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Ouabain is a cardiac glycoside long studied for treating heart diseases, but the attempts to evaluate its anti-psoriatic activity have not been reported. We aimed to explore the effects of ouabain on proliferation and metabolism towards psoriatic keratinocytes. In human HaCaT keratinocytes, ouabain potently decreased viability, promoted apoptosis and caused G2/M cycle arrest. Metabolomics analysis indicated that ouabain markedly impaired glutathione metabolism. The solute carrier family 7 member 11 (SLC7A11) is an amino acid transporter highly specific to cysteine, which is critical for glutathione synthesis. Ouabain downregulated SLC7A11, reduced cysteine uptake and subsequently inhibited glutathione synthesis, probably through inhibiting Akt/mTOR/beclin axis that regulate protein activity of SLC7A11. The impaired glutathione synthesis and oxidative stress caused by ouabain may contribute to its cytotoxicity towards psoriatic keratinocytes. Our results provide experimental evidence supporting further study of ouabain as a potential anti-psoriatic agent.
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Antineoplásicos , Psoriasis , Humanos , Ouabaína/farmacología , Ouabaína/metabolismo , Ouabaína/uso terapéutico , Cisteína/metabolismo , Cisteína/farmacología , Cisteína/uso terapéutico , Queratinocitos/metabolismo , Antineoplásicos/farmacología , Apoptosis , Glutatión/metabolismo , Psoriasis/tratamiento farmacológico , Psoriasis/genética , Proliferación CelularRESUMEN
BACKGROUND: Gastrointestinal stromal tumors (GISTs) are the prevailing sarcomas of the gastrointestinal tract. Tyrosine kinase inhibitors (TKIs) therapy, exemplified by Imatinib mesylate (IM), constitutes the established adjuvant therapy for GISTs. Nevertheless, post-treatment resistance poses a challenge that all patients must confront. The presence of tumor heterogeneity and secondary mutation mechanisms fail to account for some instances of acquired drug resistance. Certain investigations suggest a strong association between tumor drug resistance and mesenchymal stromal cells (MSC) in the tumor microenvironment, but the underlying mechanism remains obscure. Scarce research has explored the connection between GIST drug resistance and the tumor microenvironment, as well as the corresponding mechanism. METHODS: Immunofluorescence and fluorescence-activated cell sorting (FACS) methodologies were employed to detect the presence of MSC in GIST samples. The investigation encompassed the examination of MSC migration towards tumor tissue and the impact of MSC on the survival of GIST cells under IM treatment. Through ELISA, western blotting, and flow cytometry analyses, it was confirmed that Transforming Growth Factor Beta 2 (TGF-ß2) triggers the activation of the PI3K-AKT pathway by MSC, thereby facilitating drug resistance in GIST. RESULTS: Our findings revealed a positive correlation between a high proportion of MSC and both GIST resistance and a poor prognosis. In vitro studies demonstrated the ability of MSC to migrate towards GIST. Additionally, MSC were observed to secrete TGF-ß2, consequently activating the PI3K-AKT pathway and augmenting GIST resistance. CONCLUSIONS: Our investigation has revealed that MSC within GISTs possess the capacity to augment drug resistance, thereby highlighting their novel mechanism and offering a promising target for intervention in GIST therapy.
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Antineoplásicos , Neoplasias Gastrointestinales , Tumores del Estroma Gastrointestinal , Células Madre Mesenquimatosas , Humanos , Tumores del Estroma Gastrointestinal/tratamiento farmacológico , Tumores del Estroma Gastrointestinal/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas , Factor de Crecimiento Transformador beta2/farmacología , Factor de Crecimiento Transformador beta2/uso terapéutico , Proteínas Proto-Oncogénicas c-kit/metabolismo , Mesilato de Imatinib/farmacología , Mesilato de Imatinib/uso terapéutico , Resistencia a Antineoplásicos/genética , Células Madre Mesenquimatosas/metabolismo , Neoplasias Gastrointestinales/tratamiento farmacológico , Antineoplásicos/uso terapéutico , Microambiente TumoralRESUMEN
OBJECTIVES: To evaluate the pharmacokinetics, pharmacodynamics, and tolerability of JY09, a long-acting glucagon-like peptide-1 (GLP-1) receptor agonist, in healthy subjects. MATERIALS AND METHODS: Healthy subjects were enrolled into 5 cohorts (0.3, 0.7, 1.5, 3.0, and 6.0 mg JY09) and received subcutaneous JY09 or placebo according to a randomized, double-blind, placebo-controlled, single-center, dose-escalating phase I trial design. Blood samples were collected over a 42-day period, and JY09 in plasma was determined by an electrochemical luminescence method. For the pharmacodynamic evaluation, oral glucose tolerance tests (OGTTs) were conducted predose and on day 5 after the target dose, during which plasma glucose, insulin, C-peptide, and glucagon concentrations were analyzed. Tolerability was assessed using physical examination and queries, vital sign measurements, laboratory analysis, and detection of immunogenicity. RESULTS: In healthy Chinese subjects, JY09 exhibited a dose-dependent increase in AUC0-inf and Cmax from 0.7 to 6.0 mg JY09. The half-life of JY09 was ~ 9.3 days, and the peak concentration was reached at ~ 60 - 72 hours. Following the OGTT, an increase in C-peptide concentration was observed after exposure to JY09 at the dose of 6.0 mg compared to the placebo group. JY09 was well tolerated in healthy Chinese subjects following a single dose of up to 6.0 mg. No symptomatic hypoglycemia was reported, and the most commonly observed adverse event was suppressed appetite, and its incidence was dose-dependent. Four subjects (13%) developed anti-JY09 antibodies. CONCLUSION: JY09 has a long half-life of ~ 9.3 days, with an acceptable safety profile.
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Voluntarios Sanos , Área Bajo la Curva , Glucemia , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Péptido 1 Similar al Glucagón , Semivida , Humanos , Hipoglucemiantes/efectos adversosRESUMEN
Psoriasis is an autoimmune skin disease characterized by keratinocyte hyperproliferation and chronic inflammation. The pathogenesis of psoriasis involves proinflammatory cytokines, such as tumor necrosis factor (TNF), but the mechanism of keratinocyte activation is not well understood. Here, we show that TNF (10 or 50 ng/mL) stimulates a significant (P < .0001) gene expression and secretion of proinflammatory IL-6, CXCL8 and VEGF from both cultured human HaCaT and normal epidermal human keratinocytes (NHEKs). This effect occurs via activation of the mammalian target of rapamycin (mTOR) signalling complex as shown by Western blot analysis and phospho-ELISAs. Pretreatment with the novel natural flavonoid tetramethoxyluteolin (10-100 µmol L-1 ) significantly (P < .0001) inhibits gene expression and secretion (P < .0001) of all 3 mediators in a concentration-dependent manner. Moreover, tetramethoxyluteolin (50 µmol L-1 ) appears to be a potent inhibitor of the phosphorylated mTOR substrates (pmTORSer2448 , pp70S6KThr389 and p4EBP1Thr37/46 ) as compared to known mTOR inhibitors in keratinocytes. The present findings indicate that TNF stimulates skin inflammation via mTOR signalling. Inhibition by tetramethoxyluteolin may be used in the treatment for psoriasis.
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Interleucina-6/metabolismo , Interleucina-8/metabolismo , Queratinocitos/metabolismo , Luteolina/farmacología , Serina-Treonina Quinasas TOR/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Supervivencia Celular , Células Cultivadas , Humanos , Inflamación , Queratinocitos/citología , Macrófagos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Psoriasis/tratamiento farmacológico , Proteínas Recombinantes/metabolismoRESUMEN
BACKGROUND: Mast cells (MCs) are hematopoietic cells that mature in tissues and are involved in allergy, immunity, and inflammation by secreting multiple mediators. The natural flavone luteolin has anti-inflammatory actions and inhibits human mast cells (MCs). OBJECTIVE: We sought to investigate the ability of luteolin and its novel structural analog 3',4',5,7-tetramethoxyluteolin (methlut) to inhibit human MC mediator expression and release in vitro and in vivo. METHODS: Human LAD2 cells and umbilical primary human cord blood-derived cultured mast cells were stimulated with substance P (SP) or IgE/anti-IgE with or without preincubation with luteolin, methlut, or cromolyn (1-100 µmol/L) for 2 or 24 hours, after which mediator secretion was measured. The effect of the compounds on MC intracellular calcium levels and nuclear factor κB activation was also investigated. Pretreatment with methlut was also studied in mice passively sensitized with dinitrophenol-human serum albumin and challenged intradermally. RESULTS: Methlut is a more potent inhibitor than luteolin or cromolyn for ß-hexosaminidase and histamine secretion from LAD2 cells stimulated by either SP or IgE/anti-IgE, but only methlut and luteolin significantly inhibit preformed TNF secretion. Methlut is also a more potent inhibitor than luteolin of de novo-synthesized TNF from LAD2 cells and of CCL2 from human cord blood-derived cultured MCs. This mechanism of action for methlut might be due to its ability to inhibit intracellular calcium level increases, as well as nuclear factor κB induction, at both the transcriptional and translational levels in LAD2 cells stimulated by SP without affecting cell viability. Intraperitoneal treatment with methlut significantly decreases skin vascular permeability of Evans blue dye in mice passively sensitized to dinitrophenol-human serum albumin and challenged intradermally. CONCLUSION: Methlut is a promising MC inhibitor for the treatment of allergic and inflammatory conditions.
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Flavonas/farmacología , Luteolina/farmacología , Mastocitos/efectos de los fármacos , Mastocitos/inmunología , Anticuerpos Antiidiotipos/inmunología , Calcio/metabolismo , Permeabilidad Capilar/efectos de los fármacos , Degranulación de la Célula/efectos de los fármacos , Degranulación de la Célula/inmunología , Células Cultivadas , Quimiocina CCL2/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Hexosaminidasas/metabolismo , Humanos , Inmunoglobulina E/inmunología , Mastocitos/metabolismo , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Factores de Necrosis Tumoral/metabolismoRESUMEN
Introduction: TT-01025-CL is an oral, irreversible small molecule that potently inhibits vascular adhesion protein-1 (VAP-1) for the treatment of inflammation associated with non-alcoholic steatohepatitis (NASH). The objectives of this study were to evaluate the safety/tolerability, pharmacokinetics, and pharmacodynamics of TT-01025-CL, a VAP-1 inhibitor, in healthy Chinese volunteers. Methods: Double-blind, placebo-controlled, dose-escalation studies were conducted in subjects randomized to receive oral once-daily TT-01025-CL (ranges: 10-300 mg [single dose]; 20-100 mg for 7 days [multiple doses]) or placebo under fasting conditions. Safety and tolerability were monitored throughout the study. Pharmacokinetic (PK) parameters were determined using non-compartment analysis. The activity of semicarbazide-sensitive amine oxidase (SSAO)-specific amine oxidase and the accumulation of methylamine in plasma were evaluated as pharmacodynamic (PD) biomarkers. Results: A total of 36 (single-dose group) and 24 (multiple-dose group) subjects were enrolled in the study. No serious adverse events (AEs) were reported, and no subject discontinued due to an AE. All treatment-emergent adverse events (TEAEs) were mild and moderate in intensity. No dose-dependent increase in the intensity or frequency of events was observed. TT-01025-CL was rapidly absorbed after administration. In the single-ascending dose (SAD) study, median Tmax ranged from 0.5 to 2 h and mean t1/2z ranged from 2.09 to 4.39 h. PK was linear in the range of 100-300 mg. The mean Emax of methylamine ranged from 19.167 to 124.970 ng/mL, with mean TEmax ranging from 13.5 to 28.0 h. The complete inhibition (>90%) of SSAO activity was observed at 0.25-0.5 h post-dose and was maintained 48-168 h post-dose. In the multiple-ascending dose (MAD) study, a steady state was reached by day 5 in the 40 mg and 100 mg dose groups. Negligible accumulation was observed after repeated dosing. PK was linear in the range of 20-100 mg. Plasma methylamine appeared to plateau at doses of 20 mg and above, with mean Emax ranging from 124.142 to 156.070 ng/mL and mean TEmax ranging from 14.2 to 22.0 h on day 7. SSAO activity in plasma was persistently inhibited throughout the treatment period. No evident change in methylamine and SSAO activity was observed in the placebo groups. Conclusion: TT-01025-CL was safe and well-tolerated at a single dose of up to 300 mg and multiple doses of up to 100 mg once daily for 7 consecutive days. Absorption and elimination occurred rapidly in healthy volunteers. Linearity in plasma exposure was observed. TT-01025-CL inhibited SSAO activity rapidly and persistently in humans. The profile of TT-01025-CL demonstrates its suitability for further clinical development.
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Hepatocellular carcinoma (HCC) is one of the deadliest cancers of human, the tumor-related death of which ranks third among the common malignances. N6-methyladenosine (m6A) methylation, the most abundant internal modification of RNA in mammals, participates in the metabolism of mRNA and interrelates with ncRNAs. In this paper, we overviewed the complex function of m6A regulators in HCC, including regulating the tumorigenesis, progression, prognosis, stemness, metabolic reprogramming, autophagy, ferroptosis, drug resistance and tumor immune microenvironment (TIME). Furthermore, we elucidated the interplay between m6A modification and non-coding RNAs (ncRNAs), including microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs). Finally, we summarized the potential of m6A regulators as diagnostic biomarkers. What's more, we reviewed the inhibitors targeting m6A enzymes as promising therapeutic targets of HCC. We aimed to help understand the function of m6A methylation in HCC systematically and comprehensively so that more effective strategies for HCC treatment will be developed.
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Adenosina/análogos & derivados , Carcinoma Hepatocelular , Neoplasias Hepáticas , MicroARNs , Humanos , Animales , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Mamíferos , Microambiente TumoralRESUMEN
Mast cells are well known for their role in allergic and anaphylactic reactions, as well as their involvement in acquired and innate immunity. Increasing evidence now implicates mast cells in inflammatory diseases where they are activated by non-allergic triggers, such as neuropeptides and cytokines, often exerting synergistic effects as in the case of IL-33 and neurotensin. Mast cells can also release pro-inflammatory mediators selectively without degranulation. In particular, IL-1 induces selective release of IL-6, while corticotropin-releasing hormone secreted under stress induces the release of vascular endothelial growth factor. Many inflammatory diseases involve mast cells in cross-talk with T cells, such as atopic dermatitis, psoriasis and multiple sclerosis, which all worsen by stress. How mast cell differential responses are regulated is still unresolved. Preliminary evidence suggests that mitochondrial function and dynamics control mast cell degranulation, but not selective release. Recent findings also indicate that mast cells have immunomodulatory properties. Understanding selective release of mediators could explain how mast cells participate in numerous diverse biologic processes, and how they exert both immunostimulatory and immunosuppressive actions. Unraveling selective mast cell secretion could also help develop unique mast cell inhibitors with novel therapeutic applications. This article is part of a Special Issue entitled: Mast cells in inflammation.
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Inflamación/patología , Mastocitos/metabolismo , Animales , Hormona Liberadora de Corticotropina/metabolismo , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Mastocitos/inmunología , Mastocitos/patología , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología , Enfermedades de la Piel/inmunología , Enfermedades de la Piel/patología , Estrés FisiológicoRESUMEN
BACKGROUND: Mast cells are immune cells derived from hematopoietic precursors that mature in the tissue microenvironment. Mast cells are critical for allergic, immune and inflammatory processes, many of which involve tumor necrosis factor (TNF). These cells uniquely store TNF in their secretory granules. Upon stimulation, mast cells rapidly (30 min) secrete ß-hexosaminidase and granule-stored TNF through degranulation, but also increase TNF mRNA and release de novo synthesized TNF 24 h later. The regulation of these two distinct pathways is poorly understood. METHODS: Human LAD2 leukemic mast cells are stimulated by substance P. TNF secretion and gene expression were measured by ELISA and real-time PCR, and mitochondrial dynamics was observed in live cells under confocal microscopy. Cell energy consumption was measured in terms of oxygen consumption rate. RESULTS: Here, we show that granule-stored TNF is preformed, and its secretion from LAD2 mast cells stimulated by substance P (1) exhibits higher energy consumption and is inhibited by the mitochondrial ATP pump blocker oligomycin, (2) shows rapid increase in intracellular calcium levels, and (3) exhibits reversible mitochondrial translocation, from a perinuclear distribution to the cell surface, as compared to de novo synthesized TNF release induced by lipopolysaccharide. This mitochondrial translocation is confirmed using primary human umbilical cord blood-derived mast cells stimulated by an allergic trigger (IgE/streptavidin). CONCLUSION: Our findings indicate that unique mitochondrial functions distinguish granule-stored from newly synthesized TNF release from human mast cells, thus permitting the versatile involvement of mast cells in different biological processes.
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Mastocitos/fisiología , Mitocondrias/inmunología , Vesículas Secretoras/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Calcio/metabolismo , Degranulación de la Célula/efectos de los fármacos , Línea Celular Tumoral , Células Cultivadas , Humanos , Lipopolisacáridos/farmacología , Mastocitos/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Oligomicinas/farmacología , ARN Mensajero/metabolismo , Sustancia P/farmacología , Factor de Necrosis Tumoral alfa/genética , Desacopladores/farmacologíaRESUMEN
BACKGROUND: Mast cells derive from hematopoietic cell precursors and participate in tissue allergic, immune, and inflammatory processes. They secrete many mediators, including preformed TNF, in response to allergic, neuropeptide, and environmental triggers. However, regulation of mast cell degranulation is not well understood. OBJECTIVE: We investigated the role of mitochondrial dynamics in degranulation of human cultured mast cells. METHODS: Human umbilical cord blood-derived mast cells (hCBMCs) and Laboratory of Allergic Diseases 2 (LAD2) mast cells were examined by confocal and differential interference contrast microscopy during activation by IgE/antigen and substance P (SP). Mast cells in control and atopic dermatitis (AD) skin were evaluated by transmission electron microscopy. LAD2 cells were pretreated with mitochondrial division inhibitor, a dynamin-related protein 1 (Drp1) inhibitor, and small interfering RNA for Drp1, which is necessary for mitochondrial fission and translocation. Calcineurin and Drp1 gene expression was analyzed in stimulated LAD2 cells and AD skin biopsies. RESULTS: Stimulation of hCBMCs with IgE/antigen or LAD2 cells with SP leads to rapid (30 minutes) secretion of preformed TNF. Degranulation is accompanied by mitochondrial translocation from a perinuclear location to exocytosis sites. Extracellular calcium depletion prevents these effects, indicating calcium requirement. The calcium-dependent calcineurin and Drp1 are activated 30 minutes after SP stimulation. Reduction of Drp1 activity by mitochondrial division inhibitor and decrease of Drp1 expression using small interfering RNA inhibit mitochondrial translocation, degranulation, and TNF secretion. Mitochondrial translocation is also evident by transmission electron microscopy in skin mast cells from AD biopsies, in which gene expression of calcineurin, Drp1, and SP is higher than in normal skin. CONCLUSION: Human mast cell degranulation requires mitochondrial dynamics, also implicated in AD.
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Degranulación de la Célula/fisiología , Dermatitis Atópica/fisiopatología , Mastocitos/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Adolescente , Adulto , Antígenos/administración & dosificación , Transporte Biológico Activo , Calcineurina/genética , Calcineurina/metabolismo , Calcio/metabolismo , Estudios de Casos y Controles , Degranulación de la Célula/efectos de los fármacos , Degranulación de la Célula/inmunología , Células Cultivadas , Niño , Dermatitis Atópica/genética , Dermatitis Atópica/inmunología , Dinaminas , Exocitosis/fisiología , Femenino , GTP Fosfohidrolasas/antagonistas & inhibidores , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Humanos , Inmunoglobulina E/administración & dosificación , Masculino , Mastocitos/efectos de los fármacos , Mastocitos/inmunología , Mastocitos/ultraestructura , Microscopía Electrónica de Transmisión , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Persona de Mediana Edad , Mitocondrias/fisiología , Proteínas Mitocondriales/antagonistas & inhibidores , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , ARN Interferente Pequeño/genética , Sustancia P/administración & dosificación , Sustancia P/genética , Adulto JovenRESUMEN
Long noncoding RNAs (lncRNAs) have crucial functions in the tumorigenesis and metastasis of cancers. N6-methyladenosine (m6A) modification of RNA is an important epigenetic regulatory mechanism in various malignancies. Nevertheless, the mechanism of m6A-modified lncRNA in diffuse large B cell lymphoma (DLBCL) has remained poorly defined. In the present study, we showed that lncRNA TRERNA1 was associated with the poor prognosis of DLBCL patients. TRERNA1 with internal m6A modification was highly correlated with the demethylase ALKBH5 expression. We further demonstrated that TRERNA1 was a potential downstream target of ALKBH5-mediated m6A modification by m6A-RNA sequencing and m6A-RIP assays. Decreased m6A methylation of TRERNA1 regulated by ALKBH5 was shown to regulate cell proliferation in vitro and in vivo. The results of mechanism analyses revealed that TRERNA1 recruited EZH2 to epigenetically silence the expression of the cyclin-dependent kinases inhibitor p21 by H3K27me3 modification of its promoter region. In addition, ALKBH5 further inhibited p21 expression. Taken together, our results elucidate the functional roles and epigenetic alterations of TRERNA1 through m6A modification in DLBCL. TRERNA1, the expression of which is upregulated by ALKBH5, acts as a scaffold that decreases p21 expression. The results of the present study provide novel targets for the diagnosis and treatment of DLBCL.
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BACKGROUND: Coronary artery ectasia (CAE) is a cardiovascular disorder characterized by abnormal coronary artery dilation and disturbed coronary flow. The exact pathophysiology of CAE is still unclear. We aimed to investigate differences in metabolomic profiles between CAE patients and healthy controls. METHODS: Radial artery blood samples were collected from 14 pure CAE patients, 12 mixed CAE patients with atherosclerosis, and 14 controls with normal angiography. Differential serum metabolites were analyzed by untargeted ultra-high performance liquid chromatography-mass spectrometry. Serum ICAM-1, VEGF, ROS, and glutathione levels were also measured. RESULTS: Ten metabolites distinguished pure CAE patients from controls and mixed CAE, including 1-cyano-2-hydroxy-3-butene, 2,3-dihydro-6-methyl-5-(5-methyl-2-furanyl)-1H-pyrrolizine, 2-propionylpyrrole, 2-pyrrolidinone, 3-(2-furanylmethylene)pyrrolidine, D-alanine, furanofukinin, o-ethyltoluene, rotundine A, and SM(d18:1/18:1(9Z)). Related metabolic pathways include amino acid metabolism, sphingolipid dysfunction, energy metabolism, mitochondrial dysfunction, and oxidative stress. Serum concentrations of ICAM-1, VEGF and ROS were significantly elevated in CAE patients compared to controls, while glutathione decreased significantly in CAE patients. Moreover, ICAM-1 levels were negatively correlated with 2-propionylpyrrole, and VEGF levels were negatively correlated with SM(d18:1/18:1(9Z)), while GSH and ROS levels were correlated with the abundance of SM(d18:1/18:1(9Z)), further confirming systemic inflammation and oxidative stress in CAE. CONCLUSIONS: This is the first report describing differential serum metabolomic profiles of pure CAE patients compared to mixed CAE and healthy controls, which revealed 10 potential biomarkers that can provide an early diagnosis of pure CAE. These discriminatory metabolites and related metabolic pathways can help to better understand the pathogenesis of pure CAE.
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Enfermedad de la Arteria Coronaria , Vasos Coronarios , Estudios de Casos y Controles , Angiografía Coronaria , Vasos Coronarios/metabolismo , Dilatación Patológica/metabolismo , Dilatación Patológica/patología , Glutatión/metabolismo , Humanos , Molécula 1 de Adhesión Intercelular , Especies Reactivas de Oxígeno/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: The pathogenesis of psoriasis is still not fully understood. Dynamin-related protein 1 (Drp1) regulates mitochondrial fission and is implicated in various inflammatory conditions, but research regarding Drp1 in the skin is scarce. OBJECTIVES: To examine Drp1 expression in psoriasis vulgaris lesional skin and the effect of Drp1 expression on keratinocyte proliferation and inflammatory mediator release in vitro. MATERIALS AND METHODS: Skin biopsies were collected, and the expression of Drp1 and TNF-α was investigated in the skin. Serum TNF-α level was also examined. The effect of Drp1 on keratinocyte proliferation and inflammatory mediator release was evaluated in HaCaT cells following Drp1 transfection. The effect of TNF-α on Drp1 expression was also studied in HaCaT cells. RESULTS: Drp1 expression was significantly increased and positively correlated with PASI score and TNF-α expression in skin. In HaCaT cells, Drp1 transfection altered cellular ATP and proliferation, induced G2/M arrest, and affected inflammatory mediator (TNF-α, IL-6 and CXCL8) release. Moreover, TNF-α induced Drp1 expression and recovered cellular ATP content and mediator release. CONCLUSION: Drp1 is significantly elevated in psoriasis vulgaris skin and positively correlates with disease severity, moreover, in vitro, Drp1 regulates keratinocyte function.
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Proliferación Celular , Dinaminas/metabolismo , Queratinocitos/fisiología , Psoriasis/metabolismo , Psoriasis/fisiopatología , Piel/metabolismo , Adenosina Trifosfato/metabolismo , División Celular , Dinaminas/genética , Puntos de Control de la Fase G2 del Ciclo Celular/fisiología , Células HaCaT , Humanos , Mediadores de Inflamación/metabolismo , Dinámicas Mitocondriales , Índice de Severidad de la Enfermedad , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia ArribaAsunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Síndrome de Fatiga Crónica/tratamiento farmacológico , Inhibidores Selectivos de la Recaptación de Serotonina/uso terapéutico , Animales , Antiinflamatorios no Esteroideos/efectos adversos , Antiinflamatorios no Esteroideos/metabolismo , Antidepresivos/efectos adversos , Antidepresivos/metabolismo , Antidepresivos/uso terapéutico , Interacciones Farmacológicas/fisiología , Síndrome de Fatiga Crónica/metabolismo , Hemorragia Gastrointestinal/inducido químicamente , Hemorragia Gastrointestinal/metabolismo , Humanos , Inhibidores Selectivos de la Recaptación de Serotonina/efectos adversos , Inhibidores Selectivos de la Recaptación de Serotonina/metabolismo , Resultado del TratamientoRESUMEN
PURPOSE: Tunneling nanotubes (TNTs) are extremely thin (50-200 nm), actin-containing cell surface protrusions up to a few microns in length that can develop rapidly and connect various cell types. Mast cells (MCs) are unique immunomodulatory cells that are found perivascularly in all tissues. MCs communicate with many other cell types through the release of inflammatory, neurosensitizing, and vasoactive molecules, through which they are involved in the pathogenesis of many inflammatory diseases. We, therefore, investigated the possibility that MCs may form TNTs and communicate among themselves and with glioblastoma cells. METHODS: Laboratory Allergic Diseases (LAD)-2 human MCs were cultured in medium supplemented with 100 U/mL penicillin/streptomycin and 100 ng/mL recombinant human stem cell factor. They were incubated with 20 nmol/L deep red probe for 20 minutes and 50 nmol/L green probe for 30 minutes. Human glioblastoma cells were incubated with 20 nmol/L deep red probe only, moved to glass-bottom culture dishes, and observed using a substance P 2 confocal microscope. LAD2 MCs were stimulated with 2 µmol/L of the peptide substance P for 30 minutes at 37ºC. Confocal digital images were processed. FINDINGS: MCs can rapidly (within 5 minutes) form TNTs, which appear to transport mitochondrial and secretory granule particles among themselves and with cocultured glioblastoma cells. IMPLICATIONS: MCs are now accepted as having an important role in many diseases with an inflammatory component. TNTs provide a rapid and direct way for MCs to "alarm" other cell types with specificity not present when mediators are secreted into the tissue microenvironment. The identification of TNTs and their cargo could be important in the diagnosis and possible treatment of many inflammatory diseases.
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Comunicación Celular/inmunología , Microambiente Celular/inmunología , Nanotubos , Transducción de Señal/inmunología , Animales , Línea Celular Tumoral , Humanos , Mastocitos/inmunología , RatonesRESUMEN
Diabetic foot ulceration is a severe complication of diabetes that lacks effective treatment. Mast cells (MCs) contribute to wound healing, but their role in diabetes skin complications is poorly understood. Here we show that the number of degranulated MCs is increased in unwounded forearm and foot skin of patients with diabetes and in unwounded dorsal skin of diabetic mice (P < 0.05). Conversely, postwounding MC degranulation increases in nondiabetic mice, but not in diabetic mice. Pretreatment with the MC degranulation inhibitor disodium cromoglycate rescues diabetes-associated wound-healing impairment in mice and shifts macrophages to the regenerative M2 phenotype (P < 0.05). Nevertheless, nondiabetic and diabetic mice deficient in MCs have delayed wound healing compared with their wild-type (WT) controls, implying that some MC mediator is needed for proper healing. MCs are a major source of vascular endothelial growth factor (VEGF) in mouse skin, but the level of VEGF is reduced in diabetic mouse skin, and its release from human MCs is reduced in hyperglycemic conditions. Topical treatment with the MC trigger substance P does not affect wound healing in MC-deficient mice, but improves it in WT mice. In conclusion, the presence of nondegranulated MCs in unwounded skin is required for proper wound healing, and therapies inhibiting MC degranulation could improve wound healing in diabetes.
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Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Neuropatías Diabéticas/metabolismo , Mastocitos/metabolismo , Piel/metabolismo , Cicatrización de Heridas/fisiología , Anciano , Animales , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 1/patología , Neuropatías Diabéticas/patología , Femenino , Humanos , Masculino , Mastocitos/patología , Ratones , Persona de Mediana Edad , Piel/patologíaRESUMEN
Psoriasis (Ps) is an autoimmune disease characterized by keratinocyte hyperproliferation and chronic inflammation, with increased expression of tumor necrosis factor (TNF) and vascular endothelial growth factor (VEGF). Anti-TNF biologic agents are effective in treating Ps, but are associated with increased risk of infections and blood malignancies. Moreover, keratinocyte hyperproliferation and activation have yet to be addressed. Flavonoids, such as luteolin, are natural compounds with potent anti-inflammatory properties, but their actions on keratinocytes remain unknown. We show that TNF (50 ng/mL) triggers significant production of inflammatory mediators interleukin-6, interleukin-8 and VEGF from both human HaCaT and primary keratinocytes. Pretreatment with the flavonoid luteolin (10-100 µM) significantly inhibits mRNA expression and release of all three mediators in a concentration-dependent manner. More importantly, luteolin decreases TNF-induced phosphorylation, nuclear translocation and DNA binding of the nuclear factor-kappa B (NF-κB) typically involved in inflammatory mediator transcription. We also report that luteolin reduces TNF-induced mRNA expression of two genes (NFKB1 and RELA) encoding two NF-κB subunits (NF-κB p50 and NF-κB p65, respectively). Interestingly, we show that gene expression of RELA is increased in human psoriatic skin. Keratinocyte proliferation, which is a characteristic feature of psoriatic skin, is effectively reduced by luteolin in HaCaT cells, but not in primary keratinocytes. Finally, luteolin does not affect intracellular ATP production or viability. Appropriate formulations of luteolin and related flavones may be promising candidates to be developed into local and systemic treatments for Ps and other inflammatory skin diseases.
Asunto(s)
Queratinocitos/efectos de los fármacos , Luteolina/farmacología , FN-kappa B/metabolismo , Psoriasis/metabolismo , Piel/efectos de los fármacos , Transporte Activo de Núcleo Celular/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Adulto , Western Blotting , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Expresión Génica/efectos de los fármacos , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Queratinocitos/citología , Queratinocitos/metabolismo , FN-kappa B/genética , Fosforilación/efectos de los fármacos , Psoriasis/genética , Psoriasis/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/metabolismo , Piel/patología , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Autoimmune diseases continue to increase, but the reason(s) remain obscure and infections have not proven to be major contributors. Mast cells are tissue immune cells responsible for allergies, but have been increasingly shown to be involved in innate and acquired immunity, as well as inflammation. This involvement is possible because of their ability to release multiple mediators in response to a great variety of triggers. We recently published that activation of mast cells is accompanied by mitochondrial fission and translocation to the cell surface from where they secrete at least ATP and DNA outside the cell without cell damage. These extracellular mitochondrial components are misconstrued by the body as "innate pathogens" leading to powerful autocrine and paracrine auto-immune/auto-inflammatory responses. We also showed that mitochondrial DNA is increased in the serum of young children with autism spectrum disorders (ASD), a condition that could involve "focal brain allergy/encephalitits". Blocking the secretion of extracellular mitochondrial components could present unique possibilities for the therapy of ASD and other autoimmune diseases. Unique formulation of the flavonoid luteolin offers unique advantages.
Asunto(s)
Trastorno Autístico/inmunología , Trastorno Autístico/metabolismo , Autoinmunidad , Encéfalo/patología , Mastocitos/metabolismo , Mitocondrias/metabolismo , Encéfalo/inmunología , Encéfalo/metabolismo , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Mastocitos/inmunología , Mastocitos/patología , Mitocondrias/patologíaRESUMEN
Mast cells are hematopoietically-derived tissue immune cells that participate in acquired and innate immunity, as well as in inflammation through release of many chemokines and cytokines, especially in response to the pro-inflammatory peptide substance P (SP). Inflammation is critical in the pathogenesis of many diseases, but the trigger(s) is often unknown. We investigated if mast cell stimulation leads to secretion of mitochondrial components and whether these could elicit autocrine and/or paracrine inflammatory effects. Here we show that human LAD2 mast cells stimulated by IgE/anti-IgE or by the SP led to secretion of mitochondrial particles, mitochondrial (mt) mtDNA and ATP without cell death. Mitochondria purified from LAD2 cells and, when mitochondria added to mast cells trigger degranulation and release of histamine, PGD(2), IL-8, TNF, and IL-1ß. This stimulatory effect is partially inhibited by an ATP receptor antagonist and by DNAse. These results suggest that the mitochondrial protein fraction may also contribute. Purified mitochondria also stimulate IL-8 and vascular endothelial growth factor (VEGF) release from cultured human keratinocytes, and VEGF release from primary human microvascular endothelial cells. In order to investigate if mitochondrial components could be secreted in vivo, we injected rats intraperiotoneally (ip) with compound 48/80, which mimicks the action of SP. Peritoneal mast cells degranulated and mitochondrial particles were documented by transimission electron microscopy outside the cells. We also wished to investigate if mitochondrial components secreted locally could reach the systemic circulation. Administration ip of mtDNA isolated from LAD2 cells in rats was detected in their serum within 4 hr, indicating that extravascular mtDNA could enter the systemic circulation. Secretion of mitochondrial components from stimulated live mast cells may act as "autopathogens" contributing to the pathogenesis of inflammatory diseases and may be used as targets for novel treatments.
Asunto(s)
Comunicación Autocrina , Mastocitos/citología , Mastocitos/metabolismo , Mitocondrias/metabolismo , Comunicación Paracrina , Animales , Comunicación Autocrina/efectos de los fármacos , Degranulación de la Célula/efectos de los fármacos , Citocinas/metabolismo , ADN Mitocondrial/metabolismo , Exosomas/efectos de los fármacos , Exosomas/metabolismo , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Humanos , Inflamasomas/metabolismo , Inflamación/inmunología , Inflamación/metabolismo , Masculino , Mastocitos/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Comunicación Paracrina/efectos de los fármacos , Ratas , Receptores Purinérgicos P2X7/metabolismo , p-Metoxi-N-metilfenetilamina/farmacologíaRESUMEN
Mast cells are immune cells critical in the pathogenesis of allergic, but also inflammatory and autoimmune diseases through release of many pro-inflammatory cytokines such as IL-8 and TNF. Contact dermatitis and photosensitivity are skin conditions that involve non-immune triggers such as substance P (SP), and do not respond to conventional treatment. Inhibition of mast cell cytokine release could be effective therapy for such diseases. Unfortunately, disodium cromoglycate (cromolyn), the only compound marketed as a mast cell "stabilizer", is not particularly effective in blocking human mast cells. Instead, flavonoids are potent anti-oxidant and anti-inflammatory compounds with mast cell inhibitory actions. Here, we first compared the flavonoid quercetin (Que) and cromolyn on cultured human mast cells. Que and cromolyn (100 µM) can effectively inhibit secretion of histamine and PGD(2). Que and cromolyn also inhibit histamine, leukotrienes and PGD(2) from primary human cord blood-derived cultured mast cells (hCBMCs) stimulated by IgE/Anti-IgE. However, Que is more effective than cromolyn in inhibiting IL-8 and TNF release from LAD2 mast cells stimulated by SP. Moreover, Que reduces IL-6 release from hCBMCs in a dose-dependent manner. Que inhibits cytosolic calcium level increase and NF-kappa B activation. Interestingly, Que is effective prophylactically, while cromolyn must be added together with the trigger or it rapidly loses its effect. In two pilot, open-label, clinical trials, Que significantly decreased contact dermatitis and photosensitivity, skin conditions that do not respond to conventional treatment. In summary, Que is a promising candidate as an effective mast cell inhibitor for allergic and inflammatory diseases, especially in formulations that permit more sufficient oral absorption.