RESUMO
Parkinson's disease (PD) is a neurodegenerative disease characterized by dopaminergic neuron death and neuroinflammation. Emerging evidence points to the involvement of the transient receptor potential melastatin 2 (TRPM2) channel in neuron death and glial activation in several neurodegenerative diseases. However, the involvement of TRPM2 in PD and specifically its relation to the neuroinflammation aspect of the disease remains poorly understood. Here, we hypothesized that AG490, a TRPM2 inhibitor, can be used as a treatment in a mouse model of PD. Mice underwent stereotaxic surgery for 6-hydroxydopamine (6-OHDA) administration in the right striatum. Motor behavioral tests (apomorphine, cylinder, and rotarod) were performed on day 3 post-injection to confirm the PD model induction. AG490 was then daily injected i.p. between days 3 to 6 after surgery. On day 6, motor behavior was assessed again. Substantia nigra (SNc) and striatum (CPu) were collected for immunohistochemistry, immunoblotting, and RT-qPCR analysis on day 7. Our results revealed that AG490 post-treatment reduced motor behavior impairment and nigrostriatal neurodegeneration. In addition, the compound prevented TRPM2 upregulation and changes of the Akt/GSK-3ß/caspase-3 signaling pathway. The TRPM2 inhibition also avoids the glial morphology changes observed in the PD group. Remarkably, the morphometrical analysis revealed that the ameboid-shaped microglia, found in 6-OHDA-injected animals, were no longer present in the AG490-treated group. These results indicate that AG490 treatment can reduce dopaminergic neuronal death and suppress neuroinflammation in a PD mouse model. Inhibition of TRPM2 by AG490 could then represent a potential therapeutical strategy to be evaluated for PD treatment.
Assuntos
Camundongos Endogâmicos C57BL , Neuroglia , Canais de Cátion TRPM , Tirfostinas , Animais , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/metabolismo , Camundongos , Masculino , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neuroglia/patologia , Tirfostinas/farmacologia , Tirfostinas/uso terapêutico , Progressão da Doença , Oxidopamina/toxicidade , Modelos Animais de Doenças , Degeneração Neural/patologia , Degeneração Neural/tratamento farmacológico , Transtornos Parkinsonianos/patologia , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/prevenção & controle , Substância Negra/efeitos dos fármacos , Substância Negra/patologia , Substância Negra/metabolismo , Doença de Parkinson/patologia , Doença de Parkinson/metabolismo , Doença de Parkinson/tratamento farmacológicoRESUMO
Chikungunya virus (CHIKV) is a globally public health threat. There are currently no medications available to treat CHIKV infection. High-throughput screening of 419 kinase inhibitors was performed based on the cytopathic effect method, and six kinase inhibitors with reduced cytopathic effects, including tyrphostin AG879 (AG879), tyrphostin 9 (A9), sorafenib, sorafenib tosylate, regorafenib, and TAK-632, were identified. The anti-CHIKV activities of two receptor tyrosine kinase inhibitors, AG879 and A9, that have not been previously reported, were selected for further evaluation. The results indicated that 50% cytotoxic concentration (CC50) of AG879 and A9 in Vero cells were greater than 30 µM and 6.50 µM, respectively and 50% effective concentration (EC50) were 0.84 µM and 0.36 µM, respectively. The time-of-addition and time-of-removal assays illustrated that both AG879 and A9 function in the middle stage of CHIKV life cycle. Further, AG879 and A9 do not affect viral attachment; however, they inhibit viral RNA replication, and exhibit antiviral activity against CHIKV Eastern/Central/South African and Asian strains, Ross River virus and Sindbis virus in vitro.
Assuntos
Antineoplásicos , Febre de Chikungunya , Vírus Chikungunya , Animais , Chlorocebus aethiops , Humanos , Vírus Chikungunya/genética , Células Vero , Tirfostinas/farmacologia , Tirfostinas/uso terapêutico , Linhagem Celular , Antivirais/farmacologia , Antivirais/uso terapêutico , Replicação Viral , Inibidores de Proteínas Quinases/farmacologia , Antineoplásicos/farmacologiaRESUMO
Osteoarthritis (OA) is a complex chronic inflammatory disease characterized by articular degeneration and pain. Recent studies have identified interleukin 6 (IL-6) as a potential mediator leading to OA, but the therapeutic effects of inhibiting IL-6 signaling in intreating OA need to be further clarified. Here, we identified the intracellular signal transduction induced by recombinant IL-6 and focused on the impact of tyrphostin AG490 (a JAK2 inhibitor) on cartilage degeneration and OA pain. We found that IL-6 increased the inflammatory cytokines production and hypertrophic markers expression of primary mouse chondrocytes by activating JAK2/STAT3. Meanwhile, tyrphostin AG490 significantly attenuated articular degeneration and osteophyte formation in experimental mice with anterior cruciate ligament transection (ACLT) surgery. In vivo electrophysiological experiments showed that articular stimulation of IL-6 induced spinal hyperexcitability, which was prevented by coinjection of tyrphostin AG490. Specifically, compared with DMSO-treated ACLT mice, tyrphostin AG490 improved ambulate activity of mice and abolished the enhancement of serum bradykinin induced by IL-6. Together, we suggest that tyrphostin AG490 protected against progression of OA and improved OA prognosis by reducing cartilage degeneration and arthritis pain. Our findings provide further evidence for targeting IL-6 signaling in the treatment of OA.
Assuntos
Cartilagem Articular , Osteoartrite , Animais , Tirfostinas/farmacologia , Tirfostinas/uso terapêutico , Interleucina-6/metabolismo , Cartilagem Articular/metabolismo , Osteoartrite/tratamento farmacológico , Osteoartrite/metabolismo , Condrócitos , Dor/tratamento farmacológico , Dor/metabolismo , Modelos Animais de DoençasRESUMO
Ulcerative colitis (UC) is a chronic inflammatory bowel disease. Several studies have demonstrated that α7 nicotinic acetylcholine receptors (α7nAChRs) exert anti-inflammatory effects on immune cells and nicotine suppress UC onset and relapse. Plasmacytoid dendritic cells (pDCs) reportedly accumulate in the colon of UC patients. Therefore, we investigated the pathophysiological roles of α7nAChRs on pDCs in the pathology of UC using oxazolone (OXZ)-induced Th2-type colitis with BALB/c mice. 2-deoxy-D-glucose, a central vagal stimulant suppressed OXZ colitis, and nicotine also ameliorated OXZ colitis with suppressing Th2 cytokines, which was reversed by α7nAChR antagonist methyllycaconitine. Additionally, α7nAChRs were expressed on pDCs, which were located very close to cholinergic nerve fibers in the colon of OXZ mice. Furthermore, nicotine suppressed CCL21-induced bone marrow-derived pDC migration due to Rac 1 inactivation, which was reversed by methyllycaconitine, a JAK2 inhibitor AG490 or caspase-3 inhibitor AZ-10417808. CCL21 was mainly expressed in the isolated lymphoid follicles (ILFs) of the colon during OXZ colitis. The therapeutic effect of cholinergic pathway on OXZ colitis probably through α7nAChRs on pDCs were attributed to the suppression of pDC migration toward the ILFs. Therefore, the activation of α7nAChRs has innovative therapeutic potential for the treatment of UC.
Assuntos
Neurônios Colinérgicos/efeitos dos fármacos , Colite Ulcerativa/tratamento farmacológico , Células Dendríticas/efeitos dos fármacos , Neuroimunomodulação , Células Th2/metabolismo , Aconitina/análogos & derivados , Aconitina/farmacologia , Aconitina/uso terapêutico , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Caspase 3/metabolismo , Inibidores de Caspase/farmacologia , Inibidores de Caspase/uso terapêutico , Estimulantes do Sistema Nervoso Central/farmacologia , Estimulantes do Sistema Nervoso Central/uso terapêutico , Colite Ulcerativa/induzido quimicamente , Colo/metabolismo , Células Dendríticas/metabolismo , Desoxiglucose/farmacologia , Desoxiglucose/uso terapêutico , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Janus Quinase 2/metabolismo , Camundongos Endogâmicos BALB C , Neuropeptídeos/metabolismo , Nicotina/farmacologia , Nicotina/uso terapêutico , Oxazolona/toxicidade , Fator de Transcrição STAT3/metabolismo , Células Th2/efeitos dos fármacos , Tirfostinas/farmacologia , Tirfostinas/uso terapêutico , Nervo Vago/efeitos dos fármacos , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Receptor Nicotínico de Acetilcolina alfa7/antagonistas & inibidores , Receptor Nicotínico de Acetilcolina alfa7/genética , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
BACKGROUND: Hippo-Yes-associated protein (YAP) signaling is a key regulator of organ size and tumorigenesis, yet the underlying molecular mechanism is still poorly understood. At present, the significance of the Hippo-YAP pathway in diffuse large B-cell lymphoma (DLBCL) is ill-defined. METHODS: The expression of YAP in DLBCL was determined in public database and clinical specimens. The effects of YAP knockdown, CRISPR/Cas9-mediated YAP deletion, and YAP inhibitor treatment on cell proliferation and the cell cycle were evaluated both in vitro and in vivo. RNA sequencing was conducted to detect dysregulated RNAs in YAP-knockout DLBCL cells. The regulatory effects of insulin-like growth factor-1 receptor (IGF-1R) on Hippo-YAP signaling were explored by targeted inhibition and rescue experiments. RESULTS: High expression of YAP was significantly correlated with disease progression and poor prognosis. Knockdown of YAP expression suppressed cell proliferation and induced cell cycle arrest in DLBCL cells. Verteporfin (VP), a benzoporphyrin derivative, exerted an anti-tumor effect by regulating the expression of YAP and the downstream target genes, CTGF and CYR61. In vitro and in vivo studies revealed that deletion of YAP expression with a CRISPR/Cas9 genome editing system significantly restrained tumor growth. Moreover, downregulation of IGF-1R expression led to a remarkable decrease in YAP expression. In contrast, exposure to IGF-1 promoted YAP expression and reversed the inhibition of YAP expression induced by IGF-1R inhibitors. CONCLUSIONS: Our study highlights the critical role of YAP in the pathogenesis of DLBCL and uncovers the regulatory effect of IGF-1R on Hippo-YAP signaling, suggesting a novel therapeutic strategy for DLBCL.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Linfoma Difuso de Grandes Células B/metabolismo , Proteínas de Neoplasias/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Receptor IGF Tipo 1/fisiologia , Transdução de Sinais/fisiologia , Fatores de Transcrição/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Idoso , Animais , Apoptose/efeitos dos fármacos , Linfócitos B/metabolismo , Sistemas CRISPR-Cas , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Via de Sinalização Hippo , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Linfoma Difuso de Grandes Células B/etiologia , Linfoma Difuso de Grandes Células B/patologia , Masculino , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Proteínas de Neoplasias/antagonistas & inibidores , Podofilotoxina/análogos & derivados , Podofilotoxina/farmacologia , Pseudolinfoma/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Distribuição Aleatória , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor IGF Tipo 1/biossíntese , Receptor IGF Tipo 1/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/deficiência , Tirfostinas/farmacologia , Tirfostinas/uso terapêutico , Verteporfina/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas de Sinalização YAPRESUMO
BACKGROUND: Congenital obstructive nephropathy is the main cause of end-stage renal disease in infants and children. Renal insufficiency is due to impaired growth and maturation in the developing kidney with obstruction. Congenital obstructive nephropathy leads to cytokine mediated inflammation and the development of interstitial fibrosis. The Janus kinase-2 (JAK-2) and Signal Transducer and Activator of Transcription'-3 (STAT3) are involved in cytokine production, inflammation, and interstitial fibrosis. METHODS: We studied the role of JAK2/STAT3 in a model of congenital obstructive nephropathy using unilateral ureteral obstruction (UUO) in neonatal mice at the second day of life. Cytokine production, inflammation, and interstitial fibrosis were analyzed in obstructed and sham operated kidneys of neonatal mice treated with or without JAK2/STAT3 inhibitor Tyrphostin AG490. To mimic obstruction and distension, proximal tubular cells were stretched in vitro. RESULTS: We show that STAT3 is highly activated in the developing kidney with obstruction and in proximal tubular cells following stretch. JAK2/STAT3 activation mediates cytokine release and leukocyte recruitment into neonatal kidneys after UUO. Pharmacological blockade of JAK2/STAT3 by Tyrphostin AG490 reduced inflammation, tubular apoptosis, and interstitial fibrosis. JAK2/STAT3 blockade decreased pro-inflammatory and profibrotic mediators in tubular cells. CONCLUSION: Our findings provide evidence that JAK2/STAT3 mediates inflammation and fibrosis in the developing kidney with obstruction. Blocking JAK2/STAT3 may prove beneficial in congenital obstructive nephropathy in children.
Assuntos
Fibrose/prevenção & controle , Inflamação/prevenção & controle , Tirfostinas/uso terapêutico , Obstrução Ureteral/tratamento farmacológico , Animais , Animais Recém-Nascidos , Inibidores Enzimáticos/uso terapêutico , Janus Quinase 2/antagonistas & inibidores , Camundongos , Fator de Transcrição STAT3/antagonistas & inibidores , Obstrução Ureteral/patologiaRESUMO
In infants and children, neonatal hypoxic-ischemic (HI) brain injury represents a major cause of chronic neurological morbidity. The transient receptor potential melastatin 2 (TRPM2), a non-selective cation channel that conducts calcium, can mediate neuronal death following HI brain injury. An important endogenous activator of TRPM2 is H2O2, which has previously been reported to be upregulated in the neonatal brain after hypoxic ischemic injury. Here, incorporating both in vitro (H2O2-induced neuronal cell death model) and in vivo (mouse HI brain injury model) approaches, we examined the effects of AG490, which can inhibit the H2O2-induced TRPM2 channel. We found that AG490 elicited neuroprotective effects. We confirmed that AG490 reduced H2O2-induced TRPM2 currents. Specifically, application of AG490 to neurons ameliorated H2O2-induced cell injury in vitro. In addition, AG490 administration reduced brain damage and improved neurobehavioral performance following HI brain injury in vivo. The neuroprotective benefits of AG490 suggest that pharmacological inhibition of H2O2-activated TRPM2 currents can be exploited as a potential therapeutic strategy to treat HI-induced neurological complications.
Assuntos
Hipóxia-Isquemia Encefálica/metabolismo , Hipóxia-Isquemia Encefálica/prevenção & controle , Fármacos Neuroprotetores/uso terapêutico , Canais de Cátion TRPM/metabolismo , Tirfostinas/uso terapêutico , Animais , Animais Recém-Nascidos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Células HEK293 , Humanos , Hipóxia-Isquemia Encefálica/induzido quimicamente , Camundongos , Fármacos Neuroprotetores/farmacologia , Oxidantes/toxicidade , Distribuição Aleatória , Canais de Cátion TRPM/antagonistas & inibidores , Tirfostinas/farmacologiaRESUMO
A variety of osteolytic factors have been identified from breast cancer cells leading to osteolysis, but less is known about which factor plays an essential role in the initiation process prior to the overt vicious osteolytic cycle. Here, we present in vitro and in vivo evidences to clarify the role of interleukin-11 (IL-11) as an essential contributor to breast cancer bone metastasis mediated osteolysis. Animal studies showed that bone specific metastatic BoM-1833 cells induce earlier onset of osteolysis and faster tumor growth compared with MCF7 and parental MDA-MB-231 cells in BALB/c-nu/nu nude mice. IL-11 was further screened and identified as the indispensable factor secreted by BoM-1833 cells inducing osteoclastogenesis independently of receptor activator of nuclear factor κB ligand (RANKL). Mechanistic investigation revealed that the JAK1/STAT3 signaling pathway as a downstream effector of IL-11, STAT3 activation further induces the expression of c-Myc, a necessary factor required for osteoclastogenesis. By inhibiting STAT3 phosphorylation, AG-490 was shown effective in reducing osteolysis and tumor growth in the metastatic niche. Overall, our results revealed the essential role and the underlying molecular mechanism of IL-11 in breast cancer bone metastasis mediated osteolysis. STAT3 targeting through AG-490 is a potential therapeutic strategy for mitigating osteolysis and tumor growth of bone metastatic breast cancer.
Assuntos
Neoplasias Ósseas/patologia , Neoplasias da Mama/patologia , Interleucina-11/farmacologia , Osteogênese/efeitos dos fármacos , Osteólise/prevenção & controle , Ligante RANK/farmacologia , Animais , Neoplasias Ósseas/mortalidade , Neoplasias Ósseas/secundário , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Interleucina-11/metabolismo , Interleucina-11/uso terapêutico , Janus Quinase 1/metabolismo , Estimativa de Kaplan-Meier , Fator Estimulador de Colônias de Macrófagos/farmacologia , Fator Estimulador de Colônias de Macrófagos/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Osteólise/patologia , Ligante RANK/uso terapêutico , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tirfostinas/farmacologia , Tirfostinas/uso terapêuticoRESUMO
Interstitial cystitis (IC) is a heterogeneous syndrome with unknown etiology, and microRNAs (miRs) were found to be involved in IC. In our study, we aim to explore the role of miR-132 in the inflammatory response and detrusor fibrosis in IC through the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway in rat models. A rat model of IC was established and treated with the miR-132 mimic, miR-132 inhibitor, and/or JAK-STAT signaling pathway inhibitor AG490. Enzyme-linked immunosorbent assay was applied to measure the expression of interleukin (IL)-6, IL-10, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α), and intercellular adhesion molecule-1 (ICAM-1). The urodynamic test was performed to assess urodynamic parameters, and reverse transcription quantitative polymerase chain reaction and Western blot analysis for the expression of miR-132, STAT4, suppressors of cytokine signaling 3 (SOCS3), JAK2, vascular endothelial growth factor (VEGF), IFN-γ, and TNF-α. IC rats treated with miR-132 inhibitor and AG490 had decreased collagen fiber, inflammatory cell infiltration, and mast cells, lower expression of IL-6, IL-10, IFN-γ, TNF-α, ICAM-1, collagens I and III, and alleviated urodynamic parameters and decreased expression of STAT4, VEGF, JAK2, IFN-γ, TNF-α, and increased expression of SOCS3. Taken together, our data indicate that downregulation of miR-132 alleviates inflammatory response and detrusor fibrosis in IC via the inhibition of the JAK-STAT signaling pathway.
Assuntos
Cistite Intersticial/metabolismo , Inflamação/metabolismo , Janus Quinases/metabolismo , MicroRNAs/metabolismo , Animais , Cistite Intersticial/tratamento farmacológico , Feminino , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Inflamação/tratamento farmacológico , Janus Quinase 2/metabolismo , MicroRNAs/genética , Ratos , Ratos Sprague-Dawley , Fator de Transcrição STAT4/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fator de Necrose Tumoral alfa/metabolismo , Tirfostinas/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Subarachnoid hemorrhage (SAH) is a devastating disease. Cerebral vasospasm is still an important cause of post-SAH poor outcomes, but its mechanisms remain unveiled. Activation of epidermal growth factor receptor (EGFR) is suggested to cause vasoconstriction in vitro, but no report has demonstrated the involvement of EGFR in vasospasm development after SAH in vivo. Cross-talk of EGFR and vascular endothelial growth factor (VEGF) receptor, which may affect post-SAH vasospasm, was also reported in cancer cells, but has not been demonstrated in post-SAH vasospasm. The aim of this study was to investigate whether EGFR as well as EGFR-VEGF receptor cross-talk engage in the development of cerebral vasospasm in a mouse SAH model. C57BL6 mice underwent endovascular perforation SAH or sham modeling. At 30 min post-modeling, mice were randomly administrated vehicle or 2 doses of selective EGFR inhibitors intracerebroventricularly. A higher dose of the inhibitor significantly prevented post-SAH neurological impairments at 72 h and vasospasm at 24 h associated with suppression of post-SAH activation of EGFR and extracellular signal-regulated kinase (ERK) 1/2 in the cerebral artery wall, especially in the smooth muscle cell layers. Anti-EGFR neutralizing antibody also showed similar effects. However, neither expression levels of VEGF nor activation levels of a major receptor of VEGF, VEGF receptor-2, were affected by SAH and two kinds of EGFR inactivation. Thus, this study first showed that EGFR-ERK1/2 pathways may be involved in post-SAH vasospasm development, and that EGFR-VEGF receptor cross-talk may not play a significant role in the development of vasospasm in mice.
Assuntos
Receptores ErbB/antagonistas & inibidores , Inibidores de Proteínas Quinases/uso terapêutico , Hemorragia Subaracnóidea/complicações , Hemorragia Subaracnóidea/tratamento farmacológico , Vasoespasmo Intracraniano/complicações , Vasoespasmo Intracraniano/tratamento farmacológico , Animais , Cetuximab/farmacologia , Cetuximab/uso terapêutico , Receptores ErbB/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Quinazolinas/administração & dosagem , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , Tirfostinas/administração & dosagem , Tirfostinas/farmacologia , Tirfostinas/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Oxytocin (OT) has a postconditioning effect against the ischemia-reperfusion (I/R) injury. However, its precise cardioprotection mechanism at the early reperfusion phase remains under debate. Our previous study revealed that OT postconditioning (OTpost) is cardioprotective by activating the Reperfusion Injury Salvage Kinase (RISK) pathway. Therefore, the present study is aimed to determine the biological effects of OTpost via the OT receptor and the activation of the JAK/STAT3 signaling pathway, mitochondrial adenosine triphosphate-dependent potassium channel (mitoKATP), nitric oxide (NO) release, and its anti-apoptotic effects against I/R injury in an isolated rat heart model. Sixty-three rats were randomly allocated to one of nine groups. OT was perfused 40â¯min prior to the regional ischemia or 15â¯min at the early reperfusion phase. AG490 (a JAK/STAT3 inhibitor), 5HD (a mitoKATP blocker), atosiban (an OT receptor antagonist), L-NAME (a nonspecific nitric oxide synthase inhibitor) were applied either alone or in combination with OT during the pre-ischemia phase and/or in the early reperfusion phase. Myocardial infarct size, hemodynamic factor, ventricular arrhythmia, coronary flow, cardiac biochemical marker, and the apoptosis index were determined at the end of reperfusion. Oxytocin postconditioning reduced infarct size, lactate dehydrogenase activity, arrhythmia score, ventricular fibrillation, and apoptosis. Moreover, AG490, 5HD, atosiban, and L-NAME abrogated the cardioprotective effects of OT. Our results demonstrated that the cardioprotective effects of OT are mediated by NO release, and the activation of mitoKATP and the SAFE pathway through the JAK/STAT3 signaling cascade that finally lead to decrease in the apoptosis index during the early reperfusion phase.
Assuntos
Coração/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Ocitocina/farmacologia , Ocitocina/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Coração/fisiologia , Pós-Condicionamento Isquêmico/métodos , L-Lactato Desidrogenase/metabolismo , Masculino , NG-Nitroarginina Metil Éster/farmacologia , NG-Nitroarginina Metil Éster/uso terapêutico , Óxido Nítrico/metabolismo , Ratos , Tirfostinas/farmacologia , Tirfostinas/uso terapêutico , Vasotocina/análogos & derivados , Vasotocina/farmacologia , Vasotocina/uso terapêuticoRESUMO
Diabetic retinopathy (DR) is one of the most frequently occurring microvascular complications of diabetes. Recent evidence indicates that epidermal growth factor receptors (EGFRs) are critical pathogenic players in non-neoplastic diseases, including diabetic cardiomyopathy and DR. However, the precise pathogenic mechanism of EGFR in DR has yet to be fully understood. In this study, we developed a type 1 diabetic early-stage retinopathy mouse model using injections of streptozotocin and an oxygen-induced end-stage diabetic retinopathy (OIR) model characterized by hypoxia-induced revascularization. We tested the hypothesis that the pathogenesis of DR can be reduced by the classic EGFR inhibitor, AG1478, in the mouse models. Our data indicated that treatment of AG1478 prevented retinal dysfunction, and reduced impairment of retinal structures as well as mitochondrial structures in retinal blood vessels in diabetic mice. Furthermore, AG1478 reduced neovascular tufts formation but had no effects on revascularization at the avascular sites when compared to untreated littermates in the OIR model. Our findings provide strong evidence that EGFR critically promoted retinal dysfunction, retinal structural impairment, and retinal vascular abnormalities in models of DR. We conclude that EGFR can be a potential important therapeutic target for treatment of DR.
Assuntos
Retinopatia Diabética/patologia , Retinopatia Diabética/fisiopatologia , Receptores ErbB/antagonistas & inibidores , Neovascularização Patológica/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Tirfostinas/farmacologia , Animais , Progressão da Doença , Feminino , Inflamação/patologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Quinazolinas/uso terapêutico , Vasos Retinianos/efeitos dos fármacos , Vasos Retinianos/patologia , Tirfostinas/uso terapêuticoRESUMO
Although the molecular therapeutics targeting key biomarkers such as epithelial growth factor receptor (EGFR), PI3K/AKT/mTOR, and vascular endothelial growth factor (VEGF) shows some success in clinical trials, some internally existing challenges in endothelial cancer biology hinder the drug effects. One of the major challenges stems from cancer stem cell-derived drug resistance. CD133 positive cells are well believed as cancer stem cells (CSC) in endometrial cancers and NOTCH pathway plays a critical role in retaining CD133+ cells by promoting CSC self-renewal and chemoresistance. Here, we initiated a therapeutic strategy to improve effects of EGFR inhibition by targeting NOTCH pathway of CD133+ cells in endometrial cancers. We first detected and purified the CD133+ cell fraction in endometrial cancer cell line Ishikawa (IK), and validated activation of NOTCH pathway in the CD133+ cells that have higher proliferation rate and lower apoptosis rate, comparing to CD133- cells. Results of nude mouse xenograft experiments further demonstrated CD133+ cells retain higher tumorigenesis capacity than CD133- cells, indicating their tumor-initiating property. Last, we applied both NOTCH inhibitor DAPT and EGFR inhibitor AG1478 treatment on endometrial cancer lines IK and HEC-1A and the results suggested improvement effects of the combination therapy compared to the treatments of DAPT or AG1478 alone. These findings indicated targeting NOTCH pathway in CD133+ cells, combining with EGFR inhibition, which provides a novel therapeutic strategy for endometrial cancer diseases.
Assuntos
Antígeno AC133/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias do Endométrio/tratamento farmacológico , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinogênese/efeitos dos fármacos , Carcinogênese/patologia , Linhagem Celular Tumoral , Diaminas/farmacologia , Diaminas/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Neoplasias do Endométrio/patologia , Receptores ErbB/antagonistas & inibidores , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , Receptores Notch/antagonistas & inibidores , Tiazóis/farmacologia , Tiazóis/uso terapêutico , Tirfostinas/farmacologia , Tirfostinas/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Angiogenesis is an essential event in tumor growth and metastasis, and immune system also contributes to the tumor evasion. Emerging evidences have suggested the bidirectional link between angiogenesis and immunosuppression. Myeloid-derived suppressor cell (MDSC) is a kind of immunosuppressive cells and plays an important role in this process. However, the actual regulatory mechanisms of angiogenesis and MDSCs in head and neck squamous cell carcinoma (HNSCC) were unclear. In this study, through analyzing the immunohistochemistry staining of human HNSCC tissue microarray, we found that the microvascular density (MVD) was significantly increased in HNSCC patients. We also characterized angiogenic factors p-STAT3, VEGFA, CK2, and MDSCs marker CD11b in HNSCC tissue array, and found the close expression correlation among these markers. To determine the role of JAK2/STAT3 pathway in tumor microenvironment of HNSCC, we utilized AG490 (an inhibitor of JAK2/STAT3) for further research. Results showed that inhibition of JAK2/STAT3 suppressed angiogenesis by decreasing VEGFA and HIF1-α both in vitro and vivo. Moreover, in HNSCC transgenic mouse model, inhibiting JAK2/STAT3 not only suppressed angiogenesis but also reduced MDSCs in the tumor microenvironment through suppressing VEGFA and CK2. Our findings demonstrated the close relationship between angiogenesis and MDSCs in HNSCC, and inhibition of JAK2/STAT3 could reduce tumor-induced angiogenesis and decrease MDSCs.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Janus Quinase 2/antagonistas & inibidores , Células Supressoras Mieloides/efeitos dos fármacos , Neovascularização Patológica/tratamento farmacológico , Fator de Transcrição STAT3/antagonistas & inibidores , Tirfostinas/uso terapêutico , Inibidores da Angiogênese/farmacologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carcinoma de Células Escamosas/irrigação sanguínea , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Neoplasias de Cabeça e Pescoço/irrigação sanguínea , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Janus Quinase 2/metabolismo , Camundongos Transgênicos , Células Supressoras Mieloides/metabolismo , Células Supressoras Mieloides/patologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Tirfostinas/farmacologiaRESUMO
We previously reported that EGFR tyrosine kinase (EGFRtk) activity and endoplasmic reticulum (ER) stress are enhanced in type 2 diabetic (T2D) mice and cause vascular dysfunction. In the present study, we determined the in vivo contribution of EGFRtk and ER stress in acute myocardial infarction induced by acute ischemia (40 min)-reperfusion (24 h) (I/R) injury in T2D (db-/db-) mice. We treated db-/db- mice with EGFRtk inhibitor (AG1478, 10 mg/kg/day) for 2 weeks. Mice were then subjected to myocardial I/R injury. The db-/db- mice developed a significant infarct after I/R injury. The inhibition of EGFRtk significantly reduced the infarct size and ER stress induction. We also determined that the inhibition of ER stress (tauroursodeoxycholic acid, TUDCA, 150 mg/kg per day) in db-/db- significantly decrease the infarct size indicating that ER stress is a downstream mechanism to EGFRtk. Moreover, AG1478 and TUDCA reduced myocardium p38 and ERK1/2 MAP-kinases activity, and increased the activity of the pro-survival signaling cascade Akt. Additionally, the inhibition of EGFRtk and ER stress reduced cell apoptosis and the inflammation as indicated by the reduction in macrophages and neutrophil infiltration. We determined for the first time that the inhibition of EGFRtk protects T2D heart against I/R injury through ER stress-dependent mechanism. The cardioprotective effect of EGFRtk and ER stress inhibition involves the activation of survival pathway, and inhibition of apoptosis, and inflammation. Thus, targeting EGFRtk and ER stress has the potential for therapy to overcome myocardial infarction in T2D.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Cardiomiopatias Diabéticas/metabolismo , Estresse do Retículo Endoplasmático , Receptores ErbB/metabolismo , Infarto do Miocárdio/metabolismo , Animais , Apoptose , Cardiotônicos/farmacologia , Cardiotônicos/uso terapêutico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Cardiomiopatias Diabéticas/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , Ácido Tauroquenodesoxicólico/farmacologia , Ácido Tauroquenodesoxicólico/uso terapêutico , Tirfostinas/farmacologia , Tirfostinas/uso terapêuticoRESUMO
BACKGROUND AND OBJECTIVE: Tyrphostin "AG1024" is an insulin growth factor-1 receptor (IGF-1R) inhibitor that displayed an effect on the viability of larval and mature schistosomes in vitro. We sought to investigate the possible in vivo role of AG1024 as a potential new anti-Schistosoma drug against immature and adult stages of Schistosoma mansoni and its effect on the degree of hepatic fibrosis and insulin pathway. METHODS: The study included a control non-infected group and 5 groups of S. manosoni-infected CD-1 albino mice (20 mice each) assigned to treatment as follows: vehicle-treated, early AG1024, 30µg/100µl DMSO, IP for 10days started 30days post-infection (dpi), early praziquantel (PZQ), 500mg/kg orally for 2days (30dpi), late AG1024 (60dpi), and late PZQ (60dpi). All mice were sacrificed 12 weeks post-infection. Parasitological, chemical and histopathological parameters were studied. Immunohistochemistry of TGF-ß and GLUT4 in liver sections was done to further evaluate the effect of AG1024 on the degree of hepatic fibrosis and insulin signaling pathway, respectively. RESULTS: Early administration of AG1024 (30dpi) resulted in significant reduction of hepatic and intestinal tissue egg count with a reduction of 79.99% and 89.1% respectively. Late administration of AG1024 (60dpi) led to 77.78% reduction of intestinal eggs count; however, hepatic egg count wasn't reduced significantly. No reduction in worm burden was recorded for both administration regimens. Both regimens lead to significant decrease of both ALT and AST, mean hepatic granuloma diameter but an increase in fibrosis percentage (65.2% and 55% respectively). Both early and late treatment with AG1024 showed a significant increment of TGF-ß expression by 71.4% and 39.3%, respectively (p<0.0001) compared to PZQ-treated and infected non-treated groups. Hepatic GLUT4 expression was significantly decreased compared to infected non-treated group (p<0.001) and the corresponding PZQ-treated group. CONCLUSION: Early AG1024 administration induced more significant results compared to early PZQ with a promising activity against egg production and subsequent reduction of tissue egg load rather than direct schistosomicidal effect; however, it induced granuloma fibrosis, TGF-ß expression, and disrupted the insulin signaling pathway.
Assuntos
Receptor IGF Tipo 1/antagonistas & inibidores , Esquistossomicidas/uso terapêutico , Tirfostinas/farmacologia , Animais , Antígenos de Helmintos/urina , Peso Corporal , Feminino , Transportador de Glucose Tipo 4/metabolismo , Homeostase/efeitos dos fármacos , Resistência à Insulina , Fígado/efeitos dos fármacos , Fígado/parasitologia , Fígado/patologia , Camundongos , Tamanho do Órgão , Praziquantel/farmacologia , Receptor IGF Tipo 1/metabolismo , Schistosoma mansoni/efeitos dos fármacos , Esquistossomose mansoni/tratamento farmacológico , Esquistossomose mansoni/parasitologia , Esquistossomose mansoni/urina , Esquistossomicidas/administração & dosagem , Esquistossomicidas/farmacologia , Fator de Crescimento Transformador beta/metabolismo , Tirfostinas/administração & dosagem , Tirfostinas/uso terapêuticoRESUMO
Sepsis-associated encephalopathy (SAE) is a common complication associated with poor prognosis in septic patients, but the underlying mechanism remains unclear. We hypothesized that disturbed neuregulin 1 (NRG1)-ErbB4 signaling in the parvalbumin interneurons was involved in sepsis-induced cognitive impairment in a mouse model of SAE. The SAE model was induced by cecal ligation/perforation (CLP). Animals were randomly divided into the following six groups: sham + vehicle group, sham + NRG1 group, CLP + vehicle group, CLP + NRG1 group, CLP + NRG1 + AG1478 (ErbB4 inhibitor) group, and CLP + minocycline group. Behavioral tests and in vivo electrophysiology were performed at the indicated time points. The brain tissues were harvested to determine the levels of hippocampcal cytokines, IBA1-positive cells, NRG1, ErbB4, and parvalbumin. In the present study, sepsis induced the anxiety-like behavior and hippocampal-dependent cognitive impairment, as reflected by significantly increased distance spent in the open field test and decreased freezing time to context in the fear conditioning test. The abnormal behavioral changes co-occurred with significant increases in hippocampal IBA1-positive cells, IL-1ß and IL-6 levels, and decreased NRG1, ErbB4, parvalbumin expressions, and evoked gamma activity. NRG1 treatment attenuated the sepsis-induced cognitive impairment and the associated biochemical markers, which were abolished by AG1478 administration. Notably, minocycline treatment attenuated neuroinflammation and mimicked the beneficial effects of NRG1 treatment. In summary, we provided additional evidence that the disruption of NRG1-ErbB4 signaling in the parvalbumin interneurons mediated by neuroinflammation might lead to abnormal gamma oscillations and thus contribute to cognitive impairment in a mouse model of SAE.
Assuntos
Encefalite/patologia , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Neuregulina-1/metabolismo , Receptor ErbB-4/metabolismo , Encefalopatia Associada a Sepse/fisiopatologia , Animais , Disfunção Cognitiva/tratamento farmacológico , Disfunção Cognitiva/etiologia , Modelos Animais de Doenças , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Ritmo Gama , Hipocampo/química , Interneurônios/metabolismo , Camundongos , Parvalbuminas , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , Receptor ErbB-4/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Tirfostinas/farmacologia , Tirfostinas/uso terapêuticoRESUMO
Prostaglandin E2 (PGE2) is one of the major signaling molecules involved in hyperalgesia, acting directly on nociceptors and resulting in the activation of PKA and PKC. Once active, these kinases phosphorylate many cellular proteins, resulting in changes on nociceptors sensorial transduction properties. The Janus Kinases (JAKs) are a family of intracellular signaling molecules generally associated with cytokine signaling, and their activity can be increased in nociceptors after peripheral inflammation. However, there are no evidences of JAKs direct involvement in PGE2 mediated sensitization of nociceptors. Therefore, the aim of the present study was to explore a possible role for JAKs in PGE2 mediated sensitization. In cultured dorsal root ganglion (DRG) neurons, we observed that the administration of PGE2 increases capsaicin induced calcium transients, and a pre-incubation of DRG cells with the JAK inhibitor AG490 blocks this PGE2 in vitro effect. Intrathecal administration of AG490 to ten-weeks-old male Wistar rats reduces the hyperalgesia induced by the intraplantar administration of PGE2 or carrageenan in the right hind paw. We also observed that carrageenan administration in the right hind paw induced an increase in membrane associated PKCepsilon in the ipsilateral L5 DRG, and this increase was blocked by intrathecal AG490 administration. In conclusion the present study indicates that the JAKs expressed in the DRG and spinal cord may have a role in the sensitization of nociceptors by a peripheral inflammatory event. Moreover, the inhibition of JAKs may be a possible novel pharmacological target for the control of the inflammatory hyperalgesia.
Assuntos
Dinoprostona/imunologia , Hiperalgesia/imunologia , Janus Quinase 2/imunologia , Nociceptores/imunologia , Animais , Carragenina/imunologia , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/imunologia , Gânglios Espinais/patologia , Hiperalgesia/tratamento farmacológico , Hiperalgesia/patologia , Janus Quinase 2/antagonistas & inibidores , Masculino , Nociceptores/efeitos dos fármacos , Nociceptores/patologia , Ratos Wistar , Tirfostinas/farmacocinética , Tirfostinas/uso terapêuticoRESUMO
Obesity is often associated with increased risk of cardiovascular diseases. Previous studies suggest that epidermal growth factor receptor (EGFR) antagonism may be effective for the treatment of angiotensin II-induced cardiac hypertrophy and diabetic cardiomyopathy. This study was performed to demonstrate if EGFR plays a role in the pathogenesis of hyperlipidemia/obesity-related cardiac injuries. The in vivo studies using both wild type (WT) and apolipoprotein E (ApoE) knockout mice fed with high fat diet (HFD) showed the beneficial effects of small-molecule EGFR inhibitors, AG1478 and 542, against obesity-induced myocardial injury. Administration of AG1478 and 542 significantly reduced myocardial inflammation, fibrosis, apoptosis, and dysfunction in both two obese mouse models. In vitro, EGFR signaling was blocked by either siRNA silencing or small-molecule EGFR inhibitors in palmitic acid (PA)-stimulated cardiomyocytes. EGFR inhibition attenuated PA-induced inflammatory response and apoptosis in H9C2 cells. Furthermore, we found that PA-induced EGFR activation was mediated by the upstream TLR4 and c-Src. This study has confirmed the detrimental effect of EGFR activation in the pathogenesis of obesity-induced cardiac inflammatory injuries in experimental mice, and has demonstrated the TLR4/c-Src-mediated mechanisms for PA-induced EGFR activation. Our data suggest that EGFR may be a therapeutic target for obesity-related cardiovascular diseases.
Assuntos
Doenças Cardiovasculares/metabolismo , Receptores ErbB/antagonistas & inibidores , Hiperlipidemias/complicações , Miócitos Cardíacos/metabolismo , Animais , Apolipoproteínas E/genética , Apoptose , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/etiologia , Linhagem Celular , Dieta Hiperlipídica/efeitos adversos , Receptores ErbB/genética , Receptores ErbB/metabolismo , Hiperlipidemias/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Ácido Palmítico/toxicidade , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , Ratos , Receptor 4 Toll-Like/metabolismo , Tirfostinas/farmacologia , Tirfostinas/uso terapêutico , Quinases da Família src/metabolismoRESUMO
BACKGROUND: Mucus hypersecretion and neutrophil infiltration are important characteristics of airway inflammation. Epidermal growth factor receptor (EGFR) transactivation induces mucus and inflammatory cytokine secretion from airway epithelial cells. To elucidate the roles of EGFR in airway inflammation, the in vitro effects on mucin production and interleukin (IL) 8 secretion from cultured airway epithelial cells and the in vivo effects on mucus hypersecretion and neutrophil infiltration in rat nasal mucosa of the EGFR tyrosine kinase inhibitor AG1478 were examined. METHODS: The in vitro effects of AG1478 treatment of cultured NCI-H292 cells on lipopolysaccharide (LPS) induced or tumor necrosis factor (TNF) α induced MUC5AC mucin and IL-8 secretion were evaluated. Hypertrophic and metaplastic changes of goblet cells, mucus production and neutrophil infiltration in rat nasal epithelium were induced by intranasal instillation of LPS in vivo, and the inhibitory effects of AG1478 by intraperitoneal injection or intranasal instillation were examined. RESULTS: AG1478 (1-1000 nM) significantly inhibited both LPS-induced and TNF-α-induced secretion of MUC5AC and IL-8 from cultured NCI-H292 cells in a dose-dependent manner. The expression of MUC5AC and IL-8 messenger RNAs was also significantly inhibited. Intranasal instillation of AG1478 one hour after intranasal LPS instillation significantly inhibited LPS-induced goblet cell metaplasia, mucus production, and neutrophil infiltration in rat nasal epithelium, as did intraperitoneal injection of AG1478 one hour before LPS instillation. CONCLUSIONS: These results indicated that EGFR transactivation plays an important role in mucin and IL-8 secretion from airway epithelial cells. Intranasal instillation of an EGFR tyrosine kinase inhibitor may be a new therapeutic approach for the treatment of upper airway inflammation.