RESUMEN
The prevalence of Alzheimer's disease (AD) is increasing globally due to population aging. However, effective clinical treatment strategies for AD still remain elusive. The mechanisms underlying AD onset and the interplay between its pathological factors have so far been unclear. Evidence indicates that AD progression is ultimately driven by neuronal loss, which in turn is caused by neuroapoptosis and neuroinflammation. Therefore, the inhibition of neuroapoptosis and neuroinflammation could be a useful anti-AD strategy. Nonetheless, the delivery of active drug agents into the brain parenchyma is hindered by the blood-brain barrier (BBB). To address this challenge, we fabricated a black phosphorus nanosheet (BP)-based methylene blue (MB) delivery system (BP-MB) for AD therapy. After confirming the successful preparation of BP-MB, we proved that its BBB-crossing ability was enhanced under near-infrared light irradiation. In vitro pharmacodynamics analysis revealed that BP and MB could synergistically scavenge excessive reactive oxygen species (ROS) in okadaic acid (OA)-treated PC12 cells and lipopolysaccharide (LPS)-treated BV2 cells, thus efficiently reversing neuroapoptosis and neuroinflammation. To study in vivo pharmacodynamics, we established a mouse model of AD mice, and behavioral tests confirmed that BP-MB treatment could successfully improve cognitive function in these animals. Notably, the results of pathological evaluation were consistent with those of the in vitro assays. The findings demonstrated that BP-MB could scavenge excessive ROS and inhibit Tau hyperphosphorylation, thereby alleviating downstream neuroapoptosis and regulating the polarization of microglia from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype. Overall, this study highlights the therapeutic potential of a smart nanomedicine with the capability of reversing neuroapoptosis and neuroinflammation for AD treatment.
Asunto(s)
Enfermedad de Alzheimer , Apoptosis , Barrera Hematoencefálica , Azul de Metileno , Nanomedicina , Enfermedades Neuroinflamatorias , Animales , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/patología , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Apoptosis/efectos de los fármacos , Células PC12 , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Ratas , Ratones , Nanomedicina/métodos , Azul de Metileno/farmacología , Azul de Metileno/uso terapéutico , Masculino , Especies Reactivas de Oxígeno/metabolismo , Ratones Endogámicos C57BLRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Melastoma dodecandrum Lour. (MD), a traditional Chinese medicine used by the She ethnic group, has been used to treat cerebral ischemia-reperfusion (CIR) injury due to its efficacy in promoting blood circulation and removing blood stasiss; however, the therapeutic effects and mechanisms of MD in treating CIR injury remain unclear. AIM: To investigate the protective effects of MD on CIR injury, in addition to its impact on oxidative stress, endoplasmic reticulum (ER) stress, and cell apoptosis. MATERIALS AND METHODS: The research was conducted using both cell experiments and animal experiments. The CCK-8 method, immunofluorescence staining, and flow cytometry were used to analyze the effects of MD-containing serum on oxygen-glucose deprivation/reperfusion (OGD/R)-induced PC12 cell viability, reactive oxygen species (ROS) clearance, anti-inflammatory, neuroprotection and inhibition of apoptosis. Furthermore, 2,3,5-Triphenyl tetrazolium chloride staining, hematoxylin and eosin staining, Nissl staining, and immunohistochemistry were used to detect infarct size, pathological changes, Nissl corpuscula and neuronal protein expression in middle cerebral artery occlusion (MCAO) rats. Polymerase chain reaction and Western Blotting were conducted in cell and animal experiments to detect the expression levels of ER stress-related genes and proteins. RESULTS: The MD extract enhanced the viability of PC12 cells under OGD/R modeling, reduced ROS and IL-6 levels, increased MBP levels, and inhibited cell apoptosis. Furthermore, MD improved the infarct area in MCAO rats, increased the number of Nissl bodies, and regulated neuronal protein levels including Microtubule-Associated Protein 2 (MAP-2), Myelin Basic Protein (MBP), Glial Fibrillary Acidic Protein (GFAP), and Neurofilament 200 (NF200). Additionally, MD could regulate the expression levels of oxidative stress proteins malondialdehyde (MDA), nitric oxide (NO), superoxide dismutase (SOD), and catalase (CAT). Both cell and animal experiments demonstrated that MD could inhibit ER stress-related proteins (GRP78, ATF4, ATF6, CHOP) and reduce cell apoptosis. CONCLUSION: This study confirmed that the therapeutic mechanism of the MD extract on CIR injury was via the inhibition of oxidative stress and the ER stress pathway, in addition to the inhibition of apoptosis.
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Apoptosis , Estrés del Retículo Endoplásmico , Fármacos Neuroprotectores , Estrés Oxidativo , Ratas Sprague-Dawley , Daño por Reperfusión , Animales , Estrés del Retículo Endoplásmico/efectos de los fármacos , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/metabolismo , Daño por Reperfusión/prevención & control , Estrés Oxidativo/efectos de los fármacos , Ratas , Células PC12 , Masculino , Fármacos Neuroprotectores/farmacología , Apoptosis/efectos de los fármacos , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Especies Reactivas de Oxígeno/metabolismo , Supervivencia Celular/efectos de los fármacos , Isquemia Encefálica/tratamiento farmacológico , Extractos Vegetales/farmacología , Extractos Vegetales/química , Medicamentos Herbarios Chinos/farmacología , Medicamentos Herbarios Chinos/uso terapéuticoRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: The incidence and mortality of cerebrovascular diseases are increasing year by year. Cerebral ischemia-reperfusion injury (CIRI) is common in patients with ischemic stroke. Naoxintong (NXT) is composed of a variety of Chinese medicines and has the ability to treat CIRI. AIM OF THE STUDY: The aim of this study is to investigate whether NXT regulates mitophagy in CIRI based on network pharmacology analysis and experimental validation. MATERIALS AND METHODS: Oxygen and glucose deprivation/re-oxygenation (OGD/R, 2/22 h) model of PC12 cells and transient middle cerebral artery occlusion (tMCAO, 2/22 h) model of rats were established. Pharmacodynamic indicators include neurological deficit score, 2,3,5-triphenyte-trazoliumchloride (TTC) staining, hematoxylin-eosin (HE) staining and cell viability. Network pharmacology was used to predict pharmacological mechanisms. Pharmacological mechanism indexes include transmission electron microscopy (TEM), drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), immunohistochemistry (IHC), western blot (WB) and immunofluorescence (IF). Kevetrin (an agonists of p53) and pifithrin-α (an inhibitor of p53) used to detect the key role of p53 in mitophagy of NXT. RESULTS: NXT (1% serum containing NXT and 110 mg/kg) improved the damage of OGD/R PC12 cells and tMCAO rats, and this protective effect was related to the anti-oxidation and ability to promote mitophagy of NXT. NXT and pifithrin-α increased the expression of promoting-mitophagy targets (PINK1, PRKN and LC3B) and inhibited the expression of inhibiting-mitophagy targets (p52) via restraining p53, and finally accelerated mitophagy caused by CIRI. CONCLUSION: This study demonstrates that NXT promotes mitophagy in CIRI through restraining p53 and promoting PINK1/PRKN in vivo and in vitro.
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Medicamentos Herbarios Chinos , Mitofagia , Farmacología en Red , Proteínas Quinasas , Daño por Reperfusión , Proteína p53 Supresora de Tumor , Animales , Masculino , Ratas , Isquemia Encefálica/tratamiento farmacológico , Medicamentos Herbarios Chinos/farmacología , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/patología , Mitofagia/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Células PC12 , Proteínas Quinasas/metabolismo , Ratas Sprague-Dawley , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína LigasasRESUMEN
BACKGROUND: Dysfunction of the cholinergic system and increased oxidative stress have a crucial role in cognitive disorders including Alzheimer's disease (AD). Here, we have investigated the protective effects of betanin, a novel acetylcholinesterase (AChE) inhibitor, on hydrogen peroxide (H2O2)-induced cell death in PC12 cells. METHODS AND RESULTS: The protective effects were assessed by measuring cell viability, the amount of reactive oxygen species (ROS) production, AChE activity, cell damage, and apoptosis using resazurin, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), Ellman method, lactate dehydrogenase (LDH) release, propidium iodide (PI) staining and flow cytometry, and Western blot analysis. H2O2 (150 µM) resulted in cell viability reduction and apoptosis induction while, pretreatment with the betanin (10, 20, and 50 µM) and N-Acetyl-L-cysteine (NAC) (2.5 and 5 mM) significantly increased the viability (P < 0.05, P < 0.01 and P < 0.001) and at 5-50 µM betanin decreased ROS amount (P < 0.05, P < 0.01 and P < 0.001). Whereas, pretreatment with the betanin (10, 20, and 50 µM) decreased AChE activity (P < 0.001), also at 20 and 50 µM betanin reduced the release of LDH (P < 0.001), and at 10-50 µM decreased the percentage of apoptotic cells (P < 0.001). Apoptosis biomarkers such as cleaved poly (ADP-ribose) polymerase (PARP) (P < 0.01 and P < 0.001) and cytochrome c (P < 0.05 and P < 0.001) were attenuated after pretreatment of PC12 cells with betanin at 10-20 µM and 10-50 µM respectively. Indeed, survivin (P < 0.001) increased after pretreatment of cells with betanin at 10-20 µM. CONCLUSIONS: Overall, betanin may use the potential to delay or prevent cell death caused by AD through decreasing the activity of AChE as well as attenuating the expression of proteins involved in the apoptosis pathway.
Asunto(s)
Acetilcolinesterasa , Apoptosis , Betacianinas , Supervivencia Celular , Inhibidores de la Colinesterasa , Peróxido de Hidrógeno , Estrés Oxidativo , Especies Reactivas de Oxígeno , Células PC12 , Animales , Ratas , Apoptosis/efectos de los fármacos , Inhibidores de la Colinesterasa/farmacología , Peróxido de Hidrógeno/farmacología , Especies Reactivas de Oxígeno/metabolismo , Betacianinas/farmacología , Supervivencia Celular/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Acetilcolinesterasa/metabolismo , Fármacos Neuroprotectores/farmacologíaRESUMEN
Neonatal hypoxic-ischemic encephalopathy (HIE) is a severe disease with a poor prognosis, whose clinical treatment is still limited to therapeutic hypothermia with limited efficacy. Perillyl alcohol (POH), a natural monoterpene found in various plant essential oils, has shown neuroprotective properties, though its effects on HIE are not well understood. This study investigates the neuroprotective effects of POH on HIE both in vitro and in vivo. We established an in vitro model using glucose deprivation and hypoxia/reperfusion (OGD/R) in PC12 cells, alongside an in vivo model via the modified Rice-Vannucci method. Results indicated that POH acted as an indirect antioxidant, reducing inducible nitric oxide synthase and malondialdehyde production, maintaining content of antioxidant molecules and enzymes in OGD/R-induced PC12 cells. In vivo, POH remarkably lessened infarct volume, reduced cerebral edema, accelerated tissue regeneration, and blocked reactive astrogliosis after hypoxic-ischemic brain injury. POH exerted antiapoptotic activities through both the intrinsic and extrinsic apoptotic pathways. Mechanistically, POH activated Nrf2 and inactivated its negative regulator Keap1. The use of ML385, a Nrf2 inhibitor, reversed these effects. Overall, POH mitigates neuronal damage in HIE by combating oxidative stress, reducing inflammation, and inhibiting apoptosis via the Nrf2/Keap1 pathway, suggesting its potential for HIE treatment.
Asunto(s)
Animales Recién Nacidos , Hipoxia-Isquemia Encefálica , Proteína 1 Asociada A ECH Tipo Kelch , Monoterpenos , Factor 2 Relacionado con NF-E2 , Fármacos Neuroprotectores , Transducción de Señal , Animales , Factor 2 Relacionado con NF-E2/metabolismo , Ratas , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Hipoxia-Isquemia Encefálica/tratamiento farmacológico , Hipoxia-Isquemia Encefálica/metabolismo , Transducción de Señal/efectos de los fármacos , Monoterpenos/farmacología , Monoterpenos/uso terapéutico , Células PC12 , Estrés Oxidativo/efectos de los fármacos , Ratas Sprague-Dawley , Antioxidantes/farmacología , Apoptosis/efectos de los fármacosRESUMEN
Cytopathology induced by methamphetamine (METH) is reminiscent of degenerative disorders such as Parkinson's disease, and it is characterized by membrane organelles arranged in tubulo-vesicular structures. These areas, appearing as clusters of vesicles, have never been defined concerning the presence of specific organelles. Therefore, the present study aimed to identify the relative and absolute area of specific membrane-bound organelles following a moderate dose (100 µM) of METH administered to catecholamine-containing PC12 cells. Organelles and antigens were detected by immunofluorescence, and they were further quantified by plain electron microscopy and in situ stoichiometry. This analysis indicated an increase in autophagosomes and damaged mitochondria along with a decrease in lysosomes and healthy mitochondria. Following METH, a severe dissipation of hallmark proteins from their own vesicles was measured. In fact, the amounts of LC3 and p62 were reduced within autophagy vacuoles compared with the whole cytosol. Similarly, LAMP1 and Cathepsin-D within lysosomes were reduced. These findings suggest a loss of compartmentalization and confirm a decrease in the competence of cell clearing organelles during catecholamine degeneration. Such cell entropy is consistent with a loss of energy stores, which routinely govern appropriate subcellular compartmentalization.
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Autofagosomas , Lisosomas , Metanfetamina , Metanfetamina/farmacología , Animales , Células PC12 , Ratas , Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Autofagosomas/metabolismo , Autofagosomas/efectos de los fármacos , Autofagia/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Catepsina D/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismoRESUMEN
BACKGROUND: Atrazine (ATR), a commonly used herbicide, is linked to dopaminergic neurotoxicity, which may cause symptoms resembling Parkinson's disease (PD). This study aims to reveal the molecular regulatory networks responsible for ATR exposure and its effects on dopaminergic neurotoxicity based on an integration strategy. METHODS: Our approach involved network toxicology, construction of protein-protein interaction (PPI) networks, gene ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, as well as molecular docking techniques. Subsequently, we validated the predicted results in PC12 cells in vitro. RESULTS: An integrated analysis strategy indicating that 5 hub targets, including mitogen-activated protein kinase 3 (Mapk3), catalase (Cat), heme oxygenase 1 (Hmox1), tumor protein p53 (Tp53), and prostaglandin-endoperoxide synthase 2 (Ptgs2), may play a crucial role in ATR-induced dopaminergic injury. Molecular docking indicated that the 5 hub targets exhibited certain binding activity with ATR. Cell counting kit-8 (CCK8) results illustrated a dose-response relationship in PC12 cells. Real-time quantitative polymerase chain reaction (RT-qPCR) displayed notable changes in the expression of hub targets mRNA levels, with the exception of Mapk3. Western blotting results suggested that ATR treatment in PC12 cells resulted in an upregulation of the Cat, Hmox1, and p-Mapk3 protein expression levels while causing a downregulation in Tp53, Ptgs2, and Mapk3. CONCLUSION: Our findings indicated that 5 hub targets identified could play a vital role in ATR-induced dopaminergic neurotoxicity in PC12 cells. These results provide preliminary support for further investigation into the molecular mechanism of ATR-induced toxicity.
Asunto(s)
Atrazina , Neuronas Dopaminérgicas , Herbicidas , Simulación del Acoplamiento Molecular , Atrazina/toxicidad , Animales , Células PC12 , Ratas , Herbicidas/toxicidad , Neuronas Dopaminérgicas/efectos de los fármacos , Mapas de Interacción de Proteínas , Dopamina/metabolismoRESUMEN
Elemental analysis at the single-cell level is an emerging technique in the field of inductively coupled plasma mass spectrometry (ICP-MS). In comparison to the analysis of cell suspensions by fast time-resolved analysis, single-cell sampling by laser ablation (LA) allows the discriminatory analysis of single cells according to their size and morphology. In this study, we evaluated the changes in elemental contents in a single cell through differentiation of rat adrenal pheochromocytoma into neuron-like cells by LA-ICP-MS. The contents of seven essential minerals were increased about 2-3 times after the differentiation. In addition, we evaluated the degree of differentiation at the single-cell level by means of imaging cytometry after immunofluorescence staining of microtubule-associated protein 2 (Map2), a neuron-specific protein. The fluorescence intensities of Alexa Fluor 488-conjugated secondary antibody against the anti-Map2 primary antibody showed large variations among the cells after the onset of differentiation. Although the average fluorescence intensity was increased through the differentiation, there were still less-matured neuron-like cells that exhibited a lower fluorescence intensity after 5 days of differentiation. Since a positive correlation between the fluorescence intensity and the cell size in area was found, we separately measured the elemental contents in the less-matured smaller cells and well-matured larger cells by LA-ICP-MS. The larger cells had higher elemental contents than the smaller cells, indicating that the essential minerals are highly required at a later stage of differentiation.
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Diferenciación Celular , Espectrometría de Masas , Análisis de la Célula Individual , Animales , Ratas , Análisis de la Célula Individual/métodos , Células PC12 , Espectrometría de Masas/métodos , Terapia por Láser/métodos , Neuronas/química , Neuronas/citología , Minerales/análisis , Minerales/químicaRESUMEN
Oltipraz (OPZ) is a synthetic dithiolethione and is considered a novel activator of nuclear factor E2-related factor 2 (Nrf2). Increasing evidence indicates that Nrf2 protects against cerebral ischemia/reperfusion (I/R) injury by antagonizing ferroptosis and lipid peroxidation. However, the protective effects of OPZ on cerebral I/R injury remain to be elucidated. We investigated the in vitro and in vivo neuroprotective effects of OPZ. Mice were subjected to middle cerebral artery occlusion/reperfusion (MCAO/R) to construct an in vivo model and PC12 cells were exposed to oxygen and glucose deprivation/reoxygenation (OGD/R) to establish an in vitro model. OPZ administration reduced the infarct volume and brain water content, and alleviated the neurological deficit of MCAO/R mice. Moreover, OPZ ameliorated MCAO/R-induced oxidative stress by decreasing the levels of 4-HNE and MDA and increasing the activities of SOD and GSH. We also found that OPZ ameliorated MCAO/R-induced ferroptosis by increasing SLC7A11 and GPX4 protein expression and downregulating ACSL4 protein expression. Similarly, the in vitro results revealed that OGD/R-induced oxidative stress and ferroptosis. Finally, mechanistic analysis revealed that OPZ significantly upregulated the Nrf2 expression and Nrf2 knockout (Nrf2 KO) abolished the OPZ-mediated protective effects. Taken together, these findings demonstrate that OPZ ameliorates cerebral I/R injury by suppressing the oxidative stress and ferroptosis.
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Ferroptosis , Infarto de la Arteria Cerebral Media , Ratones Endogámicos C57BL , Factor 2 Relacionado con NF-E2 , Fármacos Neuroprotectores , Estrés Oxidativo , Daño por Reperfusión , Tionas , Tiofenos , Animales , Ferroptosis/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/metabolismo , Tionas/farmacología , Tionas/uso terapéutico , Células PC12 , Factor 2 Relacionado con NF-E2/metabolismo , Ratones , Masculino , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Tiofenos/farmacología , Tiofenos/uso terapéutico , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/patología , Ratas , Coenzima A Ligasas/metabolismo , Coenzima A Ligasas/genética , Sistema de Transporte de Aminoácidos y+/metabolismo , Sistema de Transporte de Aminoácidos y+/genética , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Fosfolípido Hidroperóxido Glutatión Peroxidasa/genética , Modelos Animales de Enfermedad , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/patología , Encéfalo/metabolismo , PirazinasRESUMEN
EXPRESSION OF CONCERN: J.-Z. Yu, J. Kuret, and M. M. Rasenick, "Transient Expression of Fluorescent Tau Proteins Promotes Process Formation in PC12 Cells: Contributions of the Tau C-terminus to This Process," Journal of Neuroscience Research 67, no. 5 (2002): 625-633, https://doi.org/10.1002/jnr.10152. This Expression of Concern for the above article published online on 16 January 2002, in Wiley Online Library (wileyonlinelibrary.com), has been published by agreement between the journal Editors-in-Chief, Cristina A. Ghiani and J. Paula Warrington; and Wiley Periodicals LLC. The Expression of Concern has been agreed following concerns raised regarding suspected duplication between the two images, Tau23-GFP (72 hours) presented in Figure 4a and Tau 24 (174-383)-GFP (24 hours) presented in Figure 5a. The authors acknowledge the duplication but due to the length of time that has elapsed since the study was conducted and published, they were unable to provide an explanation or the original data. The journal has decided to issue an Expression of Concern to alert the readers.
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Proteínas tau , Proteínas tau/metabolismo , Proteínas tau/genética , Células PC12 , Animales , Ratas , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Neuritas/metabolismoRESUMEN
Spinal cord injury (SCI) is one of the most serious health problems, with no effective therapy. Recent studies indicate that Fisetin, a natural polyphenolic flavonoid, exhibits multiple functions, such as life-prolonging, antioxidant, antitumor, and neuroprotection. However, the restorative effects of Fisetin on SCI and the underlying mechanism are still unclear. In the present study, we found that Fisetin reduced LPS-induced apoptosis and oxidative damage in PC12 cells and reversed LPS-induced M1 polarization in BV2 cells. Additionally, Fisetin safely and effectively promoted the motor function recovery of SCI mice by attenuating neurological damage and promoting neurogenesis at the lesion. Moreover, Fisetin administration inhibited glial scar formation, modulated microglia/macrophage polarization, and reduced neuroinflammation. Network pharmacology, RNA-seq, and molecular biology revealed that Fisetin inhibited the activation of the JAK2/STAT3 signaling pathway. Notably, Colivelin TFA, an activator of JAK2/STAT3 signaling, attenuated Fis-mediated neuroinflammation inhibition and therapeutic effects on SCI mice. Collectively, Fisetin promotes functional recovery after SCI by inhibiting microglia/macrophage M1 polarization and the JAK2/STAT3 signaling pathway. Thus, Fisetin may be a promising therapeutic drug for the treatment of SCI.
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Flavonoles , Janus Quinasa 2 , Macrófagos , Microglía , Factor de Transcripción STAT3 , Transducción de Señal , Traumatismos de la Médula Espinal , Animales , Humanos , Masculino , Ratones , Ratas , Polaridad Celular/efectos de los fármacos , Flavonoides/farmacología , Flavonoides/administración & dosificación , Flavonoles/farmacología , Janus Quinasa 2/metabolismo , Janus Quinasa 2/genética , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Microglía/metabolismo , Microglía/inmunología , Células PC12 , Recuperación de la Función/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/genética , Traumatismos de la Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/inmunología , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/genéticaRESUMEN
The therapy of large defects in peripheral nerve injury (PNI) suffers from several drawbacks, especially the lack of autologous nerve donors. Nerve conduits are considered as a solution for nerve injury treatment, but biocompatibility improvements is still required for conduits prepared with synthetic materials. Cell-derived extracellular matrix (ECM) has drawn attention due to its lower risk of immunogenic response and independence from donor availability. The goal of this study is to coat bone mesenchymal stem cell-derived ECMs on poly(lactic-co-glycolic) acid (PLGA) conduits to enhance their ability to support neural growth and neurite extensions. The ECM-coated conduits have better hydrophilic properties than the pure PLGA conduits. A marked increase on PC12 and RSC96 cells' viability, proliferation and dorsal root ganglion neurite extension was observed. Quantitative PCR analysis exhibited a significant increase in markers for cell proliferation (GAP43), neurite extension (NF-H, MAP2, andßIII-tubulin) and neural function (TREK-1). These results show the potential of ECM-coated PLGA conduits in PNI therapy.
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Proliferación Celular , Supervivencia Celular , Matriz Extracelular , Células Madre Mesenquimatosas , Regeneración Nerviosa , Neuritas , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Animales , Ratas , Neuritas/metabolismo , Células PC12 , Matriz Extracelular/metabolismo , Células Madre Mesenquimatosas/citología , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Regeneración Nerviosa/efectos de los fármacos , Andamios del Tejido/química , Materiales Biocompatibles Revestidos/química , Materiales Biocompatibles Revestidos/farmacología , Ganglios Espinales , Traumatismos de los Nervios Periféricos/terapia , Ingeniería de Tejidos/métodos , Polímeros/química , Ensayo de MaterialesRESUMEN
A series of scutellarein 7-l-amino acid carbamate-4'-cycloalkylamine propyl ether conjugates were designed and synthesized for the first time as multifunctional agents for Alzheimer's disease (AD) therapy. The designed compounds exhibited more balanced and effective multi-target potency. Among them, compound 11l, l-Valine carbamate derivative of scutellarein cycloheptylamine ether, exhibited the most potent inhibition of electric eel AChE enzymes and human AChE enzymes, with an IC50 values of 7.04 µM and 9.73 µM, respectively. Moreover, 11l exhibited more potent H3R antagonistic activities than clobenpropit, with an IC50 value of 1.09 nM. Compound 11l not only displayed excellent inhibition of self- and Cu2+-induced Aß1-42 aggregation (95.48 % and 88.63 % inhibition, respectively) but also induced the disassembly of self- and Cu2+-induced Aß fibrils (80.16 % and 89.30 % disaggregation, respectively). Moreover, 11l significantly reduced tau protein hyperphosphorylation induced by Aß25-35. It exhibited effective antioxidant activity and neuroprotective potency, and inhibited RSL3-induced PC12 cell ferroptosis. Assays of hCMEC/D3 and hPepT1-MDCK cell line permeability indicated that 11l would have optimal blood-brain barrier permeability and intestinal absorption characteristics. In addition, in vivo studies revealed that compound 11l significantly attenuated learning and memory impairment in an AD mouse model. Finally, a pharmacokinetic characterization of 11l indicated favorable druggability and pharmacokinetic properties. Taken together, our results suggest that 11l is a potential candidate for AD treatment and merits further investigation.
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Acetilcolinesterasa , Enfermedad de Alzheimer , Apigenina , Inhibidores de la Colinesterasa , Antagonistas de los Receptores Histamínicos H3 , Receptores Histamínicos H3 , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Inhibidores de la Colinesterasa/farmacología , Inhibidores de la Colinesterasa/química , Inhibidores de la Colinesterasa/síntesis química , Humanos , Animales , Relación Estructura-Actividad , Acetilcolinesterasa/metabolismo , Ratones , Antagonistas de los Receptores Histamínicos H3/farmacología , Antagonistas de los Receptores Histamínicos H3/química , Antagonistas de los Receptores Histamínicos H3/síntesis química , Ligandos , Apigenina/farmacología , Apigenina/química , Apigenina/síntesis química , Receptores Histamínicos H3/metabolismo , Estructura Molecular , Relación Dosis-Respuesta a Droga , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/antagonistas & inhibidores , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/síntesis química , Electrophorus , Ratas , Fragmentos de Péptidos/metabolismo , Masculino , Células PC12RESUMEN
Plant-derived exosome-like nanovesicles (ELNs) are nano-sized vesicles extracted from edible plants. Lycium ruthenicum Murray (LRM) has been gaining increasing attention due to its nutritional and medicinal value, but the ELNs in LRM has not been reported. In this study, LRM-ELNs were obtained, and the proteins, lipids, microRNAs (miRNAs) and active components in LRM tissues and LRM-ELNs was analyzed by LC-MS/MS, LC-MS, high-throughput sequencing techniques, and physical and chemical analysis. LRM-ELNs can be uptaken by PC12 cells through macropinocytosis and caveolin-mediated endocytosis primarily. Transcriptomic and western blot experiments indicate that LRM-ELNs can inhibit Aß-induced apoptosis in PC12 cells through the MAPK and PI3K/AKT signaling pathways, with miRNAs playing a crucial role. These results indicate that LRM-ELNs have the protection effect on PC12 cells and can be considered as dietary supplements for alleviating neurodegenerative diseases.
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Péptidos beta-Amiloides , Apoptosis , Exosomas , Lycium , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Animales , Células PC12 , Apoptosis/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Exosomas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Lycium/química , Transducción de Señal/efectos de los fármacos , Péptidos beta-Amiloides/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , MicroARNs/genética , MicroARNs/metabolismoRESUMEN
OBJECTIVE: This study aimed to establish a neural cell injury model in vitro by stimulating PC12 cells with lipopolysaccharide (LPS) and to examine the effects of astragaloside IV on key targets using high-throughput sequence technology and bioinformatics analyses. METHODS: PC12 cells in the logarithmic growth phase were treated with LPS at final concentrations of 0.25, 0.5, 0.75, 1, and 1.25 mg/mL for 24 h. Cell morphology was evaluated, and cell survival rates were calculated. A neurocyte inflammatory model was established with LPS treatment, which reached a 50% cell survival rate. PC12 cells were treated with 0.01, 0.1, 1, 10, or 100 µmol/L astragaloside IV for 24 h. The concentration of astragaloside IV that did not affect the cell survival rate was selected as the treatment group for subsequent experiments. NOS activity was detected by colorimetry; the expression levels of ERCC2, XRCC4, XRCC2, TNF-α, IL-1ß, TLR4, NOS and COX-2 mRNA and protein were detected by RT-qPCR and Western blotting. The differentially expressed genes (DEGs) between the groups were screened using a second-generation sequence (fold change>2, P<0.05) with the following KEGG enrichment analysis, RT-qPCR and Western blotting were used to detect the mRNA and protein expression of DEGs related to the IL-17 pathway in different groups of PC12 cells. RESULTS: The viability of PC12 cells was not altered by treatment with 0.01, 0.1, or 1 µmol/L astragaloside IV for 24 h (P>0.05). However, after treatment with 0.5, 0.75, 1, or 1.25 mg/mL LPS for 24 h, the viability steadily decreased (P<0.01). The mRNA and protein expression levels of ERCC2, XRCC4, XRCC2, TNF-α, IL-1ß, TLR4, NOS, and COX-2 were significantly increased after PC12 cells were treated with 1 mg/mL LPS for 24 h (P<0.01); however, these changes were reversed when PC12 cells were pretreated with 0.01, 0.1, or 1 µmol/L astragaloside IV in PC12 cells and then treated with 1 mg/mL LPS for 24 h (P<0.05). Second-generation sequencing revealed that 1026 genes were upregulated, while 1287 genes were downregulated. The DEGs were associated with autophagy, TNF-α, interleukin-17, MAPK, P53, Toll-like receptor, and NOD-like receptor signaling pathways. Furthermore, PC12 cells treated with a 1 mg/mL LPS for 24 h exhibited increased mRNA and protein expression of CCL2, CCL11, CCL7, MMP3, and MMP10, which are associated with the IL-17 pathway. RT-qPCR and Western blotting analyses confirmed that the DEGs listed above corresponded to the sequence assay results. CONCLUSION: LPS can damage PC12 cells and cause inflammatory reactions in nerve cells and DNA damage. astragaloside IV plays an anti-inflammatory and DNA damage protective role and inhibits the IL-17 signaling pathway to exert a neuroprotective effect in vitro.
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Antiinflamatorios , Supervivencia Celular , Reparación del ADN , Lipopolisacáridos , Saponinas , Triterpenos , Animales , Células PC12 , Ratas , Lipopolisacáridos/farmacología , Triterpenos/farmacología , Saponinas/farmacología , Antiinflamatorios/farmacología , Supervivencia Celular/efectos de los fármacos , Reparación del ADN/efectos de los fármacosRESUMEN
Inhibiting O-GlcNAcase and thereby up-regulation of the O-GlcNAc levels of tau was a potential approach for discovering AD treatments. Herein, a series of novel highly potent OGA inhibitors embracing a 4-(arylethynyl)piperidine moiety was achieved by capitalizing on the substrate recognition domain. Extensive structure-activity relationships resulted in compound 81 with significant enzymatic inhibition (IC50 = 4.93 ± 2.05 nM) and cellular potency (EC50 = 7.47 ± 3.96 nM in PC12 cells). It markedly increased the protein O-GlcNAcylation levels and reduced the phosphorylation on Ser199, Thr205, and Ser396 of tau in the OA-injured SH-SY5Y cell model, suggesting its potential role for AD treatment. In fact, an in vivo efficacy of ameliorating cognitive impairment was observed following treatment of APP/PS1 mice with compound 81 (100 mg/kg). Additionally, the appropriate plasma PK and beneficial BBB penetration properties were also observed. Compound 81 deserves to be further explored as an anti-AD agent.
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Enfermedad de Alzheimer , Inhibidores Enzimáticos , Piperidinas , beta-N-Acetilhexosaminidasas , Enfermedad de Alzheimer/tratamiento farmacológico , Animales , Piperidinas/farmacología , Piperidinas/uso terapéutico , Piperidinas/síntesis química , Piperidinas/química , Piperidinas/farmacocinética , Humanos , Relación Estructura-Actividad , beta-N-Acetilhexosaminidasas/antagonistas & inhibidores , beta-N-Acetilhexosaminidasas/metabolismo , Ratas , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/uso terapéutico , Inhibidores Enzimáticos/farmacocinética , Ratones , Células PC12 , Descubrimiento de Drogas , Ratones Transgénicos , Proteínas tau/metabolismo , Proteínas tau/antagonistas & inhibidores , MasculinoRESUMEN
Providing antioxidants and targeting acetylcholinesterase (AChE) are key strategies in treating neurocognitive dysfunction. In this study, bioactive sturgeon (Acipenser schrenckii) spinal cord peptides (SSCPs) with antioxidant and AChE inhibitory potency were extracted and separated from sturgeon spinal cord by enzymatic hydrolysis and ultrafiltration, and targeted peptide PGGW was screened via computer simulated molecular docking. Further, the molecular dynamic interactions of the PGGW with superoxide dismutase (SOD) and AChE were analyzed, and the protective effect of PGGW on glutamate-induced PC12 cells in vitro was evaluated. The <3 kDa fraction of SSCPs displays the most potent antioxidative efficacy (1 mg/mL, DPPHâ¢: 89.07%, ABTS+: 76.35%). Molecular dynamics simulation showed that PGGW was stable within AChE and tightly bound to residues SER203, PHE295, ILE294 and TRP236. When combined with SOD, the indole group of PGGW was stuck inside SOD, but the tail chain PGG fluctuated greatly outside. Surface plasmon resonance demonstrated that PGGW has a high binding affinity for AChE (KD = 1.4 mM) and 0.01 mg/mL PGGW provided good protection against glutamate-induced apoptosis. The findings suggest a promising strategy for drug research on neurodegenerative diseases.
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Acetilcolinesterasa , Antioxidantes , Inhibidores de la Colinesterasa , Proteínas de Peces , Peces , Simulación del Acoplamiento Molecular , Péptidos , Médula Espinal , Animales , Antioxidantes/química , Antioxidantes/farmacología , Péptidos/química , Péptidos/farmacología , Acetilcolinesterasa/química , Acetilcolinesterasa/metabolismo , Inhibidores de la Colinesterasa/farmacología , Inhibidores de la Colinesterasa/química , Proteínas de Peces/química , Proteínas de Peces/farmacología , Ratas , Médula Espinal/química , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Células PC12 , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa/químicaRESUMEN
Controlling biomolecular-cell interactions is crucial for the design of scaffolds for tissue engineering (TE). Regenerated silk fibroin (RSF) has been extensively used as TE scaffolds, however, RSF showed poor attachment of neuronal cells, such as rat pheochromocytoma (PC12) cells. In this work, amphiphilic peptides containing a hydrophobic isoleucine tail (I3) and laminin or fibronectin derived peptides (IKVAV, PDSGR, YIGSR, RGDS and PHSRN) were designed for promoting scaffold-cell interaction. Three of them (I3KVAV, I3RGDS and I3YIGSR) can self-assemble into nanofibers, therefore, were used to enhance the application of RSF in neuron TE. Live / dead assays revealed that the peptides exhibited negligible cytotoxicity against PC12 cells. The specific interaction between PC12 cells and the peptides were investigate using atomic force microscopy (AFM). The results indicated a synergistic effect in the designed peptides, promoting cellular attachment, proliferation and morphology changes. In addition, AFM results showed that co-assembling peptides I3KVAV and I3YIGSR possesses the best regulation of proliferation and attachment of PC12 cells, consistent with immunofluorescence staining results. Moreover, cell culture with hydrogels revealed that a mixture of peptides I3KVAV and I3YIGSR can also promote 3D neurites outgrowth. The approach of combining two different self-assembling peptides shows great potential for nerve regeneration applications.
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Nanofibras , Proyección Neuronal , Péptidos , Seda , Andamios del Tejido , Animales , Células PC12 , Ratas , Nanofibras/química , Andamios del Tejido/química , Proyección Neuronal/efectos de los fármacos , Péptidos/química , Péptidos/farmacología , Seda/química , Proliferación Celular/efectos de los fármacos , Fibroínas/química , Fibroínas/farmacología , Ingeniería de Tejidos/métodos , Adhesión Celular/efectos de los fármacosRESUMEN
Alzheimer's disease is associated both with imbalances in Al3+ production and changes in viscosity in cells. Their simultaneous measurement could therefore provide valuable insights into Alzheimer's disease pathology. Their simultaneous measurement would therefore be of great value in investigating the pathological mechanism of Alzheimer's disease. We designed a fluorescent probe YM2T with AIE effect that is capable of selectively responding to Al3+ by fluorescence colormetrics and to viscosity by fluorescence "turn on" modes. Additionally, Al3+ and viscosity were simultaneously detected in PC12 cells using the low cytotoxic probe YM2T via blue and green fluorescence channels. More importantly, the YM2T probe was used to image mice with AD. Hence, the YM2T probe shows potential as a useful molecular instrument for studying the pathological impact of Al3+ and viscosity.
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Aluminio , Enfermedad de Alzheimer , Colorantes Fluorescentes , Imagen Óptica , Enfermedad de Alzheimer/diagnóstico por imagen , Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Viscosidad , Animales , Células PC12 , Ratones , Aluminio/análisis , Aluminio/química , Estructura Molecular , Ratas , Relación Dosis-Respuesta a Droga , Relación Estructura-Actividad , Modelos Animales de EnfermedadRESUMEN
Dendrobium nobile is a species of the genus Dendrobium that can be used as both a medicinal herb and healthy food. The sesquiterpenes in D. nobile have attracted extensive attention in recent years. In this study, Amide × RP offline two-dimensional chromatography separation tandem high-resolution mass spectrometry combined with GNPS (Global Natural Product Social Molecular Networking) was developed for the characterization of sesquiterpenes in D. nobile. After first-dimensional amide separation, the 70% ethanol extract of D. nobile was divided into 40 fractions, which were analyzed by second-dimensional reverse-phase system separation and LTQ-Orbitrap detection. The raw data was imported into the GNPS, resulting in the efficient clustering of similar substances. Finally, 594 sesquiterpene compounds were characterized, and 25 compounds were isolated based on molecular network analysis, including six new compounds. In vitro bioassays, the isolated compounds decreased NO production in the LPS-induced microglial BV-2 cell model and the content of MDA in PC12 cells, demonstrating neuroprotective activity. These findings unraveled the underlying material and provided valuable insights into the quality control of D. nobile.