Resveratrol Prevents Cell Swelling Through Inhibition of SUR1 Expression in Brain Micro Endothelial Cells Subjected to OGD/Recovery.

The SUR1-TRPM4-AQP4 complex is overexpressed in the initial phase of edema induced after cerebral ischemia, allowing the massive internalization of Na+ and water within the brain micro endothelial cells (BMEC) of the blood-brain barrier. The expression of the Abcc8 gene encoding SUR1 depends on transcriptional factors that are responsive to oxidative stress. Because reactive oxygen species (ROS) are generated during cerebral ischemia, we hypothesized that antioxidant compounds might be able to regulate the expression of SUR1. Therefore, the effect of resveratrol (RSV) on SUR1 expression was evaluated in the BMEC cell line HBEC-5i subjected to oxygen and glucose deprivation (OGD) for 2 h followed by different recovery times. Different concentrations of RSV were administered. ROS production was detected with etidine, and protein levels were evaluated by Western blotting and immunofluorescence. Intracellular Na+ levels and cellular swelling were detected by imaging; cellular metabolic activity and rupture of the cell membrane were detected by MTT and LDH release, respectively; and EMSA assays measured the activity of transcriptional factors. OGD/recovery increased ROS production induced the AKT kinase activity and the activation of SP1 and NFκB. SUR1 protein expression and intracellular Na+ concentration in the HBEC-5i cells increased after a few hours of OGD. These effects correlated with cellular swelling and necrotic cell death, responses that the administration of RSV prevented. Our results indicate that the ROS/AKT/SP1-NFκB pathway is involved in SUR1 expression during OGD/recovery in BMEC of the blood-brain barrier. Thus, RSV prevented cellular edema formation through modulation of SUR1 expression.

The short form of the SUR1 and its functional implications in the damaged brain.

Sulfonylurea receptor (SUR) belongs to the adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporter family; however, SUR is associated with ion channels and acts as a regulatory subunit determining the opening or closing of the pore. Abcc8 and Abcc9 genes code for the proteins SUR1 and SUR2, respectively. The SUR1 transcript encodes a protein of 1582 amino acids with a mass around 140-177 kDa expressed in the pancreas, brain, heart, and other tissues. It is well known that SUR1 assembles with Kir6.2 and TRPM4 to establish KATP channels and non-selective cation channels, respectively. Abbc8 and 9 are alternatively spliced, and the resulting transcripts encode different isoforms of SUR1 and SUR2, which have been detected by different experimental strategies. Interestingly, the use of binding assays to sulfonylureas and Western blotting has allowed the detection of shorter forms of SUR (~65 kDa). Identity of the SUR1 variants has not been clarified, and some authors have suggested that the shorter forms are unspecific. However, immunoprecipitation assays have shown that SUR2 short forms are part of a functional channel even coexisting with the typical forms of the receptor in the heart. This evidence confirms that the structure of the short forms of the SURs is fully functional and does not lose the ability to interact with the channels. Since structural changes in short forms of SUR modify its affinity to ATP, regulation of its expression might represent an advantage in pathologies where ATP concentrations decrease and a therapeutic target to induce neuroprotection. Remarkably, the expression of SUR1 variants might be induced by conditions associated to the decrease of energetic substrates in the brain (e.g. during stroke and epilepsy). In this review, we want to contribute to the knowledge of SUR1 complexity by analyzing evidence that shows the existence of short SUR1 variants and its possible implications in brain function.

Resveratrol Prevents GLUT3 Up-Regulation Induced by Middle Cerebral Artery Occlusion.

Glucose transporter (GLUT)3 up-regulation is an adaptive response activated to prevent cellular damage when brain metabolic energy is reduced. Resveratrol is a natural polyphenol with anti-oxidant and anti-inflammatory features that protects neurons against damage induced in cerebral ischemia. Since transcription factors sensitive to oxidative stress and inflammation modulate GLUT3 expression, the purpose of this work was to assess the effect of resveratrol on GLUT3 expression levels after ischemia. Male Wistar rats were subjected to 2 h of middle cerebral artery occlusion (MCAO) followed by different times of reperfusion. Resveratrol (1.9 mg/kg; i. p.) was administered at the onset of the restoration of the blood flow. Quantitative-PCR and Western blot showed that MCAO provoked a substantial increase in GLUT3 expression in the ipsilateral side to the lesion of the cerebral cortex. Immunofluorescence assays indicated that GLUT3 levels were upregulated in astrocytes. Additionally, an important increase in GLUT3 occurred in other cellular types (e.g., damaged neurons, microglia, or infiltrated macrophages). Immunodetection of the microtubule-associated protein 2 (MAP2) showed that MCAO induced severe damage to the neuronal population. However, the administration of resveratrol at the time of reperfusion resulted in injury reduction. Resveratrol also prevented the MCAO-induced increase of GLUT3 expression. In conclusion, resveratrol protects neurons from damage induced by ischemia and prevents GLUT3 upregulation in the damaged brain that might depend on AMPK activation.

Resveratrol Activates Neuronal Autophagy Through AMPK in the Ischemic Brain.

During cerebral ischemia, oxygen and glucose levels decrease, producing many consequences such as the generation of reactive oxygen species, tissue injury, and the general metabolism collapse. Resveratrol triggers signaling dependent on the protein kinase activated by adenosine monophosphate (AMPK), the sensor of cellular energy metabolism that regulates autophagy, eliminates damaged mitochondria, and increases energy sources. In the present study, we investigated the participation of AMPK activation in the protective effect of resveratrol on cerebral ischemia and excitotoxicity. We found that resveratrol increased the levels of phosphorylated AMPK in the cerebral cortex of rats subjected to middle cerebral artery occlusion (MCAO) and in primary cultured neurons exposed to glutamate-induced excitotoxicity. Resveratrol (1.8 mg/Kg; i. v.; administered at the beginning of reperfusion) decreased the infarct area and increased survival of rats subjected to MCAO. In neuronal cultures, resveratrol treatment (40 µM, after excitotoxicity) reduced the production of superoxide anion, prevented the overload of intracellular Ca+2 associated to mitochondrial failure, reduced the release of the lactate dehydrogenase enzyme, and reduced death. It also promoted mitophagy (increased Beclin 1 level, favored the recruitment of LC3-II, reduced LAMP1, and reduced mitochondrial matrix protein HSP60 levels). In both models, inhibition of AMPK activation with Compound C obstructed the effect of resveratrol, showing that its protective effect depends, partially, on the activation of the AMPK/autophagy pathway.

Aged garlic extract and S-allylcysteine increase the GLUT3 and GCLC expression levels in cerebral ischemia.

BACKGROUND: During cerebral ischemia, energy restoration through the regulation of glucose transporters and antioxidant defense mechanisms is essential to maintain cell viability. Antioxidant therapy has been considered effective to attenuate brain damage; moreover, the regulation of transcription factors that positively regulate the expression of glucose transporters is associated with this therapy. Recently, it has been reported that the use of antioxidants such as S-allylcysteine (SAC), a component of aged garlic extract (AGE), improves survival in experimental models of cerebral ischemia. OBJECTIVES: The aim of this study was to determine the effect of AGE and SAC on the level of mRNA expression of the main neuronal glucose transporter (GLUT3) and the glutamate cysteine ligase catalytic subunit (GCLC) in rats with transient focal cerebral ischemia. MATERIAL AND METHODS: Cerebral ischemia was induced in male Wistar rats by middle cerebral artery occlusion (MCAO) for 2 h. The animals were sacrificed after different reperfusion times (0-48 h). Animals injected with AGE (360 mg/kg, intraperitoneally (i.p.)) and SAC (300 mg/kg, i.p.) at the beginning of reperfusion were sacrificed after 2 h. The mRNA expression level was analyzed in the fronto-parietal cortex using quantitative polymerase chain reaction (qPCR). RESULTS: Two major increases in GLUT3 expression at 1 h and 24 h of reperfusion were found. Both treatments increased GLUT3 and GCLC mRNA levels in control and under ischemic/reperfusion injury animals. CONCLUSIONS: This data suggests that SAC and AGE might induce neuroprotection, while controlling reactive oxygen species (ROS) levels, as indicated by the increase in GCLC expression, and regulating the energy content of the cell by increasing glucose transport mediated by GLUT3.

Histamine H1 and H3 receptor activation increases the expression of Glucose Transporter 1 (GLUT-1) in rat cerebro-cortical astrocytes in primary culture.

Astrocytes take up glucose via the 45 kDa isoform of the Glucose Transporter 1 (GLUT-1), and in this work we have investigated whether histamine regulates GLUT-1 expression in rat cerebro-cortical astrocytes in primary culture. Cultured astrocytes expressed histamine H1 and H3 receptors (H1Rs and H3Rs) as evaluated by radioligand binding. Receptor functionality was confirmed by the increase in the intracellular concentration of Ca2+ (H1R) and the inhibition of forskolin-induced cAMP accumulation (H3R). Quantitative RT-PCR showed that histamine and selective H1R and H3R agonists (1 h incubation) significantly increased GLUT-1 mRNA to 153 ±â€¯7, 163 ±â€¯2 and 168 ±â€¯13% of control values, respectively. In immunoblot assays, incubation (3 h) with histamine or H1R and H3R agonists increased GLUT-1 protein levels to 224 ±â€¯12, 305 ±â€¯11 and 193 ±â€¯13% of control values, respectively, an action confirmed by inmunocytochemistry. The effects of H1R and H3R agonists were blocked by the selective antagonists mepyramine (H1R) and clobenpropit (H3R). The pharmacological inhibition of protein kinase C (PKC) prevented the increase in GLUT-1 protein induced by either H1R or H3R activation. Furthermore, histamine increased ERK-1/2 phosphorylation, and the effect of H1R and H3R activation on GLUT-1 protein levels was reduced or prevented, respectively, by MEK-1/2 inhibition. These results indicate that by activating H1Rs and H3Rs histamine regulates the expression of GLUT-1 by astrocytes. The effect appears to involve the phospholipase C (PLC) → diacylglycerol (DAG)/Ca2+→ PKC and PLC → DAG/Ca2+ → PKC → MAPK pathways.

Combined Administration of Streptozotocin and Sucrose Accelerates the Appearance of Type 2 Diabetes Symptoms in Rats.

Type 2 diabetes is a disease with a high global prevalence, characterized by chronic hyperglycemia, insulin resistance, polyphagia, polydipsia, polyuria, and changes in body weight. Animal models have been very useful for the study of this disease and to search for new therapeutic targets that delay, attenuate, or avoid diabetic complications. The purpose of this work was to establish a model of type 2 diabetes and exhibit the majority of the characteristics of the disease. Two-day-old male and female Wistar rats were treated once with streptozotocin (70 or 90 mg/kg body weight). After weaning, they were given a sucrose-sweetened beverage (SSB; sucrose at 10 or 30%) during 7 or 11 weeks; their body weight and food intake were measured daily. With the rats at 14 weeks of age, we determined the following: (a) fasting blood glucose, (b) oral glucose tolerance, and (c) insulin tolerance. We found that the supplementation of sucrose at 10% for 7 weeks in male rats which had previously been given streptozotocin (70 mg/kg) at neonatal stage leads to the appearance of the signs and symptoms of the characteristic of type 2 diabetes in adulthood.

Current evidence for AMPK activation involvement on resveratrol-induced neuroprotection in cerebral ischemia.

OBJECTIVES: Cerebral ischemia is a neurological condition in which energetics and oxidative stress are dysregulated. Resveratrol is a stilbene with potent pharmacological effects associated with its antioxidant properties. In the brain, resveratrol produces protective responses against ischemia, decreases infarct volume and improves neurological function. Adenosine monophosphate-activated protein kinase (AMPK) is a cellular sensor that acts as a switch to initiate adaptive changes in response to fluctuations in energy metabolism. RESULTS: In ischemia, AMPK is activated, nevertheless conflicting results about its contribution to protection have become apparent, and this matter continues without resolution. Interestingly, AMPK activation by resveratrol has been implicated in regulating cell survival in different experimental models. Although resveratrol's ability to regulate AMPK directly or after signaling is only beginning to be understood, targeting this enzyme by resveratrol in brain suggest that it could contribute to the amelioration of some pathologic features induced after an energetic deficit. CONCLUSION: The present review discusses the potential role of resveratrol in regulating AMPK activity on brain before, during, or after ischemia and offer suggestions for feasible future studies.

[Detection of cytomegalovirus by real-time PCR in HIV-positive plasm]. / Detección de citomegalovirus por PCR en tiempo real en plasmas positivos a VIH.

BACKGROUND: Cytomegalovirus is a betaherpesvirus responsible for persistent infections that are generally asymptomatic in healthy individuals. In the absence of an effective immune response, as in neonates, cancer patients, organ transplant recipients, individuals with AIDS, etc., cytomegalovirus may cause severe disease. Early detection of this virus would prevent serious health consequences in immunocompromised patients; it is important to employ sensitive methods and accurate detection to support treatment-related decision making. Real-time molecular methods, such as the polymerase chain reaction, possess higher sensitivity to detect positive samples. METHODS: We compared the sensitivity and specificity of the following detection methods: the endpoint PCR trade-validated method (Pol, viral gene target) and real-time PCR, which detects viral genes Pol (early gene), and pp65 (late gene). We performed a cross-sectional study of 43 human immunodeficiency virus-positive samples. RESULTS: The molecular detection methods in real-time detected a greater number of cytomegalovirus-positive samples than those at the endpoint. CONCLUSIONS: There must be at least two independent cytomegalovirus target-genes in order to make the detection by real-time PCR.

INTRODUCCIÓN: el citomegalovirus es responsable de infecciones persistentes, generalmente asintomáticas en personas sanas pero que en ausencia de una respuesta inmune efectiva puede causar enfermedad severa, por ello es muy importante su detección temprana en los individuos con trastornos de la inmunidad. El objetivo de esta investigación fue hacer un análisis del límite de detección, sensibilidad y concordancia de la reacción en cadena de la polimerasa (PCR) en punto final con los obtenidos con la PCR en tiempo real. MÉTODOS: se realizó un estudio transversal con 43 muestras de plasma humano positivas al virus de la inmunodeficiencia humano, provenientes de individuos de 18 o más años de edad, de uno u otro sexo. Todas las muestras tuvieron una carga viral-VIH mayor a 100 000 copias/mL. Para la PCR en punto final se empleó un método comercial para identificar UL54 (gen viral blanco) y para la PCR en tiempo real se amplificaron fragmentos de los genes UL54 (gen temprano) y UL83 (gen tardío) del citomegalovirus humano. RESULTADOS: mediante PCR en punto final (método comercial-validado) solo tres individuos fueron positivos a citomegalovirus humano (7 %), con una la carga viral de 1500 a 1670 copias/mL. Las muestras positivas a citomegalovirus humano mediante PCR en tiempo real tuvieron un rango de 4.36 a 4692.86 copias de citomegalovirus humano CONCLUSIONES: es necesario tener al menos dos genes blancos de citomegalovirus humano para detectarlo de manera ratificada mediante PCR en tiempo real.

Is human cytomegalovirus associated with breast cancer progression?

BACKGROUND: It has been hypothesized that human cytomegalovirus (HCMV) may be associated with breast cancer progression. However, the role of HCMV infection in breast cancer remains controversial. We aimed to assess whether HCMV genes (UL122 and UL83) could be detected in breast carcinomas and reinvestigated their possible association with breast cancer progression. DNA from paraffin-embedded tissues was analyzed by real-time PCR. We investigated 20 fibroadenomas and 27 primary breast carcinomas (stages II, III, and IV). FINDINGS: Two carcinomas were positive for HCMV, one was positive for two TaqMan viral detection probes, and one was positive for a sole TaqMan viral detection probe (UL83), whereas the remainder of the samples was negative. CONCLUSIONS: Samples studied showed no association between HCMV infection and breast cancer progression.

Comparison of antioxidant activity of hydroethanolic fresh and aged garlic extracts and their effects on cerebral ischemia.

Antioxidant properties and protective effect of aged garlic extract (AGE) and of 20% hydroethanolic fresh extracts from garlic clove (GCE) and skin (GSE) on cerebral ischemia were evaluated by administering extracts at the beginning of reperfusion in a rat model of stroke. All three extracts scavenged superoxide anion, peroxynitrite anion, and peroxyl radicals, but with different efficiencies; furthermore, GCE and GSE scavenged hydroxyl radicals and GSE scavenged singlet oxygen. These extracts significantly prevented reduction of neuronal nuclear antigen in the infarcted area, although no improvement in neurological function was observed. Importantly, GCE and GSE contained S-allylcystein, a compound associated with AGE's neuroprotective effect against damage induced by cerebral ischemia. Extracts decreased mRNA expression of NR1- and NR2B-NMDA-receptor subunits and prevented ischemia-induced reduction in mitochondrial potential and in ATP synthesis. These results indicate that antioxidants present in garlic extracts may regulate ROS concentrations during ischemia, favour pro-survival pathways, and attenuate mitochondrial dysfunction.

Glucose transporters regulation on ischemic brain: possible role as therapeutic target.

Ischemic stroke is a major cause of death worldwide that provokes a high society cost. Deprivation of blood supply, with the subsequent deficiency of glucose and oxygen, triggers an important number of mechanisms (e.g. excitotoxicity, oxidative stress and inflammation) leading to irreversible neuronal injury. Consequently, ischemia increases the energy demand which is associated with profound changes in brain energy metabolism. Glucose transport activity may adapt to ensure the delivery of glucose to maintain normal cellular function, even at the low glucose levels observed in plasma during ischemia. In the brain, the main glucose transporters (GLUTs) are GLUT3 in neurons and GLUT1 in the microvascular endothelial cells of the blood brain barrier and glia. The intracellular signaling pathways involved in GLUT regulation in cerebral ischemia remain unclear; however, it has been established that ischemia induces changes in their expression. In this review, we describe the effect of glutamate-induced excitotoxicity, mitochondrial damage, glucose deprivation, and hypoxia on GLUTs expression in the brain. Additionally, we discuss the possible role of GLUTs as therapeutic target for ischemia. Despite of the intense research, current therapeutics options for stroke are very limited, therefore it is especially important to find new options. Few studies have examined the neuroprotective potential of GLUT up-regulation in ischemic stroke; however, evidence suggests that augmented GLUTs could be related to a protective mechanism. Increased understanding of the beneficial effects of GLUTs activation provides the rationale for targeting GLUT in the development of new therapeutic strategies.

S-Allylcysteine, a garlic-derived antioxidant, ameliorates quinolinic acid-induced neurotoxicity and oxidative damage in rats.

Excitotoxicity elicited by overactivation of N-methyl-D-aspartate receptors is a well-known characteristic of quinolinic acid-induced neurotoxicity. However, since many experimental evidences suggest that the actions of quinolinic acid also involve reactive oxygen species formation and oxidative stress as major features of its pattern of toxicity, the use of antioxidants as experimental tools against the deleterious effects evoked by this neurotoxin becomes more relevant. In this work, we investigated the effect of a garlic-derived compound and well-characterized free radical scavenger, S-allylcysteine, on quinolinic acid-induced striatal neurotoxicity and oxidative damage. For this purpose, rats were administered S-allylcysteine (150, 300 or 450 mg/kg, i.p.) 30 min before a single striatal infusion of 1 microl of quinolinic acid (240 nmol). The lower dose (150 mg/kg) of S-allylcysteine resulted effective to prevent only the quinolinate-induced lipid peroxidation (P < 0.05), whereas the systemic administration of 300 mg/kg of this compound to rats decreased effectively the quinolinic acid-induced oxidative injury measured as striatal reactive oxygen species formation (P < 0.01) and lipid peroxidation (P < 0.05). S-Allylcysteine (300 mg/kg) also prevented the striatal decrease of copper/zinc-superoxide dismutase activity (P < 0.05) produced by quinolinate. In addition, S-allylcysteine, at the same dose tested, was able to reduce the quinolinic acid-induced neurotoxicity evaluated as circling behavior (P < 0.01) and striatal morphologic alterations. In summary, S-allylcysteine ameliorates the in vivo quinolinate striatal toxicity by a mechanism related to its ability to: (a) scavenge free radicals; (b) decrease oxidative stress; and (c) preserve the striatal activity of Cu,Zn-superoxide dismutase (Cu,Zn-SOD). This antioxidant effect seems to be responsible for the preservation of the morphological and functional integrity of the striatum.