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Mutation or disruption of the SH3 and ankyrin repeat domains 3 (SHANK3) gene represents a highly penetrant, monogenic risk factor for autism spectrum disorder, and is a cause of Phelan-McDermid syndrome. Recent advances in gene editing have enabled the creation of genetically engineered non-human-primate models, which might better approximate the behavioural and neural phenotypes of autism spectrum disorder than do rodent models, and may lead to more effective treatments. Here we report CRISPR-Cas9-mediated generation of germline-transmissible mutations of SHANK3 in cynomolgus macaques (Macaca fascicularis) and their F1 offspring. Genotyping of somatic cells as well as brain biopsies confirmed mutations in the SHANK3 gene and reduced levels of SHANK3 protein in these macaques. Analysis of data from functional magnetic resonance imaging revealed altered local and global connectivity patterns that were indicative of circuit abnormalities. The founder mutants exhibited sleep disturbances, motor deficits and increased repetitive behaviours, as well as social and learning impairments. Together, these results parallel some aspects of the dysfunctions in the SHANK3 gene and circuits, as well as the behavioural phenotypes, that characterize autism spectrum disorder and Phelan-McDermid syndrome.
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Conducta Animal , Encéfalo/fisiopatología , Macaca fascicularis/genética , Macaca fascicularis/psicología , Mutación , Proteínas del Tejido Nervioso/genética , Vías Nerviosas/fisiopatología , Animales , Encéfalo/patología , Movimientos Oculares/genética , Femenino , Mutación de Línea Germinal/genética , Herencia/genética , Relaciones Interpersonales , Imagen por Resonancia Magnética , Masculino , Tono Muscular/genética , Vías Nerviosas/patología , Sueño/genética , Vocalización AnimalRESUMEN
Ginsenoside Rg1 (Rg1), one of the active ingredients in Panax ginseng, has been known to regulate many cellular processes. The purpose of this study was to investigate the protective effects of Rg1 on apoptosis in mouse cultured astrocytes in vitro and a mouse model of cerebral ischemia and reperfusion in vivo. The cell apoptosis was measured by fluorescence microplate reader and xCELLigence system and the Ca(2+) overload was recorded by confocal microscopy. The mitochondrial membrane potential and reactive oxygen species (ROS) were determined by flow cytometry. BALB/c mice were subjected to transient middle cerebral artery occlusion (MCAO) and randomly divided into four groups: Sham (sham-operated +0.9% saline), MCAO (MCAO+0.9% saline), Rg1-L (MCAO+Rg1 20 mg/kg) and Rg1-H (MCAO+Rg1 40 mg/kg). Neurological deficit scores, brain water content and infarct volume were evaluated at 24 h after reperfusion. The results showed that Rg1 significantly attenuated H2O2-induced apoptosis in astrocytes. Rg1 efficiently inhibited intracellular Ca(2+) overload, loss of mitochondrial membrane potential, and ROS production in astrocytes. In vivo study, it was also observed that Rg1 markedly reduced the neurological deficit scores, brain edema, and infarct volume in the model mice. These results suggest that Rg1 possesses significant neuroprotective effects, which might be related to the prevention of astrocytes from apoptosis.
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Astrocitos/efectos de los fármacos , Isquemia Encefálica/complicaciones , Ginsenósidos/farmacología , Daño por Reperfusión/tratamiento farmacológico , Animales , Calcio/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Peróxido de Hidrógeno/toxicidad , Masculino , Ratones , Ratones Endogámicos BALB C , Fármacos Neuroprotectores/farmacología , Especies Reactivas de OxígenoRESUMEN
This study investigated the effect of arctigenin (Arc) on the cell activation, cytokines expression, proliferation, and cell-cycle distribution of mouse T lymphocytes. Mouse lymphocytes were prepared from lymph node and treated with Phorbol-12-myristate-13-acetate (PMA)/Ionimycin (Ion) and/or Arc. CD69, CD25, cytokines, proliferation and cell cycle were assayed by flow cytometry. The results showed that, at concentrations of less than 1.00 micromol x L(-1), Arc expressed non-obvious cell damage to cultured lymphocytes, however, it could significantly down-regulate the expression of CD69 and CD25, as well as TNF-alpha, IFN-gamma, IL-2, IL-4, IL-6 and IL-10 on PMA/Ion stimulated lymphocytes. At the same time, Arc could also inhibit the proliferation of PMA/Ion-activated lymphocytes and exhibited lymphocyte G 0/G1 phase cycle arrest. These results suggest that Arc possesses significant anti-inflammatory effects that may be mediated through the regulation of cell activation, cytokines expression and cell proliferation.
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Antiinflamatorios/farmacología , Citocinas/metabolismo , Furanos/farmacología , Lignanos/farmacología , Activación de Linfocitos/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Animales , Antiinflamatorios/aislamiento & purificación , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Arctium/química , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Femenino , Furanos/aislamiento & purificación , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Interleucina-2/metabolismo , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Interleucina-4/metabolismo , Interleucina-6/metabolismo , Ionomicina/farmacología , Lectinas Tipo C/metabolismo , Lignanos/aislamiento & purificación , Ratones , Ratones Endogámicos BALB C , Plantas Medicinales/química , Linfocitos T/citología , Linfocitos T/inmunología , Acetato de Tetradecanoilforbol/farmacología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVE: FTY720 is a potent drug for multiple sclerosis treatment. To biologically address its possible applications to more generalized diseases with aberrant inflammation, we are testing whether FTY720 can function as a lymphocyte cell cycle blocker and activation suppressor via a concanavalin A (ConA)-mediated mouse lymphocyte pan-activation model. METHODS: Mouse lymphocytes were obtained from lymph nodes and subjected to ConA and/or FTY720 treatment. Cell viability was assayed by MTT and mitochondrial assays. Early and late activation, cell cycle, proliferation and intracellular Ca(2+) concentration ([Ca(2+)](i)) were analyzed by flow cytometry. RESULTS: At concentrations of less than 500 nM, FTY720 significantly down-regulated both CD69 and CD25 expressions of T cells, as well as inhibiting proliferation of activated lymphocytes. In addition, FTY720 blocked the ConA-induced mitogenesis, exhibiting lymphocyte G(0)/G(1) phase cell cycle arrest with significant reduction of cells in S and G(2)/M phases. Meanwhile, a significant decline in [Ca(2+)](i) was observed. The correlation of [Ca(2+)](i) and cell cycle arrest were validated by employing a [Ca(2+)](i) inhibitor SK&F 96365 and testing with and without FTY720 treatment. CONCLUSION: We demonstrated that FTY720 induces G(0)/G(1) phase cell cycle arrest, resulting in proliferation inhibition upon lymphocyte pan-activation, which may be related to reduction of overall intracellular Ca(2+) load.
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Inmunosupresores/farmacología , Activación de Linfocitos/inmunología , Glicoles de Propileno/farmacología , Esfingosina/análogos & derivados , Linfocitos T/efectos de los fármacos , Animales , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Concanavalina A/inmunología , Regulación hacia Abajo , Femenino , Clorhidrato de Fingolimod , Subunidad alfa del Receptor de Interleucina-2/inmunología , Lectinas Tipo C/inmunología , Ratones , Ratones Endogámicos BALB C , Esfingosina/farmacología , Linfocitos T/inmunologíaRESUMEN
Mesenchymal stromal cells (MSCs) have shown significant heterogeneity in terms of therapeutic efficacy for inflammatory bowel disease (IBD) treatment, which may be due to an insufficient number of MSCs homing to the damaged tissue of the colon. Engineering MSCs with specific chemokine receptors can enhance the homing ability by lentiviral transduction. However, the unclear specific chemokine profile related to IBD and the safety concerns of viral-based gene delivery limit its application. Thus, a new strategy to modify MSCs to express specific chemokine receptors using mRNA engineering is developed to evaluate the homing ability of MSCs and its therapeutic effects for IBD. We found that CXCL2 and CXCL5 were highly expressed in the inflammatory colon, while MSCs minimally expressed the corresponding receptor CXCR2. Transient expression of CXCR2 in MSC was constructed and exhibited significantly enhanced migration to the inflamed colons, leading to a robust anti-inflammatory effect and high efficacy. Furthermore, the high expression of semaphorins7A on MSCs were found to induce the macrophages to produce IL-10, which may play a critical therapeutic role. This study demonstrated that the specific chemokine receptor CXCR2 mRNA-engineered MSCs not only improves the therapeutic efficacy of IBD but also provides an efficient and safe MSC modification strategy.
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PURPOSE: The present study investigated whether routine baseline platelet parameters(BPPs) detected before clopidogrel therapy in acute non-cardioembolic ischemic stroke(NCIS) could predict early platelet response and future clinical outcomes. RESULTS: The CYP2C19 polymorphisms constituted independent risk factors for LCR. The number of female patients, the incidence of diabetes mellitus (DM), the level of low-density lipoprotein(LDL) cholesterol, and the neutrophil-to-lymphocyte ratio(NLR) were significantly high in the clinical clopidogrel resistance (CCR) group. However, none of the BPPs had a significant association with laboratory clopidogrel resistance (LCR) or discriminated with the cut-off values regarding LCR or CCR. The patients were divided into two groups according to the average mean platelet volume(MPV) or platelet count(PC). We found that the HbA1c level, the number of female patients, and the CCR were higher in the groups with elevated MPV (≥ 10.6fL) and PC (≥ 235 × 109/L); the LCR, the NIHSS score at discharge, and elevated MPV and PC were risk predictors for CCR. MATERIALS AND METHODS: This study included 196 patients with acute NCIS who underwent routine blood tests upon admission, were treated with clopidogrel, and were followed up for 6 months. Early platelet response was assessed and the CYP2C19 genetic variants were screened for. All participants were categorized into either laboratory clopidogrel resistance(LCR) or clinical clopidogrel resistance (CCR) groups. CONCLUSIONS: Elevated baseline MPV and PC before clopidogrel therapy, as well as CYP2C19 gene variants, should be included in a risk algorithm for NCIS. Furthermore, other nongenetic clinical risk factors should be assessed for optimal prediction of the risk for thrombotic events because of individual variability in platelet response to clopidogrel.
RESUMEN
BACKGROUND: Clopidogrel resistance(CR)is found in non-cardioembolic ischemic stroke (NCIS) patients. However, it is still largely unknown how to identify CR in NCIS patients by laboratory and genetic characteristics. METHODS: A total of 95 patients with acute NCIS were recruited. Phosphorylation of the vasodilator stimulated phosphoprotein (VASP) was detected using flow cytometry, and genes(CYP2C19,CYP3A4) were detected using the Sanger method. The baseline of platelet reactivity index (BPRI) before clopidogrel treatment and the platelet reactivity index with clopidogrel treatment (CPRI) for 7 days were measured. Laboratory clopidogrel resistance (LCR) was defined as CPRI of ≥ 50%. Clinical clopidogrel resistance (CCR) was defined as the presence of progressive stroke during hospitalization, stroke recurrence or occurrence of other ischemic vascular events within 6 months. RESULTS: The incidence of LCR was 41.05% and 18.95% developed CCR. The incidence of LCR was significantly higher in GA/AA patients with CYP2C19 (681G > A) (χ2 = 11.16, P = 0.001) and CYP2C19 (636G > A) (χ2 = 4.829, P = 0.028) than in wildtype GG patients. CYP2C19 (681G > A) (OR 6.272, 95%CI 2.162,18.199,P = 0.001) and CYP2C19 (636G > A) (OR: 5.625,95%CI 1.439, 21.583,P = 0.013) were risk factors for LCR. patients with LCR were more likely to develop CCR (χ2 = 6.021, P = 0.014). The probability of CCR was markedly increased in GA/AA patients with CYP2C19 (681G > A) (χ2 = 10.341, P = 0.001). We identified CYP2C19 (681G > A) (OR 7.814, 95%CI 1.816, 33.618 P = 0.006), Essen score (OR 8.351, 95%CI 1.848, 37.745 P = 0.006), and LCR (OR 5.881, 95%CI 1.373, 25.192, P = 0.017) as risk factors for CCR. CONCLUSION: In clinical practice,LCR and CYP2C19 gene polymorphism should be assessed in NCIS patients receiving clopidogrel treatment.
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Isquemia Encefálica/tratamiento farmacológico , Moléculas de Adhesión Celular/genética , Resistencia a Medicamentos/genética , Proteínas de Microfilamentos/genética , Fosfoproteínas/genética , Polimorfismo Genético/genética , Accidente Cerebrovascular/tratamiento farmacológico , Ticlopidina/análogos & derivados , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Isquemia Encefálica/epidemiología , Isquemia Encefálica/genética , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/metabolismo , China/epidemiología , Clopidogrel , Femenino , Marcadores Genéticos/genética , Humanos , Masculino , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/metabolismo , Persona de Mediana Edad , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilación , Inhibidores de Agregación Plaquetaria/uso terapéutico , Polimorfismo de Nucleótido Simple/genética , Prevalencia , Reproducibilidad de los Resultados , Factores de Riesgo , Sensibilidad y Especificidad , Accidente Cerebrovascular/epidemiología , Accidente Cerebrovascular/genética , Ticlopidina/uso terapéutico , Insuficiencia del Tratamiento , Adulto JovenRESUMEN
OBJECTIVE: To investigate the effect of anhydroicaritin (AHI) against zymosan-induced peritonitis in mice. METHODS: Peritonitis was induced in mice by intraperitoneal injection of zymosan. All mice were monitored for systemic toxicity and mortality for 13 d after zymosan or saline administration. In another set of experiments, the peritoneal exudates were collected. The leukocyte numbers and the production of inflammatory cytokines (IL-6, IL-10, TNF-α, MCP-1) were determined by flow cytometry. The release of nitric oxide (NO) was measured by a Griess reagent system. The Ca(2+); influx in bone marrow-derived macrophages was recorded by laser scanning confocal microscopy with Fluo4-AM loading. The expression of iNOS was determined by Western blotting. RESULTS: AHI (4 mg/kg) prolonged survival of peritonitis mice, inhibited massive leukocyte transmigration into the peritoneal cavity, and decreased the overproduction of NO, IL-10, TNF-α, MCP-1 and IL-6. In LPS-stimulated mouse macrophages, AHI (5 µmol/L) pretreatment significantly inhibited the elevation of intracellular Ca(2+);, and markedly decreased iNOS protein expression. CONCLUSION: AHI possesses significant protective effects on the zymosan-induced peritonitis mice, which might be associated with the regulation of Ca(2+); influx in macrophages and iNOS expression.
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Benzopiranos/uso terapéutico , Peritonitis/prevención & control , Animales , Calcio/metabolismo , Citocinas/análisis , Femenino , Recuento de Leucocitos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Óxido Nítrico Sintasa de Tipo II/análisisRESUMEN
Icaritin, an intestinal metabolite of prenylflavonoids from Herba Epimedii, has been known to regulate many cellular processes. The purpose of this study was to investigate the protective effects of icaritin on inflammation in lipopolysaccharide (LPS) stimulated mouse peritoneal macrophages in vitro and zymosan induced peritonitis model in vivo. The release of Nitric oxide (NO) was measured by a Griess reagent system. The phagocytosis, the expression of CD69, the production of inflammatory cytokines and the leukocytes numbers were determined by flow cytometry. The Ca(2+) influx was recorded by confocal microscopy. The phosphorylation of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) was determined by Western blot. The results showed that icaritin significantly inhibited the NO, IL-6, IL-10 TNF-α, and MCP-1 production both in vitro and in vivo. Icaritin efficiently diminished the uptake of nonopsonized pHrodo™-labeled Escherichia coli bacteria on the LPS-stimulated macrophages. In addition, icaritin significantly inhibited the expression of CD69 on CD11b(+) macrophages. Icaritin pretreatment significantly inhibited the elevation of intracellular Ca(2+) induced by LPS. Furthermore, icaritin markedly decreased phospho-p38 and JNK protein expression in LPS-stimulated mouse peritoneal macrophages. In vivo study, it was also observed that icaritin prolonged survival of peritonitis mice, and inhibited massive leukocyte influx into the peritoneal cavity. These results suggest that icaritin possesses significant anti-inflammatory effects that may be mediated through the regulation of inflammatory cytokines and phosphorylation of p38 and JNK.
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Antiinflamatorios/uso terapéutico , Flavonoides/uso terapéutico , Peritonitis/tratamiento farmacológico , Animales , Antiinflamatorios/farmacología , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Calcio/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Flavonoides/farmacología , Lectinas Tipo C/metabolismo , Recuento de Leucocitos , Lipopolisacáridos , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/fisiología , Ratones , Ratones Endogámicos BALB C , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Óxido Nítrico/metabolismo , Peritonitis/metabolismo , Fagocitosis/efectos de los fármacosRESUMEN
AIM: To investigate the effects of anhydroicaritin (AHI) on the immunologic function of mouse macrophages stimulated by lipopolysaccharide (LPS) in vitro and its related immunosuppressive mechanism. METHODS: Mouse bone marrow-derived macrophages were isolated. Then, the drug toxicology of different concentrations of AHI on macrophages was measured by CCK-8 assay. The amount of NO produced in macrophages was detected by Griess kit. The phagocytosis of macrophages to E.coli BioParticles was assayed by flow cytometry (FCM). The expression of CD69, which was the marker of early activation of macrophages, was measured by FCM combined with two-color immunofluorescent staining of cell surface antigen. Cytometric bead array (CBA) kit was used to detect the production of cytokines of macrophages stimulated by LPS. RESULTS: AHI (2.5, 5, 10 µmol/L) significantly reduced the production of NO in macrophages stimulated by LPS, and inhibited the phagocytosis of activated macrophages. The results of FCM analysis showed that AHI decreased proportions of CD69 on LPS-stimulated macrophages. Furthermore, AHI downregulated the secretion of cytokines of LPS-induced macrophages. CONCLUSION: AHI, which exhibits immunosuppressive effect on the mouse macrophages stimulated by LPS, is promising to be developed as an immunosuppressive reagent.
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Flavonoides/farmacología , Flavonoles/farmacología , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Animales , Antígenos CD/inmunología , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/inmunología , Antígenos de Diferenciación de Linfocitos T/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Escherichia coli/inmunología , Flavonoides/química , Citometría de Flujo , Lectinas Tipo C/inmunología , Lectinas Tipo C/metabolismo , Lipopolisacáridos/farmacología , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Óxido Nítrico/inmunología , Óxido Nítrico/metabolismo , Fagocitosis/inmunologíaRESUMEN
Abnormal inflammations are central therapeutic targets in numerous infectious and autoimmune diseases. Dendritic cells (DCs) are involved in these inflammations, serving as both antigen presenters and proinflammatory cytokine providers. As an immuno-suppressor applied to the therapies of multiple sclerosis and allograft transplantation, fingolimod (FTY720) was shown to affect DC migration and its crosstalk with T cells. We posit FTY720 can induce an anergy-polarized phenotype switch on DCs in vitro, especially upon endotoxic activation. A lipopolysaccharide (LPS)-induced mouse bone marrow-derived dendritic cell (BMDC) activation model was employed to test FTY720-induced phenotypic changes on immature and mature DCs. Specifically, methods for morphology, nanostructure, cytokine production, phagocytosis, endocytosis and specific antigen presentation studies were used. FTY720 induced significant alterations of surface markers, as well as decline of shape indices, cell volume, surface roughness in LPS-activated mature BMDCs. These phenotypic, morphological and topographical changes were accompanied by FTY720-mediated down-regulation of proinflammatory cytokines, including IL-6, TNF-α, IL-12 and MCP-1. Together with suppressed nitric oxide (NO) production and CCR7 transcription in FTY720-treated BMDCs with or without LPS activation, an inhibitory mechanism of NO and cytokine reciprocal activation was suggested. This implication was supported by the impaired phagocytotic, endocytotic and specific antigen presentation abilities observed in the FTY720-treated BMDCs. In conclusion, we demonstrated FTY720 can induce anergy-polarization in both immature and LPS-activated mature BMDCs. A possible mechanism is FTY720-mediated reciprocal suppression on the intrinsic activation pathway and cytokine production with endpoint exhibitions on phagocytosis, endocytosis, antigen presentation as well as cellular morphology and topography.
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Células de la Médula Ósea/citología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Glicoles de Propileno/farmacología , Esfingosina/análogos & derivados , Animales , Presentación de Antígeno/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citocinas/biosíntesis , Células Dendríticas/citología , Células Dendríticas/metabolismo , Femenino , Clorhidrato de Fingolimod , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos BALB C , Óxido Nítrico/biosíntesis , Fagocitosis/efectos de los fármacos , Fenotipo , Receptores CCR7/biosíntesis , Esfingosina/farmacologíaRESUMEN
A rise in intracellular Ca(2+) ([Ca(2+)](i)) is crucial for the activation of macrophages, however, the mechanisms and consequences of this [Ca(2+)](i) increase remain unclear. This study investigated the role of calcium in mouse peritoneal macrophages stimulated with LPS plus IFN-γ by using the store-operated Ca(2+) channel (SOCC) blocker SK&F 96365. Our results showed that SK&F 96365 pretreatment significantly inhibited the elevation of [Ca(2+)](i) induced by ionomycin, thapsigargin, and LPS plus IFN-γ, respectively. Phagocytosis analyzing results showed that SK&F 96365 efficiently diminished the uptake of nonopsonized 1 µM yellow-green beads or pHrodo™-labeled Escherichia coli bacteria both on the resting and LPS plus IFN-γ-stimulated macrophages. In addition, SK&F 96365 significantly inhibited the LPS plus IFN-γ-induced brisk uptake of NO and ROS. The CBA analyzing results showed that SK&F 96365 pretreatment efficiently inhibited the production of LPS plus IFN-γ-induced inflammatory cytokines of IL-6, MCP-1, TNF, INF-γ, and IL-10. However, SK&F 96365 pretreatment did not inhibit but augment the production of LPS plus IFN-γ-induced IL-1ß secretion. Furthermore, SK&F 96365 pretreatment inhibited the LPS plus IFN-γ-induced translocation of NF-κB to the nucleus, and induced a decrease in mitochondrial membrane potential (ΔΨm) in LPS plus IFN-γ-activated macrophages. This study provides insight into the role of calcium in the activation of peritoneal macrophages induced by LPS plus IFN-γ, and blocking the calcium influx by SK&F 96365 exhibited a domain inhibitory effect on the LPS plus IFN-γ-induced inflammatory response in macrophages.
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Bloqueadores de los Canales de Calcio/farmacología , Calcio/metabolismo , Imidazoles/farmacología , Inflamación/inmunología , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/efectos de los fármacos , Animales , Canales de Calcio/metabolismo , Células Cultivadas , Quimiocina CCL2/inmunología , Quimiocina CCL2/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/inmunología , Femenino , Inflamación/metabolismo , Interferón gamma/inmunología , Interferón gamma/metabolismo , Interleucina-10/inmunología , Interleucina-10/metabolismo , Interleucina-1beta/inmunología , Interleucina-1beta/metabolismo , Interleucina-6/inmunología , Interleucina-6/metabolismo , Ionomicina/farmacología , Lipopolisacáridos/inmunología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , FN-kappa B/metabolismo , Óxido Nítrico/metabolismo , Fagocitosis/efectos de los fármacos , Fagocitosis/inmunología , Transporte de Proteínas/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Tapsigargina/farmacología , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
PURPOSE: Melanoma is a malignant tumor and causes majority of deaths related to skin cancer. Fibroblast growth factor 2 (FGF2) greatly contributes to melanoma growth and progress. In this paper, we attempt to evaluate the therapeutic potential of FGF2-binding peptide (named P7) using as a potent FGF2 antagonist via exploration of its antitumor effect on melanoma in vitro and in vivo. METHODS: Cell viability was measured by WST-1. Cell cycle progression was determined by propidium iodide staining and flow cytometry. Western blotting was carried out to detect the activation of Erk1/2, P38, Akt, and MEK, and the expression of apoptosis-associated proteins. The influence of P7 on FGF2 internalization was assessed by separation of nuclear and cytoplasmic protein fractions followed by Western blotting. Female C57BL/6 mice bearing xenograft melanoma were established and used to evaluate the antitumor effect of P7 in vivo. RESULTS: In this study, we first proved that P7 peptides significantly inhibited proliferation of FGF2-induced melanoma cell line B16-F10. Further investigations revealed that the mechanisms of P7 peptides inhibiting cell proliferation of melanoma cells stimulated with FGF2 in vitro involved cell cycle arrest at the G0/G1 phase, blockade of the activation of Erk1/2, P38, and Akt cascades, and inhibition of FGF2 internalization. Finally, treatment of P7 peptides in a murine melanoma model resulted in significant inhibition of tumor growth and angiogenesis in vivo, which was associated with blockade of mitogen-activated protein kinase signal activation, and suppression of the expressions of anti-apoptotic Bcl-2 protein and angiogenic factor in the melanoma tumors. CONCLUSIONS: The FGF2-binding peptide with potent antiproliferation and anti-angiogenic activity may have therapeutic potential in melanoma.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Melanoma Experimental/prevención & control , Oligopéptidos/farmacología , Secuencia de Aminoácidos , Animales , Western Blotting , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Endocitosis/efectos de los fármacos , Femenino , Factor 2 de Crecimiento de Fibroblastos/antagonistas & inhibidores , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Citometría de Flujo , Fase G1/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Neovascularización Patológica/prevención & control , Oligopéptidos/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fase de Descanso del Ciclo Celular/efectos de los fármacos , Carga Tumoral/efectos de los fármacosRESUMEN
SK&F 96365, 51-(beta-[3-(p-methoxyphenyl)-propyloxy]-p-methoxyphenethyl)-1H-imidazole hydrochloride, has emerged as a useful pharmacological tool in the study of store-operated Ca²âº entry (SOCE). But the precise molecular mechanism and effect of SK&F 96365 on mouse lymphocytes are still not well determined. This study investigated the pharmacological profile of SK&F 96365 on mouse lymphocytes stimulated by mitogen concanavalin A (Con A) or by a combination of a protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA) and a calcium ionophore, ionomycin in vitro. Our results showed that SK&F 96365 pre-treatment diminished the cytosolic calcium rise on lymphocytes induced by ionomycin, PMA/ionomycin, and thapsigargin (TG), respectively. CFDA-SE staining results showed that SK&F 96365 (5-20 µM) inhibited both Con A- and PMA/ionomycin-induced lymphocytes proliferation in a time- and dose-dependent manner. Upon the same stimulation, SK&F 96365 inhibited the expression of CD69 and CD25 on CD3⺠T lymphocytes in a dose-dependent manner. The cell cycle analyzing results showed that SK&F 96365 caused a G0/G1 phase cell cycle arrest on both Con A- and PMA/ionomycin-activated lymphocytes in a dose-dependent manner. In addition, SK&F 96365 induced a decrease in mitochondrial membrane potential (ΔΨm) and promoted mitochondrial permeability transition (MPT) in both Con A- and PMA/ionomycin-activated lymphocytes. Furthermore, SK&F 96365 significantly inhibited the production of proinflammatory cytokines (interferon (IFN)-γ and tumor necrosis factor (TNF)), and the anti-inflammatory cytokine (IL-10) on both Con A- and PMA/ionomycin-activated lymphocytes. SK&F 96365 did not induce a statistically significant increase in levels of proinflammatory IL-6 and monocyte chemoattractant protein-1 (MCP-1) but of IL-12p70 upon the stimulation of Con A, whereas these three cytokines were markedly inhibited by it upon the stimulation of PMA/ionomycin. This finding revealed that SK&F 96365 exhibited an anti-inflammatory effect on mouse lymphocytes both upon the stimulation of Con A and PMA/ionomycin, and the precise mechanism of SK&F 96365 inhibiting Con A-activated lymphocytes proliferation is different from PMA/ionomycin.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Ciclo Celular/efectos de los fármacos , Imidazoles/farmacología , Inflamación/inmunología , Transducción de Señal/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Animales , Antiinflamatorios/inmunología , Antiinflamatorios/farmacología , Antígenos CD/biosíntesis , Antígenos CD/inmunología , Calcio/inmunología , Bloqueadores de los Canales de Calcio/inmunología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/inmunología , Ciclo Celular/inmunología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Concanavalina A/inmunología , Concanavalina A/farmacología , Citocinas/biosíntesis , Citocinas/inmunología , Relación Dosis-Respuesta a Droga , Femenino , Fluoresceínas/análisis , Colorantes Fluorescentes/análisis , Imidazoles/inmunología , Inflamación/tratamiento farmacológico , Inflamación/patología , Ionomicina/inmunología , Ionomicina/farmacología , Activación de Linfocitos/efectos de los fármacos , Recuento de Linfocitos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Potencial de la Membrana Mitocondrial/inmunología , Ratones , Ratones Endogámicos BALB C , Mitocondrias/efectos de los fármacos , Mitocondrias/inmunología , Transducción de Señal/inmunología , Succinimidas/análisis , Linfocitos T/inmunología , Acetato de Tetradecanoilforbol/inmunología , Acetato de Tetradecanoilforbol/farmacología , Tapsigargina/inmunología , Tapsigargina/farmacologíaRESUMEN
The objective of this study is to illuminate the mechanism of biodegradation of triphenyltin (TPhT). The removal of TPhT by Klebsiella pneumoniae was, therefore, investigated through characteristics studies. The influences of the various parameters were also discussed. The results demonstrated that the cell, extracellular secretion and intracellular enzyme were the effective biomasses for the biodegradation of TPhT. At initial concentration of 3 mg x L(-1), 10.9%, 5.3% and 47.9% of TPhT could be degraded by these biomasses respectively at 30 degrees C within 2 hours under an rotary shaker at 120 r x min(-1). The experimental results also showed that the enzyme activity could be affected by the buffers, pH, temperature, metals and the concentration of TPhT. The degradation efficiency would reach the highest point at pH 8, and at the optimal temperature of 50 degrees C. Metals including Mg2+, Mn2+, Fe2+ and Fe3+ improved the enzyme activity at certain concentrations. In the presence of 15 mg x L(-1) of Mg2+, the removal percentage of TPhT was up to 73.8%. It suggested that the metals activated the enzyme and interacted with the TPhT enabling its removal during the biodegradation process. Linear plots of removal ratios versus concentrations of TPhT meant that the biodegradation fitted the Michaelis-Menten model. The Vmax and Km of this biodegradation were 0. 15 mg x (L x min)(-1) and 47.1 mg x L(-1), respectively.