RESUMEN
Surfactant protein-D (SP-D) is a regulator of pulmonary innate immunity whose oligomeric state can be altered through S-nitrosylation to regulate its signaling function in macrophages. Here, we examined how nitrosylation of SP-D alters the phenotypic response of macrophages to stimuli both in vivo and in vitro. Bronchoalveolar lavage (BAL) from C57BL6/J and SP-D-overexpressing (SP-D OE) mice was incubated with RAW264.7 cells ± LPS. LPS induces the expression of the inflammatory genes Il1b and Nos2, which is reduced 10-fold by SP-D OE-BAL. S-nitrosylation of the SP-D OE-BAL (SNO-SP-D OE-BAL) abrogated this inhibition. SNO-SP-D OE-BAL alone induced Il1b and Nos2 expression. PCR array analysis of macrophages incubated with SP-D OE-BAL (±LPS) shows increased expression of repair genes, Ccl20, Cxcl1, and Vcam1, that was accentuated by LPS. LPS increases inflammatory gene expression, Il1a, Nos2, Tnf, and Ptgs2, which was accentuated by SNO-SP-D OE-BAL but inhibited by SP-D OE-BAL. The transcription factor NF-κB was identified as a target for SNO-SP-D by IPA, which was confirmed by Trans-AM ELISA in vitro. In vivo, SP-D overexpression increases the burden of infection in a Pneumocystis model while increasing cellular recruitment. Expression of iNOS and the production of NO metabolites were significantly reduced in SP-D OE mice relative to C57BL6/J. Inflammatory gene expression was increased in infected C57BL6/J mice but decreased in SP-D OE. SP-D oligomeric structure was disrupted in C57BL6/J infected mice but unaltered within SP-D OE. Thus SP-D modulates macrophage phenotype and the balance of multimeric to trimeric SP-D is critical to this regulation.
Asunto(s)
Macrófagos Alveolares/inmunología , Compuestos Nitrosos/metabolismo , Infecciones por Pneumocystis/genética , Procesamiento Proteico-Postraduccional , Proteína D Asociada a Surfactante Pulmonar/metabolismo , Animales , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/inmunología , Quimiocina CCL20/genética , Quimiocina CCL20/inmunología , Quimiocina CXCL1/genética , Quimiocina CXCL1/inmunología , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/inmunología , Femenino , Inmunidad Innata , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Lipopolisacáridos/farmacología , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/microbiología , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , FN-kappa B/genética , FN-kappa B/inmunología , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/inmunología , Compuestos Nitrosos/inmunología , Fenotipo , Pneumocystis/crecimiento & desarrollo , Pneumocystis/patogenicidad , Infecciones por Pneumocystis/inmunología , Infecciones por Pneumocystis/metabolismo , Infecciones por Pneumocystis/microbiología , Proteína D Asociada a Surfactante Pulmonar/genética , Proteína D Asociada a Surfactante Pulmonar/inmunología , Células RAW 264.7 , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/inmunologíaRESUMEN
Pulmonary lymphangioleiomyomatosis (LAM) is a rare lung disease caused by mutations of the tumor suppressor genes, tuberous sclerosis complex (TSC) 1 or TSC2. LAM affects women almost exclusively, and it is characterized by neoplastic growth of atypical smooth muscle-like TSC2-null LAM cells in the pulmonary interstitium, cystic destruction of lung parenchyma, and progressive decline in lung function. In this study, we hypothesized that TSC2-null lesions promote a proinflammatory environment, which contributes to lung parenchyma destruction. Using a TSC2-null female murine LAM model, we demonstrate that TSC2-null lesions promote alveolar macrophage accumulation, recruitment of immature multinucleated cells, an increased induction of proinflammatory genes, nitric oxide (NO) synthase 2, IL-6, chemokine (C-C motif) ligand 2 (CCL2)/monocyte chemotactic protein 1 (MCP1), chemokine (C-X-C motif) ligand 1 (CXCL1)/keratinocyte chemoattractant (KC), and up-regulation of IL-6, KC, MCP-1, and transforming growth factor-ß1 levels in bronchoalveolar lavage fluid. Bronchoalveolar lavage fluid also contained an increased level of surfactant protein (SP)-D, but not SP-A, significant reduction of SP-B levels, and a resultant increase in alveolar surface tension. Consistent with the growth of TSC2-null lesions, NO levels were also increased and, in turn, modified SP-D through S-nitrosylation, forming S-nitrosylated SP-D, a known consequence of lung inflammation. Progressive growth of TSC2-null lesions was accompanied by elevated levels of matrix metalloproteinase-3 and -9. This report demonstrates a link between growth of TSC2-null lesions and inflammation-induced surfactant dysfunction that might contribute to lung destruction in LAM.
Asunto(s)
Linfangioleiomiomatosis/metabolismo , Linfangioleiomiomatosis/patología , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/patología , Animales , Lavado Broncoalveolar , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Linfangioleiomiomatosis/genética , Metaloproteinasa 3 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Mutantes , Óxido Nítrico/genética , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Proteína A Asociada a Surfactante Pulmonar/genética , Proteína A Asociada a Surfactante Pulmonar/metabolismo , Proteína D Asociada a Surfactante Pulmonar/genética , Proteína D Asociada a Surfactante Pulmonar/metabolismo , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Pulmonary lymphangioleiomyomatosis (LAM), a rare progressive lung disease associated with mutations of the tuberous sclerosis complex 2 (Tsc2) tumor suppressor gene, manifests by neoplastic growth of LAM cells, induction of cystic lung destruction, and respiratory failure. LAM severity correlates with upregulation in serum of the prolymphangiogenic vascular endothelial growth factor D (VEGF-D) that distinguishes LAM from other cystic diseases. The goals of our study was to determine whether Tsc2 deficiency upregulates VEGF-D, and whether axitinib, the Food and Drug Administration-approved small-molecule inhibitor of VEGF receptor (VEGFR) signaling, will reduce Tsc2-null lung lesion growth in a mouse model of LAM. Our data demonstrate upregulation of VEGF-D in the serum and lung lining in mice with Tsc2-null lesions. Progressive growth of Tsc2-null lesions induces recruitment and activation of inflammatory cells and increased nitric oxide production. Recruited cells isolated from the lung lining of mice with Tsc2-null lesions demonstrate upregulated expression of provasculogenic Vegfa, prolymphangiogenic Figf, and proinflammatory Nos2, Il6, and Ccl2 genes. Importantly, axitinib is an effective inhibitor of Tsc2-null lesion growth and inflammatory cell recruitment, which correlates with reduced VEGF-D levels in serum and lung lining. Our data demonstrate that pharmacological inhibition of VEGFR signaling with axitinib inhibits Tsc2-null lesion growth, attenuates recruitment and activation of inflammatory cells, and reduces VEGF-D levels systemically and in the lung lining. Our study suggests a potential therapeutic benefit of inhibition of VEGFR signaling for treatment of LAM.
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Proliferación Celular/efectos de los fármacos , Imidazoles/farmacología , Indazoles/farmacología , Pulmón/efectos de los fármacos , Linfangioleiomiomatosis/tratamiento farmacológico , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Animales , Axitinib , Línea Celular Tumoral , Quimiocina CCL2/metabolismo , Modelos Animales de Enfermedad , Femenino , Pulmón/metabolismo , Enfermedades Pulmonares/tratamiento farmacológico , Enfermedades Pulmonares/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Linfangioleiomiomatosis/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Óxido Nítrico Sintasa de Tipo II/metabolismo , Proteína 2 del Complejo de la Esclerosis Tuberosa , Regulación hacia Arriba/efectos de los fármacos , Factor D de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Surfactant protein D (SP-D) modulates the lung's immune system. Its absence leads to NOS2-independent alveolar lipoproteinosis and NOS2-dependent chronic inflammation, which is critical for early emphysematous remodeling. With aging, SP-D knockout mice develop an additional interstitial fibrotic component. We hypothesize that this age-related interstitial septal wall remodeling is mediated by NOS2. Using invasive pulmonary function testing such as the forced oscillation technique and quasistatic pressure-volume perturbation and design-based stereology, we compared 29-wk-old SP-D knockout (Sftpd(-/-)) mice, SP-D/NOS2 double-knockout (DiNOS) mice, and wild-type mice (WT). Structural changes, including alveolar epithelial surface area, distribution of septal wall thickness, and volumes of septal wall components (alveolar epithelium, interstitial tissue, and endothelium) were quantified. Twenty-nine-week-old Sftpd(-/-) mice had preserved lung mechanics at the organ level, whereas elastance was increased in DiNOS. Airspace enlargement and loss of surface area of alveolar epithelium coexist with increased septal wall thickness in Sftpd(-/-) mice. These changes were reduced in DiNOS, and compared with Sftpd(-/-) mice a decrease in volumes of interstitial tissue and alveolar epithelium was found. To understand the effects of lung pathology on measured lung mechanics, structural data were used to inform a computational model, simulating lung mechanics as a function of airspace derecruitment, septal wall destruction (loss of surface area), and septal wall thickening. In conclusion, NOS2 mediates remodeling of septal walls, resulting in deposition of interstitial tissue in Sftpd(-/-). Forward modeling linking structure and lung mechanics describes the complex mechanical properties by parenchymatous destruction (emphysema), interstitial remodeling (septal wall thickening), and altered recruitability of acinar airspaces.
Asunto(s)
Remodelación de las Vías Aéreas (Respiratorias) , Modelos Biológicos , Óxido Nítrico Sintasa de Tipo II/metabolismo , Alveolos Pulmonares/metabolismo , Proteína D Asociada a Surfactante Pulmonar/deficiencia , Mecánica Respiratoria , Animales , Ratones , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo II/genética , Alveolos Pulmonares/patología , Proteína D Asociada a Surfactante Pulmonar/metabolismoRESUMEN
Current clinical strategies for the delivery of pulmonary therapeutics to the lung are primarily targeted to the upper portions of the airways. However, targeted delivery to the lower regions of the lung is necessary for the treatment of parenchymal lung injury and disease. Here, we have developed an mRNA therapeutic for the lower lung using one-component Ionizable Amphiphilic Janus Dendrimers (IAJDs) as a delivery vehicle. We deliver an anti-inflammatory cytokine mRNA, transforming growth factor-beta (TGF-ß), to produce transient protein expression in the lower regions of the lung. This study highlights IAJD's potential for precise, effective, and safe delivery of TGF-ß mRNA to the lung. This delivery system offers a promising approach for targeting therapeutics to the specific tissues, a strategy necessary to fill the current clinical gap in treating parenchymal lung injury and disease.
RESUMEN
BACKGROUND: Increasing evidence suggests seizures cause blood-brain barrier (BBB) dysfunction including decreased seizure threshold and higher onset potential of future seizures. However, the mechanisms underlying BBB damage in seizures remains poorly understood. Evidence in human and animal models shows BBB disruption is associated with activation of matrix metalloproteinase-9 (MMP-9) after cerebral ischemia and inflammation. The objective of this study was to determine whether MMP-9 concentrations in cerebral spinal fluid (CSF) are associated with BBB disruption in patients after epileptic seizures. METHODS: Thirty-one patients with generalized tonic-clonic (GTC) seizures were included in the study: 20 had recurrent GTC seizures (RS), and 11 had a single GTC seizure (SS) episode. Twenty-five adult non-seizure patients were used as controls. CSF samples were collected by lumbar puncture within 24 h after seizure cessation (range: 3-15 h, mean 6.2 h). CSF MMP-9 levels were determined by an enzyme-linked immunosorbent assay (ELISA). MMP enzyme activity was measured by gelatin zymography. The CSF/serum albumin ratio (albumin quotient, QAlb) was used as a measure of blood-brain barrier permeability. RESULTS: We found significantly higher CSF MMP-9 concentrations in seizure patients compared with controls (P < 0.001). CSF MMP-9 levels and QAlb values were higher in RS patients compared with SS and controls. Moreover, CSF MMP-9 concentration showed strong correlation between QAlb values (r = 0.76, P < 0.0001) and between CSF leukocyte counts (r = 0.77, P < 0.0001) in patients after seizures. Gelatin zymography showed MMP-9 proteolytic activity only in GTC seizure patients. CONCLUSIONS: Our results suggest MMP-9 plays a role in BBB dysfunction, characterized by invasion of leukocytes into the CSF during seizures.
Asunto(s)
Barrera Hematoencefálica/patología , Epilepsia Tónico-Clónica/líquido cefalorraquídeo , Epilepsia Tónico-Clónica/patología , Metaloproteinasa 9 de la Matriz/líquido cefalorraquídeo , Convulsiones/líquido cefalorraquídeo , Convulsiones/patología , Adolescente , Adulto , Anciano , Anticonvulsivantes/uso terapéutico , Recuento de Células , Electroencefalografía , Femenino , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Permeabilidad , Albúmina Sérica/análisis , Tomografía Computarizada por Rayos X , Adulto JovenRESUMEN
Bleomycin causes acute lung injury through production of reactive species and initiation of inflammation. Previous work has shown alteration to the production of reactive oxygen species results in attenuation of injury. Vitamin E, in particular, γ-tocopherol, isoform, has the potential to scavenge reactive oxygen and nitrogen species. This study examines the utility of dietary supplementation with tocopherols in reducing bleomycin-mediated acute lung injury. Male C57BL6/J mice were intratracheally instilled with PBS or 2 units/kg bleomycin. Animals were analyzed 3 and 8 days post instillation at the cellular, tissue, and organ levels. Results showed successful delivery of tocopherols to the lung via dietary supplementation. Also, increases in reactive oxygen and nitrogen species due to bleomycin are normalized in those mice fed tocopherol diet. Injury was not prevented but inflammation progression was altered, in particular macrophage activation and function. Inflammatory scores based on histology demonstrate limited progression of inflammation in those mice treated with bleomycin and fed tocopherol diet compared to control diet. Upregulation of enzymes and cytokines involved in pro-inflammation were limited by tocopherol supplementation. Day 3 functional changes in elastance in response to bleomycin are prevented, however, 8 days post injury the effect of the tocopherol diet is lost. The effect of tocopherol supplementation upon the inflammatory process is demonstrated by a shift in the phenotype of macrophage activation. The effect of these changes on resolution and the progression of pulmonary fibrosis has yet to be elucidated.
Asunto(s)
Antioxidantes/farmacología , Bleomicina/toxicidad , Pulmón/efectos de los fármacos , Óxido Nítrico/metabolismo , Neumonía/metabolismo , Tocoferoles/farmacología , Administración Oral , Animales , Líquido del Lavado Bronquioalveolar/citología , Ciclooxigenasa 2/metabolismo , Pulmón/metabolismo , Pulmón/patología , Lesión Pulmonar/inducido químicamente , Lesión Pulmonar/tratamiento farmacológico , Lesión Pulmonar/metabolismo , Lesión Pulmonar/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa de Tipo II/metabolismo , Neumonía/tratamiento farmacológico , Neumonía/patología , Especies Reactivas de Oxígeno/metabolismo , Pruebas de Función RespiratoriaRESUMEN
Microglia are resident immune cells of the central nervous system. Their persistent activation in neurodegenerative diseases, traditionally attributed to neuronal dysfunction, may be due to a microglial failure to modulate the release of cytotoxic mediators such as nitric oxide (NO). The persistent activation of microglia with the subsequent release of NO vis-á-vis the accumulation of redox transition metals such as copper (Cu) in neurodegenerative diseases, prompted the hypothesis that copper would alter NO signaling by changing the redox environment of the cell and that, by altering the fate of NO, microglia would adopt a different phenotype. We have used the microglial cell model, BV2, to examine the effects of Cu(I) on NO production and activation as they have been shown to be phenotypically plastic. Our results show that cell viability is not affected by Cu(I) in BV2 microglia and that it has no effect on iNOS mRNA, protein expression and nitrite release. However, when LPS is added to Cu(I)-treated medium, nitrite release is abrogated while iNOS expression is not significantly altered. This effect is Cu(I)-specific and it is not observed with other non-redox metals, suggesting that Cu(I) modulates NO reactivity. Immunofluorescence analysis shows that the M1 (inflammatory) phenotype of BV2 microglia observed in response to LPS, is shifted to an M2 (adaptive) phenotype when Cu(I) is administered in combination with LPS. This same shift is not observed when iNOS function is inhibited by 1400W. In the present study we show that Cu(I) modulates the release of NO to the media, without altering iNOS expression, and produces phenotypic changes in BV2 microglia.
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Cobre/farmacología , Microglía/metabolismo , Óxido Nítrico/metabolismo , Animales , Línea Celular , Supervivencia Celular , Técnica del Anticuerpo Fluorescente , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Ratones , Enfermedades Neurodegenerativas/metabolismo , FenotipoRESUMEN
RATIONALE: The pulmonary phenotype of Hermansky-Pudlak syndrome (HPS) in adults includes foamy alveolar type 2 cells, inflammation, and lung remodeling, but there is no information about ontogeny or early disease mediators. OBJECTIVES: To establish the ontogeny of HPS lung disease in an animal model, examine disease mediators, and relate them to patients with HPS1. METHODS: Mice with mutations in both HPS1/pale ear and HPS2/AP3B1/pearl (EPPE mice) were studied longitudinally. Total lung homogenate, lung tissue sections, and bronchoalveolar lavage (BAL) were examined for phospholipid, collagen, histology, cell counts, chemokines, surfactant protein D (SP-D), and S-nitrosylated SP-D. Isolated alveolar epithelial cells were examined for expression of inflammatory mediators, and chemotaxis assays were used to assess their importance. Pulmonary function test results and BAL from patients with HPS1 and normal volunteers were examined for clinical correlation. MEASUREMENTS AND MAIN RESULTS: EPPE mice develop increased total lung phospholipid, followed by a macrophage-predominant pulmonary inflammation, and lung remodeling including fibrosis. BAL fluid from EPPE animals exhibited early accumulation of both SP-D and S-nitrosylated SP-D. BAL fluid from patients with HPS1 exhibited similar changes in SP-D that correlated inversely with pulmonary function. Alveolar epithelial cells demonstrated expression of both monocyte chemotactic protein (MCP)-1 and inducible nitric oxide synthase in juvenile EPPE mice. Last, BAL from EPPE mice and patients with HPS1 enhanced migration of RAW267.4 cells, which was attenuated by immunodepletion of SP-D and MCP-1. CONCLUSIONS: Inflammation is initiated from the abnormal alveolar epithelial cells in HPS, and S-nitrosylated SP-D plays a significant role in amplifying pulmonary inflammation.
Asunto(s)
Modelos Animales de Enfermedad , Síndrome de Hermanski-Pudlak , Ratones , Neumonía/etiología , Alveolos Pulmonares/fisiopatología , Proteína D Asociada a Surfactante Pulmonar/metabolismo , Mucosa Respiratoria/fisiopatología , Envejecimiento/metabolismo , Animales , Movimiento Celular , Quimiocina CCL2/metabolismo , Factores Quimiotácticos/metabolismo , Citocinas/metabolismo , Fibrosis , Síndrome de Hermanski-Pudlak/fisiopatología , Humanos , Pulmón/metabolismo , Macrófagos/patología , Ratones Endogámicos C57BL , Ratones Mutantes , Compuestos Nitrosos/metabolismo , Fosfolípidos/metabolismo , Alveolos Pulmonares/patología , Índice de Severidad de la Enfermedad , Factores de TiempoRESUMEN
OBJECTIVE: To investigate the protective effects of three Polyphenolic compounds on intestinal microbial communities in mice exposed intermittent plateau hypoxia. METHODS: In this study, 60 healthy male Balb/c mice were randomly divided into plain control group, plateau control group, primary anthocyanin intervention group, quercetin intervention group and resveratrol intervention group, 12 mice in each group. Primary anthocyanin, quercetin and resveratrol were administrated by gavage at the doses of 50, 100 and 20 mg/kg in pharmacological intervention group, respectively. After exposure of the mice to simulation plateau-condition for 30 days, the serum samples were collected for DAO testing, sterile feces were collected in mice, and the diversity and genus level of the mouse gut bacteria were detected by using 16S rRNA technology. Ileum tissue was fixed and stained with HE. RESULTS: HE staining showed that the plateau control group had significant damage to the intestinal tissue structure compared to the plain control group, and the serum DAO concentration was increased (Pï¼0.05), but there was no statistical difference in the abundance and diversity of intestinal flora species. Contrast to simulated intermittent plateau hypoxia group, the structure of the intestine tissue and the level of DAO in the quercetin intervention group and resveratrol intervention group were improved(Pï¼0.05), the abundance and α diversity of the intestinal flora were decreased, the relative abundance of Bacteroidetes was reduced(Pï¼0.05), and the Firmicutes was increased. Concomitantly, significant decreases in relative abundance were observed for Corynebacterium glutamicum and Lactobacillus reuteri(Pï¼ 0.05). CONCLUSION: Quercetin and resveratrol showed some degree of protection to mice intestinal microbial communities, and increased the diversity and the abundance of the dominant flora and inhibited the growth of conditional pathogenic bacteria.
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Microbioma Gastrointestinal , Ratones , Masculino , Animales , Microbioma Gastrointestinal/genética , ARN Ribosómico 16S/genética , Quercetina/farmacología , Resveratrol/farmacología , Antocianinas/farmacología , Bacterias/genética , HipoxiaRESUMEN
Objective: To investigate the effects of a self-designed nutritional preparation on hypothalamic-pituitary-ovarian (HPO) axis function and energy metabolism in female SD rats exposed to intermittent cold. Methods: Female SD rats were divided into control group, cold exposure group and nutritional preparation group. The control group and cold exposure group were given distilled water by daily gavage, and the nutritional preparation group was given nutritional preparation intragastrically. After the treatment, the cold exposure group and nutritional preparation group were exposed to -10â in a cabin for 4 h every day. After being treated for 14 days, the serum, uterus and ovary of rats were collected. The serum follicle stimulating hormone (FSH), luteinizing hormone (LH) and other hormone indicators were detected by enzyme-linked immunosorbent assay (ELISA) and colorimetry was used to detect ATPase and other energy metabolism related indicators. Results: Compared with the control group, cold exposure significantly up-regulated the protein expressions of FSHR and LHR, and notably enhanced the activity of Na+-K+-ATPase and Ca2+-Mg2+-ATPase in ovary and uterus (P<0.05). Nutritional preparation down-regulated the protein expressions of FSHR and LHR, and inhibited the activity of ATPase in ovary and uterus (P<0.05) compared with the cold exposure group. Conclusion: Nutritional preparations can effectively improve the expressions of HPO axis related receptors and abnormal energy metabolism in uterus and ovary caused by intermittent cold exposure.
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Ovario , Útero , Animales , Femenino , Ratas , Adenosina Trifosfatasas/metabolismo , Metabolismo Energético , Ratas Sprague-Dawley , Útero/metabolismo , FríoRESUMEN
Objective: To investigate the anti-fatigue effects of composition of Moringa oleifera leaves and Polygonatum polysaccharide, and to explore the mechanisms. Methods: Thirty male Kunming mice were randomly divided into control (C) and composition of Moringa oleifera leaves and Polygonatum polysaccharide group (MP). There were 15 mice in each group. Group C was given distilled water and the group MP was given composition intragastriclly every day. The volume was 0.5 ml. After 14 days of treatment, weight-bearing swimming experiment was conducted, and exhaustive swimming time was recorded. The bearing weight was 3% of the body weight. In another experiment, 48 male Kunming mice were randomly divided into quiet control group (QC), swimming control group (SC) and composition group (MP). There were 16 mice in each group. The QC and SC groups were given distilled water intragastrically, and the group MP was treated with composition every day for 14 days. The volume was 0.5 ml. On the day 15, 30 minutes after intragastriclly administration of distilled water, blood, liver and hind leg muscle of the QC group were collected immediately. The SC and MP groups were subjected non-weight-bearing swimming experiment, and blood, liver and hind leg muscle were collected after swimming. The fatigue related indexes, oxidant/antioxidant parameters and energy metabolism indicators in serum and tissues were determined by commercial kits. Results: The exhaustive swimming time of mice in MP group was significantly longer than that in the C group (P<0.05). Compared with the control group, non-weight-bearing swimming decreased the contents of serum glucose and GSH, the contents of hepatic glycogen and ATP, the hepatic activities of SOD, LDH and ATPase, and muscle activity of GSH-Px (P< 0.05). However, serum levels of BUN and MDA were increased (P<0.05). Compared with the SC group, the composition remarkably increased the contents of serum glucose and hepatic glycogen, increased serum content of GSH, enhanced hepatic activities of SOD, LDH and ATPase and muscle activity of GSH-Px, and increased the hepatic content of ATP (P<0.05). However, the serum level of BUN was decreased (P<0.05). Conclusion: The Moringa oleifera leaves and Polygonatum polysaccharide composition possesses anti-fatigue effects. Anti-oxidant and improving energy metabolism could be the important mechanisms.
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Moringa oleifera , Polygonatum , Masculino , Ratones , Animales , Moringa oleifera/metabolismo , Polygonatum/metabolismo , Glucógeno Hepático , Polisacáridos/farmacología , Antioxidantes , Superóxido Dismutasa/metabolismo , Adenosina Trifosfatasas , Glucosa , Agua , Adenosina TrifosfatoRESUMEN
The pulmonary collectins, surfactant proteins A and D (SP-A and SP-D) have been implicated in the regulation of the innate immune system within the lung. In particular, SP-D appears to have both pro- and anti-inflammatory signaling functions. At present, the molecular mechanisms involved in switching between these functions remain unclear. SP-D differs in its quaternary structure from SP-A and the other members of the collectin family, such as C1q, in that it forms large multimers held together by the N-terminal domain, rather than aligning the triple helix domains in the traditional "bunch of flowers" arrangement. There are two cysteine residues within the hydrophobic N terminus of SP-D that are critical for multimer assembly and have been proposed to be involved in stabilizing disulfide bonds. Here we show that these cysteines exist within the reduced state in dodecameric SP-D and form a specific target for S-nitrosylation both in vitro and by endogenous, pulmonary derived nitric oxide (NO) within a rodent acute lung injury model. S-nitrosylation is becoming increasingly recognized as an important post-translational modification with signaling consequences. The formation of S-nitrosothiol (SNO)-SP-D both in vivo and in vitro results in a disruption of SP-D multimers such that trimers become evident. SNO-SP-D but not SP-D, either dodecameric or trimeric, is chemoattractive for macrophages and induces p38 MAPK phosphorylation. The signaling capacity of SNO-SP-D appears to be mediated by binding to calreticulin/CD91. We propose that NO controls the dichotomous nature of this pulmonary collectin and that posttranslational modification by S-nitrosylation causes quaternary structural alterations in SP-D, causing it to switch its inflammatory signaling role. This represents new insight into both the regulation of protein function by S-nitrosylation and NO's role in innate immunity.
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Lesión Pulmonar Aguda/inmunología , Inflamación/inmunología , Óxido Nítrico/metabolismo , Proteína D Asociada a Surfactante Pulmonar/metabolismo , S-Nitrosotioles/metabolismo , Transducción de Señal , Animales , Cisteína/química , Cisteína/metabolismo , Dimerización , Regulación de la Expresión Génica , Humanos , Inmunidad Innata , Inflamación/metabolismo , Pulmón/inmunología , Pulmón/metabolismo , Macrófagos , Masculino , Ratones , Proteína D Asociada a Surfactante Pulmonar/química , Proteína D Asociada a Surfactante Pulmonar/genética , Proteína D Asociada a Surfactante Pulmonar/inmunología , Ratas , Ratas Sprague-Dawley , Mucosa Respiratoria/inmunologíaRESUMEN
Nitric oxide and secondary oxides of nitrogen react with unsaturated fatty acids such as linoleic acid to yield oxidized and nitrated products. Fatty acid nitroalkene derivatives, (e.g. nitrolinoleate [LNO(2)]) are produced by oxidative inflammatory reactions, detected clinically, display potent electrophilic reactivity and induce post-translational protein modifications that mediate adaptive inflammatory signaling responses. LNO(2) signaling was examined in lung epithelial cells because the alveolar compartment is a rich site for the transduction of redox and inflammatory reactions. LNO(2) did not directly induce Ca(2+) influx in cultured lung epithelial cells, but inhibited bradykinin-induced Ca(2+) influx in a cGMP-independent manner. In contrast, LNO(2) activated MAP kinase (Erk1/2) by a mechanism independent of bradykinin. It was hypothesized that these unique responses were transduced by activation of different protein kinase C isotypes, supported by the observation that LNO(2)-mediated inhibition of Ca(2+) influx was blocked by the non-selective PKC inhibitors chelerythine chloride and calphostin C, but not by the calcium dependent "classic" PKC inhibitor Gö6976. Western blot analysis showed that atypical PKCζ was activated by LNO(2) stimulation, with PKCζ and Erk activation also demonstrated in primary culture of human lung type II cells. Addition of pseudotypical PKCζ substrate peptide reversed LNO(2)-mediated induction of Ca(2+) influx and MAP kinase activation. Finally, the electrophilic nature of LNO(2) resulted in a novel mode of PKCζ activation, covalent adduction of the enzyme. In summary, LNO(2) mediated signaling in lung type II epithelial cells occurs via a unique pathway involving PKCζ.
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Células Epiteliales/metabolismo , Ácidos Grasos/metabolismo , Pulmón/citología , Proteína Quinasa C/metabolismo , Transducción de Señal , Alquenos/metabolismo , Humanos , Células Tumorales CultivadasRESUMEN
Pneumocystis pneumonia (PCP), the most common opportunistic pulmonary infection associated with HIV infection, is marked by impaired gas exchange and significant hypoxemia. Immune reconstitution disease (IRD) represents a syndrome of paradoxical respiratory failure in patients with active or recently treated PCP subjected to immune reconstitution. To model IRD, C57BL/6 mice were selectively depleted of CD4(+) T cells using mAb GK1.5. Following inoculation with Pneumocystis murina cysts, infection was allowed to progress for 2 wk, GK1.5 was withdrawn, and mice were followed for another 2 or 4 wk. Flow cytometry of spleen cells demonstrated recovery of CD4(+) cells to >65% of nondepleted controls. Lung tissue and bronchoalveolar lavage fluid harvested from IRD mice were analyzed in tandem with samples from CD4-depleted mice that manifested progressive PCP for 6 wks. Despite significantly decreased pathogen burdens, IRD mice had persistent parenchymal lung inflammation, increased bronchoalveolar lavage fluid cellularity, markedly impaired surfactant biophysical function, and decreased amounts of surfactant phospholipid and surfactant protein (SP)-B. Paradoxically, IRD mice also had substantial increases in the lung collectin SP-D, including significant amounts of an S-nitrosylated form. By native PAGE, formation of S-nitrosylated SP-D in vivo resulted in disruption of SP-D multimers. Bronchoalveolar lavage fluid from IRD mice selectively enhanced macrophage chemotaxis in vitro, an effect that was blocked by ascorbate treatment. We conclude that while PCP impairs pulmonary function and produces abnormalities in surfactant components and biophysics, these responses are exacerbated by IRD. This worsening of pulmonary inflammation, in response to persistent Pneumocystis Ags, is mediated by recruitment of effector cells modulated by S-nitrosylated SP-D.
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Síndrome Inflamatorio de Reconstitución Inmune/inmunología , Neumonía por Pneumocystis/inmunología , Proteína D Asociada a Surfactante Pulmonar/metabolismo , Animales , Western Blotting , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/inmunología , Linfocitos T CD4-Positivos/inmunología , Electroforesis en Gel de Poliacrilamida , Citometría de Flujo , Síndrome Inflamatorio de Reconstitución Inmune/complicaciones , Síndrome Inflamatorio de Reconstitución Inmune/metabolismo , Pulmón/inmunología , Pulmón/microbiología , Pulmón/patología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Neumonía por Pneumocystis/complicaciones , Neumonía por Pneumocystis/metabolismo , Proteína B Asociada a Surfactante Pulmonar/inmunología , Proteína B Asociada a Surfactante Pulmonar/metabolismo , Proteína D Asociada a Surfactante Pulmonar/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
OBJECTIVE: To explore metabolic changes after acute hypoxia and modulating effect of vitamins B1, B2, and PP supplementation in mice exposed to acute hypoxia. METHODS: Fifty male Kunming mice were randomly divided into 5 groups: normal, acute hypoxia, acute hypoxia with 2, 4 and 8 time-vitamins B1, B2, and PP supplementation. All mice were fed with corresponding diets for two weeks and then were exposed to a simulated altitude of 6,000 meters for 8 h, except for the normal group. Nuclear magnetic resonance analysis was used to identify the changes of serum metabolic profiles. RESULTS: There were significant changes in some serum metabolites under induced acute hypoxia, essentially relative increase in the concentrations of lactate, sugar and lipids and decrease in ethanol. The serum levels of choline, succinate, taurine, alanine, and glutamine also increased and phosphocholine decreased in the acute hypoxia group. After vitamins B1, B2, and PP supplementation, all these metabolic changes gradually recovered. CONCLUSIONS: Significant changes in serum metabolic profile were observed by metabolomics in mice exposed to acute hypoxia, and vitamins B1, B2, and PP supplementation proved to be beneficial to improving some metabolic pathways. It is suggested that the dietary intakes of vitamins B1, B2, and PP should be increased under hypoxia condition.
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Hipoxia/sangre , Hipoxia/metabolismo , Metabolómica/métodos , Fenómenos Fisiológicos de la Nutrición/efectos de los fármacos , Complejo Vitamínico B/uso terapéutico , Enfermedad Aguda , Animales , Relación Dosis-Respuesta a Droga , Metabolismo de los Lípidos/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Masculino , Ratones , Ratones Endogámicos , Niacinamida/administración & dosificación , Niacinamida/uso terapéutico , Análisis de Componente Principal , Riboflavina/administración & dosificación , Riboflavina/uso terapéutico , Tiamina/administración & dosificación , Tiamina/uso terapéutico , Complejo Vitamínico B/administración & dosificaciónRESUMEN
OBJECTIVE: To investigate whether the gene transfer of phospholamban antisense RNA could inhibit remodeling and preserve cardiac function after myocardial infarction. METHODS: Wistar rats received a ligation of left coronary with a direct intramyocardial injection of phospholamban antisense RNA eukaryote vector PcDNA4-asPLB. The cardiac function, hemodynamics and ventricular geometry of three groups (shame, saline injection and PcDNA4-asPLB injection) were studied by echocardiography and left ventricle hemodynamic recording. The levels of phospholamban (PLB) and sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) were analyzed by Western blot and the expressions of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) examined by RT-PCR. The histological study was performed to evaluate the collage content and cardiomyocyte fiber size. RESULTS: The PcDNA4-asPLB injection group had significantly better systolic cardiac function and diastolic function [LVEF (39.4 +/- 7.8)% vs (30.9 +/- 7.4)%, P < 0.05; dp/dt Max (1545 +/- 127) mm Hg x s(-1) vs (1172 +/- 91) mm Hg x s(-1), P < 0.05)]. Compared with saline injection, the PLB expression was inhibited by 50% in PcDNA4-asPLB injection group (PLB/beta-actin ratio, 0.28 +/- 0.07 vs 0.57 +/- 0.11, P < 0.05) and the function of SERCA2a was enhanced [(1.47 +/- 0.21) micromol x min(-1) x g(-1) protein vs (0.34 +/- 0.13) micromol x min(-1) x g(-1) protein, P < 0.05]. The expressions of ANP and BNP in the saline injection group were elevated as compared to those in the PcDNA4-asPLB injection group. Histological study also showed that the collage density and the cardiomyocyte fiber size in the saline injection group were worse than those in the PcDNA4-asPLB injection group. CONCLUSION: Intramyocardial injection of phospholamban antisense RNA eukaryote vector PcDNA4-asPLB after myocardial infarction results in PLB expression inhibition, attenuates ventricular remodeling and improves systolic and diastolic cardiac functions.
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Proteínas de Unión al Calcio/genética , Insuficiencia Cardíaca/etiología , Infarto del Miocardio/complicaciones , Infarto del Miocardio/fisiopatología , ARN sin Sentido , Animales , Insuficiencia Cardíaca/fisiopatología , Masculino , Ratas , Ratas Wistar , Transfección , Remodelación VentricularRESUMEN
OBJECTIVE: To investigate the effects of mitogen-activated protein kinases (MAPKs) inhibitors on glutathione (GSH) metabolism, and to explore the pathway related to GSH metabolism. METHODS: BRL rat hepatocytes were treated by c-Jun NH2-terminal kinase (JNK),p38, and extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitors:SP600125, SB203580 and PD98659, respectively, for 24 h. MTT method was used to measure hepatocytes viability. The content of GSH was determined by high performance liquid chromatography. The protein expressions of JNK and phosphorylated JNK (p-JNK) was tested by Luminex method. Activities of GSH metabolic enzymes were detected by commercial kits. RESULTS: Hepatocytes vitality was inhibited when the concentrations of SP600125, SB203580 and PD98659 were higher than 10 µmol/L, 20 µmol/L, and 40 µmol/L, respectively; SP600125 decreased the content of GSH in hepatocytes, while SB203580 and PD98659 had no effect. SP600125 reduced p-JNK protein expression, and enhanced GSH-Px activity significantly. CONCLUSIONS: JNK MAPK pathway takes part in the GSH metabolism in hepatocytes.
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Inhibidores Enzimáticos/farmacología , Glutatión/metabolismo , Hepatocitos/enzimología , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Animales , Células Cultivadas , Hepatocitos/efectos de los fármacos , RatasRESUMEN
Microglia, the primary resident immune cells of the central nervous system (CNS), responds rapidly to pathogens and injury by secreting immune mediators including nitric oxide (NO). The reaction of NO with the anti-oxidant glutathione forms S-nitrosoglutathione (GSNO), the major pool of biologic NO in the body. GSNO is degraded by GSNO reductase (GSNOR). Recently, we have shown that copper (Cu(I)) inhibits the release of NO in lipopolysaccharide (LPS)-stimulated BV2 microglia and induces BV2 microglia to acquire a mixed a profile with both pro- and anti-inflammatory characteristics. Since GSNOR is the critical enzyme in GSNO metabolism, we sought to determine whether Cu(I) affects GSNOR activity and S-nitrosothiol (SNO) accumulation in activated BV2 microglia. Our results show that GSNOR protein expression is reduced by Cu(I) treatment in LPS-stimulated BV2 microglia. Our results also show a decrease in S-nitrosothiol content despite a reduced GSNOR expression. This effect is most likely due to Cu(I) reacting with the central thiol of the SNO bond resulting in the degradation of SNO. A dose of 1 µM Cu(I) did not affect SNO protein accumulation in LPS-stimulated BV2 microglia, however, a dose of 100 µM Cu(I) inhibited SNO protein in accordance with inhibition of S-nitrosothiols. These data provide direct evidence that Cu(I) disrupts S-nitrosothiol homeostasis and NO metabolism, and, thus, provide new insights into the mechanisms involved in microglia-mediated-CNS disorders.
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Cobre/toxicidad , Microglía/metabolismo , S-Nitrosotioles/antagonistas & inhibidores , S-Nitrosotioles/metabolismo , Transducción de Señal/fisiología , Animales , Línea Celular Transformada , Glutatión/análogos & derivados , Glutatión/antagonistas & inhibidores , Glutatión/metabolismo , Ratones , Microglía/efectos de los fármacos , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico/metabolismo , Nitritos/antagonistas & inhibidores , Nitritos/metabolismo , Nitrocompuestos/antagonistas & inhibidores , Nitrocompuestos/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
OBJECTIVE: To observe the effects of fruit juices with different antioxidant capacity on antioxidant system function of aged rats. METHODS: Thirty Wistar rats were randomly divided into three groups: pomegranate juice and apple juice as two experimental groups, while distilled water as normal control group. They were administrated fruit juices or distilled water respectively by gavage daily for 4 weeks. At the end of experiment, the antioxidant system function was assessed. RESULTS: The aged rats in pomegranate juice group showed significantly higher serum antioxidant capacity (0.90 +/- 0.13) mmol/L than that in control group (0.79 +/- 0.10) mmol/L (P < 0.05). The concentrations of serum carbonyl and oxLDL were decreased significantly in pomegranate juice group as compared to the control group (P < 0.05). The percentage of injured blood lymphocyte DNA and the ratio of tail length/total length were declined significantly in pomegranate juice group (P < 0.05 and P < 0.01 respectively). The apple juice showed no effects except decreased ratio of tail length/total length of injured lymphocyte DNA. There were no changes in concentrations of serum vitamin C, vitamin E, urinary 8-OH-dG excretion and the activities of serum SOD, GSH-Px, CAT among three groups. CONCLUSIONS: The pomegranate juice should possess higher antioxidant capacity and might improve the antioxidant system function of aged rats, while the apple juice is relatively lower in antioxidant capacity and not very effective. The polyphenols in pomegranate juice might be the important functional components.