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Some species of the Gentianaceae family are a valuable source of secondary metabolites. However, the phytochemical knowledge of some of these species remains insufficient. Therefore, this work focused on the isolation of the two main secondary metabolites in the methanolic extract from a Gentiana capitata cell suspension using preparative HPLC and the determination of their structure using UHPLC-DAD-IT-MS/MS and NMR methods. Their content in the methanolic extract was quantified using a previously validated HPLC method. The toxicity of the extract and two isolated compounds was also tested on the PC-12 cell line. The structures of the main secondary metabolites were identified as isosaponarin and 3,7,8-Trimethoxy-9-oxo-9H-xanthen-1-yl 6-O-ß-D-ribopyranosyl-ß-D-allopyranoside by comparing the UHPLC-DAD-IT-MS/MS and NMR results with the literature data. The content of isosaponarin was determined to be 0.76 ± 0.04%, and the content of 3,7,8-trimethoxy-9-oxo-9H-xanthen-1-yl 6-O-ß-D-ribopyranosyl-ß-D-allopyranoside was found to be 0.31 ± 0.02% in the dry extract. Additionally, a two-fold increase in the viability of the PC-12 cell line was observed compared to the control after treatment with the methanolic extract at a concentration of 500 µg/mL. These results suggest the potential use of G. capitata cell suspension methanolic extract as a new source of isosaponarin and 3,7,8-trimethoxy-9-oxo-9H-xanthen-1-yl 6-O-ß-D-ribopyranosyl-ß-D-allopyranoside, highlighting their lack of toxicity to the PC-12 (rat pheochromocytoma) cell line.
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Sobrevivência Celular , Gentiana , Extratos Vegetais , Animais , Ratos , Células PC12 , Sobrevivência Celular/efeitos dos fármacos , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Gentiana/química , Saponinas/farmacologia , Saponinas/química , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas em Tandem , Espectroscopia de Ressonância MagnéticaRESUMO
Elemental analysis at the single-cell level is an emerging technique in the field of inductively coupled plasma mass spectrometry (ICP-MS). In comparison to the analysis of cell suspensions by fast time-resolved analysis, single-cell sampling by laser ablation (LA) allows the discriminatory analysis of single cells according to their size and morphology. In this study, we evaluated the changes in elemental contents in a single cell through differentiation of rat adrenal pheochromocytoma into neuron-like cells by LA-ICP-MS. The contents of seven essential minerals were increased about 2-3 times after the differentiation. In addition, we evaluated the degree of differentiation at the single-cell level by means of imaging cytometry after immunofluorescence staining of microtubule-associated protein 2 (Map2), a neuron-specific protein. The fluorescence intensities of Alexa Fluor 488-conjugated secondary antibody against the anti-Map2 primary antibody showed large variations among the cells after the onset of differentiation. Although the average fluorescence intensity was increased through the differentiation, there were still less-matured neuron-like cells that exhibited a lower fluorescence intensity after 5 days of differentiation. Since a positive correlation between the fluorescence intensity and the cell size in area was found, we separately measured the elemental contents in the less-matured smaller cells and well-matured larger cells by LA-ICP-MS. The larger cells had higher elemental contents than the smaller cells, indicating that the essential minerals are highly required at a later stage of differentiation.
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Providing antioxidants and targeting acetylcholinesterase (AChE) are key strategies in treating neurocognitive dysfunction. In this study, bioactive sturgeon (Acipenser schrenckii) spinal cord peptides (SSCPs) with antioxidant and AChE inhibitory potency were extracted and separated from sturgeon spinal cord by enzymatic hydrolysis and ultrafiltration, and targeted peptide PGGW was screened via computer simulated molecular docking. Further, the molecular dynamic interactions of the PGGW with superoxide dismutase (SOD) and AChE were analyzed, and the protective effect of PGGW on glutamate-induced PC12 cells in vitro was evaluated. The <3 kDa fraction of SSCPs displays the most potent antioxidative efficacy (1 mg/mL, DPPHâ¢: 89.07%, ABTS+: 76.35%). Molecular dynamics simulation showed that PGGW was stable within AChE and tightly bound to residues SER203, PHE295, ILE294 and TRP236. When combined with SOD, the indole group of PGGW was stuck inside SOD, but the tail chain PGG fluctuated greatly outside. Surface plasmon resonance demonstrated that PGGW has a high binding affinity for AChE (KD = 1.4 mM) and 0.01 mg/mL PGGW provided good protection against glutamate-induced apoptosis. The findings suggest a promising strategy for drug research on neurodegenerative diseases.
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OBJECTIVE: This study aimed to establish a neural cell injury model in vitro by stimulating PC12 cells with lipopolysaccharide (LPS) and to examine the effects of astragaloside IV on key targets using high-throughput sequence technology and bioinformatics analyses. METHODS: PC12 cells in the logarithmic growth phase were treated with LPS at final concentrations of 0.25, 0.5, 0.75, 1, and 1.25 mg/mL for 24 h. Cell morphology was evaluated, and cell survival rates were calculated. A neurocyte inflammatory model was established with LPS treatment, which reached a 50% cell survival rate. PC12 cells were treated with 0.01, 0.1, 1, 10, or 100 µmol/L astragaloside IV for 24 h. The concentration of astragaloside IV that did not affect the cell survival rate was selected as the treatment group for subsequent experiments. NOS activity was detected by colorimetry; the expression levels of ERCC2, XRCC4, XRCC2, TNF-α, IL-1ß, TLR4, NOS and COX-2 mRNA and protein were detected by RT-qPCR and Western blotting. The differentially expressed genes (DEGs) between the groups were screened using a second-generation sequence (fold change>2, P<0.05) with the following KEGG enrichment analysis, RT-qPCR and Western blotting were used to detect the mRNA and protein expression of DEGs related to the IL-17 pathway in different groups of PC12 cells. RESULTS: The viability of PC12 cells was not altered by treatment with 0.01, 0.1, or 1 µmol/L astragaloside IV for 24 h (P>0.05). However, after treatment with 0.5, 0.75, 1, or 1.25 mg/mL LPS for 24 h, the viability steadily decreased (P<0.01). The mRNA and protein expression levels of ERCC2, XRCC4, XRCC2, TNF-α, IL-1ß, TLR4, NOS, and COX-2 were significantly increased after PC12 cells were treated with 1 mg/mL LPS for 24 h (P<0.01); however, these changes were reversed when PC12 cells were pretreated with 0.01, 0.1, or 1 µmol/L astragaloside IV in PC12 cells and then treated with 1 mg/mL LPS for 24 h (P<0.05). Second-generation sequencing revealed that 1026 genes were upregulated, while 1287 genes were downregulated. The DEGs were associated with autophagy, TNF-α, interleukin-17, MAPK, P53, Toll-like receptor, and NOD-like receptor signaling pathways. Furthermore, PC12 cells treated with a 1 mg/mL LPS for 24 h exhibited increased mRNA and protein expression of CCL2, CCL11, CCL7, MMP3, and MMP10, which are associated with the IL-17 pathway. RT-qPCR and Western blotting analyses confirmed that the DEGs listed above corresponded to the sequence assay results. CONCLUSION: LPS can damage PC12 cells and cause inflammatory reactions in nerve cells and DNA damage. astragaloside IV plays an anti-inflammatory and DNA damage protective role and inhibits the IL-17 signaling pathway to exert a neuroprotective effect in vitro.
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Anti-Inflamatórios , Sobrevivência Celular , Reparo do DNA , Lipopolissacarídeos , Saponinas , Triterpenos , Animais , Células PC12 , Ratos , Lipopolissacarídeos/farmacologia , Triterpenos/farmacologia , Saponinas/farmacologia , Anti-Inflamatórios/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacosRESUMO
Controlling biomolecular-cell interactions is crucial for the design of scaffolds for tissue engineering (TE). Regenerated silk fibroin (RSF) has been extensively used as TE scaffolds, however, RSF showed poor attachment of neuronal cells, such as rat pheochromocytoma (PC12) cells. In this work, amphiphilic peptides containing a hydrophobic isoleucine tail (I3) and laminin or fibronectin derived peptides (IKVAV, PDSGR, YIGSR, RGDS and PHSRN) were designed for promoting scaffold-cell interaction. Three of them (I3KVAV, I3RGDS and I3YIGSR) can self-assemble into nanofibers, therefore, were used to enhance the application of RSF in neuron TE. Live / dead assays revealed that the peptides exhibited negligible cytotoxicity against PC12 cells. The specific interaction between PC12 cells and the peptides were investigate using atomic force microscopy (AFM). The results indicated a synergistic effect in the designed peptides, promoting cellular attachment, proliferation and morphology changes. In addition, AFM results showed that co-assembling peptides I3KVAV and I3YIGSR possesses the best regulation of proliferation and attachment of PC12 cells, consistent with immunofluorescence staining results. Moreover, cell culture with hydrogels revealed that a mixture of peptides I3KVAV and I3YIGSR can also promote 3D neurites outgrowth. The approach of combining two different self-assembling peptides shows great potential for nerve regeneration applications.
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Nymphaea candida Presl (NC), traditionally used in medicine for heat syndrome-related ailments, possesses antioxidative, anti-inflammatory, hepatoprotective, and neuroprotective properties. This research investigates the antidepressant and neuroprotective effects and mechanisms of Nymphaea candida Presl ethyl acetate (NCEA). Primary components of NCEA were identified as phenolic acids and flavonoids through UPLC-MS/MS analysis. The depression mouse model was induced via intracerebroventricular injection of Lipopolysaccharide (LPS), followed by oral administration of fluoxetine and NCEA for one week. Biochemical assays and HE staining confirmed NCEA's non-toxicity and protective effects on the liver and lungs. NCEA administration mitigated LPS-induced depressive behaviors, decreased IL-1ß, TNF-α levels in the hippocampus, suppressed microglial activation, reduced Iba-1 expression, and increased NA, brain-derived neurotrophic factor (BDNF), and dendritic spine density in the hippocampus. Furthermore, NCEA enhanced cell viability in a CORT-induced PC12 cell model, decreased lactate dehydrogenase (LDH) release rate, total superoxide dismutase (SOD) inhibition rate, intracellular nitric oxide (NO) release, and reduced reactive oxygen species (ROS) production. Our research findings suggest that NCEA exhibits significant antidepressant effects, which may be attributed to its reduction of neuroinflammation, improvement in neurotransmitter levels, neuronal protection, and antioxidative stress properties.
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Thyroid hormones (THs) are essential in neuronal and glial cell development and differentiation, synaptogenesis, and myelin sheath formation. In addition to nuclear receptors, TH acts through αvß3-integrin on the plasma membrane, influencing transcriptional regulation of signaling proteins that, in turn, affect adhesion and survival of nerve cells in various neurologic disorders. TH exhibits protective properties during brain hypoxia; however, precise intracellular mechanisms responsible for the preventive effects of TH remain unclear. In this study, we investigated the impact of TH on integrin αvß3-dependent downstream systems in normoxic and hypoxic conditions of pheochromocytoma PC12 cells. Our findings reveal that triiodothyronine (T3), acting through αvß3-integrin, induces activation of the JAK2/STAT5 pathway and suppression of the SHP2 in hypoxic PC12 cells. This activation correlates with the downregulation of the expression palmitoyltransferase-ZDHHC2 and ZDHHC9 genes, leading to a subsequent decrease in palmitoylation and phosphorylation of Fyn tyrosine kinase. We propose that these changes may occur due to STAT5-dependent epigenetic silencing of the palmitoyltransferase gene, which in turn reduces palmitoylation/phosphorylation of Fyn with a subsequent increase in the survival of cells. In summary, our study provides the first evidence demonstrating the involvement of integrin-dependent JAK/STAT pathway, SHP2 suppression, and altered post-translational modification of Fyn in protective effects of T3 during hypoxia.
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Neuritin plays an important role in promoting nerve injury repair and maintaining synaptic plasticity, making it a potential therapeutic target for the treatment of nerve injury and neurodegenerative diseases. The present study aimed to obtain an active, unlabeled neuritin protein. Initially, a neuritin protein expression system with an enterokinase site was constructed in Escherichia coli. After optimizing induction conditions and screening for high expression, a neuritin recombinant protein with purity exceeding 85 % was obtained through Ni-affinity chromatography. Subsequently, unlabeled neuritin with a molecular weight of 11 kDa was obtained through the enzymatic cleavage of the His label using an enterokinase. Furthermore, a neuritin recombinant protein with purity exceeding 95 % was obtained using gel chromatography. Functional investigations revealed that neurite outgrowth of PC12 cells was stimulated by the isolated neuritin. This study establishes a method to obtain active and unlabeled neuritin protein, providing a foundation for subsequent research on its biological functions.
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Escherichia coli , Proteínas Ligadas por GPI , Proteínas Recombinantes , Animais , Células PC12 , Ratos , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Escherichia coli/genética , Escherichia coli/metabolismo , Neuropeptídeos/genética , Neuropeptídeos/química , Neuropeptídeos/metabolismo , Enteropeptidase/metabolismo , Enteropeptidase/genética , Enteropeptidase/química , Cromatografia de Afinidade , Crescimento Neuronal/efeitos dos fármacos , Cromatografia em Gel , Expressão GênicaRESUMO
AIMS: This study investigates the neuroprotective effects of lipidized analogues of 2-SS-CART(61-102) derived from anorexigenic neuropeptide cocaine- and amphetamine-regulated transcript peptide (CARTp) in light of the link between obesity, its comorbidities, and the development of Alzheimer's disease. METHODS: We introduce novel lipidized analogues derived from 2-SS-CART(61-102), a specific analogue of natural CART(61-102), with two disulfide bridges. Using hypothermic PC12 cells, we tested the effect of the most potent analogues on Tau phosphorylation. We further described the anorexigenic and neuroprotective potential of subcutaneously (SC) injected lipidized CARTp analogue in a mouse model with prediabetes and obesity induced by neonatal monosodium glutamate (MSG) administration. RESULTS: Compared to the non-lipidized 2-SS-CART(61-102), all lipidized analogues exhibited a potent binding affinity to PC12 cells and enhanced in vitro stability in rat plasma. Two most potent lipidized analogues attenuated hypothermia-induced Tau hyperphosphorylation at multiple epitopes. Subsequently, chronic SC treatment with palm-2-SS-CART(61-102) significantly decreased body weight and food intake, improved metabolic parameters, decreased level of pTau and increased neurogenesis in hippocampi of obese MSG mice. CONCLUSION: Our unique CARTp analogue palm-2-SS-CART(61-102) shows promise as a potent anti-obesity and neuroprotective agent.
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Modelos Animais de Doenças , Proteínas do Tecido Nervoso , Fármacos Neuroprotetores , Obesidade , Glutamato de Sódio , Proteínas tau , Animais , Fármacos Neuroprotetores/farmacologia , Obesidade/metabolismo , Obesidade/tratamento farmacológico , Células PC12 , Ratos , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Proteínas tau/metabolismo , Masculino , Fosforilação/efeitos dos fármacos , Depressores do Apetite/farmacologia , Camundongos Endogâmicos C57BL , Lipídeos/química , Lipídeos/sangueRESUMO
Alzheimer's disease (AD) is a progressive neurodegenerative disorder. Accumulation of ß-amyloid (Aß) in the brain has been recognized as a key factor in the onset and progression of Alzheimer's disease (AD).The accumulation of Aß in the brain catalyzes the production of reactive oxygen species (ROS), which in turn triggers oxidative damage to cellular components such as DNA, lipids, and proteins. In the present study, we investigated the protective effect of Ganoderic acid A (GA.A) against Aß42-induced apoptosis in PC12 cells. Changes in mitochondrial membrane potential indicated that GA.A treats mitochondrial dysfunction by decreasing Aß42 deposition and inhibiting neural protofiber tangle formation. Changes in intracellular Ca2+ and caspase-3 indicated that GA.A reduced mitochondrial damage by Aß42 in PC12 cells, thereby decreasing ROS accumulation and reducing Aß protofiber-induced cytotoxicity. These features suggest that GA.A has great potential as an effective neuroprotective drug in the treatment of Alzheimer's disease.
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Peptídeos beta-Amiloides , Apoptose , Lanosterol , Potencial da Membrana Mitocondrial , Mitocôndrias , Fármacos Neuroprotetores , Fragmentos de Peptídeos , Espécies Reativas de Oxigênio , Animais , Peptídeos beta-Amiloides/toxicidade , Peptídeos beta-Amiloides/metabolismo , Células PC12 , Ratos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fragmentos de Peptídeos/toxicidade , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Apoptose/efeitos dos fármacos , Lanosterol/farmacologia , Lanosterol/análogos & derivados , Cálcio/metabolismo , Caspase 3/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Ácidos HeptanoicosRESUMO
Elevated homocysteine (Hcy) levels are detrimental to neuronal cells and contribute to cognitive dysfunction in rats. Mitochondria plays a crucial role in cellular energy metabolism. Interestingly, the damaging effects of Hcy in vivo and in vitro conditions exhibit distinct results. Herein, we aimed to investigate the effects of Hcy on mitochondrial function in primary neurons and PC12 cells and explore the underlying mechanisms involved. The metabolic intermediates of Hcy act as methyl donors and play important epigenetic regulatory roles. N6-methyldeoxyadenosine (6 mA) modification, which is enriched in mitochondrial DNA (mtDNA), can be mediated by methylase METTL4. Our study suggested that mitochondrial perturbation caused by Hcy in primary neurons and PC12 cells may be attributable to mtDNA 6 mA modification difference. Hcy could activate the expression of METTL4 within mitochondria to facilitate mtDNA 6 mA status, and repress mtDNA transcription, then result in mitochondrial dysfunction.
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Desoxiadenosinas , Hipocampo , Homocisteína , Mitocôndrias , Neurônios , Animais , Ratos , Células PC12 , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Homocisteína/farmacologia , Homocisteína/análogos & derivados , Homocisteína/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Desoxiadenosinas/farmacologia , Hipocampo/metabolismo , Hipocampo/efeitos dos fármacos , Ratos Sprague-Dawley , DNA Mitocondrial/metabolismo , DNA Mitocondrial/genética , Células Cultivadas , Metiltransferases/metabolismo , Metiltransferases/genéticaRESUMO
The gene product of ocular albinism 1 (OA1)/G-protein-coupled receptor (GPR)143 is a receptor for L-3,4-dihydroxyphenylanine (l-DOPA), the most effective agent for Parkinson's disease. When overexpressed, human wild-type GPR143, but not its mutants, inhibits neurite outgrowth in PC12 cells. We investigated the downstream signaling pathway for GPR143-induced inhibition of neurite outgrowth. Nifedipine restored GPR143-induced neurite outgrowth inhibition to the level of control transfectant but did not affect outgrowth in GPR143-knockdown cells. Cilnidipine and flunarizine also suppressed the GPR143-induced inhibition, but their effects at higher concentrations still occurred even in GPR143-knockdown cells. These results suggest that GPR143 regulates neurite outgrowth via L-type calcium channel(s).
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Canais de Cálcio Tipo L , Crescimento Neuronal , Nifedipino , Receptores Acoplados a Proteínas G , Células PC12 , Animais , Ratos , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo L/genética , Nifedipino/farmacologia , Crescimento Neuronal/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/fisiologia , Humanos , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Proteínas do Olho/farmacologia , Flunarizina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Levodopa/farmacologia , Técnicas de Silenciamento de Genes , Neuritos/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , Glicoproteínas de MembranaRESUMO
INTRODUCTION: The proline-rich decapeptide 10c (Bj-PRO-10c; ENWPHPQIPP) from the Bothrops jararaca snake modulates argininosuccinate synthetase (AsS) activity to stimulate L-arginine metabolite production and neuroprotection in the SH-SY5Y cell line. The relationships between structure, interactions with AsS, and neuroprotection are little known. We evaluated the neuroprotective effects of Bj-PRO-10c and three other PROs (Bn-PRO-10a,
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Rasagiline (Azilect®) is a selective monoamine oxidase B (MAO-B) inhibitor that provides symptomatic benefits in Parkinson's disease (PD) treatment and has been found to exert preclinical neuroprotective effects. Here, we investigated the neuroprotective signaling pathways of acute rasagiline treatment for 22 h in PC12 neuronal cultures exposed to oxygen-glucose deprivation (OGD) for 4 h, followed by 18 h of reoxygenation (R), causing 40% aponecrotic cell death. In this study, 3-10 µM rasagiline induced dose-dependent neuroprotection of 20-80%, reduced the production of the neurotoxic reactive oxygen species by 15%, and reduced the nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by 75-90%. In addition, 10 µM rasagiline increased protein kinase B (Akt) phosphorylation by 50% and decreased the protein expression of the ischemia-induced α-synuclein protein by 50% in correlation with the neuroprotective effect. Treatment with 1-5 µM rasagiline induced nuclear shuttling of transcription factor Nrf2 by 40-90% and increased the mRNA levels of the antioxidant enzymes heme oxygenase-1, (NAD (P) H- quinone dehydrogenase, and catalase by 1.8-2.0-fold compared to OGD/R insult. These results indicate that rasagiline provides neuroprotection to the ischemic neuronal cultures through the inhibition of α-synuclein and GAPDH-mediated aponecrotic cell death, as well as via mitochondrial protection, by increasing mitochondria-specific antioxidant enzymes through a mechanism involving the Akt/Nrf2 redox-signaling pathway. These findings may be exploited for neuroprotective drug development in PD and stroke therapy.
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Dopamine is an important neurotransmitter in the body and closely related to many neurodegenerative diseases. Therefore, the detection of dopamine is of great significance for the diagnosis and treatment of diseases, screening of drugs and unraveling of relevant pathogenic mechanisms. However, the low concentration of dopamine in the body and the complexity of the matrix make the accurate detection of dopamine challenging. Herein, an electrochemical sensor is constructed based on ternary nanocomposites consisting of one-dimensional Pt nanowires, two-dimensional MXene nanosheets, and three-dimensional porous carbon. The Pt nanowires exhibit excellent catalytic activity due to the abundant grain boundaries and highly undercoordinated atoms; MXene nanosheets not only facilitate the growth of Pt nanowires, but also enhance the electrical conductivity and hydrophilicity; and the porous carbon helps induce significant adsorption of dopamine on the electrode surface. In electrochemical tests, the ternary nanocomposite-based sensor achieves an ultra-sensitive detection of dopamine (S/N = 3) with a low limit of detection (LOD) of 28 nM, satisfactory selectivity and excellent stability. Furthermore, the sensor can be used for the detection of dopamine in serum and in situ monitoring of dopamine release from PC12 cells. Such a highly sensitive nanocomposite sensor can be exploited for in situ monitoring of important neurotransmitters at the cellular level, which is of great significance for related drug screening and mechanistic studies.
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Carbono , Dopamina , Técnicas Eletroquímicas , Nanocompostos , Nanofios , Platina , Dopamina/análise , Dopamina/sangue , Dopamina/química , Platina/química , Células PC12 , Nanofios/química , Nanocompostos/química , Animais , Carbono/química , Ratos , Porosidade , Técnicas Eletroquímicas/métodos , Neurônios/metabolismo , Limite de Detecção , EletrodosRESUMO
Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) is a pleiotropic peptide known to promote many beneficial processes following neural damage and cell death after stroke. Despite PACAP's known neurotrophic and anti-inflammatory properties, it has not realized its translational potential due to a poor pharmacokinetic profile (non-linear PK/PD), and limited Blood-Brain Barrier Penetration (BBB) permeability. We have previously shown that glycosylation of PACAP increases stability and enhances BBB penetration. In addition, our prior studies showed reduced neuronal cell death and neuroinflammation in models of Parkinson's disease and Traumatic Brain Injury (TBI). In this study we show that a PACAP(1-27) glucoside retains the known neurotrophic activity of native PACAP(1-27)in vitro and a 5-day daily treatment regimen (100 nM) leads to neurite-like extensions in PC12 cells. In addition, we show that intraperitoneal injection of a PACAP(1-27) lactoside (10 mg/kg) with improved BBB-penetration, given 1-hour after reperfusion in a Transient Middle Cerebral Artery Occlusion (tMCAO) mouse model, reduces the infarct size after the ischemic injury in males significantly by â¼ 36 %, and the data suggest a dose-dependency. In conclusion, our data support further development of PACAP glycopeptides as promising novel drug candidates for the treatment of stroke, an area with an urgent clinical need.
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Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Animais , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/administração & dosagem , Masculino , Ratos , Camundongos , Células PC12 , Camundongos Endogâmicos C57BL , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/patologia , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/patologia , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/uso terapêutico , Modelos Animais de Doenças , Glicosídeos/farmacologia , Glicosídeos/uso terapêutico , Glicosídeos/administração & dosagem , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Neuritos/efeitos dos fármacos , Neuritos/patologiaRESUMO
Rapid and accurate in situ determination of dopamine is of great significance in the study of neurological diseases. In this work, poly (3,4-ethylenedioxythiophene): poly (styrenesulfonic acid) (PEDOT: PSS)/graphene oxide (GO) fibers were fabricated by an effective method based on microfluidic wet spinning technology. The composite microfibers with stratified and dense arrangement were continuously prepared by injecting PEDOT: PSS and GO dispersion solutions into a microfluidic chip. PEDOT: PSS/GO fiber microelectrodes with high electrochemical activity and enhanced electrochemical oxidation activity of dopamine were constructed by controlling the structure composition of the microfibers with varying flow rate. The fabricated fiber microelectrode had a low detection limit (4.56 nM) and wide detection range (0.01-8.0 µM) for dopamine detection with excellent stability, repeatability, and reproducibility. In addition, the PEDOT: PSS/GO fiber microelectrode prepared was successfully used for the detection of dopamine in human serum and PC12 cells. The strategy for the fabrication of multi-component fiber microelectrodes is a new and effective approach for monitoring the intercellular neurotransmitter dopamine and has high potential as an implantable neural microelectrode.
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Dopamina , Grafite , Microeletrodos , Poliestirenos , Células PC12 , Dopamina/sangue , Humanos , Ratos , Animais , Poliestirenos/química , Grafite/química , Limite de Detecção , Técnicas Eletroquímicas/métodos , Técnicas Eletroquímicas/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Compostos Bicíclicos Heterocíclicos com Pontes/química , Tiofenos/química , Dispositivos Lab-On-A-Chip , PolímerosRESUMO
Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by the accumulation of amyloid beta (Aß) plaques in the brain. Aß1-42 is the main component of Aß plaque, which is toxic to neuronal cells. Si nanowires (Si NWs) have the advantages of small particle size, high specific surface area, and good biocompatibility, and have potential application prospects in suppressing Aß aggregation. In this study, we employed the vapor-liquid-solid (VLS) growth mechanism to grow Si NWs using Au nanoparticles as catalysts in a plasma-enhanced chemical vapor deposition (PECVD) system. Subsequently, these Si NWs were transferred to a phosphoric acid buffer solution (PBS). We found that Si NWs significantly reduced cell death in PC12 cells (rat adrenal pheochromocytoma cells) induced by Aß1-42 oligomers via double staining with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and fluorescein diacetate/propyl iodide (FDA/PI). Most importantly, pre-incubated Si NWs largely prevented Aß1-42 oligomer-induced PC12 cell death, suggesting that Si NWs exerts an anti-Aß neuroprotective effect by inhibiting Aß aggregation. The analysis of Fourier Transform Infrared (FTIR) results demonstrates that Si NWs reduce the toxicity of fibrils and oligomers by intervening in the formation of ß-sheet structures, thereby protecting the viability of nerve cells. Our findings suggest that Si NWs may be a potential therapeutic agent for AD by protecting neuronal cells from the toxicity of Aß1-42.
Assuntos
Peptídeos beta-Amiloides , Nanofios , Fármacos Neuroprotetores , Silício , Animais , Ratos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/toxicidade , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/antagonistas & inibidores , Sobrevivência Celular/efeitos dos fármacos , Nanofios/química , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/química , Células PC12 , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/toxicidade , Fragmentos de Peptídeos/farmacologia , Agregados Proteicos/efeitos dos fármacos , Silício/químicaRESUMO
Endosomal trafficking of TrkA is a critical process for nerve growth factor (NGF)-dependent neuronal cell survival and differentiation. The small GTPase ADP-ribosylation factor 6 (Arf6) is implicated in NGF-dependent processes in PC12 cells through endosomal trafficking and actin cytoskeleton reorganization. However, the regulatory mechanism for Arf6 in NGF signaling is largely unknown. In this study, we demonstrated that EFA6A, an Arf6-specific guanine nucleotide exchange factor, was abundantly expressed in PC12 cells and that knockdown of EFA6A significantly inhibited NGF-dependent Arf6 activation, TrkA recycling from early endosomes to the cell surface, prolonged ERK1/2 phosphorylation, and neurite outgrowth. We also demonstrated that EFA6A forms a protein complex with TrkA through its N-terminal region, thereby enhancing its catalytic activity for Arf6. Similarly, we demonstrated that EFA6A forms a protein complex with TrkA in cultured dorsal root ganglion (DRG) neurons. Furthermore, cultured DRG neurons from EFA6A knockout mice exhibited disturbed NGF-dependent TrkA trafficking compared with wild-type neurons. These findings provide the first evidence for EFA6A as a key regulator of NGF-dependent TrkA trafficking and signaling.
Assuntos
Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP , Endossomos , Fatores de Troca do Nucleotídeo Guanina , Fator de Crescimento Neural , Crescimento Neuronal , Receptor trkA , Animais , Camundongos , Ratos , Fatores de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/genética , Endossomos/metabolismo , Gânglios Espinais/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Camundongos Knockout , Fator de Crescimento Neural/metabolismo , Células PC12 , Transporte Proteico , Receptor trkA/metabolismoRESUMO
We have previously shown that unconventional myosin VI (MVI), a unique actin-based motor protein, shuttles between the cytoplasm and nucleus in neurosecretory PC12 cells in a stimulation-dependent manner and interacts with numerous proteins involved in nuclear processes. Among the identified potential MVI partners was nucleolin, a major nucleolar protein implicated in rRNA processing and ribosome assembly. Several other nucleolar proteins such as fibrillarin, UBF (upstream binding factor), and B23 (also termed nucleophosmin) have been shown to interact with MVI. A bioinformatics tool predicted the presence of the nucleolar localization signal (NoLS) within the MVI globular tail domain, and immunostaining confirmed the presence of MVI within the nucleolus. Depletion of MVI, previously shown to impair PC12 cell proliferation and motility, caused disorganization of the nucleolus and rough endoplasmic reticulum (rER). However, lack of MVI does not affect nucleolar transcription. In light of these data, we propose that MVI is important for nucleolar and ribosome maintenance but not for RNA polymerase 1-related transcription.