RESUMEN
BACKGROUND: Alcohol intake predisposes to infections and sepsis. Alcohol and sepsis inhibit the expression of milk fat globule epidermal growth factor-factor VIII (MFG-E8), a glycoprotein essential for optimal efferocytosis, resulting in the release of proinflammatory molecules and increased sepsis severity. We previously reported that recombinant mouse (rm) MFG-E8 attenuates sepsis-induced organ injury in rats with acute alcohol intoxication. In order to develop a therapy that can be safely used in humans, we have produced recombinant human (rh) MFG-E8 and evaluated its efficacy to ameliorate sepsis after acute exposure to alcohol. METHODS: We induced acute alcohol intoxication with a bolus injection of alcohol (1.75 g/kg BW) followed by an intravenous infusion of 300 mg/kg/h alcohol for 10 h. Sepsis was then induced by cecal ligation and puncture (CLP). At -10, 0, and 10 h relative to CLP, rats received MFG-E8 or vehicle (albumin) intravenously. Animals were euthanized at 20 h after CLP for blood and tissue collection. Additional groups of animals were used for a survival study. RESULTS: Compared to vehicle, rhMFG-E8 treatment ameliorated blood levels of proinflammatory cytokines (% improvement: TNF-α 49.8%, IL-6 34.7%) and endotoxin (61.7%), as well as of transaminases (AST 36.2%, ALT 40.1%) and lactate (18.4%). Rats treated with rhMFG-E8 also had a significant histological attenuation of the acute lung injury, as well as a reduction in the number of apoptotic cells in the thymus (43.4%) and cleaved caspase 3 (38.7%) in the spleen. In addition, rhMFG-E8 improved the 10-day sepsis survival rate from 45 to 80% CONCLUSION: rhMFG-E8 significantly ameliorated sepsis in rats with acute alcohol exposure, demonstrating rhMFG-E8's potential to be developed as an effective therapy for sepsis in alcohol abusers.
Asunto(s)
Alcoholes/efectos adversos , Antígenos de Superficie/farmacología , Proteínas de la Leche/farmacología , Proteínas Recombinantes/farmacología , Sepsis , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/patología , Animales , Apoptosis/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratas , Ratas Sprague-Dawley , Sepsis/inducido químicamente , Sepsis/metabolismo , Sepsis/mortalidadRESUMEN
BACKGROUND: Cardiovascular dysfunction, characterized by reduced cardiac contractility and depressed endothelium-dependent vascular relaxation, is common in severe sepsis. Although it is known that ghrelin produces beneficial effects following various adverse circulatory conditions, it remains unknown whether ghrelin increases cardiac contractility and improves vascular responsiveness to vasoactive agents in severe sepsis. METHODS: Male adult rats were subjected to sepsis by cecal ligation and puncture (CLP). At 5 h after CLP, a bolus intravenous injection of 2 nmol ghrelin was followed by a continuous infusion of 12 nmol ghrelin via a primed mini-pump over 15 h. At 20 h after CLP (i.e., severe sepsis), the maximal rates of ventricular pressure increase (+dP/dt(max)) and decrease (-dP/dt(max)) were determined in vivo. In additional groups of animals, the thoracic aortae were isolated at 20 h after CLP. The aortae were cut into rings, and placed in organ chambers. Norepinephrine (NE) was used to induce vascular contraction. Dose responses for an endothelium-dependent vasodilator, acetylcholine (ACh), and an endothelium-independent vasodilator, nitroglycerine (NTG) were carried out. RESULTS: +dP/dt(max) and -dP/dt(max) decreased significantly at 20 h after CLP. Treatment with ghrelin significantly increased +dP/dt(max) and -dP/dt(max) by 36% (P < 0.05) and 35% (P < 0.05), respectively. Moreover, NE-induced vascular contraction and endothelium-dependent (ACh-induced) vascular relaxation decreased significantly at 20 h after CLP. Administration of ghrelin, however, increased NE-induced vascular contraction and ACh-induced vascular relaxation. In contrast, no significant reduction in NTG-induced vascular relaxation was seen in rats with severe sepsis irrespective of ghrelin treatment. CONCLUSIONS: Ghrelin may be further developed as a useful agent for maintaining cardiovascular stability in severe sepsis.
Asunto(s)
Presión Sanguínea/efectos de los fármacos , Ghrelina/farmacología , Contracción Miocárdica/efectos de los fármacos , Sepsis/tratamiento farmacológico , Vasodilatación/efectos de los fármacos , Acetilcolina/farmacología , Animales , Análisis de los Gases de la Sangre , Presión Sanguínea/fisiología , Ciego/lesiones , Modelos Animales de Enfermedad , Hematócrito , Hemoglobinas/metabolismo , Bombas de Infusión , Masculino , Contracción Miocárdica/fisiología , Norepinefrina/farmacología , Ratas , Ratas Sprague-Dawley , Sepsis/fisiopatología , Índice de Severidad de la Enfermedad , Vasoconstrictores/farmacología , Vasodilatación/fisiología , Vasodilatadores/farmacología , Función Ventricular Izquierda/efectos de los fármacos , Función Ventricular Izquierda/fisiología , Heridas Punzantes/fisiopatologíaRESUMEN
Stroke is a leading cause of death and the primary medical cause of acquired adult disability worldwide. The progressive brain injury after acute stroke is partly mediated by ischemia-elicited inflammatory responses. The vasoactive hormone adrenomedullin (AM), upregulated under various inflammatory conditions, counterbalances inflammatory responses. However, regulation of AM activity in ischemic stroke remains largely unknown. Recent studies have demonstrated the presence of a specific AM binding protein (that is, AMBP-1) in mammalian blood. AMBP-1 potentiates AM biological activities. Using a rat model of focal cerebral ischemia induced by permanent middle cerebral artery occlusion (MCAO), we found that plasma levels of AM increased significantly, whereas plasma levels of AMBP-1 decreased significantly after stroke. When given peripherally early after MCAO, exogenous human AM in combination with human AMBP-1 reduced brain infarct volume 24 and 72 h after MCAO, an effect not observed after the treatment by human AM or human AMBP-1 alone. Furthermore, treatment of human AM/AMBP-1 reduced neuron apoptosis and morphological damage, inhibited neutrophil infiltration in the brain and decreased serum levels of S100B and lactate. Thus, human AM/AMBP-1 has the ability to reduce stroke-induced brain injury in rats. AM/AMBP-1 can be developed as a novel therapeutic agent for patients with ischemic stroke.
Asunto(s)
Adrenomedulina/farmacología , Apoptosis/efectos de los fármacos , Lesiones Encefálicas/prevención & control , Factor H de Complemento/farmacología , Adrenomedulina/sangre , Adrenomedulina/genética , Animales , Western Blotting , Lesiones Encefálicas/etiología , Lesiones Encefálicas/metabolismo , Isquemia Encefálica/complicaciones , Cardiotónicos/sangre , Cardiotónicos/metabolismo , Cardiotónicos/farmacología , Factor H de Complemento/genética , Factor H de Complemento/metabolismo , Sinergismo Farmacológico , Humanos , Inmunohistoquímica , Infarto de la Arteria Cerebral Media/etiología , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/prevención & control , Lactatos/metabolismo , Masculino , Factores de Crecimiento Nervioso/metabolismo , Peroxidasa/metabolismo , Ratas , Ratas Sprague-Dawley , Subunidad beta de la Proteína de Unión al Calcio S100 , Proteínas S100/metabolismo , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/fisiopatologíaRESUMEN
Alcohol-induced liver disease is associated with unacceptable morbidity and mortality. When activated, Kupffer cells (KCs), the resident macrophages in the liver, release proinflammatory cytokine TNF-α, a key mediator of hepatic damage. Although chronic alcohol causes increase in norepinephrine (NE) release leading to hepatic dysfunction, the mechanism of NE-induced hepatic injury in chronic alcohol exposure has not been elucidated. This study was conducted to determine whether chronic alcohol exposure increases NE and upregulates KC α(2A)-adrenoceptors (α(2A)-AR) to cause TNF-α release. We also examined the role of mitogen activated protein kinase (MAPK) phosphatase-1 (MKP-1) in this process. Male adult rats were fed the Lieber-DeCarli liquid diet containing alcohol as 36% of total calories. The animals were sacrificed after 6 weeks and blood and liver samples were harvested for further analysis. KCs from healthy male rats were cultured with alcohol for 7 days, and cells then harvested for RNA and protein analyses. Chronic alcohol exposure resulted in hepatic damage. Alcohol caused a 276% increase in circulating NE and 86% increase in TNF-α in the liver. There was a 75% and 62% decrease in MKP-1 mRNA and protein levels, respectively in the liver. In-vitro experiments revealed 121% and 98% increase in TNF-α and α(2A)-AR mRNA levels with alcohol exposure, respectively, and a 32% decrease in MKP-1 mRNA compared to controls. In summary, chronic alcohol exposure elevates NE and upregulates KC α(2A)-AR to release TNF-α. Alcohol induced downregulation of MKP-1 leads to further release of TNF-α and hepatic injury.
Asunto(s)
Fosfatasa 1 de Especificidad Dual/metabolismo , Etanol/administración & dosificación , Macrófagos del Hígado/efectos de los fármacos , Hepatopatías Alcohólicas/metabolismo , Norepinefrina/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Animales , Regulación hacia Abajo , Macrófagos del Hígado/metabolismo , Hígado/efectos de los fármacos , Hígado/patología , Masculino , Ratas , Ratas Sprague-Dawley , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia ArribaRESUMEN
BACKGROUND: Despite advances in our understanding of excessive alcohol-intake-related tissue injury and modernization of the management of septic patients, high morbidity and mortality caused by infectious diseases in alcohol abusers remain a prominent challenge. Our previous studies have shown that milk fat globule epidermal growth factor-factor VIII (MFG-E8), a protein required to opsonize apoptotic cells for phagocytosis, is protective in inflammation. However, it remains unknown whether MFG-E8 ameliorates sepsis-induced apoptosis and organ injury in alcohol-intoxicated rats. The purpose of this study was to determine whether recombinant murine MFG-E8 (rmMFG-E8) attenuates organ injury after acute alcohol exposure and subsequent sepsis. METHODS: Acute alcohol intoxication was induced in male adult rats by a bolus injection of intravenous alcohol at 1.75 g/kg BW, followed by an intravenous infusion of 300 mg/kg BW/h of alcohol for 10 hours. Sepsis was induced at the end of 10-hour alcohol infusion by cecal ligation and puncture (CLP). rmMFG-E8 or vehicle (normal saline) was administered intravenously 3 times (i.e., at the beginning of alcohol injection, the beginning of CLP, and 10 hours post-CLP) at a dose of 20 microg/kg BW each. Blood and tissue samples were collected 20 hours after CLP in alcoholic animals for various measurements. RESULTS: Acute alcohol exposure per se did not affect the production of MFG-E8; however, it primed the animal and enhanced sepsis-induced MFG-E8 downregulation in the spleen. Administration of rmMFG-E8 reduces alcohol/sepsis-induced apoptosis in the spleen, lungs, and liver. In addition, administration of rmMFG-E8 after alcohol exposure and subsequent sepsis decreases circulating levels of TNF-alpha and interleukin-6 and attenuates organ injury. CONCLUSIONS: rmMFG-E8 attenuates sepsis-induced apoptosis and organ injury in alcohol-intoxicated rats.
Asunto(s)
Intoxicación Alcohólica/prevención & control , Antígenos de Superficie/farmacología , Apoptosis/efectos de los fármacos , Proteínas de la Leche/farmacología , Proteínas Recombinantes/farmacología , Sepsis/patología , Intoxicación Alcohólica/sangre , Intoxicación Alcohólica/metabolismo , Intoxicación Alcohólica/patología , Animales , Antígenos de Superficie/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Interleucina-6/sangre , Hígado/efectos de los fármacos , Hígado/patología , Pulmón/efectos de los fármacos , Pulmón/patología , Masculino , Proteínas de la Leche/metabolismo , Ratas , Ratas Sprague-Dawley , Sepsis/sangre , Sepsis/metabolismo , Bazo/efectos de los fármacos , Bazo/metabolismo , Bazo/patología , Factor de Necrosis Tumoral alfa/sangreRESUMEN
We have previously demonstrated the involvement of milk fat globule-epidermal growth factor-factor 8 (MFGE8) in reducing neutrophil infiltration in a murine model of acute lung injury (ALI). In the present study, we aimed to delineate the mechanisms through which MFGE8 attenuates neutrophil migration. Recombinant human MFGE8 (rhMFGE8) was expressed and purified in our facility. The human differentiated neutrophil cell line, dHL60, was treated with rhMFGE8 and cell migration assay was performed in a Boyden chamber using recombinant interleukin8 (IL8) as the chemoattractant. Surface CXCR2 and intracellular G proteincoupled receptor kinase 2 (GRK2) levels were evaluated by flow cytometry or western blot analysis. The levels of mitogenactivated protein (MAP) kinases were determined by western blot analysis. Treatment with rhMFGE8 resulted in a significant inhibition of dHL60 cell migration in a dosedependent manner. There was a 46% decrease in CXCR2 expression in the rhMFGE8treated dHL60 cells, which was associated with a 32% increase in GRK2 expression. In the dHL60 cells, treatment with rhMFGE8 promoted the phosphorylation of p38 and extracellular signal-regulated kinase (ERK) within 1030 min. The use of SB203580, a p38 inhibitor, and PD98059, an ERK inhibitor, resulted in the restoration of dHL60 cell migration which was significantly inhibited treatment with rhMFGE8. Furthermore, blocking the MFGE8 receptors, αvß3/αvß5integrins, by antiαvintegrin neutralizing antibody (Ab) inhibited the activation of p38 and ERK, and reversed the rhMFGE8induced inhibition of dHL60 cell migration. Finally, treatment of the dHL60 cells with SB203580 and PD98059 neutralized the rhMFGE8induced downregulation of CXCR2 expression and upregulation of GRK2 expression, as well as the inhibitory effects on cell migration. Our findings reveal a novel mechanism of action of MFGE8 through which it inhibits neutrophil migration through αvß3-integrin-dependent MAP kinase activation.
Asunto(s)
Antígenos de Superficie/farmacología , Movimiento Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Integrina alfaVbeta3/antagonistas & inhibidores , Proteínas de la Leche/farmacología , Neutrófilos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/farmacología , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Flavonoides , Citometría de Flujo , Quinasa 2 del Receptor Acoplado a Proteína-G/biosíntesis , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Células HL-60 , Humanos , Imidazoles/farmacología , Integrina alfaVbeta3/metabolismo , Interleucina-8/inmunología , Infiltración Neutrófila/efectos de los fármacos , Infiltración Neutrófila/inmunología , Neutrófilos/inmunología , Fosforilación/efectos de los fármacos , Piridinas/farmacología , Receptores de Interleucina-8B/biosíntesis , Receptores de Interleucina-8B/metabolismo , Receptores de Vitronectina/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Endogenous molecules released by dying cells [i.e., damage-associated molecular patterns (DAMPs)] after trauma and severe blood loss can activate pattern recognition receptors, leading to a cascade of inflammatory responses and organ injury. Mitochondrial transcription factor A (TFAM) is a transcription factor for mitochondrial DNA. TFAM is structurally related to high mobility group box 1 (HMGB1), an important member of DAMPs. We, therefore, hypothesized that TFAM can be released into the circulation after hemorrhage to initiate inflammatory responses. In order to examine this hypothesis, male Sprague-Dawley rats were bled to and maintained at a mean arterial pressure of 40 mmHg for 90 min. They were then resuscitated with an equal volume of shed blood in the form of Ringer's lactate (i.e., low-volume resuscitation) over 60 min. TFAM levels in the serum were measured at 4 h after hemorrhage and resuscitation. Our results showed that serum levels of TFAM were more than doubled after hemorrhage and resuscitation. To further characterize TFAM's biological activity, we expressed recombinant rat TFAM with a GST-tag (GST-TFAM) in an E. coli expression system. The purity of GST-TFAM was over 99% and it was immunoreactive for specific anti-TFAM antibodies. Using RAW 264.7 cells and primary rat peritoneal macrophages, we showed that GST-TFAM dose-dependently increased TNF-α release. To determine the biological activity of GST-TFAM in vivo, GST-TFAM was intravenously injected in healthy male adult rats. Our results demonstrated that intravenous injection of GST-TFAM, not GST alone, upregulated circulating levels of pro-inflammatory cytokines, increased neutrophil infiltration to the lungs and caused organ injury in healthy animals. Thus, TFAM can act as a DAMP and may contribute to the initiation of inflammatory responses in hemorrhagic shock.
Asunto(s)
Proteínas de Unión al ADN/sangre , Proteínas de Unión al ADN/inmunología , Mediadores de Inflamación/inmunología , Proteínas Mitocondriales/sangre , Proteínas Mitocondriales/inmunología , Choque Hemorrágico/inmunología , Factores de Transcripción/sangre , Factores de Transcripción/inmunología , Animales , Línea Celular , Proteínas de Unión al ADN/administración & dosificación , Proteínas de Unión al ADN/genética , Interleucina-6/sangre , Macrófagos/metabolismo , Masculino , Ratones , Proteínas Mitocondriales/administración & dosificación , Proteínas Mitocondriales/genética , Infiltración Neutrófila , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/administración & dosificación , Choque Hemorrágico/metabolismo , Choque Hemorrágico/patología , Factores de Transcripción/administración & dosificación , Factores de Transcripción/genética , Factor de Necrosis Tumoral alfa/sangreRESUMEN
Excessive inflammation and apoptosis contribute to the pathogenesis of ischemic stroke. MFG-E8 is a 66-kDa glycoprotein that has shown tissue protection in various models of organ injury. However, the potential role of MFG-E8 in cerebral ischemia has not been investigated. We found that levels of MFG-E8 protein in the brain were reduced at 24 h after cerebral ischemia. To assess the potential role of MFG-E8 in cerebral ischemia, adult male Sprague-Dawley rats were subjected to permanent middle cerebral artery occlusion (MCAO). At 1 h post-stroke onset, an intravenous administration of 1 ml saline as vehicle or 160 µg/kg BW recombinant human MFG-E8 (rhMFG-E8) as treatment was given. The optimal dose of rhMFG-E8 was obtained from previous dose-response organ protection in rat sepsis studies. Neurological scores were determined at 24 h and 48 h post-MCAO. Rats were sacrificed thereafter and brains rapidly removed and analyzed for infarct size, histopathology, and markers of inflammation and apoptosis. Compared with saline vehicle, rhMFG-E8 treatment led to significant decreases in sensorimotor and vestibulomotor deficits, and infarct size at 24 h and 48 h post-MCAO. Measures associated with improved outcome included reduced microglial inflammatory cytokine secretion, adhesion molecules and neutrophil influx, cleaved caspase-3, and upregulation of peroxisome proliferator activated receptor-γ (PPAR-γ), and Bcl-2/Bax ratio leading to decreased apoptosis. Thus, rhMFG-E8 treatment is neuroprotective against cerebral ischemia through suppression of inflammation and apoptosis. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.
Asunto(s)
Antígenos de Superficie/uso terapéutico , Apoptosis/efectos de los fármacos , Isquemia Encefálica/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Proteínas de la Leche/uso terapéutico , Animales , Antígenos de Superficie/metabolismo , Antígenos de Superficie/farmacología , Presión Sanguínea/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Inflamación/metabolismo , Inflamación/patología , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Interleucina-6/metabolismo , Masculino , Microglía/efectos de los fármacos , Microglía/metabolismo , Microglía/patología , Proteínas de la Leche/metabolismo , Proteínas de la Leche/farmacología , Necrosis , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Neutrófilos/patología , Ratas , Ratas Sprague-Dawley , Recuperación de la Función/efectos de los fármacosRESUMEN
Ethanol has been known to cause injury to the liver and other tissues; however the molecular factors responsible for alcohol induced liver injury has not been fully understood. Recent studies indicate that reactive oxygen species (ROS) may play an important role in alcohol induced liver injury. Peroxisome proliferator activated receptor-gamma (PPAR-gamma)-coactivator 1alpha (PGC-1alpha) has been shown to be involved in defenses against ROS by inducing many ROS-detoxifying enzymes. However, the role of PGC-1alpha in alcohol induced liver injury has not been elucidated. Therefore, in this study, we examined the effect of alcohol on gene and protein expression of PGC-1alpha in H4-IIE cells (in vitro) and hepatic tissues (in vivo) by real-time PCR and Western blot, respectively. Our results show that exposure to 500 mM ethanol in H4-IIE cells for 24 h significantly decreased both gene and protein expression of PGC-1alpha. PGC-1alpha gene expression was significantly decreased in cells exposed to 100 ng/ml LPS or 1% hypoxia for 24 h. In addition, PGC-1alpha gene and protein expressions were slightly lower in hepatic tissues of rats exposed to ethanol for 15 h, at the level equivalent to the 500 mM used in culture cells, in comparison to sham rats. In contrast, serum LDH and AST levels in ethanol exposed rats were 1.9 fold and 2.8 fold higher than that of sham rats, respectively, which suggest significant organ injury in these rats following ethanol exposure. Likewise, catalase, an enzyme that hydrolyzes peroxide to water, is significantly increased in ethanol exposed H4-IIE cells which further confirms ROS generation due to ethanol exposure. Thus, our results show that oxidative stress conditions such as acute alcohol consumption, LPS or hypoxia suppresses PGC-1alpha expression in the liver, thereby presumably downregulates pertinent ROS-scavenging enzymes and enhances liver injury.