RESUMEN
Parkinson's disease (PD) is an incurable neurodegenerative disease characterized by motor and non-motor disabilities resulting from neuronal cell death in the substantia nigra and striatum. Microglial activation and oxidative stress are two of the primary mechanisms driving that neuronal death. Here, we evaluated the effects of geranium oil on 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPTP) mouse model for PD, on microglial activation, and oxidative stress. We demonstrate that oral treatment with geranium oil improved motor performance in this model. The therapeutic effects of geranium oil were observed as a significant increase in rotarod latency and distance among the mice treated with geranium oil, as compared to vehicle-treated MPTP mice. Geranium oil also prevented dopaminergic neuron death in the substantia nigra of the treated mice. These therapeutic effects can be partially attributed to the antioxidant and anti-inflammatory properties of geranium oil, which were observed as attenuated accumulation of reactive oxygen species and inhibition of the secretion of proinflammatory cytokines from geranium oil-treated activated microglial cells. A repeated-dose oral toxicity study showed that geranium oil is not toxic to mice. In light of that finding and since geranium oil is defined by the FDA as generally recognized as safe (GRAS), we do not foresee any toxicity problems in the future and suggest that geranium oil may be a safe and effective oral treatment for PD. Since the MPTP model is only one of the preclinical models for PD, further studies are needed to confirm that geranium oil can be used to prevent or treat PD.
Asunto(s)
Geranium , Enfermedades Neurodegenerativas , Enfermedad de Parkinson , Animales , Ratones , Enfermedades Neurodegenerativas/metabolismo , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismo , Sustancia Negra/metabolismo , Muerte Celular , Ratones Endogámicos C57BL , Modelos Animales de EnfermedadRESUMEN
INTRODUCTION: Extracorporeal photopheresis (ECP) is considered an effective treatment for patients with chronic graft vs host disease (cGVHD) and demonstrates efficacy in ameliorating GVHD. The mechanism by which ECP acts against cGVHD is not fully understood. Preliminary observations have hinted at the potential involvement of neutrophil extracellular traps (NETs) formation in the pathogenesis of cGVHD. We aimed to assess the influence of ECP on the formation of NETs in patients with cGVHD as a potential mechanism in this setting. METHODS: Patients treated with ECP for cGVHD at the Rabin Medical Center were included in this study. Blood samples were obtained at three different time points: before starting an ECP cycle, at the end of the first day of treatment, and 24 h following the initiation of the ECP treatment cycle. Neutrophils were harvested from all blood samples. NET formation was assessed by measurement of NET-bound specific neutrophil elastase activity and by immunofluorescence staining. RESULTS: Six patients (two females and four males) with cGVHD were included in the study. We observed a significant increase in NET formation among all six patients following ECP. Net-bound specific neutrophil elastase activity was elevated from a median value of 2.23 mU/mL (interquartile range [IQR] 2.06-2.47 mU/mL) at baseline to a median value of 13.06 mU/mL (IQR 10.27-15.97 mU/mL) immediately after the treatment and to a peak median value of 14.73 mU/mL (IQR 9.6-22.38 mU/mL) 24 h following the initiation of the ECP cycle. A qualitative assessment of NET formation using immunofluorescence staining has demonstrated markedly increased expression of citrullinated histone H3, a marker of NET formation, following ECP treatment. CONCLUSIONS: Our preliminary data indicate that ECP induces NET formation among patients with cGVHD. The contribution of increased NET formation to the therapeutic effect of cGVHD should be further investigated.
RESUMEN
Multiple endocrine neoplasia 4 (MEN4) is a rare multiglandular endocrine neoplasia syndrome clinically hallmarked by primary hyperparathyroidism (PHPT), pituitary adenoma (PitAd), and neuroendocrine tumors (NET), clinically overlapping MEN1. The underlying mutated gene - CDKN1B, encodes for the cell-cycle regulator p27. Possible genotype-phenotype correlations in MEN4 have not been thoroughly assessed. Prompted by the findings in three Israeli MEN4 kindreds, we performed a literature review on published and unpublished data from previously reported MEN4/CDKN1B cases. Univariate analysis analyzed time-dependent risks for developing PHPT, PitAd, or NET by variant type and position along the gene. Overall, 74 MEN4 cases were analyzed. PHPT risk was 53.4% by age 60 years (mean age at diagnosis age 50.6 ± 13.9 years), risk for PitAd was 23.2% and risk for NET was 16.2% (34.4 ± 21.4 and 52.9 ± 13.9 years, respectively). The frameshift variant p.Q107fs was the most common variant identified (4/41 (9.7%) kindreds). Patients with indels had higher risk for PHPT vs point mutations (log-rank, P = 0.029). Variants in codons 94-96 were associated with higher risk for PHPT (P < 0.001) and PitAd (P = 0.031). To conclude, MEN4 is clinically distinct from MEN1, with lower risk and older age for PHPT diagnosis. We report recurrent CDKN1B frameshift variants and possible genotype-phenotype correlations.
Asunto(s)
Adenoma , Neoplasia Endocrina Múltiple Tipo 1 , Neoplasia Endocrina Múltiple , Tumores Neuroendocrinos , Neoplasias Hipofisarias , Humanos , Neoplasia Endocrina Múltiple/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Mutación de Línea Germinal , Fenotipo , Neoplasias Hipofisarias/genética , Adenoma/genética , Tumores Neuroendocrinos/patología , Neoplasia Endocrina Múltiple Tipo 1/genéticaRESUMEN
Cardiovascular complications are increasingly reported with the use of certain tyrosine kinase inhibitors (TKIs) to treat chronic myeloid leukemia (CML). We studied neutrophil extracellular trap (NET) formation in CML and evaluated the effect of TKIs on NET formation. Neutrophils isolated from treatment-naïve patients with CML showed a significant increase in NET formation compared to matched controls at baseline and after stimulation with ionomycin (IO) and phorbol 12-myristate 13-acetate (PMA). Expression of citrullinated histone H3 (H3cit), peptidyl arginine deiminase 4 (PAD4) and reactive oxygen species (ROS) was significantly higher in CML samples compared to controls. Pre-treatment of neutrophils with TKIs was associated with a differential effect on NET formation, and ponatinib significantly augmented NET-associated elastase and ROS levels as compared to controls and other TKIs. BCR-ABL1 retroviral transduced HoxB8-immortalized mouse hematopoietic progenitors, which differentiate into neutrophils in-vitro, demonstrated increased H3cit & myeloperoxidase (MPO) expression consistent with excess NET formation. This was inhibited by Cl-amidine, a PAD4 inhibitor, but not by the NADPH inhibitor diphenyleneiodonium (DPI). Ponatinib pre-exposure significantly increased H3cit expression in HoxB8-BCR-ABL1 cells after stimulation with IO. In summary, CML is associated with increased NET formation, which is augmented by ponatinib, suggesting a possible role for NETs in promoting vascular toxicity in CML.
RESUMEN
Previous studies indicated that microglia cells upregulate the expression of aldolase C (ALDOC) in melanoma cells. The present study using brain-metastasizing variants from three human melanomas explores the functional role of ALDOC in the formation and maintenance of melanoma brain metastasis (MBM). ALDOC overexpression impacted differentially the malignant phenotype of these three variants. In the first variant, ALDOC overexpression promoted cell viability, adhesion to and transmigration through a layer of brain endothelial cells, and amplified brain micrometastasis formation. The cross-talk between this MBM variant and microglia cells promoted the proliferation and migration of the latter cells. In sharp contrast, ALDOC overexpression in the second brain-metastasizing melanoma variant reduced or did not affect the same malignancy features. In the third melanoma variant, ALDOC overexpression augmented certain characteristics of malignancy and reduced others. The analysis of biological functions and disease pathways in the ALDOC overexpressing variants clearly indicated that ALDOC induced the expression of tumor progression promoting genes in the first variant and antitumor progression properties in the second variant. Overall, these results accentuate the complex microenvironment interactions between microglia cells and MBM, and the functional impact of intertumor heterogeneity. Since intertumor heterogeneity imposes a challenge in the planning of cancer treatment, we propose to employ the functional response of tumors with an identical histology, to a particular drug or the molecular signature of this response, as a predictive indicator of response/nonresponse to this drug.
Asunto(s)
Neoplasias Encefálicas/secundario , Fructosa-Bifosfato Aldolasa/fisiología , Melanoma/patología , Microambiente Tumoral/fisiología , Animales , Variación Biológica Poblacional/genética , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Movimiento Celular/genética , Supervivencia Celular/genética , Fructosa-Bifosfato Aldolasa/genética , Células HEK293 , Humanos , Masculino , Melanoma/genética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Fenotipo , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Microambiente Tumoral/genéticaRESUMEN
Melanoma has the highest propensity to metastasize to the brain compared to other cancers, as brain metastases are found frequently high in patients who have prolonged survival with visceral metastasis. Once disseminated in the brain, melanoma cells communicate with brain resident cells that include astrocytes and microglia. Microglia cells are the resident macrophages of the brain and are the main immunological cells in the CNS involved in neuroinflammation. Data on the interactions between brain metastatic melanoma cells and microglia and on the role of microglia-mediated neuroinflammation in facilitating melanoma brain metastasis are lacking. To elucidate the role of microglia in melanoma brain metastasis progression, we examined the bidirectional interactions between microglia and melanoma cells in the tumor microenvironment. We identified the molecular and functional modifications occurring in brain-metastasizing melanoma cells and microglia cells after the treatment of each cell type with supernatants of the counter cell type. Both cells induced alteration in gene expression programs, cell signaling, and cytokine secretion in the counter cell type. Moreover, melanoma cells exerted significant morphological changes on microglia cells, enhanced proliferation, induced matrix metalloproteinase-2 (MMP-2) activation, and cell migration. Microglia cells induced phenotypic changes in melanoma cells increasing their malignant phenotype: increased melanoma proliferation, MMP-2 activity, cell migration, brain endothelial penetration, and tumor cells ability to grow as spheroids in 3D cultures. Our work provides a novel insight into the bidirectional interactions between melanoma and micoglia cells, suggesting the contribution of microglia to melanoma brain metastasis formation.
Asunto(s)
Neoplasias Encefálicas/genética , Melanoma/genética , Microglía/metabolismo , Neoplasias Cutáneas/genética , Microambiente Tumoral/genética , Animales , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/secundario , Comunicación Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Melanoma/metabolismo , Melanoma/patología , Ratones Desnudos , Microglía/patología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Trasplante HeterólogoRESUMEN
The development of melanoma brain metastasis is largely dependent on mutual interactions between the melanoma cells and cells in the brain microenvironment. Here, we report that the extracellular cysteine protease inhibitor cystatin C (CysC) is involved in these interactions. Microglia-derived factors upregulated CysC secretion by melanoma. Similarly, melanoma-derived factors upregulated CysC secretion by microglia. Whereas CysC enhanced melanoma cell migration through a layer of brain endothelial cells, it inhibited the migration of microglia cells toward melanoma cells. CysC was also found to promote the formation of melanoma three-dimensional structures in matrigel. IHC analysis revealed increased expression levels of CysC in the brain of immune-deficient mice bearing xenografted human melanoma brain metastasis compared to the brain of control mice. Based on these in vitro and in vivo experiments we hypothesize that CysC promotes melanoma brain metastasis. Increased expression levels of CysC were detected in the regenerating brain of mice after stroke. Post-stroke brain with melanoma brain metastasis showed an even stronger expression of CysC. The in vitro induction of stroke-like conditions in brain microenvironmental cells increased the levels of CysC in the secretome of microglia cells, but not in the secretome of brain endothelial cells. The similarities between melanoma brain metastasis and stroke with respect to CysC expression by and secretion from microglia cells suggest that CysC may be involved in shared pathways between brain metastasis and post-stroke regeneration. This manifests the tendency of tumor cells to highjack physiological molecular pathways in their progression.
Asunto(s)
Neoplasias Encefálicas/genética , Cistatina C/genética , Melanoma/genética , Microglía/metabolismo , Animales , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/secundario , Línea Celular Tumoral , Movimiento Celular/genética , Colágeno/farmacología , Inhibidores de Cisteína Proteinasa/metabolismo , Combinación de Medicamentos , Regulación Neoplásica de la Expresión Génica , Humanos , Laminina/farmacología , Melanoma/patología , Ratones , Microglía/patología , Metástasis de la Neoplasia , Proteoglicanos/farmacología , Transducción de Señal , Microambiente Tumoral/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Alzheimer's disease (AD) is the most prevalent cause of dementia in adults. Current available drugs for AD transiently alleviate some of the symptoms, but do not modify the disease mechanism or cure it. Therefore, new drugs are desperately needed. Key contributors to AD are amyloid beta (Aß)- and reactive oxygen species (ROS)-induced cytotoxicities. Plant-derived substances have been shown to affect various potential targets in various diseases including AD. Therefore, phytochemicals which can protect neuronal cells against these insults might help in preventing and treating this disease. In the following research, we have isolated the sesquiterpene lactone achillolide A from the plant Achillea fragrantissima and, for the first time, characterized its effects on Aß-treated neuroblastoma cells. Aß is a peptide derived from the sequential cleavage of amyloid precursor protein, and is part of the pathogenesis of AD. Our current study aimed to determine whether achillolide A can interfere with Aß-induced processes in Neuro2a cells, and protect them from its toxicity. Our results show that achillolide A decreased Aß-induced death and enhanced the viability of Neuro2a cells. In addition, achillolide A reduced the accumulation of Aß-induced ROS and inhibited the phosphorylation of stress-activated protein kinase/c-Jun N-terminal kinase and p44/42 mitogen-activated protein kinase in these cells. We therefore suggest that achillolide A may have therapeutic potential for the treatment of AD.
Asunto(s)
Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/efectos de los fármacos , Extractos Vegetales/química , Especies Reactivas de Oxígeno/metabolismo , Sesquiterpenos/uso terapéutico , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Humanos , Extractos Vegetales/farmacología , Sesquiterpenos/farmacologíaRESUMEN
BACKGROUND: Alzheimer's disease is a neurodegenerative disease, characterized by progressive decline in memory and cognitive functions, that results from loss of neurons in the brain. Amyloid beta (Aß) protein and oxidative stress are major contributors to Alzheimer's disease, therefore, protecting neuronal cells against Aß-induced toxicity and oxidative stress might form an effective approach for treatment of this disease. 3,5,4'-trihydroxy-6,7,3'-trimethoxyflavone (TTF) is a flavonoid we have purified from the plant Achillea fragrantissima; and the present study examined, for the first time, the effects of this compound on Aß-toxicity to neuronal cells. METHODS: Various chromatographic techniques were used to isolate TTF from the plant Achillea fragrantissima, and an N2a neuroblastoma cell line was used to study its activities. The cellular levels of total and phosphorylated stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and of total and phosphorylated extracellular signal-regulated kinase (ERK 1/2) were determined by enzyme-linked immunosorbent assay (ELISA). Intracellular reactive oxygen species (ROS) levels were measured by using 2',7'-dichlorofluorescein diacetate (DCF-DA). Cytotoxicity and cell viability were assessed by using lactate dehydrogenase (LDH) activity in cell-conditioned media, or by crystal violet cell staining, respectively. RESULTS: TTF prevented the Aß-induced death of neurons and attenuated the intracellular accumulation of ROS following treatment of these cells with Aß. TTF also inhibited the Aß-induced phosphorylation of the signaling proteins SAPK/JNK and ERK 1/2, which belong to the mitogen-activated protein kinase (MAPK) family. CONCLUSION: TTF should be studied further as a potential therapeutic means for the treatment of Alzheimer's disease.
Asunto(s)
Achillea/química , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/toxicidad , Antioxidantes/farmacología , Flavonas/farmacología , Fármacos Neuroprotectores/farmacología , Extractos Vegetales/farmacología , Transducción de Señal/efectos de los fármacos , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/fisiopatología , Péptidos beta-Amiloides/metabolismo , Antioxidantes/química , Apoptosis/efectos de los fármacos , Línea Celular , Supervivencia Celular , Flavonas/química , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fármacos Neuroprotectores/química , Estrés Oxidativo/efectos de los fármacos , Fosforilación , Extractos Vegetales/química , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Glutamate toxicity is a major contributor to the pathophysiology of numerous neurodegenerative diseases including amyotrophic lateral sclerosis and Alzheimer's disease. Therefore, protecting neuronal cells against glutamate-induced cytotoxicity might be an effective approach for the treatment of these diseases. We have previously purified from the medicinal plant Achillea fragrantissima two bioactive compounds which were not studied before: the sesquiterpene lactone achillolide A and the flavonoid 3,5,4'-trihydroxy-6,7,3'-trimethoxyflavone (TTF). We have shown that these compounds protect astrocytes from oxidative stress-induced cell death and inhibit microglial activation. The current study examined for the first time their effects on differentiated mouse neuroblastoma N2a cells and on glutamate toxicity. We have found that, although these compounds belong to different chemical families, they protect neuronal cells from glutamate toxicity. We further demonstrate that this protective effect might be, at least partially, due to inhibitory effects of these compounds on the levels of reactive oxygen species produced following treatment with glutamate.
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Achillea/química , Antioxidantes/farmacología , Flavonoides/farmacología , Fármacos Neuroprotectores/farmacología , Sesquiterpenos/farmacología , Animales , Antioxidantes/química , Línea Celular Tumoral , Flavonoides/química , Ácido Glutámico/toxicidad , Ratones , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/química , Sesquiterpenos/químicaRESUMEN
We previously identified the chemokine receptor CCR4 as part of the molecular signature of melanoma brain metastasis. The aim of this study was to determine the functional significance of CCR4 in melanoma brain metastasis. We show that CCR4 is more highly expressed by brain metastasizing melanoma cells than by local cutaneous cells from the same melanoma. Moreover, we found that the expression of CCR4 is significantly higher in paired clinical specimens of melanoma metastases than in samples of primary tumors from the same patients. Notably, the expression of the CCR4 ligands, Ccl22 and Ccl17 is upregulated at the earliest stages of brain metastasis, and precedes the infiltration of melanoma cells to the brain. In-vitro, CCL17 induced migration and transendothelial migration of melanoma cells. Functionally, human melanoma cells over-expressing CCR4 were more tumorigenic and produced a higher load of spontaneous brain micrometastasis than control cells. Blocking CCR4 with a small molecule CCR4 antagonist in-vivo, reduced the tumorigenicity and micrometastasis formation of melanoma cells. Taken together, these findings implicate CCR4 as a driver of melanoma brain metastasis.
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Neoplasias Encefálicas/secundario , Melanoma/metabolismo , Melanoma/patología , Receptores CCR4/metabolismo , Animales , Biomarcadores , Neoplasias Encefálicas/tratamiento farmacológico , Línea Celular Tumoral , Movimiento Celular , Supervivencia Celular/genética , Quimiocina CCL17/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Expresión Génica , Humanos , Inmunofenotipificación , Ligandos , Masculino , Melanoma/tratamiento farmacológico , Melanoma/genética , Ratones , Fenotipo , Receptores CCR4/antagonistas & inhibidores , Receptores CCR4/genética , Células del Estroma/metabolismo , Carga Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Achillolide A is a natural sesquiterpene lactone that we have previously shown can inhibit microglial activation. In this study we present evidence for its beneficial effects on astrocytes under oxidative stress, a situation relevant to neurodegenerative diseases and brain injuries. Viability of brain astrocytes (primary cultures) was determined by lactate dehydrogenase (LDH) activity, intracellular ROS levels were detected using 2',7'-dichlorofluorescein diacetate, in vitro antioxidant activity was measured by differential pulse voltammetry, and protein phosphorylation was determined using specific ELISA kits. We have found that achillolide A prevented the H2O2-induced death of astrocytes, and attenuated the induced intracellular accumulation of reactive oxygen species (ROS). These activities could be attributed to the inhibition of the H2O2-induced phosphorylation of MAP/ERK kinase 1 (MEK1) and p44/42 mitogen-activated protein kinases (MAPK), and to the antioxidant activity of achillolide A, but not to H2O2 scavenging. This is the first study that demonstrates its protective effects on brain astrocytes, and its ability to interfere with MAPK activation. We propose that achillolide A deserves further evaluation for its potential to be developed as a drug for the prevention/treatment of neurodegenerative diseases and brain injuries where oxidative stress is part of the pathophysiology.
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Achillea/química , Astrocitos/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Extractos Vegetales/farmacología , Especies Reactivas de Oxígeno/metabolismo , Animales , Astrocitos/citología , Astrocitos/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Peróxido de Hidrógeno/efectos adversos , Lactonas/química , Lactonas/farmacología , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/farmacología , Extractos Vegetales/química , Ratas , Sesquiterpenos/química , Sesquiterpenos/farmacologíaRESUMEN
Chronic inflammation has been implicated in the pathogenesis of various neurodegenerative diseases. During the neuroinflammatory process, microglial cells release neurotoxic and proinflammatory mediators. In the present study, using activity-guided fractionation, we have purified an anti-inflammatory compound determined by spectroscopic methods to be a sesquiterpene lactone named achillolide A from Achillea fragrantissima (Forsk.) Sch. Bip. In primary cultures of lipopolysaccharide-activated microglial cells, achillolide A inhibited the lipopolysaccharide-induced levels of proinflammatory and toxic mediators including glutamate, nitric oxide, matrix metalloproteinase-9, cyclooxygenase-2, induced nitric oxide synthase, interleukin-1ß, and tumor necrosis factor-α. Achillolide A also exhibited an antioxidant capacity, as was shown in a cell free system as well as by its ability to reduce intracellular reactive oxygen species levels in microglial cells. Thus, achillolide A might have therapeutic potential for treatment of neurodegenerative diseases and deserves further studies.
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Achillea/química , Antiinflamatorios/farmacología , Mediadores de Inflamación/metabolismo , Lactonas/farmacología , Microglía/efectos de los fármacos , Extractos Vegetales/farmacología , Sesquiterpenos/farmacología , Animales , Antiinflamatorios/uso terapéutico , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , Regulación hacia Abajo , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Lactonas/uso terapéutico , Microglía/metabolismo , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/metabolismo , Fitoterapia , Extractos Vegetales/uso terapéutico , Ratas , Especies Reactivas de Oxígeno/metabolismo , Sesquiterpenos/uso terapéuticoRESUMEN
Oxidative stress is tightly involved in various neurodegenerative diseases such as Parkinson's and Alzheimer's diseases, and conditions such as ischemia. Astrocytes, the most abundant glial cells in the brain, protect neurons from reactive oxygen species (ROS) and provide them with trophic support. Therefore, any damage to astrocytes will affect neuronal survival. In a previous study we have demonstrated that an extract prepared from the plant Achillea fragrantissima (Af) prevented the oxidative stress-induced death of astrocytes and attenuated the intracellular accumulation of ROS in astrocytes under oxidative stress. In the present study, using activity guided fractionation, we have purified from this plant the active compound, determined to be a flavonoid named 3,5,4'-trihydroxy-6,7,3'-trimethoxyflavone (TTF). The effects of TTF in any biological system have not been studied previously, and this is the first study to characterize the anti-oxidant and protective effects of this compound in the context of neurodegenerative diseases. Using primary cultures of astrocytes we have found that TTF prevented the hydrogen peroxide (H2O2)-induced death of astrocytes, and attenuated the intracellular accumulation of ROS following treatment of these cells with H2O2 or the peroxyl radicals generating molecule 2,2'-Azobis(amidinopropane) (ABAP). TTF also interfered with cell signaling events and inhibited the phosphorylation of the signaling proteins stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), extracellular signal regulated kinase (ERK 1/2) and mitogen activated protein kinase kinase (MEK1) and the phosphorylation of the transcription factor cyclic AMP response element-binding protein (CREB). The mechanism of the protective effect of TTF against H2O2-cytotoxicity could not be attributed to a direct H2O2 scavenging but rather to the scavenging of free radicals as was shown in cell free systems. Thus, TTF might be a therapeutic candidate for the prevention/treatment of neurodegenerative diseases where oxidative stress is part of the pathophysiology.
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Astrocitos/metabolismo , Flavonas/farmacología , Líquido Intracelular/metabolismo , Estrés Oxidativo/fisiología , Extractos Vegetales/farmacología , Especies Reactivas de Oxígeno/metabolismo , Achillea , Animales , Astrocitos/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Citoprotección/efectos de los fármacos , Citoprotección/fisiología , Flavonas/aislamiento & purificación , Peróxido de Hidrógeno/toxicidad , Líquido Intracelular/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Extractos Vegetales/aislamiento & purificación , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Oxidative stress is involved in the pathogenesis of neurodegenerative diseases such as Parkinson's and Alzheimer's diseases. Astrocytes, the most abundant glial cells in the brain, protect neurons from reactive oxygen species (ROS) and provide them with trophic support, such as glial-derived neurotrophic factor (GDNF). Thus, any damage to astrocytes will affect neuronal survival. In the present study, by activity-guided fractionation, we have purified from the desert plant Pulicaria incisa two protective compounds and determined their structures by spectroscopic methods. The compounds were found to be new chalcones-pulichalconoid B and pulichalconoid C. This is the first study to characterize the antioxidant and protective effects of these compounds in any biological system. Using primary cultures of astrocytes, we have found that pulichalconoid B attenuated the accumulation of ROS following treatment of these cells with hydrogen peroxide by 89% and prevented 89% of the H2O2-induced death of astrocytes. Pulichalconoid B exhibited an antioxidant effect both in vitro and in the cellular antioxidant assay in astrocytes and microglial cells. Pulichalconoid B also caused a fourfold increase in GDNF transcription in these cells. Thus, this chalcone deserves further studies in order to evaluate if beneficial therapeutic effect exists.
Asunto(s)
Antioxidantes/farmacología , Astrocitos/efectos de los fármacos , Encéfalo/citología , Pulicaria/química , Animales , Antioxidantes/química , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Espectroscopía de Resonancia Magnética , Ratas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Oxidative stress is involved in the pathogenesis of neurodegenerative diseases such as Parkinson's and Alzheimer's diseases. Astrocytes, the most abundant glial cells in the brain, protect neurons from reactive oxygen species (ROS) and provide them with trophic support, such as glial-derived neurotrophic factor (GDNF). Thus, any damage to astrocytes will affect neuronal survival. In the present study, an infusion prepared from the desert plant Pulicaria incisa (Pi) was tested for its protective and antioxidant effects on astrocytes subjected to oxidative stress. The Pi infusion attenuated the intracellular accumulation of ROS following treatment with hydrogen peroxide and zinc and prevented the H(2)O(2)-induced death of astrocytes. The Pi infusion also exhibited an antioxidant effect in vitro and induced GDNF transcription in astrocytes. It is proposed that this Pi infusion be further evaluated for use as a functional beverage for the prevention and/or treatment of brain injuries and neurodegenerative diseases in which oxidative stress plays a role.
Asunto(s)
Antioxidantes/farmacología , Astrocitos/citología , Astrocitos/efectos de los fármacos , Citoprotección/efectos de los fármacos , Extractos Vegetales/farmacología , Pulicaria/química , Amidinas/farmacología , Animales , Astrocitos/metabolismo , Compuestos de Bifenilo/metabolismo , Muerte Celular/efectos de los fármacos , Cloruros/farmacología , Depuradores de Radicales Libres/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Peróxido de Hidrógeno/farmacología , Oxidación-Reducción/efectos de los fármacos , Peróxidos/metabolismo , Picratos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Factores de Tiempo , Compuestos de Zinc/farmacologíaRESUMEN
BACKGROUND: The neuroinflammatory process plays a central role in the initiation and progression of neurodegenerative diseases such as Parkinson's and Alzheimer's diseases, and involves the activation of brain microglial cells. During the neuroinflammatory process, microglial cells release proinflammatory mediators such as cytokines, matrix metalloproteinases (MMP), Reactive oxygen species (ROS) and nitric oxide (NO). In the present study, extracts from 66 different desert plants were tested for their effect on lipopolysaccharide (LPS) - induced production of NO by primary microglial cells. The extract of Achillea fragrantissima (Af), which is a desert plant that has been used for many years in traditional medicine for the treatment of various diseases, was the most efficient extract, and was further studied for additional anti-neuroinflammatory effects in these cells. METHODS: In the present study, the ethanolic extract prepared from Af was tested for its anti-inflammatory effects on lipopolysaccharide (LPS)-activated primary cultures of brain microglial cells. The levels of the proinflammatory cytokines interleukin1ß (IL-1ß) and tumor necrosis factor-α (TNFα) secreted by the cells were determined by reverse transcriptase-PCR and Enzyme-linked immunosorbent assay (ELISA), respectively. NO levels secreted by the activate cells were measured using Griess reagent, ROS levels were measured by 2'7'-dichlorofluorescein diacetate (DCF-DA), MMP-9 activity was measured using gel zymography, and the protein levels of the proinflammatory enzymes cyclooxygenase-2 (COX-2) and induced nitric oxide synthase (iNOS) were measured by Western blot analysis. Cell viability was assessed using Lactate dehydrogenase (LDH) activity in the media conditioned by the cells or by the crystal violet cell staining. RESULTS: We have found that out of the 66 desert plants tested, the extract of Af was the most efficient extract and inhibited ~70% of the NO produced by the LPS-activated microglial cells, without affecting cell viability. In addition, this extract inhibited the LPS - elicited expression of the proinflammatory mediators IL-1ß, TNFα, MMP-9, COX-2 and iNOS in these cells. CONCLUSIONS: Thus, phytochemicals present in the Af extract could be beneficial in preventing/treating neurodegenerative diseases in which neuroinflammation is part of the pathophysiology.
Asunto(s)
Achillea/química , Antiinflamatorios/farmacología , Microglía/efectos de los fármacos , Microglía/inmunología , Enfermedades Neurodegenerativas/inmunología , Extractos Vegetales/farmacología , Animales , Células Cultivadas , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/inmunología , Humanos , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Masculino , Microglía/enzimología , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/enzimología , Enfermedades Neurodegenerativas/genética , Óxido Nítrico/inmunología , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/inmunología , Ratas , Ratas WistarRESUMEN
To determine the effect of the tolerogenic peptide hCDR1 on hippocampal neurogenesis, we treated SLE-afflicted (NZBxNZW)F1 mice with hCDR1 (once a week for 10weeks). The treatment resulted in the up-regulation of neurogenesis in the dentate gyrus and restored the NeuN immunoreactivity in brain hippocampi of the mice in association with increased gene expression of IGF-1, NGF and BDNF. Furthermore, hCDR1 treatment significantly up-regulated p-ERK and p-Akt that are suggested to be key components in mediating growth factor-induced neurogenesis. The observed effects of hCDR1 on hippocampal-neurogenesis and on associated signaling pathways suggest a potential role for hCDR1 in CNS lupus.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Hipocampo/efectos de los fármacos , Vasculitis por Lupus del Sistema Nervioso Central/tratamiento farmacológico , Neurogénesis/efectos de los fármacos , Fragmentos de Péptidos/uso terapéutico , Animales , Western Blotting , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Femenino , Expresión Génica/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Inmunohistoquímica , Vasculitis por Lupus del Sistema Nervioso Central/metabolismo , Vasculitis por Lupus del Sistema Nervioso Central/patología , Ratones , Factores de Crecimiento Nervioso/efectos de los fármacos , Proteínas Quinasas/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacosRESUMEN
Systemic lupus erythematosus (SLE) is an autoimmune disease that involves dysregulation of B and T cells. A tolerogenic peptide, designated hCDR1, ameliorates disease manifestations in SLE-afflicted mice. In the present study, the effect of treatment with hCDR1 on the CD74/macrophage migration inhibitory factor (MIF) pathway was studied. We report here that B lymphocytes from SLE-afflicted mice express relatively elevated levels of CD74, compared with B cells from healthy mice. CD74 is a receptor found in complex with CD44, and it binds the pro-inflammatory cytokine MIF. The latter components were also up-regulated in B cells from the diseased mice, and treatment with hCDR1 resulted in their down-regulation and in reduced B-cell survival. Furthermore, up-regulation of CD74 and CD44 expression was detected in brain hippocampi and kidneys, two target organs in SLE. Treatment with hCDR1 diminished the expression of those molecules to the levels determined for young healthy mice. These results suggest that the CD74/MIF pathway plays an important role in lupus pathology.