RESUMEN
Neurodegenerative diseases are characterized by the progressive loss of selectively vulnerable populations of neurons, and many factors are involved in its causes. Neurotoxicity and oxidative stress, are the main related factors. The octapeptide Ile-Ile-Ala-Val-Glu-Ala-Gly-Cys (IEC) was identified from the microalgae Isochrysis zhanjiangensis and exhibited potential anti-oxidative stress activity. In this study, the stability of α-synaptic protein binding to IEC was modeled using molecular dynamics, and the results indicated binding stabilization within 60â ns. Oxidative stress in neurons is the major cause of α-synaptic protein congestion. Therefore, we next evaluated the protective effects of IEC against oxidative stress and neurotoxicity in 6-ohdainduced Parkinson's disease (PD) model SH-SY5Y cells inâ vitro. In oxidative stress, IEC appeared to increase the expression of the antioxidant enzymes HO-1 and GPX through the antioxidant pathway of Nrf2, and molecular docking of IEC with Nrf2 and GPX could generate hydrogen bonds. Regarding apoptosis, IEC protected cells by increasing the Bcl-2/Bax ratio, inhibiting the caspase cascade, acting on p53, and modulating the Jak2/Stat3 pathway. The results indicated that IEC exerted neuroprotective effects through the inhibition of α-synaptic protein aggregation and antioxidant activity. Therefore, microalgal peptides have promising applications in the prevention and treatment of neurodegenerative diseases.
Asunto(s)
Janus Quinasa 2 , Microalgas , Factor 2 Relacionado con NF-E2 , Estrés Oxidativo , Oxidopamina , Factor de Transcripción STAT3 , Estrés Oxidativo/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , Humanos , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/antagonistas & inhibidores , Janus Quinasa 2/metabolismo , Janus Quinasa 2/antagonistas & inhibidores , Microalgas/química , Microalgas/metabolismo , Oxidopamina/farmacología , Oxidopamina/antagonistas & inhibidores , Hemo-Oxigenasa 1/metabolismo , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/aislamiento & purificación , Oligopéptidos/farmacología , Oligopéptidos/química , Transducción de Señal/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Apoptosis/efectos de los fármacosRESUMEN
Since there is no disease-modifying treatment discovered yet for Parkinson's disease (PD), there is still a vital need to develop novel selective monoamine oxidase B (MAO-B) inhibitors as promising therapeutically active candidates for PD patients. Herein, we report the design, synthesis, and full characterization of new twenty-six indole derivatives as potential human MAO-B (hMAO-B) selective inhibitors. Six compounds (2i, 3b-e, and 5) exhibited low micromolar to nanomolar inhibitory activities over hMAO-B; compared to our recently reported N-substituted indole-based lead compound VIII (hMAO-B IC50 = 777 nM), compound 5 (3,4-dichloro-N-(1H-indol-5-yl)benzamide) exhibited 18-fold increase in potency (IC50 = 42 nM). A selectivity study over hMAO-A revealed an excellent selectivity index of compound 5 (SI > 2375) with a 47-fold increase compared to rasagiline (II, a well-known MAO-B inhibitor, SI > 50). A further kinetic evaluation of compound 5 over hMAO-B showed a reversible and competitive mode of inhibition with Ki value of 7 nM. Highly effective permeability and high CNS bioavailability of compound 5 with Pe = 54.49 × 10-6 cm/s were demonstrated. Compound 5 also exhibited a low cytotoxicity profile and a promising neuroprotective effect against the 6-hydroxydopamine-induced neuronal cell damage in PC12 cells, which was more effective than that of rasagiline. Docking simulations on both hMAO-B and hMAO-A supported the in vitro data and served as further molecular evidence. Accordingly, we report the discovery of compound 5 as one of the most potent indole-based MAO-B inhibitors to date which is noteworthy to be further evaluated as a promising agent for PD treatment.
Asunto(s)
Descubrimiento de Drogas , Inhibidores de la Monoaminooxidasa/farmacología , Monoaminooxidasa/metabolismo , Fármacos Neuroprotectores/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Inhibidores de la Monoaminooxidasa/síntesis química , Inhibidores de la Monoaminooxidasa/química , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/química , Oxidopamina/antagonistas & inhibidores , Oxidopamina/farmacología , Células PC12 , Ratas , Relación Estructura-ActividadRESUMEN
Accumulating evidence has demonstrated that cellular antioxidant systems play essential roles in retarding oxidative stress-related diseases, such as Parkinson's disease. Because nuclear factor erythroid 2-related factor 2 (Nrf2) is a chief regulator of cellular antioxidant systems, small molecules with Nrf2-activating ability may be promising neuroprotective agents. Avenanthramide-2c (Aven-2c), avenanthramide-2f (Aven-2f) and avenanthramide-2p (Aven-2p) are the most abundant avenanthramides in oats, and they have been documented to possess multiple pharmacological benefits. In this work, we synthesized these three compounds and evaluated their cytoprotective effect against oxidative stress-induced PC12 cell injuries. Aven-2c displayed the best protective potency among them. Aven-2c conferred protection on PC12 cells by scavenging free radicals and activating the Nrf2-ARE signaling pathway. Pretreatment of PC12 cells with Aven-2c efficiently enhanced Nrf2 nuclear accumulation and evoked the expression of a set of cytoprotective molecules. The mechanistic study also supports that Nrf2 activation is the molecular basis for the cellular action of Aven-2c. Collectively, this study demonstrates that Aven-2c is a potent Nrf2 agonist, shedding light on the potential usage of Aven-2c in the treatment of neuroprotective diseases.
Asunto(s)
Elementos de Respuesta Antioxidante , Antioxidantes/farmacología , Factor 2 Relacionado con NF-E2/genética , Fármacos Neuroprotectores/farmacología , Transducción de Señal/genética , ortoaminobenzoatos/farmacología , Animales , Avena/química , Supervivencia Celular/efectos de los fármacos , Regulación de la Expresión Génica , Glutamato-Cisteína Ligasa/genética , Glutamato-Cisteína Ligasa/metabolismo , Hemo Oxigenasa (Desciclizante)/genética , Hemo Oxigenasa (Desciclizante)/metabolismo , Peróxido de Hidrógeno/antagonistas & inhibidores , Peróxido de Hidrógeno/farmacología , NAD(P)H Deshidrogenasa (Quinona)/genética , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/efectos de los fármacos , Oxidopamina/antagonistas & inhibidores , Oxidopamina/farmacología , Células PC12 , Extractos Vegetales/química , Ratas , Tiorredoxina Reductasa 1/genética , Tiorredoxina Reductasa 1/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMEN
Parkinson's disease (PD) is one of the most common central nervous system (CNS) degenerative disease and is characterized by a progressive loss of midbrain substantia nigra dopamine (DA) neurons. Dendrobium nobileLindl alkaloid (DNLA) is an active component extracted from D. nobile Lindl, which is a traditional Chinese herb. The various pharmacological effects of D. nobile are beneficial for human health. Recently, DNLA-mediated neuroprotective effects have been reported. However, the neuroprotection of DNLA on 6-hydroxydopamine (6-OHDA)-induced DA neurotoxicity is still unknown. This study aimed to explore the neuroprotective effects of DNLA on DA neurotoxicity induced by 6-OHDA. In PD rat model, continuous intragastric administration of DNLA (20 mg/kg) for 7 days significantly ameliorated 6-OHDA-induced DA neurons loss in the midbrain substantia nigra. In addition, primary rat midbrain neuron-glia cocultures were used to explore the mechanisms underlying DNLA-related DA neuroprotection. The studies on neuron-glia cocultures revealed that neuroprotective effects of DNLA (2.5 ng/mL) were mediated by inhibiting the release of proinflammatory cytokines. Taken together, DNLA holds neuroprotective effect on 6-OHDA-induced neurons neurodegeneration by selectively inhibiting the production of proinflammatory factors and could be a potential compound for PD treatment.
Asunto(s)
Alcaloides/farmacología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Oxidopamina/antagonistas & inhibidores , Alcaloides/administración & dosificación , Animales , Dopamina/toxicidad , Masculino , Neuronas/patología , Fármacos Neuroprotectores/administración & dosificación , Oxidopamina/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
Daidzein, one of the important isoflavones, is extensively metabolized in the human body following consumption. In particular, 6,7,4'-trihydroxyisoflavone (THIF), a major metabolite of daidzein, has been the focus of recent investigations due to its various health benefits, such as anti-cancer and anti-obesity effects. However, the protective effects of 6,7,4'-THIF have not yet been studied in models of Parkinson's disease (PD). Therefore, the present study aimed to investigate the protective activity of 6,7,4'-THIF on 6-hydroxydopamine (OHDA)-induced neurotoxicity in SH-SY5Y human neuroblastoma cells. Pretreatment of SH-SY5Y cells with 6,7,4'-THIF significantly inhibited 6-OHDA-induced neuronal cell death, lactate dehydrogenase release, and reactive oxygen species production. In addition, 6,7,4'-THIF significantly attenuated reductions in 6-OHDA-induced superoxide dismutase activity and glutathione content. Moreover, 6,7,4'-THIF attenuated alterations in Bax and Bcl-2 expression and caspase-3 activity in 6-OHDA-induced SH-SY5Y cells. Furthermore, 6,7,4'-THIF significantly reduced 6-OHDA-induced phosphorylation of c-Jun N-terminal kinase, p38 mitogen-activated protein kinase, and extracellular signal-regulated kinase 1/2. Additionally, 6,7,4'-THIF effectively prevented 6-OHDA-induced loss of tyrosine hydroxylase. Taken together, these results suggest that 6,7,4'-THIF, a major metabolite of daidzein, may be an attractive option for treating and/or preventing neurodegenerative disorders such as PD.
Asunto(s)
Antineoplásicos/farmacología , Isoflavonas/metabolismo , Isoflavonas/farmacología , Neuroblastoma/tratamiento farmacológico , Neuronas/efectos de los fármacos , Oxidopamina/antagonistas & inhibidores , Antineoplásicos/química , Antineoplásicos/metabolismo , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Isoflavonas/química , Estructura Molecular , Neuroblastoma/metabolismo , Neuroblastoma/patología , Neuronas/metabolismo , Neuronas/patología , Estrés Oxidativo/efectos de los fármacos , Oxidopamina/farmacología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Enfermedad de Parkinson/prevención & control , Células Tumorales CultivadasRESUMEN
Alzheimer's disease is an irreversible, complex and progressive neurodegenerative disorder associated with oxidative stress and mitochondrial dysfunction. Exogenous antioxidants can be beneficial for decreasing oxidative stress, as they are able to reward the lack of efficacy of the endogenous defense systems and raise the overall antioxidant response in a pathological condition. Along our overarching project related with the design and development of potent and safe multi-target mitochondriotropic antioxidants, based on dietary antioxidants, novel derivatives were obtained. Overall, mitochondriotropic antioxidants showed remarkable antioxidant and chelating properties, presenting low cytotoxic effects on human differentiated neuronal (SH-SY5Y) and hepatocarcinoma (HepG2) cells and exhibited neuroprotective properties on SH-SY5Y cells against 6-hydroxydopamine (6-OHDA) or hydrogen peroxide (H2O2) oxidative insults. Moreover, compounds 58, 59, 62, 63, 66 and 67 were able to permeate a layer of hCMEC/D3 cells in a time-dependent manner. Mitochondriotropic antioxidant 67 stands out by its remarkable iron chelating and neuroprotective properties toward both H2O2 and 6-OHDA-induced oxidative damage, drug-like properties and BBB permeability.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/fisiopatología , Antioxidantes/síntesis química , Enfermedades Mitocondriales/prevención & control , Enfermedad de Alzheimer/patología , Antioxidantes/farmacología , Barrera Hematoencefálica/metabolismo , Línea Celular , Línea Celular Tumoral , Humanos , Peróxido de Hidrógeno/antagonistas & inhibidores , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/efectos de los fármacos , Oxidopamina/antagonistas & inhibidoresRESUMEN
Palythoa caribaeorum (class Anthozoa) is a zoanthid that together jellyfishes, hydra, and sea anemones, which are venomous and predatory, belongs to the Phyllum Cnidaria. The distinguished feature in these marine animals is the cnidocytes in the body tissues, responsible for toxin production and injection that are used majorly for prey capture and defense. With exception for other anthozoans, the toxin cocktails of zoanthids have been scarcely studied and are poorly known. Here, on the basis of the analysis of P. caribaeorum transcriptome, numerous predicted venom-featured polypeptides were identified including allergens, neurotoxins, membrane-active, and Kunitz-like peptides (PcKuz). The three predicted PcKuz isotoxins (1-3) were selected for functional studies. Through computational processing comprising structural phylogenetic analysis, molecular docking, and dynamics simulation, PcKuz3 was shown to be a potential voltage gated potassium-channel inhibitor. PcKuz3 fitted well as new functional Kunitz-type toxins with strong antilocomotor activity as in vivo assessed in zebrafish larvae, with weak inhibitory effect toward proteases, as evaluated in vitro. Notably, PcKuz3 can suppress, at low concentration, the 6-OHDA-induced neurotoxicity on the locomotive behavior of zebrafish, which indicated PcKuz3 may have a neuroprotective effect. Taken together, PcKuz3 figures as a novel neurotoxin structure, which differs from known homologous peptides expressed in sea anemone. Moreover, the novel PcKuz3 provides an insightful hint for biodrug development for prospective neurodegenerative disease treatment.
Asunto(s)
Antozoos/química , Venenos de Cnidarios/aislamiento & purificación , Neurotoxinas/aislamiento & purificación , Péptidos/aislamiento & purificación , Bloqueadores de los Canales de Potasio/aislamiento & purificación , Transcriptoma , Alérgenos/química , Alérgenos/aislamiento & purificación , Animales , Antozoos/patogenicidad , Antozoos/fisiología , Sitios de Unión , Venenos de Cnidarios/química , Venenos de Cnidarios/toxicidad , Secuenciación de Nucleótidos de Alto Rendimiento , Larva/efectos de los fármacos , Larva/fisiología , Locomoción/efectos de los fármacos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Neurotoxinas/química , Neurotoxinas/toxicidad , Oxidopamina/antagonistas & inhibidores , Oxidopamina/farmacología , Péptidos/química , Péptidos/toxicidad , Bloqueadores de los Canales de Potasio/química , Bloqueadores de los Canales de Potasio/toxicidad , Canales de Potasio con Entrada de Voltaje/antagonistas & inhibidores , Canales de Potasio con Entrada de Voltaje/química , Canales de Potasio con Entrada de Voltaje/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Pez CebraRESUMEN
Acacetin (5,7-dihydroxy-4'-methoxyflavone), a flavonoid compound isolated from Flos Chrysanthemi Indici, chrysanthemum, safflower, and Calamintha and Linaria species has been shown to have anti-cancer activity, indicating its potential clinical value in cancer treatment. In this study, we sought to study the potentials of acacetin in preventing human dopaminergic neuronal death via inhibition of 6-hydroxydopamine (6-OHDA)-induced neuronal cell death in the SH-SY5Y cells. Our results suggest that acacetin was effective in preventing 6-OHDA-induced neuronal cell death through regulation of mitochondrial-mediated cascade apoptotic cell death. Pretreatment with acacetin significantly inhibited neurotoxicity and neuronal cell death through reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP) dysfunction. Acacetin also markedly acted on key molecules in apoptotic cell death pathways and reduced phosphorylation of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinases (PI3K)/Akt, and glycogen synthase kinase-3beta (GSK-3ß). These results suggested that acacetin could inhibit 6-OHDA-induced neuronal cell death originating from ROS-mediated cascade apoptosis pathway. Thus, the results of our study suggest that acacetin is a potent therapeutic agent for PD progression.
Asunto(s)
Flavonas/farmacología , Neuronas/efectos de los fármacos , Oxidopamina/antagonistas & inhibidores , Enfermedad de Parkinson/tratamiento farmacológico , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Flavonas/química , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Estructura Molecular , Neuronas/patología , Oxidopamina/farmacología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Especies Reactivas de Oxígeno/metabolismo , Relación Estructura-ActividadRESUMEN
Parkinson's disease (PD) is one of the most common neurodegenerative diseases. Recent epidemiological studies suggest that echinacoside (ECH), a phenylethanoid glycoside found in Cistanche deserticola, has a protective effect against the development of PD. However, the detailed mechanisms of how ECH suppresses neuronal death have not been fully elucidated. In this study, we confirmed that ECH protects nigrostriatal neurons against 6-hydroxydopamine (6-OHDA)-induced endoplasmic reticulum stress (ERS) in vivo and in vitro. ECH rescued cell viability in damaged cells and decreased 6-OHDA-induced reactive oxygen species accumulation in vitro. It also rescued tyrosine hydroxylase and dopamine transporter expression in the striatum, and decreased α-synuclein aggregation following 6-OHDA treatment in vivo. The validated mechanism of ECH activity was the reduction in the 6-OHDA-induced accumulation of seipin (Berardinelli-Seip congenital lipodystrophy 2). Seipin has been shown to be a key molecule related to motor neuron disease and was tightly associated with ERS in a series of in vivo studies. ECH attenuated seipinopathy by promoting seipin degradation via ubiquitination. ERS was relieved by ECH through the Grp94/Bip-ATF4-CHOP signal pathway.
Asunto(s)
Cuerpo Estriado/efectos de los fármacos , Glicósidos/farmacología , Proteínas de Unión al GTP Heterotriméricas/genética , Fármacos Neuroprotectores/farmacología , Oxidopamina/antagonistas & inhibidores , Porción Compacta de la Sustancia Negra/efectos de los fármacos , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Adrenérgicos/farmacología , Animales , Línea Celular Tumoral , Cistanche/química , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Regulación de la Expresión Génica , Glicósidos/aislamiento & purificación , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Unión al GTP Heterotriméricas/antagonistas & inhibidores , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Inyecciones Intraventriculares , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Fármacos Neuroprotectores/aislamiento & purificación , Oxidopamina/farmacología , Porción Compacta de la Sustancia Negra/metabolismo , Porción Compacta de la Sustancia Negra/patología , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Técnicas Estereotáxicas , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismoRESUMEN
Cystatin C (CYS C, Cst3) is an endogenous cysteine protease inhibitor that plays neuroprotective roles in neurodegenerative diseases. We aimed to explore the association of CYS C with Parkinson's disease (PD) models and investigate its involvement in the role of neurovascular units (NVUs) in PD neuro-pathogenesis. We used A53T α-synuclein (SNCA) transgenic mice and 6-hydroxydopamine-lesioned DAergic PC12 cells as experimental PD models to investigate the mechanisms behind this association. The injections of CYS C were administered to the right substantia nigra (SN) of A53T SNCA transgenic mice to measure the effects of CYS C in transgenic A53T SNCA mice. To explore the angiogenesis in vivo and in vitro, we used the chick embryo chorioallantoic membrane (CAM) assay and tube formation (TF) assay. We found that CYS C has a neuroprotective effect in this in vivo PD model. We observed increased VEGF, NURR1 and autophagy markers LC3B and decreased SNCA and apoptosis marker cleaved CASP3 in different brain regions of CYS C-treated A53T SNCA transgenic mice. In vitro, we observed that CYS C-induced VEGF, a secreted protein, attenuated 6-OHDA-lesioned DAergic PC12 cell degeneration by regulating p-PKC-α/p-ERK1/2-Nurr1 signaling and inducing autophagy. VEGF-mediated angiogenesis was markedly enhanced in the conditioned media of 6-OHDA-lesioned PC12 cells with CYS C-overexpression, whereas blockage of autophagy in CYS C-overexpressing PC12 cells significantly downregulated VEGF expression and the associated angiogenesis. Our data indicate that CYS C displays dual neuronal-vascular functions, promoting PC12 cell survival and angiogenesis via regulating the level of secreted VEGF in NVUs. Our study provides evidence that may aid in the development of an alternative approach for the treatment of PD through modulation of CYS C-mediated neuronal-vascular pathways.
Asunto(s)
Cistatina C/farmacología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Enfermedad de Parkinson/genética , Factor A de Crecimiento Endotelial Vascular/genética , alfa-Sinucleína/genética , Animales , Autofagia/efectos de los fármacos , Caspasa 3/genética , Caspasa 3/metabolismo , Embrión de Pollo , Membrana Corioalantoides/irrigación sanguínea , Membrana Corioalantoides/efectos de los fármacos , Membrana Corioalantoides/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Inyecciones Intraventriculares , Ratones , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Oxidopamina/antagonistas & inhibidores , Oxidopamina/farmacología , Células PC12 , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Ratas , Transducción de Señal , Sustancia Negra/efectos de los fármacos , Sustancia Negra/metabolismo , Sustancia Negra/patología , Factor A de Crecimiento Endotelial Vascular/metabolismo , alfa-Sinucleína/metabolismoRESUMEN
This study investigated the effects of asarinin on dopamine biosynthesis and 6-hydroxydopamine (6-OHDA)-induced cytotoxicity in rat adrenal pheochromocytoma (PC12) cells. Treatment with asarinin (25-50 µM) increased intracellular dopamine levels and enhanced L-DOPA-induced increases in dopamine levels. Asarinin (25 µM) induced cyclic AMP-dependent protein kinase A (PKA) signaling, leading to increased cyclic AMP-response element binding protein (CREB) and tyrosine hydroxylase (TH) phosphorylation, which in turn stimulated dopamine production. Asarinin (25 µM) also activated transient phosphorylation of extracellular signal-regulated kinase (ERK1/2) and Bad phosphorylation at Ser 112, both of which have been shown to promote cell survival. In contrast, asarinin (25 µM) inhibited sustained ERK1/2, Bax, c-Jun N-terminal kinase (JNK1/2) and p38 mitogen-activated protein kinase (p38MAPK) phosphorylation and caspase-3 activity, which were induced by 6-OHDA (100 µM). These results suggest that asarinin induces dopamine biosynthesis via activation of the PKA-CREB-TH system and protects against 6-OHDA-induced cytotoxicity by inhibiting the sustained activation of the ERK-p38MAPK-JNK1/2-caspase-3 system in PC12 cells.
Asunto(s)
Dioxoles/farmacología , Dopamina/biosíntesis , Lignanos/farmacología , Oxidopamina/antagonistas & inhibidores , Animales , Asarum/química , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Dioxoles/química , Dioxoles/aislamiento & purificación , Relación Dosis-Respuesta a Droga , Lignanos/química , Lignanos/aislamiento & purificación , Estructura Molecular , Oxidopamina/toxicidad , Células PC12 , Ratas , Relación Estructura-ActividadRESUMEN
Research on Parkinson's disease (PD) and drug development is hampered by the lack of suitable human in vitro models that simply and accurately recreate the disease conditions. To counteract this, many attempts to differentiate cell lines, such as the human SH-SY5Y neuroblastoma, into dopaminergic neurons have been undertaken since they are easier to cultivate when compared with other cellular models. Here, we characterized neuronal features discriminating undifferentiated and retinoic acid (RA)-differentiated SH-SYSY cells and described significant differences between these cell models in 6-hydroxydopamine (6-OHDA) cytotoxicity. In contrast to undifferentiated cells, RA-differentiated SH-SY5Y cells demonstrated low proliferative rate and a pronounced neuronal morphology with high expression of genes related to synapse vesicle cycle, dopamine synthesis/degradation, and of dopamine transporter (DAT). Significant differences between undifferentiated and RA-differentiated SH-SY5Y cells in the overall capacity of antioxidant defenses were found; although RA-differentiated SH-SY5Y cells presented a higher basal antioxidant capacity with high resistance against H2O2 insult, they were twofold more sensitive to 6-OHDA. DAT inhibition by 3α-bis-4-fluorophenyl-methoxytropane and dithiothreitol (a cell-permeable thiol-reducing agent) protected RA-differentiated, but not undifferentiated, SH-SY5Y cells from oxidative damage and cell death caused by 6-OHDA. Here, we demonstrate that undifferentiated and RA-differentiated SH-SY5Y cells are two unique phenotypes and also have dissimilar mechanisms in 6-OHDA cytotoxicity. Hence, our data support the use of RA-differentiated SH-SY5Y cells as an in vitro model of PD. This study may impact our understanding of the pathological mechanisms of PD and the development of new therapies and drugs for the management of the disease.
Asunto(s)
Antioxidantes/metabolismo , Diferenciación Celular/efectos de los fármacos , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/antagonistas & inhibidores , Neuronas Dopaminérgicas/fisiología , Tretinoina/farmacología , Muerte Celular/efectos de los fármacos , Células Cultivadas , Ditiotreitol/farmacología , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Neuronas Dopaminérgicas/metabolismo , Humanos , Peróxido de Hidrógeno , Oxidación-Reducción/efectos de los fármacos , Oxidopamina/antagonistas & inhibidores , Fosfinas/farmacologíaRESUMEN
Dopamine (DA) contributes to the regulation of voluntary movement, and a deficiency in DAergic neurons leads to movement disorders. The objective of this study was to examine the neuroprotective effect of DA D2-like receptor agonist, lisuride, and the role of DA receptors in this protection. Treatment with lisuride alleviated loss of tyrosine hydroxylase (TH) both direct and intraperitoneal injection in 6-hydroxydopamine (6-OHDA) mouse model. Similar results were obtained in primary neuronal cultures treated with lisuride. Lisuride protected TH expression against 6-OHDA-induced cytotoxicity in a concentration-dependent manner. Then, we evaluated the role of DA D2 and D3 receptor in neuroprotective effect of lisuride. Treatment of neuronal cultures with L-741,626, a DA D2 receptor-selective antagonist, did not alter neuroprotective effect of lisuride. However, protective effect of lisuride on TH expression was abolished when cells were treated with GR103691, a D3 receptor selective antagonist. Furthermore, whether lisuride can alleviate mitochondrial damage of DAergic neurons induced by 6-OHDA, we investigated the expression of the mitochondrial regulatory protein, paraplegin, and changes in mitochondria morphology. Treatment with lisuride countered a 6-OHDA-induced reduction in paraplegin and TH expression, and co-treatment with GR103691 blocked this effect of lisuride. Transmission electron microscopy confirmed the lisuride mitigation of 6-OHDA-induced damage to the mitochondrial membrane and cristae. These results suggest that the DA D3 receptor mediates the neuroprotective effects of lisuride by preventing mitochondrial damage.
Asunto(s)
Lisurida/farmacología , Fármacos Neuroprotectores/farmacología , Receptores de Dopamina D3/agonistas , ATPasas Asociadas con Actividades Celulares Diversas , Animales , Compuestos de Bifenilo/farmacología , Antagonistas de los Receptores de Dopamina D2/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Indoles/farmacología , Lisurida/antagonistas & inhibidores , Masculino , Metaloendopeptidasas/metabolismo , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Fármacos Neuroprotectores/antagonistas & inhibidores , Oxidopamina/antagonistas & inhibidores , Piperazinas/farmacología , Piperidinas/farmacología , Cultivo Primario de Células , Receptores de Dopamina D3/antagonistas & inhibidores , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Human Hsp70-2 is a chaperone expressed mainly in the nervous system. Up to now, no study has reported on the recombinant expression of this important human chaperone. Herein, we describe the successful purification and characterization of recombinant human Hsp70-2 in Escherichia coli in both the full-length and the chimeric protein containing the protein transduction domain corresponding to the trans-activator of transcription (Tat) from HIV. Under optimized conditions, the Tat-Hsp70-2 was expressed in a soluble form and purified by two chromatographic steps (in a 3.6 mg/L fermentation broth yield): recombinant Tat-Hsp70-2 was folded and showed ATPase activity. In contrast, the full-length recombinant protein was only expressed in the form of inclusion bodies and thus was purified following a refolding procedure. The refolded Hsp70-2 protein was inactive and the protein conformation slightly altered as compared to the corresponding Tat-fused variant. The Tat-Hsp70-2 protein (100 nM), when added to human neuroblastoma SH-SY5Y cells subjected to hydrogen peroxide or 6-hydroxydopamine stress, partially protected from the deleterious effect of these treatments. This work describes an approach for the functional expression of human Tat-Hsp70-2 that provides sufficient material for detailed structure-function studies and for testing its ability to protect neuroblastoma cells from oxidative stress.
Asunto(s)
Adenosina Trifosfatasas/biosíntesis , Proteínas HSP70 de Choque Térmico/biosíntesis , Fármacos Neuroprotectores/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/biosíntesis , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/aislamiento & purificación , Adenosina Trifosfatasas/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/aislamiento & purificación , Proteínas HSP70 de Choque Térmico/farmacología , Humanos , Peróxido de Hidrógeno/antagonistas & inhibidores , Peróxido de Hidrógeno/farmacología , Cuerpos de Inclusión/química , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fármacos Neuroprotectores/aislamiento & purificación , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo , Oxidopamina/antagonistas & inhibidores , Oxidopamina/farmacología , Pliegue de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/farmacología , Solubilidad , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/aislamiento & purificación , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/farmacologíaRESUMEN
The Portulaca oleracea L. (Portulacaceae) is a cosmopolitan species with a wide range of biological activities, including antioxidant and neuroprotective actions. We investigated the effects of P. oleracea extracts in a 6-hydroxydopamine rat model of Parkinson's disease, a debilitating disorder without effective treatments. Chemical profiles of aqueous and ethanolic extracts of whole plant were analyzed by thin layer chromatography and the antioxidant activity was assessed by 2,2-diphenyl-1-picrilhidrazila method. Male Wistar rats received intrastriatal 6-hydroxydopamine and were treated with vehicle or extracts (oral, 200 and 400 mg/kg) daily for two weeks. The behavioral open field test was conducted at days 1 and 15. Immunohistochemical analysis was performed 4 weeks after surgery to quantify tyrosine-hydroxylase cell counts in the substantia nigra pars compacta. Extracts presented antioxidant activity in concentrations above 300 µg/kg. The chromatographic analysis revealed the presence of Levodopa, alkaloids, flavonoids, saponins, tannins, terpenoids and polysaccharides. Both extracts improved motor recovery 15 days after lesion and protected from tyrosine-hydroxylase cell loss after 4 weeks, but these effects were more evident for the aqueous extract. Because the dopamine precursor is present, in addition to antioxidant compounds and neuroprotective effects, P. oleracea can be considered as potential strategy for treating Parkinson's disease.
Asunto(s)
Neuronas Dopaminérgicas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Oxidopamina/toxicidad , Portulaca/química , Simpaticolíticos/toxicidad , Animales , Neuronas Dopaminérgicas/enzimología , Masculino , Oxidopamina/antagonistas & inhibidores , Enfermedad de Parkinson/tratamiento farmacológico , Porción Compacta de la Sustancia Negra/citología , Porción Compacta de la Sustancia Negra/enzimología , Ratas , Ratas Wistar , Simpaticolíticos/antagonistas & inhibidores , Tirosina 3-Monooxigenasa/análisisRESUMEN
Glial cell line-derived neurotrophic factor (GDNF) has strong neuroprotective and neurorestorative effects on dopaminergic (DA) neurons in the substantia nigra (SN); however, the underlying molecular mechanisms remain to be fully elucidated. In this study, we found that the expression level of transcription factor Six2 was increased in damaged DA neurons after GDNF rescue in vivo and in vitro. Knockdown of Six2 resulted in decreased cell viability and increased the apoptosis of damaged DA neurons after GDNF treatment in vitro. In contrast, Six2 overexpression increased cell viability and decreased cell apoptosis. Furthermore, genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) indicated that Six2 directly bound to the promoter CAGCTG sequence of smad ubiquitylation regulatory factor 1 (Smurf1). ChIP-quantitative polymerase chain reaction (qPCR) analysis showed that Smurf1 expression was significantly upregulated after GDNF rescue. Moreover, knockdown of Six2 decreased Smurf1 expression, whereas overexpression of Six2 increased Smurf1 expression in damaged DA neurons after GDNF rescue. Meanwhile, knockdown and overexpression of Smurf1 increased and decreased p53 expression, respectively. Taken together, our results from in vitro and in vivo analysis indicate that Six2 mediates the protective effects of GDNF on damaged DA neurons by regulating Smurf1 expression, which could be useful in identifying potential drug targets for injured DA neurons.
Asunto(s)
Neuronas Dopaminérgicas/efectos de los fármacos , Factor Neurotrófico Derivado de la Línea Celular Glial/farmacología , Proteínas de Homeodominio/genética , Fármacos Neuroprotectores/farmacología , Porción Compacta de la Sustancia Negra/efectos de los fármacos , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas/genética , Animales , Apoptosis/efectos de los fármacos , Sitios de Unión , Línea Celular , Supervivencia Celular/efectos de los fármacos , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/metabolismo , Regulación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Humanos , Motivos de Nucleótidos , Oxidopamina/antagonistas & inhibidores , Oxidopamina/toxicidad , Porción Compacta de la Sustancia Negra/citología , Porción Compacta de la Sustancia Negra/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Técnicas Estereotáxicas , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Hydroxytyrosol (3,4-dihydroxyphenylethanol, HT), a major polyphenol in olive oils, has received increasing attention due to its multiple pharmacological activities. However, it is not well understood how HT works on the neuronal system. We report herein that HT efficiently scavenges free radicals in vitro and displays cytoprotection against oxidative stress-induced damage in PC12 cells. HT completely protects the cells from hydrogen peroxide-induced death and rescues the cells from 6-hydroxydopamine-induced damage. Mechanistic studies reveal that Nrf2 is a prerequisite for the neuroprotection of HT as knocking down Nrf2 eliminated this action. HT, via activation of the Keap1-Nrf2 pathway, elevates a panel of cytoprotective enzymes, including glutamate-cysteine ligase, HO-1, NQO1 and thioredoxin reductase. Our study reveals that HT provides dual neuroprotection and cellular antioxidant defense as both a free radical scavenger and Nrf2 activator, suggesting the potential pharmaceutical usage of HT for the treatment of neurodegenerative disorders.
Asunto(s)
Antioxidantes , Neuronas , Fármacos Neuroprotectores , Aceite de Oliva , Estrés Oxidativo , Alcohol Feniletílico , Animales , Ratas , Adrenérgicos/efectos adversos , Adrenérgicos/química , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Depuradores de Radicales Libres/farmacología , Peróxido de Hidrógeno/antagonistas & inhibidores , Peróxido de Hidrógeno/toxicidad , Cinética , Proteínas del Tejido Nervioso/agonistas , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Aceite de Oliva/química , Oxidantes/antagonistas & inhibidores , Oxidantes/toxicidad , Estrés Oxidativo/efectos de los fármacos , Oxidopamina/efectos adversos , Oxidopamina/antagonistas & inhibidores , Células PC12 , Alcohol Feniletílico/análogos & derivados , Alcohol Feniletílico/farmacología , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Interferencia de ARN , ARN Interferente Pequeño , Transducción de Señal/efectos de los fármacos , Factor 2 Relacionado con NF-E2RESUMEN
BACKGROUND: Parkinson disease (PD) is a common adult-onset neurodegenerative disorder, and PD related neuronal injury is associated with oxidative stress and mitochondrial dysfunction. Allicin, the main biologically active compound derived from garlic, has been shown to exert various anti-oxidative and anti-apoptotic activities in in vitro and in vivo studies. METHODS: The present study aimed to investigate the potential protective role of allicin in an in vitro PD model induced by 6-hydroxydopamine (6-OHDA) in PC12 cells. The protective effects were measured by cell viability, decreased lactate dehydrogenase (LDH) release and flow cytometry, and the anti-oxidative activity was determined by reactive oxygen species (ROS) generation, lipid peroxidation and the endogenous antioxidant enzyme activities. Mitochondrial function in PC12 cells was detected by mitochondrial membrane potential (MMP) collapse, cytochrome c release, mitochondrial ATP synthesis, and the mitochondrial Ca(2+) buffering capacity. To investigate the potential mechanism, we also measured the expression of mitochondrial biogenesis factors, mitochondrial morphological dynamic changes, as well as detected mitochondrial dynamic proteins by western blot. RESULTS: We found that allicin treatment significant increased cell viability, and decreased LDH release and apoptotic cell death after 6-OHDA exposure. Allicin also inhibited ROS generation, reduced lipid peroxidation and preserved the endogenous antioxidant enzyme activities. These protective effects were associated with suppressed mitochondrial dysfunction, as evidenced by decreased MMP collapse and cytochrome c release, preserved mitochondrial ATP synthesis, and the promotion of mitochondrial Ca(2+) buffering capacity. In addition, allicin significantly enhanced mitochondrial biogenesis and prevented fragmentation of mitochondrial network after 6-OHDA treatment. The results of western blot analysis showed that the 6-OHDA induced decrease in the expression of optic atrophy type 1 (Opa-1), increase in mitochondrial fission 1 (Fis-1) and dynamin-related protein 1 (Drp-1) were all partially revised by allicin. CONCLUSION: In summary, our data strongly suggested that allicin treatment can exert protective effects against PD related neuronal injury through inhibiting oxidative stress and mitochondrial dysfunction with dynamic changes.
Asunto(s)
Adrenérgicos/efectos adversos , Antioxidantes/farmacología , Dinámicas Mitocondriales/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Oxidopamina/efectos adversos , Ácidos Sulfínicos/farmacología , Adenosina Trifosfato/biosíntesis , Animales , Apoptosis/efectos de los fármacos , Calcio/metabolismo , Supervivencia Celular/efectos de los fármacos , Citocromos c/metabolismo , Disulfuros , Dinaminas/agonistas , Dinaminas/genética , Dinaminas/metabolismo , GTP Fosfohidrolasas/antagonistas & inhibidores , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Expresión Génica , L-Lactato Deshidrogenasa/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Proteínas Mitocondriales/agonistas , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Estrés Oxidativo/efectos de los fármacos , Oxidopamina/antagonistas & inhibidores , Células PC12 , Ratas , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: The aim of this study was to investigate the protective role of intranasally administered substance P-loaded gelatin nanoparticles (SP-GNPs) against 6-hydroxydopamine (6-OHDA)-induced apoptosis in vitro and in vivo, and to provide a new strategy for treating brain pathology, such as Parkinson's disease. METHODS: SP-GNPs were prepared by a water-in-water emulsion method, and their stability, encapsulating efficiency, and loading capacity were evaluated. PC-12 cells were used to examine the enhancement of growth and inhibition of apoptosis by SP-GNPs in vitro using MTT assays. In the in vivo study, hemiparkinsonian rats were created by intracerebroventricular injection of 6-OHDA. The rats then received intranasal SP-GNPs daily for 2 weeks. Functional improvement was assessed by quantifying rotational behavior, and the degree of apoptosis was assessed by immunohistochemical staining for caspase-3 in the substantia nigra region. RESULTS: PC-12 cells with 6-OHDA-induced disease treated with SP-GNPs showed higher cell viability than their untreated counterparts, and cell viability increased as the concentration of substance P (SP) increased, indicating that SP could enhance cell growth and inhibit the cell apoptosis induced by 6-OHDA. Rats with 6-OHDA-induced hemiparkinsonism treated with SP-GNPs made fewer rotations and showed less staining for caspase-3 than their counterparts not treated with SP, indicating that SP protects rats with 6-OHDA-induced hemiparkinsonism from apoptosis and therefore demonstrates their functional improvement. CONCLUSION: Intranasal delivery of SP-GNPs protects against 6-OHDA-induced apoptosis both in vitro and in vivo.
Asunto(s)
Apoptosis/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Gelatina/química , Nanopartículas/química , Oxidopamina/antagonistas & inhibidores , Sustancia P/administración & dosificación , Sustancia P/farmacología , Administración Intranasal , Animales , Caspasa 3/análisis , Caspasa 3/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Masculino , Estructura Molecular , Oxidopamina/administración & dosificación , Oxidopamina/farmacología , Células PC12 , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Sustancia P/química , Sustancia Negra/efectos de los fármacos , Sustancia Negra/metabolismo , Sustancia Negra/patologíaRESUMEN
The present study was conducted to investigate the effect of the neurotoxins 6-hydroxydopamine and lipopolysaccharide on astrocytes. Rat astrocyte C6 cells were treated with different concentration of 6-hydroxydopamine (6-OHDA)/lipopolysaccharides (LPS) for 24 h. Both neurotoxins significantly decreased the viability of astrocytes, augmented the expression of inducible nitric oxide synthase (iNOS) and the astrocyte marker--glial fibrillar acidic protein. A significantly decreased mitochondrial dehydrogenase activity, mitochondrial membrane potential, augmented reactive oxygen species (ROS) level, caspase-3 mRNA level, chromatin condensation and DNA damage was observed in 6-OHDA/LPS treated astroglial cells. 6-OHDA/LPS treatment also caused the significantly increased expression of iNOS and nitrite level. Findings showed that 6-OHDA/LPS treatment caused mitochondrial dysfunction mediated death of astrocytes, which significantly involve the nitric oxide. Since we have observed significantly increased level of iNOS along with mitochondrial impairment and apoptotic cell death in astrocytes, therefore to validate the role of iNOS, the cells were co-treated with iNOS inhibitor aminoguanidine (AG, 100 µM). Co-treatment of AG significantly attenuated the 6-OHDA/LPS induced cell death, mitochondrial activity, augmented ROS level, chromatin condensation and DNA damage. GFAP and caspase-3 expression were also inhibited with co-treatment of AG, although the extent of inhibition was different in both experimental sets. In conclusion, the findings showed that iNOS mediated increased level of nitric oxide acts as a key regulatory molecule in 6-OHDA/LPS induced mitochondrial dysfunction, DNA damage and apoptotic death of astrocytes.