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Both epidemiological investigations and animal studies have linked arsenic-contaminated water to cancers, including skin, liver and lung cancers. Besides genotoxicity, arsenic exposure-related pathogenesis of disease is widely considered through epigenetic mechanisms; however, the underlying mechanism remains to be determined. Herein we explore the initial epigenetic changes via acute sodium arsenite (As) exposures of mouse embryonic fibroblast (MEF) cells and histone H3K79 methyltransferase Dot1L knockout (Dot1L-/-) MEF cells. Our RNA-seq and Western blot data demonstrated that, in both cell lines, acute As exposure abolished histone acetyltransferase p300 at the RNA level and subsequent protein level. Consequently, p300-specific main target histone H3K27ac, a marker separating active from poised enhancers, decreased dramatically as validated by both Western blot and ChIP-qPCR/seq analyses. Concomitantly, H3K4me1 as another well-known marker for enhancers also showed significant decreases, suggesting an underappreciated crosstalk between H3K4me1 and H3K27ac involved in As exposure. Significantly, As exposure-reduced H3K27ac and H3K4me1 inhibited the expression of genes including EP300 itself and Kruppel Like Factor 4(Klf4) that both are tumor suppressor genes. Collectively, our investigations identified p300 as an internal bridging factor within cells to sense external environmental As exposure to alter chromatin, thereby changing gene transcription for disease pathogenesis.
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Arsenitos/toxicidad , Fibroblastos/efectos de los fármacos , Histonas/metabolismo , Compuestos de Sodio/toxicidad , Factores de Transcripción p300-CBP/antagonistas & inhibidores , Animales , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Factor 4 Similar a Kruppel , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción p300-CBP/metabolismoRESUMEN
Multixenobiotic resistance (MXR) transporters, which belong to ATP-binding cassette (ABC) family proteins, are present in living organisms as a first line of defense system against xenobiotics and environmental contaminants. The effects of six organic UV filters (4-methyl -benzylidene camphor, 4-MBC; benzophenone-3, BP-3; butyl methoxydibenzoyl-methane, BM-DBM; ethylhexyl methoxy cinnamate, EHMC; octocrylene, OC and homosalate, HMS) on multixenobiotic resistance (MXR) in Tetrahymena thermophila were investigated in this study. It was found that 4-MBC, BP-3 and BM-DBM could significantly inhibit activity of the MXR system, causing concentration dependent accumulation of rhodamine 123; while EHMC, OC and HMS had weak MXR inhibition. The IC50 (50 % inhibition concentration) values of 4-MBC, BP-3 and BM-DBM were 23.54, 40.59 and 26.37 µM, respectively, with inhibitory potentials of 23.1, 13.4 and 20.6 % relative to verapamil (VER, a model inhibitor of P-glycoprotein). Our results firstly provide the evidence for UV filters inhibition effect on MXR in aquatic organisms. In addition, it was revealed by molecular docking analysis that the selected six UV filters can occupy the same binding site on T. thermophila P-gp as VER does; and form H-bonds with residues Ser 328 and/or Asn 281. This study raises the awareness of aquatic ecological risk from the organic UV filters exposure, as they would be involved in potentiating toxic effects by chemosensitizing.
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Resistencia a Medicamentos/efectos de los fármacos , Rodamina 123/metabolismo , Protectores Solares/toxicidad , Tetrahymena thermophila/fisiología , Contaminantes Químicos del Agua/toxicidad , Subfamilia B de Transportador de Casetes de Unión a ATP , Bioensayo , Simulación del Acoplamiento MolecularRESUMEN
OBJECTIVE AND DESIGN: Activations of the complement C5a (C5a) and the urokinase-type plasminogen activator (uPA) are commonly seen together during sepsis. However, the mechanism linking these two important pathways remains elusive. MATERIAL, METHODS AND TREATMENT: We used the C57BL/6 J mice model of sepsis induced by cecal ligation puncture (CLP) procedure, injected anti-C5aR or rottlerin through the tail vein to neutralize C5aR or PKC-δ, and then isolated peritoneal macrophages. Total RNA was isolated from the cells and analyzed by quantitative PCR. RESULTS: Our study revealed that neutralizing C5aR markedly inhibited sepsis-induced uPA receptor (uPAR) expression and its downstream signaling in macrophage. Similarly, neutralizing uPAR suppressed sepsis activation of C5a signaling. Importantly, inhibition of PKC-δ largely blocked sepsis-induced expression of C5aR and uPAR. CONCLUSIONS: Our study demonstrates a crosstalk between the complement C5a signaling and the fibrinolytic uPA pathways, which may depend on each other to maintain their expression and signaling, and reveals a central role of PKC-δ in mediating sepsis-induced activation of these pathways.
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Complemento C5a/inmunología , Macrófagos Peritoneales/inmunología , Proteína Quinasa C-delta/inmunología , Sepsis/inmunología , Activador de Plasminógeno de Tipo Uroquinasa/inmunología , Animales , Células Cultivadas , Complemento C5a/genética , Femenino , Masculino , Ratones Endogámicos C57BL , Transducción de Señal , Activador de Plasminógeno de Tipo Uroquinasa/genéticaRESUMEN
Epidemiological studies have shown that maternal hormone exposure is associated with autism spectrum disorders (ASD). The hormone oxytocin (OXT) is a central nervous neuropeptide that plays an important role in social behaviors as well as ASD etiology, although the detailed mechanism remains largely unknown. In this study, we aim to investigate the potential role and contribution of OXT to prenatal progestin exposure-mediated mouse offspring. Our in vitro study in the hypothalamic neurons that isolated from paraventricular nuclei area of mice showed that transient progestin exposure causes persistent epigenetic changes on the OXT promoter, resulting in dissociation of estrogen receptor ß (ERß) and retinoic acid-related orphan receptor α (RORA) from the OXT promoter with subsequent persistent OXT suppression. Our in vivo study showed that prenatal exposure of medroxyprogesterone acetate (MPA) triggers social deficits in mouse offspring; prenatal OXT deficiency in OXT knockdown mouse partly mimics, while postnatal ERß expression or postnatal OXT peptide injection partly ameliorates, prenatal MPA exposure-mediated social deficits, which include impaired social interaction and social abilities. On the other hand, OXT had no effect on prenatal MPA exposure-mediated anxiety-like behaviors. We conclude that prenatal MPA exposure-mediated oxytocin suppression contributes to social deficits in mouse offspring.
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Oxitocina , Progestinas , Animales , Femenino , Ratones , Neuronas/metabolismo , Oxitocina/metabolismo , Embarazo , Conducta Social , EsteroidesRESUMEN
BACKGROUND: Delayed wound healing is one of the major complications of diabetes mellitus and is characterized by prolonged inflammation, delayed re-epithelialization and consistent oxidative stress, although the detailed mechanism remains unknown. In this study, we aimed to investigate the potential role and effect of pterostilbene (PTE) and hematopoietic stem cells (HSCs) on diabetic wound healing. METHODS: Diabetic rats were used to measure the epigenetic changes in both HSCs and peripheral blood mononuclear cells (PBMCs). A cutaneous burn injury was induced in the rats and PTE-treated diabetic HSCs were transplanted for evaluation of wound healing. In addition, several biomedical parameters, including gene expression, oxidative stress, mitochondrial function and inflammation in macrophages, were also measured. RESULTS: Our data showed that PTE had a much stronger effect than resveratrol on accelerating diabetic wound healing, likely because PTE can ameliorate diabetes-induced epigenetic changes to estrogen receptor ß promoter in HSCs, while resveratrol cannot. Further investigation showed that bone marrow transplantation of PTE-treated diabetic HSCs restores diabetes-induced suppression of estrogen receptor ß and its target genes, including nuclear respiratory factor-1 and superoxide dismutase 2, and protects against diabetes-induced oxidative stress, mitochondrial dysfunction and elevated pro-inflammatory cytokines in both PBMCs and macrophages, subsequently accelerating cutaneous wound healing. CONCLUSIONS: HSC may play an important role in wound healing through transferring epigenetic modifications to subsequent PBMCs and macrophages by differentiation, while PTE accelerates diabetic wound healing by modulating diabetes-induced epigenetic changes in HSCs. Thus, PTE may be a novel therapeutic strategy for diabetic wound healing.
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C1 inhibitor (C1INH), a member of the serine proteinase inhibitor (serpin) family, functions as an inhibitor of the complement and contact systems. Cleavage of the reactive center loop (RCL) within the carboxyl-terminal domain of C1INH (iC1INH), lacking of serpin function, induces a conformational change in the molecule. Our previous data demonstrated that active, intact C1INH prevents vascular permeability induced by gram-negative bacterial lipopolysaccharide (LPS). In this study, we investigate the role of RCL-cleaved, inactive C1INH (iC1INH) in vascular endothelial activation. In the cultured primary human umbilical vein endothelial cell (HUVEC) monolayer, iC1INH blocked LPS-induced cell injury by evaluated as transendothelial flux, cell detachment, and cytoskeletal disorganization. LPS-induced upregulation of vascular cell adhesion molecule-1 (VCAM-1) could be suppressed by treatment with iC1INH. Studies exploring the underlying mechanism of iC1INH-mediated suppression in VCAM-1 expression were related to reduction of NF-kappaB activation and nuclear translocation in an I kappa B alpha-dependent manner. The inhibitory effect was associated with stabilization of the NF-kappaB inhibitor I kappa B and reduction of inhibitor I kappa B kinase activity. In the model of endotoxin-induced mice, increased plasma leakage in local abdominal skin in response to LPS was reversed by treatment with iC1INH. Furthermore, systemic administration of LPS to mice resulted in increased microvascular permeability in multiple organs, which was reduced by iC1INH. These data provide evidence that iC1INH has an anti-vascular permeability independent on the serpin function.
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Permeabilidad Capilar , Proteína Inhibidora del Complemento C1/fisiología , Células Endoteliales/fisiología , Secuencias de Aminoácidos , Animales , Adhesión Celular , Células Cultivadas , Proteína Inhibidora del Complemento C1/farmacología , Citoesqueleto/metabolismo , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/citología , Humanos , Proteínas I-kappa B/fisiología , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos BALB C , FN-kappa B/fisiología , Venas Umbilicales/citología , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Delayed wound healing in diabetic patients is a serious diabetic complication, resulting in major health problems as well as high mortality and disability. The detailed mechanism still needs to be fully understood. In this study, we aim to investigate potential mechanisms and explore an efficient strategy for clinical treatment of diabetic wound healing. Human umbilical endothelial cells were exposed to hyperglycemia for 4 days, then switched to normoglycemia for an additional 4 days. The cells were harvested for the analysis of reactive oxygen species (ROS) generation, gene expression and VEGF signaling pathway. Furthermore, the diabetic wound model was established in rats for the evaluation of wound healing rates under the treatment of either ERß agonist/antagonist or SOD mimetic MnTBAP. Our results show that transient hyperglycemia exposure results in persistent ROS overgeneration after the switch to normoglycemia, along with suppressed expression of ERß, SOD2, and the VEGF signaling pathway. Either ERß expression or activation diminishes ROS generation. In vivo experiments with diabetic rats show that ERß activation or SOD mimetic MnTBAP diminishes ROS generation in tissues and accelerates diabetic wound healing. Transient hyperglycemia exposure induces ROS generation and suppresses ERß expression, subsequently resulting in SOD2 suppression with additional elevated ROS generation. This forms a positive-feed forward loop for ROS generation with persistent oxidative stress. ERß expression or activation breaks this loop and ameliorates this effect, thereby accelerating diabetic wound healing. We conclude that ERß accelerates diabetic wound healing by ameliorating hyperglycemia-induced persistent oxidative stress. This provides a new strategy for clinical treatment of diabetic wound healing based on ERß activation.
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Acute myocardial infarction (AMI) is associated with inflammation and apoptosis. Emodin plays an anti-inflammatory role in several inflammatory diseases. Recent studies have demonstrated that emodin protects against myocardial ischemia/reperfusion injury. However, its mechanism underlying its effects remains unknown. In a murine model of AMI, based on ligation of the left coronary artery, administration of emodin reduced myocardial infarct size (MIS) in a dose-dependent manner. Emodin significantly suppressed TNF-alpha expression and NF-kappaB activation in the local myocardial infarction area. Treatment with emodin inhibited myocardial cell apoptosis by inhibiting caspase-3 activation. Therefore, these studies demonstrate that emodin protects against myocardial cell injury via suppression of local inflammation and apoptosis.
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Antiinflamatorios no Esteroideos/farmacología , Apoptosis/efectos de los fármacos , Emodina/farmacología , Infarto del Miocardio , Miocardio , Animales , Antiinflamatorios no Esteroideos/uso terapéutico , Western Blotting , Modelos Animales de Enfermedad , Ensayo de Cambio de Movilidad Electroforética , Emodina/uso terapéutico , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Masculino , Ratones , Ratones Endogámicos BALB C , Infarto del Miocardio/inmunología , Infarto del Miocardio/patología , Infarto del Miocardio/prevención & control , Miocardio/enzimología , Miocardio/inmunología , Miocardio/patología , FN-kappa B/metabolismo , Peroxidasa/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Histone modification including H3 lysine 79 methylation (H3K79me) plays a key role during gene transcription and DNA damage repair. DOT1L, the sole methyltransferase for three states of H3K79me, is implicated in leukemia, co-lorectal cancer, and dilated cardiomyopathy. However, understanding of DOT1L and H3K79me in these pathways and disease pathogenesis has been limited due to the difficulty of working with DOT1L protein. For instance, locus-specific or genome-wide binding sites of DOT1L revealed by chromatin immunoprecipitation (ChIP)-based methods are necessary for inferring its functions, but high-quality ChIP-grade antibodies are currently not available. Herein we have developed a knock-in approach to tag endogenous DOT1L with 3 × Flag at its C-terminal domain to follow functional analyses. The knock-in was facilitated by using TALENs to induce a targeted double-strand break at the endogenous DOTIL to stimulate local homologous recombination at that site. The single cell colonies with successful knock-in were isolated and verified by different methods. We also demonstrated that tagged DOT1L maintains its normal function in terms of methylation and that the engineered cells would be very useful for further studies.
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Recent clinical cohort study shows that testosterone therapy increases cardiovascular diseases in men with low testosterone levels, excessive circulating androgen levels may play a detrimental role in the vascular system, while the potential mechanism and effect of testosterone exposure on the vascular function in offspring is still unknown. Our preliminary results showed that perinatal testosterone exposure in mice induces estrogen receptor ß (ERß) suppression in endothelial progenitor cells (EPCs) in offspring but not mothers, while estradiol (E2) had no effect. Further investigation showed that ERß suppression is due to perinatal testosterone exposure-induced epigenetic changes with altered DNA methylation on the ERß promoter. During aging, EPCs with ERß suppression mobilize to the vascular wall, differentiate into ERß-suppressed mouse endothelial cells (MECs) with downregulated expression of SOD2 (mitochondrial superoxide dismutase) and ERRα (estrogen-related receptor α). This results in reactive oxygen species (ROS) generation and DNA damage, and the dysfunction of mitochondria and fatty acid metabolism, subsequently potentiating vascular dysfunction. Bone marrow transplantation of EPCs that overexpressed with either ERß or a SIRT1 single mutant SIRT1-C152(D) that could modulate SIRT1 phosphorylation significantly ameliorated vascular dysfunction, while ERß knockdown worsened the problem. We conclude that perinatal testosterone exposure potentiates vascular dysfunction through ERß suppression in EPCs.
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Células Progenitoras Endoteliales/efectos de los fármacos , Células Progenitoras Endoteliales/metabolismo , Receptor beta de Estrógeno/metabolismo , Testosterona/farmacología , Animales , Western Blotting , Inmunoprecipitación de Cromatina , Daño del ADN , Metilación de ADN , Estradiol/farmacología , Receptor beta de Estrógeno/genética , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Fosforilación/efectos de los fármacos , ARN Interferente Pequeño/genética , Radioinmunoensayo , Especies Reactivas de Oxígeno/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Superóxido Dismutasa/metabolismo , Receptor Relacionado con Estrógeno ERRalfaRESUMEN
The information about adverse effects of emerging contaminants on aquatic protozoa is very scarce. The growth inhibition effect, cell viability, genotoxicity and multixenobiotic resistance (MXR) responses of two commonly used antimicrobial agents, triclosan (TCS) and triclocarban (TCC) to protozoan Tetrahymena thermophila were investigated in this study. The results revealed that TCS and TCC can inhibit the growth of T. thermophila with 24h EC50 values of 1063 and 295µgL(-1), respectively. The impairment of plasma membrane was observed after 2h exposure of TCS or TCC at the level of mg/L. Furthermore, it is noticeable that at environmentally relevant concentration (1.0µgL(-1)), both TCS and TCC can lead to statistically significant DNA damage in T. thermophila, while the inhibition of growth and change of cell viability cannot be observed. Our results firstly provide the evidence for genotoxic effects of TCS and TCC on the freshwater protozoan. Additionally, both TCS and TCC were found to inhibit the efflux transporter activities, with the inhibitory potencies of 39% and 40% (using verapamil as a model inhibitor), respectively. Particularly, TCC could significantly down-regulate the expression of MXR related gene Abcb15, which encodes the membrane efflux protein that acting as P-gp in T. thermophila. The results raise the awareness of potential aquatic ecological and human health risks from the exposure of TCS and TCC, as they might potentiate the toxic effects by chemosensitizing with co-existing toxicants.
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Carbanilidas/toxicidad , Resistencia a Medicamentos/efectos de los fármacos , Tetrahymena thermophila/citología , Tetrahymena thermophila/genética , Triclosán/toxicidad , Xenobióticos/toxicidad , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Supervivencia Celular/efectos de los fármacos , Interacciones Farmacológicas , Monitoreo del Ambiente/métodos , Agua Dulce/química , Mutágenos/toxicidad , Tetrahymena thermophila/efectos de los fármacos , Tetrahymena thermophila/crecimiento & desarrollo , Contaminantes Químicos del Agua/toxicidad , Xenobióticos/metabolismoRESUMEN
Epidemiological studies have shown that estrogens have protective effects in cardiovascular diseases, even though the results from human clinical trials remain controversial, while most of the animal experiments confirmed this effect, but the detailed mechanism remains unclear. In this study, we found that estradiol (E2) treatment significantly increases the expression of mitochondrial superoxide dismutase (SOD2) in mice and in vitro in human aorta endothelial cells. Further investigation shows that E2 up-regulates SOD2 through tethering of estrogen receptor (ER) to Sp1 and the increased binding of Sp1 to GC-box on the SOD2 promoter, where ERα responses E2-mediated gene activation, and ERß maintains basal gene expression level. The E2/ER-mediated SOD2 up-regulation results in minimized ROS generation, which highly favors healthy cardiovascular function. Gene therapy through lentivirus-carried endothelium-specific delivery to the vascular wall in high-fat diet (HFT) mice shows that the SOD2 expression in endothelial cells normalizes E2 deficiency-induced ROS generation with ameliorated mitochondrial dysfunction and vascular damage, while SOD2 knockdown worsens the problem despite the presence of E2, indicating that E2-induced SOD2 expression plays an important vasculoprotective role. To our knowledge, this is the first report for the mechanism by which E2 improves cardiovascular function through up-regulation of SOD2 in endothelial cells. In turn, this suggests a novel gene therapy through lentivirus-carried gene delivery to vascular wall for E2 deficiency-induced cardiovascular damage in postmenopausal women.
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Endotelio Vascular/metabolismo , Estradiol/metabolismo , Regulación de la Expresión Génica , Superóxido Dismutasa/genética , Animales , Sitios de Unión , Línea Celular Transformada , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Endotelio Vascular/efectos de los fármacos , Estradiol/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Modelos Biológicos , Regiones Promotoras Genéticas , Unión Proteica , Especies Reactivas de Oxígeno/metabolismo , Receptor TIE-2/genética , Receptores de Estrógenos/antagonistas & inhibidores , Elementos de Respuesta , Factores Sexuales , Factor de Transcripción Sp1/metabolismo , Superóxido Dismutasa/metabolismo , Superóxidos/metabolismo , Transducción GenéticaRESUMEN
UV filters are increasingly used in sunscreens and other personal care products. Although their residues have been widely identified in aquatic environment, little is known about the influences of UV filters to protozoan. The growth inhibition effects, cell viability and oxidative stress responses of four commonly used UV filters, 2-ethylhexyl 4-methoxycinnamate (EHMC), benzophenone-3 (BP-3), 4-methyl-benzylidene camphor (4-MBC) and octocrylene (OC), to protozoan Tetrahymena thermophila were investigated in this study. The 24-h EC50 values with 95% confidence intervals for BP-3 and 4-MBC were 7.544 (6.561-8.675) mg L(-1) and 5.125 (4.874-5.388) mg L(-1), respectively. EHMC and OC did not inhibit the growth of T. thermophila after 24h exposure at the tested concentrations. The results of cell viability assays with propidium iodide (PI) staining were consistent with that of the growth inhibition tests. As for BP-3 and 4-MBC, the relatively higher concentrations, i.e. of 10.0 and 15.0 mg L(-1), could lead to the cell membranes impairment after 4h exposure. With the increase of the exposure time to 6h, their adverse effects on cell viability of T. thermophila were observed at the relatively lower concentration groups (1.0 mg L(-1) and 5.0 mg L(-1)). In addition, it is noticeable that at environmentally relevant concentration (1.0 µg L(-1)), BP-3 and 4-MBC could lead to the significant increase of catalase (CAT) activities of the T. thermophila cells. Especially for the BP-3, the oxidative injuries were further confirmed by the reduction of glutathione (GSH) content. It is imperative to further investigate the additive action of UV filters and seek other sensitive endpoint, especially at environmentally relevant concentration.
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Estrés Oxidativo , Protectores Solares/toxicidad , Tetrahymena thermophila/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidad , Acrilatos/toxicidad , Benzofenonas/toxicidad , Alcanfor/análogos & derivados , Alcanfor/toxicidad , Supervivencia Celular , Cinamatos/toxicidad , Tetrahymena thermophila/fisiología , Rayos UltravioletaRESUMEN
Food safety is of extreme importance to human health. Vanillin and ethyl vanillin are the widely used food additives and spices in foods, beverages, cosmetics and drugs. The objective of the present work was to evaluate the impact of vanillin and ethyl vanillin on the activities of CYP2C9, CYP2E1, CYP3A4, CYP2B6 and CYP1A2 in human liver microsomes (HLM) in vitro, and impact on the activities of CYP1A2, CYP2C, CYP3A and CYP2E1 in rat liver microsomes (RLM) in vivo. The in vitro results demonstrated that vanillin and ethyl vanillin had no significant effect on the activity of five human CYP450 enzymes with concentration ranged from 8 to 128 µM. However, after rats were orally administered vanillin or ethyl vanillin once a day for seven consecutive days, CYP2E1 activity was increased and CYP1A2 activity was decreased in RLM. The in vivo results revealed that drug interaction between vanillin/ethyl vanillin and the CYP2E1/CYP1A2-metabolizing drugs might be possible, and also suggested that the application of the above additives in foods and drugs should not be unlimited so as to avoid the adverse interaction.
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Antioxidantes/farmacología , Benzaldehídos/farmacología , Sistema Enzimático del Citocromo P-450/metabolismo , Animales , Humanos , Técnicas In Vitro , Indicadores y Reactivos , Isoenzimas/metabolismo , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Ratas , Ratas Wistar , Reproducibilidad de los ResultadosRESUMEN
Endothelial barrier dysfunction leading to increased permeability and vascular leakage is an underlying cause of several pathological conditions. Whereas these changes have been shown to be associated with activation of the complement system, leading to the release of C5a and interaction of C5a-C5a receptor (C5aR), the role of C5aR in endothelial cells remain(s) ill-defined. Here, we report an essential role of C5aR in endothelial cell injury and vascular permeability through silencing of the C5aR gene using siRNA. In the cultured mouse dermal microvascular endothelial cells (MEMECs) monolayer transfected with C5aR-siRNA, endotoxin-induced cell injury by evaluated as transendothelial flux, cell detachment, and cytoskeletal disorganization was inhibited. Upregulation of vascular cell adhesion molecule-1 (VCAM-1) was also suppressed. Studies exploring the underlying mechanism of siRNA-mediated suppression in VCAM-1 expression were related to reduction of NF-kappaB activation and nuclear localization of both p50 and p65. The effect was associated with inhibition in activation of protein kinase Cdelta(PKC-delta) and induction of PKC-mediated mitogen-activated protein kinase phosphatases-1 (MKP-1) leading to the increased activity of p42/p44 mitogen-activated protein (MAP) kinase cascade. In the model of mice administrated with C5aR-siRNA, endotoxin-induced plasma leakage was inhibited in local abdominal skin. Systemic administration of endotoxin to mice resulted in increased microvascular permeability in multiple organs was reduced. These studies demonstrate that the C5aR responsible for vascular endothelial cell injury and plasma permeability is an important factor, and that blockade of C5aR may be useful therapeutic targets for the prevention of vascular permeability in pathogenic condition.
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Permeabilidad Capilar/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Bacterias Gramnegativas/química , Lipopolisacáridos/farmacología , ARN Interferente Pequeño/metabolismo , Receptor de Anafilatoxina C5a/genética , Animales , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/enzimología , Endotelio Vascular/patología , Técnicas de Silenciamiento del Gen , Quinasa I-kappa B/metabolismo , Proteínas I-kappa B/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Microvasos/efectos de los fármacos , Microvasos/metabolismo , Microvasos/patología , Subunidad p50 de NF-kappa B/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Receptor de Anafilatoxina C5a/metabolismo , Piel/irrigación sanguínea , Piel/efectos de los fármacos , Factor de Transcripción ReIA/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
The betulinic acid (BetA) purified from Pulsatilla chinensis (PC) has been found to have selective inhibitory effects on hepatitis B virus (HBV). In hepatocytes from HBV-transgenic mice, we showed that BetA substantially inhibited HBV replication by downregulation of manganese superoxide dismutase (SOD2) expression, with subsequent reactive oxygen species generation and mitochondrial dysfunction. Also, the HBV X protein (HBx) is suppressed and translocated into the mitochondria followed by cytochrome c release. Further investigation revealed that SOD2 expression was suppressed by BetA-induced cAMP-response element-binding protein dephosphorylation at Ser133, which subsequently prevented SOD2 transcription through the cAMP-response element-binding protein-binding motif on the SOD2 promoter. SOD2 overexpression abolished the inhibitory effect of BetA on HBV replication, whereas SOD2 knockdown mimicked this effect, indicating that BetA-mediated HBV clearance was due to modulation of the mitochondrial redox balance. This observation was further confirmed in HBV-transgenic mice, where both BetA and PC crude extracts suppressed SOD2 expression, with enhanced reactive oxygen species generation in liver tissues followed by substantial HBV clearance. We conclude that BetA from PC could be a good candidate for anti-HBV drug development.
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Antivirales/farmacología , Virus de la Hepatitis B/efectos de los fármacos , Superóxido Dismutasa/antagonistas & inhibidores , Superóxido Dismutasa/genética , Triterpenos/farmacología , Animales , Supervivencia Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Virus de la Hepatitis B/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Masculino , Ratones , Ratones Transgénicos , Mitocondrias/metabolismo , Modelos Genéticos , Triterpenos Pentacíclicos , Fosforilación , Regiones Promotoras Genéticas , Pulsatilla/química , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismo , Replicación Viral/efectos de los fármacos , Ácido BetulínicoRESUMEN
A major cause of tumor treatment failure is cancer cell metastasis. Toll-like receptor 4 (TLR4)-mediated signaling has been implicated in tumor cell invasion, survival, and metastasis in a variety of cancers. In this study, we investigated the biological roles of TLR4 in prostate metastatic cell invasion and survival, and the potential of gene silencing of TLR4 using small interfering RNA (siRNA) for treatment of cancer. In cultured human prostate cancer cell lines, TLR4 were higher PC3 and DU145 as compared with the poorly metastatic LNCaP indicating that up-regulation of TLR4 was positively correlated with metastasis of tumor cell. In the highly metastatic cancer cell PC3, gene silencing of TLR4 using siRNA significantly inhibited TLR4 mRNA expression and protein level. Knockdown of TLR4 in PC3 cells resulted in a dramatic reduction of tumor cell migration and invasion as indicated by a Matrigel invasion assay. Furthermore, TLR4 siRNA suppressed cell viability and ultimately caused the induction of apoptotic cell death. The effects were associated with abrogating TLR4-mediated signaling to downstream target molecules such as myeloid differentiation factor 88 (MyD88), adaptor-inducing IFN-beta (TRIF), and interferon regulatory factor-1 (IRF-1). In a mouse prostate cancer model, administration with the plasmid construct expressing siRNA for TLR4 obviously inhibited established tumor growth and survival. These studies revealed evidence of a multifaceted signaling network operating downstream of TLR4-mediated tumor cell invasion, proliferation, and survival. Thus, RNA interference-directed targeting of TLR4 may raise the potential of its application for cancer therapy.
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Neoplasias de la Próstata/metabolismo , ARN Interferente Pequeño/metabolismo , Receptor Toll-Like 4/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Apoptosis/fisiología , Línea Celular Tumoral , Movimiento Celular/fisiología , Proliferación Celular , Supervivencia Celular/fisiología , Regulación hacia Abajo/fisiología , Técnicas de Silenciamiento del Gen , Silenciador del Gen/fisiología , Humanos , Factor 1 Regulador del Interferón/metabolismo , Masculino , Ratones , Ratones Desnudos , Factor 88 de Diferenciación Mieloide/metabolismo , Invasividad Neoplásica , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Receptor Toll-Like 4/genética , Regulación hacia Arriba/fisiología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
C1 inhibitor (C1INH), a complement regulatory protein, prevents endotoxin shock via a direct interaction of the amino-terminal domain with gram-negative bacterial lipopolysaccharide (LPS). Importantly, the cleaved, inactive C1INH still is an anti-endotoxin effector indicating the anti-endotoxin peptide that generates from the amino-terminal domain of C1INH. In this study, we first identified that a cleaved fragment within the major part of the amino-terminal domain in in vitro proteolytic analysis of C1INH had an ability to bind to LPS. We synthesized several peptides overlapping the C1INH cleaved fragment. Among these synthetic peptides, a 13-mer derivative peptide at position from 18 to 30, named N2((18-30)), exhibited the most powerful anti-endotoxin activity in vitro, enlightening that it was most strong at binding to LPS, inhibiting the interaction of LPS with LPS-binding protein (LBP), blocking LPS binding to CD14(+) cells, and suppressing production of tumor necrosis factor (TNF)-alpha by murine macrophages, RAW 264.7. In the murine endotoxin shock model, the peptide N2((18-30)) protected mice from LPS-induced lethal septic shock by inhibiting macrophage activation. These data indicate that the peptide N2((18-30)) derived from the amino-terminal region of C1INH is anti-endotoxin.
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Proteína Inhibidora del Complemento C1/química , Endotoxinas/química , Péptidos/química , Secuencia de Aminoácidos , Animales , Relación Dosis-Respuesta a Droga , Endotoxinas/metabolismo , Inflamación , Receptores de Lipopolisacáridos/biosíntesis , Receptores de Lipopolisacáridos/química , Lipopolisacáridos/química , Lipopolisacáridos/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Choque/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Increased expression of adhesion molecules by activated endothelium is a critical feature of vascular inflammation associated with the several diseases such as endotoxin shock and sepsis/septic shock. Our data demonstrated complement regulatory protein C1 inhibitor (C1INH) prevents endothelial cell injury. We hypothesized that C1INH has the ability of an anti-endothelial activation associated with suppression of expression of adhesion molecule(s). C1INH blocked leukocyte adhesion to endothelial cell monolayer in both static assay and flow conditions. In inflammatory condition, C1INH reduced vascular cell adhesion molecule (VCAM-1) expression associated with its cytoplasmic mRNA destabilization and nuclear transcription level. Studies exploring the underlying mechanism of C1INH-mediated suppression in VCAM-1 expression were related to reduction of NF-kappaB activation and nuclear translocation in an IkappaBalpha-dependent manner. The inhibitory effects were associated with reduction of inhibitor IkappaB kinase activity and stabilization of the NF-kappaB inhibitor IkappaB. These findings indicate a novel role for C1INH in inhibition of vascular endothelial activation. These observations could provide the basis for new therapeutic application of C1INH to target inflammatory processes in different pathologic situations.
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Proteínas Inactivadoras del Complemento 1/inmunología , Células Endoteliales/inmunología , Lipopolisacáridos/administración & dosificación , Serpinas/inmunología , Molécula 1 de Adhesión Celular Vascular/inmunología , Células Cultivadas , Proteína Inhibidora del Complemento C1 , Relación Dosis-Respuesta a Droga , Células Endoteliales/efectos de los fármacos , HumanosRESUMEN
The complement system activation can mediate myocardial ischemia and reperfusion (I/R). Inhibition of C5a activity reveals attenuation of I/R-induced myocardial infarct size. However, the contribution of C5a receptor (C5aR) to I/R injury remains to be unknown. Here, we reported that both mRNA and protein for the C5aR were constitutively expressed on cardiomyocytes and upregulated as a function of time after I/R-induced myocardial cell injury in mice. Blockade of C5aR markedly decreased microvascular permeability in ischemic myocardial area and leukocyte adherence to coronary artery endothelium. Importantly, the blocking of C5aR with an anti-C5aR antibody was associated with inhibition in activation of protein kinase C delta (PKC-delta) and induction of PKC-mediated mitogen-activated protein kinase phosphatases-1 (MKP-1) leading to the increased activity of p42/p44 mitogen-activated protein (MAP) kinase cascade. These data provide evidence that C5aR-mediated myocardial cell injury is an important pathogenic factor, and that C5aR blockade may be useful therapeutic targets for the prevention of myocardial I/R injury.