RESUMEN
Orexins are neuronal peptides that play a prominent role in sleep behavior and feeding behavior in the central nervous system, though their receptors also exist in peripheral organs, including the adrenal gland. In this study, the effects of orexins on catecholamine synthesis in the rat adrenomedullary cell line PC12 were investigated by focusing on their interaction with the adrenomedullary bone morphogenetic protein (BMP)-4. Orexin A treatment reduced the mRNA levels of key enzymes for catecholamine synthesis, including tyrosine hydroxylase (Th), 3,4-dihydroxyphenylalanie decarboxylase (Ddc) and dopamine ß-hydroxylase (Dbh), in a concentration-dependent manner. On the other hand, treatment with BMP-4 suppressed the expression of Th and Ddc but enhanced that of Dbh with or without co-treatment with orexin A. Of note, orexin A augmented BMP-receptor signaling detected by the phosphorylation of Smad1/5/9 through the suppression of inhibitory Smad6/7 and the upregulation of BMP type-II receptor (BMPRII). Furthermore, treatment with BMP-4 upregulated the mRNA levels of OX1R in PC12 cells. Collectively, the results indicate that orexin and BMP-4 suppress adrenomedullary catecholamine synthesis by mutually upregulating the pathway of each other in adrenomedullary cells.
Asunto(s)
Proteínas Morfogenéticas Óseas , Catecolaminas , Orexinas , Animales , Ratas , Proteínas Morfogenéticas Óseas/metabolismo , Catecolaminas/metabolismo , Orexinas/farmacología , Orexinas/metabolismo , ARN Mensajero , Transducción de Señal , Tirosina 3-Monooxigenasa/genética , Tirosina 3-Monooxigenasa/metabolismo , Células PC12/metabolismoRESUMEN
Beauvericin (BEA), a naturally occurring cyclic peptide with good pharmacological activity, has been widely explored in anticancer research. Although BEA is toxic, studies have demonstrated its antioxidant activity. However, to date, the antioxidant mechanisms of BEA remain unclear. Herein, we conducted a comprehensive and detailed study of the antioxidant mechanism of BEA using an untargeted metabolomics approach, subsequently validating the results. BEA concentrations of 0.5 and 1 µM significantly inhibited H2O2-induced oxidative stress (OS), decreased reactive oxygen species levels in PC-12 cells, and restored the mitochondrial membrane potential. Untargeted metabolomics indicated that BEA was primarily involved in lipid-related metabolism, suggesting its role in resisting OS in PC-12 cells by participating in lipid metabolism. BEA combated OS damage by increasing phosphatidylcholine, phosphatidylethanolamine, and sphingolipid levels. In the current study, BEA upregulated proteins related to the PI3K/AKT/mTOR pathway, thereby promoting cell survival. These findings support the antioxidant activity of BEA at low concentrations, warranting further research into its pharmacological effects.
Asunto(s)
Antioxidantes , Apoptosis , Depsipéptidos , Metabolismo de los Lípidos , Antioxidantes/farmacología , Supervivencia Celular , Depsipéptidos/farmacología , Peróxido de Hidrógeno/toxicidad , Metabolismo de los Lípidos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Serina-Treonina Quinasas TOR , Células PC12/efectos de los fármacos , Células PC12/metabolismo , Animales , RatasRESUMEN
Autophagy is a major clearance pathway for misfolded α-synuclein which promotes ferroptosis through NCOA4-mediated ferritin degradation. The regulation of these two processes to achieve improved neuroprotection in Parkinson's disease (PD) must be elucidated. Transcription factor EB (TFEB) is a master regulator of both autophagy and lysosome biogenesis, and lysosomes are important cellular iron storage organelles; however, the role of TFEB in ferroptosis and iron metabolism remains unclear. In this study, TFEB overexpression promoted the clearance of misfolded α-synuclein and prevented ferroptosis and iron overload. TFEB overexpression up-regulated transferrin receptor 1 (TfR1) synthesis and increased the localization of TfR1 in the lysosome, facilitating lysosomal iron import and transient lysosomal iron storage. TFEB overexpression increased the levels of cellular iron-safe storage proteins (both ferritin light and heavy chains). These functions in iron metabolism maintain the cellular labile iron at a low level and electrical activity, even under iron overload conditions. Notably, lower levels of cellular labile iron and the upregulation of ferritin light and heavy chains were reversed after TfR1 knockdown in cells overexpressing TFEB, indicating that TFEB regulates cellular labile iron and suppresses ferroptosis in a TfR1 dependent manner. Taken together, this evidence of the regulation of iron metabolism enriches our understanding of the function of TFEB. In addition, TFEB overexpression protects against ferroptosis and iron overload and provides a new direction and perspective for autophagy regulation in PD.
Asunto(s)
Ferroptosis , Sobrecarga de Hierro , Enfermedad de Parkinson , alfa-Sinucleína/metabolismo , Autofagia/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Ferritinas/metabolismo , Ferroptosis/genética , Hierro/metabolismo , Sobrecarga de Hierro/metabolismo , Lisosomas/metabolismo , Enfermedad de Parkinson/metabolismo , Receptores de Transferrina/genética , Receptores de Transferrina/metabolismo , Animales , Ratones , Ratas , Células PC12/metabolismoRESUMEN
BACKGROUND: Alzheimer's disease (AD) is the most common type of dementia and has a complex pathogenesis with no effective treatment. Energy metabolism disorders, as an early pathological event of AD,have attracted attention as a promising area of AD research. Codonopsis pilosula Polysaccharides are the main effective components of Codonopsis pilosula, which have been demonstrated to regulate energy metabolism. METHODS: In order to further study the roles and mechanisms of Codonopsis pilosula polysaccharides in AD, this study used an Aß1-40-induced PC12 cells model to study the protective effects of Codonopsis pilosula polysaccharides and their potential mechanisms in improving energy metabolism dysfunction. RESULTS: The results showed that Aß1-40 induced a decrease in PC12 cells viability, energy metabolism molecules (ATP, NAD+, and NAD+/NADH) and Mitochondrial Membrane Potential (MMP) and an increase in ROS. Additionally, it was found that Aß1-40 increased CD38 expression related to NAD+ homeostasis, whereas Silent Information Regulation 2 homolog1 (SIRT1, SIRT3), Peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) and SIRT3 activity were decreased. Codonopsis pilosula polysaccharides increased NAD+, NAD+/NADH, SIRT3, SIRT1, and PGC-1α related to NAD+, thus partially recovering ATP. CONCLUSION: Our findings reveal that Codonopsis pilosula polysaccharides protected PC12 cells from Aß1-40-induced damage, suggesting that these components of the Codonopsis pilosula herb may represent an early treatment option for AD patients.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Codonopsis/metabolismo , NAD , Células PC12/metabolismo , Fragmentos de Péptidos/metabolismo , Polisacáridos/farmacología , Animales , Metabolismo Energético , Humanos , NAD/farmacología , Extractos Vegetales/farmacología , Ratas , Transducción de Señal/efectos de los fármacosRESUMEN
Neuritogenesis is the process underling nervous system regeneration; however, optimal extracellular signals that can promote neuronal regenerative activities require further investigation. Previously, we developed a novel method for inducing neuronal differentiation in rat PC12 cells using temperature-controlled repeated thermal stimulation (TRTS) with a heating plate. Based on neurogenic sensitivity to TRTS, PC12 cells were classified as either hyper- or hyposensitive. In this study, we aimed to investigate the mechanism of hyposensitivity by establishing two PC12-derived subclones according to TRTS sensitivity during differentiation: PC12-P1F1, a hypersensitive subclone, and PC12-P1D10, a hyposensitive subclone. To characterize these subclones, cell size and neuritogenesis were evaluated in subclones treated with nerve growth factor (NGF), bone morphogenetic protein (BMP), or various TRTS. No significant differences in cell size were observed among the parental cells and subclones. BMP4- or TRTS-induced neuritogenesis was increased in PC12-P1F1 cells compared to that in the parental cells, while no neuritogenesis was observed in PC12-P1D10 cells. In contrast, NGF-induced neuritogenesis was observed in all three cell lines. Furthermore, a BMP inhibitor, LDN-193189, considerably inhibited TRTS-induced neuritogenesis. These results suggest that the BMP pathway might be required for TRTS-induced neuritogenesis, demonstrating the useful aspects of these novel subclones for TRTS research.
Asunto(s)
Regeneración Nerviosa/fisiología , Células PC12/metabolismo , Sensación Térmica/fisiología , Animales , Diferenciación Celular/fisiología , Neuritas/metabolismo , Neurogénesis/fisiología , Neuronas/metabolismo , Células PC12/fisiología , Ratas , TemperaturaRESUMEN
Drug discovery from complex mixtures, like Chinese herbs, is challenging and extensive false positives make it difficult to obtain compounds with anti-Alzheimer's activity. In this study, a continuous method comprised of accelerated solvent extraction coupled with online two-dimensional countercurrent chromatography was developed for the efficient, scaled-up extraction and separation of six bioactive compounds from Citrus limon peels: neoeriocitrin, isonaringin, naringin, hesperidin, neohesperidin, and limonin. These active compounds were isolated and purified from the raw plant materials by two-dimensional countercurrent chromatography separation via two sets of an n-hexane/n-butanol/methanol/water solvent system: 0.23:1.00:0.25:1.13 and 0.47:1.00:0.38:1.46, v/v/v/v. The compounds were collected in yields of 0.22, 0.25, 0.10, 0.31, 0.29, and 0.28 mg/g, respectively, with purities of 95.79, 96.47, 97.69, 97.22, 98.11, and 98.82%, respectively. Subsequently, a simple and efficient in vitro method was developed for rapidly evaluating the acetylcholinesterase inhibitory activities of six bioactive components. Furthermore, the PC12 cell model and the in vitro metabolism of cytochromes P450 were employed to verify the monomers obtained from the continuous method. The results demonstrated that these six bioactive extracts from the C. limon peels were strong acetylcholinesterase inhibitors.
Asunto(s)
Citrus/química , Distribución en Contracorriente/métodos , Flavanonas/aislamiento & purificación , Extractos Vegetales/química , Animales , Inhibidores de la Colinesterasa/aislamiento & purificación , Inhibidores de la Colinesterasa/farmacología , Sistema Enzimático del Citocromo P-450/efectos de los fármacos , Sistema Enzimático del Citocromo P-450/metabolismo , Disacáridos/aislamiento & purificación , Disacáridos/farmacología , Flavanonas/farmacología , Flavonoides/aislamiento & purificación , Flavonoides/farmacología , Hesperidina/análogos & derivados , Hesperidina/aislamiento & purificación , Hesperidina/farmacología , Células PC12/efectos de los fármacos , Células PC12/metabolismo , Ratas , Solventes/químicaRESUMEN
Ferulic acid (FA) has been shown to have a neuroprotective effect on Alzheimer's disease induced by amyloid-beta (Aß) neurotoxicity. This work aims to ascertain the structure-activity relationship of FA and its alkyl esters (FAEs) for evaluating the antioxidant activities in PC12 cells and Aß1-42 aggregation inhibitory activities in vitro, as well as the signaling mechanisms against oxidative stress elicited by Aß1-42 in PC12 cells. Our data showed that alterations in the subcellular localization and cytotoxicity of FAEs caused by the lipophilicity of FA were crucial when evaluating their antioxidant capacities. Pre-treating cells with butyl ferulate (FAC4) significantly attenuated Aß1-42-evoked intracellular ROS formation. Besides, FAC4 exhibited the highest Aß1-42 aggregation inhibitory effectiveness. The molecular docking results showed that FAC4 binds to amide NH in Gln15 and Lys16 via a hydrogen bond. Notably, FAC4 could upregulate antioxidant defense systems by modulating the Keap1-Nrf2-ARE signaling pathway. Identification of the functions of FAEs could be useful in developing food supplements or drugs for treating AD.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/efectos de los fármacos , Ácidos Cumáricos/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Péptidos beta-Amiloides/metabolismo , Animales , Ácidos Cumáricos/administración & dosificación , Ácidos Cumáricos/farmacología , Humanos , Simulación del Acoplamiento Molecular , Factor 2 Relacionado con NF-E2/metabolismo , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/efectos de los fármacos , Células PC12/efectos de los fármacos , Células PC12/metabolismo , RatasRESUMEN
The PC12 cell line is one of the most commonly used in neuroscience research, including studies on neurotoxicity, neuroprotection, neurosecretion, neuroinflammation, and synaptogenesis. Two types of this line are available in the ATCC collection: traditional PC12 cells grown in suspension and well-attached adherent phenotype. PC12 cells grown in suspension tend to aggregate and adhere poorly to non-coated surfaces. Therefore, it is necessary to modify the surface of culture vessels. This paper aims to characterise the use of two distinct variants of PC12 cells as well as describe their differentiation and neuronal outgrowth with diverse NGF concentrations (rat or human origin) on various surfaces. In our study, we evaluated cell morphology, neurite length, density and outgrowth (measured spectrofluorimetrically), and expression of neuronal biomarkers (doublecortin and NeuN). We found that the collagen coating was the most versatile method of surface modification for both cell lines. For adherent cells, the coating was definitely less important, and the poly-d-lysine surface was as good as collagen. We also demonstrated that the concentration of NGF is of great importance for the degree of differentiation of cells. For suspension cells, we achieved the best neuronal characteristics (length and density of neurites) after 14 days of incubation with 100 ng/mL NGF (change every 48 h), while for adherent cells after 3-5 days, after which they began to proliferate. In the PC12 cell line, doublecortin (DCX) expression in the cytoplasm and NeuN in the cell nucleus were found. In turn, in the PC12 Adh line, DCX was not expressed, and NeuN expression was located in the entire cell (both in the nucleus and cytoplasm). Only the traditional PC12 line grown in suspension after differentiation with NGF should be used for neurobiological studies, especially until the role of the NeuN protein, whose expression has also been noted in the cytoplasm of adherent cells, is well understood.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Neuritas/metabolismo , Células PC12/metabolismo , Animales , Diferenciación Celular , Proteína Doblecortina , Humanos , RatasRESUMEN
Zinc, a suspected potentiator of learning and memory, is shown to affect exocytotic release and storage in neurotransmitter-containing vesicles. Structural and size analysis of the vesicular dense core and halo using transmission electron microscopy was combined with single-cell amperometry to study the vesicle size changes induced after zinc treatment and to compare these changes to theoretical predictions based on the concept of partial release as opposed to full quantal release. This powerful combined analytical approach establishes the existence of an unsuspected strong link between vesicle structure and exocytotic dynamics, which can be used to explain the mechanism of regulation of synaptic plasticity by Zn2+ through modulation of neurotransmitter release.
Asunto(s)
Neurotransmisores/genética , Células PC12/metabolismo , Transmisión Sináptica/genética , Zinc/química , Animales , Transporte Biológico , RatasRESUMEN
Neuronal cell death induced by amyloid-ß (Aß) oligomers is implicated in neuronal degeneration and is a leading cause of Alzheimer's disease (AD). Therefore, to identify effective therapeutic agents for AD, we investigated the neuroprotective effects of two naturally occurring retinoid X receptor (RXR) agonists (SPF1 and SPF2), isolated from the root of Sophora tonkinensis Gagnep., on the Aß25-35-induced cytotoxicity against nerve growth factor-differentiated rat pheochromocytoma (PC12) cells. Pretreatment with SPFs significantly prevented Aß25-35-induced apoptosis in PC12 cells, similarly to the synthetic RXR agonist bexarotene. These effects were blocked by the RXR antagonist PA452. When the effects of SPFs were studied in the presence of the liver X receptor (LXR) agonist T0901317, the protective effects of SPFs were enhanced, suggesting that RXR/LXR heterodimers may play a key role in the neuroprotective effects of SPFs. SPFs and T0901317 induced ATP-binding cassette transporter 1 (ABCA1) protein expression in PC12 cells when administered alone or in combination. Intriguingly, a functional inhibitor of ABCA1 cyclosporine A negated the neuroprotective effects of SPFs or T0901317. Taken together, these results demonstrate that the RXR agonists SPF1 and SPF2 protect PC12 cells from Aß25-35-induced neurotoxicity in an RXR-dependent manner and that their effects are markedly enhanced by the LXR agonist T0901317, in part related to ABCA1 function. These results suggest a novel approach to the treatment or prevention of AD.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/efectos adversos , Fármacos Neuroprotectores/uso terapéutico , Células PC12/metabolismo , Fragmentos de Péptidos/efectos adversos , Receptores X Retinoide/uso terapéutico , Sophora/química , Enfermedad de Alzheimer/patología , Animales , Humanos , Fármacos Neuroprotectores/farmacología , Ratas , Receptores X Retinoide/agonistas , Receptores X Retinoide/farmacologíaRESUMEN
Diffusible amyloid-ß (Aß) oligomers are currently presumed to be the most cytotoxic Aß assembly and held responsible to trigger the pathogenesis of Alzheimer's disease (AD). Thus, Aß oligomers are a prominent target in AD drug development. Previously, we reported on our solely D-enantiomeric peptide D3 and its derivatives as AD drug candidates. Here, we compare one of the most promising D3 derivatives, ANK6, with its tandem version (tANK6), and its head-to-tail cyclized isoform (cANK6r). In vitro tests investigating the D-peptides' potencies to inhibit Aß aggregation, eliminate Aß oligomers, and reduce Aß-induced cytotoxicity revealed that all three D-peptides efficiently target Aß. Subsequent preclinical pharmacokinetic studies of the three all-D-peptides in wildtype mice showed promising blood-brain barrier permeability with cANK6r yielding the highest levels in brain. The peptides' potencies to lower Aß toxicity and their remarkable brain/plasma ratios make them promising AD drug candidates.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Oligopéptidos/farmacocinética , Oligopéptidos/uso terapéutico , Fragmentos de Péptidos/metabolismo , Agregado de Proteínas/efectos de los fármacos , Péptidos beta-Amiloides/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Humanos , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Oligopéptidos/líquido cefalorraquídeo , Oligopéptidos/química , Células PC12/efectos de los fármacos , Células PC12/metabolismo , Fragmentos de Péptidos/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Isoformas de Proteínas/farmacocinética , Ratas , Estereoisomerismo , Distribución Tisular/efectos de los fármacos , Tritio/líquido cefalorraquídeo , Tritio/farmacocinéticaRESUMEN
6'-O-galloylpaeoniflorin (GPF), a galloylated derivative of paeoniflorin isolated from peony root, has been proven to possess antioxidant potential. In this present study, we revealed that GPF treatment exerted significant neuroprotection of PC12 cells following OGD, as evidenced by a reduction of oxidative stress, inflammatory response, cellular injury, and apoptosis in vitro. Furthermore, treatment with GPF increased the levels of phosphorylated Akt (p-Akt) and nuclear factor-erythroid 2-related factor 2 (Nrf2), as well as promoted Nrf2 translocation in PC12 cells, which could be inhibited by Ly294002, an inhibitor of phosphoinositide 3-kinase (PI3K). In addition, Nrf2 knockdown or Ly294002 treatment significantly attenuated the antioxidant, anti-inflammatory, and antiapoptotic activities of GPF in vitro. In vivo studies indicated that GPF treatment significantly reduced infarct volume and improved neurological deficits in rats subjected to CIRI, as well as decreased oxidative stress, inflammation, and apoptosis, which could be inhibited by administration of Ly294002. In conclusion, these results revealed that GPF possesses neuroprotective effects against oxidative stress, inflammation, and apoptosis after ischemia-reperfusion insult via activation of the PI3K/Akt/Nrf2 pathway.
Asunto(s)
Isquemia Encefálica/tratamiento farmacológico , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Glucósidos/uso terapéutico , Monoterpenos/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Células PC12/metabolismo , Daño por Reperfusión/tratamiento farmacológico , Animales , Isquemia Encefálica/patología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular Tumoral , Glucósidos/farmacología , Masculino , Monoterpenos/farmacología , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Ratas , Ratas WistarRESUMEN
Amphetamine (AMPH) abuse can influence neuropsychiatric disorders and cell apoptosis by interfering with the protein kinase B/ glycogen synthase kinase 3 beta (AKT/GSK3ß) pathway. However, the mechanisms underlying this regulation are poorly understood. Using PC12 cells, we found that AMPH inhibited AKT and GSK-3ß phosphorylation levels and increased total GSK-3ß levels. Furthermore, AMPH caused an increase in the activity of protein phosphatase 2 (PP2A), a signaling protein upstream of AKT, which in turn inhibited phosphorylated AKT levels. Okadaic acid, a PP2A inhibitor, protected PC12 cells against AMPH-induced apoptosis. Together, our results suggest that the PP2A/AKT/GSK3ß pathway plays an important role in AMPH-induced neurotoxicity.
Asunto(s)
Anfetamina/farmacología , Estimulantes del Sistema Nervioso Central/farmacología , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Proteína Oncogénica v-akt/metabolismo , Proteína Fosfatasa 2/metabolismo , Transducción de Señal/efectos de los fármacos , Anfetamina/toxicidad , Animales , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Glucógeno Sintasa Quinasa 3 beta/genética , Ácido Ocadaico/farmacología , Proteína Oncogénica v-akt/genética , Células PC12/efectos de los fármacos , Células PC12/metabolismo , Proteína Fosfatasa 2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , RatasRESUMEN
This study aimed to explore the efficacy of propofol to treat malignant pheochromocytoma (PCC) in vitro and in vivo. In vitro, PC12 cells were treated with different concentrations of propofol (0, 1, 5, and 10 µg/mL) for specific times followed by a MTT assay to detect cell proliferation. Transwell assays were performed to assess the function of propofol on the migration and invasion of PC12 cells, and flow cytometry to analyze cell apoptosis and cell cycle progression. Quantitative real-time polymerase chain reaction was carried out to analyze the expression level of mRNA (Bcl-2, Bax, and CyclinE). The levels of Bcl-2, Bax, CyclinE, FOXO1, FOXO3, Bim, procaspase-3, and active caspase-3 were determined by western blotting. In vivo, the effects of propofol on PCC tumor growth were detected by transplanted mouse model. Transferase dUTP nick-end labeling was performed to detect tissue cell apoptosis. The results indicated that propofol inhibited PC12 cell proliferation, prevented cell migration and invasion, and induced the apoptosis of PC12 cells in a dose- and time-dependent manner. Propofol treatment increased the expression of Bax and decreased that of Bcl-2. In addition, propofol significantly induced the G1/S phase arrest in PC12 cells, and the expression of Cyclin E was reduced. Moreover, the levels of FOXO1, FOXO3, Bim, procaspase-3, and active caspase-3 were enhanced by propofol treatment. In vivo, propofol treatment significantly reduced the PCC tumor growth and induced tissue cell apoptosis. In conclusion, propofol has potent anti-PCC activity in vitro and in vivo, and is a potential small-molecule drug for treating malignant PCC.
Asunto(s)
Feocromocitoma/tratamiento farmacológico , Propofol/farmacología , Propofol/uso terapéutico , Neoplasias de las Glándulas Suprarrenales/tratamiento farmacológico , Neoplasias de las Glándulas Suprarrenales/metabolismo , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Ratones , Ratones Desnudos , Células PC12/efectos de los fármacos , Células PC12/metabolismo , Feocromocitoma/metabolismo , Propofol/metabolismo , Ratas , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Levodopa (L-dopa) remains the best treatment for Parkinson's disease (PD). However, long-term L-dopa treatment induces dyskinesia. The mechanism of L-dopa-induced dyskinesia (LID) is not fully understood. Enhanced activity of protein kinase A (PKA) and pulsatile dopamine (DA) stimulation plays an important role in LID. Tyrosine hydroxylase (TH) is the rate-limiting enzyme for DA synthesis. Decreased TH activity causes reduced pulsatile DA stimulation, which in turn reduces LID. Moreover, TH is a substrate of CaMKII. However, it is unknown whether inhibition of CaMKII reduces LID by downregulating the activity of TH. In this study, we found that CaMKII antagonist KN-93 reduced DA released in PC12 cells; in the meantime, KN-93 reduced phosphorylated levels of CaMKIIα and TH at Ser 40. Intrastriatal administration of KN-93 reduced LID without affecting the antiparkinsonian effect of L-dopa in PD mice. Mechanistically, KN-93 treatmentreduced phosphorylated CaMKIIα levels and subsequently downregulated phosphorylated TH at Ser 40 expression. Consequently, extracellular DA efflux was reduced andthe activation threshold of the PKA pathway was lowered. Moreover, KN-93 treatment reduced the expression of Arc and Penk, two immediate early genes, induced by chronic L-dopa. These data indicate that inhibition of CaMKIIα decreases LID at least partially by suppressing TH activity and subsequently reducing extracellular DA efflux and the activity of the PKA pathway, suggesting that CaMKIIα may be an alternative target for the treatment of LID.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Discinesia Inducida por Medicamentos/metabolismo , Enfermedad de Parkinson Secundaria/tratamiento farmacológico , Tirosina 3-Monooxigenasa/metabolismo , Animales , Antiparkinsonianos , Bencilaminas/uso terapéutico , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Modelos Animales de Enfermedad , Dopamina/farmacología , Discinesia Inducida por Medicamentos/tratamiento farmacológico , Discinesia Inducida por Medicamentos/etiología , Levodopa/efectos adversos , Masculino , Ratones Endogámicos C57BL , Oxidopamina/toxicidad , Células PC12/efectos de los fármacos , Células PC12/metabolismo , Enfermedad de Parkinson , Enfermedad de Parkinson Secundaria/inducido químicamente , Inhibidores de Proteínas Quinasas/uso terapéutico , Ratas , Transducción de Señal/efectos de los fármacos , Sulfonamidas/uso terapéutico , Simpaticolíticos/toxicidadRESUMEN
Neurodegenerative diseases (NDD) are typically associated with neuron loss in nervous system areas. Interventions with related death mechanisms may ameliorate NDD progression. Oxidative stress plays an important role in NDD cell death routines. However, tert-butylhydroperoxide (t-BHP), a widely used oxidative stress stimulus, induces neural cell death through a mechanism that remains elusive. In our study, the ferroptosis marker events occurred after co-treatment with 100 µM t-BHP for 1 h, all of which were reversed in the presence of the ferroptosis inhibitor ferrostatin-1 (Fer-1) and the iron chelator deferoxamine, implying the occurrence of ferroptosis. Moreover, mitochondrial dysfunction accompanied by a decreased in membrane potential and ATP production, increased mitochondrial ROS generation. Furthermore, this mitochondrial dysfunction could be reversed by Fer-1. In addition, JNK1/2 and ERK1/2 were activated upstream of the ferroptosis and mitochondrial dysfunction. In summary, these data suggest that ferroptosis, coupled with mitochondrial dysfunction, was involved in t-BHP-induced PC12 death. JNK1/2 and ERK1/2 played important roles in t-BHP-induced cell death. Overall, this study might provide clues to the oxidative stress-based strategies for cell protection in NDD.
Asunto(s)
Adenosina Trifosfato/metabolismo , Mitocondrias/efectos de los fármacos , Células PC12/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , terc-Butilhidroperóxido/farmacología , Animales , Muerte Celular , Supervivencia Celular/efectos de los fármacos , Ciclohexilaminas/farmacología , Deferoxamina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/metabolismo , Estrés Oxidativo , Células PC12/citología , Células PC12/metabolismo , Fenilendiaminas/farmacología , RatasRESUMEN
Huntington's disease (HD) is a monogenic inherited polyglutamine-mediated neurodegenerative disorder for which effective therapies are currently unavailable. Neuropeptide Y (NPY) has been implicated as a potential therapeutic target in several neurodegenerative diseases, including HD. However, its mechanisms of action in the context of HD pathology remain unknown. Here, we investigated the beneficial effects of Y2 receptor (Y2R) activation with NPY or Y2R selective agonist NPY13-36 in the R6/2 mouse and PC12 cell models of HD. Also, we explored the effects of selective pharmacological blockage of Y2R using selective non-peptide small molecule Y2R antagonist SF31 in vivo and in vitro. Our results showed that activation of Y2R with intranasal NPY or NPY13-36 led to an improved motor function in R6/2 mice as revealed by rotarod performance, vertical pole test, and hindlimb clasping behaviour. Also, intranasal NPY or NPY13-36 led to a decrease in aggregated mHtt and mediated increase in dopamine and cAMP-regulated phosphoprotein, 32kDa (DARPP-32), brain-derived neurotrophic factor (BDNF), and activated extracellular signal-regulated protein kinases (pERK1/2) levels in R6/2 mice. Intranasal NPY or NPY13-36 had no effect on body weight but showed positive effects on survival in R6/2 mice. Furthermore, intranasal NPY or NPY13-36 attenuated induction of proinflammatory cytokine and inflammatory mediators in R6/2 mice. In contrast, antagonizing by using SF31 exacerbates phenotypic severity in R6/2 mice and treatment effects with either intranasal NPY or NPY13-36 were significantly blocked.In vitro, using inducible PC12/HttQ103-EGFP cells, treatment with NPY or NPY13-36 protected against mHtt-mediated neuromorphological defects (neurite length and soma area) and neurotoxicity but had no effect on mHtt inclusion body formation. Conversely, co-treatment with SF31 significantly inhibited these effects. Together, our findings extend previous evidence of the beneficial effects of NPY in R6/2 mice, and more importantly, suggest that targeted activation of Y2R receptor might be a promising disease-modifying target for HD and other neurodegenerative diseases.
Asunto(s)
Encéfalo/patología , Encefalitis/etiología , Regulación de la Expresión Génica/genética , Enfermedad de Huntington/complicaciones , Receptores de Neuropéptido Y/metabolismo , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Fosfoproteína 32 Regulada por Dopamina y AMPc/metabolismo , Encefalitis/tratamiento farmacológico , Encefalitis/genética , Inhibidores Enzimáticos/farmacología , Fluoresceínas/farmacocinética , Regulación de la Expresión Génica/efectos de los fármacos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/tratamiento farmacológico , Enfermedad de Huntington/genética , Enfermedad de Huntington/mortalidad , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Ratones , Ratones Transgénicos , Actividad Motora/efectos de los fármacos , Actividad Motora/genética , Fuerza Muscular/efectos de los fármacos , Fuerza Muscular/genética , Neuropéptido Y/uso terapéutico , Células PC12/efectos de los fármacos , Células PC12/metabolismo , Fragmentos de Péptidos/uso terapéutico , Trastornos Psicomotores/tratamiento farmacológico , Trastornos Psicomotores/etiología , Ratas , Receptores de Neuropéptido Y/genética , Repeticiones de Trinucleótidos/genéticaRESUMEN
Hydroxylated polychlorobiphenyls (OH-PCBs) are major metabolites of PCBs that are widely distributed in the environment. While the effects of penta- to hepta-chlorinated OH-PCBs on neuronal differentiation have been widely reported, those of lower chlorinated OH-PCBs have not been extensively studied. To investigate the effects of lower chlorinated OH-PCBs on neuronal development, we studied the effects of mono- to hexa-chlorinated OH-PCBs on PC12 cells. Morphological changes were examined using an automatic system IN Cell Analyzer. Seventeen of the 20 OH-PCBs investigated promoted neuronal elongation in an OH-PCB concentration-dependent manner, while three OH-PCB congeners suppressed neuronal elongation based on Dunnett's analysis. In particular, the top five OH-PCBs (4OH-PCB2, 4'OH-PCB3, 4'OH-PCB25, 4'OH-PCB68, and 4'OH-PCB159), which have hydroxyl groups at the para-position and chlorine substitutions at the 2, 4, or 3' positions, significantly promoted neuronal elongation. Moreover, these neuronal elongations were suppressed by U0126, and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 was observed in PC12 cells treated with 4OH-PCB2, 4'OH-PCB25, and 4'OH-PCB159. Taken together, our results indicate that the effect of OH-PCB on neuronal development is not dependent on the number of chlorine groups but on the chemical structure, and the mitogen-activated kinase kinase (MEK)-ERK1/2 signaling pathway is involved in this process.
Asunto(s)
Butadienos/química , Nitrilos/química , Células PC12/química , Bifenilos Policlorados/química , Transducción de Señal/efectos de los fármacos , Animales , Butadienos/metabolismo , Contaminantes Ambientales/farmacología , Halogenación , Hidroxilación , Nitrilos/metabolismo , Células PC12/metabolismo , RatasRESUMEN
Objective: To investigate the regulation of AMPK-mTOR signal transduction pathway in paraquat-induced autophagy of pheochromocytoma cells (PC12) . Methods: The PC12 cell were treated with terminal concentrations of 0, 25, 50, 100, 200, 300 and 400 µmol/L PQ for 24 hours, and the cells were induced by 300 µmol/L PQ for different time (6, 12, 24, 48 h) . MTT was used to detect the relative survival rate of cells, and the dose/time-effect relationship was determined respectively. The cells were treated with PQ at concentrations of 0, 100, 200 and 300 µmol/L PQ for 24 hours, the lactate dehydrogenase (LDH) activity in the culture supernatant was detected by spectrophotometry. The expression and distribution of autophagic lysosomes were observed by MDC staining. The intracellular reactive oxygen species (ROS) was detected by dichlorofluorescein diacetate (DCFH-DA) . The expression of microtubule-related protein 1 light chain 3 (LC3) was measured by immunofluorescence. The protein level of LC3â ¡, p62, Beclin1 and p-AMPK, p-mTOR were detected by Western blot. Results: Compared with the control group, the cell survival rate of the 100, 200, 300, 400 µmol/L PQ group decreased significantly, and showed a dose-dependent pattern (P<0.05) . The survival rate of cells treated with 300 µmol/L PQ decreased significantly with the prolongation of exposure time (12, 24, 48 h) (P<0.05) . Compared with the control group, the activity of LDH in 100, 200, 300 µmol/L PQ-treated group were significantly higher while The fluorescence intensity of ROS was significantly increased (P<0.05) . MDC staining showed the density of autophagic lysosomes and fluorescence intensity in PQ-treated group significantly decreased (P<0.05) . Immunofluorescence results showed the LC3 fluorescence intensity of PQ-treated group decreased which was consistent with MDC staining results. Western blot showed that compared with the control group, the expression levels of autophagy related proteins LC3â ¡and Beclin1 in PQ-treated group were significantly lower, while the expression level of p62 protein was higher (P<0.05) . p-AMPK protein level decreased and p-mTOR protein expression increased in 200 and 300µmol/L PQ-treatd groups, with statistically significant difference (P<0.05) . Conclusion: AMPK-mTOR signaling pathway played a regulatory role in PQ-induced decreased autophagy of PC12 cell.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/metabolismo , Animales , Autofagia/efectos de los fármacos , Células PC12/metabolismo , Paraquat/toxicidad , RatasRESUMEN
Decursin, the major bioactive component of Angelica gigas Nakai, exhibited neuroprotective properties. Our previous studies showed that decursin conferred neuroprotective effects in PC12 cells induced by Amyloid-ß (Aß)25-35 via antiapoptosis and antioxidant. In this study, the antiinflammatory effects of decursin against PC12 cells injury stimulated by Aß25-35 were assessed. Our results demonstrated that decursin suppressed the expression of cyclooxygenase-2 protein and prostaglandin E2 content which was stimulated by Aß25-35 in PC12 cells. Meanwhile, the nuclear translocation of nuclear factor-κB in Aß25-35 -treated PC12 cells was also inhibited by decursin. In addition, decursin suppressed phosphorylation of the two upstream pathway kinases, p38 and c-Jun N-terminal kinase. Overall, our findings indicate that decursin exerts protective effects against neuroinflammation stimulated by Aß25-35 in PC12 cells by abolishing cyclooxygenase-2 protein expression through inactivation of nuclear factor-κB via the upstream kinases including p38 and c-Jun N-terminal kinase. This work provides a new insight into the pharmacological mode of decursin and should facilitate its therapeutic application in treatment of inflammatory disorders.