RESUMO
In this study, the effects of 3,5,7,3',4'-pentamethoxyflavone (KP1), a major bioactive ingredient isolated from the Kaempferia parviflora rhizomes, on a neurite outgrowth in Neuro2a cells and its mechanism have been investigated. KP1 increased concentration-dependently the percentage of neurite-bearing cells. KP1 showed a remarkable capability to elicit neurite outgrowth in Neuro2a cells, as evidenced by morphological alterations and immunostaining using anti-class III ß-tubulin and anti-NeuN antibodies. KP1 also displayed a higher neurogenic activity than retinoic acid (RA), a promoter of neurite outgrowth in Neuro2a cells. KP1 treatment caused significant elevation in phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38 MAPK) and glycogen synthase kinase-3ß (GSK-3ß). However, KP1-triggered neurite outgrowth was markedly inhibited by treatment with the ERK inhibitor U0126, whereas p38 MAPK inhibitor SB203580 and GSK-3ß inhibitor SB216763 did not influence KP1-induced neurite outgrowth. These results demonstrate that KP1 elicits neurite outgrowth and triggers cell differentiation of Neuro2a cells through ERK signal pathway.
Assuntos
Sistema de Sinalização das MAP Quinases , Crescimento Neuronal , Animais , Crescimento Neuronal/efeitos dos fármacos , Camundongos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Neuritos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Flavonoides/farmacologia , Flavonas/farmacologia , Flavonas/química , Linhagem Celular Tumoral , Glicogênio Sintase Quinase 3 beta/metabolismo , Linhagem CelularRESUMO
Aberrant insulin-like growth factor 1 (IGF-1) signaling has been proposed as a contributing factor to the development of neurodegenerative disorders including diabetic neuropathy, and delivery of exogenous IGF-1 has been explored as a treatment for Alzheimer's disease and amyotrophic lateral sclerosis. However, the role of autocrine/paracrine IGF-1 in neuroprotection has not been well established. We therefore used in vitro cell culture systems and animal models of diabetic neuropathy to characterize endogenous IGF-1 in sensory neurons and determine the factors regulating IGF-1 expression and/or affecting neuronal health. Single-cell RNA sequencing (scRNA-Seq) and in situ hybridization analyses revealed high expression of endogenous IGF-1 in non-peptidergic neurons and satellite glial cells (SGCs) of dorsal root ganglia (DRG). Brain cortex and DRG had higher IGF-1 gene expression than sciatic nerve. Bidirectional transport of IGF-1 along sensory nerves was observed. Despite no difference in IGF-1 receptor levels, IGF-1 gene expression was significantly (P < 0.05) reduced in liver and DRG from streptozotocin (STZ)-induced type 1 diabetic rats, Zucker diabetic fatty (ZDF) rats, mice on a high-fat/ high-sugar diet and db/db type 2 diabetic mice. Hyperglycemia suppressed IGF-1 gene expression in cultured DRG neurons and this was reversed by exogenous IGF-1 or the aldose reductase inhibitor sorbinil. Transcription factors, such as NFAT1 and CEBPß, were also less enriched at the IGF-1 promoter in DRG from diabetic rats vs control rats. CEBPß overexpression promoted neurite outgrowth and mitochondrial respiration, both of which were blunted by knocking down or blocking IGF-1. Suppression of endogenous IGF-1 in diabetes may contribute to neuropathy and its upregulation at the transcriptional level by CEBPß can be a promising therapeutic approach.
Assuntos
Envelhecimento/metabolismo , Axônios/patologia , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Metabolismo Energético , Fator de Crescimento Insulin-Like I/metabolismo , Células Receptoras Sensoriais/metabolismo , Animais , Anticorpos Neutralizantes/farmacologia , Axônios/efeitos dos fármacos , Axônios/metabolismo , Sequência de Bases , Proteína beta Intensificadora de Ligação a CCAAT/genética , Respiração Celular/efeitos dos fármacos , Células Cultivadas , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Metabolismo Energético/efeitos dos fármacos , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Células HEK293 , Humanos , Fator de Crescimento Insulin-Like I/genética , Fígado/metabolismo , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fatores de Transcrição NFATC/metabolismo , Crescimento Neuronal/efeitos dos fármacos , Polímeros/metabolismo , Regiões Promotoras Genéticas/genética , Transporte Proteico/efeitos dos fármacos , Ratos Sprague-Dawley , Células Receptoras Sensoriais/patologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Gongjin-dan (GJD) is a multiherbal formula produced from 10 medicinal herbs and has been traditonally used as an oriental medicine to treat cardiovascular diseases, alcoholic hepatitis, mild dementia, and anemia. Additionally, increasing evidence suggests that GJD exerts neuroprotective effects by suppressing inflammation and oxidative stress-induced events to prevent neurological diseases. However, the mechanism by which GJD prevents oxidative stress-induced neuronal injury in a mature neuron remains unknown. Here, we examined the preventive effect and mechanism of GJD on primary cortical neurons exposed to hydrogen peroxide (H2O2). In the neuroprotection signaling pathway, Sirtuin1 is involved in neuroprotective action as a therapeutic target for neurological diseases. After pre-treatment with GJD at three concentrations (10, 25, and 50 µg/mL) and stimulation by H2O2 (30 µM) for 24 h, the influence of GJD on Sirtuin1 activation was assessed using immunocytochemistry, real-time PCR, western blotting, and flow cytometry. GJD effectively ameliorated H2O2-induced neuronal death against oxidative damage through Sirtuin1 activation. In addition, GJD-induced Sirtuin1 activation accelerated elongation of new axons and formation of synapses via increased expression of nerve growth factor and brain-derived neurotrophic factor, as well as regeneration-related genes. Thus, GJD shows potential for preventing neurological diseases via Sirtuin1 activation.
Assuntos
Crescimento Neuronal/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1/metabolismo , Animais , Córtex Cerebral/crescimento & desenvolvimento , Peróxido de Hidrogênio/efeitos adversos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Characterization of new pharmacological targets is a promising approach in research of neurorepair mechanisms. The G protein-coupled receptor 17 (GPR17) has recently been proposed as an interesting pharmacological target, e.g., in neuroregenerative processes. Using the well-established ex vivo model of organotypic slice co-cultures of the mesocortical dopaminergic system (prefrontal cortex (PFC) and substantia nigra/ventral tegmental area (SN/VTA) complex), the influence of GPR17 ligands on neurite outgrowth from SN/VTA to the PFC was investigated. The growth-promoting effects of Montelukast (MTK; GPR17- and cysteinyl-leukotriene receptor antagonist), the glial cell line-derived neurotrophic factor (GDNF) and of two potent, selective GPR17 agonists (PSB-16484 and PSB-16282) were characterized. Treatment with MTK resulted in a significant increase in mean neurite density, comparable with the effects of GDNF. The combination of MTK and GPR17 agonist PSB-16484 significantly inhibited neuronal growth. qPCR studies revealed an MTK-induced elevated mRNA-expression of genes relevant for neuronal growth. Immunofluorescence labelling showed a marked expression of GPR17 on NG2-positive glia. Western blot and RT-qPCR analysis of untreated cultures suggest a time-dependent, injury-induced stimulation of GPR17. In conclusion, MTK was identified as a stimulator of neurite fibre outgrowth, mediating its effects through GPR17, highlighting GPR17 as an interesting therapeutic target in neuronal regeneration.
Assuntos
Acetatos/farmacologia , Ciclopropanos/farmacologia , Antagonistas de Leucotrienos/farmacologia , Crescimento Neuronal/efeitos dos fármacos , Quinolinas/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Sulfetos/farmacologia , Animais , Animais Recém-Nascidos , Técnicas de Cocultura , Avaliação Pré-Clínica de Medicamentos , Feminino , Masculino , Regeneração Nervosa/efeitos dos fármacos , Crescimento Neuronal/genética , RatosRESUMO
Alzheimer's disease (AD) is an incurable neurodegenerative disease. Repairing damaged nerves and promoting nerve regeneration are key ways to relieve AD symptoms. However, due to the lack of effective strategies to deliver nerve growth factor (NGF) to the brain, achieving neuron regeneration is a major challenge for curing AD. Herein, a ROS-responsive ruthenium nanoplatform (R@NGF-Se-Se-Ru) drug delivery system for AD management by promoting neuron regeneration and Aß clearance was investigated. Under near-infrared (NIR) irradiation, nanoclusters have good photothermal properties, which can effectively inhibit the aggregation of Aß and disaggregate Aß fibrils. Interestingly, the diselenide bond in the nanoclusters is broken, and the nanoclusters are degraded into small ruthenium nanoparticles in the high reactive oxygen species (ROS) environment of the diseased area. Besides, NGF can promote neuronal regeneration and repair damaged nerves. Furthermore, R@NGF-Se-Se-Ru efficiently crosses the blood-brain barrier (BBB) owing to the covalently grafted target peptides of RVG (R). In vivo studies demonstrate that R@NGF-Se-Se-Ru nanoclusters decrease Aß deposits, inhibit Aß-induced cytotoxicity, and promote neurite outgrowth. The study confirms that promoting both Aß clearance and neuron regeneration is an important therapeutic target for anti-AD drugs and provides a novel insight for AD therapy.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Portadores de Fármacos/química , Nanoestruturas/química , Fator de Crescimento Neural/uso terapêutico , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Materiais Biocompatíveis/farmacologia , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular Tumoral , Hemólise/efeitos dos fármacos , Humanos , Raios Infravermelhos , Fator de Crescimento Neural/química , Fator de Crescimento Neural/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Crescimento Neuronal/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Rutênio/química , Selênio/químicaRESUMO
The mold Monascus has been used as a natural food coloring agent and food additive for more than 1000 years in Asian countries. In Chinese herbology, it was also used for easing digestion and antiseptic effects. Through a thorough investigation of a citrinin-free strain: M. purpureus BCRC 38110, four azaphilones, three benzenoids, one benzofuranone, one 5',6'-dihydrospiro[isochromane-1,2'-pyran]-4'(3'H)-one derivative, two steroids, and six tetralones have been successfully identified. Among them, monapyridine A (1), monatetralones A-E (2-6), and monabenzofuranone (7) were first reported. Their structures were characterized by 1D and 2D NMR, UV, IR, and HRESIMS analyses. With a series of bioactivity screening, monascuspirolide B (14) and ergosterol peroxide (16) exhibited concentration-dependent attenuation of the paclitaxel-induced neurite damage of mouse dorsal root ganglion neurons. The interleukin (IL)-1ß-induced release of inflammatory cytokines IL-8 and tumor necrosis factor (TNF)-α in human chondrosarcoma cells was inhibited by monapurpureusone (8) and monascuspirolide B (14). Altogether, M. purpureus BCRC 38110 possessed potentials as natural therapeutics against inflammatory osteoarthritis and paclitaxel-induced neurotoxicity.
Assuntos
Anti-Inflamatórios/isolamento & purificação , Anti-Inflamatórios/farmacologia , Monascus/química , Fármacos Neuroprotetores/isolamento & purificação , Fármacos Neuroprotetores/farmacologia , Animais , Anti-Inflamatórios/química , Linhagem Celular Tumoral , Células Cultivadas , Gânglios Espinais/efeitos dos fármacos , Humanos , Interleucina-1beta/imunologia , Interleucina-8/metabolismo , Camundongos , Estrutura Molecular , Monascus/metabolismo , Crescimento Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/química , Osteoartrite/tratamento farmacológico , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Amyloid ß (Aß) plays a major role in the neurodegeneration of Alzheimer's disease (AD). The accumulation of misfolded Aß causes oxidative stress and inflammatory damage leading to apoptotic cell death. Traditional Chinese herbal medicine (CHM) has been widely used in treating neurodegenerative diseases by reducing oxidative stress and neuroinflammation. We examined the neuroprotective effect of formulated CHM Shaoyao Gancao Tang (SG-Tang, made of Paeonia lactiflora and Glycyrrhiza uralensis at 1:1 ratio) in AD cell and mouse models. In Aß-GFP SH-SY5Y cells, SG-Tang reduced Aß aggregation and reactive oxygen species (ROS) production, as well as improved neurite outgrowth. When the Aß-GFP-expressing cells were stimulated with conditioned medium from interferon (IFN)-γ-activated HMC3 microglia, SG-Tang suppressed expressions of inducible nitric oxide synthase (iNOS), NLR family pyrin domain containing 1 (NLRP1) and 3 (NLRP3), tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and IL-6, attenuated caspase-1 activity and ROS production, and promoted neurite outgrowth. In streptozocin-induced hyperglycemic APP/PS1/Tau triple transgenic (3×Tg-AD) mice, SG-Tang also reduced expressions of NLRP1, NLRP3, Aß and Tau in hippocampus and cortex, as well as improved working and spatial memories in Y maze and Morris water maze. Collectively, our results demonstrate the potential of SG-Tang in treating AD by moderating neuroinflammation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Cognição , Medicamentos de Ervas Chinesas/uso terapêutico , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Neuroproteção , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/metabolismo , Animais , Linhagem Celular , Cognição/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/farmacologia , Proteínas de Fluorescência Verde/metabolismo , Humanos , Interferon gama/metabolismo , Memória/efeitos dos fármacos , Transtornos da Memória/complicações , Transtornos da Memória/fisiopatologia , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Modelos Biológicos , Crescimento Neuronal/efeitos dos fármacos , Neuroproteção/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Agregados Proteicos/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Aprendizagem Espacial/efeitos dos fármacos , Proteínas tau/metabolismoRESUMO
It has been suggested that the intelligence quotient of children born to pregnant women taking 1000 mg or more of valproic acid per day is lower than that of children born to pregnant women taking other antiepileptic drugs. However, the mechanism whereby intelligence quotient is decreased in children exposed to valproic acid during the fetal period has not yet been elucidated. Therefore, we used the human neuroblastoma cell line SH-SY5Y to evaluate the effects of antiepileptic drugs containing valproic acid on nerve cells. We assessed the anti-proliferative effects of drugs in these cells via WST-8 colorimetric assay, using the Cell Counting Kit-8. We also quantified drug effects on axonal elongation from images using ImageJ software. We also evaluated drug effects on mRNA expression levels on molecules implicated in nervous system development and folic acid uptake using real-time PCR. We observed that carbamazepine and lamotrigen were toxic to SH-SY5Y cells at concentrations >500 µM. In contrast, phenytoin and valproic acid were not toxic to these cells. Carbamazepine, lamotrigen, phenytoin, and valproic acid did not affect axonal outgrowth in SH-SY5Y cells. Sodium channel neuronal type 1a (SCN1A) mRNA expression-level ratios increased when valproic acid was supplemented to cells. The overexpression of SCN1A mRNA due to high valproic acid concentrations during the fetal period may affect neurodevelopment. However, since detailed mechanisms have not yet been elucidated, it is necessary to evaluate it by comparing cell axon elongation and SCN1A protein expression due to high-concentration valproic acid exposure.
Assuntos
Anticonvulsivantes/farmacologia , Epilepsia/tratamento farmacológico , Canal de Sódio Disparado por Voltagem NAV1.1/metabolismo , Neuroblastoma/tratamento farmacológico , Crescimento Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal/tratamento farmacológico , Carbamazepina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Criança , Epilepsia/complicações , Epilepsia/metabolismo , Feminino , Humanos , Lamotrigina/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Neuroblastoma/complicações , Neuroblastoma/metabolismo , Fenitoína/farmacologia , Gravidez , Ácido Valproico/farmacologiaRESUMO
To investigate the therapeutic efficacy of Scutellarin (SCU) on neurite growth and neurological functional recovery in neonatal hypoxic-ischemic (HI) rats. Primary cortical neurons were cultured to detect the effect of SCU on cell viability of neurons under oxygen-glucose deprivation (OGD). Double immunofluorescence staining of Tuj1 and TUNEL then observed the neurite growth and cell apoptosis in vitro,and double immunofluorescence staining of NEUN and TUNEL was performed to examine the neuronal apoptosis and cell apoptosis in brain tissues after HI in vivo. Pharmacological efficacy of SCU was also evaluated in HI rats by neurobehavioral tests, triphenyl tetrazolium chloride staining, Hematoxylin and eosin staining and Nissl staining. Astrocytes and microglia expression in damaged brain tissues were detected by immunostaining of GFAP and Iba1. A quantitative real-time polymerase chain reaction and western blot were applied to investigate the genetic expression changes and the protein levels of autophagy-related proteins in the injured cortex and hippocampus after HI. We found that SCU administration preserved cell viability, promoted neurite outgrowth and suppressed apoptosis of neurons subjected to OGD both in vitroand in vivo. Meanwhile, 20 mg/kg SCU treatment improved neurological functions and decreased the expression of astrocytes and microglia in the cortex and hippocampus of HI rats. Additionally, SCU treatment depressed the elevated levels of autophagy-related proteins and the p75 neurotrophin receptor (p75NTR) in both cortex and hippocampus. This study demonstrated the potential therapeutic efficacy of SCU by enhancing neurogenesis and restoring long-term neurological dysfunctions, which might be associated with p75NTR depletion in HI rats.
Assuntos
Animais Recém-Nascidos , Apigenina/farmacologia , Apigenina/uso terapêutico , Encéfalo/fisiopatologia , Glucuronatos/farmacologia , Glucuronatos/uso terapêutico , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Hipóxia-Isquemia Encefálica/genética , Neurogênese/efeitos dos fármacos , Crescimento Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Autofagia/genética , Encéfalo/citologia , Encéfalo/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Hipóxia-Isquemia Encefálica/fisiopatologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Ratos , Receptores de Fatores de Crescimento/metabolismoRESUMO
Achyranthes bidentata polypeptide k (ABPPk), a powerful active component from a traditional Chinese medicinal herb-Achyranthes bidentata Bl., has exhibited promising neuroprotective activity due to its multiple-targeting capability. However, the effect of ABPPk on the survival, growth and axonal regeneration of spinal cord motor neurons remains unclear. Here, a modified method, which is more optimized for embryonic cells in ambient carbon dioxide levels, was used for acquisition of rat embryonic spinal cord motor neurons with high survival and purity. ABPPk concentration-dependently enhanced the neuronal viability and promoted the neurite outgrowth. Co-culture of motor neurons and skeletal myocytes model indicated that ABPPk enhanced the neuromuscular junction development and maturation. A microfluidic axotomy model was further established for the axonal disconnection, and ABPPk significantly accelerated the axonal regeneration of motor neurons. Furthermore, we demonstrated that the upregulation of three neurofilament protein subunits in motor neurons might be relevant to the mechanisms of the growth-promoting effect of ABPPk. Our findings provide an experimental and theoretical basis for the development of ABPPk as a potential application in the development of treatment strategy for nerve injury diseases.
Assuntos
Achyranthes , Axônios/efeitos dos fármacos , Neurônios Motores/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Regeneração Nervosa/efeitos dos fármacos , Junção Neuromuscular/efeitos dos fármacos , Crescimento Neuronal/efeitos dos fármacos , Extratos Vegetais/farmacologia , Animais , Axotomia , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Proteína GAP-43/efeitos dos fármacos , Proteína GAP-43/metabolismo , Técnicas In Vitro , Proteínas de Neurofilamentos/efeitos dos fármacos , Proteínas de Neurofilamentos/metabolismo , Peptídeos/farmacologia , Traumatismos dos Nervos Periféricos , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Medula Espinal/citologiaRESUMO
Two new sesquiterpenoids, including a kessane-type sesquiterpenoid (1) and one bisabolane derivative (2), together with fourteen known sesquiterpenoids (3-16), were isolated from the roots and rhizomes of Valeriana amurensis. The structures of new compounds were established on the basis of extensive spectroscopic analysis. All isolates were evaluated for their effects on nerve growth factor (NGF)-mediated neurite outgrowth in pheochromocytoma (PC12) cells. As a results, four compounds including 10-12 and 15 showed potent promoting effects at the concentration of 10 µM on NGF-induced neurite outgrowth in PC12 cells with the differentiation rate of 11.84%, 12.21%, 13.77% and 12.16%, respectively.
Assuntos
Fator de Crescimento Neural/farmacologia , Crescimento Neuronal/efeitos dos fármacos , Raízes de Plantas/química , Rizoma/química , Sesquiterpenos/isolamento & purificação , Sesquiterpenos/farmacologia , Valeriana/química , Animais , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Fator de Crescimento Neural/metabolismo , Células PC12 , Espectroscopia de Prótons por Ressonância Magnética , Ratos , Sesquiterpenos/químicaRESUMO
AIMS: Our work aims to revealing the underlying microtubule mechanism of neurites outgrowth during neuronal development and also proposes a feasible intervention pathway for reconstructing neural network connections after nerve injury. BACKGROUND: Microtubule polymerization and severing form the basis for neurite outgrowth and branch formation. However, the mechanisms that underlie the dynamic instability of microtubules are unclear. Here, we showed that neurite outgrowth mediated by collapsing response mediator protein 2 (CRMP2) can be enhanced by spastin, which had an effect on the severing of microtubule cytoskeleton. OBJECTIVE: To explore whether neurite outgrowth was mediated by coordination of CRMP2 and spastin. METHODS: Hippocampal neurons were cultured in vitro in 24-well culture plates for 4 days before being used to perform the transfection. Calcium phosphate was used to transfect the CRMP2 and spastin constructs and their control into the neurons. An interaction between CRMP2 and spastin was examined by using pull down, CoIP and immunofluorescence colocalization assays. And immunostaining was also performed to determine the morphology of neurites. RESULTS: We first demonstrated that CRMP2 interacted with spastin to promote neurite outgrowth and branch formation. Then our results identified that CRMP2 interacted with the microtubule- binding domain of spastin via its C-terminus, and deleting these binding sites inhibited neurite outgrowth and branch formation. In addition, we confirmed one phosphorylation site at S210 of spastin in hippocampal neurons. Spastin phosphorylation at S210 failed to alter the binding affinity of CRMP2 but inhibited its binding to microtubules. Further study showed that phosphorylation spastin at S210 inhibited the neurite outgrowth induced by CRMP2 and spastin interaction through downregulation of microtubule-severing activity. CONCLUSION: Taken together, our data demonstrated that both CRMP2 and spastin interaction and the microtubule-severing activity of spastin were required for neurite outgrowth and branch formation.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Microtúbulos/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Crescimento Neuronal/efeitos dos fármacos , Espastina/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Humanos , Proteínas Associadas aos Microtúbulos , Neuritos/efeitos dos fármacos , FosforilaçãoRESUMO
The embryonic rat dorsal root ganglion (DRG) neuron-derived 50B11 cell line is a promising sensory neuron model expressing markers characteristic of NGF and GDNF-dependent C-fibre nociceptors. Whether these cells have the capacity to develop into distinct nociceptive subtypes based on NGF- or GDNF-dependence has not been investigated. Here we show that by augmenting forskolin (FSK) and growth factor supplementation with NGF or GDNF, 50B11 cultures can be driven to acquire differential functional responses to common nociceptive agonists capsaicin and ATP respectively. In addition, to previous studies, we also demonstrate that a differentiated neuronal phenotype can be maintained for up to 7 days. Western blot analysis of nociceptive marker proteins further demonstrates that the 50B11 cells partially recapitulate the functional phenotypes of classical NGF-dependent (peptidergic) and GDNF-dependent (non-peptidergic) neuronal subtypes described in DRGs. Further, 50B11 cells differentiated with NGF/FSK, but not GDNF/FSK, show sensitization to acute prostaglandin E2 treatment. Finally, RNA-Seq analysis confirms that differentiation with NGF/FSK or GDNF/FSK produces two 50B11 cell subtypes with distinct transcriptome expression profiles. Gene ontology comparison of the two subtypes of differentiated 50B11 cells to rodent DRG neurons studies shows significant overlap in matching or partially matching categories. This transcriptomic analysis will aid future suitability assessment of the 50B11 cells as a high-throughput nociceptor model for a broad range of experimental applications. In conclusion, this study shows that the 50B11 cell line is capable of partially recapitulating features of two distinct types of embryonic NGF and GDNF-dependent nociceptor-like cells.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Gânglios Espinais/citologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Fator de Crescimento Neural/farmacologia , Nociceptores/citologia , Potenciais de Ação/efeitos dos fármacos , Trifosfato de Adenosina/farmacologia , Animais , Biomarcadores/metabolismo , Capsaicina/farmacologia , Diferenciação Celular/genética , Linhagem Celular , Forma Celular/efeitos dos fármacos , Colforsina/farmacologia , Dinoprostona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Variação Genética , Crescimento Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Nociceptores/efeitos dos fármacos , Fenótipo , Análise de Componente Principal , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Canais de Sódio/metabolismoRESUMO
In the current super-aging society, the establishment of methods for prevention and treatment of Alzheimer's disease (AD) is an urgent task. One of the causes of AD is thought to be a decrease in the revel of nerve growth factor (NGF) in the brain. Compounds showing NGF-mimicking activity and NGF-enhancing activity have been examined as possible agents for improving symptoms. In the present study, sunflower seed extract was found to have neurite outgrowth-promoting activity, which is an NGF-enhancing activity, in PC12 cells. To investigate neurite outgrowth-promoting compounds from sunflower seed extract, bioassay-guided purification was carried out. The purified active fraction was obtained by liquid-liquid partition followed by some column chromatographies. Proton nuclear magnetic resonance and gas chromatography-mass spectrometry analyses of the purified active fraction indicated that the fraction was a mixture of ß-sitosterol, stigmasterol and campesterol, with ß-sitosterol being the main component. Neurite outgrowth-promoting activities of ß-sitosterol, stigmasterol, campesterol and cholesterol were evaluated in PC12 cells. ß-Sitosterol and stigmasterol showed the strongest activity of the four sterol compounds (ß-sitosterol ≈ stigmasterol > campesterol > cholesterol), and cholesterol did not show any activity. The results indicated that ß-sitosterol was the major component responsible for the neurite outgrowth-promoting activity of sunflower seeds. Results of immunostaining also showed that promotion by ß-sitosterol of neurite formation induced by NGF was accompanied by neurofilament expression. ß-Sitosterol, which showed NGF-enhancing activity, might be a candidate ingredient in food for prevention of AD.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Helianthus/química , Extratos Vegetais/farmacologia , Doença de Alzheimer/genética , Animais , Encéfalo/efeitos dos fármacos , Colesterol/análogos & derivados , Colesterol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Fator de Crescimento Neural/genética , Neuritos/efeitos dos fármacos , Crescimento Neuronal/efeitos dos fármacos , Células PC12 , Fitosteróis/farmacologia , Extratos Vegetais/química , Ratos , Sementes/química , Sitosteroides/farmacologia , Estigmasterol/farmacologiaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Herbal formulation Buyang Huanwu Decoction (BYHWD) has been used to treat cardiovascular disorders including cerebral ischemia. Recent studies showed its effects on promoting axonal regeneration after nerve injury. However, compositional reformulation supplemented with herbal components that regulates inflammation may increase its efficacy for nerve repair. AIM OF THE STUDY: We prepared a new herbal decoction by adding selected herbal components to BYHWD (augmented BYHWD; ABHD) and investigated the effect of ABHD on the production of inflammatory cytokines and axonal regeneration using an animal model of nerve transection and coaptation (NTC). MATERIALS AND METHODS: A rat model of NTC was performed on the sciatic nerve. The sciatic nerve and dorsal root ganglion (DRG) were isolated and used for immunofluorescence staining and western blot analysis. DRG tissue was also used to prepare primary neuron culture and the length of neurites was analyzed. Sensorimotor nerve activities were assessed by rotarod and von Frey tests. RESULTS: Three herbal components that facilitated neurite outgrowth were chosen to formulate ABHD. ABHD administration into the sciatic nerve 1 week or 3 months after NTC facilitated axonal regeneration. Cell division cycle 2 (Cdc2) and brain-derived neurotrophic factor (BDNF) proteins were induced from the reconnected distal portion of the sciatic nerve and the levels were further elevated by in vivo administration of ABHD. Phospho-Erk1/2 level was increased by ABHD treatment as well, implying its role in mediating retrograde transport of BDNF signals into the neuronal cell body. Production of inflammatory cytokines IL-1ß and TNF-α was induced in the reconnected nerve but attenuated by ABHD treatment. Behavioral tests revealed that ABHD treatment improved functional recovery of sensorimotor activities. CONCLUSIONS: A newly formulated ABHD is effective at regulating the production of inflammatory cytokines and promoting axonal regeneration after nerve transection and may be considered to develop therapeutic strategies for peripheral nerve injury disorders.
Assuntos
Anti-Inflamatórios/farmacologia , Axônios/efeitos dos fármacos , Citocinas/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Gânglios Espinais/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Regeneração Nervosa/efeitos dos fármacos , Nervo Isquiático/efeitos dos fármacos , Neuropatia Ciática/tratamento farmacológico , Animais , Axônios/metabolismo , Comportamento Animal/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Gânglios Espinais/metabolismo , Gânglios Espinais/fisiopatologia , Masculino , Camundongos Endogâmicos ICR , Atividade Motora/efeitos dos fármacos , Crescimento Neuronal/efeitos dos fármacos , Percepção da Dor/efeitos dos fármacos , Ratos Sprague-Dawley , Nervo Isquiático/metabolismo , Nervo Isquiático/fisiopatologia , Nervo Isquiático/cirurgia , Neuropatia Ciática/metabolismo , Neuropatia Ciática/fisiopatologia , Transdução de SinaisRESUMO
Cortex Mori Radicis extract (CMR) has various pharmacological properties, such as antiinflammatory, antiallergic and antihyperglycemic effects. However, the effects and mechanisms of CMR in the neuroregeneration of diabetic peripheral neuropathy (DPN) are unclear. In the present study, the effects of CMR on neurite outgrowth of dorsal root ganglia (DRG) neurons in diabetic rats were investigated and its underlying mechanisms were explored. SD rats were subjected to a highfat diet with lowdose streptozotocin to induce a Type II diabetes model with peripheral neuropathy. CMR was then applied for four weeks continuously with or without injection of small interfere (si)RNA targeting the transient receptor potential canonical channel 1 (TRPC1) via the tail vein. Blood glucose levels, the number of Nissl bodies, neurite outgrowth and growth cone turning in DRG neurons were evaluated. The expression of TRPC1 protein, Ca2+ influx and activation of the PI3K/AKT signaling pathway were also investigated. The results of the present study showed that CMR significantly lowered blood glucose levels, reversed the loss of Nissl bodies, induced neurite outgrowth and restored the response of the growth cone of DRG neurons in diabetic rats. CMR exerted neurite outgrowthpromoting effects by increasing TRPC1 expression, reducing Ca2+ influx and enhancing AKT phosphorylation. siRNA targeting TRPC1 in the CMR group abrogated its antidiabetic and neuroregenerative effects, suggesting the involvement of TRPC1 in the biological effects of CMR on DPN.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Neuropatias Diabéticas/tratamento farmacológico , Morus , Neuritos/metabolismo , Crescimento Neuronal/efeitos dos fármacos , Extratos Vegetais/farmacologia , Animais , Glicemia/efeitos dos fármacos , Cálcio/metabolismo , Células Cultivadas , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Neuropatias Diabéticas/sangue , Neuropatias Diabéticas/genética , Neuropatias Diabéticas/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/crescimento & desenvolvimento , Gânglios Espinais/metabolismo , Masculino , Neuritos/efeitos dos fármacos , Neuritos/patologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Corpos de Nissl/efeitos dos fármacos , Corpos de Nissl/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Extratos Vegetais/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Canais de Cátion TRPC/genética , Canais de Cátion TRPC/metabolismo , Regulação para CimaRESUMO
Murraya koenigii is a medicinal plant that contains several carbazole-type alkaloids as its characteristic constituents. Blood-brain barrier permeable constituents of M. koenigii accelerated neurite outgrowth in PC-12 cells. Nine compounds were isolated from M. koenigii and their effects on neurite outgrowth were examined. Murrayamine-E (8) at 10 µM showed significant effect. Focusing on the carbazole skeleton, we synthesized derivatives to attenuate cytotoxicity. 9-Benzyl-9H-carbazol-4-ol (15) exhibited strong neurite outgrowth accelerative effect. In addition, the novel object recognition test and the Morris water maze test were performed to evaluate memory improvement of 15 in APdE9 mice. Compound 15 tended to improve spatial memory in the Morris water maze test. These results suggest that carbazole derivative 15 would be a seed compound for Alzheimer's disease drug.
Assuntos
Alcaloides/química , Doença de Alzheimer/tratamento farmacológico , Carbazóis/química , Murraya/química , Crescimento Neuronal/efeitos dos fármacos , Extratos Vegetais/química , Plantas Medicinais/efeitos dos fármacos , Memória Espacial/efeitos dos fármacos , Animais , Feminino , Camundongos , Células PC12 , RatosRESUMO
Pleurotus eryngii (king oyster mushroom) is a renowned culinary mushroom with various medicinal properties that may be beneficial for health maintenance and disease prevention. However, its effect on the nervous system remains elusive. In this study, hot water (PE-HWA) and ethanol (PE-ETH) extracts of P. eryngii were investigated and compared for their neuroprotective, anti-inflammatory, and neurite outgrowth activities in vitro. Based on the results, both extracts up to 400 µg/mL were nontoxic to PC12 cells and BV2 microglia (p > 0.05). Treatment with 250 µM hydrogen peroxide (H2O2) markedly (p < 0.0001) reduced the PC12 cell viability to 67.74 ± 6.47%. Coincubation with 200 µg/mL and 400 µg/mL of PE-ETH dose-dependently increased the cell viability to 85.34 ± 1.91% (p < 0.001) and 98.37 ± 6.42% (p < 0.0001) respectively, while PE-HWA showed no activity. Nitric oxide (NO) released by BV2 microglia was notably (p < 0.0001) increased by 1 µg/mL lipopolysaccharides (LPS) from 7.46 ± 0.73 µM to 80.00 ± 3.78 µM indicating an inflammatory reaction. However, coincubation with 200 and 400 µg/mL of PE-ETH significantly (p < 0.0001) reduced the NO level to 58.57 ± 6.19 µM and 52.86 ± 3.43 µM respectively, while PE-HWA was noneffective. PE-ETH and PE-HWA at 40 µg/mL significantly increased the neurite-bearing cells from 4.70 ± 3.36% to 13.12 ± 2.82% (p < 0.01) and 20.93 ± 5.37% (p < 0.0001) respectively. Pleurotus eryngii, particularly the ethanol extract (PE-ETH) and its potentially bioactive compounds, could be explored as a neurohealth promoting agent, due to its collective neuroprotective, anti-inflammatory, and neurite outgrowth activities.
Assuntos
Anti-Inflamatórios/farmacologia , Neuritos/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Extratos Vegetais/farmacologia , Pleurotus/química , Animais , Anti-Inflamatórios/isolamento & purificação , Peróxido de Hidrogênio/toxicidade , Microglia/efeitos dos fármacos , Neuritos/fisiologia , Crescimento Neuronal/efeitos dos fármacos , Fármacos Neuroprotetores/isolamento & purificação , Células PC12 , Extratos Vegetais/isolamento & purificação , RatosRESUMO
Blood-brain barrier (BBB)-permeable components in the methanolic extract of Nelumbo nucifera flowers showed accelerative effects on neurite outgrowth in PC-12 cells. Among the constituents isolated from N. nucifera flowers in our previous study, aporphine-type alkaloids, lirinidine, asimilobine, N-methylasimilobine, and pronuciferine, showed accelerative effects. Lirinidine, N-methylasimilobine, and an alkaloid-rich diethyl ether fraction at low concentrations increased the expression of mRNAs coding for TrkA, Vav3, and Rac1. In addition, good permeability of asimilobine and N-methylasimilobine was confirmed using an in vitro BBB model. Asimilobine and N-methylasimilobine are considered to be suitable as seed compounds of drugs for Alzheimer's disease, because of their activity and BBB permeability.
Assuntos
Alcaloides/farmacologia , Aporfinas/farmacologia , Barreira Hematoencefálica/efeitos dos fármacos , Nelumbo/química , Neuritos/metabolismo , Doença de Alzheimer/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Flores/química , Metanol , Crescimento Neuronal/efeitos dos fármacos , Células PC12 , Extratos Vegetais/farmacologia , Ratos , Compostos de Espiro/farmacologiaRESUMO
A new ester (1) and a terpenoid (2) were isolated from the dried whole plant of Disporopsis aspersa (HUA) ENGL. ex DIELS for the first time and their structures were elucidated, as well as their biological activities are described. The two compounds all showed good antifungal activities, especially furanone (2) exhibited better antifungal activity against Pseudoperonospora cubensis and Phytophthora infestans with EC50 value of 22.82, 18.90 µg/mL, respectively. Compound 1 exhibited a significant promotion on the neurite outgrowth in NGF-induced PC-12 cells, and moderate inhibition on the NO production induced by lipopolysaccharide (LPS) in BV-2 microglial cells.