that the N459-glycan is relatively flexible while the N263-glycan is more rigid. CONCLUSIONS: We conclude that the N459-glycan of IgA1-Fc is essential for dimer formation and prevention of higher-order aggregates while those at N263 (Fc) and N49 (J-chain) stabilize the Fc-J-chain complex. GENERAL SIGNIFICANCE: Site-specific role for N-glycan in molecular assembly is addressed.
Asunto(s)
Inmunoglobulina A , Polisacáridos , Humanos , Inmunoglobulina A/química , Polisacáridos/química , Espectrometría de MasasRESUMEN
The gut microbiome is essential for human health. Mouse microbiota models, including gnotobiotic mice, are the most prominent tools to elucidate the functions of gut bacteria. Here, we propose a targeted-bacterium-depleted (TBD) model using lytic bacteriophage to selectively deplete gut bacterium of healthy or otherwise defined mice. These phage-treated animals should have a near-complete spectrum of gut bacteria except for the depleted bacterium. To prove the concept, we employed Escherichia coli-specific phage T7 to repress E. coli in the healthy mice. Our results showed that the E. coli-depleted mice exhibited bravery-like behaviors, correlated to the presence of E. coli rather than the equilibrium among gut bacteria. Thus, we demonstrate that the TBD model is a powerful tool to elucidate the function of a specific bacterial species within a near-intact gut microbiota environment and complements gnotobiotic mice models.
Asunto(s)
Bacteriófagos , Microbioma Gastrointestinal , Microbiota , Humanos , Animales , Ratones , Bacteriófagos/genética , Escherichia coli , BacteriasRESUMEN
The oncogene c-Jun is activated by Jun N-terminal kinase (JNK). Exosomes are nanometer-sized membrane vesicles released from a variety of cell types, and are essential for cell-to-cell communication. By using specific JNK inhibitor SP600125 or CRISPR/Cas9 to delete c-Jun, we found that exosomes from SP600125-treated A549 cancer cells (Exo-SP) or from c-Jun-KO-A549 cells (Exo-c-Jun-KO) dramatically inhibited tube formation of HUVECs. And the miR-494 levels in SP600125 treated or c-Jun-KO A549 cells, Exo-SP or Exo-c-Jun-KO, and HUVECs treated with Exo-SP or Exo-c-Jun-KO were significantly decreased. Meanwhile, Exo-SP and Exo-c-Jun-KO enhanced expression of phosphatase and tensin homolog deleted on chromosome ten (PTEN). Addition of miR-494 agomir in Exo-c-Jun-KO treated HUVECs inhibited PTEN expression and promoted tube formation, suggesting the target of miR-494 might be PTEN in HUVECs. Moreover, A549 tumor xenograft model and Matrigel plug assay demonstrated that Exo-c-Jun-KO attenuated tumor growth and angiogenesis through reducing miR-494. Taken together, inhibition of c-Jun in A549 cancer cells exhibited antiangiogenic activity in vitro and in vivo through exosome/miRNA-494-3p/PTEN signal pathway.
RESUMEN
Cancer cells secrete abundant exosomes, and the secretion can be promoted by an increase of intracellular Ca2+. Stromal interaction molecule 1 (STIM1) plays a key role in shaping Ca2+ signals. MicroRNAs (miRNAs) have been reported to be potential therapeutic targets for many diseases, including breast cancer. Recently, we investigated the effect of exosomes from STIM1-knockout breast cancer MDA-MB-231 cells (Exo-STIM1-KO), and from SKF96365-treated MDA-MB-231 cells (Exo-SKF) on angiogenesis in human umbilical vein endothelial cells (HUVECs) and nude mice. The exosomes Exo-STIM1-KO and Exo-SKF inhibited tube formation by HUVECs remarkably. The miR-145 was increased in SKF96365 treated or STIM1-knockout MDA-MB-231 cells, Exo-SKF and Exo-STIM1-KO, and HUVECs treated with Exo-SKF or Exo-STIM1-KO. Moreover, the expressions of insulin receptor substrate 1 (IRS1), which is the target of miR-145, and the downstream proteins such as Akt/mammalian target of rapamycin (mTOR), Raf/extracellular signal regulated-protein kinase (ERK), and p38 were markedly inhibited in HUVECs treated with Exo-SKF or Exo-STIM1-KO. Matrigel plug assay in vivo showed that tumor angiogenesis was suppressed in Exo-STIM1-KO, but promoted when miR-145 antagomir was added. Taken together, our findings suggest that STIM1 promotes angiogenesis by reducing exosomal miR-145 in breast cancer MDA-MB-231 cells.
Asunto(s)
Neoplasias de la Mama/metabolismo , Exosomas/metabolismo , MicroARNs/metabolismo , Proteínas de Neoplasias/fisiología , Neovascularización Patológica/metabolismo , Molécula de Interacción Estromal 1/fisiología , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones DesnudosRESUMEN
The aim of the present study is to investigate the anti-inflammatory and anti-allergic effects of taxifolin on mast cells and mast cell-mediated allergic reaction. We assessed the effect of taxifolin on the activation of bone marrow-derived mast cells (BMMCs) and rat basophilic leukemia (RBL)-2H3 cells induced by immunoglobulin E (IgE)/antigen (Ag), and the activation of human mast cell line (HMC-1) induced by PMA plus A23187. Taxifolin inhibited degranulation, generation of leukotriene C4 (LTC4), production of interlukin-6 (IL-6), and expression of cyclooxygenase-2 (COX-2) through blocking intracellular Ca2+ mobilization, phosphorylation of phospholipase Cγ (PLCγ) and mitogen-activated protein kinases (MAPKs), translocation of cytosolic phospholipase A2 (cPLA2) and 5-lipoxygenase (5-LO), and Akt/IKK/NF-κB pathway, in BMMC cells. Furthermore, taxifolin suppressed phosphorylation of Syk, but without effect on Fyn and Lyn. Taxifolin also inhibited activation of RBL-2H3 and HMC-1 cells via Akt/IKK/NF-κB and MAPKs/cPLA2 signal pathway. Treatment with taxifolin attenuated the mast cell-mediated passive cutaneous anaphylaxis (PCA) reaction. Our results suggest that taxifolin might become a potential drug candidate for the treatment of allergic and inflammatory diseases.
Asunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Hipersensibilidad/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Mastocitos/efectos de los fármacos , Anafilaxis Cutánea Pasiva/efectos de los fármacos , Quercetina/análogos & derivados , Animales , Degranulación de la Célula/efectos de los fármacos , Línea Celular , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Humanos , Inmunoglobulina E/metabolismo , Leucotrieno C4/metabolismo , Ratones , Ratones Endogámicos BALB C , FN-kappa B/metabolismo , Quercetina/uso terapéutico , Ratas , Transducción de SeñalRESUMEN
We previously reported the anti-inflammatory and anti-asthmatic activities of the extract of the Inula japonica Thunb. Aiming for discovery of a novel anti-inflammatory compound, we isolated spinacetin from the extract and investigated its in vitro and in vivo anti-inflammatory effect and the related mechanism. Effect of spinacetin on the Syk signaling pathway was studied in bone marrow-derived mast cells (BMMCs), and that on the nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) was investigated in Rat basophilic leukemia (RBL)-2H3 cells and human mast cell line (HMC-1). The in vivo anti-inflammatory activity was assessed with passive cutaneous anaphylaxis (PCA) reaction assay. Spinacetin significantly inhibited the release of histamine, and production of inflammatory mediators such as leukotriene C4 (LTC4) and interlukin-6 (IL-6) in IgE/Ag stimulated BMMCs. Analysis of the signaling pathways demonstrated that spinacetin inhibited activation of Syk, linker of activated T cells (LAT), phospholipase Cγ (PLCγ), cytosolic phospholipase A2 (cPLA2), MAPKs, Akt/NF-κB, and intracellular Ca2+ mobilization but with no effect on Fyn and Lyn. On the other hand, spinacetin suppressed IgE/Ag-induced activation of RBL-2H3 cells with inhibition against phosphorylation of extracellular signal regulated-protein kinase (ERK), c-Jun-NH2-terminal kinase (JNK), p38 MAPKs, PLCγ, translocation of cPLA2, and Akt/IκBα/NF-κB signal. However, spinacetin had no effect on PMA and A23187-induced activation of HMC-1. Furthermore, oral administration of spinacetin dose-dependently attenuated IgE/Ag-mediated PCA reaction in mouse model. Taken together, spinacetin showed the activities in preventing inflammatory processes, which might be at least partially attributed to the abolishment of Syk-dependent activation of IgE/Ag-mediated mast cells.
RESUMEN
In the present study, we investigated the anti-inflammatory effect of roburic acid on production of nitric oxide (NO) and interlukin-6 (IL-6) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. We found that roburic acid reduced production of NO and IL-6, and the expression of inducible nitric oxide synthases (iNOS). Meanwhile, phosphorylation of inhibitor of κBα (IκBα) and IκB kinase α/ß (IKKα/ß), as well as translocation of nuclear factor-κB (NF-κB) to the nucleus, was suppressed by roburic acid treatment. In addition, phosphorylation of mitogen-activated protein kinase (MAPKs) including p38 and c-Jun-NH2-terminal kinase (JNK) was inhibited. Roburic acid exhibited inhibitory activities on production of NO and IL-6 via blocking IKK/IκB/NF-κB and MAPKs pathway, suggesting the potential application as a drug candidate for therapy of inflammatory diseases.
Asunto(s)
Interleucina-6/biosíntesis , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , FN-kappa B/efectos de los fármacos , Óxido Nítrico/biosíntesis , Triterpenos/farmacología , Animales , Gentianaceae/química , Quinasa I-kappa B/efectos de los fármacos , Proteínas I-kappa B/efectos de los fármacos , Interleucina-6/antagonistas & inhibidores , Lipopolisacáridos , Macrófagos/metabolismo , Ratones , Óxido Nítrico/antagonistas & inhibidores , Células RAW 264.7RESUMEN
Background: The aim of this study was to evaluate the inhibitory effect of tamarixetin on the production of inflammatory mediators in IgE/antigen-induced mouse bone marrow-derived mast cells (BMMCs). Materials and methods: The effects of tamarixetin on mast cell activation were investigated with regard to degranulation, eicosanoid generation, Ca2+ influx, and immunoblotting of various signaling molecules. Results: Tamarixetin effectively decreased degranulation and the eicosanoid generation such as leukotriene C4 and prostaglandin D2 in BMMCs. To elucidate the mechanism involved, we investigated the effect of tamarixetin on the phosphorylation of signal molecules. Tamarixetin inhibited the phosphorylation of Akt and its downstream signal molecules including IKK and nuclear factor κB. In addition, tamarixetin downregulated the phosphorylation of cytosolic phospholipase A2 (cPLA2) and p38 mitogen-activated protein kinase. Conclusions: Taken together, this study suggests that tamarixetin inhibits degranulation and eicosanoid generation through the PLCγ1 as well as Akt pathways in BMMCs, which would be potential for the prevention of allergic inflammatory diseases (AU)