RESUMEN
A mild ischemic stroke may cause both debilitating locomotor and cognitive decline, for which the mechanism is not fully understood, and no therapies are currently available. In this study, a nonfatal stroke model was constructed in mice by a modified middle cerebral artery occlusion (MCAO) procedure, allowing an extended recovery period up to 28 days. The extended MCAO model successfully mimicked phenotypes of a recovery phase post-stroke, including locomotor motor and cognitive deficiencies, which were effectively improved after Shuxuening injection (SXNI) treatment. Tissue slices staining showed that SXNI repaired brain injury and reduced neuronal apoptosis, especially in the hippocampus CA3 region. Transcriptomics sequencing study revealed 565 differentially expressed genes (DEGs) in the ischemic brain after SXNI treatment. Integrated network pharmacological analysis identified Neurotrophin/Trk Signaling was the most relevant pathway, which involves 15 key genes. Related DEGs were further validated by RT-PCR. Western-blot analysis showed that SXNI reversed the abnormal expression of BDNF, TrkB, Mek3 and Jnk1after stroke. ELISA found that SXNI increased brain level of p-Erk and Creb. At sub-brain level, the expression of BDNF and TrkB was decreased and GFAP was increased on the hippocampal CA3 region in the post-stroke recovery phase and this abnormality was improved by SXNI. In vitro experiments also found that oxygen glucose deprivation reduced the expression of BDNF and TrkB, which was reversed by SXNI. In summary, we conclude that SXNI facilitates the recovery of cognitive and locomotor dysfunction by modulating Neurotrophin/Trk Signaling in a mouse model for the recovery phase of post-ischemic stroke.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/efectos de los fármacos , Trastornos del Conocimiento/tratamiento farmacológico , Trastornos del Conocimiento/etiología , Medicamentos Herbarios Chinos/uso terapéutico , Hipocampo/metabolismo , Trastornos del Movimiento/tratamiento farmacológico , Trastornos del Movimiento/etiología , Factores de Crecimiento Nervioso/efectos de los fármacos , Receptor trkA/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Rehabilitación de Accidente Cerebrovascular/métodos , Accidente Cerebrovascular/complicaciones , Animales , Línea Celular , Medicamentos Herbarios Chinos/administración & dosificación , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Desempeño Psicomotor/efectos de los fármacos , Recuperación de la Función , Accidente Cerebrovascular/psicología , TranscriptomaRESUMEN
BACKGROUND: Pyrrolizidine alkaloids (PAs) are commonly found in many plants including those used in medical therapeutics. The hepatotoxicities of PAs have been demonstrated both in vivo and in vitro; however, the neurotoxicities of PAs are rarely mentioned. PURPOSE: In this study, we aimed to investigate in vitro neurotoxicities of clivorine, one of the PAs found in various Ligularia species, in cultured PC12 cells. STUDY DESIGN: PC12 cell line was employed to first elucidate the neurotoxicity and the underlying mechanism of clivorine, including cell viability and morphology change, neuronal differentiation marker and signaling pathway. METHODS: PC12 cells were challenged with series concentrations of clivorine and/or nerve growth factor (NGF). The cell lysates were collected for MTT assay, trypan blue staining, immunocytofluorescent staining, qRT-PCR and western blotting. RESULTS: Clivorine inhibited cell proliferation and neuronal differentiation evidenced by MTT assay and dose-dependently reducing neurite outgrowth, respectively. In addition, clivorine decreased the level of mRNAs encoding for neuronal differentiation markers, e.g. neurofilaments and TrkA (NGF receptor). Furthermore, clivorine reduced the NGF-induced the phosphorylations of TrkA, protein kinase B and cAMP response element-binding protein in cultured PC12 cells. CONCLUSION: Taken together, our results suggest that clivorine might possess neurotoxicities in PC12 cells via down-regulating the NGF/TrkA/Akt signaling pathway. PAs not only damage the liver, but also possess neurotoxicities, which could possibly result in brain disorders, such as depression.
Asunto(s)
Asteraceae/química , Factor de Crecimiento Nervioso/farmacología , Neuronas/efectos de los fármacos , Alcaloides de Pirrolicidina/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Proteína Oncogénica v-akt/efectos de los fármacos , Células PC12 , Fosforilación , Ratas , Receptor trkA/efectos de los fármacosRESUMEN
p75(NTR) is a neurotrophin receptor that can mediate either survival or death of neurons depending on the cell context. Modulation of p75(NTR) is a promising strategy to promote neuronal survival for treatment of cognitive disorders such as Alzheimer's disease. Despite years of investigation into the signaling mechanisms of p75(NTR), no p75(NTR) signaling assay has yet been developed that is compatible with efficient screening of small-molecule modulators. In this work, we developed a homogeneous cell-based assay for screening p75(NTR) modulators and studying p75(NTR) function. Stimulation of p75(NTR)-transfected cells using either nerve growth factor (NGF) or Pro-NGF resulted in an enhanced caspase-3 activity as assessed by cleavage of a fluorescent caspase-3 substrate. Optimization of the assay with respect to time, cell density, NGF and Pro-NGF concentration, and other factors provided a twofold increase in the caspase-3 activity compared to background. Withdrawal of serum during the NGF or Pro-NGF treatment period was found to be essential for p75(NTR)-dependent caspase-3 activation. We validated the method by demonstrating that a signaling-incompetent p75(NTR) mutant could not substitute for wild-type p75(NTR) in mediating caspase-3 activation. A focused library screen identified new inhibitors of p75(NTR) signaling. This method will be useful for identifying small-molecule modulators of p75(NTR) as well as further characterizing downstream signaling events.
Asunto(s)
Caspasa 3/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Activación Enzimática/efectos de los fármacos , Proteínas del Tejido Nervioso/fisiología , Receptores de Factor de Crecimiento Nervioso/fisiología , Transducción de Señal/efectos de los fármacos , Animales , Supervivencia Celular/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Células HEK293 , Humanos , Microscopía Fluorescente , Factor de Crecimiento Nervioso/farmacología , Ratas , Receptor trkA/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas , TransfecciónRESUMEN
Targeted gene delivery to diseased subtypes of neurons will be beneficial to the success of gene therapy of neurological disorders. We designed a recombinant cationic polypeptide to facilitate gene delivery to neuronal-like PC12 cells that express the nerve growth factor (NGF) receptors. The recombinant polypeptide was composed of a targeting moiety derived from loop 4-containing hairpin motif of NGF and a DNA-binding moiety of 10-lysine sequence and expressed in Escherichia coli. It activated NGF receptor, TrkA and its downstream signaling pathways in PC12 and promoted the survival of neuronally differentiated PC12 cells deprived of serum. The polypeptide could also bind plasmid DNA and enhance polycation-mediated gene delivery in NGF receptor-expressing PC12 cells, but not in COS7 cells lacking NGF receptors. The enhancement of gene transfer in PC12 was inhibited by pretreatment of free, unbound polypeptides, suggesting a NGF-receptor-specific effect of the polypeptide. These observations demonstrated the concept of using receptor-mediated mechanism for targeted gene delivery to neurons.
Asunto(s)
ADN Recombinante/administración & dosificación , Marcación de Gen/métodos , Células PC12/efectos de los fármacos , Receptor trkA/efectos de los fármacos , Proteínas Recombinantes de Fusión/farmacología , Transfección/métodos , Secuencias de Aminoácidos , Animales , Células COS/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , ADN Recombinante/farmacología , Portadores de Fármacos , Evaluación Preclínica de Medicamentos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Especificidad de Órganos , Células PC12/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Ratas , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/química , Transducción de Señal/efectos de los fármacosRESUMEN
Melatonin is an endogenously generated potent antioxidant. Our previous studies indicate that melatonin improved learning and memory deficits in APP695 transgenic mouse of Alzheimer's disease. An ovariectomized (OVX) rat model which is characterized by progressive memory deficits, central cholinergic nerve system degeneration and differentiation/apoptosis imbalance is the ideal in vivo model in which to test the neuroprotective effects of melatonin. OVX Sprague-Dawley rats received daily injections of melatonin (5, 10 and 20 mg/kg) or 17 beta-estradiol (E2, 80 microg/kg) or sesame oil for 16 wk. Morris water maze results showed that ovarian steroid deprivation resulted in spatial memory impairment, while melatonin and E2 significantly ameliorated spatial memory deficits in OVX rats. The latency to find the hidden platform and the distance to reach the platform become shorter in both melatonin and E2-treated rats compared with those that were only OVX. Four months after OVX, the choline acetyltransferase activity in the frontal cortex and hippocampus were greatly decreased in comparison with the controls. Melatonin and E2 antagonized the effects induced by OVX. Interestingly, the activity of the acetylcholinesterase was not altered in any group of rats. DNA fragmentation was presented in the front cortex of the OVX rats. Melatonin and E2 reduced the number of apoptotic neurons. These findings demonstrate the important effects of melatonin and E2 on cholinergic neurons and support the potential application of melatonin in the treatment of dementia in postmenopausal women. Our results indicate that neuroprotection by melatonin partly correlated to modulation of apoptosis and protection of the cholinergic system. Early long-term melatonin application is a promising strategy which could potentially be applied in a clinic setting.
Asunto(s)
Enfermedad de Alzheimer/fisiopatología , Trastornos del Conocimiento/tratamiento farmacológico , Estradiol/farmacología , Melatonina/farmacología , Trastornos de la Memoria/tratamiento farmacológico , Enfermedad de Alzheimer/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Colina O-Acetiltransferasa/efectos de los fármacos , Colina O-Acetiltransferasa/metabolismo , Trastornos del Conocimiento/patología , Modelos Animales de Enfermedad , Femenino , Lóbulo Frontal/efectos de los fármacos , Lóbulo Frontal/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Aprendizaje por Laberinto/efectos de los fármacos , Trastornos de la Memoria/patología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Ovariectomía , Posmenopausia , Ratas , Ratas Sprague-Dawley , Receptor trkA/efectos de los fármacos , Receptor trkA/metabolismoRESUMEN
Three new macrocyclic diterpenes, kansuinins F (1), G (2), and H (3), together with four known jatrophane diterpenes, kansuinins D (4), E (5), and A (6) and 3beta,5alpha,7beta,15beta-tetraacetoxy-9alpha-nicotinoyloxyjatropha-6(17)-11E-dien-14-one, were isolated from the roots of Euphorbia kansui. Compounds 1 and 2 were assigned as 6(17)-en-11,12-epoxy-14-one-type jatrophane diterpenes, and compound 3 as a 6(17)-en-11,14-epoxy-12-one jatrophane diterpene. The structures of compounds 1-3 and the relative configurations of compounds 4 and 5 were determined by spectral data analysis. Kansuinin E (5) exhibited a specific survival effect on fibroblasts that expressed TrkA, a high-affinity receptor for nerve growth factor.
Asunto(s)
Diterpenos/aislamiento & purificación , Euphorbia/química , Plantas Medicinales/química , Diterpenos/química , Diterpenos/farmacología , Fibroblastos/metabolismo , Medicina Tradicional China , Estructura Molecular , Factor de Crecimiento Nervioso/efectos de los fármacos , Raíces de Plantas/química , Receptor trkA/efectos de los fármacosRESUMEN
Factors limiting the therapeutic application of neurotrophins to neurodegenerative diseases include poor stability and CNS penetration. Moreover, certain neurotrophin effects, such as promotion of neuronal death via interaction with the p75NTR receptor, might further limit their application. We have proposed that development of small molecule mimetics of neurotrophins might serve to overcome these limitations. In previous work, our laboratory established the proof-of-principle that mimetics of specific nerve growth factor (NGF) domains could prevent neuronal death. Peptidomimetics of the loop 1 domain prevent death via p75NTR-dependent signaling and peptidomimetics of the loop 4 domain prevent death via Trk-related signaling. In current work we are designing pharmacophore queries corresponding to loop domains 1 or 4 that incorporate features of the NGF crystal structure along with features derived from peptidomimetic structure-activity-relationships. Screening of in silico databases containing non-peptide, small molecules has identified a number of candidate NGF domain mimetics. Preliminary assessment of these compounds using neurotrophin bioassays indicates that several are capable of preventing neuronal death. Ongoing studies will determine whether these compounds act via p75NTR or Trk receptors.