RESUMEN
Carvedilol, a third-generation and nonselective ß-adrenoceptor antagonist, is a licensed drug for treating patients suffering from heart failure in clinics. It has been shown that Carvedilol protects cells against mitochondrial dysfunction. However, it's unknown whether Carvedilol affects mitochondrial biogenesis. In this study, we found that treatment with Carvedilol in HUVECs resulted in a significant increase of PGC-1α, NRF1, and TFAM. Notably, Carvedilol significantly increased mtDNA contents and the two mitochondrial proteins, cytochrome C and COX IV. In addition, MitoTracker Red staining results indicated that treatment with Carvedilol increased mitochondria mass. Mechanistically, we found that the effect of Carvedilol on the expression of PGC-1α is mediated by the PKA-CREB pathway. Importantly, our results revealed that stimulation of mitochondrial biogenesis by carvedilol resulted in functional gain of the mitochondria by showing increased oxygen consumption and mitochondrial respiratory rate. The increased expression of PGC-1α and mitochondrial biogenesis induced by Carvedilol might suggest a new mechanism of the therapeutic effects of Carvedilol in heart failure.
Asunto(s)
Carbazoles/administración & dosificación , Proteínas de Unión al ADN/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/ultraestructura , Mitocondrias/fisiología , Proteínas Mitocondriales/metabolismo , Propanolaminas/administración & dosificación , Factores de Transcripción/metabolismo , Antagonistas Adrenérgicos beta/administración & dosificación , Carvedilol , Aumento de la Célula/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Mitocondrias/efectos de los fármacos , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
OBJECTIVE: The mechanisms of contrast-induced nephropathy are not fully understood and sensitive biomarkers of contrast-induced nephropathy are yet to be found. We investigated whether urinary fibrinogen could be a potential biomarker for contrast-induced nephropathy. METHODS: To create a contrast-induced nephropathy model, mice received a prostaglandin synthesis inhibitor (indomethacin) and a nitric oxide synthase inhibitor (Nω-Nitro-L-arginine methyl ester) intraperitoneally followed by a different dose of iodixanol. In the control group, normal saline was administered. Urinary fibrinogen and serum creatinine were analyzed using enzyme-linked immunosorbent assay. Kidneys were used to quantify fibrinogen using qRT-PCR and Western blot and for histopathological examination. RESULTS: Histopathological examination demonstrated mild renal injury in the low-dose group, and moderate renal injury in the high-dose group. Urinary fibrinogen levels were significantly increased in an iodixanol dose-dependent manner (control vs. low-dose group, P < 0.05; control vs. high-dose group P < 0.01). Serum creatinine levels were only increased in the high-dose group (P < 0.01 compared to control), but not in the low-dose group. For fibrinogen-gene expression, in the low-dose group, Fgγ increased (qRT-PCR, Western blot, P < 0.05) in the high-dose group, Fgß and Fgγ decreased (qRT-PCR, P < 0.01; Western blot, P < 0.05), and Fgα increased (qRT-PCR, P < 0.05; Western blot, P < 0.05). CONCLUSIONS: We propose that urinary fibrinogen could be used as a potential biomarker for early contrast-induced nephropathy diagnosis.
Asunto(s)
Lesión Renal Aguda/orina , Fibrinógeno/orina , Riñón/metabolismo , Ácidos Triyodobenzoicos , Lesión Renal Aguda/sangre , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/genética , Animales , Biomarcadores/sangre , Biomarcadores/orina , Creatinina/sangre , Modelos Animales de Enfermedad , Diagnóstico Precoz , Fibrinógeno/genética , Regulación de la Expresión Génica , Indometacina , Riñón/patología , Masculino , Ratones Endogámicos C57BL , NG-Nitroarginina Metil Éster , Valor Predictivo de las Pruebas , Factores de Tiempo , UrinálisisRESUMEN
Production of IL-1beta typically requires two-separate signals. The first signal, from a pathogen-associated molecular pattern, promotes intracellular production of immature cytokine. The second signal, derived from a danger signal such as extracellular ATP, results in assembly of an inflammasome, activation of caspase-1 and secretion of mature cytokine. The inflammasome component, Nalp3, plays a non-redundant role in caspase-1 activation in response to ATP binding to P2X(7) in macrophages. Gingival epithelial cells (GECs) are an important component of the innate-immune response to periodontal bacteria. We had shown that GECs express a functional P2X(7) receptor, but the ability of GECs to secrete IL-1beta during infection remained unknown. We find that GECs express a functional Nalp3 inflammasome. Treatment of GECs with LPS or infection with the periodontal pathogen, Porphyromonas gingivalis, induced expression of the il-1beta gene and intracellular accumulation of IL-1beta protein. However, IL-1beta was not secreted unless LPS-treated or infected cells were subsequently stimulated with ATP. Conversely, caspase-1 is activated in GECs following ATP treatment but not P. gingivalis infection. Furthermore, depletion of Nalp3 by siRNA abrogated the ability of ATP to induce IL-1beta secretion in infected cells. The Nalp3 inflammasome is therefore likely to be an important mediator of the inflammatory response in gingival epithelium.
Asunto(s)
Adenosina Trifosfato/farmacología , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Encía/citología , Encía/microbiología , Porphyromonas gingivalis/fisiología , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Caspasa 1/metabolismo , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Lipopolisacáridos/farmacología , Proteína con Dominio Pirina 3 de la Familia NLR , Reacción en Cadena de la Polimerasa , Interferencia de ARN , ARN Interferente Pequeño , Transducción de Señal/efectos de los fármacosRESUMEN
Interferon Regulatory Factor-3 (IRF-3) plays a central role in the induction of interferon (IFN) production and succeeding interferon-stimulated genes (ISG) expression en route for restraining hepatitis C virus (HCV) infection. Here, we established a stable Huh7.5-IRF3ER cell line expressing a fusion protein of IRF-3 and mouse estrogen receptor (ER) to examine IFN production and anti-HCV effects of IRF-3 in retinoic acid inducible-gene-I (RIG-I) deficient Huh 7.5 cells. Homodimerization of the IRF-3ER fusion protein was detected by Western blotting after treatment with the estrogen receptor agonist 4-hydrotamoxifen (4-HT) in Huh7.5-IRF3ER cells. Expression of IFN-α, IFN-ß, and their inhibitory effects on HCV replication were demonstrated by real-time polymerase chain reaction (PCR). Peak expression of IFN-α and IFN-ß was achieved 24-hours post 4-HT treatment, coinciding with the appearance of phosphorylated signal transducer and activator of transcription (STAT) proteins. Additionally, HCV viral replication declined in time-dependent fashion. In previous studies, a novel IFN-mediated pathway regulating expression of 1-8U and heterogeneous nuclear ribonucleoprotein M (hnRNP M) inhibited HCV internal ribosomal entry site (IRES)-dependent translation. When expression of ISGs such as 1-8U and hnRNP M were measured in 4-HT-treated Huh7.5-IRF3ER cells, both genes were positively regulated by activation of the IRF-3ER fusion protein. In conclusion, the anti-HCV effects of IRF-3ER homodimerization inhibited HCV RNA replication as well as HCV IRES-dependent translation in Huh7.5-IRF3ER cells. The results of this study indicate that IRF-3ER homodimerization is a key step to restore IFN expression in Huh7.5-IRF3ER cells and in achieving its anti-HCV effects.
Asunto(s)
ARN Helicasas DEAD-box/deficiencia , Hepacivirus/fisiología , Hepatocitos/virología , Factor 3 Regulador del Interferón/biosíntesis , Receptores de Estrógenos/biosíntesis , Replicación Viral , Animales , Línea Celular , Proteína 58 DEAD Box , Expresión Génica , Hepacivirus/crecimiento & desarrollo , Hepacivirus/inmunología , Humanos , Factor 3 Regulador del Interferón/genética , Interferón-alfa/biosíntesis , Interferón-alfa/inmunología , Interferón beta/biosíntesis , Interferón beta/inmunología , Ratones , Biosíntesis de Proteínas , Multimerización de Proteína , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Estrógenos/genética , Receptores Inmunológicos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Transcripción GenéticaRESUMEN
The purinergic receptor P2X(7) is involved in cell death, inhibition of intracellular infection and secretion of inflammatory cytokines. The role of the P2X(7) receptor in bacterial infection has been primarily established in macrophages. Here we show that primary gingival epithelial cells, an important component of the oral innate immune response, also express functional P2X(7) and are sensitive to ATP-induced apoptosis. Porphyromonas gingivalis, an intracellular bacterium and successful colonizer of oral tissues, can inhibit gingival epithelial cell apoptosis induced by ATP ligation of P2X(7) receptors. A P. gingivalis homologue of nucleoside diphosphate kinase (NDK), an ATP-consuming enzyme, is secreted extracellularly and is required for maximal suppression of apoptosis. An ndk-deficient mutant was unable to prevent ATP-induced host-cell death nor plasma membrane permeabilization in the epithelial cells. Treatment with purified recombinant NDK inhibited ATP-mediated host-cell plasma membrane permeabilization in a dose-dependent manner. Therefore, NDK promotes survival of host cells by hydrolysing extracellular ATP and preventing apoptosis-mediated through P2X(7).
Asunto(s)
Adenosina Trifosfato/farmacología , Apoptosis/efectos de los fármacos , Porphyromonas gingivalis/fisiología , Receptores Purinérgicos P2/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/metabolismo , Células Cultivadas , Citometría de Flujo , Encía/citología , Encía/efectos de los fármacos , Encía/microbiología , Humanos , Etiquetado Corte-Fin in Situ , Microscopía Fluorescente , Nucleósido-Difosfato Quinasa/metabolismo , Porphyromonas gingivalis/metabolismo , Receptores Purinérgicos P2X7RESUMEN
BACKGROUND: Chemotherapeutic resistance is a main problem in clinical breast cancer therapy. The purpose of our study is to investigate whether the combination of neoadjuvant chemotherapy and survivin siRNA treatment could enhance the therapeutic effect of neoadjuvant chemotherapy using paclitaxel or epirubicin. MATERIALS AND METHODS: The molecular cloning technique was applied to construct the expression vector of siRNA against survivin. Effectene Transfection Reagent was used to transfect plasmids to MCF-7 cells. Survivin expressions were detected by quantitative real-time PCR (qRT-PCR) and Western blot methods. The effect of paclitaxel or epirubicin, with or without the combination of survivin siRNA treatment, on drug susceptibility of MCF-7 cells was detected by CCK-8 assay. MCF-7 cell apoptosis was detected by Flow Cytometry. RESULTS: Survivin siRNA effectively inhibited the expression of Survivin RNA and protein levels (P < 0.05). Both paclitaxel and epirubicin can suppress the proliferation of MCF-7 cells and induce apoptosis to a certain degree respectively. The combination of survivin siRNA with the two chemotherapy drugs significantly enhanced both effects of the two chemotherapeutics respectively (P < 0.05). CONCLUSION: Survivin siRNA combined with the neoadjuvant chemotherapy can significantly enhance the sensitivity of MCF-7 cells to chemotherapeutics and cell apoptosis. This technology has important potential value in the therapeutic study of breast cancer.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias de la Mama/patología , Resistencia a Antineoplásicos/genética , Epirrubicina/farmacología , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Terapia Neoadyuvante , ARN Interferente Pequeño/genética , Antibióticos Antineoplásicos/farmacología , Western Blotting , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Terapia Combinada , Femenino , Citometría de Flujo , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Survivin , Células Tumorales CultivadasRESUMEN
PURPOSE: To investigate the effect of Iodixanol on kidney proximal tubular cell line human kidney 2 (HK-2). METHODS: The HK-2 cells were treated with Iodixanol. A Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was used to evaluate apoptosis. Cell viability was measured by proliferation assay kit. Cell permeability changes were assessed by transwell assay and intercellular gaps measurement. Expression of claudin-2 was assessed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and Western blot. RESULTS: Iodixanol reduced tubule cell viability (P < .01) but did not cause apoptosis. The intercellular gap formation (P < .01) and transwell (P < .05) assays revealed that cell permeability significantly increased after Iodixanol treatment of monolayer cells. Western blot and qRT-PCR showed significant upregulation of claudin-2 protein (P < .05) and messenger RNA expression (P < .01). CONCLUSIONS: Our in vitro data do not support the hypothesis that direct kidney cell death from Iodixanol is a major mechanism of contrast-induced nephropathy (CIN). However, increased permeability of proximal tubule epithelium caused by Iodixanol may play an important role in CIN.