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1.
Nat Commun ; 11(1): 4061, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792541

RESUMO

Adhesions are fibrotic scars that form between abdominal organs following surgery or infection, and may cause bowel obstruction, chronic pain, or infertility. Our understanding of adhesion biology is limited, which explains the paucity of anti-adhesion treatments. Here we present a systematic analysis of mouse and human adhesion tissues. First, we show that adhesions derive primarily from the visceral peritoneum, consistent with our clinical experience that adhesions form primarily following laparotomy rather than laparoscopy. Second, adhesions are formed by poly-clonal proliferating tissue-resident fibroblasts. Third, using single cell RNA-sequencing, we identify heterogeneity among adhesion fibroblasts, which is more pronounced at early timepoints. Fourth, JUN promotes adhesion formation and results in upregulation of PDGFRA expression. With JUN suppression, adhesion formation is diminished. Our findings support JUN as a therapeutic target to prevent adhesions. An anti-JUN therapy that could be applied intra-operatively to prevent adhesion formation could dramatically improve the lives of surgical patients.


Assuntos
Aderências Teciduais/metabolismo , Aderências Teciduais/patologia , Animais , Benzofenonas/farmacologia , Sistemas CRISPR-Cas , Células Cultivadas , Doxiciclina/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Imunofluorescência , Gastroenteropatias/metabolismo , Gastroenteropatias/patologia , Humanos , Imuno-Histoquímica , Isoxazóis/farmacologia , Lipossomos/metabolismo , Camundongos , Células NIH 3T3 , Parabiose , RNA Mensageiro/metabolismo , Tamoxifeno/farmacologia
2.
Science ; 369(6505): 842-846, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32792398

RESUMO

How is neuropathic pain regulated in peripheral sensory neurons? Importins are key regulators of nucleocytoplasmic transport. In this study, we found that importin α3 (also known as karyopherin subunit alpha 4) can control pain responsiveness in peripheral sensory neurons in mice. Importin α3 knockout or sensory neuron-specific knockdown in mice reduced responsiveness to diverse noxious stimuli and increased tolerance to neuropathic pain. Importin α3-bound c-Fos and importin α3-deficient neurons were impaired in c-Fos nuclear import. Knockdown or dominant-negative inhibition of c-Fos or c-Jun in sensory neurons reduced neuropathic pain. In silico screens identified drugs that mimic importin α3 deficiency. These drugs attenuated neuropathic pain and reduced c-Fos nuclear localization. Thus, perturbing c-Fos nuclear import by importin α3 in peripheral neurons can promote analgesia.


Assuntos
Dor Crônica/fisiopatologia , Neuralgia/fisiopatologia , Células Receptoras Sensoriais/fisiologia , alfa Carioferinas/fisiologia , Transporte Ativo do Núcleo Celular , Animais , Benzofenonas/farmacologia , Dor Crônica/genética , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Isoxazóis/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Neuralgia/genética , Proteínas Proto-Oncogênicas c-fos/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fator de Transcrição AP-1/metabolismo , alfa Carioferinas/genética
3.
Mol Pharmacol ; 98(1): 49-60, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32358164

RESUMO

Negative allosteric modulation of the metabotropic glutamate 5 (mGlu5) receptor has emerged as a potential strategy for the treatment of neurologic disorders. Despite the success in preclinical studies, many mGlu5 negative allosteric modulators (NAMs) that have reached clinical trials failed due to lack of efficacy. In this study, we provide a detailed in vitro pharmacological characterization of nine clinically and preclinically tested NAMs. We evaluated inhibition of l-glutamate-induced signaling with Ca2+ mobilization, inositol monophosphate (IP1) accumulation, extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, and real-time receptor internalization assays on rat mGlu5 expressed in HEK293A cells. Moreover, we determined association rates (kon) and dissociation rates (koff), as well as NAM affinities with [3H]methoxy-PEPy binding experiments. kon and koff values varied greatly between the nine NAMs (34- and 139-fold, respectively) resulting in long receptor residence times (>400 min) for basimglurant and mavoglurant, medium residence times (10-30 min) for AZD2066, remeglurant, and (RS)-remeglurant, and low residence times (<10 mins) for dipraglurant, F169521, F1699611, and STX107. We found that all NAMs inhibited l-glutamate-induced mGlu5 receptor internalization, generally with a similar potency to IP1 accumulation and ERK1/2 phosphorylation, whereas Ca2+ mobilization was less potently inhibited. Operational model of allosterism analyses revealed that dipraglurant and (RS)-remeglurant were biased toward (affinity) receptor internalization and away (cooperativity) from the ERK1/2 phosphorylation pathway, respectively. Our study is the first to measure mGlu5 NAM binding kinetics and negative allosteric modulation of mGlu5 receptor internalization and adds significant new knowledge about the molecular pharmacology of a diverse range of clinically relevant NAMs. SIGNIFICANCE STATEMENT: The metabotropic glutamate 5 (mGlu5) receptor is important in many brain functions and implicated in several neurological pathologies. Negative allosteric modulators (NAMs) have shown promising results in preclinical models but have so far failed in human clinical trials. Here we provide the most comprehensive and comparative molecular pharmacological study to date of nine preclinically/clinically tested NAMs at the mGlu5 receptor, which is also the first study to measure ligand binding kinetics and negative allosteric modulation of mGlu5 receptor internalization.


Assuntos
Imidazóis/farmacologia , Indóis/farmacologia , Isoxazóis/farmacologia , Piridinas/farmacologia , Receptor de Glutamato Metabotrópico 5/antagonistas & inibidores , Triazóis/farmacologia , Regulação Alostérica/efeitos dos fármacos , Animais , Cálcio/metabolismo , Células HEK293 , Humanos , Imidazóis/química , Indóis/química , Fosfatos de Inositol/metabolismo , Isoxazóis/química , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Estrutura Molecular , Fosforilação/efeitos dos fármacos , Piridinas/química , Ratos , Fatores de Tempo , Triazóis/química
4.
PLoS One ; 15(3): e0230753, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32218595

RESUMO

Rabbits (Oryctolagus cuniculi) are very popular as pets. However, problems of otitits caused by Psoroptes cuniculi are one of the main reasons to visit the veterinarian. Isoxazolines are an alternative treatment to treat this mite, and therefore, an evaluation of the effectiveness of oral afoxalaner with milbemycin oxime in rabbits infected with P. cuniculi was carried out. Nineteen rabbits, of New Zealand breed, with otitis due to an infection with P. cuniculi, were treated, whereas six rabbits were left untreated and formed the control group. The ear canals of each individual were examined, through the collection of otic exudate samples with cotton swabs. These were visualized under the microscope to identify the ectoparasite. Each animal was treated with a single oral dose of 2.50 mg / kg of afoxolaner, and 0.50 mg / kg of milbemycin oxime. Clinical signs and lesions associated with the infection, such as the presence of detritus, cerumen and / or scabs, and erythema, were evaluated. After receiving the treatment, all the lesions were classified as: mild, moderate and intense, with a visual analog scale. A week after providing medication, there was a decrease in the lesions of the group treated with Nexgard Spectra®, without further topical or systemic treatment. The decrease was gradual in the treated group and no recurrence was detected of P. cuniculi infection in both ears. Thus, the administration of a single oral dose of afoxolaner with milbemycin oxime was effective for the treatment of P. cuniculi infection in rabbits.


Assuntos
Isoxazóis/farmacologia , Macrolídeos/farmacologia , Infestações por Ácaros/tratamento farmacológico , Naftalenos/farmacologia , Psoroptidae/fisiologia , Animais , Relação Dose-Resposta a Droga , Interações Medicamentosas , Isoxazóis/uso terapêutico , Macrolídeos/uso terapêutico , Naftalenos/uso terapêutico , Coelhos
5.
Am J Physiol Gastrointest Liver Physiol ; 318(4): G682-G693, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32003602

RESUMO

Postprandial dyslipidemia is a common feature of insulin-resistant states and contributes to increased cardiovascular disease risk. Recently, bile acids have been recognized beyond their emulsification properties as important signaling molecules that promote energy expenditure, improve insulin sensitivity, and lower fasting lipemia. Although bile acid receptors have become novel pharmaceutical targets, their effects on postprandial lipid metabolism remain unclear. Here, we investigated the potential role of bile acids in regulation of postprandial chylomicron production and triglyceride excursion. Healthy C57BL/6 mice were given an intraduodenal infusion of taurocholic acid (TA) under fat-loaded conditions, and circulating lipids were measured. Targeting of bile acid receptors was achieved with GW4064, a synthetic agonist to the farnesoid X receptor (FXR), and deoxycholic acid (DCA), an activator of the Takeda G-protein-coupled receptor 5. TA, GW4064, and DCA treatments all lowered postprandial lipemia. FXR agonism also reduced intestinal triglyceride content and activity of microsomal triglyceride transfer protein, involved in chylomicron assembly. Importantly, TA (but not DCA) effects were largely lost in FXR knockout mice. These bile acid effects are reminiscent of the antidiabetic hormone glucagon-like peptide-1 (GLP-1). Although the GLP-1 receptor agonist exendin-4 retained its ability to acutely lower postprandial lipemia during bile acid sequestration and FXR deficiency, it did raise hepatic expression of the rate-limiting enzyme for bile acid synthesis. Bile acid signaling may be an important mechanism of controlling dietary lipid absorption, and bile acid receptors may constitute novel targets for the treatment of postprandial dyslipidemia.NEW & NOTEWORTHY We present new data suggesting potentially important roles for bile acids in regulation of postprandial lipid metabolism. Specific bile acid species, particularly secondary bile acids, were found to markedly inhibit absorption of dietary lipid and reduce postprandial triglyceride excursion. These effects appear to be mediated via bile acid receptors, farnesoid X receptor (FXR) and Takeda G protein-coupled receptor 5 (TGR5). Importantly, bile acid signaling may trigger glucagon-like peptide-1 (GLP-1) secretion, which may in turn mediate the marked inhibitory effects on dietary fat absorption.


Assuntos
Ácido Desoxicólico/farmacologia , Hiperlipidemias/tratamento farmacológico , Isoxazóis/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Período Pós-Prandial , Receptores Citoplasmáticos e Nucleares/agonistas , Animais , Ácidos e Sais Biliares/metabolismo , Ácidos e Sais Biliares/farmacologia , Colesterol 7-alfa-Hidroxilase/genética , Colesterol 7-alfa-Hidroxilase/metabolismo , Exenatida/farmacologia , Esvaziamento Gástrico/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Insulina/sangue , Mucosa Intestinal , Intestinos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores Acoplados a Proteínas-G/agonistas , Ácido Taurocólico/farmacologia
6.
Arch Biochem Biophys ; 682: 108281, 2020 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-32001246

RESUMO

Upregulation of nerve growth factor (NGF) in parenchymal hepatocytes has been shown to exert hepatoprotective function during cholestatic liver injury. However, the modulatory role of NGF in regulation of liver autophagy remains unclear. This study aimed to scrutinize the regulatory role of NGF in hepatic expression of farnesoid X receptor (FXR), a bile acid (BA)-activated nuclear receptor, and to determine its cytoprotective effect on BA-induced autophagy and cytotoxicity. Livers of human hepatolithiasis and bile duct ligation (BDL)-induced mouse cholestasis were used for histopathological and molecular detection. The regulatory roles of NGF in autophagy flux and FXR expression, as well as its hepatoprotection against BA cytotoxicity were examined in cultured hepatocytes. FXR downregulation in human hepatolithiasis livers showed positive correlation with hepatic NGF levels. NGF administration upregulated hepatic FXR levels, while neutralization of NGF decreased FXR expression in BDL-induced cholestatic mouse livers. In vitro studies demonstrated that NGF upregulated FXR expression, increased cellular LC3 levels, and exerted hepatoprotective effect in cultured primary rat hepatocytes. Conversely, autophagy inhibition abrogated NGF-driven cytoprotection under BA exposure, suggesting involvement of NGF-modulated auophagy flux. Although FXR agonistic GW4064 stimulation did not affect auophagic LC3 levels, FXR activity inhibition significantly potentiated BA-induced cytotoxicity and increased cellular p62/SQSTM1 and Rab7 protein in SK-Hep1 hepatocytes. Moreover, FXR gene silencing abolished the protective effect of NGF under BA exposure. These findings support that NGF modulates autophagy flux via FXR upregulation and protects hepatocytes against BA-induced cytotoxicity. NGF/FXR axis is a novel therapeutic target for treatment of cholestatic liver diseases.


Assuntos
Autofagia , Colestase/metabolismo , Hepatócitos/metabolismo , Fígado/metabolismo , Fator de Crescimento Neural/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Linhagem Celular Tumoral , Colestase/patologia , Citoproteção , Hepatócitos/citologia , Humanos , Isoxazóis/farmacologia , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Substâncias Protetoras/farmacologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Ativação Transcricional
7.
BMC Genomics ; 21(1): 120, 2020 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-32013879

RESUMO

BACKGROUND: Fluralaner is a novel isoxazoline insecticide with a unique action site on the γ-aminobutyric acid receptor (GABAR), shows excellent activity on agricultural pests including the common cutworm Spodoptera litura, and significantly influences the development and fecundity of S. litura at either lethal or sublethal doses. Herein, Illumina HiSeq Xten (IHX) platform was used to explore the transcriptome of S. litura and to identify genes responding to fluralaner exposure. RESULTS: A total of 16,572 genes, including 451 newly identified genes, were observed in the S. litura transcriptome and annotated according to the COG, GO, KEGG and NR databases. These genes included 156 detoxification enzyme genes [107 cytochrome P450 enzymes (P450s), 30 glutathione S-transferases (GSTs) and 19 carboxylesterases (CarEs)] and 24 insecticide-targeted genes [5 ionotropic GABARs, 1 glutamate-gated chloride channel (GluCl), 2 voltage-gated sodium channels (VGSCs), 13 nicotinic acetylcholine receptors (nAChRs), 2 acetylcholinesterases (AChEs) and 1 ryanodine receptor (RyR)]. There were 3275 and 2491 differentially expressed genes (DEGs) in S. litura treated with LC30 or LC50 concentrations of fluralaner, respectively. Among the DEGs, 20 related to detoxification [16 P450s, 1 GST and 3 CarEs] and 5 were growth-related genes (1 chitin and 4 juvenile hormone synthesis genes). For 26 randomly selected DEGs, real-time quantitative PCR (RT-qPCR) results showed that the relative expression levels of genes encoding several P450s, GSTs, heat shock protein (HSP) 68, vacuolar protein sorting-associated protein 13 (VPSAP13), sodium-coupled monocarboxylate transporter 1 (SCMT1), pupal cuticle protein (PCP), protein takeout (PT) and low density lipoprotein receptor adapter protein 1-B (LDLRAP1-B) were significantly up-regulated. Conversely, genes encoding esterase, sulfotransferase 1C4, proton-coupled folate transporter, chitinase 10, gelsolin-related protein of 125 kDa (GRP), fibroin heavy chain (FHC), fatty acid synthase and some P450s were significantly down-regulated in response to fluralaner. CONCLUSIONS: The transcriptome in this study provides more effective resources for the further study of S. litura whilst the DEGs identified sheds further light on the molecular response to fluralaner.


Assuntos
Isoxazóis/farmacologia , Spodoptera/efeitos dos fármacos , Spodoptera/genética , Transcriptoma/genética , Animais , Sistema Enzimático do Citocromo P-450/genética , Perfilação da Expressão Gênica/métodos , Inativação Metabólica/efeitos dos fármacos , Inativação Metabólica/genética , Proteínas de Insetos/genética , Inseticidas/farmacologia , Larva/efeitos dos fármacos , Larva/genética , Pupa/efeitos dos fármacos , RNA-Seq/métodos , Regulação para Cima/genética , Sequenciamento Completo do Exoma/métodos
8.
Toxicol Appl Pharmacol ; 391: 114916, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-32035996

RESUMO

Fibroblast-to-myofibroblast differentiation is one of the most important characteristics of pulmonary fibrosis, and screening natural compounds targeting fibroblast differentiation is always a promising approach to discover drug candidates for treatment of pulmonary fibrosis. Trehalose reportedly has many potential medical applications, especially in treating neurodegeneration diseases. However, it remains unclear whether trehalose suppresses lung fibroblast differentiation. In this work, we found that trehalose decreased the expression levels of α-smooth muscle actin (α-SMA) following the induction of transforming growth factor ß1 (TGF-ß1) in pretreatment, co-treatment, and post-treatment groups. Trehalose also reduced the production of type I collagen, lung fibroblast-containing gel contractility and cell filament formation in TGF-ß1-stimulated MRC-5 cells. Although trehalose is a known autophagy inducer, our results showed that its suppressive effect on fibroblast differentiation was not via trehalose-induced autophagy. And it did not affect canonical TGFß/Smad2/3 pathway. By applying proteomic profiling technology, we demonstrated that the downregulation of ß-catenin was involved in the trehalose-repressive action on fibroblast differentiation. The ß-catenin agonist, SKL2001, reversed the suppressive effect of trehalose on fibroblast differentiation. Overall, these experiments demonstrated that trehalose suppressed fibroblast differentiation via the downregulation of ß-catenin, but not through canonical autophagy and TGFß/Smad2/3 pathway, which is not only a novel understanding of trehalose, but also quite helpful for in vivo research of trehalose on pulmonary fibrosis in future.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Pulmão/citologia , Miofibroblastos/efeitos dos fármacos , Proteômica/métodos , Fator de Crescimento Transformador beta1/genética , Trealose/farmacologia , Actinas/biossíntese , Actinas/genética , Autofagia/efeitos dos fármacos , Linhagem Celular , Colágeno Tipo I/biossíntese , Regulação para Baixo , Humanos , Imidazóis/farmacologia , Isoxazóis/farmacologia , Fibrose Pulmonar/patologia , Transdução de Sinais/efeitos dos fármacos , Trealose/antagonistas & inibidores , beta Catenina/agonistas , beta Catenina/antagonistas & inibidores
9.
J Med Chem ; 63(5): 2470-2488, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-31972093

RESUMO

Two series of saccharin/isoxazole and saccharin/isoxazoline hybrids were synthesized by 1,3-dipolar cycloaddition. The new compounds showed to be endowed with potent and selective inhibitory activity against the cancer-related human carbonic anhydrase (hCA) IX and XII isoforms in the nanomolar range, while no affinity was encountered for off-targets, such as hCA I and II. Successive enantioseparation on a milligram scale of the most representative compounds led to the discovery that (S)-isomers were more potent than their corresponding (R)-enantiomers. Lastly, molecular modeling studies were conducted to define those structural requirements that were responsible for the discrimination among selected human isoforms of carbonic anhydrases. Two nanomolar hCA IX and XII inhibitors were also screened for their selective toxicity against non tumoral primary cells (fibroblasts) and against a breast adenocarcinoma cell line (MCF7) in hypoxic environment. The efficacious combination of these compounds with doxorubicin on MCF7 cells was demonstrated after 72 h of treatment.


Assuntos
Antígenos de Neoplasias/metabolismo , Anidrase Carbônica IX/metabolismo , Inibidores da Anidrase Carbônica/química , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Sacarina/química , Sacarina/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Anidrase Carbônica IX/antagonistas & inibidores , Inibidores da Anidrase Carbônica/síntese química , Linhagem Celular , Reação de Cicloadição , Humanos , Isoxazóis/síntese química , Isoxazóis/química , Isoxazóis/farmacologia , Células MCF-7 , Simulação de Acoplamento Molecular , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/metabolismo , Sacarina/síntese química
10.
Nat Commun ; 11(1): 562, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992715

RESUMO

Cancer immunotherapy has emerged as a promising cancer treatment. However, the presence of immune-refractory tumor cells limits its clinical success by blocking amplification of anti-tumor immunity. Previously, we found that immune selection by immunotherapy drives the evolution of tumors toward multi-modal resistant and stem-like phenotypes via transcription induction of AKT co-activator TCL1A by NANOG. Here, we report a crucial role of HSP90A at the crossroads between NANOG-TCL1A axis and multi-aggressive properties of immune-edited tumor cells by identifying HSP90AA1 as a NANOG transcriptional target. Furthermore, we demonstrate that HSP90A potentiates AKT activation through TCL1A-stabilization, thereby contributing to the multi-aggressive properties in NANOGhigh tumor cells. Importantly, HSP90 inhibition sensitized immune-refractory tumor to adoptive T cell transfer as well as PD-1 blockade, and re-invigorated the immune cycle of tumor-reactive T cells. Our findings implicate that the HSP90A-TCL1A-AKT pathway ignited by NANOG is a central molecular axis and a potential target for immune-refractory tumor.


Assuntos
Proteínas de Choque Térmico HSP90/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/metabolismo , Imunidade , Imunoterapia , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Isoxazóis/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Proteína Homeobox Nanog/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Resorcinóis/farmacologia
11.
Eur J Pharmacol ; 868: 172886, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31866407

RESUMO

Lysophosphatidic acid (LPA), as a bioactive lipid, plays a variety of physiological and pathological roles via activating six types of G-protein-coupled LPA receptors (LPA1-6). Our preliminary study found that LPA1 is highly expressed in lung cancer tissues compared with paracancerous tissues, but the role of LPA1 in lung carcinoma is unclear. This study aimed to elucidate the association between LPA1 and lung tumour behaviour at the cellular and animal model levels. We found that LPA promoted the migration, proliferation and colony formation of a lung cancer cell line (A549). LPA1 and LPA3 are preferentially expressed in A549 cells, and both Ki16425 (LPA1 and LPA3 antagonist) and ono7300243 (LPA1 antagonist) completely blocked the LPA-induced actions. These results were further verified by experiments of the LPA1/3 overexpression and LPA1 knockdown A549 cells. Furthermore, LPA1 overexpression and knockdown A549 cells were used to assess the in vivo tumour-bearing animal model and the mechanism underlying LPA-induced actions. In the animal model, A549 cell-derived tumour volume was significantly increased by LPA1 overexpression and significantly decreased by LPA1 knockdown respectively, suggesting that LPA1 is a regulator of in vivo tumour formation. Our results also indicated that the LPA1/Gi/MAP kinase/NF-κB pathway is involved in LPA-induced oncogenic actions in A549 cells. Thus, targeting LPA1 may be a novel strategy for treating lung carcinoma.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Células A549 , Animais , Antineoplásicos/uso terapêutico , Movimento Celular/efeitos dos fármacos , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Isoxazóis/farmacologia , Isoxazóis/uso terapêutico , Neoplasias Pulmonares/patologia , Lisofosfolipídeos/metabolismo , Masculino , Camundongos , NF-kappa B/metabolismo , Propionatos/farmacologia , Propionatos/uso terapêutico , Receptores de Ácidos Lisofosfatídicos/genética , Receptores de Ácidos Lisofosfatídicos/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Eur J Pharmacol ; 869: 172875, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31877279

RESUMO

There is growing evidence for the contribution of the activated coagulation factor X (FXa) in the development of chronic inflammatory lung diseases. Therefore, we aimed to investigate effects of exogenous FXa on mitochondrial and metabolic function as well as the induction of inflammatory molecules in type II alveolar epithelial cells. Effects of FXa on epithelial cells were investigated in A549 cell line. Activation of extracellular signal-regulated kinase (ERK) and induction of inflammatory molecules were examined by immunoblot and gene expression analysis. Mitochondrial function was assessed by the measurement of oxygen consumption during maximal oxidative phosphorylation and quantitative determination of cardiolipin oxidation. Apoptosis was tested using a caspase 3 antibody. Metabolic activity and lactate dehydrogenase assay were applied for the detection of cellular viability. FXa activated ERK1/2 and induced an increase in the expression of pro-inflammatory cytokines, which was prevented by an inhibitor of FXa, edoxaban, or an inhibitor of protease-activated receptor 1, vorapaxar. Exposure to FXa caused mitochondrial alteration with restricted capacity for ATP generation, which was effectively prevented by edoxaban, vorapaxar and GB83 (inhibitor of protease-activated receptor 2). Of note, exposure to FXa did not initiate apoptosis in epithelial cells. FXa-dependent pro-inflammatory state and impairment of mitochondria did not reach the level of significance in lung epithelial cells. However, these effects might limit regenerative potency of lung epithelial cells, particular under clinical circumstances where lung injury causes exposure to clotting factors.


Assuntos
Células Epiteliais/metabolismo , Fator Xa/metabolismo , Inflamação/metabolismo , Mitocôndrias/metabolismo , Receptores Ativados por Proteinase/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Dipeptídeos/farmacologia , Células Epiteliais/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Inibidores do Fator Xa/farmacologia , Humanos , Isoxazóis/farmacologia , Mitocôndrias/efeitos dos fármacos , Piridinas/farmacologia , Receptores Ativados por Proteinase/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Tiazóis/farmacologia
13.
Respir Res ; 20(1): 285, 2019 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-31852500

RESUMO

BACKGROUND: ß2 receptor agonists induce airway smooth muscle relaxation by increasing intracellular cAMP production. PKA is the traditional downstream signaling pathway of cAMP. Exchange protein directly activated by cAMP (Epac) was identified as another important signaling molecule of cAMP recently. The role of Epac in asthmatic airway inflammation and airway remodeling is unclear. METHODS: We established OVA-sensitized and -challenged acute and chronic asthma mice models to explore the expression of Epac at first. Then, airway inflammation and airway hyperresponsiveness in acute asthma mice model and airway remodeling in chronic asthma mice model were observed respectively after treatment with Epac-selective cAMP analogue 8-pCPT-2'-O-Me-cAMP (8pCPT) and Epac inhibitor ESI-09. Next, the effects of 8pCPT and ESI-09 on the proliferation and apoptosis of in vitro cultured mouse airway smooth muscle cells (ASMCs) were detected with CCK-8 assays and Annexin-V staining. Lastly, the effects of 8pCPT and ESI-09 on store-operated Ca2+ entry (SOCE) of ASMCs were examined by confocal Ca2+ fluorescence measurement. RESULTS: We found that in lung tissues of acute and chronic asthma mice models, both mRNA and protein expression of Epac1 and Epac2, two isoforms of Epac, were lower than that of control mice. In acute asthma mice model, the airway inflammatory cell infiltration, Th2 cytokines secretion and airway hyperresponsiveness were significantly attenuated by 8pCPT and aggravated by ESI-09. In chronic asthma mice model, 8pCPT decreased airway inflammatory cell infiltration and airway remodeling indexes such as collagen deposition and airway smooth muscle cell proliferation, while ESI-09 increased airway inflammation and airway remodeling. In vitro cultured mice ASMCs, 8pCPT dose-dependently inhibited, whereas ESI-09 promoted ASMCs proliferation. Interestingly, 8pCPT promoted the apoptosis of ASMCs, whereas ESI-09 had no effect on ASMCs apoptosis. Lastly, confocal Ca2+ fluorescence examination found that 8pCPT could inhibit SOCE in ASMCs at 100 µM, and ESI-09 promoted SOCE of ASMCs at 10 µM and 100 µM. In addition, the promoting effect of ESI-09 on ASMCs proliferation was inhibited by store-operated Ca2+ channel blocker, SKF-96365. CONCLUSIONS: Our results suggest that Epac has a protecting effect on asthmatic airway inflammation and airway remodeling, and Epac reduces ASMCs proliferation by inhibiting SOCE in part.


Assuntos
Remodelação das Vias Aéreas , Asma/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Pulmão/metabolismo , Pneumonia/metabolismo , Hipersensibilidade Respiratória/metabolismo , Remodelação das Vias Aéreas/efeitos dos fármacos , Animais , Apoptose , Asma/induzido quimicamente , Asma/tratamento farmacológico , Asma/fisiopatologia , Sinalização do Cálcio , Proliferação de Células , Células Cultivadas , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Modelos Animais de Doenças , Feminino , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/genética , Hidrazonas/farmacologia , Mediadores da Inflamação/metabolismo , Isoxazóis/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/fisiopatologia , Camundongos Endogâmicos BALB C , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Ovalbumina , Pneumonia/induzido quimicamente , Pneumonia/fisiopatologia , Pneumonia/prevenção & controle , Hipersensibilidade Respiratória/induzido quimicamente , Hipersensibilidade Respiratória/tratamento farmacológico , Hipersensibilidade Respiratória/fisiopatologia
14.
Molecules ; 24(22)2019 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-31744088

RESUMO

The modulators of farnesoid X receptor (FXR), a bile acid receptor, regulate various biological processes including bile acid metabolism, and are associated with the control of fatty liver and osteoporosis. Thus, the control of FXR activity and development of FXR modulators are critical not only for research, but also for clinical application. In this study, we synthesized novel FXR agonists 1-4 possessing isoxazole and N-substituted benzimidazole moieties, and compared their effects on osteoblast differentiation with the known FXR agonists, chenodeoxycholic acid and a synthetic compound, GW4064. Two (3 and 4) of the four novel FXR agonists 1-4 showed high specificities for FXR. Computer-assisted modeling suggested that the binding of the FXR agonist 3 with ligand binding domain of FXR was similar to GW4064. FXR was expressed in mouse bone marrow-derived mesenchymal stem cell (MSC)-like ST2 cells (ST-2 MSCs). The FXR agonists activated the BMP-2-induced differentiation of ST-2 MSCs into osteoblasts and enhanced the expression of RUNX2. Moreover, the potency of the FXR agonist 3 was comparable to GW4064 in promoting osteoblast differentiation of ST-2 MSCs. These results indicate that FXR activation enhanced the BMP-2-induced differentiation of MSCs into osteoblasts through activating RUNX2 expression. FXR could be a potential therapeutic target for the treatment of bone diseases such as osteoporosis.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Isoxazóis/síntese química , Isoxazóis/farmacologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoblastos/citologia , Receptores Citoplasmáticos e Nucleares/agonistas , Animais , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Química Sintética , Fluorimunoensaio , Genes Reporter , Humanos , Isoxazóis/química , Camundongos , Modelos Moleculares , Receptores Citoplasmáticos e Nucleares/química , Relação Estrutura-Atividade
15.
Molecules ; 24(22)2019 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-31698778

RESUMO

Homonucleoside analogues cis-16 and trans-17 having a (5-methoxycarbonyl)isoxazolidine framework were synthesized via the 1,3-dipolar cycloaddition of nucleobase-derived nitrones with methyl acrylate. Hydrogenolysis of the isoxazolidines containing thymine, dihydrouracil, theophylline and adenine moieties efficiently led to the formation of the respective γ-lactam analogues. γ-Lactam analogues having 5-bromouracil and 5-chlorouracil fragments were synthesized by treatment of uracil-containing γ-lactams with NBS and NCS. Isoxazolidine and γ-lactam analogues of homonucleosides obtained herein were evaluated for activity against a broad range of DNA and RNA viruses. None of the compounds that were tested exhibited antiviral or cytotoxic activity at concentrations up to 100 µM. The cytostatic activities of all compounds toward nine cancerous cell lines was tested. γ-Lactams trans-15e (Cl-Ura) and cis-15h (Theo) appeared the most active toward pancreatic adenocarcinoma cells (Capan-1), showing IC50 values 21.5 and 18.2 µM, respectively. Isoxazolidine cis-15e (Cl-Ura) inhibited the proliferation of colorectal carcinoma (HCT-116).


Assuntos
Isoxazóis/química , Lactamas/química , Nucleosídeos/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Isoxazóis/farmacologia , Lactamas/farmacologia , Estrutura Molecular , Análise Espectral
16.
Yakugaku Zasshi ; 139(11): 1397-1402, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31685736

RESUMO

Patients with epilepsy are often affected by not only seizures but also a variety of cognitive and psychiatric comorbidities that further impair their quality of life. However, it is unclear whether epilepsy is associated with psychic function. The aim of the present study was to clarify the effects of kindling-induced epileptic seizures on psychic functioning, using behavioral pharmacological tests. Pentylenetetrazol (PTZ)-kindled mice displayed impaired motor coordination (in the rotarod test), and social approach impairment (in the three-chamber social test) compared with vehicle mice. Intraperitoneal ABT-418 treatment (0.05 mg/kg) alleviated these behavioral abnormalities in PTZ-kindled mice. Immunolabeling of tissue sections demonstrated that expression of the α4 subunit of the α4ß2 nicotinic acetylcholine receptor in the piriform cortex was significantly decreased in PTZ-kindled mice. In contrast, expression of the synaptic adhesion molecule neuroligin 3 (NLG3) was significantly higher in the piriform cortex of PTZ-kindled mice compared with vehicle mice. Collectively, our findings suggest that attention deficit/hyperactivity disorder (ADHD)-like or autistic-like behavioral abnormalities associated with epilepsy are closely linked to downregulation of the α4 subunit of the α4ß2 receptor and upregulation of NLG3 in the mouse piriform cortex. In summary, this study indicates that ABT-418 is a good candidate for the treatment of patients with epilepsy complicated by psychiatric symptoms such as autism and ADHD.


Assuntos
Epilepsia/psicologia , Excitação Neurológica , Transtornos Mentais/tratamento farmacológico , Transtornos Mentais/etiologia , Pentilenotetrazol , Receptores Nicotínicos/metabolismo , Convulsões/psicologia , Animais , Moléculas de Adesão Celular Neuronais/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Epilepsia/induzido quimicamente , Epilepsia/tratamento farmacológico , Epilepsia/etiologia , Isoxazóis/farmacologia , Isoxazóis/uso terapêutico , Proteínas de Membrana/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Córtex Piriforme/metabolismo , Desempenho Psicomotor/efeitos dos fármacos , Pirrolidinas/farmacologia , Pirrolidinas/uso terapêutico , Qualidade de Vida , Convulsões/induzido quimicamente , Convulsões/tratamento farmacológico , Convulsões/etiologia , Comportamento Social
17.
Molecules ; 24(20)2019 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-31635044

RESUMO

As a continuation of our efforts to discover and develop "me-better" active molecules, in this study, a series of novel isoxazole-amide derivatives containing an acylhydrazone moiety were synthesized and evaluated for their antiviral activities against tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV). Antiviral bioassays indicated that some of the target compounds exhibited better in vivo antiviral activities against TMV and CMV than those of Ningnanmycin (NNM). Especially, the compound 7t exhibited the best curative, protection, and inactivation activities against TMV and CMV which were superior to those of NNM. Meanwhile, our present work also revealed that compound 7t could enhance the defense-related enzyme activity and increase the chlorophyll content in tobacco leaves to induce resistance and enhance plant tolerance to TMV infection.


Assuntos
Antivirais/síntese química , Hidrazonas/química , Isoxazóis/síntese química , Antivirais/química , Antivirais/farmacologia , Clorofila/metabolismo , Cucumovirus/efeitos dos fármacos , Resistência à Doença , Isoxazóis/química , Isoxazóis/farmacologia , Estrutura Molecular , Tabaco/metabolismo , Tabaco/virologia , Vírus do Mosaico do Tabaco/efeitos dos fármacos
18.
Respir Res ; 20(1): 228, 2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31640701

RESUMO

BACKGROUND: Antiviral drugs such as rupintrivir may have an immune-modulatory effect in experimentally induced allergic asthma with subsequent RV infection. We infected lung slices of house-dust mite (HDM)-sensitized asthmatic mice ex vivo with human rhinovirus (RV) and investigated the effect of the antiviral drug rupintrivir on RV-induced cytokine response in lung tissue of HDM-sensitized mice ex vivo. METHODS: Mice were sensitized with HDM. Precision-cut lung slices (PCLS) were prepared from HDM-sensitized or non-sensitized mice. Lung slices were infected ex vivo with RV or RV together with rupintrivir. Modulation of immune responses was evaluated by cytokine secretion 48 h post infection. RESULTS: In vivo HDM sensitization resulted in a TH-2/TH-17-dominated cytokine response that persisted in PCLS ex vivo. RV infection of PCLS from non-sensitized mice resulted in the induction of an antiviral and pro-inflammatory immune response, as indicated by the secretion of IFN-α, IFN-ß, IFN-γ, TNF-α, MCP-1, IP-10, IL-10, and IL-17A. In contrast, PCLS from HDM-sensitized mice showed an attenuated antiviral response, but exaggerated IL-4, IL-6, and IL-10 secretion upon infection. Rupintrivir inhibited exaggerated pro-inflammatory cytokine IL-6 and TH-2 cytokine IL-4 in HDM-sensitized mice. CONCLUSIONS: In summary, this study demonstrates that treatment with rupintrivir influences virus-induced IL-4 and IL-6 cytokine release under experimental conditions ex vivo.


Assuntos
Interleucina-4/imunologia , Isoxazóis/farmacologia , Pulmão/imunologia , Pyroglyphidae/imunologia , Pirrolidinonas/farmacologia , Rhinovirus , Células Th2/imunologia , Animais , Antivirais/farmacologia , Citocinas/antagonistas & inibidores , Citocinas/imunologia , Feminino , Interleucina-4/antagonistas & inibidores , Pulmão/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Técnicas de Cultura de Órgãos , Células Th2/efeitos dos fármacos
19.
J Pharmacol Sci ; 141(1): 83-85, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31607444

RESUMO

Dysregulation of the blood brain barrier due to oxidative stress causes neurological disorders, such as Alzheimer's and Parkinson's disease. We employed brain microvascular endothelial cells; to investigate the effects of P53 towards the lipid oxidation of the BBB. P53 reduction by LPS, siRNA for P53 and Pifithrin increased the expression levels of malondialdehyde, a marker of oxidative stress and lipid peroxidation. Furthermore, P53 suppression impaired the permeability of the BBB monolayers. In contrast, P53 induction by Nutlin and Hsp90 inhibitor AUY922 enhanced the BBB function. In conclusion, P53 supports BBB integrity, at least in part, by reducing lipid peroxidation.


Assuntos
Barreira Hematoencefálica/metabolismo , Peroxidação de Lipídeos/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Células Cultivadas , Células Endoteliais , Humanos , Isoxazóis/farmacologia , Malondialdeído/metabolismo , Estresse Oxidativo , Resorcinóis/farmacologia
20.
J Enzyme Inhib Med Chem ; 34(1): 1716-1721, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31571509

RESUMO

A series of 4-arylamido 5-methylisoxazole derivatives incorporating benzimidazole was designed and synthesised by conformational restriction of an in-house type II FMS inhibitor. Kinase profiling of one compound revealed interesting features, with increased inhibitory potency towards FLT3 and concomitant loss of potency towards FMS. Several benzimidazole derivatives 5a-5g and 6a-6c containing various hydrophobic moieties were synthesised, and their inhibitory activity against FLT3 was evaluated. Specifically, 5a, 5-methyl-N-(2-(3-(4-methylpiperazin-1-yl)-5-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-5-yl) isoxazole-4-carboxamide, exhibited the most potent inhibitory activity against FLT3 (IC50 = 495 nM), with excellent selectivity profiles.


Assuntos
Benzimidazóis/química , Isoxazóis/química , Inibidores de Proteínas Quinases/química , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Benzimidazóis/síntese química , Benzimidazóis/farmacologia , Descoberta de Drogas , Interações Hidrofóbicas e Hidrofílicas , Concentração Inibidora 50 , Isoxazóis/síntese química , Isoxazóis/farmacologia , Conformação Molecular , Simulação de Acoplamento Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Relação Estrutura-Atividade
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