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1.
Chem Biol Interact ; 311: 108772, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31351049

RESUMO

Atherosclerosis is a common type of cardiovascular disease (CVD), remaining one of the leading causes of global death. Tripartite motif-containing 28 (TRIM28) is a member of TRIM family that has been found to be involved in atherosclerosis. However, the role of TRIM28 in atherosclerosis remains unknown. This study aimed to investigate the effects of TRIM28 on the phenotypic switching of human aortic smooth muscle cells (HASMCs), which is considered as a fundamental event during the development of atherosclerosis. The results showed that TRIM28 was highly expressed in human atherosclerotic tissues, as well in cultured HASMCs stimulated by platelet-derived growth factor subunit B homodimer (PDGF-BB). Knockdown of TRIM28 by transfection with siRNA targeting TRIM28 (si-TRIM28) significantly suppressed the PDGF-BB-induced cell proliferation and migration of HASMCs. Besides, knockdown of TRIM28 inhibited the expressions of matrix metalloproteinase (MMP)-2 and MMP-9. The VSMC markers including α-smooth muscle actin (α-SMA), calponin and SM22α were upregulated in TRIM28 knocked down HASMCs. Furthermore, knockdown of TRIM28 blocked PDGF-BB-induced NF-κB activation in HASMCs. Collectively, knockdown of TRIM28 prevented PDGF-BB-induced phenotypic switching of HASMCs, which might be mediated by the regulation of NF-κB signaling pathway.


Assuntos
Becaplermina/farmacologia , Proliferação de Células/efeitos dos fármacos , Proteína 28 com Motivo Tripartido/metabolismo , Aterosclerose/metabolismo , Aterosclerose/patologia , Proteínas de Ligação ao Cálcio/metabolismo , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Proteínas dos Microfilamentos/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fator de Transcrição RelA/metabolismo , Proteína 28 com Motivo Tripartido/antagonistas & inibidores , Proteína 28 com Motivo Tripartido/genética , Regulação para Cima/efeitos dos fármacos
2.
Life Sci ; 231: 116554, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31194992

RESUMO

AIMS: Several adipokines have been proven to improve the therapeutic efficacy of mesenchymal stromal cells (MSCs) when used to treat ischemic heart disease. Asprosin (ASP) is a newly-discovered adipokine. ASP might also predict the severity of coronary pathology. We investigated the role of ASP on MSCs and the effects of ASP-pretreated MSCs on myocardial infarction (MI). MAIN METHODS: MSCs were labelled with a lentivirus carrying green fluorescent protein (GFP). For in vivo study, after pretreatment with vehicle or ASP, MSCs were injected into infarcted hearts. Cardiac function and fibrosis were then evaluated 4 weeks after the induction of MI and survival of MSCs evaluated after 1 week. MSCs proliferation and migration were investigated after ASP treatment in vitro. MSCs apoptosis induced by hydrogen peroxide (H2O2) was assessed using flow cytometry. KEY FINDINGS: Compared to vehicle-pretreated MSCs, ASP-pretreated MSCs significantly improved the left ventricular ejection fraction (LVEF), and inhibited myocardial fibrosis 4 weeks after MI. ASP pretreatment may have promoted homing of transplanted MSCs. In vitro results showed that ASP had no significant effect on MSC proliferation and migration, but protected these cells from H2O2-induced apoptosis. Among 21 molecules associated with antioxidation and cell death, the antioxidant enzyme SOD2 was significantly upregulated by ASP. Furthermore, ASP treatment inhibited H2O2-induced ROS generation and apoptosis via the activated ERK1/2-SOD2 pathway. SIGNIFICANCE: This is the first evidence that ASP can regulate MSCs function and enhance MSCs therapy for ischemic heart disease. Furthermore, we demonstrate that ASP protects MSCs from oxidative stress-induced apoptosis via the ERK1/2-SOD2 pathway.


Assuntos
Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Proteínas dos Microfilamentos/metabolismo , Infarto do Miocárdio/terapia , Fragmentos de Peptídeos/metabolismo , Hormônios Peptídicos/metabolismo , Superóxido Dismutase/metabolismo , Animais , Apoptose/fisiologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Coração/fisiopatologia , Peróxido de Hidrogênio/farmacologia , Masculino , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patologia , Espécies Reativas de Oxigênio/metabolismo , Função Ventricular Esquerda
3.
Life Sci ; 232: 116600, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31251998

RESUMO

Neuroinflammation is one of the significant neuropathological conditions in Parkinson's disease (PD) which is due to microglial and astrocytes activation leads to progressive dopaminergic neuronal loss. To date, Current PD drugs offers only symptomatic relief with adverse effects and lack of ability to prevent the progression of neurodegeneration. Therefore, a better approach to develop a multi potent drug of natural origin would be beneficial in managing the disease. Therefore, the present study aimed to investigate the neuroprotective and anti-inflammatory effects of PHL by exploring its neuroprotective mechanism in 1-methyl-4-phenyl-1,2,3,6-tetrahydro pyridine (MPTP) induced PD in mice. MPTP intoxication in mice cause motor abnormalities, decreased dopamine (DA) levels, reduced tyrosine hydroxylase (TH) enzyme protein expression and inflammation which were effectively restored by PHL. Moreover gliotic specific inflammatory markers like glial fibrillary acidic protein (GFAP), ionized calcium-binding adaptor protein-1 (Iba-1), iNOS and COX-2 were found to be expressed more in MPTP intoxicated mice, Further the levels of proinflammatory cytokines like IL-ß, IL-6, and TNF-α were significantly upregulated in MPTP intoxicated mice, these deleterious responses were diminished to extend neuroprotection by PHL treatment. Our findings strongly suggest PHL as a potent therapeutic agent in treating PD.


Assuntos
Transtornos Parkinsonianos/tratamento farmacológico , Floretina/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/metabolismo , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Dopamina/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Microglia/efeitos dos fármacos , Neuroimunomodulação/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Óxido Nítrico Sintase Tipo II/metabolismo , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/patologia , Tirosina 3-Mono-Oxigenase/metabolismo
5.
Microb Pathog ; 133: 103545, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31112772

RESUMO

Commensal enteric microbes under specific conditions viz. immunocompromised system, altered microbiota or uncompetitive niche induce their otherwise dormant pathogenic phenotype to distort host cellular functioning. Here we investigate how under in vitro environment established by using Caco-2 cells, commensal gut microbe E. coli K12 (ATCC 14849) disrupt intestinal epithelial barrier function. Caco-2 cells exposed to E. coli showed the time dependent significant (P < 0.01) decrease in transepithelial electrical resistance (TEER) and concomitantly increased phenol red flux across cell monolayer in contrast to non infected control cells. E. coli infected intestinal cells were observed with suppressed (p < 0.05) mRNA levels of ZO-1, Claudin-1, Occludin and Cingulin-1 in contrast to significantly (p < 0.05) higher PIgR and hbd-2 mRNA fold changes. Immunofluorescent and electron micrographs revealed the disrupted distribution and localisation of specific tight junction proteins (Zo-1 and Claudin-1) and actin filament in E. coli infected Caco-2 cells that ultimately resulted in deformed cellular morphology. Taken together, E. coli K12 under compromised in vitro milieu disrupted the intestinal barrier functions by decreasing the expression of important tight junction genes along with the altered distribution of associated proteins that increased the intestinal permeability as reflected by phenol red flux and TEER values.


Assuntos
Escherichia coli K12/fisiologia , Escherichia coli K12/patogenicidade , Microbioma Gastrointestinal , Infecções Oportunistas/microbiologia , Simbiose , Células CACO-2/citologia , Células CACO-2/microbiologia , Claudina-1/metabolismo , Proteínas do Citoesqueleto , Impedância Elétrica , Células Epiteliais/metabolismo , Expressão Gênica , Interações entre Hospedeiro e Microrganismos , Humanos , Mucosa Intestinal/metabolismo , Intestinos/microbiologia , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Ocludina/genética , Ocludina/metabolismo , Permeabilidade , RNA Mensageiro , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo , beta-Defensinas/metabolismo
6.
PLoS Pathog ; 15(5): e1007737, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31071198

RESUMO

Streptococcus equi subsp. zooepidemicus (SEZ) is a zoonotic pathogen capable of causing meningitis in humans. The mechanisms that enable pathogens to traverse the blood-brain barrier (BBB) are incompletely understood. Here, we investigated the role of a newly identified Fic domain-containing protein, BifA, in SEZ virulence. BifA was required for SEZ to cross the BBB and to cause meningitis in mice. BifA also enhanced SEZ translocation across human Brain Microvascular Endothelial Cell (hBMEC) monolayers. Purified BifA or its Fic domain-containing C-terminus alone were able to enter into hBMECs, leading to disruption of monolayer barrier integrity. A SILAC-based proteomic screen revealed that BifA binds moesin. BifA's Fic domain was required for its binding to this regulator of host cell cytoskeletal processes. BifA treatment of hBMECs led to moesin phosphorylation and downstream RhoA activation. Inhibition of moesin activation or moesin depletion in hBMEC monolayers abrogated BifA-mediated increases in barrier permeability and SEZ's capacity to translocate across monolayers. Thus, BifA activation of moesin appears to constitute a key mechanism by which SEZ disrupts endothelial monolayer integrity to penetrate the BBB.


Assuntos
Proteínas de Bactérias/metabolismo , Barreira Hematoencefálica/patologia , Encéfalo/patologia , Endotélio Vascular/patologia , Proteínas dos Microfilamentos/metabolismo , Streptococcus/fisiologia , Virulência , Animais , Proteínas de Bactérias/genética , Transporte Biológico , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/microbiologia , Encéfalo/metabolismo , Encéfalo/microbiologia , Permeabilidade da Membrana Celular , Células Cultivadas , Endotélio Vascular/metabolismo , Endotélio Vascular/microbiologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C
7.
Nat Commun ; 10(1): 2356, 2019 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-31142743

RESUMO

Centrosomes control cell motility, polarity and migration that is thought to be mediated by their microtubule-organizing capacity. Here we demonstrate that WNT signalling drives a distinct form of non-directional cell motility that requires a key centrosome module, but not microtubules or centrosomes. Upon exosome mobilization of PCP-proteins, we show that DVL2 orchestrates recruitment of a CEP192-PLK4/AURKB complex to the cell cortex where PLK4/AURKB act redundantly to drive protrusive activity and cell motility. This is mediated by coordination of formin-dependent actin remodelling through displacement of cortically localized DAAM1 for DAAM2. Furthermore, abnormal expression of PLK4, AURKB and DAAM1 is associated with poor outcomes in breast and bladder cancers. Thus, a centrosomal module plays an atypical function in WNT signalling and actin nucleation that is critical for cancer cell motility and is associated with more aggressive cancers. These studies have broad implications in how contextual signalling controls distinct modes of cell migration.


Assuntos
Aurora Quinase B/metabolismo , Movimento Celular , Centrossomo/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Desgrenhadas/metabolismo , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Via de Sinalização Wnt , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias da Mama/metabolismo , Carcinoma de Células de Transição/metabolismo , Linhagem Celular Tumoral , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Prognóstico , Mapas de Interação de Proteínas , Reação em Cadeia da Polimerase em Tempo Real , Neoplasias da Bexiga Urinária/metabolismo
8.
Nat Commun ; 10(1): 2193, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31097705

RESUMO

Filamentous actin (F-actin) networks facilitate key processes like cell shape control, division, polarization and motility. The dynamic coordination of F-actin networks and its impact on cellular activities are poorly understood. We report an antagonistic relationship between endosomal F-actin assembly and cortical actin bundle integrity during Drosophila airway maturation. Double mutants lacking receptor tyrosine phosphatases (PTP) Ptp10D and Ptp4E, clear luminal proteins and disassemble apical actin bundles prematurely. These defects are counterbalanced by reduction of endosomal trafficking and by mutations affecting the tyrosine kinase Btk29A, and the actin nucleation factor WASH. Btk29A forms protein complexes with Ptp10D and WASH, and Btk29A phosphorylates WASH. This phosphorylation activates endosomal WASH function in flies and mice. In contrast, a phospho-mimetic WASH variant induces endosomal actin accumulation, premature luminal endocytosis and cortical F-actin disassembly. We conclude that PTPs and Btk29A regulate WASH activity to balance the endosomal and cortical F-actin networks during epithelial tube maturation.


Assuntos
Proteínas de Drosophila/metabolismo , Endossomos/metabolismo , Morfogênese/fisiologia , Proteínas Tirosina Quinases/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Actinas/metabolismo , Animais , Animais Geneticamente Modificados , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster , Embrião não Mamífero/diagnóstico por imagem , Epitélio/diagnóstico por imagem , Epitélio/crescimento & desenvolvimento , Fibroblastos , Microscopia Intravital , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microscopia Confocal , Fosforilação/fisiologia , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/metabolismo , Sistema Respiratório/diagnóstico por imagem , Sistema Respiratório/crescimento & desenvolvimento , Proteínas de Transporte Vesicular/genética
9.
Reprod Domest Anim ; 54(6): 882-891, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30974481

RESUMO

Dairy cow mastitis is a detrimental factor in milk quality and food safety. Mastitis generally refers to inflammation caused by infection by pathogenic microorganisms. Our studies in recent years have revealed the role of miRNA regulation in Staphylococcus aureus-induced mastitis. In the present study, we overexpressed and suppressed miR-145 to investigate the function of miR-145 in Mac-T cells. Flow cytometry, ELISA and EdU staining were used to detect changes in the secretion of several Mac-T cytokines and in cell proliferation. We found that overexpression of miR-145 in Mac-T cells significantly reduced the secretion of IL-12 and TNF-α, but increased the secretion of IFN-γ; the proliferation of bovine mammary epithelial cells was also inhibited. Using quantitative real-time PCR (qRT-PCR), Western blotting and luciferase multiplex verification techniques, we found that miR-145 targeted and regulated FSCN1. Knock-down of FSCN1 significantly increased the secretion of IL-12, while the secretion of TNF-α was significantly downregulated in Mac-T cells. Upon S. aureus infection of mammary gland tissue, the body initiated inflammatory responses; Bta-miR-145 expression was downregulated, which reduced the inhibitory effect on the FSCN1 gene; and upregulation of FSCN1 expression promoted mammary epithelial cell proliferation to allow the recovery of damaged tissue. The results of the present study will aid in understanding the immune mechanism opposing S. aureus infection in dairy cows and will provide a laboratory research basis for the prevention and treatment of mastitis.


Assuntos
Proteínas de Transporte/metabolismo , Mastite Bovina/imunologia , MicroRNAs/metabolismo , Proteínas dos Microfilamentos/metabolismo , Infecções Estafilocócicas/veterinária , Animais , Proteínas de Transporte/genética , Bovinos , Linhagem Celular , Proliferação de Células , Citocinas/metabolismo , Células Epiteliais/fisiologia , Feminino , Proteínas dos Microfilamentos/genética , Infecções Estafilocócicas/imunologia , Staphylococcus aureus
10.
J Neuroinflammation ; 16(1): 67, 2019 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-30927920

RESUMO

BACKGROUND: Patients diagnosed with chronic fatigue syndrome (CFS) or fibromyalgia experience chronic pain. Concomitantly, the rat model of CFS exhibits microglial activation in the lumbar spinal cord and pain behavior without peripheral tissue damage and/or inflammation. The present study addressed the mechanism underlying the association between pain and chronic stress using this rat model. METHODS: Chronic or continuous stress-loading (CS) model rats, housed in a cage with a thin level of water (1.5 cm in depth), were used. The von Frey test and pressure pain test were employed to measure pain behavior. The neuronal and microglial activations were immunohistochemically demonstrated with antibodies against ATF3 and Iba1. Electromyography was used to evaluate muscle activity. RESULTS: The expression of ATF3, a marker of neuronal hyperactivity or injury, was first observed in the lumbar dorsal root ganglion (DRG) neurons 2 days after CS initiation. More than 50% of ATF3-positive neurons simultaneously expressed the proprioceptor markers TrkC or VGluT1, whereas the co-expression rates for TrkA, TrkB, IB4, and CGRP were lower than 20%. Retrograde labeling using fluorogold showed that ATF3-positive proprioceptive DRG neurons mainly projected to the soleus. Substantial microglial accumulation was observed in the medial part of the dorsal horn on the fifth CS day. Microglial accumulation was observed around a subset of motor neurons in the dorsal part of the ventral horn on the sixth CS day. The motor neurons surrounded by microglia were ATF3-positive and mainly projected to the soleus. Electromyographic activity in the soleus was two to three times higher in the CS group than in the control group. These results suggest that chronic proprioceptor activation induces the sequential activation of neurons along the spinal reflex arc, and the neuronal activation further activates microglia along the arc. Proprioceptor suppression by ankle joint immobilization significantly suppressed the accumulation of microglia in the spinal cord, as well as the pain behavior. CONCLUSION: Our results indicate that proprioceptor-induced microglial activation may be a key player in the initiation and maintenance of abnormal pain in patients with CFS.


Assuntos
Citocinas/metabolismo , Síndrome de Fadiga Crônica/complicações , Microglia/patologia , Dor/etiologia , Dor/patologia , Distúrbios Somatossensoriais/etiologia , Fator 3 Ativador da Transcrição/genética , Fator 3 Ativador da Transcrição/metabolismo , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Gânglios Espinais/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Proteínas dos Microfilamentos/metabolismo , Microglia/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Medição da Dor , Ratos , Ratos Sprague-Dawley , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/metabolismo , Distúrbios Somatossensoriais/patologia , Estilbamidinas/metabolismo , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo
11.
Int J Mol Sci ; 20(8)2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-31018575

RESUMO

The merlin-ERM (ezrin, radixin, moesin) family of proteins plays a central role in linking the cellular membranes to the cortical actin cytoskeleton. Merlin regulates contact inhibition and is an integral part of cell-cell junctions, while ERM proteins, ezrin, radixin and moesin, assist in the formation and maintenance of specialized plasma membrane structures and membrane vesicle structures. These two protein families share a common evolutionary history, having arisen and separated via gene duplication near the origin of metazoa. During approximately 0.5 billion years of evolution, the merlin and ERM family proteins have maintained both sequence and structural conservation to an extraordinary level. Comparing crystal structures of merlin-ERM proteins and their complexes, a picture emerges of the merlin-ERM proteins acting as switchable interaction hubs, assembling protein complexes on cellular membranes and linking them to the actin cytoskeleton. Given the high level of structural conservation between the merlin and ERM family proteins we speculate that they may function together.


Assuntos
Membrana Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Neurofibromina 2/metabolismo , Citoesqueleto de Actina/metabolismo , Sequência de Aminoácidos , Animais , Membrana Celular/química , Inibição de Contato , Proteínas do Citoesqueleto/química , Humanos , Proteínas de Membrana/química , Proteínas dos Microfilamentos/química , Modelos Moleculares , Neurofibromina 2/química , Conformação Proteica , Domínios Proteicos , Mapas de Interação de Proteínas , Alinhamento de Sequência
12.
J Neuroinflammation ; 16(1): 79, 2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-30971251

RESUMO

BACKGROUND: Microglia play crucial roles in the maintenance of brain homeostasis. Activated microglia show a biphasic influence, promoting beneficial repair and causing harmful damage via M2 and M1 microglia, respectively. It is well-known that microglia are initially activated to the M2 state and subsequently switch to the M1 state, called M2-to-M1 class switching in acute ischemic models. However, the activation process of microglia in chronic and sporadic hypertension remains poorly understood. We aimed to clarify the process using a chronic hypertension model, the deoxycorticosterone acetate (DOCA)-salt-treated Wistar rats. METHODS: After unilateral nephrectomy, the rats were randomly divided into DOCA-salt, placebo, and control groups. DOCA-salt rats received a weekly subcutaneous injection of DOCA (40 mg/kg) and were continuously provided with 1% NaCl in drinking water. Placebo rats received a weekly subcutaneous injection of vehicle and were provided with tap water. Control rats received no administration of DOCA or NaCl. To investigate the temporal expression profiles of M1- and M2-specific markers for microglia, the animals were subjected to the immunohistochemical and biochemical studies after 2, 3, or 4 weeks DOCA-salt treatment. RESULTS: Hypertension occurred after 2 weeks of DOCA and salt administration, when round-shaped microglia with slightly shortened processes were observed juxtaposed to the vessels, although the histopathological findings were normal. After 3 weeks of DOCA and salt administration, M1-state perivascular and parenchyma microglia significantly increased, when local histopathological findings began to be observed but cerebrovascular destruction did not occur. On the other hand, M2-state microglia were never observed around the vessels at this period. Interestingly, prior to M1 activation, about 55% of perivascular microglia transiently expressed Ki-67, one of the cell proliferation markers. CONCLUSIONS: We concluded that the resting perivascular microglia directly switched to the pro-inflammatory M1 state via a transient proliferative state in DOCA-salt rats. Our results suggest that the activation machinery of microglia in chronic hypertension differs from acute ischemic models. Proliferative microglia are possible initial key players in the development of hypertension-induced cerebral vessel damage. Fine-tuning of microglia proliferation and activation could constitute an innovative therapeutic strategy to prevent its development.


Assuntos
Encéfalo/patologia , Proliferação de Células/fisiologia , Hipertensão/complicações , Hipertensão/patologia , Microglia/classificação , Microglia/patologia , Animais , Antígenos CD/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/metabolismo , Carboximetilcelulose Sódica/farmacologia , Proliferação de Células/efeitos dos fármacos , Acetato de Desoxicorticosterona/toxicidade , Modelos Animais de Doenças , Lateralidade Funcional , Hipertensão/diagnóstico por imagem , Hipertensão/etiologia , Antígeno Ki-67/metabolismo , Imagem por Ressonância Magnética , Masculino , Proteínas dos Microfilamentos/metabolismo , Microglia/efeitos dos fármacos , Mineralocorticoides/toxicidade , Nefrectomia/efeitos adversos , Ratos , Ratos Wistar , Cloreto de Sódio/toxicidade , Fatores de Tempo
13.
J Neuroinflammation ; 16(1): 78, 2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-30971286

RESUMO

BACKGROUND: Central sensitization is an important mechanism of chronic migraine (CM) and is related to the inflammatory response of microglia. The NOD-like receptor protein 3 (NLRP3) inflammasome may regulate the inflammatory process of microglia in several neurological diseases, but its role in CM is largely unknown. Therefore, the aim of this study was to identify the precise role of microglial NLRP3 in CM. METHODS: An experimental CM mouse model was established by repeated intraperitoneal (i.p) injection with nitroglycerin (NTG). We evaluated the expression levels of NLRP3 and its downstream interleukin (IL)-1ß protein in the trigeminal nucleus caudalis (TNC; which is a central area relevant to migraine pain) at different time points. To further examine the effects of the NLRP3 inflammasome pathway on central sensitization of CM, we examined MCC950, an NLRP3 inflammasome-specific inhibitor, and IL-1ra, an IL-1ß antagonist, whether altered NTG-induced mechanical hyperalgesia of the periorbital area and hind paw. The effect of MCC950 and IL-1ra on c-Fos, phosphorylated extracellular signal-regulated kinase (p-ERK) and calcitonin gene-related peptide (CGRP) expression in the TNC were also analyzed. The cell localization of NLRP3 and IL-1ß in the TNC was evaluated by immunofluorescence staining. RESULTS: Repeated NTG administration induced acute and chronic mechanical hyperalgesia and increased expression of NLRP3 and IL-1ß. Blockade of NLRP3 or IL-1ß reduced NTG-induced hyperalgesia, and this effect was accompanied by a significant inhibition of the NTG-induced increase in p-ERK, c-Fos and CGRP levels in the TNC. Immunofluorescence staining revealed that NLRP3 and IL-1ß were mainly expressed in microglia in the TNC, and the IL-1ß receptor, IL-1R, was mainly expressed in neurons in the TNC. CONCLUSIONS: These results indicate that NLRP3 activation in the TNC participates in the microglial-neuronal signal by mediating the inflammatory response. This process contributes to the central sensitization observed in CM.


Assuntos
Sensibilização do Sistema Nervoso Central/fisiologia , Compostos Heterocíclicos de 4 ou mais Anéis/uso terapêutico , Interleucina-1beta/metabolismo , Transtornos de Enxaqueca/tratamento farmacológico , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Sulfonas/uso terapêutico , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Sensibilização do Sistema Nervoso Central/efeitos dos fármacos , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Hiperalgesia/induzido quimicamente , Injeções Intraventriculares , Proteína Antagonista do Receptor de Interleucina 1/uso terapêutico , Interleucina-1beta/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Transtornos de Enxaqueca/induzido quimicamente , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Nitroglicerina/toxicidade , Medição da Dor , Limiar da Dor/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Vasodilatadores/toxicidade
14.
J Neuroinflammation ; 16(1): 82, 2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30975169

RESUMO

BACKGROUND: Neuropathic pain is caused by sensory nerve injury, but effective treatments are currently lacking. Microglia are activated in the spinal dorsal horn after sensory nerve injury and contribute to neuropathic pain. Accordingly, molecules expressed by these cells are considered potential targets for therapeutic strategies. Our previous gene screening study using a mouse model of motor nerve injury showed that the G-protein-coupled receptor 34 gene (GPR34) is induced by nerve injury. Because GPR34 is now considered a microglia-enriched gene, we explored the possibility that it might be involved in microglial activation in the dorsal horn in a mouse model of neuropathic pain. METHODS: mRNA expression of GPR34 and pro-inflammatory molecules was determined by quantitative real-time PCR in wild-type and GPR34-deficient mice with L4 spinal nerve injury. In situ hybridization was used to identify GPR34 expression in microglia, and immunohistochemistry with the microglial marker Iba1 was performed to examine microglial numbers and morphology. Mechanical sensitivity was evaluated by the von Frey hair test. Liquid chromatography-tandem mass spectrometry quantified expression of the ligand for GPR34, lysophosphatidylserine (LysoPS), in the dorsal horn, and a GPR34 antagonist was intrathecally administrated to examine the effect of inhibiting LysoPS-GPR34 signaling on mechanical sensitivity. RESULTS: GPR34 was predominantly expressed by microglia in the dorsal horn after L4 nerve injury. There were no histological differences in microglial numbers or morphology between WT and GPR34-deficient mice. However, nerve injury-induced pro-inflammatory cytokine expression levels in microglia and pain behaviors were significantly attenuated in GPR34-deficient mice. Furthermore, the intrathecal administration of the GPR34 antagonist reduced neuropathic pain. CONCLUSIONS: Inhibition of GPR34-mediated signal by GPR34 gene deletion reduced nerve injury-induced neuropathic pain by suppressing pro-inflammatory responses of microglia without affecting their morphology. Therefore, the suppression of GPR34 activity may have therapeutic potential for alleviating neuropathic pain.


Assuntos
Microglia/metabolismo , Neuralgia/metabolismo , Neuralgia/patologia , Receptores de Lisofosfolipídeos/metabolismo , Medula Espinal/patologia , Análise de Variância , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Fatores Reguladores de Interferon/metabolismo , Lisofosfolipídeos/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Neuralgia/tratamento farmacológico , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Medição da Dor , Limiar da Dor/fisiologia , Proteína Quinase C/metabolismo , RNA Mensageiro/metabolismo , Receptores de Lisofosfolipídeos/antagonistas & inibidores , Receptores de Lisofosfolipídeos/genética , Fatores de Tempo
15.
Am J Vet Res ; 80(5): 505-512, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31034271

RESUMO

OBJECTIVE: To determine pharmacokinetics and pharmacodynamics after oral administration of a single dose of clopidogrel to horses. ANIMALS: 6 healthy adult horses. PROCEDURES: Blood samples were collected before and at various times up to 24 hours after oral administration of clopidogrel (2 mg/kg). Reactivity of platelets from each blood sample was determined by optical aggregometry and phosphorylation of vasodilator-stimulated phosphoprotein (VASP). Concentrations of clopidogrel and the clopidogrel active metabolite derivative (CAMD) were measured in each blood sample by use of liquid chromatography-tandem mass spectrometry, and pharmacokinetic parameters were determined with a noncompartmental model. RESULTS: Compared with results for preadministration samples, platelet aggregation in response to 12.5µM ADP decreased significantly within 4 hours after clopidogrel administration for 5 of 6 horses. After 24 hours, platelet aggregation was identical to that measured before administration. Platelet aggregation in response to 25µM ADP was identical between samples obtained before and after administration. Phosphorylation of VASP in response to ADP (20µM) and prostaglandin E1 (3.3µM) was also unchanged by administration of clopidogrel. Time to maximum concentration of clopidogrel and CAMD was 0.54 and 0.71 hours, respectively, and calculated terminal-phase half-life of clopidogrel and CAMD was 1.81 and 0.97 hours, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Clopidogrel or CAMD caused competitive inhibition of ADP-induced platelet aggregation during the first 24 hours after clopidogrel administration. Because CAMD was rapidly eliminated from horses, clopidogrel administration may be needed more frequently than in other species in which clopidogrel causes irreversible platelet inhibition. (Am J Vet Res 2019;80:505-512).


Assuntos
Plaquetas/efeitos dos fármacos , Clopidogrel/farmacocinética , Cavalos/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação de Plaquetas/farmacocinética , Difosfato de Adenosina/farmacologia , Administração Oral , Animais , Área Sob a Curva , Plaquetas/metabolismo , Moléculas de Adesão Celular/metabolismo , Clopidogrel/administração & dosagem , Feminino , Masculino , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação de Plaquetas/administração & dosagem
16.
J Biol Regul Homeost Agents ; 33(2): 439-445, 2019 Mar-Apr,.
Artigo em Inglês | MEDLINE | ID: mdl-30971068

RESUMO

In this study, phosphorylation levels of vasodilator stimulated phosphoprotein (VASP) were detected by flow cytometry (FCM) to investigate the effects of ticagrelor and clopidogrel on platelet aggregation function (PAF) after percutaneous coronary intervention (PCI) in patients with acute coronary syndrome (ACS).


Assuntos
Síndrome Coronariana Aguda/terapia , Moléculas de Adesão Celular/metabolismo , Clopidogrel/uso terapêutico , Proteínas dos Microfilamentos/metabolismo , Intervenção Coronária Percutânea , Fosfoproteínas/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Ticagrelor/uso terapêutico , Humanos , Fosforilação , Inibidores da Agregação de Plaquetas/uso terapêutico , Resultado do Tratamento
17.
Mol Med Rep ; 19(6): 4919-4926, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30942422

RESUMO

Cervical cancer is a malignancy that threatens female health. The present study aimed to investigate the role of transgelin 2 (TAGLN2) in cervical cancer. Reverse transcription­quantitative polymerase chain reaction and western blotting were conducted to detect the mRNA and protein expression levels of particular factors in HeLa cells. Cell Counting kit­8, wound healing and Transwell assays were conducted to determine cell viability, and migratory and invasive abilities, respectively. The results demonstrated that the expression levels of TAGLN2 were decreased in cervical cancer tissues and were associated with the survival time of patients with cervical cancer. In addition, the expression of TAGLN2 was significantly reduced in three cervical cancer cell lines (HeLa, SiHa and C­33A) compared with in a normal cervical cell line. The present study also demonstrated that TAGLN2 overexpression in HeLa cells could inhibit cell viability, migration and invasion, and it was suggested that this may occur via upregulation of the expression levels of E­cadherin and inhibitor of nuclear factor κ­light­chain­enhancer of activated B cells (NF­κB) (IκB), and downregulation of C­X­C chemokine receptor type 4, matrix metalloproteinase (MMP)­2, MMP­9, p50 and transcription factor p65. In conclusion, TAGLN2 was revealed to inhibit cell viability, and the migratory and invasive abilities of HeLa cervical cancer cells via regulating the expression of metastasis­associated factors and the NF­κB signaling pathway. The present study proposed a novel target gene for the diagnosis, treatment and prognosis of cervical cancer.


Assuntos
Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Neoplasias do Colo do Útero/patologia , Adulto , Idoso , Caderinas/metabolismo , Linhagem Celular , Movimento Celular , Sobrevivência Celular , Feminino , Células HeLa , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Proteínas dos Microfilamentos/genética , Pessoa de Meia-Idade , Proteínas Musculares/genética , NF-kappa B/metabolismo , Estadiamento de Neoplasias , Receptores CXCR4/metabolismo , Transdução de Sinais , Regulação para Cima , Neoplasias do Colo do Útero/metabolismo
18.
Gene Expr Patterns ; 32: 53-66, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30940554

RESUMO

We have cloned and characterized an intronic fragment of zebrafish lymphocyte cytosolic protein 1 (lcp1, also called L-plastin) that drives expression to the zebrafish enveloping layer (EVL). L-plastin is a calcium-dependent actin-bundling protein belonging to the plastin/fimbrin family of proteins, and is necessary for the proper migration and attachment of several adult cell types, including leukocytes and osteoclasts. However, in zebrafish lcp1 is abundantly expressed much earlier, during differentiation of the EVL. The cells of this epithelial layer migrate collectively, spreading vegetally over the yolk. L-plastin expression persists into the larval periderm, a transient epithelial tissue that forms the first larval skin. This finding establishes that L-plastin is activated in two different embryonic waves, with a distinct regulatory switch between the early EVL and the later leukocyte. To better study L-plastin expressing cells we attempted CRISPR/Cas9 homology-driven recombination (HDR) to insert a self-cleaving peptide (Cre-P2A-EGFP-CAAX) downstream of the native lcp1 promoter. This produced a stable zebrafish line expressing Cre recombinase in EVL nuclei and green fluorescence in EVL cell membranes. In vivo tracking of these labeled cells provided enhanced views of EVL migration behavior, membrane extensions, and mitotic events. Finally, we experimentally dissected key elements of the targeted lcp1 locus, discovering a ∼300 bp intronic sequence sufficient to drive EVL expression. The lcp1: Cre-P2A-EGFP-CAAX zebrafish should be useful for studying enveloping layer specification, gastrulation movements and periderm development in this widely used vertebrate model. In addition, the conserved regulatory sequences we have isolated predict that L-plastin orthologs may have a similar early expression pattern in other vertebrate embryos.


Assuntos
Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Animais , Animais Geneticamente Modificados , Diferenciação Celular , Embrião não Mamífero/metabolismo , Epitélio/crescimento & desenvolvimento , Gastrulação , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Sequências Reguladoras de Ácido Nucleico/genética , Transcriptoma/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
19.
PLoS Genet ; 15(3): e1008012, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30865627

RESUMO

orb is a founding member of the CPEB family of translational regulators and is required at multiple steps during Drosophila oogenesis. Previous studies showed that orb is required during mid-oogenesis for the translation of the posterior/germline determinant oskar mRNA and the dorsal-ventral determinant gurken mRNA. Here, we report that orb also functions upstream of these axes determinants in the polarization of the microtubule network (MT). Prior to oskar and gurken translational activation, the oocyte MT network is repolarized. The MT organizing center at the oocyte posterior is disassembled, and a new MT network is established at the oocyte anterior. Repolarization depends upon cross-regulatory interactions between anterior (apical) and posterior (basal) Par proteins. We show that repolarization of the oocyte also requires orb and that orb is needed for the proper functioning of the Par proteins. orb interacts genetically with aPKC and cdc42 and in egg chambers compromised for orb activity, Par-1 and aPKC protein and aPKC mRNA are mislocalized. Moreover, like cdc42-, the defects in Par protein localization appear to be connected to abnormalities in the cortical actin cytoskeleton. These abnormalities also disrupt the localization of the spectraplakin Shot and the microtubule minus-end binding protein Patronin. These two proteins play a critical role in the repolarization of the MT network.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Oócitos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Animais Geneticamente Modificados , Polaridade Celular/genética , Polaridade Celular/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Citoesqueleto/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Feminino , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Genes de Insetos , Quinase 3 da Glicogênio Sintase/genética , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Oócitos/citologia , Oogênese/genética , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Transporte de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador alfa/genética , Fator de Crescimento Transformador alfa/metabolismo
20.
PLoS Genet ; 15(3): e1008050, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30856164

RESUMO

CENP-F is a large, microtubule-binding protein that regulates multiple cellular processes including chromosome segregation and mitochondrial trafficking at cytokinesis. This multiplicity of functions is mediated through the binding of various partners, like Bub1 at the kinetochore and Miro at mitochondria. Due to the multifunctionality of CENP-F, the cellular phenotypes observed upon its depletion are difficult to interpret and there is a need to genetically separate its different functions by preventing binding to selected partners. Here we engineer a CENP-F point-mutant that is deficient in Miro binding and thus is unable to localize to mitochondria, but retains other localizations. We introduce this mutation in cultured human cells using CRISPR/Cas9 system and show it causes a defect in mitochondrial spreading similar to that observed upon Miro depletion. We further create a mouse model carrying this CENP-F variant, as well as truncated CENP-F mutants lacking the farnesylated C-terminus of the protein. Importantly, one of these truncations leads to ~80% downregulation of CENP-F expression. We observe that, despite the phenotypes apparent in cultured cells, mutant mice develop normally. Taken together, these mice will serve as important models to study CENP-F biology at organismal level. In addition, because truncations of CENP-F in humans cause a lethal disease termed Strømme syndrome, they might also be relevant disease models.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Anormalidades do Olho/genética , Humanos , Atresia Intestinal/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Microcefalia/genética , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Mutação Puntual , Prenilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas rho de Ligação ao GTP/química , Proteínas rho de Ligação ao GTP/genética
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