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Background: L-ergothioneine (EGT), an antioxidative and anti-inflammatory amino acid, is abundant in various mushroom fruiting bodies. Meanwhile, the effects of EGT-containing mushrooms on human skin are unknown. This study investigated the effects of oral ingestion of a novel EGT-rich strain of Pleurotus species (hiratake) on skin conditions in humans. Methods: We conducted a 12-week, randomized, double-blind, placebo-controlled, parallel-group trial to evaluate skin moisturizing functions and facial conditions in 80 healthy women who were randomly assigned to either a group that was supplemented with hiratake tablets containing 25 mg of EGT/day or a placebo group. Skin moisture content, transepidermal water loss (TEWL), and facial scores (VISIA scores) were measured at baseline, 8 weeks, and 12 weeks of supplementation. Results: At 8 weeks, the skin moisture content was significantly higher on the temple in the hiratake group than in the placebo group. The hiratake group also exhibited a significant increase in skin moisture content on the arm at 8 and 12 weeks compared with baseline. At 12 weeks, wrinkle and texture scores were significantly better in the hiratake group than in the placebo group, and plasma EGT concentrations in the hiratake group were 4.7-fold higher than baseline (from 3.4 to 15.9 µM). Furthermore, EGT concentrations in plasma were significantly correlated with improvements in skin moisture content and TEWL on the arm, implying that these skin moisturizing benefits could be partly attributed to EGT. A stratified analysis of participants with a low baseline plasma EGT concentration (< 3.3 µM) revealed that skin moisture content on the temple was significantly higher at 8 and 12 weeks, and skin moisture content on the arm at 12 weeks tended to be higher (p = 0.074), in the hiratake group than in the placebo group. These findings suggested that oral ingestion of EGT-rich hiratake can improve skin moisturizing functions. Conclusion: EGT-rich hiratake may help maintain skin conditions in healthy women, and EGT may play a role in these beneficial effects.
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Healthy aging has emerged as a crucial issue with the increase in the geriatric population worldwide. Food-derived sulfur-containing amino acid ergothioneine (ERGO) is a potential dietary supplement, which exhibits various beneficial effects in experimental animals although the preventive effects of ERGO on aging and/or age-related impairments such as frailty and cognitive impairment are unclear. We investigated the effects of daily oral supplementation of ERGO dissolved in drinking water on lifespan, frailty, and cognitive impairment in male mice from 7 weeks of age to the end of their lives. Ingestion of 4 ~ 5 mg/kg/day of ERGO remarkably extended the lifespan of male mice. The longevity effect of ERGO was further supported by increase in life and non-frailty spans of Caenorhabditis elegans in the presence of ERGO. Compared with the control group, the ERGO group showed significantly lower age-related declines in weight, fat mass, and average and maximum movement velocities at 88 weeks of age. This was compatible with dramatical suppression by ERGO of the age-related increments in plasma biomarkers (BMs) such as the chemokine ligand 9, creatinine, symmetric dimethylarginine, urea, asymmetric dimethylarginine, quinolinic acid, and kynurenine. The oral intake of ERGO also rescued age-related impairments in learning and memory ability, which might be associated with suppression of the age-related decline in hippocampal neurogenesis and TDP43 protein aggregation and promotion of microglial shift to the M2 phenotype by ERGO ingestion. Ingestion of ERGO may promote longevity and healthy aging in male mice, possibly through multiple biological mechanisms.
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Caenorhabditis elegans , Ergotioneína , Envejecimiento Saludable , Longevidad , Animales , Ergotioneína/farmacología , Masculino , Longevidad/efectos de los fármacos , Envejecimiento Saludable/efectos de los fármacos , Caenorhabditis elegans/efectos de los fármacos , Ratones , Suplementos Dietéticos , Ratones Endogámicos C57BL , FragilidadRESUMEN
Oral administration of the food-derived antioxidant amino acid ergothioneine (ERGO) results in its efficient distribution in the brain and enhances cognitive function. However, effect of ERGO deficiency on cognitive impairment and the underlying mechanisms remain unknown. We revealed that cognitive function and hippocampal neurogenesis were lower in mice fed an ERGO-free diet than in those fed the control diet. Furthermore, ERGO supplementation to achieve the control diet ERGO levels reversed these effects and restored ERGO concentrations in the plasma and hippocampus. The ERGO-induced recovery of cognitive function and hippocampal neurogenesis was blocked by inhibiting the neurotrophic factor receptor tropomyosin receptor kinase B (TrkB), with a concomitant reduction in hippocampal phosphorylated TrkB, suggesting the involvement of TrkB in these events in mice. Phosphorylated TrkB was also detected in extracellular vesicles (EVs) derived from serum of volunteers who had been orally administered placebo or ERGO-containing tablets. Importantly, the ratio of serum EV-derived phosphorylated TrkB was significantly higher in the ERGO-treated group than in the placebo-treated group and was positively correlated with both serum ERGO concentrations and several cognitive domain scores from Cognitrax. Altogether, TrkB phosphorylation is involved in ERGO-induced cognitive enhancement in mice, and TrkB phosphorylation levels in serum EVs may quantitatively represent ERGO-induced cognitive enhancement in humans.
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Breast cancer resistance protein (BCRP) is expressed on hepatic bile canalicular membranes; however, its impact on substrate drug disposition is limited. This study proposes an in vivo knockdown approach using adeno-associated virus encoding short hairpin RNA (shRNA) targeting the bcrp gene (AAV-shBcrp) to clarify the substrate, the overall disposition of which is largely governed by hepatic Bcrp. The disposition of the tyrosine kinase inhibitor, regorafenib, was first examined in bcrp gene knockout (Bcrp-/-) and wild-type (WT) mice, as it was sequentially converted to active metabolites M - 2 and M - 5, which are BCRP substrates. After oral administration of regorafenib, plasma and liver concentrations of M - 5, but not regorafenib, were higher in Bcrp-/- than WT mice. To directly examine the role of hepatic Bcrp in M - 5 disposition, M - 5 was intravenously injected into mice three weeks after the intravenous injection of AAV-shBcrp, when mRNA of Bcrp in the liver (but not the small intestine) was downregulated. AAV-shBcrp-treated mice showed higher M - 5 concentration in plasma and liver, but lower biliary excretion than the control mice, indicating the fundamental role of hepatic Bcrp in M - 5 disposition. This is the first application of AAV-knockdown strategy to clarify the pharmacokinetic role of xenobiotic efflux transporters in the liver.
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Dependovirus , Ratones , Animales , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Proteínas de Neoplasias/metabolismo , Hígado/metabolismo , Inhibidores de Proteínas Quinasas/metabolismo , ARN Interferente Pequeño/metabolismo , Ratones NoqueadosRESUMEN
Ergothioneine (ERGO) is a naturally occurring food-derived antioxidant. Despite its extremely hydrophilic properties, ERGO is easily absorbed from the gastrointestinal tract and distributed to various organs, including the brain. This is primarily because its entry into brain cells is mediated by the ERGO-specific transporter OCTN1/SLC22A4. Octn1 gene knockout mice do not have ERGO in the brain, due to the absence of OCTN1 in neurons, neural stem cells, and microglia. The existence of OCTN1 and uptake of ERGO into the brain parenchymal cells may suggest that ERGO and its transporter play a pivotal role in brain function. Oral administration of ERGO has antidepressant activities in mice. Furthermore, repeated oral administration of ERGO and ERGO-containing food extract tablets enhance memory function in mice and humans, respectively. ERGO also protects against stress-induced sleep disturbance and neuronal injury induced by amyloid ß in rodents. In vitro observations suggest that ERGO benefits brain function through both its antioxidative activity and by promoting neurogenesis and neuronal maturation. This review discusses the possible involvement of ERGO in brain function and its potential therapeutic properties.
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Ergotioneína , Células-Madre Neurales , Simportadores , Péptidos beta-Amiloides , Animales , Antioxidantes/farmacología , Encéfalo/metabolismo , Ergotioneína/farmacología , Ratones , Células-Madre Neurales/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismoRESUMEN
Potential inhibition of the breast cancer resistance protein (BCRP), a drug efflux transporter, is a key issue during drug development, and the use of its physiologic substrates as biomarkers can be advantageous to assess inhibition. In this study, we aimed to identify BCRP substrates by an untargeted metabolomic approach. Mice were orally administered lapatinib to inhibit BCRP in vivo, and plasma samples were assessed by liquid chromatography/time of flight/mass spectrometry with all-ion fragmentation acquisition and quantified by liquid chromatography with tandem mass spectrometry. A differential metabolomic analysis was also performed for plasma from Bcrp -/- and wild-type mice. Plasma peaks of food-derived isoflavone metabolites, daidzein sulfate (DS), and genistein sulfate (GS) increased after lapatinib administration and in Bcrp -/- mice. Administration of lapatinib and another BCRP inhibitor febuxostat increased the area under the plasma concentration-time curve (AUC) of DS, GS, and equol sulfate (ES) by 3.6- and 1.8-, 5.6- and 4.1-, and 1.6- and 4.8-fold, respectively. BCRP inhibitors also increased the AUC and maximum plasma concentration of DS and ES after coadministration with each parent compound. After adding parent compounds to the apical side of induced pluripotent stem cell-derived small intestinal epithelial-like cells, DS, GS, and ES in the basal compartment significantly increased in the presence of lapatinib and febuxostat, suggesting the inhibition of intestinal BCRP. ATP-dependent uptake of DS and ES in BCRP-expressing membrane vesicles was reduced by both inhibitors, indicating inhibition of BCRP-mediated DS and ES transport. Thus, we propose the first evidence of surrogate markers for BCRP inhibition. SIGNIFICANCE STATEMENT: This study performed untargeted metabolomics to identify substrates of BCRP/ABCG2 to assess changes in its transport activity in vivo by BCRP/ABCG2 inhibitors. Food-derived isoflavone sulfates were identified as useful markers for evaluating changes in BCRP-mediated transport in the small intestine by its inhibitors.
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Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/efectos de los fármacos , Neoplasias de la Mama/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Isoflavonas/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Animales , Biomarcadores , Neoplasias de la Mama/metabolismo , Cromatografía Líquida de Alta Presión , Resistencia a Antineoplásicos/genética , Genisteína/farmacología , Humanos , Células Madre Pluripotentes Inducidas , Isoflavonas/química , Lapatinib/farmacología , Ratones , Ratones Noqueados , Sulfatos/farmacología , Espectrometría de Masas en TándemRESUMEN
Neurogenesis is the process by which new neurons are generated from neural stem cells (NSCs), which are cells that have the ability to proliferate and differentiate into neurons, astrocytes, and oligodendrocytes. The process is essential for homeostatic tissue regeneration and the coordination of neural plasticity throughout life, as neurons cannot regenerate once injured. Therefore, defects in neurogenesis are related to the onset and exacerbation of several neuropsychiatric disorders, and therefore, the regulation of neurogenesis is considered to be a novel strategy for treatment. Neurogenesis is regulated not only by NSCs themselves, but also by the functional microenvironment surrounding the NSCs, known as the "neurogenic niche." The neurogenic niche consists of several types of neural cells, including neurons, glial cells, and vascular cells. To allow communication with these cells, transporters may be involved in the secretion and uptake of substrates that are essential for signal transduction. This chapter will focus on the involvement of polyspecific solute carriers transporting organic cations in the possible regulation of neurogenesis by controlling the concentration of several organic cation substrates in NSCs and the neurogenic niche. The potential therapeutic implications of neurogenesis regulation by these transporters will also be discussed.
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Células-Madre Neurales , Neurogénesis , Neuroglía , Neuronas , Transducción de SeñalRESUMEN
BACKGROUND: The enhancement of learning and memory through food-derived ingredients is of great interest to healthy individuals as well as those with diseases. Ergothioneine (ERGO) is a hydrophilic antioxidant highly contained in edible golden oyster mushrooms (Pleurotus cornucopiae var. citrinopileatus), and systemically absorbed by its specific transporter, carnitine/organic cation transporter OCTN1/SLC22A4. OBJECTIVE: This study aims to examine the possible enhancement of object recognition memory by oral administration of ERGO in normal mice. METHODS: Novel object recognition test, spatial recognition test, LC-MS/MS, Golgi staining, neuronal culture, western blotting, immunocytochemistry, and quantitative RT-PCR were utilized. RESULT: After oral administration of ERGO (at a dose of 1-50 mg/kg) three times per week for two weeks in ICR mice, the novel object recognition test revealed a longer exploration time for the novel object than for the familiar object. After oral administration of ERGO, the spatial recognition test also revealed a longer exploration time for the spatially moved object than the unmoved one in mice fed ERGO-free diet. The discrimination index was significantly higher in the ERGO-treated group than the control in both behavioral tests. ERGO administration led to an increase in its concentration in the plasma and hippocampus. The systemic concentration reached was relevant to those found in humans after oral ERGO administration. Golgi staining revealed that ERGO administration increased the number of matured spines in the hippocampus. Exposure of cultured hippocampal neurons to ERGO elevated the expression of the synapse formation marker, synapsin I. This elevation of synapsin I was inhibited by the tropomyosin receptor kinase inhibitor, K252a. Treatment with ERGO also increased the expression of neurotrophin-3 and -5, and phosphorylated mammalian target of rapamycin in hippocampal neurons. CONCLUSION: Oral intake of ERGO may enhance object recognition memory at its plasma concentration achievable in humans, and this enhancement effect could occur, at least in part, through the promotion of neuronal maturation in the hippocampus.
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Antioxidantes/química , Conducta Animal/efectos de los fármacos , Inhibidores Enzimáticos/química , Ergotioneína/química , Fenómenos Fisiológicos de la Nutrición/efectos de los fármacos , Pleurotus/química , Administración Oral , Animales , Antioxidantes/administración & dosificación , Antioxidantes/análisis , Carbazoles/farmacología , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/sangre , Ergotioneína/administración & dosificación , Ergotioneína/sangre , Hipocampo/metabolismo , Humanos , Alcaloides Indólicos/farmacología , Masculino , Ratones Endogámicos ICR , Neurogénesis/efectos de los fármacos , Neuronas/metabolismo , Sinapsinas/metabolismo , Espectrometría de Masas en TándemRESUMEN
Understanding of the underlying mechanism of epilepsy is desired since some patients fail to control their seizures. The carnitine/organic cation transporter OCTN1/SLC22A4 is expressed in brain neurons and transports food-derived antioxidant ergothioneine (ERGO), L-carnitine, and spermine, all of which may be associated with epilepsy. This study aimed to clarify the possible association of this transporter with epileptic seizures. In both pentylenetetrazole (PTZ)-induced acute seizure and kindling models, ocnt1 gene knockout mice (octn1-/-) showed lower seizure scores compared with wild-type mice. Up-regulation of the epilepsy-related genes, c-fos and Arc, and the neurotrophic factor BDNF following PTZ administration was observed in the hippocampus of wild-type, but not octn1-/- mice. To find the OCTN1 substrate associated with the seizure, untargeted metabolomics analysis using liquid chromatography-quadrupole time-of-flight mass spectrometry was conducted on extracts from the hippocampus, frontal cortex, and plasma of both strains, leading to the identification of a plant alkaloid homostachydrine as a compound present in a lower concentration in octn1-/- mice. OCTN1-mediated uptake of deuterium-labeled homostachydrine was confirmed in OCTN1-transfected HEK293 cells, suggesting that this compound is a substrate of OCTN1. Homostachydrine administration increased PTZ-induced acute seizure scores and the expression of Arc in the hippocampus and that of Arc, Egr1, and BDNF in the frontal cortex. Conversely, administration of the OCTN1 substrate/inhibitor ERGO inhibited PTZ-induced kindling and reduced the plasma homostachydrine concentration. Thus, these results suggest that OCTN1 is at least partially associated with PTZ-induced seizures, which is potentially deteriorated by treatment with homostachydrine, a newly identified food-derived OCTN1 substrate.
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Epilepsia/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Piperidinas/farmacología , Convulsiones/metabolismo , Simportadores/metabolismo , Xenobióticos/farmacología , Animales , Antioxidantes/farmacología , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Proteínas del Citoesqueleto/metabolismo , Epilepsia/inducido químicamente , Ergotioneína/farmacología , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Masculino , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Transporte de Catión Orgánico/genética , Pentilenotetrazol , Piperidinas/metabolismo , Piperidinas/orina , Convulsiones/inducido químicamente , Simportadores/genética , Xenobióticos/metabolismo , Xenobióticos/orinaRESUMEN
The aim of the present study is to construct and characterize a novel three-dimensional culture system for mouse neurons using the functional polymer, FP001. Stereoscopically extended neurites were found in primary mouse cortical neurons cultured in the FP001-containing medium. Neurons cultured with FP001 were distributed throughout the medium of the observation range whereas neurons cultured without FP001 were distributed only on the bottom of the dish. These results demonstrated that neurons can be three-dimensionally cultured using the FP001-containing medium. The mRNA expression of the glutamatergic neuronal marker vesicular glutamate transporter 1 in neurons cultured in the FP001-containing medium were higher than that in neurons cultured in the FP001-free medium. Expression of the matured neuronal marker, microtubule-associated protein 2 (MAP2) a,b, and the synapse formation marker, Synapsin I, in neurons cultured with FP001 was also higher than that in neurons cultured without FP001. The expression pattern of MAP2a,b in neurons cultured with FP001, but not that in neurons cultured without FP001, was similar to that in the embryonic cerebral cortex. Exposure to glutamate significantly increased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction activity in neurons cultured with FP001 compared to that in neurons cultured without FP001. These results suggested that glutamatergic neurotransmission in neurons three-dimensionally cultured in the FP001-containing medium may be upregulated compared to neurons two-dimensionally cultured in the FP001-free medium. Thus, neurons with the properties close to those in the embryonic brain could be obtained by three-dimensionally culturing neurons using FP001, compared to two-dimensional culture with a conventional adhesion method.
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Técnicas de Cultivo de Célula/métodos , Corteza Cerebral/citología , Medios de Cultivo/química , Neuronas/citología , Polisacáridos Bacterianos/química , Animales , Corteza Cerebral/embriología , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Proteínas Asociadas a Microtúbulos/metabolismo , Neuritas/metabolismo , Neuronas/metabolismo , Sinapsinas/metabolismoRESUMEN
The promotion of neurogenesis is considered to be an effective therapeutic strategy for neuropsychiatric disorders because impairment of neurogenesis is associated with the onset and progression of these disorders. We have previously demonstrated that orally ingested ergothioneine (ERGO), a naturally occurring antioxidant and hydrophilic amino acid, promotes neurogenesis in the hippocampal dentate gyrus (DG) with its abundant neural stem cells (NSCs) and exerts antidepressant-like effects in mice. Independent of its antioxidant activities, ERGO induces in cultured NSCs this differentiation through induction of the basic helix-loop-helix transcription factor Math1. However, the upstream signaling of Math1 in the mechanisms underlying ERGO-induced neuronal differentiation remains unclear. The purpose of the present study was to elucidate the upstream signaling with the aim of discovering novel targets for the treatment of neuropsychiatric disorders. We focused on neurotrophic factor signaling, as it is important for the promotion of neurogenesis and the induction of antidepressant effects. We also focused on the signaling of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1), a known amino acid sensor, and the members of this signaling pathway, mTOR and p70 ribosomal protein S6 kinase 1 (S6K1). Exposure of cultured NSCs to ERGO significantly increased the expression of phosphorylated S6K1 (p-S6K1) at Thr389 in only 1â¯h, of phosphorylated mTOR (p-mTOR) in 6â¯h, and of the gene product of neurotrophin 4/5 (NT5) which activates tropomyosin receptor kinase B (TrkB) in 24â¯h. ERGO increased the population of ßIII-tubulin-positive neurons, and this effect was suppressed by the inhibitors of S6K1 (PF4708671), mTORC1 (rapamycin), and TrkB (GNF5837). Oral administration of ERGO to mice significantly increased in the DG the expression of p-S6K1 at Thr389, the gene product of NT5, and phosphorylated TrkB but not that of p-mTOR. Thus, neuronal differentiation of NSCs induced by ERGO is mediated, at least in part, through phosphorylation of S6K1 at Thr389 and subsequent activation of TrkB signaling through the induction of NT5. Thus, S6K1 and NT5 might be promising target molecules for the treatment of neuropsychiatric disorders.
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Antioxidantes/farmacología , Ergotioneína/farmacología , Células-Madre Neurales/efectos de los fármacos , Neurogénesis/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Células Cultivadas , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos ICR , Factores de Crecimiento Nervioso/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismoRESUMEN
Brain immune cells, i.e., microglia, play an important role in the maintenance of brain homeostasis, whereas chronic overactivation of microglia is involved in the development of various neurodegenerative disorders. Therefore, the regulation of microglial activation may contribute to their treatment. The aim of the present study was to clarify the functional expression of carnitine/organic cation transporter OCTN1/SLC22A4, which recognizes the naturally occurring food-derived antioxidant ergothioneine (ERGO) as a substrate in vivo, in microglia and its role in regulation of microglial activation. Primary cultured microglia derived from wild-type mice (WT-microglia) and mouse microglial cell line BV2 exhibited time-dependent uptake of [3H]- or d9-labeled ERGO. The uptake was markedly decreased in cultured microglia from octn1 gene knockout mice (octn1 -/--microglia) and BV2 cells transfected with small interfering RNA targeting the mouse octn1 gene (siOCTN1). These results demonstrate that OCTN1 is functionally expressed in murine microglial cells. Exposure of WT-microglia to ERGO led to a significant decrease in cellular hypertrophy by LPS-stimulation with concomitant attenuation of intracellular reactive oxygen species (ROS), suggesting that OCTN1-mediated ERGO uptake may suppress cellular hypertrophy via the inhibition of ROS production with microglial activation. The expression of mRNA for interleukin-1ß (IL-1ß) after LPS-treatment was significantly increased in octn1 -/--microglia and siOCTN1-treated BV2 cells compared to the control cells. Meanwhile, treatment of ERGO minimally affected the induction of IL-1ß mRNA by LPS-stimulation in cultured microglia and BV2 cells. Thus, OCTN1 negatively regulated the induction of inflammatory cytokine IL-1ß, at least in part, via the transport of unidentified substrates other than ERGO in microglial cells.
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Transporte Biológico/efectos de los fármacos , Proteínas Portadoras/metabolismo , Ergotioneína/farmacología , Proteínas de la Membrana/metabolismo , Microglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Animales , Antioxidantes/metabolismo , Antioxidantes/farmacología , Carnitina , Proteínas Portadoras/genética , Células Cultivadas , Interleucina-1beta/metabolismo , Lipopolisacáridos/farmacología , Proteínas de la Membrana/genética , Ratones , Microglía/metabolismo , Neuronas/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , SimportadoresRESUMEN
The aim of the present study is to discover multidrug resistance-associated protein (MRP) inhibitors with neuroblastoma-selective cytotoxicity by means of fluorescence assay with a membrane-permeable fluorescent dye, Fluo-8 AM, based on our observation that gene expression of Mrp3 in neuroblastoma Neuro2a cells was remarkably higher than that in primary cultured cortical neurons, as determined by real-time PCR. Neuro2a cells showed minimal fluorescence upon incubation with Fluo-8 AM. However, blocking of Mrp3 efflux function by small interfering RNA (siRNA) transfection or inhibition with probenecid resulted in significant dye accumulation, observed as an increase of fluorescence. Interestingly, Mrp3 siRNA or probenecid treatment also resulted in increased cytotoxicity, as evidenced by decreased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-reducing activity of Neuro2a, with a concomitant increase in release of lactate dehydrogenase. On the other hand, primary cultured neurons exhibited higher fluorescence intensity after incubation with Fluo-8 AM regardless of addition of probenecid. Also, probenecid only minimally affected MTT-reducing activity. Thus, probenecid showed selective cytotoxicity towards Neuro2a cells. Based on these findings, we screened a series of established therapeutic agents for ability to induce Fluo-8 accumulation in Neuro2a cells. Several uricosuric and nonsteroidal anti-inflammatory drugs were identified, and these drugs were confirmed to decrease MTT-reducing activity selectively in Neuro2a. There was a negative linear correlation between Fluo-8 accumulation and cytotoxicity of these agents. Although the compounds identified here are insufficiently potent for practical application, further screening to discover higher-affinity MRP3 inhibitors using larger chemical libraries may uncover drug candidates with potent neuroblastoma-selective cytotoxicity.
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Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Neuroblastoma/tratamiento farmacológico , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Antiinflamatorios no Esteroideos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Femenino , Ratones , Ratones Endogámicos ICR , Neuroblastoma/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Embarazo , Uricosúricos/farmacologíaRESUMEN
Pharmacological outcome and a certain side effects of therapeutic drugs generally depend on concentration of the drugs and/or their active metabolites in the body. Physiologically-based pharmacokinetics is quantitative tool to understand the drug concentration in the body. Drug efficacy is sometimes affected by subjective factors and cannot be clearly quantified. Even in such cases, it could be possible to quantitatively understand possible pharmacological events occurred in the patients by understanding pharmacokinetics of the corresponding drug. Here, we have attempted to summarize the basis of physiologically-based pharmacokinetics to understand which factors will determine drug concentration in the body and how to predict/speculate the drug concentration in the body in a quantitative manner. For easier understanding by the readers, we introduce some examples of pharmacokinetic property of several osteoporosis drugs.
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Farmacocinética , Disponibilidad Biológica , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Humanos , Absorción Intestinal , Modelos Biológicos , Preparaciones Farmacéuticas/metabolismoRESUMEN
BACKGROUND: Clinically used antidepressants suffer from various side effects. Therefore, we searched for a safe antidepressant with minimal side effects among food ingredients that are distributed to the brain. Here, we focused on ERGO (ergothioneine), which is a hydrophilic antioxidant and contained at high levels in edible golden oyster mushrooms. ERGO is a typical substrate of carnitine/organic cation transporter OCTN1/SLC22A4, which is expressed in the brain and neuronal stem cells, although little is known about its permeation through the BBB (blood-brain barrier) or its neurological activity. METHODS: To clarify the exposure of ERGO to brain and the possible antidepressant-like effect after oral ingestion, ERGO or GOME (golden oyster mushroom extract) which contains 1.2% (w/w) ERGO was mixed with feed and provided to mice for 2 weeks, and then ERGO concentration and antidepressant-like effect were evaluated by LC-MS/MS and FST (forced swimming test) or TST (tail suspension test), respectively. RESULTS: Diet containing ERGO or GOME greatly increased the ERGO concentrations in plasma and brain, and significantly decreased the immobility time in both FST and TST. The required amount of GOME (~37 mg/day) to show the antidepressant-like effect corresponds to at most 8 g/day in humans. In mice receiving GOME-containing diet, doublecortin-positive cells showed a significant increase from the basal level, suggesting promotion of neuronal differentiation. CONCLUSION: Thus, orally ingested ERGO is transported across the BBB into the brain, where it may promote neuronal differentiation and alleviate symptoms of depression at plausibly achieved level of daily ingestion.
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Antidepresivos/farmacología , Antioxidantes/farmacología , Conducta Animal/efectos de los fármacos , Encéfalo/metabolismo , Ergotioneína/farmacología , Extractos Vegetales/farmacología , Pleurotus , Animales , Antidepresivos/administración & dosificación , Antidepresivos/sangre , Antioxidantes/administración & dosificación , Antioxidantes/metabolismo , Encéfalo/efectos de los fármacos , Depresión/dietoterapia , Depresión/tratamiento farmacológico , Ergotioneína/administración & dosificación , Ergotioneína/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Extractos Vegetales/administración & dosificación , Extractos Vegetales/sangreRESUMEN
The high prevalence/incidence of hearing loss (HL) in humans makes it the most common sensory defect. The majority of the cases are of genetic origin. Non-syndromic hereditary HL is extremely heterogeneous. Genetic approaches have been instrumental in deciphering genes that are crucial for auditory function. In this study, we first used NADf chip to exclude the implication of known North-African mutations in HL in a large consanguineous Tunisian family (FT13) affected by autosomal recessive non-syndromic HL (ARNSHL). We then performed genome-wide linkage analysis and assigned the deafness gene locus to ch:5q23.2-31.1, corresponding to the DFNB60 ARNSHL locus. Moreover, we performed whole exome sequencing on FT13 patient DNA and uncovered amino acid substitution p.Cys113Tyr in SLC22A4, a transporter of organic cations, cosegregating with HL in FT13 and therefore the cause of ARNSHL DFNB60. We also screened a cohort of small Tunisian HL families and uncovered an additional deaf proband of consanguineous parents that is homozygous for p.Cys113Tyr carried by the same microsatellite marker haplotype as in FT13, indicating that this mutation is ancestral. Using immunofluorescence, we found that Slc22a4 is expressed in stria vascularis (SV) endothelial cells of rodent cochlea and targets their apical plasma membrane. We also found Slc22a4 transcripts in our RNA-seq library from purified primary culture of mouse SV endothelial cells. Interestingly, p.Cys113Tyr mutation affects the trafficking of the transporter and severely alters ergothioneine uptake. We conclude that SLC22A4 is an organic cation transporter of the SV endothelium that is essential for hearing, and its mutation causes DFNB60 form of HL.
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Cóclea/patología , Consanguinidad , Endotelio/patología , Genes Recesivos/genética , Pérdida Auditiva/genética , Mutación/genética , Proteínas de Transporte de Catión Orgánico/genética , Secuencia de Aminoácidos , Animales , Células Cultivadas , Cóclea/metabolismo , Endotelio/metabolismo , Exoma/genética , Femenino , Células HEK293 , Pérdida Auditiva/patología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Linaje , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Ratas , Ratas Sprague-Dawley , Homología de Secuencia de Aminoácido , SimportadoresRESUMEN
Understanding the specific survival of the rare chronic myelogenous leukaemia (CML) stem cell population could provide a target for therapeutics aimed at eradicating these cells. However, little is known about how survival signalling is regulated in CML stem cells. In this study, we survey global metabolic differences between murine normal haematopoietic stem cells (HSCs) and CML stem cells using metabolomics techniques. Strikingly, we show that CML stem cells accumulate significantly higher levels of certain dipeptide species than normal HSCs. Once internalized, these dipeptide species activate amino-acid signalling via a pathway involving p38MAPK and the stemness transcription factor Smad3, which promotes CML stem cell maintenance. Importantly, pharmacological inhibition of dipeptide uptake inhibits CML stem cell activity in vivo. Our results demonstrate that dipeptide species support CML stem cell maintenance by activating p38MAPK-Smad3 signalling in vivo, and thus point towards a potential therapeutic target for CML treatment.
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Dipéptidos/clasificación , Regulación Neoplásica de la Expresión Génica/fisiología , Leucemia Mielógena Crónica BCR-ABL Positiva , Células Madre Neoplásicas/metabolismo , Proteína smad3/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , ADN Complementario , Dipéptidos/metabolismo , Femenino , Masculino , Ratones , Ratones Transgénicos , Retroviridae , Transducción de Señal/fisiología , Proteína smad3/genética , Simportadores/genética , Simportadores/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
5-Aminosalicylic acid (5-ASA) is an orally administered therapeutic agent for inflammatory bowel diseases, such as ulcerative colitis and Crohn's disease. We hypothesized that the absorption of 5-ASA is mediated by the polyspecific carnitine/organic cation transporter (OCTN1/SLC22A4), based on the similarity of chemical structure between 5-ASA and other OCTN1 substrates. Therefore, we examined the involvement of this transporter in the disposition of 5-ASA in vivo by using octn1 gene knockout (octn1(-/-)) mice. After oral administration of 5-ASA, the plasma concentrations of 5-ASA and its primary metabolite, N-acetyl-5-aminosalicylate (Ac-5-ASA), in octn1(-/-) mice were much lower than those in wild-type mice. The time required to reach maximum plasma concentration was also delayed in octn1(-/-) mice. On the other hand, the plasma concentration profiles of both 5-ASA and Ac-5-ASA after intravenous administration of 5-ASA (bolus or infusion) were similar in the two strains. Uptake of 5-ASA from the apical to the basal side of isolated small-intestinal tissues of octn1(-/-) mice, determined in an Ussing-type chamber, was lower than that in wild-type mice. Further, uptake of 5-ASA in HEK293 cells stably transfected with the OCTN1 gene, assessed as the sum of cell-associated 5-ASA and Ac-5-ASA, was higher than that in HEK293 cells transfected with the vector alone. Overall, these results indicate that OCTN1 is involved, at least in part, in the gastrointestinal absorption of 5-ASA.
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Carnitina/metabolismo , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Absorción Intestinal , Intestino Delgado/metabolismo , Mesalamina/farmacocinética , Transportador 1 de Catión Orgánico/metabolismo , Animales , Disponibilidad Biológica , Transporte Biológico Activo , Células HEK293 , Humanos , Masculino , Proteínas de la Membrana , Mesalamina/sangre , Mesalamina/uso terapéutico , Ratones , Ratones Noqueados , Transportador 1 de Catión Orgánico/genética , Eliminación de Secuencia , TransfecciónRESUMEN
The aim of the present study is to clarify the functional expression and physiological role in neural progenitor cells (NPCs) of carnitine/organic cation transporter OCTN1/SLC22A4, which accepts the naturally occurring food-derived antioxidant ergothioneine (ERGO) as a substrate in vivo. Real-time PCR analysis revealed that mRNA expression of OCTN1 was much higher than that of other organic cation transporters in mouse cultured cortical NPCs. Immunocytochemical analysis showed colocalization of OCTN1 with the NPC marker nestin in cultured NPCs and mouse embryonic carcinoma P19 cells differentiated into neural progenitor-like cells (P19-NPCs). These cells exhibited time-dependent [(3)H]ERGO uptake. These results demonstrate that OCTN1 is functionally expressed in murine NPCs. Cultured NPCs and P19-NPCs formed neurospheres from clusters of proliferating cells in a culture time-dependent manner. Exposure of cultured NPCs to ERGO or other antioxidants (edaravone and ascorbic acid) led to a significant decrease in the area of neurospheres with concomitant elimination of intracellular reactive oxygen species. Transfection of P19-NPCs with small interfering RNA for OCTN1 markedly promoted formation of neurospheres with a concomitant decrease of [(3)H]ERGO uptake. On the other hand, exposure of cultured NPCs to ERGO markedly increased the number of cells immunoreactive for the neuronal marker ßIII-tubulin, but decreased the number immunoreactive for the astroglial marker glial fibrillary acidic protein (GFAP), with concomitant up-regulation of neuronal differentiation activator gene Math1. Interestingly, edaravone and ascorbic acid did not affect such differentiation of NPCs, in contrast to the case of proliferation. Knockdown of OCTN1 increased the number of cells immunoreactive for GFAP, but decreased the number immunoreactive for ßIII-tubulin, with concomitant down-regulation of Math1 in P19-NPCs. Thus, OCTN1-mediated uptake of ERGO in NPCs inhibits cellular proliferation via regulation of oxidative stress, and also promotes cellular differentiation by modulating the expression of basic helix-loop-helix transcription factors via an unidentified mechanism different from antioxidant action.
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Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Ergotioneína/metabolismo , Neuronas/fisiología , Proteínas de Transporte de Catión Orgánico/metabolismo , Células Madre/fisiología , Animales , Antioxidantes/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Carnitina/genética , Carnitina/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Diferenciación Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , Células Cultivadas , Regulación hacia Abajo/genética , Regulación hacia Abajo/fisiología , Ergotioneína/genética , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Células-Madre Neurales/metabolismo , Células-Madre Neurales/fisiología , Neuronas/metabolismo , Proteínas de Transporte de Catión Orgánico/genética , ARN Mensajero/genética , Especies Reactivas de Oxígeno/metabolismo , Células Madre/metabolismo , Simportadores , Regulación hacia Arriba/genética , Regulación hacia Arriba/fisiologíaRESUMEN
Metformin is a widely used oral anti-diabetic, but the molecular mechanism(s) of its gastrointestinal membrane permeation remains unclear. Here, we examined the role of carnitine/organic cation transporter OCTN1/SLC22A4, which is localized on apical membranes of small intestine in mice and humans, in metformin absorption. The maximum plasma concentration (Cmax ) after oral administration of metformin (50 mg/kg) in octn1 gene knockout mice (octn1 (-/-) ) was higher than that in wild-type mice, with only a minimal difference in terminal half-life, but Cmax in octn1(-/-) mice given a higher dose (175 mg/kg) was lower than that in wild-type mice. Systemic elimination of metformin after intravenous administration was similar in the two strains, suggesting the possible involvement of OCTN1 in the gastrointestinal absorption. OCTN1-mediated uptake of metformin was observed in human embryonic kidney 293 cells transfected with mouse OCTN1 gene, but much lower than the uptake of the typical substrate [(3) H]ergothioneine (ERGO). In particular, the distribution volume for OCTN1-mediated uptake increased markedly and then tended to decrease as the metformin concentration was increased. Efflux of metformin preloaded in intestinal epithelial cell line Caco-2 was inhibited by ERGO. Overall, the present findings suggest that OCTN1 transports metformin and may be involved in its oral absorption in small intestine.