RESUMEN
BACKGROUND: Although the changes of mitogen-activated protein kinase (MAPK) pathway in the central nervous system (CNS) induced by excessive fluoride has been confirmed by our previous findings, the underlying mechanism(s) of the action remains unclear. Here, we investigate the possibility that microRNAs (miRNAs) are involved in the aspect. METHODS: As a model of chronic fluorosis, SD rats received different concentrations of fluoride in their drinking water for 3 or 6 months and SH-SY5Y cells were exposed to fluoride. Literature reviews and bioinformatics analyses were used to predict and real-time PCR to measure the expression of 12 miRNAs; an algorithm-based approach was applied to identify multiply potential target-genes and pathways; the dual-luciferase reporter system to detect the association of miR-132-3p with MAPK1; and fluorescence in situ hybridization to detect miR-132-3p localization. The miR-132-3p inhibitor or mimics or MAPK1 silencing RNA were transfected into cultured cells. Expression of protein components of the MAPK pathway was assessed by immunofluorescence or Western blotting. RESULTS: In the rat hippocampus exposed with high fluoride, ten miRNAs were down-regulated and two up-regulated. Among these, miR-132-3p expression was down-regulated to the greatest extent and MAPK1 level (selected from the 220 genes predicted) was corelated with the alteration of miR-132-3p. Furthermore, miR-132-3p level was declined, whereas the protein levels MAPK pathway components were increased in the rat brains and SH-SY5Y cells exposed to high fluoride. MiR-132-3p up-regulated MAPK1 by binding directly to its 3'-untranslated region. Obviously, miR-132-3p mimics or MAPK1 silencing RNA attenuated the elevated expressions of the proteins components of the MAPK pathway induced by fluorosis in SH-SY5Y cells, whereas an inhibitor of miR-132-3p just played the opposite effect. CONCLUSION: MiR-132-3p appears to modulate the changes of MAPK signaling pathway in the CNS associated with chronic fluorosis.
Asunto(s)
Fluoruros , MicroARNs , Proteína Quinasa 1 Activada por Mitógenos , Ratas Sprague-Dawley , MicroARNs/genética , Animales , Ratas , Fluoruros/toxicidad , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Masculino , Línea Celular TumoralRESUMEN
BACKGROUND To reveal the mechanism underlying the effect of alpha7 nicotinic acetylcholine receptor (nAChR) on neurodegeneration in Alzheimer disease (AD), the influence of the receptor on recognition in APP/PS1 mice was evaluated by using its selective agonist (PNU-282987). MATERIAL AND METHODS APP/PS1 and wild-type (WT) mice were treated with PNU or saline, respectively, for 7 days at the ages of 6 and 10 months. RESULTS Morris water maze analysis showed that both at 6 and 10 months of age, PNU treatment enhanced the learning and memory of APP/PS1 mice. However, PNU treatment did not alter the number of senile plaques. Furthermore, a higher protein expression of Nrf2/HO-1, ADAM10, SYP, and SNAP-25, and a lower level of oxidative stress, were observed in the hippocampus of APP/PS1 mice treated with PNU compared with the control group. CONCLUSIONS The results indicated that the activation of alpha7 nAChR by PNU improved the learning and memory of mice carrying the APP/PS1 mutation, regulated the levels of enzymes that mediate APP metabolization to reduce ß-amyloid peptide damage, and decreased the level of oxidative stress and maintained synaptic plasticity, in which the mechanism might be enhancement of the Nrf2/HO-1 pathway.
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Enfermedad de Alzheimer , Benzamidas/farmacología , Compuestos Bicíclicos con Puentes/farmacología , Hemo-Oxigenasa 1/metabolismo , Proteínas de la Membrana/metabolismo , Memoria , Factor 2 Relacionado con NF-E2/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7 , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Modelos Animales de Enfermedad , Aprendizaje/efectos de los fármacos , Aprendizaje/fisiología , Aprendizaje por Laberinto/efectos de los fármacos , Memoria/efectos de los fármacos , Memoria/fisiología , Ratones , Ratones Transgénicos , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/fisiología , Agonistas Nicotínicos/farmacología , Presenilina-1/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor Nicotínico de Acetilcolina alfa 7/agonistas , Receptor Nicotínico de Acetilcolina alfa 7/metabolismoRESUMEN
We examined the mechanism by which lithium chloride (LiCl) attenuates the impaired learning capability and memory function of dual-transgenic APP/PS1 mice. Six- or 12-month-old APP/PS1 and wild-type (WT) mice were randomized into four groups, namely WT, WT+Li (100 mg LiCl/kg body weight, gavage once daily), APP/PS1 and APP/PS1+Li. Primary rat hippocampal neurons were exposed to ß-amyloid peptide oligomers (AßOs), LiCl and/or XAV939 (inhibitor of Wnt/ß-catenin) or transfected with small interfering RNA against the ß-catenin gene. In the cerebral zone of APP/PS1 mice, the level of Aß was increased and those of α7 nicotinic acetylcholine receptors (nAChR), phosphor-GSK3ß (ser9), ß-catenin and cyclin D1 (protein and/or mRNA levels) reduced. Two-month treatment with LiCl at ages of 4 or 10 months weakened all of these effects. Similar expression variations were observed for these proteins in primary neurons exposed to AßOs, and these effects were attenuated by LiCl and aggravated by XAV939. Inhibition of ß-catenin expression lowered the level of α7 nAChR protein in these cells. LiCl attenuates the impaired learning capability and memory function of APP/PS1 mice via a mechanism that might involve elevation of the level of α7 nAChR as a result of altered Wnt/ß-catenin signalling.
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Aprendizaje/efectos de los fármacos , Cloruro de Litio/farmacología , Memoria/efectos de los fármacos , Vía de Señalización Wnt/efectos de los fármacos , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Conducta Animal , Supervivencia Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Genotipo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Ratones , Ratones Transgénicos , Fenotipo , Placa Amiloide/metabolismo , Placa Amiloide/patología , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/genéticaRESUMEN
To investigate the neuroprotective role of silent mating-type information regulation 2 homolog 1 (SIRT1) in Alzheimer disease (AD), brain tissues from patients with AD and APP/PS1 mice as well as primary rat neurons exposed to oligomers of amyloid-ß peptide were examined. The animals were treated with resveratrol (RSV) or suramin for 2 months. Cell cultures were treated with RSV, suramin, and the peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) stimulator ZLN005. Cells were transiently transfected with PGC-1α silencing RNA. The level of SIRT1 in brain tissues from patients with AD and APP/PS1 mice, including nuclear and mitochondrial proteins, as well as in primary neurons exposed to oligomers of amyloid-ß peptide, was decreased. Overexpression of APP/PS1 impaired learning and memory of mice; produced more senile plaques, disrupted membranes, and resulted in broken or absent cristae of mitochondria in the brain; decreased levels of A disintegrin and metallopeptidase domain 10, beta-secretase 2, 8-oxoguanine DNA glycosylase-1, PGC-1α, and NAD+; and increased levels of beta-secretase 1 and apoptosis. Interestingly, these changes were attenuated significantly by RSV treatment but enhanced by suramin administration. By activating PGC-1α but inhibiting SIRT1, apoptotic cell death was significantly decreased; however, by activating SIRT1 but inhibiting PGC-1α with small interfering PGC-1α, these levels remained unchanged. These findings indicate that SIRT1 may protect against AD-associated neurotoxicity, which might involve PGC-1α regulation.
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Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Sirtuina 1/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Encéfalo/metabolismo , Encéfalo/patología , Femenino , Humanos , Masculino , Ratones , RatasRESUMEN
Aim: The aim of this study is to investigate whether lithium chloride (LiCl) can regulate glycogen synthase kinase-3ß (GSK3ß)/nuclear factor E2 related factorï¼Nrf2ï¼/heme oxygenase-1 (HO-1) pathway to reduce the injury of oxidative stress in APP/PS1 double transgenic mice.Materials and Methods: The APP/PS1 double transgenic and wild-type (WT) mice were divided randomly into four groups, i.e. WT, WT + LiCl (LiCl 100 mg/kg by gavage once daily), the transgenic + LiCl and the transgenic groups. The expressions of phosphor-GSK3ß (ser9), Nrf2 and HO-1 at protein levels were detected by Western blotting. The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and the content of malondialdehyde (MDA) were measured by related detection kits. Nissl bodies in different brain regions were examined by Nissl staining.Results: The decreased protein levels of phosphor-GSK3ß (ser9), Nrf2 and HO-1, the declined activities of SOD and GSH-Px, the increased content of MDA and the decreased Nissl bodies in neurons were observed in the brains or serums of APP/PS1 mice as compared with WT. The treatment with LiCl attenuated these changes in the levels of GSK3ß/Nrf2/HO-1 pathway and oxidative stress as well as Nissl bodies induced by APP/PS1 mutation.Conclusion: LiCl reversed the declined activities of SOD and GSH-Px and the increased content of MDA as well as the decreased Nissl bodies in neurons in the brains or serums of APP/PS1 mice, the mechanism of which may be involved in the down-regulation of the activity of GSK3ß and consequently enhances the expressions of Nrf2 and HO-1.
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Enfermedad de Alzheimer/metabolismo , Encéfalo/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Hemo-Oxigenasa 1/metabolismo , Cloruro de Litio/administración & dosificación , Proteínas de la Membrana/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/efectos de los fármacos , Enfermedad de Alzheimer/sangre , Animales , Encéfalo/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Hemo-Oxigenasa 1/sangre , Masculino , Proteínas de la Membrana/sangre , Ratones Transgénicos , Factor 2 Relacionado con NF-E2/sangre , Transducción de Señal/efectos de los fármacosRESUMEN
To reveal the molecular mechanism of deficit in learning and memory induced by chronic fluorosis, the expression of muscarinic acetylcholine receptors (mAChRs) and oxidative stress were investigated. Sixty Sprague-Dawley (SD) rats were divided randomly into two groups (30 cases in each), i.e., the control group (<0.5 ppm fluoride in drinking water) and the fluoride group (50 ppm fluoride) for 10 months of treatment. The pups born from SD mothers with or without chronic fluorosis were selected at postnatal days 1, 7, 14, 21 and 28 for experiments (10 for each age). Spatial learning and memory were evaluated by Morris water maze test. The expressions of M1 and M3 mAChRs at the protein and mRNA levels were determined by Western blotting and real-time PCR, respectively. In addition, the contents of (·)OH, H2O2, O2(·-) and malondialdehyde (MDA), and activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in brains were quantitated by biochemical methods. Our results showed that as compared to controls, the abilities of learning and memory were declined in the adult rats and the offspring rats of postnatal day 28 in the fluoride groups; the expressions of both M1 and M3 mAChRs were significantly reduced at protein and mRNA levels; and the levels of (·)OH, H2O2, O2(·-) and MDA were significantly increased, while the activities of SOD and GSH-Px decreased. Interestingly, the decreased protein levels of M1 and M3 mAChRs were significantly correlated with the deficits of learning and memory and high level of oxidative stress induced by chronic fluorosis. Our results suggest that the mechanism for the deficits in learning and memory of rats with chronic fluorosis may be associated with the decreased expressions of M1 and M3 in mAChRs, in which the changes in the receptors might be the result of the high level of oxidative stress occurring in the disease.
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Fluorosis Dental/complicaciones , Trastornos de la Memoria/etiología , Receptor Muscarínico M1/genética , Receptor Muscarínico M3/genética , Animales , Enfermedad Crónica , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Glutatión Peroxidasa/metabolismo , Peróxido de Hidrógeno/metabolismo , Masculino , Malondialdehído/metabolismo , Aprendizaje por Laberinto , Estrés Oxidativo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Superóxido Dismutasa/metabolismo , Factores de TiempoRESUMEN
To examine whether resveratrol (RSV), an activator of silent mating-type information regulation 2 homolog 1 (SIRT1), can reverse the disruption of lipid metabolism caused by ß-amyloid peptide (Aß), APP/PS1 mice or cultured primary rat neurons were treated with RSV, suramin (inhibitor of SIRT1), ZLN005, a stimulator of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), or PGC-1α silencing RNA. In the brains of the APP/PS1 mice, expressions of SIRT1, PGC-1α, low-density lipoprotein receptor (LDLR) and very LDLR (VLDLR) were reduced at the protein and, in some cases, mRNA levels; while the levels of the proprotein convertase subtilisin/kexin type 9 (PCSK9), apolipoprotein E (ApoE), total cholesterol and LDL were all elevated. Interestingly, these changes were reversed by administration of RSV, while being aggravated by suramin. Furthermore, activation of PGC-1α, but inhibition of SIRT1, decreased the levels of PCSK9 and ApoE, while increased those of LDLR and VLDLR in the neurons exposed to Aß, and silencing PGC-1α, but activation of SIRT1, did not influence the levels of any of these proteins. These findings indicate that RSV can attenuate the disruption of lipid metabolism observed in the brains of APP mice and in primary neurons exposed to Aß by activating SIRT1, in which the mechanism may involve subsequently affecting PGC-1α.
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Precursor de Proteína beta-Amiloide , Proproteína Convertasa 9 , Ratas , Ratones , Animales , Resveratrol/farmacología , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Proproteína Convertasa 9/metabolismo , Sirtuina 1/metabolismo , Metabolismo de los Lípidos , Presenilina-1/metabolismo , Suramina/metabolismo , Neuronas/metabolismo , Apolipoproteínas E , Encéfalo/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismoRESUMEN
INTRODUCTION: For investigating the mechanism of brain injury caused by chronic fluorosis, this study was designed to determine whether NRH:quinone oxidoreductase 2 (NQO2) can influence autophagic disruption and oxidative stress induced in the central nervous system exposed to a high level of fluoride. METHODS: Sprague-Dawley rats drank tap water containing different concentrations of fluoride for 3 or 6 months. SH-SY5Y cells were either transfected with NQO2 RNA interference or treated with NQO2 inhibitor or activator and at the same time exposed to fluoride. The enrichment of gene signaling pathways related to autophagy was evaluated by Gene Set Enrichment Analysis; expressions of NQO2 and autophagy-related protein 5 (ATG5), LC3-II and p62, and mammalian target of rapamycin (mTOR) were quantified by Western-blotting or fluorescent staining; and the levels of malondialdehyde (MDA) and superoxide dismutase (SOD) assayed biochemically and reactive oxygen species (ROS) detected by flow cytometry. RESULTS: In the hippocampal CA3 region of rats exposed to high fluoride, the morphological characteristics of neurons were altered; the numbers of autophagosomes in the cytoplasm and the levels of NQO2 increased; the level of p-mTOR was decreased, and the levels of ATG5, LC3-II and p62 were elevated; and genes related to autophagy enriched. In vitro, in addition to similar changes in NQO2, p-mTOR, ATG5, LC3 II, and p62, exposure of SH-SY5Y cells to fluoride enhanced MDA and ROS contents and reduced SOD activity. Inhibition of NQO2 with RNAi or an inhibitor attenuated the disturbance of the autophagic flux and enhanced oxidative stress in these cells exposed to high fluoride. CONCLUSION: Our findings indicate that NQO2 may be involved in regulating autophagy and oxidative stress and thereby exerts an impact on brain injury caused by chronic fluorosis.
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Lesiones Encefálicas , Neuroblastoma , Quinona Reductasas , Ratas , Humanos , Animales , Fluoruros/farmacología , Especies Reactivas de Oxígeno/metabolismo , Ratas Sprague-Dawley , Quinona Reductasas/metabolismo , Estrés Oxidativo , Autofagia , Serina-Treonina Quinasas TOR/metabolismo , Hipocampo/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Mamíferos/metabolismoRESUMEN
BACKGROUND: Potential protection against the neurotoxic damages of high levels of fluoride on rats and SH-SY5Y cells by extract of Ginkgo biloba leaves, as well as underlying mechanisms, were examined. METHODS: The rats were divided randomly into 4 groups, i.e., control, treatment with the extract (100 mg/kg body weight, gavage once daily), treatment with fluoride (50 ppm F- in drinking water) and combined treatment with both; SH-SY5Y cells exposed to fluoride and fluoride in combination with the extract or 4-Amino-1,8-naphthalimide (4-ANI), an inhibitor of poly (ADP-ribose) polymerase-1 (PARP-1). Spatial learning and memory in the rats were assessed employing Morris water maze test; the contents of fluoride in brains and urine by fluoride ion-selective electrode; cytotoxicity of fluoride was by CCK-8 kit; the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and the content of malondialdehyde (MDA) by appropriate kits; the level of 8-hydroxydeoxyguanosine (8-OHdG) was by ELISA; the content of ROS and frequency of apoptosis by flow cytometry; the expressions of phospho-histone H2A.X(Ser139), PARP-1, poly (ADP-ribose) (PAR) and Sirtuin-1 (SIRT1) by Western blotting or immunofluorescence. RESULTS: The rats with prolong treatment of fluoride exhibited dental fluorosis, the increased contents of fluoride in brains and urine and the declined ability of learning and memory. In the hippocampus of the rats and SH-SY5Y cells exposed to fluoride, the levels of ROS, MDA, apoptosis, 8-OHdG and the protein expressions of histone H2A.X(Ser139), PARP-1 and PAR were all elevated; the activities of SOD and GSH-Px and the protein expression of SIRT1 reduced. Interestingly, the treatment of Ginkgo biloba extract attenuated these neurotoxic effects on rats and SH-SY5Y cells exposed to fluoride and the treatment of 4-ANI produced a neuroprotective effect against fluoride exposure. CONCLUSION: Ginkgo biloba extract attenuated neurotoxic damages induced by fluoride exposure to rats and SH-SY5Y cells and the underlying mechanism might involve the inhibition of PARP-1 and the promotion of SIRT1.
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Fluoruros , Neuroblastoma , Humanos , Animales , Ratas , Fluoruros/farmacología , HistonasRESUMEN
BACKGROUND: To reveal the underling molecular mechanism in brain damage induced by chronic fluorosis, the neurotoxicity and its correlation were investigated by transcriptomics and proteomics. METHODS: Sprague-Dawley rats were treated with fluoride at different concentrations (0, 5, 50 and 100â¯ppm, prepared by NaF) for 3 months. Spatial learning and memory were evaluated by Morris water maze test; neuronal morphological change in the hippocampus was observed using Nissl staining; and the level of oxidative stress including reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) were detected by biological methods. The high-throughput transcriptome sequencing (RNA-Seq) and tandem mass tag (TMT) proteomic sequencing were performed to detect the expression of differentially expressed genes and proteins, respectively. RESULTS: The results showed that compared with control group, rats exposed to high-dose fluoride exhibited declined abilities of learning and memory, decreased SOD activity and increased ROS and MDA levels, with lighter colored Nissl bodies. A total of 28 important differentially expressed genes (DEGs) were screened out by transcriptomics. Then, functional enrichment analyses showed that upregulated proteins enriched in cellular transport, while downregulated proteins enriched in synapse-related pathways. Thirteen corresponding DEGs and DAPs (cor-DEGs-DAPs) were identified by differential expressions selected with positively correlated genes/proteins, most of which were related to neurodegenerative changes and oxidative stress response. CONCLUSION: These results provide new omics evidence that rats chronically exposed to high-dose fluoride can induce neurotoxicity in the brains through changes in the cholinergic pathway and oxidative stress.
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Colinérgicos/toxicidad , Fluoruros/toxicidad , Hipocampo/efectos de los fármacos , Proteómica , Animales , Colinérgicos/administración & dosificación , Relación Dosis-Respuesta a Droga , Femenino , Fluoruros/administración & dosificación , Hipocampo/metabolismo , Masculino , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Ratas , Ratas Sprague-Dawley , TranscriptomaRESUMEN
Ligands of nicotinic acetylcholine receptors (nAChRs) are widely considered as potential therapeutic agents. The present study used primary hippocampus cells and APPswe/PSEN1dE9 double-transgenic mice models to study the possible therapeutic effect and underlying mechanism of the specific activation of α7 nAChR by PNU-282987 in the pathogenesis of Alzheimer's disease. The results indicated that activation of α7 nAChR attenuated the Aß-induced cell apoptosis, decreased the deposition of Aß, increased the expression of synaptic-associated proteins, and maintained synaptic morphology. Furthermore, in the APP/PS1_DT mice model, activation of α7 nAChR attenuated Aß-induced synaptic loss, reduced the deposition of Aß in the hippocampus, maintained the integral structure of hippocampus-derived synapse, and activated the calmodulin (CaM)-calmodulin-dependent protein kinase II (CaMKII)-cAMP response element-binding protein signaling pathway by upregulation of its key signaling proteins. In addition, activation of α7 nAChR improved the learning and memory abilities of the APP/PS1_DT mice. Collectively, the activation of α7 nAChR by PNU-282987 attenuated the toxic effect of Aß in vivo and in vitro, which including reduced deposition of Aß in the hippocampus, maintained synaptic morphology by partially reversing the expression levels of synaptic-associated proteins, activation of the Ca2+ signaling pathway, and improvement of the cognitive abilities of APP/PS1_DT mice.
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Benzamidas/farmacología , Compuestos Bicíclicos con Puentes/farmacología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Cognición/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Agonistas Nicotínicos/farmacología , Transducción de Señal , Transmisión Sináptica/efectos de los fármacos , Receptor Nicotínico de Acetilcolina alfa 7/agonistas , Enfermedad de Alzheimer/etiología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Animales , Apoptosis , Modelos Animales de Enfermedad , Hipocampo/metabolismo , Aprendizaje , Memoria , Neuronas/metabolismo , Sinapsis/metabolismo , Sinapsis/ultraestructuraRESUMEN
The aim was to determine whether the neuroprotective effect of SIRT1 in Alzheimer's disease (AD), due to inhibition of aggregation of the ß-amyloid peptide (Aß), involves activation of α7 nAChR. In present study, four-month-old APP/PS1 mice were administered resveratrol (RSV) or suramin once daily for two months, following which their spatial learning and memory were assessed using the Morris water maze test. Deposits of Aß in vivo were detected by near-infrared imaging (NIRI) and confocal laser scanning. SH-SY5Y/APPswe cells were treated with RSV, suramin, U0126 or methyllycaconitine (MLA). Levels of proteins and mRNA were determined by Western blotting and qRT-PCR, respectively. The results show that activation of SIRT1 improved their spatial learning and memory and reduced the production and aggregation of Aß in the hippocampus and cerebral cortex; whereas inhibition of SIRT1 had the opposite effects. In addition, activation of SIRT1 increased the levels of both α7 nAChR and αAPP in the brains these animals. Finally, activation of SIRT1 elevated the levels of pERK1/2, while inhibition of ERK1/2 counteracted the increase in α7 nAChR caused by RSV. These findings indicate that neuroprotection by SIRT1 may involve increasing levels of α7 nAChR through activation of the MAPK/ERK1/2 signaling pathway.
Asunto(s)
Precursor de Proteína beta-Amiloide/genética , Expresión Génica , Mutación , Neuroprotección/genética , Sirtuina 1/genética , Receptor Nicotínico de Acetilcolina alfa 7/genética , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Biomarcadores , Línea Celular Tumoral , Humanos , Sistema de Señalización de MAP Quinasas , Memoria , Ratones , Ratones Transgénicos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Interferencia de ARN , Sirtuina 1/metabolismo , Aprendizaje Espacial , Suramina/farmacologíaRESUMEN
Protection of Resveratrol (RSV) against the neurotoxicity induced by high level of fluoride was investigated. Sprague-Dawley (SD) rats and their offspring, as well as cultures of primary neurons were divided randomly into four groups: untreated (control); treated with 50 mg RSV/kg/ (once daily by gavage) or (20 M in the cultured medium); exposed to 50 ppm F- in drinking water or 4 mmol/l in the cultured medium; and exposed to fluoride then RSV as above. The adult rats were treated for 7 months and the offspring sacrificed at 28 days of age; the cultured neurons for 48 h. For general characterization, dental fluorosis was assessed and the fluoride content of the urine measured (by fluoride-electrode) in the rates and the survival of cultured neurons monitored with the CCK-8 test. The spatial learning and memory of rats were assessed with the Morris water maze test. The levels of α7 and α4 nicotinic acetylcholine receptors (nAChRs) were quantified by Western blotting; and the activities of superoxide dismutase (SOD) and catalase (CAT), and the levels of malondialdehyde (MDA) and H2O2 assayed biochemically. The results showed that chronic fluorosis resulted in the impaired learning and memory in rats and their offspring, and more oxidative stress in both rat brains and cultured neurons, which may be associated the lower levels of α7 and α4 nAChR subunits. Interestingly, RSV attenuated all of these toxic effects by fluorosis, indicating that protection against the neurotoxicity of fluoride by RSV might be in mechanism involved enhancing the expressions of these nAChRs.
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Encéfalo/efectos de los fármacos , Fluorosis Dental/tratamiento farmacológico , Neuronas/efectos de los fármacos , Sustancias Protectoras/farmacología , Receptores Nicotínicos/metabolismo , Resveratrol/farmacología , Administración Oral , Animales , Aprendizaje por Asociación/efectos de los fármacos , Encéfalo/metabolismo , Células Cultivadas , Enfermedad Crónica , Femenino , Fluoruros/administración & dosificación , Fluoruros/toxicidad , Fluoruros/orina , Fluorosis Dental/metabolismo , Masculino , Memoria/efectos de los fármacos , Neuronas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Sustancias Protectoras/administración & dosificación , Ratas , Ratas Sprague-Dawley , Resveratrol/administración & dosificaciónRESUMEN
Cognitive impairment caused by diabetes has been gradually recognized. Generally, nicotinic acetylcholine receptors (nAChRs) play an important role in the pathogenesis in dementia disorders including Alzheimer's disease (AD). However, the expression of nAChRs in the brains of type 2 diabetes mellitus (T2DM) is unexplored. This study explored the alterations of nAChRs in the postmortem brains of patients with T2DM and brains of db/db mice. Morris water maze test was used to appraise the ability of spatial learning and memory; Western blotting and RT-qPCR were performed to determine the expressions of target protein and mRNA, respectively; TUNEL was used to detect the apoptosis of neurons. We found that the protein levels of nAChR α7 and α4 subunits were significantly decreased and the apoptosis rates in neurons elevated in the hippocampus of T2DM patients and db/db mice as comparison to controls. Furthermore, the db/db mice exhibited the impaired cognition, the elevated level of pro-apoptotic protein and the reduced level of anti-apoptotic and synaptic proteins. This study shows the lowered level of nAChR α7 and α4 subunits and the elevated apoptosis in the hippocampus of T2DM patients and db/db mice, which might help explain the impaired cognition in T2DM.
Asunto(s)
Apoptosis , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Hipocampo/patología , Receptores Nicotínicos/metabolismo , Anciano , Animales , Autopsia , Disfunción Cognitiva , Femenino , Humanos , Masculino , Aprendizaje por Laberinto , Memoria , Ratones , Aprendizaje Espacial , Receptor Nicotínico de Acetilcolina alfa 7/metabolismoRESUMEN
BACKGROUND: Alzheimer's disease (AD) is responsible for 60-70% of all cases of dementia. On the other hand, the tap water consumed by hundreds of millions of people has been fluoridated to prevent tooth decay. However, little is known about the influence of fluoride on the expression of APP and subsequent changes in learning and memory and neuropathological injury. Our aim here was to determine whether exposure to fluoride aggravates the neuropathological lesions in mice carrying the amyloid precursor protein (APP)/presenilin1 (PS1) double mutation. METHODS: These transgenic or wide-type (WT) mice received 0.3 ml of a solution of fluoride (0.1 or 1 mg/ml, prepared with NaF) by intragastric administration once each day for 12 weeks. The learning and memory of these animals were assessed with the Morris water maze test. Senile plaques, ionized calcium binding adaptor molecule 1 (Iba-1), and complement component 3 (C3) expression were semi-quantified by immunohistochemical staining; the level of Aß42 was detected by Aß42 enzyme-linked immunosorbent assays (ELISAs); the levels of synaptic proteins and enzymes that cleave APP determined by Western blotting; and the malondialdehyde (MDA) content and activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) measured by biochemical procedures. RESULTS: The untreated APP mice exhibited a decline in learning and memory after 12 weeks of fluoride treatment, whereas treatment of these some animals with low or high levels of fluoride led to such declines after only 4 or 8 weeks, respectively. Exposure of APP mice to fluoride elevated the number of senile plaques and level of Aß42, Iba-1, and BACE1, while reducing the level of ADAM10 in their brains. The lower levels of synaptic proteins and enhanced oxidative stress detected in the hippocampus of APP mice were aggravated to fluoride. CONCLUSIONS: These findings indicate that exposure to fluoride, even at lower concentration, can aggravate the deficit in learning and memory and neuropathological lesions of the mice that express the high level of APP.
Asunto(s)
Enfermedad de Alzheimer/inducido químicamente , Enfermedad de Alzheimer/patología , Encéfalo/efectos de los fármacos , Encéfalo/patología , Fluoruros/toxicidad , Aprendizaje por Laberinto/efectos de los fármacos , Memoria/efectos de los fármacos , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Femenino , Hipocampo/metabolismo , Masculino , Ratones Transgénicos , Mutación , Fragmentos de Péptidos/metabolismo , Placa Amiloide , Presenilina-1/genética , Sinaptofisina/metabolismo , Proteína 25 Asociada a Sinaptosomas/metabolismoRESUMEN
In the study, we examined whether the silent information regulator 1 (SIRT1) can attenuate oxidative stress in the brains of mice carrying the APP/PS1 double mutation and/or in primary neonatal rat neurons exposed to oligomers of amyloid-ß peptide (AßOs). Starting at 4 or 8 months of age, the transgenic mice were treated with resveratrol (RSV, a stimulator of SIRT1) or suramin (an inhibitor) (each 20âmg/kg BW/day) for two months. The primary neurons were exposed to AßOs (0.5 µM) for 48âh and thereafter RSV (20 µM) or suramin (300âmg/ml) for 24âh. Cell viability was assessed by the CCK-8 assay; SIRT1 protein and mRNA determined by western blotting and real-time PCR, respectively; senile plaques examined immunohistochemically; ROS monitored by flow cytometry; and the contents of OH-, H2O2, O2·-, and MDA, and the activities of SOD and GSH-Px measured by standard biochemical procedures. In comparison to wild-type mice or untreated primary neurons, the expression of SIRT1 was significantly lower in the brains of APP/PS1 mice or neurons exposed to AßOs. In these same systems, increased numbers of senile plaques and a high level of oxidative stress were apparent. Interestingly, these two latter changes were attenuated by treatment with RSV, but enhanced by suramin. These findings indicate that SIRT1 may be neuroprotective.
Asunto(s)
Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/química , Precursor de Proteína beta-Amiloide/genética , Encéfalo/fisiopatología , Estrés Oxidativo/fisiología , Presenilina-1/genética , Sirtuina 1/metabolismo , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/toxicidad , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Animales Recién Nacidos , Encéfalo/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Glutatión Peroxidasa/metabolismo , Hipocampo/citología , Malondialdehído/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Morfolinos/toxicidad , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Estrés Oxidativo/genética , Presenilina-1/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Sirtuina 1/genética , Superóxido Dismutasa/metabolismoRESUMEN
AIMS: This study was designed to explore the expression and distribution of silent information regulator 1 (SIRT1) and superoxide dismutase 1 (SOD-1) in various regions of the brains of patients with Alzheimer's disease (AD), as well as to assess potential correlations between the levels of these proteins and also between these proteins and the Braak stage of AD. METHODS: In the temporal and frontal cortices, hippocampus and cerebellum of 10 patients with AD and 10 age-matched control subjects, expression of SIRT1 and SOD-1, together with histopathology, were assessed by immunohistochemical and immunofluorescent stainings. Relationships between variables were examined with the Pearson correlation test. RESULTS: The numbers of both SIRT1-positive and SOD-1-positive neurons and integrated optical density of immunohistochemical staining for these proteins in the temporal and frontal cortices, and hippocampus of patients with AD were significantly decreased than those in corresponding controls. In the case of the cerebellum, very weak expression of SIRT1 and obvious expression of SOD-1 were observed in granule cells, with no significant difference between AD and the control group. Interestingly, the protein levels between SIRT1 and SOD-1, as well as the level of SIRT1 or SOD-1 and Braak stage, were significantly correlated in neurons in all regions of the AD brains investigated except for the cerebellum. CONCLUSIONS: These findings indicate that the reduced level of SIRT1 in the brains of patients with AD may be related to the decline in SOD-1 and neuropathological changes of this disorder.
Asunto(s)
Enfermedad de Alzheimer/patología , Sirtuina 1/metabolismo , Superóxido Dismutasa-1/metabolismo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Estudios de Casos y Controles , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Masculino , Estrés OxidativoRESUMEN
Expressions of N-methyl-d-aspartic acid receptors (NMDARs) in the brains of rats and primary neurons exposed to high fluoride were investigated. Sprague-Dawley rats were divided randomly into a fluorosis group (50ppm fluoride in the drinking water for 6 months) and controls (<0.5ppm fluoride) and the offspring from these rats sacrificed on postnatal days 1, 7, 14, 21 and 28. The primary cultured neurons from the hippocampus of neonatal rats were treated with 5 and 50ppm fluoride for 48h. NMDAR subunits at protein or mRNA levels were quantified by Western blotting or real-time PCR. The phosphorylated calmodulin-protein kinase II (CaMKII) was determined by Western blotting, concentration of Ca2+ in neurons by laser confocal microscopy and apoptosis by flow cytometry. In the brains of adult rats and pups as well as in primary neurons exposed to high fluoride, the mRNAs encoding GluN1 and GluN2B subunits and the corresponding proteins were elevated, the GluN3A lowered and the GluN2A unchanged. In addition, the level of phosphor-CaMKII was reduced, and Ca2+ influx and apoptosis enhanced in the brains of rats and cultured neurons exposed to high fluoride. The results indicate that such modifications may involve brain damage induced by chronic fluorosis.