RESUMEN
Neurodegenerative diseases are characterized by the progressive loss of structure and/or function of both neurons and glial cells, leading to different degrees of pathology and loss of cognition. The hypothesis of circuit reconstruction in the damaged brain via direct cell replacement has been pursued extensively so far. In this context, stem cells represent a useful option since they provide tissue restoration through the substitution of damaged neuronal cells with exogenous stem cells and create a neuro-protective environment through the release of bioactive molecules for healthy neurons, as well. These peculiar properties of stem cells are opening to potential therapeutic strategies for the treatment of severe neurodegenerative disorders, for which the absence of effective treatment options leads to an increasingly socio-economic burden. Currently, the introduction of new technologies in the field of stem cells and the implementation of alternative cell tissues sources are pointing to exciting frontiers in this area of research. Here, we provide an update of the current knowledge about source and administration routes of stem cells, and review light and shadows of cells replacement therapy for the treatment of the three main neurodegenerative disorders (Amyotrophic lateral sclerosis, Parkinson's, and Alzheimer's disease).
Asunto(s)
Sistema Nervioso Central/fisiopatología , Degeneración Nerviosa , Regeneración Nerviosa , Células-Madre Neurales/trasplante , Enfermedades Neurodegenerativas/cirugía , Trasplante de Células Madre , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/fisiopatología , Enfermedad de Alzheimer/cirugía , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Esclerosis Amiotrófica Lateral/fisiopatología , Esclerosis Amiotrófica Lateral/cirugía , Animales , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Humanos , Células-Madre Neurales/inmunología , Células-Madre Neurales/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Enfermedades Neurodegenerativas/fisiopatología , Neuroinmunomodulación , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Enfermedad de Parkinson/fisiopatología , Enfermedad de Parkinson/cirugía , Fenotipo , Recuperación de la Función , Trasplante de Células Madre/efectos adversosRESUMEN
The development and commercialization of new drugs is an articulated, lengthy, and very expensive process that proceeds through several steps, starting from target identification, screening new leading compounds for testing in preclinical studies, and subsequently in clinical trials to reach the final approval for therapeutic use. Preclinical studies are usually performed using both cell cultures and animal models, although they do not completely resume the complexity of human diseases, in particular neurodegenerative conditions. To this regard, stem cells represent a powerful tool in all steps of drug discovery. The recent advancement in induced Pluripotent Stem Cells (iPSCs) technology has opened the possibility to obtain patient-specific disease models for drug screening and development. Here, we report the use of iPSCs as a disease model for drug development in the contest of neurological disorders, including Alzheimer's (AD) and Parkinson's disease (PD), Amyotrophic lateral Sclerosis (ALS), and Fragile X syndrome (FRAX).
Asunto(s)
Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/fisiología , Enfermedades del Sistema Nervioso/terapia , Esclerosis Amiotrófica Lateral/terapia , Descubrimiento de Drogas/métodos , Descubrimiento de Drogas/tendencias , Evaluación Preclínica de Medicamentos , Humanos , Modelos Biológicos , Enfermedades Neurodegenerativas/terapia , Enfermedad de Parkinson/terapia , Preparaciones Farmacéuticas , Trasplante de Células Madre/métodos , Trasplante de Células Madre/tendenciasRESUMEN
Extracellular matrix (ECM) represents an essential component of the cellular niche. In this conditioned microenvironment, the proliferation rates and differentiation states of stem cells are regulated by several factors. In contrast, in in vitro experimental models, cell growth, or induction procedures toward specific cell lines usually occur in contact with plastic, glass, or biogel supports. In this study, we evaluated the effect of a decellularized ECM, derived from bone marrow stem cells, on the neuronal differentiation of mesenchymal stem cells (MSCs) extracted from dental pulp (Dental Pulp Stem Cells - DPSCs). Since DPSCs derive from neuroectodermal embryonic precursors, they are thought to have a greater propensity toward neuronal differentiation than MSCs isolated from other sources. We hypothesized that the presence of a decellularized ECM scaffold could act positively on neuronal-DPSC differentiation through reproduction of an in vivo-like microenvironment. Results from scanning electron microscopy, immunofluorescence, and gene expression assays showed that ECM is able to positively influence the morphology of cells and their distribution and the expression of specific neuronal markers (i.e., NF-L, NF-M, NF-H, PAX6, MAP2).
RESUMEN
Regenerative medicine is a branch of translational research that aims to reestablish irreparably damaged tissues and organs by stimulating the body's own repair mechanisms via the implantation of stem cells differentiated into specialized cell types. A rich source of adult stem cells is located inside the tooth and is represented by human dental pulp stem cells, or hDPSCs. These cells are characterized by a high proliferative rate, have self-renewal and multi-lineage differentiation properties and are often used for tissue engineering and regenerative medicine. The present review will provide an overview of hDPSCs and related features with a special focus on their potential applications in regenerative medicine of the nervous system, such as, for example, after spinal cord injury. Recent advances in the identification and characterization of dental stem cells and in dental tissue engineering strategies suggest that bioengineering approaches may successfully be used to regenerate districts of the central nervous system, previously considered irreparable.
Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Pulpa Dental/citología , Células Madre Mesenquimatosas/fisiología , Regeneración Nerviosa/fisiología , Traumatismos de la Médula Espinal/terapia , Animales , Pulpa Dental/metabolismo , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismoRESUMEN
Silicon has been widely used as a material for microelectronic for more than 60 years, attracting considerable scientific interest as a promising tool for the manufacture of implantable medical devices in the context of neurodegenerative diseases. However, the use of such material involves responsibilities due to its toxicity, and researchers are pushing towards the generation of new classes of composite semiconductors, including the Silicon Carbide (3C-SiC). In the present work, we tested the biocompatibility of Silicon and 3C-SiC using an in vitro model of human neuronal stem cells derived from dental pulp (DP-NSCs) and mouse Olfactory Ensheathing Cells (OECs), a particular glial cell type showing stem cell characteristics. Specifically, we investigated the effects of 3C-SiC on neural cell morphology, viability and mitochondrial membrane potential. Data showed that both DP-NSCs and OECs, cultured on 3C-SiC, did not undergo consistent oxidative stress events and did not exhibit morphological modifications or adverse reactions in mitochondrial membrane potential. Our findings highlight the possibility to use Neural Stem Cells plated on 3C-SiC substrate as clinical tool for lesioned neural areas, paving the way for future perspectives in novel cell therapies for neuro-degenerated patients.
Asunto(s)
Materiales Biocompatibles/química , Células-Madre Neurales/efectos de los fármacos , Neuronas/efectos de los fármacos , Silicio/química , Animales , Materiales Biocompatibles/farmacología , Materiales Biocompatibles/uso terapéutico , Compuestos Inorgánicos de Carbono/química , Compuestos Inorgánicos de Carbono/farmacología , Supervivencia Celular/efectos de los fármacos , Humanos , Ratones , Neuroglía/efectos de los fármacos , Semiconductores , Silicio/farmacología , Silicio/uso terapéutico , Compuestos de Silicona/química , Compuestos de Silicona/farmacología , Propiedades de SuperficieRESUMEN
Silver nanoparticles (Ag-NPs) are one of the most popular nanotechnologies because of their unique antibacterial and antifungal properties. Given their increasing use in a wide range of commercial, biomedical and food products, exposure to Ag-NPs is now a reality in people's lives. However, there is a serious lack of information regarding their potential toxic effects in the central nervous system. In this study, we investigated the biocompatibility of "homemade" Ag-NPs in an in vitro model of human neurons derived from dental pulp mesenchymal stem cells. Our results showed that acute exposure to Ag-NPs cause cytotoxicity, by triggering cell apoptosis, damaging neuronal connections, affecting the mitochondrial activity and changing the mRNA expression level of MT3 and OSGIN2, two genes involved in heavy metals metabolism and cellular growth during oxidative stress conditions. Further studies are needed to understand the molecular mechanisms and the physiological consequences underlying Ag-NPs exposure.
Asunto(s)
Pulpa Dental/efectos de los fármacos , Células Madre Mesenquimatosas/efectos de los fármacos , Nanopartículas del Metal/toxicidad , Mitocondrias/efectos de los fármacos , Proteínas del Tejido Nervioso/biosíntesis , Nitrato de Plata/toxicidad , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Pulpa Dental/metabolismo , Relación Dosis-Respuesta a Droga , Expresión Génica , Humanos , Células Madre Mesenquimatosas/metabolismo , Metalotioneína 3 , Mitocondrias/metabolismo , Proteínas del Tejido Nervioso/genética , Neuronas/efectos de los fármacos , Neuronas/metabolismoRESUMEN
PROBLEM: Chronic endometritis (CE) is usually asymptomatic and different studies demonstrated the relation with infertility and recurrent pregnancy loss. Altered regulation of protein-encoding genes in CE has been demonstrated, but no evidence about the involvement of microRNAs in the pathology is present in literature. METHOD OF STUDY: In the endometrium from 15 women with CE and 15 healthy women, by RT-qPCR single assays, we investigated some microRNAs targeting IL11, CCL4, IGF1, and IGFBP1, which mRNAs had been found differentially expressed in endometrium of women affected by CE. The expression of IGF1 and IL11, targets of the deregulated microRNAs, has been analyzed in the same endometrium samples. We assessed the expression profiles of the deregulated microRNAs in the serum of the same patients validating their ability as biomarkers by statistical analysis. RESULTS: We demonstrated the upregulation of miR-27a-3p and miR-124-3p in the endometrium and serum from women with CE and found an anticorrelation relationship between miR-27a-3p and IGF1 in endometrium. ROC curve analysis suggested that miRNA investigation in endometrium and serum could discriminate women with CE. CONCLUSION: MiR-27a-3p and miR-124-3p could represent non-invasive markers of CE and, in a near future, could be used to assess the endometrial quality in IVF.
Asunto(s)
Aborto Espontáneo/genética , Endometritis/genética , Endometrio/fisiología , Marcadores Genéticos/genética , Infertilidad/genética , MicroARNs/genética , Adulto , Enfermedad Crónica , Femenino , Humanos , Factor I del Crecimiento Similar a la Insulina/genética , Patología Molecular , Embarazo , Regulación hacia ArribaRESUMEN
BACKGROUND: Stem cells are capable of self-renewal and differentiation into a wide range of cell types with multiple clinical and therapeutic applications. Stem cells are providing hope for many diseases that currently lack effective therapeutic methods, including strokes, Huntington's disease, Alzheimer's and Parkinson's disease. However, the paucity of suitable cell types for cell replacement therapy in patients suffering from neurological disorders has hampered the development of this promising therapeutic approach. AIM: The innovative aspect of this study has been to evaluate the neural differentiation capability of different tissue-derived stem cells coming from different tissue sources such as bone marrow, umbilical cord blood, human endometrium and amniotic fluid, cultured under the same supplemented media neuro-transcription factor conditions, testing the expression of neural markers such as GFAP, Nestin and Neurofilaments using the immunofluorescence staining assay and some typical clusters of differentiation such as CD34, CD90, CD105 and CD133 by using the cytofluorimetric test assay. RESULTS: Amniotic fluid derived stem cells showed a more primitive phenotype compared to the differentiating potential demonstrated by the other stem cell sources, representing a realistic possibility in the field of regenerative cell therapy suitable for neurodegenerative diseases.
Asunto(s)
Diferenciación Celular/fisiología , Neuronas/fisiología , Células Madre/fisiología , Adulto , Líquido Amniótico/citología , Células de la Médula Ósea/citología , Células de la Médula Ósea/fisiología , Endometrio/citología , Femenino , Sangre Fetal/citología , Citometría de Flujo , Humanos , Persona de Mediana Edad , Embarazo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Uveitis is a sight-threatening inflammatory disease of the eye which represents the third leading cause of blindness in the developed countries. The conventional pharmacological treatment includes corticosteroids and immunosuppressive agents, which are limited by their side effects. New therapeutic strategies are thus strongly needed. Exogenously-administered carbon monoxide (CO) may represent an effective treatment for conditions characterized by a dysregulated inflammatory response. Carbon monoxide-releasing molecules (CORMs) are a novel group of compounds capable of carrying and liberating controlled quantities of CO. Among CORMs, CORM-A1 represents the first example of water soluble CO releaser. We show here that CORM-A1 under a late prophylactic regime is able to significantly ameliorate the natural course of experimental autoimmune uveoretinitis, a rodent model of immunoinflammatory posterior uveitis. The present study strongly supports the development of CORM-A1 as a potential new drug for treatment of patients with non-infectious posterior uveitis.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Boranos/farmacología , Carbonatos/farmacología , ARN Mensajero/efectos de los fármacos , Retina/efectos de los fármacos , Retinitis/inmunología , Linfocitos T Reguladores/efectos de los fármacos , Úvea/efectos de los fármacos , Uveítis/inmunología , Animales , Enfermedades Autoinmunes/inducido químicamente , Enfermedades Autoinmunes/patología , Citocinas/efectos de los fármacos , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Expresión Génica/efectos de los fármacos , Fragmentos de Péptidos/toxicidad , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas Lew , Retina/inmunología , Retina/patología , Retinitis/inducido químicamente , Retinitis/patología , Proteínas de Unión al Retinol/toxicidad , Bazo/efectos de los fármacos , Bazo/metabolismo , Linfocitos T Reguladores/inmunología , Úvea/inmunología , Úvea/patología , Uveítis/inducido químicamente , Uveítis/patologíaRESUMEN
We describe a method based on fluorescence-lifetime imaging microscopy (FLIM) to assess the fluidity of various membranes in neuronal cells at different stages of development [day 12 (E12) and day 16 (E16) of gestation]. For the FLIM measurements, we use the Laurdan probe which is commonly used to assess membrane water penetration in model and in biological membranes using spectral information. Using the FLIM approach, we build a fluidity scale based on calibration with model systems of different lipid compositions. In neuronal cells, we found a marked difference in fluidity between the internal membranes and the plasma membrane, being the plasma membrane the less fluid. However, we found no significant differences between the two cell groups, E12 and E16. Comparison with NIH3T3 cells shows that the plasma membranes of E12 and E16 cells are significantly more fluid than the plasma membrane of the cancer cells.
Asunto(s)
2-Naftilamina/análogos & derivados , Membrana Celular/metabolismo , Desarrollo Embrionario , Colorantes Fluorescentes/química , Lauratos/química , Metabolismo de los Lípidos , Microscopía Fluorescente/métodos , Neuronas/citología , 2-Naftilamina/química , Animales , Colesterol/metabolismo , Femenino , Fluidez de la Membrana , Ratones , Células 3T3 NIH , Embarazo , Factores de TiempoRESUMEN
Gold nanoparticles (AuNPs) conjugated to DNA are widely used for biomedical targeting and sensing applications. DNA functionalization is easily reached on laser generated gold nanoparticles because of their unique surface chemistry, not reproducible by other methods. In this context, we present an extensive investigation concerning the attachment of DNA to the surface of laser generated nanoparticles using Dynamic Light Scattering and UV-Vis spectroscopy. The DNA conjugation is highlighted by the increase of the hydrodynamic radius and by the UV-Vis spectra behavior. Our investigation indicates that Dynamic Light Scattering is a suitable analytical tool to evidence, directly and qualitatively, the binding between a DNA molecule and a gold nanoparticle, therefore it is ideal to monitor changes in the conjugation process when experimental conditions are varied.
Asunto(s)
Dispersión Dinámica de Luz , Oro/química , Nanopartículas del Metal/química , ADN/química , Hidrodinámica , Rayos Láser , Nanotecnología/métodos , Oligonucleótidos/genética , Sales (Química) , Cloruro de Sodio , Espectrofotometría Ultravioleta , Resonancia por Plasmón de SuperficieRESUMEN
The appearance of the slow mode, revealed by dynamic light scattering (DLS) measurements in Micrococcus luteus DNA with high GC content, and the effect of guanine sequences on changes of DNA physical state and conformational transitions were investigated. We used two different spectroscopic approaches: DLS, to evidence the relatively slowly diffusing particles arising at high salt concentration, ascribable to the formation of large unspecific molecular aggregates, and circular dichroism spectroscopy, to identify these entities. Our results bring us to conclude that a peculiar, unconventional, structural transition, due to the presence of long guanine stretches, in a well-defined experimental condition, can occur. We comment on the biological implications to detect, by spectroscopic measurements, such an unusual structure involved in the stability, protection and replication maintenance along the human telomeric G-rich strand.
Asunto(s)
ADN/química , Luz , Conformación de Ácido Nucleico/efectos de los fármacos , Conformación de Ácido Nucleico/efectos de la radiación , Dispersión de Radiación , Cloruro de Sodio/farmacología , Animales , Composición de Base , Bovinos , ADN Bacteriano/química , Micrococcus luteus , Microscopía , Desnaturalización de Ácido Nucleico , Soluciones , Temperatura de Transición , Agua/químicaRESUMEN
This work reports light scattering measurements on DNA in aqueous solutions (100mM NaCl, 1mM EDTA and 10mM Tris-HCl buffer, pH 7.8) over a wide range of molecular weights (10(2)-10(5) base pairs) and shows that, in the above standard solvent, shorter chains (<10(4) base pairs) behave as a "wormlike chain" and their diffusion coefficients as obtained by dynamic light scattering measurements, confirm the prediction of standard wormlike model, whilst longer chains (>10(4) base pairs) behave in a different manner. Dynamic and static light scattering and SEM analysis indicate that DNA molecules 10(5) base pairs long, condense into compact structures in our solvent conditions. Calculations done using a wormlike model are also presented and discussed in comparison both to our experimental data and to other data reported in the literature.
Asunto(s)
ADN/química , Luz , Dispersión de Radiación , Animales , ADN Bacteriano/química , ADN Bacteriano/ultraestructura , Difusión , Humanos , Micrococcus luteus/química , Peso MolecularRESUMEN
Aerobic exercise increases free radical production as a consequence of enhanced oxygen consumption. If free radical formation exceeds antioxidant capacity, lipids, proteins, and DNA may be oxidized. Oxidative stress is widely recognized as a factor in many degenerative human diseases. The role of dietary antioxidants in protection against disease is a topic of continuing interest. In fact, there is epidemiological evidence correlating a higher intake of nutrients possessing antioxidant abilities with a lower incidence of various human diseases. This study was directed at investigating whether changes in plasma antioxidant capacity and oxidative stress markers occur in voluntary wheel runners, before and after oral supplementation with lycopene and isoflavones. For this purpose, plasma antioxidant capacity and oxidative stress markers were assessed in long distance runners at the end of a 60-minute run. Comparisons were made between runners before and after 60 days of supplementation with lycopene and isoflavones. DNA damage in blood cells of the same samples was also evaluated by comet assay. This investigation shows that oral supplementation with lycopene and soy-derived isoflavones significantly reduced lipid peroxidation and enhanced plasma nonproteic antioxidant defense.
Asunto(s)
Antioxidantes/farmacología , Carotenoides/farmacología , Ejercicio Físico/fisiología , Isoflavonas/farmacología , Peroxidación de Lípido/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Carrera/fisiología , Anciano , Antioxidantes/metabolismo , Daño del ADN , Suplementos Dietéticos , Humanos , Peróxidos Lipídicos/sangre , Licopeno , Solanum lycopersicum , Masculino , Persona de Mediana Edad , Extractos Vegetales/farmacología , Glycine max , Compuestos de Sulfhidrilo/sangreRESUMEN
BACKGROUND: Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase. It is degraded by the enzyme dimethylarginine dimethylaminohydrolase (DDAH). METHODS: Rats (n = 50) underwent to 45 min of renal ischemia followed by 30 min, 1 h, and 3 h of reperfusion. Expression of endothelial nitric oxide synthase, inducible nitric oxide synthase, DDAH-1, DDAH-2, renal DDAH activity, plasma NO2(-)/NO3(-), and ADMA levels were evaluated. RESULTS: Inducible nitric oxide synthase expression increased, as confirmed by both plasma (11.89 +/- 1.02, 15.56 +/- 0.93, 11.82 +/- 0.86, 35.05 +/- 1.28, and 43.89 +/- 1.63 nmol/ml in the control, ischemic, 30-min, 1-h, and 3-h groups, respectively) and renal (4.81 +/- 0.4, 4.85 +/- 1, 9.42 +/- 0.7, 15.42 +/- 0.85, and 22.03 +/- 1.11 nmol/mg protein) formations of NO2(-)/NO3(-). DDAH-1 expression decreased after reperfusion, whereas DDAH-2 increased after 30 min, returning to basal levels after 3 h. Total DDAH activity was reduced during all times of reperfusion. Both plasma (0.41 +/- 0.03, 0.43 +/- 0.05, 0.62 +/- 0.02, 0.71 +/- 0.02, and 0.41 +/- 0.01 nmol/ml in the control, ischemic, 30-min, 1-h, and 3-h groups, respectively) and renal (1.51 +/- 0.01, 1.5 +/- 0.01, 1.53 +/- 0.01, 2.52 +/- 0.04, and 4.48 +/- 0.03 nmol/mg protein in the control, ischemic, 30-min, 1-h, and 3-h groups, respectively) concentrations of ADMA increased. CONCLUSIONS: Results suggest that ischemia-reperfusion injury leads to reduced DDAH activity and modification of different DDAH isoform expression, thus leading to increased ADMA levels, which may lead to increased cardiovascular risk.
Asunto(s)
Amidohidrolasas/biosíntesis , Regulación Enzimológica de la Expresión Génica/fisiología , Isquemia/enzimología , Riñón/irrigación sanguínea , Riñón/enzimología , Animales , Activación Enzimática/fisiología , Isquemia/patología , Riñón/patología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Ratas , ReperfusiónRESUMEN
Many physiological effects of natural antioxidants, their extracts or their major active components, have been reported in recent decades. Most of these compounds are characterized by a phenolic structure, similar to that of alpha-tocopherol, and present antioxidant properties that have been demonstrated both in vitro and in vivo. Polyphenols may increase the capacity of endogenous antioxidant defences and modulate the cellular redox state. Changes in the cellular redox state may have wide-ranging consequences for cellular growth and differentiation. The majority of in vitro and in vivo studies conducted so far have attributed the protective effect of bioactive polyphenols to their chemical reactivity toward free radicals and their capacity to prevent the oxidation of important intracellular components. However, in recent years a possible novel aspect in the mode of action of these compounds has been suggested; that is, the ultimate stimulation of the heme oxygenase-1 (HO-1) pathway is likely to account for the established and powerful antioxidant/anti-inflammatory properties of these polyphenols. The products of the HO-catalyzed reaction, particularly carbon monoxide (CO) and biliverdin/bilirubin have been shown to exert protective effects in several organs against oxidative and other noxious stimuli. In this context, it is interesting to note that induction of HO-1 expression by means of natural compounds contributes to protection against liver damage in various experimental models. The focus of this review is on the significance of targeted induction of HO-1 as a potential therapeutic strategy to protect the liver against various stressors in several pathological conditions.
Asunto(s)
Antiinflamatorios/farmacología , Antioxidantes/farmacología , Sistema Biliar/efectos de los fármacos , Hemo-Oxigenasa 1/biosíntesis , Hepatopatías/prevención & control , Hígado/efectos de los fármacos , Animales , Antiinflamatorios/química , Antioxidantes/química , Sistema Biliar/enzimología , Inducción Enzimática , Humanos , Hígado/enzimología , Hepatopatías/enzimología , Estructura Molecular , Preparaciones de Plantas/farmacología , Relación Estructura-ActividadRESUMEN
Several lines of evidence have extensively demonstrated that peroxynitrite plays a pivotal role in Central Nervous System (CNS) injuries. The present study was aimed at elucidating the molecular mechanism by which propofol attenuates peroxynitrite-mediated injury in the brain. Primary cultured astroglial cells were incubated for 18 h with a known peroxynitrite donor (SIN-1,3 mM) in the presence or absence of propofol (40 microM, 80 microM and 160 microM). The protective effects of propofol were evaluated by MTT cytotoxicity assay, LDH release, and caspase-3 activation by Western blot analysis. Appropriate propofol concentrations (ranging from 40 microM to 160 microM) significantly increased HO-1 expression and attenuated SIN-1-mediated cytotoxicity and caspase-3 activation. The protective effects of propofol were mitigated by the addition of tin-mesoporphirin (SnMP), a potent inhibitor of HO activity. The addition of a specific synthetic inhibitor of NF-kappaB abolished propofol-mediated HO-1 induction, suggesting a possible role for this nuclear transcriptional factor in our experimental conditions. These findings indicate that propofol attenuates peroxynitrite-mediated apoptosis in astroglial cells, a property that may be relevant in both physiological and pathological processes in the CNS.
Asunto(s)
Antioxidantes/farmacología , Astrocitos/efectos de los fármacos , Astrocitos/enzimología , Inhibidores de Caspasas , Hemo Oxigenasa (Desciclizante)/metabolismo , Propofol/farmacología , Animales , Western Blotting , Caspasa 3 , Caspasas/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Hemo-Oxigenasa 1 , Molsidomina/análogos & derivados , Molsidomina/farmacología , Donantes de Óxido Nítrico/farmacología , Ácido Peroxinitroso/farmacología , Ratas , Ratas WistarRESUMEN
BACKGROUND: The concentration of peroxynitrite in the brain increases after central nervous system injuries. The authors hypothesized that propofol, because of its particular chemical structure, mitigates the effects of peroxynitrite-mediated oxidative stress and apoptosis by the induction of heme oxygenase (HO)-1 in primary cultured astroglial cells. METHODS: Primary cultured astroglial cells were incubated for 18 h with a known peroxynitrite donor (3 mm SIN-1) in the presence or absence of propofol (40 microm, 80 microm, 160 microm, and 1 mm). The protective effects of propofol were evaluated by 3(4,5-dimethyl-thiazol-2-yl)2,5-diphenyl-tetrazolium bromide cytotoxicity assay, lactic dehydrogenase release, DNA ladderization by Comet assay, and caspase-3 activation by Western blot analysis. RESULTS: Appropriate propofol concentrations (ranging from 40 microm to 1 mm) significantly increased HO-1 expression and attenuated SIN-1-mediated DNA ladderization and caspase-3 activation. The protective effects of propofol were mitigated by the addition of tin mesoporphyrin, a potent inhibitor of HO activity. The addition of a specific synthetic inhibitor of nuclear factor kappaB abolished propofol-mediated HO-1 induction, suggesting a possible role of this nuclear transcriptional factor in our experimental conditions. CONCLUSIONS: The antioxidant properties of propofol can be partially attributed to its scavenging effect on peroxynitrite as well as to its ability to increase HO-1 expression at higher concentrations, a property that might be relevant to neuroprotection during anesthesia.
Asunto(s)
Anestésicos Intravenosos/farmacología , Apoptosis/efectos de los fármacos , Astrocitos/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Molsidomina/análogos & derivados , Ácido Peroxinitroso/antagonistas & inhibidores , Propofol/farmacología , Animales , Western Blotting , Caspasa 3 , Caspasas/metabolismo , Células Cultivadas , Ensayo Cometa , ADN/química , ADN/metabolismo , Activación Enzimática/efectos de los fármacos , Hemo Oxigenasa (Desciclizante)/antagonistas & inhibidores , Hemo-Oxigenasa 1 , L-Lactato Deshidrogenasa/metabolismo , Molsidomina/farmacología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Ratas , Ratas Wistar , Sales de Tetrazolio , TiazolesRESUMEN
We discuss the use of fluorescence correlation spectroscopy for the measurement of relatively slow rotations of large macromolecules in solution or attached to other macromolecular structures. We present simulations and experimental results to illustrate the range of rotational correlation times and diffusion times that the technique can analyze. In particular, we examine various methods to analyze the polarization fluctuation data. We have found that by first constructing the polarization function and then calculating the autocorrelation function, we can obtain the rotational motion of the molecule with very little interference from the lateral diffusion of the macromolecule, as long as the rotational diffusion is significantly faster than the lateral diffusion. Surprisingly, for common fluorophores the autocorrelation of the polarization function is relatively unaffected by the photon statistics. In our instrument, two-photon excitation is used to define a small volume of illumination where a few molecules are present at any instant of time. The measurements of long DNA molecules labeled with the fluorescent probe DAPI show local rotational motions of the polymers in addition to translation motions of the entire polymer. For smaller molecules such as EGFP, the viscosity of the solution must be increased to bring the relaxation due to rotational motion into the measurable range. Overall, our results show that polarized fluorescence correlation spectroscopy can be used to detect fast and slow rotational motion in the time scale from microsecond to second, a range that cannot be easily reached by conventional fluorescence anisotropy decay methods.