RESUMO
Epidemiological studies show a coincidence between Parkinson's disease (PD) and malignant melanoma. It has been suggested that this relationship is due, at least in part, to modulation of alpha-Synuclein (αSyn/Snca). αSyn oligomers accumulate in PD, which triggers typical PD symptoms, and in malignant melanoma, which increases the proliferation of tumor cells. In addition, αSyn contributes to non-motor symptoms of PD, including pain. In this study, we investigated the role of αSyn in melanoma growth and melanoma-induced pain in a mouse model using systemic and local depletion of αSyn. B16BL6 wild-type as well as αSyn knock-down melanoma cells were inoculated into the paws of αSyn knock-out mice and wild-type mice, respectively. Tumor growth and tumor-induced pain hypersensitivity were assessed over a period of 21 days. Molecular mechanisms were analyzed by RT-PCR and Western Blot in tumors, spinal cord, and sciatic nerve. Our results indicate that both global and local ablation of Snca contribute to reduced tumor growth and to a reduction of tumor-induced mechanical allodynia, though mechanisms contributing to these effects differ. While injection of wild-type cells in Snca knock-out mice strongly increased the immune response in the tumor, local Snca knock-down decreased autophagy mechanisms and the inflammatory reaction in the tumor. In conclusion, a knockdown of αSyn might constitute a promising approach to inhibiting the progression of melanoma and reducing tumor-induced pain.
Assuntos
Dor do Câncer , Melanoma , Animais , Camundongos , alfa-Sinucleína/genética , Camundongos Knockout , Doença de Parkinson , Melanoma Maligno CutâneoRESUMO
(1) AlphαSynuclein (αSyn) is a synaptic protein which is expressed in the nervous system and has been linked to neurodegenerative diseases, in particular Parkinson's disease (PD). Symptoms of PD are mainly due to overexpression and aggregation of αSyn and include pain. However, the interconnection of αSyn and pain has not been clarified so far. (2) We investigated the potential effects of a αSyn knock-out on the nociceptive behaviour in mouse models of acute, inflammatory and neuropathic pain. Furthermore, we assessed the impact of αSyn deletion on pain-related cellular and molecular mechanisms in the spinal cord in these models. (3) Our results showed a reduction of acute cold nociception in αSyn knock-out mice while responses to acute heat and mechanical noxious stimulation were similar in wild type and knock-out mice. Inflammatory nociception was not affected by αSyn knock-out which is also mirrored by unaltered inflammatory gene expression. In contrast, in the SNI model of neuropathic pain, αSyn knock-out mice showed decreased mechanical allodynia as compared to wild type mice. This effect was associated with reduced proinflammatory mechanisms and suppressed activation of MAP kinase signalling in the spinal cord while endogenous antinociceptive mechanisms are not inhibited. (4) Our data indicate that αSyn plays a role in neuropathy and its inhibition might be useful to ameliorate pain symptoms after nerve injury.
Assuntos
Neuralgia , Animais , Modelos Animais de Doenças , Hiperalgesia/metabolismo , Camundongos , Camundongos Knockout , Neuralgia/metabolismo , Nociceptividade/fisiologiaRESUMO
Post-exercise hypotension (PEH) is the phenomenon of lowered blood pressure after a single bout of exercise. Only a fraction of people develops PEH but its occurrence correlates well with long-term effects of sports on blood pressure. Therefore, PEH has been suggested as a suitable predictor for the effectivity of exercise as therapy in hypertension. Local vascular bioactive lipids might play a potential role in this context. We performed a cross-over clinical pilot study with 18 healthy volunteers to investigate the occurrence of PEH after a single short-term endurance exercise. Furthermore, we investigated the plasma lipid profile with focus on arachidonic acid (AA)-derived metabolites as potential biomarkers of PEH. A single bout of ergometer cycling induced a significant PEH in healthy volunteers with the expected high inter-individual variability. Targeted lipid spectrum analysis revealed significant upregulation of several lipids in the direct post-exercise phase. Among these changes, only 15- hydroxyeicosatetranoic acid (HETE) correlated significantly with the extent of PEH but in an AA-independent manner, suggesting that 15-HETE might act as specific PEH-marker. Our data indicate that specific lipid modulation might facilitate the identification of patients who will benefit from exercise activity in hypertension therapy. However, larger trials including hypertonic patients are necessary to verify the clinical value of this hypothesis.
Assuntos
Ácido Araquidônico/sangue , Exercício Físico , Ácidos Hidroxieicosatetraenoicos/sangue , Hipotensão Pós-Exercício/sangue , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/sangue , Adulto , Variação Biológica da População , Pressão Sanguínea/fisiologia , Estudos Cross-Over , Dinoprostona/sangue , Feminino , Voluntários Saudáveis , Frequência Cardíaca/fisiologia , Humanos , Hipertensão/fisiopatologia , Hipertensão/terapia , Metabolismo dos Lipídeos/fisiologia , Masculino , Projetos Piloto , Tromboxanos/sangueRESUMO
Inhibitor-kappaB kinase epsilon (IKKε) and TANK-binding kinase 1 (TBK1) are non-canonical IκB kinases, both described as contributors to tumor growth and metastasis in different cancer types. Several hints indicate that they are also involved in the pathogenesis of melanoma; however, the impact of their inhibition as a potential therapeutic measure in this "difficult-to-treat" cancer type has not been investigated so far. We assessed IKKε and TBK1 expression in human malignant melanoma cells, primary tumors and the metastasis of melanoma patients. Both kinases were expressed in the primary tumor and in metastasis and showed a significant overexpression in tumor cells in comparison to melanocytes. The pharmacological inhibition of IKKε/TBK1 by the approved drug amlexanox reduced cell proliferation, migration and invasion. Amlexanox did not affect the cell cycle progression nor apoptosis induction but significantly suppressed autophagy in melanoma cells. The analysis of potential functional downstream targets revealed that NF-кB and ERK pathways might be involved in kinase-mediated effects. In an in vivo xenograft model in nude mice, amlexanox treatment significantly reduced tumor growth. In conclusion, amlexanox was able to suppress tumor progression potentially by the inhibition of autophagy as well as NF-кB and MAP kinase pathways and might therefore constitute a promising candidate for melanoma therapy.
Assuntos
Aminopiridinas/farmacologia , Melanoma/metabolismo , Aminopiridinas/metabolismo , Animais , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/metabolismo , Melanoma/tratamento farmacológico , Camundongos , Camundongos Nus , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Inhibitor-kappaB kinase epsilon (IKKε, Ikbke) constitutes an NF-κB activating kinase with high homology to the classical I-κB kinase subunits, IKKα and IKKß. It is expressed in nociceptive neurons in the spinal cord and in dorsal root ganglia (DRG) and involved in inflammatory nociception. Under inflammatory conditions, IKKε deficient mice show significantly less nociceptive behavior in comparison to wild type mice associated with reduced activation of NF-κB and attenuated NF-κB-dependent gene expression. The role of IKKε in neuropathic pain has not been investigated so far. We applied the spared nerve injury (SNI) model of neuropathic pain in mice and found an increased expression of IKKε in the spinal cord, the DRGs and the sciatic nerve after induction of neuropathy. Genetic depletion of IKKε or pharmacological inhibition by amlexanox led to a significant reduction of mechanical hyperalgesia and cold allodynia in comparison to control mice. Transcription factor ELISA indicated that the effects are mediated by reduced activation of NF-κB. Furthermore, immunofluorescence staining, qPCR and Western Blot analyses revealed that the decreased pain-like behavior was associated with a reduced activation of microglia, diminished expression of c-fos as well as a decreased activation of MAP-Kinases. In summary, we conclude that IKKε modulates mechanisms of neuropathic pain by activating NF-κB. The administration of IKKε inhibitors might therefore constitute a new and promising approach for the therapy of neuropathic pain.
Assuntos
Aminopiridinas/farmacologia , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/deficiência , Neuralgia/tratamento farmacológico , Animais , Modelos Animais de Doenças , Feminino , Gânglios Espinais/metabolismo , Hiperalgesia/tratamento farmacológico , Hiperalgesia/genética , Sistema de Sinalização das MAP Quinases/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia , NF-kappa B/metabolismo , Neuralgia/induzido quimicamente , Traumatismos dos Nervos Periféricos/tratamento farmacológico , Proteínas Serina-Treonina Quinases/metabolismo , Nervo Isquiático/metabolismo , Transdução de Sinais , Medula Espinal/metabolismoRESUMO
Physical exercise has been repeatedly associated with decreased nociceptive responses but the underlying mechanisms have still not been fully clarified. In this study, we investigated exercise-induced effects after a single bout of treadmill running on the mouse model of formalin-induced inflammatory nociception. As potential molecular mediators, we focused on endogenous endocannabinoids as well as AMP-activated protein kinase (AMPK). Our results showed that wild type mice display a reduced nociceptive response in the formalin test after treadmill running, while exercise had no effect on inflammatory nociception in AMPKα2 knockout mice. Levels of the endocannabinoid anandamide (AEA) were increased after physical activity in both wild type and AMPKα2 knockout mice, in association with decreased expression of the AEA-hydrolyzing enzyme FAAH and an increased level of the cannabinoid receptor 1 (CB1). Accordingly, treatment of wild type mice with the CB1 inverse agonist AM251 prior to the treadmill running reversed exercise-induced antinociception. However, if mice received AM251 in combination with the AMPK activator 5-amino-1-ß-d-ribofuranosyl-imidazole-4-carboxamide (AICAR), the positive effect of treadmill running on inflammatory nociception was restored, indicating that AMPK affects exercise-induced antinociception downstream of endocannabinoids. This assumption was further supported by cell culture experiments showing AMPK activation after stimulation of neuronal cells with AEA. In conclusion, our data suggest that AMPK is an intermediate effector in endocannabinoid-mediated exercise-induced antinociception. This article is part of the Special Issue entitled "A New Dawn in Cannabinoid Neurobiology".
Assuntos
Proteínas Quinases Ativadas por AMP/fisiologia , Nociceptividade/fisiologia , Condicionamento Físico Animal/fisiologia , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Amidoidrolases/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Ácidos Araquidônicos/metabolismo , Células Cultivadas , Endocanabinoides/metabolismo , Feminino , Masculino , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Nociceptividade/efeitos dos fármacos , Medição da Dor , Piperidinas/farmacologia , Alcamidas Poli-Insaturadas/metabolismo , Pirazóis/farmacologia , Receptor CB1 de Canabinoide/metabolismo , Ribonucleotídeos/farmacologiaRESUMO
Chronic pain is accompanied by production of reactive oxygen species (ROS) in various cells that are important for nociceptive processing. Recent data indicate that ROS can trigger specific redox-dependent signaling processes, but the molecular targets of ROS signaling in the nociceptive system remain largely elusive. Here, we performed a proteome screen for pain-dependent redox regulation using an OxICAT approach, thereby identifying the small GTPase Rab7 as a redox-modified target during inflammatory pain in mice. Prevention of Rab7 oxidation by replacement of the redox-sensing thiols modulates its GTPase activity. Immunofluorescence studies revealed Rab7 expression to be enriched in central terminals of sensory neurons. Knockout mice lacking Rab7 in sensory neurons showed normal responses to noxious thermal and mechanical stimuli; however, their pain behavior during inflammatory pain and in response to ROS donors was reduced. The data suggest that redox-dependent changes in Rab7 activity modulate inflammatory pain sensitivity.
Assuntos
Gânglios Espinais/metabolismo , Inflamação/metabolismo , Dor/metabolismo , Medula Espinal/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Camundongos , Camundongos Knockout , Proteômica , Espécies Reativas de Oxigênio/metabolismo , Células Receptoras Sensoriais/metabolismo , Transdução de Sinais/fisiologia , proteínas de unión al GTP Rab7RESUMO
Alterations in gene expression as a consequence of physical exercise are frequently described. The mechanism of these regulations might depend on epigenetic changes in global or gene-specific DNA methylation levels. The AMP-activated protein kinase (AMPK) plays a key role in maintenance of energy homeostasis and is activated by increases in the AMP/ATP ratio as occurring in skeletal muscles after sporting activity. To analyze whether exercise has an impact on the methylation status of the AMPK promoter, we determined the AMPK methylation status in human blood samples from patients before and after sporting activity in the context of rehabilitation as well as in skeletal muscles of trained and untrained mice. Further, we examined long interspersed nuclear element 1 (LINE-1) as indicator of global DNA methylation changes. Our results revealed that light sporting activity in mice and humans does not alter global DNA methylation but has an effect on methylation of specific CpG sites in the AMPKα2 gene. These regulations were associated with a reduced AMPKα2 mRNA and protein expression in muscle tissue, pointing at a contribution of the methylation status to AMPK expression. Taken together, these results suggest that exercise influences AMPKα2 gene methylation in human blood and eminently in the skeletal muscle of mice and therefore might repress AMPKα2 gene expression.
Assuntos
Proteínas Quinases Ativadas por AMP/sangue , Traumatismos em Atletas/fisiopatologia , Terapia por Exercício/métodos , Condicionamento Físico Animal/métodos , Condicionamento Físico Humano/métodos , Resistência Física , Adolescente , Adulto , Idoso , Animais , Traumatismos em Atletas/reabilitação , Metilação de DNA , Feminino , Treinamento Intervalado de Alta Intensidade/métodos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Resultado do Tratamento , Adulto JovemRESUMO
Inhibitor-kappaB kinase epsilon (IKKε) constitutes a non-canonical I-κB kinase, which amongst others modulates NF-κB activity. IKKε and NF-κB have both been described for their role in cell proliferation and their dysregulation has been associated with tumourigenesis and metastasis in multiple cancer types. Accordingly, overexpression and constitutive activation of NF-κB have also been shown in melanoma, however, the role of IKKε in this cancer type has not been investigated so far. Thus, we determined IKKε expression in malignant melanoma cells and we were able to show a significant overexpression of IKKε in tumour cells in comparison to melanocytes. Inhibition of IKKε either by shRNA or the pharmacological inhibitor amlexanox resulted in reduced cell proliferation associated with a cell cycle block in the G1-phase. Functional analysis indicated that NF-κB, Akt1 and MAPK pathways might be involved in the IKKε-mediated effects. In vivo, we applied a mouse melanoma skin cancer model to assess tumour growth and melanoma-associated pain in IKKε knockout mice as well as C57BL/6 mice after inoculation with IKKε-negative cells. In IKKε knockout mice, tumour growth was not altered as compared to IKKε wild type mice. However, melanoma associated pain was strongly suppressed accompanied by a reduced mRNA expression of a number of pain-relevant genes. In contrast, after inoculation of IKKε-depleted tumour cells, the development of melanoma was almost completely prevented. In conclusion, our data suggest that IKKε in the tumour plays an essential role in tumour initiation and progression while IKKε expression in tumour surrounding tissues contributes to melanoma-associated pain.
Assuntos
Quinase I-kappa B/metabolismo , Melanoma/enzimologia , Melanoma/patologia , Dor/fisiopatologia , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/patologia , Aminopiridinas/farmacologia , Animais , Ciclo Celular , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/genética , Melanócitos/enzimologia , Melanoma/fisiopatologia , Melanoma Experimental/enzimologia , Melanoma Experimental/patologia , Melanoma Experimental/fisiopatologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Cutâneas/fisiopatologiaRESUMO
The processing of pain undergoes several changes in aging that affect sensory nociceptive fibers and the endogenous neuronal inhibitory systems. So far, it is not completely clear whether age-induced modifications are associated with an increase or decrease in pain perception. In this study, we assessed the impact of age on inflammatory nociception in mice and the role of the hormonal inhibitory systems in this context. We investigated the nociceptive behavior of 12-month-old versus 6-8-week-old mice in two behavioral models of inflammatory nociception. Levels of TRP channels, and cortisol as well as cortisol targets, were measured by qPCR, ELISA, and Western blot in the differently aged mice. We observed an age-related reduction in nociceptive behavior during inflammation as well as a higher level of cortisol in the spinal cord of aged mice compared to young mice, while TRP channels were not reduced. Among potential cortisol targets, the NF-κB inhibitor protein alpha (IκBα) was increased, which might contribute to inhibition of NF-κB and a decreased expression and activity of the inducible nitric oxide synthase (iNOS). In conclusion, our results reveal a reduced nociceptive response in aged mice, which might be at least partially mediated by an augmented inflammation-induced increase in the hormonal inhibitory system involving cortisol.
Assuntos
Comportamento Animal , Inflamação/complicações , Nociceptividade , Dor Nociceptiva/etiologia , Fatores Etários , Animais , Modelos Animais de Doenças , Feminino , Hidrocortisona/metabolismo , Proteínas I-kappa B/metabolismo , Inflamação/metabolismo , Masculino , Camundongos , Inibidor de NF-kappaB alfa , Óxido Nítrico Sintase Tipo II , Medição da Dor , Medula Espinal/metabolismo , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
AMP-activated kinase (AMPK) is a cellular energy sensor, which is activated in stages of increased adenosine triphosphate (ATP) consumption. Its activation has been associated with a number of beneficial effects such as decrease of inflammatory processes and inhibition of disease progression of diabetes and obesity. A recent study suggested that salicylate, the active metabolite of the non-steroidal anti-inflammatory drug (NSAID) acetyl-salicylic acid (aspirin), is able to activate AMPK pharmacologically. This observation raised the question whether or not other NSAIDs might also act as AMPK activators and whether this action might contribute to their cyclooxygenase (COX)-independent anti-inflammatory properties. In this study, we investigated mouse and human neuronal cells and liver tissue of mice after treatment with various NSAIDs. Our results showed that the non-selective acidic NSAIDs ibuprofen and diclofenac induced AMPK activation similar to aspirin while the COX-2 selective drug etoricoxib and the non-opioid analgesic paracetamol, both drugs have no acidic structure, failed to activate AMPK. In conclusion, our results revealed that AMPK can be activated by specific non-steroidal anti-inflammatory drugs such as salicylic acid, ibuprofen or diclofenac possibly depending on the acidic structure of the drugs. AMPK might therefore contribute to their antinociceptive and anti-inflammatory properties.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Proteínas Quinases Ativadas por AMP/genética , Animais , Linhagem Celular Tumoral , Diclofenaco/farmacologia , Ativação Enzimática/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Ibuprofeno/farmacologia , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismoRESUMO
BACKGROUND: TANK-binding kinase (TBK1) is a non-canonical IκB kinase (IKK) involved in the regulation of type I interferons and of NF-κB signal transduction. It is activated by viral infections and inflammatory mediators and has therefore been associated with viral diseases, obesity, and rheumatoid arthritis. Its role in pain has not been investigated so far. Due to the important roles of NF-κB, classical IκB Kinases and the IKK-related kinase, IKKε, in inflammatory nociception, we hypothesized that TBK1, which is suggested to form a complex with IKKε under certain conditions, might also alter the inflammatory nociceptive response. METHODS: We investigated TBK1 expression and regulation in "pain-relevant" tissues of C57BL/6 mice by immunofluorescence, quantitative PCR, and Western blot analysis. Furthermore, nociceptive responses and the underlying signal transduction pathways were assessed using TBK1(-/-) mice in two models of inflammatory nociception. RESULTS: Our data show that TBK1 is expressed and regulated in the spinal cord after peripheral nociceptive stimulation and that a deletion of TBK1 alleviated the inflammatory hyperalgesia in mice while motor function and acute nociception were not altered. TBK1-mediated effects are at least partially mediated by regulation of NF-κB dependent COX-2 induction but also by alteration of expression of c-fos via modulation of MAP kinases as shown in the spinal cord of mice and in cell culture experiments. CONCLUSION: We suggest that TBK1 exerts pronociceptive effects in inflammatory nociception which are due to both modulation of NF-κB dependent genes and regulation of MAPKs and c-fos. Inhibition of TBK1 might therefore constitute a novel effective tool for analgesic therapy.
Assuntos
Hiperalgesia/etiologia , Hiperalgesia/metabolismo , Inflamação/complicações , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , NF-kappa B/genética , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Linhagem Celular Transformada , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Regulação da Expressão Gênica/genética , Inflamação/patologia , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Atividade Motora/genética , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Limiar da Dor/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Medula Espinal/metabolismo , Medula Espinal/patologia , Fatores de TempoRESUMO
AMP-activated kinase is a cellular energy sensor which is activated in stages of increased ATP consumption. Its activation has been associated with a number of beneficial effects such as decreasing inflammatory processes and the disease progress of diabetes and obesity, respectively. Furthermore, AMPK activation has been linked with induction of cell cycle arrest and apoptosis in cancer and vascular cells, indicating that it might have a therapeutic impact for the treatment of cancer and atherosclerosis. However, the impact of AMPK on the proliferation of macrophages, which also play a key role in the formation of atherosclerotic plaques and in inflammatory processes, has not been focused so far. We have assessed the influence of AICAR- and metformin-induced AMPK activation on cell viability of macrophages with and without inflammatory stimulation, respectively. In cells without inflammatory stimulation, we found a strong induction of caspase 3-dependent apoptosis associated with decreased mTOR levels and increased expression of p21. Interestingly, these effects could be inhibited by co-stimulation with bacterial lipopolysaccharide (LPS) but not by other proinflammatory cytokines suggesting that AICAR induces apoptosis via AMPK in a TLR4-pathway dependent manner. In conclusion, our results revealed that AMPK activation is not only associated with positive effects but might also contribute to risk factors by disturbing important features of macrophages. The fact that LPS is able to restore AMPK-associated apoptosis might indicate an important role of TLR4 agonists in preventing unfavorable cell death of immune cells.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Apoptose/imunologia , Caspase 3/metabolismo , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Ribonucleotídeos/metabolismo , Aminoimidazol Carboxamida/metabolismo , Aminoimidazol Carboxamida/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Metformina/farmacologia , Camundongos , Ribonucleotídeos/farmacologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
UNLABELLED: The activation of the adenosine monophosphate (AMP)-activated kinase (AMPK) has been associated with beneficial effects such as improvement of hyperglycemic states in diabetes as well as reduction of obesity and inflammatory processes. Recent studies provide evidence for a further role of AMPK in models of acute and neuropathic pain. In this study, we investigated the impact of AMPK on inflammatory nociception. Using 5-amino-1-ß-d-ribofuranosyl-imidazole-4-carboxamide (AICAR) and metformin as AMPK activators, we observed anti-inflammatory and antinociceptive effects in 2 models of inflammatory nociception. The effects were similar to those observed with the standard analgesic ibuprofen. The mechanism appears to be based on regulation of the AMPKα2 subunit of the kinase because AMPKα2 knockout mice showed increased nociceptive responses that could not be reversed by the AMPK activators. On the molecular level, antinociceptive effects are at least partially mediated by reduced activation of different MAP-kinases in the spinal cord and a subsequent decrease in pain-relevant induction of c-fos, which constitutes a reliable marker of elevated activity in spinal cord neurons following peripheral noxious stimulation. In summary, our results indicate that activation of AMPKα2 might represent a novel therapeutic option for the treatment of inflammation-associated pain, providing analgesia with fewer unwanted side effects. PERSPECTIVE: AMPK activation is associated with beneficial effects on diabetes and obesity. In addition, we have shown analgesic properties of pharmacologic AMPK activation in inflammatory nociception, indicating that AMPK might serve as a novel therapeutic target in pain with fewer unwanted side effects.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Hiperalgesia/tratamento farmacológico , Inflamação/metabolismo , Metformina/farmacologia , Nociceptividade/efeitos dos fármacos , Ribonucleotídeos/farmacologia , Proteínas Quinases Ativadas por AMP/genética , Aminoimidazol Carboxamida/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Ativação Enzimática , Hiperalgesia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Destreza Motora/efeitos dos fármacos , Nociceptividade/fisiologia , Medição da Dor , Teste de Desempenho do Rota-RodRESUMO
microRNAs (miRNAs) are small noncoding RNAs that have been linked to a number of disease-related signal transduction pathways. Several studies indicate that they are also involved in nociception. It is not clear, however, which miRNAs are important and which genes are modulated by miRNA-associated mechanisms. This study focuses on the regulation and function of the central nervous system (CNS)-specific miRNA-124a in the spinal cord of mice in a formalin model of inflammatory nociception. miRNA-124a is constitutively expressed in the spinal cord of mice, particularly in neurons of the dorsal horn. Peripheral noxious stimulation with formalin led to significant down-regulation of its expression. Knock-down of miRNA-124a by intravenous administration of a specific miRNA-124a inhibitor further increased the nociceptive behavior associated with an upregulation of the pain-relevant miRNA-124a target MeCP2 and proinflammatory marker genes. In contrast, administration of a miRNA-124a mimic counteracted these effects and decreased nociception by down-regulation of the target gene. In conclusion, our results indicate that miRNA-124a is involved in inflammatory nociception by regulation of relevant target proteins and might therefore constitute a novel target for anti-inflammatory therapy.
Assuntos
Inflamação/tratamento farmacológico , MicroRNAs/fisiologia , Percepção da Dor/fisiologia , Medula Espinal/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Fator Neurotrófico Derivado do Encéfalo/genética , Formaldeído/toxicidade , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Inflamação/induzido quimicamente , Inflamação/genética , Masculino , Proteína 2 de Ligação a Metil-CpG/biossíntese , Proteína 2 de Ligação a Metil-CpG/genética , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/antagonistas & inibidores , MicroRNAs/biossíntese , MicroRNAs/genética , Terapia de Alvo Molecular , Oligorribonucleotídeos/farmacologia , Dor/etiologia , Dor/prevenção & controle , Percepção da Dor/efeitos dos fármacos , Células do Corno Posterior/metabolismo , RNA de Cadeia Dupla/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Método Simples-Cego , Medula Espinal/fisiopatologia , TransfecçãoRESUMO
Atomoxetine (ATX), a selective norepinephrine reuptake inhibitor, is a non-stimulant approved for the treatment of attention deficit/hyperactivity disorder (ADHD). Little is known about the molecular basis for its therapeutic effect. The objective of this animal study was to determine alterations in gene expression patterns in the prefrontal cortex after long-term administration of atomoxetine. Rats were treated for 21 days during childhood and early adolescent stages of development with a once-daily oral application of 0.05 g/kg atomoxetine, which resulted in plasma levels similar to those described in children. A whole genome RNA-microarray of rat prefrontal cortical gene expression after administration of atomoxetine versus sterile water revealed an mRNA increase in 114 genes (≥2-fold) while 11 genes were down-regulated (≤0.5-fold). By applying quantitative real-time PCR (qRT-PCR) and Western Blot we confirmed a significant increase in the expression of GABA A receptor subunits as well as ubiquinol-cytochrome c reductase complex core protein 2 (Uqcrc2). SNAP-25 (synaptosomal-associated protein of 25 kDa), which is an ADHD candidate gene and an important vesicle protein involved in axonal growth, synaptic plasticity and regulation of neurotransmitter release was also significantly upregulated on RNA- and protein level after atomoxetine treatment. In summary, we could show that long-term treatment with the ADHD drug atomoxetine induces the regulation of several genes in the prefrontal cortex of young rats. Especially the increased expression of SNAP-25 and GABA-A receptor subunits may indicate additional active therapeutic mechanisms for atomoxetine.
Assuntos
Inibidores da Captação Adrenérgica/farmacologia , Estimulantes do Sistema Nervoso Central/farmacologia , Expressão Gênica/efeitos dos fármacos , Córtex Pré-Frontal/efeitos dos fármacos , Propilaminas/farmacologia , Animais , Cloridrato de Atomoxetina , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Masculino , Córtex Pré-Frontal/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/metabolismo , Análise Serial de TecidosRESUMO
p21-activated kinases (PAKs) are involved in signal cascades relevant for nociceptive processing and neuropathic pain. Particularly, the recently described group B PAKs 4, 5 and 6 regulate MAP-kinases and the rearrangement of the actin cytoskeleton, both of which have been linked to pain processing. However, a specific role of these PAKs in nociception has not yet been demonstrated. We found PAK 4, 5 and 6 expression in pain-relevant tissues in peripheral and CNS. Since viable knock-out mice only exist for the PAK isoform 5, we further assessed the impact of this PAK on acute and chronic pain using different behavioral models in mice. PAK 5 knock-out mice showed normal acute nociception and did not differ from wild type mice in their neuropathic pain behavior. However, the nociceptive response in formalin-induced paw inflammation was significantly reduced in knock-out mice associated with inhibition of MAP-kinase activation and a decreased number of formalin-induced c-Fos positive neurons in the spinal cord. Furthermore, in isolated neurons, we found a significantly reduced calcium response after stimulation of TRPA1-channels in PAK 5(-/-)- compared to PAK 5(+/+)-cells. Our results indicate that PAK 5 is involved in formalin-induced inflammatory nociception through regulation of MAPK-induced c-Fos-activation and formalin-specific TRP-channels.
Assuntos
Formaldeído/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Neuralgia/induzido quimicamente , Nociceptividade , Quinases Ativadas por p21/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Nociceptividade/fisiologia , Medição da Dor/métodosRESUMO
Inhibitor-κB kinase ε (IKKε) was only recently identified as an enzyme with high homology to the classical I-κB kinase subunits, IKKα and IKKß. Despite this similarity, it is mainly discussed as a repressor of viral infections by modulating type I IFNs. However, in vitro studies also showed that IKKε plays a role in the regulation of NF-κB activity, but the distinct mechanisms of IKKε-mediated NF-κB activation are not clear. Given the paramount role of NF-κB in inflammation, we investigated the regulation and function of IKKε in models of inflammatory hyperalgesia in mice. We found that IKKε was abundantly expressed in nociceptive neurons in the spinal cord and in dorsal root ganglia. IKKε mRNA and protein levels rapidly increased in spinal cord and dorsal root ganglia during hind paw inflammation evoked by injection of zymosan or formalin. IKKε knockout mice showed normal nociceptive responses to acute heat or mechanical stimulation. However, in inflammatory pain models, IKKε-deficient mice exhibited a significantly reduced nociceptive behavior in comparison with wild type mice, indicating that IKKε contributed to the development of inflammatory hyperalgesia. Antinociceptive effects were associated with reduced activation of NF-κB and attenuated NF-κB-dependent induction of cyclooxygenase-2, inducible NO synthase, and metalloproteinase-9. In contrast, IRF-3, which is an important IKKε target in viral infections, was not regulated after inflammatory nociceptive stimulation. Therefore, we concluded that IKKε modulates inflammatory nociceptive sensitivity by activation of NF-κB-dependent gene transcription and may be useful as a therapeutic target in the treatment of inflammatory pain.