RESUMEN
Ischemic stroke is a leading cause of disability world-wide. Mounting evidence supports neuromuscular pathology following stroke, yet mechanisms of dysfunction and therapeutic action remain undefined. The objectives of our study were to investigate neuromuscular pathophysiology following ischemic stroke and to evaluate the therapeutic effect of Robot-Assisted Mechanical massage Therapy (RAMT) on neuromuscular junction (NMJ) morphology. Using an ischemic stroke model in male rats, we demonstrated longitudinal losses of muscle contractility and electrophysiological estimates of motor unit number in paretic hindlimb muscles within 21 days of stroke. Histological characterization demonstrated striking pre- and postsynaptic alterations at the NMJ. Stroke prompted enlargement of motor axon terminals, acetylcholine receptor (AChR) area, and motor endplate size. Paretic muscle AChRs were also more homogenously distributed across motor endplates, exhibiting fewer clusters and less fragmentation. Most interestingly, NMJs in paretic muscle exhibited increased frequency of polyaxonal innervation. This finding of increased polyaxonal innervation in stroke-affected skeletal muscle suggests that reduction of motor unit number following stroke may be a spurious artifact due to overlapping of motor units rather than losses. Furthermore, we tested the effects of RAMT - which we recently showed to improve motor function and protect against subacute myokine disturbance - and found significant attenuation of stroke-induced NMJ alterations. RAMT not only normalized the post-stroke presentation of polyaxonal innervation but also mitigated postsynaptic expansion. These findings confirm complex neuromuscular pathophysiology after stroke, provide mechanistic direction for ongoing research, and inform development of future therapeutic strategies. SIGNIFICANCE: Ischemic stroke is a leading contributor to chronic disability, and there is growing evidence that neuromuscular pathology may contribute to the impact of stroke on physical function. Following ischemic stroke in a rat model, there are progressive declines of motor unit number estimates and muscle contractility. These changes are paralleled by striking pre- and postsynaptic maladaptive changes at the neuromuscular junction, including polyaxonal innervation. When administered to paretic hindlimb muscle, Robot-Assisted Mechanical massage Therapy - previously shown to improve motor function and protect against subacute myokine disturbance - prevents stroke-induced neuromuscular junction alterations. These novel observations provide insight into the neuromuscular response to cerebral ischemia, identify peripheral mechanisms of functional disability, and present a therapeutic rehabilitation strategy with clinical relevance.
Asunto(s)
Axones/fisiología , Isquemia Encefálica/rehabilitación , Accidente Cerebrovascular Isquémico/rehabilitación , Manipulaciones Musculoesqueléticas/instrumentación , Unión Neuromuscular/fisiología , Robótica/instrumentación , Animales , Isquemia Encefálica/fisiopatología , Accidente Cerebrovascular Isquémico/fisiopatología , Masculino , Fenómenos Mecánicos , Contracción Muscular/fisiología , Manipulaciones Musculoesqueléticas/métodos , Ratas , Ratas Wistar , Robótica/métodosRESUMEN
In the pathophysiologic setting of cerebral ischemia, excitotoxic levels of glutamate contribute to neuronal cell death. Our previous work demonstrated the ability of glutamate oxaloacetate transaminase (GOT) to metabolize neurotoxic glutamate in the stroke-affected brain. Here, we seek to identify small-molecule inducers of GOT expression to mitigate ischemic stroke injury. From a panel of phytoestrogen isoflavones, biochanin A (BCA) was identified as the most potent inducer of GOT gene expression in neural cells. BCA significantly increased GOT mRNA and protein expression at 24 h and protected against glutamate-induced cell death. Of note, this protection was lost when GOT was knocked down. To validate outcomes in vivo, C57BL/6 mice were intraperitoneally injected with BCA (5 and 10 mg/kg) for 4 wk and subjected to ischemic stroke. BCA levels were significantly increased in plasma and brain of mice. Immunohistochemistry demonstrated increased GOT protein expression in the brain. BCA attenuated stroke lesion volume as measured by 9.4T MRI and improved sensorimotor function-this protection was lost with GOT knockdown. BCA increased luciferase activity in cells that were transfected with the pERRE3tk-LUC plasmid, which demonstrated transactivation of GOT. This increase was lost when estrogen-related receptor response element sites were mutated. Taken together, BCA represents a natural phytoestrogen that mitigates stroke-induced injury by inducing GOT expression.-Khanna, S., Stewart, R., Gnyawali, S., Harris, H., Balch, M., Spieldenner, J., Sen, C. K., Rink, C. Phytoestrogen isoflavone intervention to engage the neuroprotective effect of glutamate oxaloacetate transaminase against stroke.
Asunto(s)
Aspartato Aminotransferasas/metabolismo , Isquemia Encefálica/tratamiento farmacológico , Ácido Glutámico/metabolismo , Isoflavonas/farmacología , Fármacos Neuroprotectores/farmacología , Fitoestrógenos/farmacología , Accidente Cerebrovascular/tratamiento farmacológico , Animales , Isquemia Encefálica/patología , Ratones , Ratones Endogámicos C57BL , Accidente Cerebrovascular/patologíaRESUMEN
Ischemic stroke results in excessive release of glutamate, which contributes to neuronal cell death. Here, we test the hypothesis that otherwise neurotoxic glutamate can be productively metabolized by glutamate oxaloacetate transaminase (GOT) to maintain cellular energetics and protect the brain from ischemic stroke injury. The GOT-dependent metabolism of glutamate was studied in primary neural cells and in stroke-affected C57-BL6 mice using magnetic resonance spectroscopy and GC-MS. Extracellular Glu sustained cell viability under hypoglycemic conditions and increased GOT-mediated metabolism in vitro Correction of stroke-induced hypoxia using supplemental oxygen in vivo lowered Glu levels as measured by 1H magnetic resonance spectroscopy. GOT knockdown abrogated this effect and caused ATP loss in the stroke-affected brain. GOT overexpression increased anaplerotic refilling of tricarboxylic acid cycle intermediates in mouse brain during ischemic stroke. Furthermore, GOT overexpression not only reduced ischemic stroke lesion volume but also attenuated neurodegeneration and improved poststroke sensorimotor function. Taken together, our results support a new paradigm that GOT enables metabolism of otherwise neurotoxic extracellular Glu through a truncated tricarboxylic acid cycle under hypoglycemic conditions.-Rink, C., Gnyawali, S., Stewart, R., Teplitsky, S., Harris, H., Roy, S., Sen, C. K., Khanna, S. Glutamate oxaloacetate transaminase enables anaplerotic refilling of TCA cycle intermediates in stroke-affected brain.
Asunto(s)
Aspartato Aminotransferasas/metabolismo , Ciclo del Ácido Cítrico , Infarto de la Arteria Cerebral Media/metabolismo , Animales , Aspartato Aminotransferasas/genética , Células Cultivadas , Glucosa/metabolismo , Ácido Glutámico/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Oxígeno/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
The objective of this study (clinicaltrials.gov NCT01858376) was to determine the effect of oral supplementation of a standardized extract of Phyllanthus emblica (CAPROS(®)) on cardiovascular disease (CVD) risk factors in overweight adult human subjects from the US population. Overweight/Class-1 obese (body-mass index: 25-35) adult subjects received 500 mg of CAPROS supplement b.i.d for 12 weeks. The study design included two baseline visits followed by 12 weeks of supplementation and then 2 weeks of washout. At all visits, peripheral venous blood was collected in sodium citrate tubes. Lipid profile measurements demonstrated a significant decrease in calculated low-density lipoprotein cholesterol and total cholesterol/high-density lipoprotein following 12 weeks of CAPROS supplementation when compared to averaged baseline visits. Circulatory high-sensitivity C reactive protein (hs-CRP) levels were significantly decreased after 12 weeks of supplementation. In addition, both ADP- and collagen-induced platelet aggregation was significantly downregulated following 12 weeks of supplementation. Overall, the study suggests that oral CAPROS supplementation may provide beneficial effects in overweight/Class-1 obese adults by lowering multiple global CVD risk factors.
Asunto(s)
Enfermedades Cardiovasculares/prevención & control , Obesidad/complicaciones , Sobrepeso/complicaciones , Phyllanthus emblica/química , Extractos Vegetales/administración & dosificación , Agregación Plaquetaria/efectos de los fármacos , Adulto , Glucemia/metabolismo , Índice de Masa Corporal , Proteína C-Reactiva/metabolismo , Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/tratamiento farmacológico , Enfermedades Cardiovasculares/fisiopatología , HDL-Colesterol/sangre , LDL-Colesterol/sangre , Suplementos Dietéticos/análisis , Femenino , Humanos , Masculino , Persona de Mediana Edad , Obesidad/sangre , Sobrepeso/sangre , Factores de RiesgoRESUMEN
The vitamin E family includes both tocopherols and tocotrienols, where α-tocopherol (αTOC) is the most bioavailable form. Clinical trials testing the therapeutic efficacy of high-dose αTOC against stroke have largely failed or reported negative outcomes when a "more is better" approach to supplementation (>400 IU/d) was used. This work addresses mechanisms by which supraphysiologic αTOC may contribute to stroke-induced brain injury. Ischemic stroke injury and the neuroinflammatory response were studied in tocopherol transfer protein-deficient mice maintained on a diet containing αTOC vitamin E at the equivalent human dose of 1680 IU/d. Ischemic stroke-induced brain injury was exacerbated in the presence of supraphysiologic brain αTOC levels. At 48 h after stroke, S100B and RAGE expression was increased in stroke-affected cortex of mice with elevated brain αTOC levels. Such increases were concomitant with aggravated microglial activation and neuroinflammatory signaling. A poststroke increase in markers of oxidative injury and neurodegeneration in the presence of elevated brain αTOC establish that at supraphysiologic levels, αTOC potentiates neuroinflammatory responses to acute ischemic stroke. Exacerbation of microglial activation by excessive αTOC likely depends on its unique cell signaling regulatory properties independent of antioxidant function. Against the background of clinical failure for high-dose αTOC, outcomes of this work identify risk for exacerbating stroke-induced brain injury as a result of supplementing diet with excessive levels of αTOC.
Asunto(s)
Antioxidantes/toxicidad , Lesiones Encefálicas/inducido químicamente , Inflamación/inducido químicamente , Isquemia/complicaciones , Microglía/patología , Accidente Cerebrovascular/complicaciones , alfa-Tocoferol/toxicidad , Animales , Biomarcadores/metabolismo , Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/patología , Humanos , Técnicas para Inmunoenzimas , Inflamación/metabolismo , Inflamación/patología , Isquemia/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Microglía/metabolismo , Accidente Cerebrovascular/patología , Superóxidos/metabolismoRESUMEN
Glutathione depletion and 12-lipoxygenase-dependent metabolism of arachidonic acid are known to be implicated in neurodegeneration associated with acute ischemic stroke. The objective of this study was to investigate the significance of miR-29 in neurodegeneration associated with acute ischemic stroke. Neural cell death caused by arachidonic acid insult of glutathione-deficient cells was preceded by a 12-lipoxygenase-dependent loss of miR-29b. Delivery of miR-29b mimic to blunt such loss was neuroprotective. miR-29b inhibition potentiated such neural cell death. 12-Lipoxygenase knockdown and inhibitors attenuated the loss of miR-29b in challenged cells. In vivo, stroke caused by middle-cerebral artery occlusion was followed by higher 12-lipoxygenase activity and loss of miR-29b as detected in laser-captured infarct site tissue. 12-Lipoxygenase knockout mice demonstrated protection against such miR loss. miR-29b gene delivery markedly attenuated stroke-induced brain lesion. Oral supplementation of α-tocotrienol, a vitamin E 12-lipoxygenase inhibitor, rescued stroke-induced loss of miR-29b and minimized lesion size. This work provides the first evidence demonstrating that loss of miR-29b at the infarct site is a key contributor to stroke lesion. Such loss is contributed by activity of the 12-lipoxygenase pathway providing maiden evidence linking arachidonic acid metabolism to miR-dependent mechanisms in stroke.
Asunto(s)
Isquemia Encefálica/genética , Isquemia Encefálica/patología , Muerte Celular/genética , Muerte Celular/fisiología , Infarto Cerebral/genética , Infarto Cerebral/patología , MicroARNs/genética , MicroARNs/fisiología , Neuronas/patología , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/patología , Animales , Antioxidantes/farmacología , Araquidonato 12-Lipooxigenasa/fisiología , Western Blotting , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Células Cultivadas , Eicosanoides/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Glutatión/metabolismo , Imagen por Resonancia Magnética , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , MicroARNs/antagonistas & inhibidores , Embarazo , ARN/biosíntesis , ARN/aislamiento & purificación , ARN Interferente Pequeño/farmacología , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Tocotrienoles/farmacología , TransfecciónRESUMEN
The natural vitamin E family is composed of 8 members equally divided into 2 classes: tocopherols (TCP) and tocotrienols (TE). A growing body of evidence suggests TE possess potent biological activity not shared by TCP. The primary objective of this work was to determine the concentrations of TE (200 mg mixed TE, b.i.d.) and TCP [200 mg α-TCP, b.i.d.)] in vital tissues and organs of adults receiving oral supplementation. Eighty participants were studied. Skin and blood vitamin E concentrations were determined from healthy participants following 12 wk of oral supplementation of TE or TCP. Vital organ vitamin E levels were determined by HPLC in adipose, brain, cardiac muscle, and liver of surgical patients following oral TE or TCP supplementation (mean duration, 20 wk; range, 1-96 wk). Oral supplementation of TE significantly increased the TE tissue concentrations in blood, skin, adipose, brain, cardiac muscle, and liver over time. α-TE was delivered to human brain at a concentration reported to be neuroprotective in experimental models of stroke. In prospective liver transplantation patients, oral TE lowered the model for end-stage liver disease (MELD) score in 50% of patients supplemented, whereas only 20% of TCP-supplemented patients demonstrated a reduction in MELD score. This work provides, to our knowledge, the first evidence demonstrating that orally supplemented TE are transported to vital organs of adult humans. The findings of this study, in the context of the current literature, lay the foundation for Phase II clinical trials testing the efficacy of TE against stroke and end-stage liver disease in humans.
Asunto(s)
Enfermedad Hepática en Estado Terminal/dietoterapia , Tocotrienoles/administración & dosificación , Tocotrienoles/farmacocinética , Adulto , Transporte Biológico Activo , Suplementos Dietéticos , Progresión de la Enfermedad , Enfermedad Hepática en Estado Terminal/metabolismo , Enfermedad Hepática en Estado Terminal/prevención & control , Femenino , Humanos , Trasplante de Hígado , Masculino , Estudios Prospectivos , Distribución Tisular , Tocoferoles/administración & dosificación , Tocoferoles/farmacocinética , Vitamina E/metabolismoRESUMEN
Vitamin E consists of tocopherols and tocotrienols, in which α-tocotrienol is the most potent neuroprotective form that is also effective in protecting against stroke in rodents. As neuroprotective agents alone are insufficient to protect against stroke, we sought to test the effects of tocotrienol on the cerebrovascular circulation during ischemic stroke using a preclinical model that enables fluoroscopy-guided angiography. Mongrel canines (mean weight=26.3±3.2 kg) were supplemented with tocotrienol-enriched (TE) supplement (200 mg b.i.d, n=11) or vehicle placebo (n=9) for 10 weeks before inducing transient middle cerebral artery (MCA) occlusion. Magnetic resonance imaging was performed 1 hour and 24 hours post reperfusion to assess stroke-induced lesion volume. Tocotrienol-enriched supplementation significantly attenuated ischemic stroke-induced lesion volume (P<0.005). Furthermore, TE prevented loss of white matter fiber tract connectivity after stroke as evident by probabilistic tractography. Post hoc analysis of cerebral angiograms during MCA occlusion revealed that TE-supplemented canines had improved cerebrovascular collateral circulation to the ischemic MCA territory (P<0.05). Tocotrienol-enriched supplementation induced arteriogenic tissue inhibitor of metalloprotease 1 and subsequently attenuated the activity of matrix metalloproteinase-2. Outcomes of the current preclinical trial set the stage for a clinical trial testing the effects of TE in patients who have suffered from transient ischemic attack and are therefore at a high risk for stroke.
Asunto(s)
Isquemia Encefálica/complicaciones , Circulación Cerebrovascular/fisiología , Circulación Colateral/fisiología , Fármacos Neuroprotectores/uso terapéutico , Accidente Cerebrovascular/prevención & control , Tocotrienoles/uso terapéutico , Animales , Isquemia Encefálica/enzimología , Isquemia Encefálica/fisiopatología , Angiografía Cerebral , Circulación Cerebrovascular/efectos de los fármacos , Circulación Colateral/efectos de los fármacos , Modelos Animales de Enfermedad , Perros , Evaluación Preclínica de Medicamentos , Fluoroscopía , Imagen por Resonancia Magnética , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/farmacología , Accidente Cerebrovascular/enzimología , Accidente Cerebrovascular/etiología , Accidente Cerebrovascular/fisiopatología , Tocotrienoles/administración & dosificación , Tocotrienoles/farmacologíaRESUMEN
This work rests on our previous report (J Cereb Blood Flow Metab 30: 1275-1287, 2010) recognizing that glutamate (Glu) oxaloacetate transaminase (GOT) is induced when brain tissue hypoxia is corrected during acute ischemic stroke (AIS). GOT can metabolize Glu into tricarboxylic acid cycle intermediates and may therefore be useful to harness excess neurotoxic extracellular Glu during AIS as a metabolic substrate. We report that in cultured neural cells challenged with hypoglycemia, extracellular Glu can support cell survival as long as there is sufficient oxygenation. This effect is abrogated by GOT knockdown. In a rodent model of AIS, supplemental oxygen (100% O(2) inhaled) during ischemia significantly increased GOT expression and activity in the stroke-affected brain tissue and prevented loss of ATP. Biochemical analyses and in vivo magnetic resonance spectroscopy during stroke demonstrated that such elevated GOT decreased Glu levels at the stroke-affected site. In vivo lentiviral gene delivery of GOT minimized lesion volume, whereas GOT knockdown worsened stroke outcomes. Thus, brain tissue GOT emerges as a novel target in managing stroke outcomes. This work demonstrates that correction of hypoxia during AIS can help clear extracellular neurotoxic Glu by enabling utilization of this amino acid as a metabolic fuel to support survival of the hypoglycemic brain tissue. Strategies to mitigate extracellular Glu-mediated neurodegeneration via blocking receptor-mediated excitotoxicity have failed in clinical trials. We introduce the concept that under hypoglycemic conditions extracellular Glu can be transformed from a neurotoxin to a survival factor by GOT, provided there is sufficient oxygen to sustain cellular respiration.
Asunto(s)
Aspartato Aminotransferasas/metabolismo , Ácido Glutámico/farmacología , Oxígeno/metabolismo , Accidente Cerebrovascular/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Aspartato Aminotransferasa Citoplasmática/genética , Aspartato Aminotransferasa Citoplasmática/metabolismo , Aspartato Aminotransferasas/genética , Encéfalo/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Ácido Glutámico/metabolismo , Hipoxia/fisiopatología , Inmunohistoquímica , Técnicas In Vitro , Imagen por Resonancia Magnética , Ratones , Reacción en Cadena de la Polimerasa , ARN Interferente Pequeño , Ratas , Ratas Sprague-Dawley , Accidente Cerebrovascular/genéticaRESUMEN
The significance of the hypoxia component of stroke injury is highlighted by hypermetabolic brain tissue enriched with arachidonic acid (AA), a 22:6n-3 polyunsaturated fatty acid. In an ischemic stroke environment in which cerebral blood flow is arrested, oxygen-starved brain tissue initiates the rapid cleavage of AA from the membrane phospholipid bilayer. Once free, AA undergoes both enzyme-independent and enzyme-mediated oxidative metabolism, resulting in the formation of number of biologically active metabolites which themselves contribute to pathological stroke outcomes. This review is intended to examine two divergent roles of molecular dioxygen in brain tissue as (1) a substrate for life-sustaining homeostatic metabolism of glucose and (2) a substrate for pathogenic metabolism of AA under conditions of stroke. Recent developments in research concerning supplemental oxygen therapy as an intervention to correct the hypoxic component of stroke injury are discussed.
Asunto(s)
Ácido Araquidónico/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Oxígeno/metabolismo , Accidente Cerebrovascular/metabolismo , Animales , Encéfalo/fisiopatología , Isquemia Encefálica/metabolismo , Isquemia Encefálica/terapia , Humanos , Estrés Oxidativo , Oxígeno/uso terapéutico , Accidente Cerebrovascular/terapiaRESUMEN
A growing body of research supports that members of the vitamin E family are not redundant with respect to their biological function. Palm oil derived from Elaeis guineensis represents the richest source of the lesser characterized vitamin E, alpha-tocotrienol. One of 8 naturally occurring and chemically distinct vitamin E analogs, alpha-tocotrienol possesses unique biological activity that is independent of its potent antioxidant capacity. Current developments in alpha-tocotrienol research demonstrate neuroprotective properties for the lipid-soluble vitamin in brain tissue rich in polyunsaturated fatty acids (PUFAs). Arachidonic acid (AA), one of the most abundant PUFAs of the central nervous system, is highly susceptible to oxidative metabolism under pathologic conditions. Cleaved from the membrane phospholipid bilayer by cytosolic phospholipase A(2), AA is metabolized by both enzymatic and nonenzymatic pathways. A number of neurodegenerative conditions in the human brain are associated with disturbed PUFA metabolism of AA, including acute ischemic stroke. Palm oil-derived alpha-tocotrienol at nanomolar concentrations has been shown to attenuate both enzymatic and nonenzymatic mediators of AA metabolism and neurodegeneration. On a concentration basis, this represents the most potent of all biological functions exhibited by any natural vitamin E molecule. Despite such therapeutic potential, the scientific literature on tocotrienols accounts for roughly 1% of the total literature on vitamin E, thus warranting further investment and investigation.
Asunto(s)
Antioxidantes/uso terapéutico , Ácido Araquidónico/metabolismo , Encéfalo/efectos de los fármacos , Enfermedades Neurodegenerativas/prevención & control , Aceites de Plantas/uso terapéutico , Vitamina E/uso terapéutico , Animales , Antioxidantes/farmacología , Arecaceae , Encéfalo/metabolismo , Humanos , Peroxidación de Lípido/efectos de los fármacos , Enfermedades Neurodegenerativas/metabolismo , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Aceite de Palma , Fitoterapia , Aceites de Plantas/química , Aceites de Plantas/farmacología , Vitamina E/análogos & derivados , Vitamina E/farmacologíaRESUMEN
Acute ischemic stroke (AIS) results in focal deprivation of blood-borne factors, one of them being oxygen. The purpose of this study was two-fold: (1) to identify therapeutic conditions for supplemental oxygen in AIS and (2) to use transcriptome-wide screening toward uncovering oxygen-sensitive mechanisms. Transient MCAO in rodents was used to delineate the therapeutic potential of normobaric (NBO, 100% O(2), 1ATA) and hyperbaric oxygen (HBO, 100% O(2), 2ATA) during ischemia (iNBO, iHBO) and after reperfusion (rNBO, rHBO). Stroke lesion was quantified using 4.7 T MRI at 48 h. Supplemental oxygen during AIS significantly attenuated percent stroke hemisphere lesion volume as compared with that in room air (RA) controls, whereas identical treatment immediately after reperfusion exacerbated lesion volume (RA=22.4+/-1.8, iNBO=9.9+/-3.6, iHBO=6.6+/-4.8, rNBO=29.8+/-3.6, rHBO=35.4+/-7.6). iNBO and iHBO corrected penumbra tissue pO(2) during AIS as measured by EPR oxymetry. Unbiased query of oxygen-sensitive transcriptome in stroke-affected tissue identified 5,769 differentially expressed genes. Candidate genes were verified by real-time PCR using neurons laser-captured from the stroke-affected somatosensory cortex. Directed microarray analysis showed that supplemental oxygen limited leukocyte accumulation to the infarct site by attenuation of stroke-inducible proinflammatory chemokine response. The findings provide key information relevant to understanding oxygen-dependent molecular mechanisms in the AIS-affected brain.
Asunto(s)
Isquemia Encefálica , Regulación de la Expresión Génica , Oxigenoterapia Hiperbárica , Oxígeno/metabolismo , Accidente Cerebrovascular , Animales , Biomarcadores/metabolismo , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Isquemia Encefálica/terapia , Modelos Animales de Enfermedad , Humanos , Infarto de la Arteria Cerebral Media , Leucocitos/citología , Leucocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Estrés Oxidativo , Distribución Aleatoria , Ratas , Ratas Wistar , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/patología , Accidente Cerebrovascular/terapiaRESUMEN
BACKGROUND: Obesity is a global public health problem. Traditional herbal medicines may have some potential in managing obesity. The dried fruit rind of Garcinia cambogia, also known as Malabar tamarind, is a unique source of (-)-hydroxycitric acid (HCA), which exhibits a distinct sour taste and has been safely used for centuries in Southeastern Asia to make meals more filling. Recently it has been demonstrated that when taken orally, a novel, highly soluble calcium/potassium salt of HCA (HCA-SX) is safe and bioavailable in the human plasma. Although HCA-SX seems to be conditionally effective in weight management in experimental animals and in humans, its mechanism of action remains unclear. METHODS: In this study, subcutaneous preadipocytes collected from obese women with body mass index>25 kg/m2 were differentiated to adipocytes for 2 weeks in culture. The effects of low-dose HCA-SX on lipid metabolism and on the adipocyte transcriptome were tested. HCA-SX augmented isoproterenol- and 3-isobutyryl-1-methylxanthine-induced lipolysis. Using oil red O, the production of lipid storage droplets by the cultured mature human adipocytes was visualized and enumerated. RESULTS: HCA-SX caused droplet dispersion facilitating lipase action on the lipids. HCA-SX markedly induced leptin expression in the adipocytes. In the microarray analyses, a total of 54,676 probe sets were screened. HCA-SX resulted in significant down-regulation of 348, and induction of 366 fat- and obesity-related genes. HCA-SX induced transactivation of hypoxia inducible factor (HIF), a novel approach in the management of obesity. CONCLUSION: Taken together, the net effects support the antilipolytic and antiadipogenic effects of HCA-SX. Further human studies are warranted.
Asunto(s)
Adipocitos/efectos de los fármacos , Fármacos Antiobesidad/farmacología , Citratos/farmacología , Garcinia cambogia/química , Perfilación de la Expresión Génica , Obesidad/tratamiento farmacológico , Transcripción Genética , Adipocitos/metabolismo , Biomarcadores/metabolismo , Índice de Masa Corporal , Supervivencia Celular , Células Cultivadas , Suplementos Dietéticos , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Lipólisis , Obesidad/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Natural vitamin E includes eight chemically distinct molecules: alpha-, beta-, gamma-, and delta-tocopherols and alpha-, beta-, gamma-, and delta-tocotrienols. More than 95% of all studies on vitamin E are directed toward the specific study of alpha-tocopherol. The other forms of natural vitamin E remain poorly understood. The abundance of alpha-tocopherol in the human body and the comparable efficiency of all vitamin E molecules as antioxidants led biologists to neglect the non-tocopherol vitamin E molecules as topics for basic and clinical research. Recent developments warrant a serious reconsideration of this conventional wisdom. The tocotrienol subfamily of natural vitamin E possesses powerful neuroprotective, anticancer, and cholesterol-lowering properties that are often not exhibited by tocopherols. Current developments in vitamin E research clearly indicate that members of the vitamin E family are not redundant with respect to their biological functions. alpha-Tocotrienol, gamma-tocopherol, and delta-tocotrienol have emerged as vitamin E molecules with functions in health and disease that are clearly distinct from that of alpha-tocopherol. At nanomolar concentration, alpha-tocotrienol, not alpha-tocopherol, prevents neurodegeneration. On a concentration basis, this finding represents the most potent of all biological functions exhibited by any natural vitamin E molecule. Recently, it has been suggested that the safe dose of various tocotrienols for human consumption is 200-1000/day. A rapidly expanding body of evidence supports that members of the vitamin E family are functionally unique. In recognition of this fact, title claims in publications should be limited to the specific form of vitamin E studied. For example, evidence for toxicity of a specific form of tocopherol in excess may not be used to conclude that high-dosage "vitamin E" supplementation may increase all-cause mortality. Such conclusion incorrectly implies that tocotrienols are toxic as well under conditions where tocotrienols were not even considered. The current state of knowledge warrants strategic investment into the lesser known forms of vitamin E. This will enable prudent selection of the appropriate vitamin E molecule for studies addressing a specific health need.
Asunto(s)
Antioxidantes , Tocotrienoles , Vitamina E , Animales , Antioxidantes/química , Antioxidantes/fisiología , Humanos , Tocotrienoles/química , Tocotrienoles/farmacología , Vitamina E/análogos & derivados , Vitamina E/química , Vitamina E/fisiologíaRESUMEN
The effects of oral niacin-bound chromium (NBC) supplementation on the subcutaneous fat tissue of type 2 Lepr(db) obese diabetic mice were examined using high-density comprehensive mouse genome (45,101 probe sets) expression arrays. The influence of such supplementation on the plasma cardiovascular risk factors of these mice was also investigated. Supplementation of NBC had no significant effect on age-dependent weight gain in the Lepr(db) obese diabetic mice. However, NBC lowered total cholesterol (TC), TC-to-HDL ratio, LDL cholesterol, and triglyceride levels while increasing HDL cholesterol in the blood plasma. No effect of NBC supplementation was observed on fasting blood glucose levels. Oral glucose tolerance test revealed a significantly improved clearance of blood glucose between 1 and 2 h of glucose challenge in NBC-supplemented mice. Unbiased genome-wide interrogation demonstrated that NBC resulted in the upregulation of muscle-specific gene expression in the fat tissue. Genes encoding proteins involved in glycolysis, muscle contraction, muscle metabolism, and muscle development were specifically upregulated in response to NBC supplementation. Genes in the adipose tissue that were downregulated in response to NBC supplementation included cell death-inducing DNA fragmentation factor (CIDEA) and uncoupling protein-1, which represent key components involved in the thermogenic role of brown adipose tissue and tocopherol transfer protein, the primary carrier of alpha-tocopherol to adipose tissue. The observation that CIDEA-null mice are resistant to obesity and diabetes suggests that the inhibitory role of NBC on CIDEA expression was favorable. Further studies testing the molecular basis of NBC function and long-term outcomes are warranted.
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Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Cromo/farmacología , Diabetes Mellitus Tipo 2/genética , Regulación de la Expresión Génica , Obesidad/genética , Administración Oral , Animales , Proteínas Reguladoras de la Apoptosis/genética , Glucemia/análisis , Colesterol/sangre , Cromo/administración & dosificación , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/metabolismo , Regulación hacia Abajo , Perfilación de la Expresión Génica , Canales Iónicos/genética , Lípidos/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Proteínas Mitocondriales/genética , Niacina/administración & dosificación , Niacina/farmacología , Obesidad/complicaciones , Obesidad/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores de Superficie Celular/genética , Receptores de Leptina , Transcripción Genética , Proteína Desacopladora 1 , Regulación hacia ArribaRESUMEN
The natural vitamin E tocotrienol (TCT) possesses biological properties not shared by tocopherols (TCP). Nanomolar alpha-TCT, not alpha-TCP, is potently neuroprotective (JBC 275:13049; 278:43508). Tocopherol-transport protein (TTP) represents the primary mechanism for maintaining normal alpha-TCP concentrations in plasma and extrahepatic tissues. TTP primarily transports alpha-TCP and has low affinity for alpha-TCT. There are no studies that have investigated tissue delivery of alpha-TCT when orally gavaged on a long-term basis. A long-term study was conducted to examine the effects of alpha-TCT or alpha-TCP supplementation, either alone or in combination, on tissue levels. Rats were maintained on a vitamin E-deficient diet and gavaged with alpha-TCT or alpha-TCP alone or in combination. Five generations of rats were studied over 60 weeks. TTP-deficient mice were supplemented with TCT and bred to examine tissue delivery of oral alpha-TCT. Orally supplemented alpha-TCT was effectively delivered to most tissues over time. When co-supplemented, alpha-TCP outcompeted alpha-TCT for transport systems delivering vitamin E to tissues. To evaluate the significance of TTP in alpha-TCT delivery to tissues, tissue levels of alpha-TCT in supplemented TTP-deficient mice were studied. alpha-TCT was transported to several vital organs in TTP-deficient mice. alpha-TCT restored fertility in TTP-deficient mice. In sum, orally supplemented alpha-TCT was successfully delivered to several vital organs. The transport efficiency of alpha-TCT to tissues may be maximized by eliminating the co-presence of alpha-TCP in the oral supplement. Examination of whether alpha-TCT may benefit humans suffering from neurological disorders because of congenital TTP deficiency is warranted.
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Tocoferoles/farmacología , Vitamina E/análogos & derivados , Administración Oral , Animales , Antioxidantes/metabolismo , Transporte Biológico , Proteínas Portadoras/metabolismo , Femenino , Humanos , Pulmón/metabolismo , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Músculo Esquelético/patología , Fármacos Neuroprotectores/farmacología , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Distribución Tisular , Tocoferoles/metabolismo , Tocotrienoles , Vitamina E/administración & dosificación , Vitamina E/metabolismo , Vitamina E/farmacologíaRESUMEN
BACKGROUND AND PURPOSE: The current work is based on our previous finding that in neuronal cells, nmol/L concentrations of alpha-tocotrienol (TCT), but not alpha-tocopherol (TCP), blocked glutamate-induced death by suppressing early activation of c-Src kinase and 12-lipoxygenase. METHODS: The single neuron microinjection technique was used to compare the neuroprotective effects of TCT with that of the more widely known TCP. Stroke-dependent brain tissue damage was studied in 12-Lox-deficient mice and spontaneously hypertensive rats orally supplemented with TCT. RESULTS: Subattomole quantity of TCT, but not TCP, protected neurons from glutamate challenge. Pharmacological as well as genetic approaches revealed that 12-Lox is rapidly tyrosine phosphorylated in the glutamate-challenged neuron and that this phosphorylation is catalyzed by c-Src. 12-Lox-deficient mice were more resistant to stroke-induced brain injury than their wild-type controls. Oral supplementation of TCT to spontaneously hypertensive rats led to increased TCT levels in the brain. TCT-supplemented rats showed more protection against stroke-induced injury compared with matched controls. Such protection was associated with lower c-Src activation and 12-Lox phosphorylation at the stroke site. CONCLUSIONS: The natural vitamin E, TCT, acts on key molecular checkpoints to protect against glutamate- and stroke-induced neurodegeneration.
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Antioxidantes/farmacología , Fármacos Neuroprotectores/farmacología , Vitamina E/análogos & derivados , Animales , Araquidonato 12-Lipooxigenasa/metabolismo , Encéfalo/patología , Proteína Tirosina Quinasa CSK , Muerte Celular , Línea Celular , Supervivencia Celular , Células Cultivadas , Corteza Cerebral/embriología , Fluoresceínas , Ácido Glutámico/química , Ácido Glutámico/metabolismo , Hipocampo/citología , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo , Compuestos Orgánicos/farmacología , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Distribución Aleatoria , Ratas , Ratas Endogámicas SHR , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/patología , Factores de Tiempo , Tocotrienoles , Transfección , Tirosina/química , Vitamina E/química , Vitamina E/metabolismo , Vitamina E/farmacología , Familia-src Quinasas/metabolismoRESUMEN
Inflammatory disorders represent a substantial health problem. Medicinal plants belonging to the Burseraceae family, including Boswellia, are especially known for their anti-inflammatory properties. The gum resin of Boswellia serrata contains boswellic acids, which inhibit leukotriene biosynthesis. A series of chronic inflammatory diseases are perpetuated by leukotrienes. Although Boswellia extract has proven to be anti-inflammatory in clinical trials, the underlying mechanisms remain to be characterized. TNF alpha represents one of the most widely recognized mediators of inflammation. One mechanism by which TNFalpha causes inflammation is by potently inducing the expression of adhesion molecules such as VCAM-1. We sought to test the genetic basis of the antiinflammatory effects of BE (standardized Boswellia extract, 5-Loxin) in a system of TNF alpha-induced gene expression in human microvascular endothelial cells. We conducted the first whole genome screen for TNF alpha- inducible genes in human microvascular cells (HMEC). Acutely, TNF alpha induced 522 genes and downregulated 141 genes in nine out of nine pairwise comparisons. Of the 522 genes induced by TNF alpha in HMEC, 113 genes were clearly sensitive to BE treatment. Such genes directly related to inflammation, cell adhesion, and proteolysis. The robust BE-sensitive candidate genes were then subjected to further processing for the identification of BE-sensitive signaling pathways. The use of resources such as GenMAPP, KEGG, and gene ontology led to the recognition of the primary BE-sensitive TNF alpha-inducible pathways. BE prevented the TNF alpha-induced expression of matrix metalloproteinases. BE also prevented the inducible expression of mediators of apoptosis. Most strikingly, however, TNF alpha-inducible expression of VCAM-1 and ICAM-1 were observed to be sensitive to BE. Realtime PCR studies showed that while TNF alpha potently induced VCAM-1 gene expression, BE completely prevented it. This result confirmed our microarray findings and built a compelling case for the anti-inflammatory property of BE. In an in vivo model of carrageenan-induced rat paw inflammation, we observed a significant antiinflammatory property of BE consistent with our in vitro findings. These findings warrant further research aimed at identifying the signaling mechanisms by which BE exerts its anti-inflammatory effects.
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Antiinflamatorios/metabolismo , Boswellia/química , Células Endoteliales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Genes/efectos de los fármacos , Genoma Humano , Triterpenos/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Apoptosis/efectos de los fármacos , Apoptosis/genética , Carragenina/toxicidad , Cartilla de ADN , Edema/inducido químicamente , Edema/tratamiento farmacológico , Edema/patología , Células Endoteliales/metabolismo , Pie/patología , Regulación de la Expresión Génica/genética , Genes/genética , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Leucotrienos/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Extractos Vegetales/metabolismo , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Pletismografía , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Triterpenos/farmacología , Triterpenos/uso terapéutico , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Obesity is a global public health problem, with about 315 million people worldwide estimated to fall into the WHO-defined obesity categories. Traditional herbal medicines may have some potential in managing obesity. Botanical dietary supplements often contain complex mixtures of phytochemicals that have additive or synergistic interactions. The dried fruit rind of Garcinia cambogia, also known as Malabar tamarind, is a unique source of (-)-hydroxycitric acid (HCA), which exhibits a distinct sour taste and has been safely used for centuries in Southeastern Asia to make meals more filling. Recently it has been demonstrated that HCA-SX or Super Citrimax, a novel derivative of HCA, is safe when taken orally and that HCA-SX is bioavailable in the human plasma as studied by GC-MS. Although HCA-SX has been observed to be conditionally effective in weight management in experimental animals as well as in humans, its mechanism of action remains to be understood. We sought to determine the effects of low-dose oral HCA-SX on the body weight and abdominal fat gene expression profile of Sprague-Dawley rats. We observed that at doses relevant for human consumption dietary HCA-SX significantly contained body weight growth. This response was associated with lowered abdominal fat leptin expression while plasma leptin levels remained unaffected. Repeated high-density microarray analysis of 9960 genes and ESTs present in the fat tissue identified a small set (approximately 1% of all genes screened) of specific genes sensitive to dietary HCA-SX. Other genes, including vital genes transcribing for mitochondrial/nuclear proteins and which are necessary for fundamental support of the tissue, were not affected by HCA-SX. Under the current experimental conditions, HCA-SX proved to be effective in restricting body weight gain in adult rats. Functional characterization of HCA-SX-sensitive genes revealed that upregulation of genes encoding serotonin receptors represent a distinct effect of dietary HCA-SX supplementation.