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PURPOSE: The purpose of this study was to compare the effect of two types of cervical collars (Philadelphia and Miami-J) on pulmonary function and ventilation in healthy volunteers through spirometry, peak flow meter, and capnograph. METHOD: Initially, subjects were randomized into two groups in which the sequence of collars' fixation was reversed. Afterward, we assessed the pulmonary parameters without a cervical collar in all participants. Each group underwent two additional test conditions, including measurements after wearing a Philadelphia and Miami-J cervical collar. In any case, we took the measurements half an hour after the collar fixation. RESULTS: The mean age of participants was 48.34 ± 1.35 years. Following either type of collars application, there was a statistically significant decrease in FEV1, FEV1/FVC, FEF25-75%, and PEF (p < .001). However, FVC was not significantly changed (p = .157). CONCLUSION: In summary, we noted a statistically significant expiratory flow obstruction after both the Philadelphia and Miami-J cervical collar. These changes were not clinically significant in healthy volunteers, albeit may have ramifications in patients with pre-existing respiratory compromise.
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Tirantes/efectos adversos , Ventilación Pulmonar , Vértebras Cervicales , Femenino , Voluntarios Sanos , Humanos , Masculino , Persona de Mediana Edad , Cuello , Estudios Prospectivos , Método Simple Ciego , EspirometríaRESUMEN
Non-ionic, low-osmolar contrast agents (CAs) used for computed tomography, such as Optiray (ioversol) and Iomeron (iomeprol), are associated with the reduced risk of adverse reactions and toxicity in comparison with ionic CAs, such as Hexabrix. Hexabrix has previously been used for imaging articular cartilage but has been commercially discontinued. This study aimed to evaluate the efficacy of Optiray and Iomeron as alternatives for visualisation of articular cartilage in small animal joints using contrast-enhanced micro-computed tomography (CECT). For this purpose, mouse femora were immersed in different concentrations (20%-50%) of Optiray 350 or Iomeron 350 for periods of time starting at five minutes. The femoral condyles were scanned ex vivo using CECT, and regions of articular cartilage manually contoured to calculate mean attenuation at each time point and concentration. For both CAs, a 30% CA concentration produced a mean cartilage attenuation optimally distinct from both bone and background signal, whilst 5-min immersion times were sufficient for equilibration of CA absorption. Additionally, plugs of bovine articular cartilage were digested by chondroitinase ABC to produce a spectrum of glycosaminoglycan (GAG) content. These samples were immersed in CA and assessed for any correlation between mean attenuation and GAG content. No significant correlation was found between attenuation and cartilage GAG content for either CAs. In conclusion, Optiray and Iomeron enable high-resolution morphological assessment of articular cartilage in small animals using CECT; however, they are not indicative of GAG content.
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Cartílago Articular/diagnóstico por imagen , Medios de Contraste , Fémur/diagnóstico por imagen , Yopamidol/análogos & derivados , Ácidos Triyodobenzoicos , Microtomografía por Rayos X/métodos , Animales , Bovinos , RatonesRESUMEN
Bone tissue is comprised of a collagen-rich matrix containing non-collagenous organic compounds, strengthened by mineral crystals. Bone strength reflects the amount and structure of bone, as well as its quality. These qualities are determined and maintained by osteoblasts (bone-forming cells) and osteoclasts (bone-resorbing cells) on the surface of the bone and osteocytes embedded within the bone matrix. Bone development and growth also involves cartilage cells (chondrocytes). These cells do not act in isolation, but function in a coordinated manner, including co-ordination within each lineage, between the cells of bone, and between these cells and other cell types within the bone microenvironment. This chapter will briefly outline the cells of bone, their major functions, and some communication pathways responsible for controlling bone development and remodeling.
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Osteoblastos , Osteoclastos , Remodelación Ósea , Huesos/fisiopatología , Osteocitos/fisiologíaRESUMEN
Alzheimer's disease (AD) is the most common neurodegenerative disorder to date, with no curative or preventive therapy. Histopathological hallmarks of AD include deposition of ß-amyloid plaques and formation of neurofibrillary tangles. Extent studies on pathology of the disease have made important discoveries regarding mechanism of disease and potential therapeutic targets. Many cellular changes including oxidative stress, disruption of Ca2+ homeostasis, inflammation, metabolic disturbances, and accumulation of unfolded/misfolded proteins can lead to programmed cell death in AD. Despite intensive research, only five approved drugs are available for the management of AD. Hence, there is a need to look at alternative therapies. Use of natural products and culinary herbs in medicine has gained popularity in recent years. Several natural substances with neuroprotective effects have been widely studied. Most of these compounds have remarkable antioxidant properties and act mainly by scavenging free radical species. Some of them increase cell survival and improve cognition by directly affecting amyloidogenesis and programmed cell death pathways. Further studies on these natural products and their mechanism of action, parallel with the use of novel pharmaceutical drug design and delivery techniques, enable us to offer an addition to conventional medicine. This review discussed some natural products with potential neuroprotective properties against Aß with respect to their mechanism of action.
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Micro-computed tomography (microCT) offers a three-dimensional (3D), high-resolution technique for the visualisation and analysis of bone microstructure. Using contrast-enhanced microCT, this capability has been expanded in recent studies to include cartilage morphometry and whole joint measures, known together as quantitative morphometric analysis (QMA). However, one of the main challenges in quantitative analysis of joint images is sensitivity to joint pose and alignment, which may influence measures related to both joint space and joint biomechanics. Thus, this study proposes a novel microCT imaging protocol for reproducible and efficient QMA of in situ mouse tibio-femoral joint. This work consists of two parts: an in situ diffusion kinetics study for a known cationic iodinated contrast agent (CA4+) for QMA of the cartilage, and a joint positioning and image processing workflow for whole joint QMA. In the diffusion kinetics study, 8 mice were injected at both of their tibio-femoral joints with distinct CA4+ concentrations and diffusion times. The mice were scanned at different time points after injection, and evaluated using attenuation and cartilage QMA measures. Results show that cartilage segmentation and QMA could be performed for CA4+ solution at a concentration of 48 mg/ml, and that reliable measurement and quantification of cartilage were achieved after 5 min of diffusion following contrast agent injection. We established the joint positioning and image processing workflow by developing a novel positioning device to control joint pose during scanning, and a spherical harmonics-based image processing workflow to ensure consistent alignment during image processing. Both legs of seven mice were scanned 10 times, 5 prior to receiving CA4+ and 5 after, and evaluated using whole joint QMA parameters. Joint QMA evaluation of the workflow showed excellent reproducibility; intraclass correlation coefficients ranged from 0.794 to 0.930, confirming that the imaging protocol enables reproducible and efficient QMA of joint structures in preclinical models, and that contrast agent injection did not cause significant alteration to the measured parameters.
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Cartílago Articular , Medios de Contraste , Ratones , Animales , Medios de Contraste/química , Microtomografía por Rayos X/métodos , Cartílago Articular/diagnóstico por imagen , Reproducibilidad de los Resultados , Fémur/diagnóstico por imagenRESUMEN
Biological heterogeneity is a primary contributor to the variation observed in experiments that probe dynamical processes, such as the internalization of material by cells. Given that internalization is a critical process by which many therapeutics and viruses reach their intracellular site of action, quantifying cell-to-cell variability in internalization is of high biological interest. Yet, it is common for studies of internalization to neglect cell-to-cell variability. We develop a simple mathematical model of internalization that captures the dynamical behaviour, cell-to-cell variation, and extrinsic noise introduced by flow cytometry. We calibrate our model through a novel distribution-matching approximate Bayesian computation algorithm to flow cytometry data of internalization of anti-transferrin receptor antibody in a human B-cell lymphoblastoid cell line. This approach provides information relating to the region of the parameter space, and consequentially the nature of cell-to-cell variability, that produces model realizations consistent with the experimental data. Given that our approach is agnostic to sample size and signal-to-noise ratio, our modelling framework is broadly applicable to identify biological variability in single-cell data from internalization assays and similar experiments that probe cellular dynamical processes.
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Endocitosis , Teorema de Bayes , Línea Celular , Citometría de Flujo , HumanosRESUMEN
There is mounting evidence implicating the role of oxidative stress induced by reactive oxygen species (ROS) in neurodegenerative disease, including Alzheimer's disease. Herein we investigated the neuroprotective potential of a natural flavonoid, calycopterin, against H(2)O(2)-induced cell death in differentiated PC12 cells. We pretreated PC12 cells with 25, 50, and 100 µM calycopterin followed by the addition of H(2)O(2) as an oxidative stress agent. We measured cell viability by the MTT test and found that 50 µM is the best protective concentration of calycopterin. Moreover, we measured six different parameters of neurite outgrowth. Interestingly, we found that calycopterin not only protects PC12 cells against H(2)O(2)-induced apoptosis but also defends against the destructive effect of oxidative stress on the criteria of neural differentiation. Calycopterin decreased ER stress-associated proteins including calpain and caspase-12, and suppressed ERK, JNK, and p38 MAPK phosphorylation. Moreover, calycopterin inhibited H(2)O(2)-induced nuclear translocation of nuclear factor-κB, a known regulator of a host of genes involved in specific stress and inflammatory responses. This observation was perfectly in agreement with the decrease of COX-2 and TNF-α levels. Calycopterin reduced intracellular ROS levels and increased catalase activity. The protective effect of this compound could represent a promising approach for the treatment of neurodegenerative diseases.
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Antiinflamatorios/farmacología , Apoptosis/efectos de los fármacos , Estrés del Retículo Endoplásmico , Flavonas/farmacología , Animales , Antiinflamatorios/química , Calpaína/metabolismo , Caspasa 12/metabolismo , Supervivencia Celular/efectos de los fármacos , Ciclooxigenasa 2/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Flavonas/química , Peróxido de Hidrógeno/toxicidad , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Células PC12 , Fosforilación , Ratas , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Parathyroid hormone-related protein (PTHrP, gene name Pthlh) is a pleiotropic regulator of tissue homeostasis. In bone, Dmp1Cre-targeted PTHrP deletion in osteocytes causes osteopenia and impaired cortical strength. We report here that this outcome depends on parental genotype. In contrast to our previous report using mice bred from heterozygous (flox/wild type) Dmp1Cre.Pthlhf/w parents, adult (16-week-old and 26-week-old) flox/flox (f/f) Dmp1Cre.Pthlhf/f mice from homozygous parents (Dmp1Cre.Pthlhf/f(hom) ) have stronger bones, with 40% more trabecular bone mass and 30% greater femoral width than controls. This greater bone size was observed in Dmp1Cre.Pthlhf/f(hom) mice as early as 12 days of age, when greater bone width was also found in male and female Dmp1Cre.Pthlhf/f(hom) mice compared to controls, but not in gene-matched mice from heterozygous parents. This suggested a maternal influence on skeletal size prior to weaning. Although Dmp1Cre has previously been reported to cause gene recombination in mammary gland, milk PTHrP protein levels were normal. The wide-bone phenotype was also noted in utero: Dmp1Cre.Pthlhf/f(hom) embryonic femurs were more mineralized and wider than controls. Closer examination revealed that Dmp1Cre caused PTHrP recombination in placenta, and in the maternal-derived decidual layer that resides between the placenta and the uterus. Decidua from mothers of Dmp1Cre.Pthlhf/f(hom) mice also exhibited lower PTHrP levels by immunohistochemistry and were smaller than controls. We conclude that Dmp1Cre leads to gene recombination in decidua, and that decidual PTHrP might, through an influence on decidual cells, limit embryonic bone radial growth. This suggests a maternal-derived developmental origin of adult bone strength. © 2021 American Society for Bone and Mineral Research (ASBMR).
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Osteocitos , Proteína Relacionada con la Hormona Paratiroidea , Animales , Desarrollo Óseo/genética , Huesos , Decidua , Femenino , Masculino , Ratones , Proteína Relacionada con la Hormona Paratiroidea/genética , EmbarazoRESUMEN
Oxidative stress has been implicated in the etiology of neurodegenerative diseases and aging. Indeed, accumulation of reactive oxygen species, such as hydrogen peroxide, generated by inflammatory cells, leads to oxidative stress, which may contribute to the neuronal degeneration observed in a wide variety of neurodegenerative disorders of the central nervous system, such as Alzheimer's disease. The present study indicates that H(2)O(2)-induced cell death can be inhibited in the presence of 1,2,4-triazine derivatives, as measured by MTT and caspase-3 activity. We further show that these compounds exert their protective effect by up-regulation of hemeoxygenase-1, glutamylcysteine synthetase, glutathione peroxidase and nuclear factor-erythroid 2 p45-related factor 2 (Nrf2), while they inhibit NF-kappaB and decrease lipid peroxidation. It shows that there is a potential cross talk between NF-kappaB and Nrf2, an important cytoprotective transcription factor in the presence of these compounds. Moreover, in order for drugs to be effective in the treatment of neurodegenerative diseases, they must be capable of penetrating the blood-brain barrier, whereas more than 98% of all potential central nervous system drugs don't cross. Using a reliable model based on the artificial neural network indicated that these compounds satisfy this requirement.
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Apoptosis/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , FN-kappa B/metabolismo , Neuronas/citología , Fármacos Neuroprotectores/farmacología , Transducción de Señal/efectos de los fármacos , Triazinas/farmacología , Regulación hacia Arriba/efectos de los fármacos , Animales , Factor 2 Relacionado con NF-E2/genética , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Células PC12 , Ratas , Activación Transcripcional/efectos de los fármacos , Triazinas/químicaRESUMEN
The role of novel triazine derivatives against oxidative stress exerted by hydrogen peroxide on differentiated rat pheochromocytoma (PC12) cell line was examined and a consistent protection from H(2)O(2)-induced cell death, associated with a marked reduction in caspase-3 activation, was observed. Moreover, activation of NF-kappaB, a known regulator of a host of genes that involves in specific stress and inflammatory responses by H(2)O(2), was greatly impaired by triazine pretreatment in differentiated PC12 cells. Neuroprotective effect of such compounds may represent a promising approach for treatment of neurodegenerative diseases.
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Fármacos Neuroprotectores/síntesis química , Triazinas/química , Animales , Caspasa 3/metabolismo , Peróxido de Hidrógeno/farmacología , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/efectos de los fármacos , Células PC12 , Ratas , Especies Reactivas de Oxígeno/metabolismo , Triazinas/síntesis química , Triazinas/farmacologíaRESUMEN
Articular joints are comprised of different tissues, including cartilage and bone, with distinctive structural and mechanical properties. Joint homeostasis depends on mechanical and biological integrity of these components and signaling exchanges between them. Chondrocytes and osteocytes actively sense, integrate, and convert mechanical forces into biochemical signals in cartilage and bone, respectively. The osteochondral interface between the bone and cartilage allows these tissues to communicate with each other and exchange signaling and nutritional molecules, and by that ensure an integrated response to mechanical stimuli. It is currently not well known how molecules are transported between these tissues. Measuring molecular transport in vivo is highly desirable for tracking cartilage degeneration and osteoarthritis progression. Since transport of contrast agents, which are used for joint imaging, also depend on diffusion through the cartilage extracellular matrix, contrast agent enhanced imaging may provide a high resolution, non-invasive method for investigating molecular transport in the osteochondral unit. Only a few techniques have been developed to track molecular transport at the osteochondral interface, and there appear opportunities for development in this field. This review will describe current knowledge of the molecular interactions and transport in the osteochondral interface and discuss the potential of using contrast agents for investigating molecular transport and structural changes of the joint.
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Mineralized bone forms when collagen-containing osteoid accrues mineral crystals. This is initiated rapidly (primary mineralization), and continues slowly (secondary mineralization) until bone is remodeled. The interconnected osteocyte network within the bone matrix differentiates from bone-forming osteoblasts; although osteoblast differentiation requires EphrinB2, osteocytes retain its expression. Here we report brittle bones in mice with osteocyte-targeted EphrinB2 deletion. This is not caused by low bone mass, but by defective bone material. While osteoid mineralization is initiated at normal rate, mineral accrual is accelerated, indicating that EphrinB2 in osteocytes limits mineral accumulation. No known regulators of mineralization are modified in the brittle cortical bone but a cluster of autophagy-associated genes are dysregulated. EphrinB2-deficient osteocytes displayed more autophagosomes in vivo and in vitro, and EphrinB2-Fc treatment suppresses autophagy in a RhoA-ROCK dependent manner. We conclude that secondary mineralization involves EphrinB2-RhoA-limited autophagy in osteocytes, and disruption leads to a bone fragility independent of bone mass.
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Autofagia/fisiología , Enfermedades del Desarrollo Óseo/genética , Calcificación Fisiológica/fisiología , Efrina-B2/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Autofagosomas/fisiología , Autofagia/genética , Enfermedades del Desarrollo Óseo/patología , Remodelación Ósea/fisiología , Línea Celular , Efrina-B2/genética , Ratones , Ratones Endogámicos C57BL , Osteocitos/metabolismo , Osteocitos/fisiología , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteína de Unión al GTP rhoARESUMEN
Parathyroid hormone-related protein (PTHrP) and parathyroid hormone (PTH) have N-terminal domains that bind a common receptor, PTHR1. N-terminal PTH (teriparatide) and now a modified N-terminal PTHrP (abaloparatide) are US Food and Drug Administration (FDA)-approved therapies for osteoporosis. In physiology, PTHrP does not normally circulate at significant levels, but acts locally, and osteocytes, cells residing within the bone matrix, express both PTHrP and the PTHR1. Because PTHR1 in osteocytes is required for normal bone resorption, we determined how osteocyte-derived PTHrP influences the skeleton. We observed that adult mice with low PTHrP in osteocytes (targeted with the Dmp1(10kb)-Cre) have low trabecular bone volume and osteoblast numbers, but osteoclast numbers were unaffected. In addition, bone size was normal, but cortical bone strength was impaired. Osteocyte-derived PTHrP therefore stimulates bone formation and bone matrix strength, but is not required for normal osteoclastogenesis. PTHrP knockdown and overexpression studies in cultured osteocytes indicate that osteocyte-secreted PTHrP regulates their expression of genes involved in matrix mineralization. We determined that osteocytes secrete full-length PTHrP with no evidence for secretion of lower molecular weight forms containing the N-terminus. We conclude that osteocyte-derived full-length PTHrP acts through both PTHR1 receptor-mediated and receptor-independent actions in a paracrine/autocrine manner to stimulate bone formation and to modify adult cortical bone strength. © 2017 American Society for Bone and Mineral Research.
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Comunicación Autocrina , Huesos/metabolismo , Osteocitos/metabolismo , Comunicación Paracrina , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Animales , Comunicación Autocrina/efectos de los fármacos , Hueso Esponjoso/patología , Diferenciación Celular/efectos de los fármacos , Línea Celular , AMP Cíclico/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Fracturas del Fémur/patología , Fémur/patología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Modelos Biológicos , Tamaño de los Órganos/efectos de los fármacos , Osteocitos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Comunicación Paracrina/efectos de los fármacos , Hormona Paratiroidea/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/farmacologíaRESUMEN
Ca(2+) is an essential second messenger, playing a fundamental role in maintaining cell viability and neuronal activity. Two specific endoplasmic reticulum calcium channels, ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3Rs) play an important role in Ca(2+) regulation. In the present study, we provided a 3D structure of RyR and IP3R by homology modeling, and we predicted their interactions with a known neuroprotective compound, 3-thiomethyl-5,6-(dimethoxyphenyl)-1,2,4-triazine (TDMT), as well as two inhibitors, dantrolene and 2-aminoethoxydiphenyl borate (2-APB). Interestingly, we found that dantrolene and 2-APB can bind to the IP3-binding domain of IP3R and RyR, while TDMT may directly block both channels by interacting with the putative resident domains in the pore. Cell culture experiments showed that these compounds could protect PC12 cells against H2O2-induced apoptosis and activate autophagic pathways. Collectively, our computational (in silico) and cell culture studies suggest that RyR and IP3R are novel and promising targets to be used against neurodegenerative diseases.
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Compuestos de Boro/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Dantroleno/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Triazinas/farmacología , Secuencia de Aminoácidos , Animales , Apoptosis , Autofagia , Compuestos de Boro/química , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/química , Dantroleno/química , Peróxido de Hidrógeno/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inhibidores , Receptores de Inositol 1,4,5-Trifosfato/química , Modelos Moleculares , Datos de Secuencia Molecular , Neuronas/citología , Neuronas/metabolismo , Fármacos Neuroprotectores/química , Células PC12 , Ratas , Canal Liberador de Calcio Receptor de Rianodina/química , Homología de Secuencia de Aminoácido , Transducción de Señal , Triazinas/químicaRESUMEN
Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is characterized by progressive loss of memory and cognitive impairment. Aggregation of amyloid-ß (Aß) peptides is the crucial factor in the onset of AD. The toxic Aß peptides Aß40 and Aß42 are produced from the Aß precursor protein (APP), a transmembrane protein which is folded and modified in endoplasmic reticulum (ER). ER is the main organelle for the synthesis and processing of nearly all proteins as well as the main cellular source of Ca2+. Under stress conditions, three main ER pathways including inositol-requiring enzyme 1, protein kinase RNA-like ER kinase, and activating transcription factor 6 become activated causing the accumulation of unfolded or misfolded proteins within ER lumen. These pathways manage the stress by regulating the expression of chaperones and enzymes involved in protein folding. Several studies have reported the dysfunction of these stress-sensing pathways in pathological conditions, including neurodegenerative diseases. Recent studies have proposed that neuronal death in AD arises from dysfunction of the ER. Here, we will review recent research findings on the interaction between ER and mitochondria, and its effect on apoptotic pathways. We further provide insights into studies which suggest the role of ER in animal and/or cellular models of AD. Therapeutic strategies that modulate ER could represent a promising approach for prevention or treatment of AD.
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Enfermedad de Alzheimer/metabolismo , Estrés del Retículo Endoplásmico , Retículo Endoplásmico/metabolismo , Terapia Molecular Dirigida , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Animales , Apoptosis , Modelos Animales de Enfermedad , Retículo Endoplásmico/patología , Humanos , Mitocondrias/metabolismo , Mitocondrias/patología , Terapia Molecular Dirigida/métodos , Terapia Molecular Dirigida/tendencias , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/uso terapéutico , Pliegue de Proteína , Desplegamiento ProteicoRESUMEN
PURPOSE: There is an increasing amount of experimental evidence that oxidative stress has a central role in the neuropathology of neurodegenerative diseases. It has been suggested that the loss of cell function results from the increased oxidative damage to proteins and DNA. Herein, we investigated the effect of a natural neuroprotective flavonoid, calycopterin, on H2O2-induced disruption of phase II detoxifying enzyme system and cAMP response element binding protein (CREB) phosphorylation. METHODS: PC12 cells were treated with 25, 50 and 100 µM of calycopterin for 3h, followed by adding H2O2 (150 µM) for 24 h. The extent of apoptosis was assessed by comet assay. The level of phosphorylated CREB, nuclear factor erythroid 2-related factor 2 (Nrf2), glutamylcysteine synthetase (γ-GCS) and heme oxygenase 1 (HO-1) were measured by western blot method. The concentration of glutathione (GSH) was determined in whole cell lysate using dithionitrobenzoic acid method. Superoxide dismutase (SOD) activity was measured by colorimetric assay. RESULT: Morphological analysis of protection induced by calycopterin, determined by comet assay, showed that calycopterin reduced DNA in tail. We found that H2O2 decreased mitochondrial membrane potential (MMP), while, calycopterin prevented this decrease in MMP in presence of H2O2. In H2O2-treated cells, calycopterin also suppressed cytochrome C release to cytosol that is necessary for maintaining mitochondrial homeostasis in survived cells. Moreover, calycopterin, in presence of H2O2 inhibited the decrease caused by oxidative stress in stress-sensing transcription factors, CREB and Nrf2, which play an important role in antioxidant capacity of the cell. There was also an increase in γ-GCS and HO-1 levels in calycopterin pretreated cells. In the presence of H2O2, calycopterin inhibited decrease in GSH level and SOD activity. CONCLUSION: We provided documentation of neuroprotective effect of a natural flavone, calycopterin, against H2O2-induced oxidative stress in differentiated PC12 cells by modulating the level of CREB phosphorylation and Nrf2 pathway.
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Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Flavonas/farmacología , Fase II de la Desintoxicación Metabólica , Factor 2 Relacionado con NF-E2/metabolismo , Fármacos Neuroprotectores/farmacología , Animales , Ensayo Cometa , Evaluación Preclínica de Medicamentos , Flavonas/administración & dosificación , Lamiaceae , Fármacos Neuroprotectores/administración & dosificación , Células PC12 , RatasRESUMEN
Much evidence supports that oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases, such as Alzheimer's disease. Herein, we studied the compensatory/adaptive mechanisms involved in 3-thiomethyl-5,6-(diphenyl)-1,2,4-triazine and 3-thiomethyl-5,6-(dichlorophenyl)-1,2,4-triazine neuroprotection. We found that these compounds could counteract H(2)O(2)-induced rupture of neurite outgrowth in differentiated PC12 cells. In addition, we found that pretreatment of cells with triazine derivatives could modulate the expression of heat shock proteins Hsp70, Hsp90, and Hsp32 in H(2)O(2)-treated PC12 cells. These compounds could also increase nuclear level of stress sensing transcription factor, NF-E2 related factor 2, which contributes to redox homeostasis and cell survival following stress. As a result, the elevated levels of glutamylcysteine synthetase, glutathione peroxidase-1, and glutathione, as well as superoxide dismutase and catalase, increased cellular antioxidant capacity. Studying the relation between structure and activity of these compounds will pave the way for exploiting preventive and/or therapeutic strategies for the management of oxidative stress-mediated disorders.
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Regulación de la Expresión Génica/efectos de los fármacos , Chaperonas Moleculares/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Fármacos Neuroprotectores/farmacología , Triazinas/farmacología , Animales , Western Blotting , Regulación hacia Abajo/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Fármacos Neuroprotectores/química , Células PC12 , Ratas , Triazinas/química , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Neurite outgrowth is an important aspect of neuronal plasticity and regeneration after neuronal injury. In this study we aimed to investigate the possible effect of 3-thiomethyl-5,6-dimethoxyphenyl-1,2,4-triazine (TDMT) on H(2)O(2)-induced impairment of neurite outgrowth. We found that TDMT could improve neurite outgrowth and neurite complexity in H(2)O(2)-exposed PC12 cells. Moreover, we found elevated levels of Hsp-70 and suppressed level of Hsp-90 in TDMT-treated cells in the presence of H(2)O(2). As another important signaling pathways that play role in neuritogenesis, as well as apoptosis, we measured the level of phosphorylated and total MAPKs proteins, JNK, ERK and p38 MAPK. We found that TDMT inhibits oxidative stress-induced phosphorylation of MAPKs. Since HSPs and MAPKs are both involved in coping with environmental changes, it will not be surprising if they can modify or augment each other's activity. Neuroprotective effect of this compound could represent a promising approach for treatment of neurodegenerative diseases.
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Peróxido de Hidrógeno/toxicidad , Neuritas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Triazinas/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factor de Crecimiento Nervioso/farmacología , Neuritas/fisiología , Células PC12 , Fosforilación/efectos de los fármacos , RatasRESUMEN
Several studies have shown that neuronal cell death due to apoptosis is the major reason for cognitive decline in Alzheimer's disease. In this study, we report the anti-apoptotic effects of three Salvia species from Iran-S. choloroleuca, S. mirzayanii and S. santolinifolia-against H(2)O(2)-induced cytotoxicity in neuron-like PC12 cells. We showed that these antioxidant species could interfere with the intrinsic pathway of apoptosis by attenuating Bax/Bcl-2 ratio, decreasing outer mitochondrial membrane break and decreasing cytochrome c release to cytoplasm. Interestingly, we found that these species were able to replenish reduced glutathione level which affects cellular redox status and cytochrome c activity. Moreover, the decreased level of caspase-3, the executioner caspase, resulted in decrease of PARP-1 cleavage. Anti-apoptotic effects of these species along with their antioxidant effects, may represent a promising approach for treatment of neurodegenerative diseases.