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1.
Anticancer Res ; 40(9): 5043-5048, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32878792

RESUMO

BACKGROUND/AIM: Eicosapentaenoic acid (EPA) inhibits NF-ĸB activation and IL-6 production in TE-1 esophageal cancer cells. NF-ĸB is related to cancer cell migration. The aim of this study is to evaluate whether EPA has a metastasis suppressing effect. Herein, we investigated EPA-treated TE-1 cell migration using TAXIScan. MATERIALS AND METHODS: EZ-TAXIScan® was used to verify whether EPA inhibits cancer cell chemotaxis. RESULTS: Using 50% fetal bovine serum (chemoattractant) without EPA (positive control), average velocity was 0.306±0.084 µm/min compared to 0.162±0.067 µm/min without chemoattraction (negative control). Directionalities of positive and negative controls were 1.039±0.152 and 0.488±0.251 radians, respectively, indicating a significant increase in migration of the positive control compared to that of the negative control. Average velocities were 0.306±0.084 (no EPA), 0.288±0.078 (100 µM EPA), and 0.240±0.054 200 µM (EPA) µm/min, indicating that EPA reduced velocity dose-dependently. Average directionalities were 1.039±0.152 (no EPA), 0.967±0.164 (100 µM EPA), and 0.901±0.146 (200 µM EPA) radians, indicating that EPA also inhibited directionality dose-dependently. CONCLUSION: EPA suppresses directional migration of TE-1 cells.


Assuntos
Antineoplásicos/farmacologia , Ácido Eicosapentaenoico/farmacologia , Antineoplásicos/química , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Ácido Eicosapentaenoico/química , Carcinoma de Células Escamosas do Esôfago , Humanos
2.
Sci Rep ; 10(1): 12491, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32719460

RESUMO

Elevated plasma concentrations of the ketone body ß-hydroxybutyrate (BHB), an endogenous agonist of the hydroxycarboxylic acid receptor 2 (HCA2), is associated with an increased incidence of inflammatory diseases during lactation in dairy cows. In the early stages of this pathology, an increase in neutrophil recruitment is observed; however, the role of BHB remains elusive. This study characterized the effect of BHB and synthetic agonists of the HCA2 receptor on bovine neutrophil chemotaxis and the signaling pathways involved in this process. We demonstrated that treatment with BHB concentrations between 1.2 and 10 mM and two full selective agonists of the HCA2 receptor, MK-1903 and nicotinic acid, increased bovine neutrophil chemotaxis. We also observed that BHB and HCA2 agonists induced calcium release and phosphorylation of AKT, ERK 1/2 and AMPKα. To evaluate the role of these pathways in bovine neutrophil chemotaxis, we used the pharmacological inhibitors BAPTA-AM, pertussis toxin, U73122, LY294002, U0126 and compound C. Our results suggest that these pathways are required for HCA2 agonist-induced bovine neutrophil chemotaxis in non-physiological condition. Concentrations around 1.4 mM of BHB after calving may exert a chemoattractant effect that is key during the onset of the inflammatory process associated with metabolic disorders in dairy cows.


Assuntos
Ácido 3-Hidroxibutírico/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Quimiotaxia , Sistema de Sinalização das MAP Quinases , Neutrófilos/citologia , Neutrófilos/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Acoplados a Proteínas-G/agonistas , Animais , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Bovinos , Quimiotaxia/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Niacina/farmacologia , Fosforilação/efeitos dos fármacos , Pirazóis/farmacologia , Receptores Acoplados a Proteínas-G/metabolismo , Fosfolipases Tipo C/metabolismo
3.
Sci Rep ; 10(1): 10515, 2020 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-32601321

RESUMO

Bone marrow mesenchymal stem cells (BMSCs) have multi-lineage differentiation potential and play an important role in tissue repair. Studies have shown that BMSCs gather at the injured tissue site after granulocyte-colony stimulating factor (G-CSF) administration. In this study, we first investigated whether G-CSF could promote BMSC homing to damaged lung tissue induced by bleomycin (BLM) and then investigated whether SDF-1/CXCR4 chemotaxis might be involved in this process. Next, we further studied the potential inhibitory effect of G-CSF administration in mice with lung fibrosis induced by bleomycin. We examined both the antifibrotic effects of G-CSF in mice with bleomycin-induced pulmonary fibrosis in vivo and its effects on the proliferation, differentiation and chemotactic movement of cells in vitro. Flow cytometry, real-time PCR, transwell and Cell Counting Kit-8 (CCK-8) assays were used in this study. The results showed that both preventative and therapeutic G-CSF administration could significantly inhibit bleomycin-induced pulmonary fibrosis. G-CSF enhanced BMSC migration to lung tissues, but this effect could be alleviated by AMD3100, which blocked the SDF-1/CXCR4 axis. We also found that BMSCs could inhibit fibroblast proliferation and transdifferentiation into myofibroblasts through paracrine actions. In conclusion, G-CSF exerted antifibrotic effects in bleomycin-induced lung fibrosis, in part by promoting BMSC homing to injured lung tissues via SDF-1/CXCR4 chemotaxis.


Assuntos
Quimiocina CXCL12/metabolismo , Quimiotaxia/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Células-Tronco Mesenquimais/efeitos dos fármacos , Fibrose Pulmonar/tratamento farmacológico , Receptores CXCR4/metabolismo , Animais , Bleomicina , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Fator Estimulador de Colônias de Granulócitos/farmacologia , Camundongos , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Transdução de Sinais/efeitos dos fármacos
4.
PLoS One ; 15(7): e0235784, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32658899

RESUMO

Soft tissue is composed of cells surrounded by an extracellular matrix that is made up of a diverse array of intricately organized proteins. These distinct components work in concert to maintain homeostasis and respond to tissue damage. During tissue repair, extracellular matrix proteins and their degradation products are known to influence physiological processes such as angiogenesis and inflammation. In this study we developed a discovery platform using a decellularized extracellular matrix biomaterial to identify new chemotrophic factors derived from the extracellular matrix. An in vitro culture of RAW.264 macrophage cells with the biomaterial ovine forestomach matrix led to the identification of a novel ~12 kDa chemotactic factor, termed 'MayDay', derived from the N-terminal 31-188 sequence of decorin. The recombinant MayDay protein was shown to be a chemotactic agent for mesenchymal stromal cells in vitro and in vivo. We hypothesize that the macrophage-induced cleavage of decorin, via MMP-12, leads to the release of the chemotactic molecule MayDay, that in turn recruits cells to the site of damaged tissue.


Assuntos
Fatores Quimiotáticos/farmacologia , Decorina/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Fatores Quimiotáticos/química , Quimiotaxia/efeitos dos fármacos , Decorina/química , Células-Tronco Mesenquimais/citologia , Camundongos , Fragmentos de Peptídeos/química , Células RAW 264.7 , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Ovinos
5.
Biochem Pharmacol ; 180: 114125, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32598947

RESUMO

BACKGROUND: Colchicine is routinely used for its anti-inflammatory properties to treat gout and Familial Mediterranean fever. More recently, it was also shown to be of therapeutic benefit for another group of diseases in which inflammation is a key component, namely, cardiovascular disease. Whilst there is considerable interest in repurposing this alkaloid, it has a narrow therapeutic index and is associated with undesirable side effects and drug interactions. We, therefore, developed a derivatives of colchicine that preferentially target leukocytes to increase their potency and diminish their side effects. The anti-inflammatory activity of the colchicine derivatives was tested in experimental models of neutrophil activation by the etiological agent of gout, monosodium urate crystals (MSU). METHODS: Using a rational drug design approach, the structure of colchicine was modified to increase its affinity for ßVI-tubulin, a colchicine ligand preferentially expressed by immune cells. The ability of the colchicine analogues with the predicted highest affinity for ßVI-tubulin to dampen neutrophil responses to MSU was determined with in vitro assays that measure MSU-induced production of ROS, release of IL-1 and CXCL8/IL-8, and the increase in the concentration of cytoplasmic calcium. The anti-inflammatory property of the derivatives was assessed in the air pouch model of MSU-induced inflammation in mice. RESULTS: The most effective compound generated, CCI, is more potent than colchicine in all the in vitro assays. It inhibits neutrophil responses to MSU in vitro at concentrations 10-100-fold lower than colchicine. Similarly, in vivo, CCI inhibits the MSU-induced recruitment of leukocytes at a 10-fold lower concentration than colchicine when administered prior to or after MSU. CONCLUSIONS: We provide evidence that colchicine can be rendered more potent atinhibiting MSU-induced neutrophil activation and inflammation using a rational drug design approach. The development of compounds such as CCI will provide more efficacious drugs that will not only alleviate gout patients of their painful inflammatory episodes at significantly lower doses than colchicine, but also be of potential therapeutic benefit for patients with other diseases treated with colchicine.


Assuntos
Anti-Inflamatórios/uso terapêutico , Colchicina/análogos & derivados , Colchicina/uso terapêutico , Gota/tratamento farmacológico , Ativação de Neutrófilo/efeitos dos fármacos , Animais , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Simulação por Computador , Desenho de Fármacos , Gota/imunologia , Humanos , Masculino , Camundongos , Simulação de Acoplamento Molecular , Terapia de Alvo Molecular , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismo , Tubulina (Proteína)/metabolismo
6.
BMC Vet Res ; 16(1): 171, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32487098

RESUMO

BACKGROUND: A healthy immune system plays a particularly important role in newborns, including in calves that are far more susceptible to infections (viral, bacterial and other) than adult individuals. Therefore, the present study aimed to evaluate the influence of HMB on the chemotactic activity (MIGRATEST® kit), phagocytic activity (PHAGOTEST® kit) and oxidative burst (BURSTTEST® kit) of monocytes and granulocytes in the peripheral blood of calves by flow cytometry. RESULTS: An analysis of granulocyte and monocyte chemotactic activity and phagocytic activity revealed significantly higher levels of phagocytic activity in calves administered HMB than in the control group, expressed in terms of the percentage of phagocytising cells and mean fluorescence intensity (MFI). HMB also had a positive effect on the oxidative metabolism of monocytes and granulocytes stimulated with PMA (4-phorbol-12-ß-myristate-13-acetate) and Escherichia coli bacteria, expressed as MFI values and the percentage of oxidative metabolism. CONCLUSION: HMB stimulates non-specific cell-mediated immunity, which is a very important consideration in newborn calves that are exposed to adverse environmental factors in the first weeks of their life. The supplementation of animal diets with HMB for both preventive and therapeutic purposes can also reduce the use of antibiotics in animal production.


Assuntos
Bovinos/sangue , Granulócitos/fisiologia , Monócitos/fisiologia , Valeratos/farmacologia , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal , Animais , Animais Recém-Nascidos , Bovinos/imunologia , Quimiotaxia/efeitos dos fármacos , Dieta/veterinária , Citometria de Fluxo/veterinária , Granulócitos/citologia , Monócitos/citologia , Fagocitose/efeitos dos fármacos , Explosão Respiratória
7.
J Med Chem ; 63(17): 9003-9019, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32407089

RESUMO

Formyl peptide receptor 2 (FPR2) agonists can stimulate resolution of inflammation and may have utility for treatment of diseases caused by chronic inflammation, including heart failure. We report the discovery of a potent and selective FPR2 agonist and its evaluation in a mouse heart failure model. A simple linear urea with moderate agonist activity served as the starting point for optimization. Introduction of a pyrrolidinone core accessed a rigid conformation that produced potent FPR2 and FPR1 agonists. Optimization of lactam substituents led to the discovery of the FPR2 selective agonist 13c, BMS-986235/LAR-1219. In cellular assays 13c inhibited neutrophil chemotaxis and stimulated macrophage phagocytosis, key end points to promote resolution of inflammation. Cardiac structure and functional improvements were observed in a mouse heart failure model following treatment with BMS-986235/LAR-1219.


Assuntos
Pirrolidinonas/química , Receptores de Formil Peptídeo/agonistas , Receptores de Lipoxinas/agonistas , Animais , Quimiotaxia/efeitos dos fármacos , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/prevenção & controle , Humanos , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Microssomos Hepáticos/metabolismo , Neutrófilos/citologia , Neutrófilos/fisiologia , Fagocitose/efeitos dos fármacos , Pirrolidinonas/metabolismo , Pirrolidinonas/farmacologia , Pirrolidinonas/uso terapêutico , Receptores de Formil Peptídeo/genética , Receptores de Formil Peptídeo/metabolismo , Receptores de Lipoxinas/genética , Receptores de Lipoxinas/metabolismo , Relação Estrutura-Atividade
8.
Biochem Biophys Res Commun ; 527(1): 194-199, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32446366

RESUMO

Peritrichously flagellated bacteria such as Escherichia coli (E. coli) perform chemotaxis by a biased random walk toward various chemicals, which was driven by the bacterial flagellar motor. Fructose, a typical monosaccharide that can attract E. coli. However, little is known about the chemotaxis and motility response of E. coli towards fructose. Here, we characterized the chemotaxis behavior of E. coli to different concentrations of fructose from 0 mM to 50 mM by using microfluidics and bead assay. We observed the wild-type cells responded to the stimulus of fructose, which suggested fructose is an attractant to E. coli, while the cells defective in chemotaxis could not sense the stimulus of fructose. The motility of wild-type cells was reduced in various concentrations of fructose, which helped the aggregation of cells near surfaces, in contrast with the result that the fructose showed no effect on the motility of the cells defective in chemotaxis. Similar phenomena are expected to be found in the effect of other monosaccharides to E. coli.


Assuntos
Quimiotaxia/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Frutose/farmacologia , Relação Dose-Resposta a Droga , Escherichia coli/metabolismo
9.
Mol Cell Biochem ; 469(1-2): 109-118, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32304005

RESUMO

Diabetes is associated with an increase in skeletal fragility and risk of fracture. However, the underlying mechanism for the same is not well understood. Specifically, the results from osteoblast cell culture studies are ambiguous due to contradicting reports. The use of supraphysiological concentrations in these studies, unachievable in vivo, might be the reason for the same. Therefore, here, we studied the effect of physiologically relevant levels of high glucose during diabetes (11.1 mM) on MC3T3-E1 osteoblast cell functions. The results showed that high glucose exposure to osteoblast cells increases their differentiation and mineralization without any effect on the proliferation. However, high glucose decreases their migratory potential and chemotaxis with a decrease in the associated cell signaling. Notably, this decrease in cell migration in high glucose conditions was accompanied by aberrant localization of Dynamin 2 in osteoblast cells. Besides, high glucose also caused a shift in mitochondrial dynamics towards the appearance of more fused and lesser fragmented mitochondria, with a concomitant decrease in the expression of DRP1, suggesting decreased mitochondrial biogenesis. In conclusion, here we are reporting for the first time that hyperglycemia causes a reduction in osteoblast cell migration and chemotaxis. This decrease might lead to an inefficient movement of osteoblasts to the erosion site resulting in uneven mineralization and skeletal fragility found in type 2 diabetes patients, in spite of having normal bone mineral density (BMD).


Assuntos
Movimento Celular/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Diabetes Mellitus Tipo 2/metabolismo , Glucose/farmacologia , Hiperglicemia/metabolismo , Dinâmica Mitocondrial/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Dinamina II/metabolismo , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Biogênese de Organelas , Osteoblastos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
10.
PLoS Negl Trop Dis ; 14(3): e0008220, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32226018

RESUMO

Human clonorchiasis, caused by Clonorchis sinensis, is endemic in East Asian countries. C. sinensis metacercariae excyst in the duodenum of mammalian hosts, migrate to the intrahepatic bile duct, and mature into adults in the milieu of bile. We have previously shown that newly excysted juvenile C. sinensis move chemotactically toward bile and bile acids. Here, the chemotactic behavior of adult C. sinensis (CsAd) toward bile and bile acids was investigated. CsAds moved toward 0.05-5% bile and were most attracted to 0.5% bile but moved away from 10% bile. Upon exposure to 1-10% bile, CsAds eventually stopped moving and then died quickly. Among bile acids, CsAds showed strong chemotaxis toward cholic acid (CA) and deoxycholic acid. On the contrary, CsAds repelled from lithocholic acid (LCA). Moreover, at higher than 10 mM LCA, CsAds became sluggish and eventually died. Dopamine D1 receptor antagonists (LE-300 and SKF-83566), D2/3 receptor antagonists (raclopride and its derivative CS-49612), and a dopamine re-uptake inhibitor inhibited CA-induced chemotaxis of CsAds almost completely. Clinically used antipsychotic drugs, namely chlorpromazine, haloperidol, and clozapine, are dopaminergic antagonists and are secreted into bile. They completely inhibited chemotaxis of CsAds toward CA. At the maximum doses used to treat patients, the three tested medicines only expelled 2-12% of CsAds from the experimentally infected rabbits, but reduced egg production by 64-79%. Thus, antipsychotic medicines with dopaminergic antagonism could be considered as new anthelmintic candidates for human C. sinensis infections.


Assuntos
Anti-Helmínticos/farmacologia , Antipsicóticos/farmacologia , Quimiotaxia/efeitos dos fármacos , Clonorchis sinensis/efeitos dos fármacos , Clonorchis sinensis/fisiologia , Antagonistas de Dopamina/farmacologia , Animais , Anti-Helmínticos/administração & dosagem , Antipsicóticos/administração & dosagem , Bile/metabolismo , Fatores Quimiotáticos/metabolismo , Ácido Cólico/metabolismo , Clonorquíase/tratamento farmacológico , Modelos Animais de Doenças , Antagonistas de Dopamina/administração & dosagem , Feminino , Fertilidade/efeitos dos fármacos , Humanos , Ácido Litocólico/metabolismo , Coelhos , Análise de Sobrevida , Resultado do Tratamento
11.
Am J Physiol Cell Physiol ; 319(1): C75-C92, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32348173

RESUMO

Cell migration is central to development, wound healing, tissue regeneration, and immunity. Despite extensive knowledge of muscle regeneration, myoblast migration during regeneration is not well understood. C2C12 mouse myoblast migration and morphology were investigated using a triple-docking polydimethylsiloxane-based microfluidic device in which cells moved under gravity-driven laminar flow on uniform (=) collagen (CN=), fibronectin (FN=), or opposing gradients (CN-FN or FN-CN). In haptotaxis experiments, migration was faster on FN= than on CN=. At 10 h, cells were more elongated on FN-CN and migration was faster than on the CN-FN substrate. Net migration distance on FN-CN at 10 h was greater than on CN-FN, as cells rapidly entered the channel as a larger population (bulk-cell movement, wave 1). Hepatocyte growth factor (HGF) stimulated rapid chemotaxis on FN= but not CN=, increasing migration speed at 10 h early in the channel at low HGF in a steep HGF gradient. HGF accelerated migration on FN= and bulk-cell movement on both uniform substrates. An HGF gradient also slowed cells in wave 2 moving on FN-CN, not CN-FN. Both opposing-gradient substrates affected the shape, speed, and net distance of migrating cells. Gradient and uniform configurations of HGF and substrate differentially influenced migration behavior. Therefore, haptotaxis substrate configuration potently modifies myoblast chemotaxis by HGF. Innovative microfluidic experiments advance our understanding of intricate complexities of myoblast migration. Findings can be leveraged to engineer muscle-tissue volumes for transplantation after serious injury. New analytical approaches may generate broader insights into cell migration.


Assuntos
Quimiotaxia/fisiologia , Colágeno/metabolismo , Fibronectinas/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Microfluídica/métodos , Mioblastos/fisiologia , Animais , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Quimiotaxia/efeitos dos fármacos , Colágeno/administração & dosagem , Fibronectinas/administração & dosagem , Fator de Crescimento de Hepatócito/administração & dosagem , Humanos , Camundongos , Mioblastos/efeitos dos fármacos
12.
Can J Microbiol ; 66(10): 562-575, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32348684

RESUMO

Root colonization by plant-growth-promoting bacteria could not be useful without the beneficial properties of the bacterium itself. Thus, it is necessary to evaluate the bacterial capacity to form biofilms and establish a successful interaction with the plant roots. We assessed the ability of growth-promoting bacterial strains to form biofilm and display chemotactic behaviour in response to organic acids and (or) root exudates of the model plant Brachypodium distachyon. This assessment was based on the evaluation of single strains of bacteria and a multispecies consortium. The strains coexisted together and formed biofilm under biotic (living root) and abiotic (glass) surfaces. Citric acid stimulated biofilm formation in all individual strains, indicating a strong chemotactic behaviour towards organic acids. Recognizing that the transition from single strains of bacteria to a "multicellular" system would not happen without the presence of adhesion, the alginate and exopolysaccharide (EPS) contents were evaluated. The EPS amounts were comparable in single strains and consortium forms. Alginate production increased 160% in the consortium subjected to drought stress (10% PEG). These findings demonstrated that (i) bacteria-bacteria interaction is the hub of various factors that would not only affect their relation but also could indirectly affect the balanced plant-microbe relation and (ii) root exudates could be very selective in recruiting a highly qualified multispecies consortium.


Assuntos
Biofilmes/crescimento & desenvolvimento , Brachypodium/química , Brachypodium/microbiologia , Quimiotaxia/fisiologia , Endófitos/fisiologia , Ácidos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Biofilmes/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Endófitos/efeitos dos fármacos , Interações entre Hospedeiro e Microrganismos , Consórcios Microbianos/efeitos dos fármacos , Exsudatos de Plantas/farmacologia , Raízes de Plantas/química , Raízes de Plantas/microbiologia
13.
J Mol Biol ; 432(10): 3137-3148, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32247761

RESUMO

Bacterial motility is related to many cellular activities, such as cell migration, aggregation, and biofilm formations. The ability to control motility and direct the bacteria to certain location could be used to guide the bacteria in applications such as seeking for and killing pathogen, forming various population-level patterns, and delivering of drugs and vaccines. Currently, bacteria motility is mainly controlled by chemotaxis (prescribed chemical stimuli), which needs physical contact with the chemical inducer. This lacks the flexibility for pattern formation as it has limited spatial control. To overcome the limitations, we developed blue light-regulated synthetic genetic circuit to control bacterial directional motility, by taking the advantage that light stimulus can be delivered to cells in different patterns with precise spatial control. The circuit developed enables programmed Escherichia coli cells to increase directional motility and move away from the blue light, i.e., that negative phototaxis is utilized. This further allows the control of the cells to form aggregation with different patterns. Further, we showed that the circuit can be used to separate two different strains. The demonstrated ability of blue light-controllable gene circuits to regulate a CheZ expression could give researchers more means to control bacterial motility and pattern formation.


Assuntos
Escherichia coli/fisiologia , Luz/efeitos adversos , Proteínas Quimiotáticas Aceptoras de Metil/genética , Fenômenos Fisiológicos Bacterianos/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regiões Promotoras Genéticas , Biologia Sintética
14.
Zhongguo Zhong Yao Za Zhi ; 45(2): 361-366, 2020 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-32237319

RESUMO

To investigate the effects of butyl alcohol extract of Baitouweng Decoction(BAEB) on neutrophil chemotaxis in vaginal mucosa of mice with vulvovaginal candidiasis(VVC). Seventy-two SPF female Kunming mice were randomly divided into normal control group, model group, fluconazole group, BAEB low-dose group, middle-dose group and high-dose group. Subcutaneous injection of estradiol benzoate was conducted to induce pseudo-estrus, and then 2×10~6 CFU·mL~(-1)of Candida albicans was inoculated into vaginal lumen, followed by drug treatment for 7 days. Gram staining was used to observe the morphological changes of C. albicans in vagina; vaginal fungal load was detected on agar plate. Histological changes of vaginal tissues in mice were observed by HE staining. Lactate dehydrogenase(LDH), interleukin-6(IL-6) and tumor necrosis factor(TNF-α) levels in mouse lavage fluid were detected by enzyme-linked immunosorbent assay(ELISA). Neutrophils in vaginal lavage fluid was observed and counted by using Pap smear. The levels of IL-8 and MIP-2 in vaginal mucosa were detected by ELISA. IL-8 and MIP-2 mRNA levels in vaginal mucosa of mice were detected by qRT-PCR. The results showed that as compared with the normal group, VVC model group had a large number of hyphae and a high level of fungal loadinvagina. The vaginal mucosa was completely destroyed, the number of neutrophils increased, and the protein and mRNA levels of IL-8 and MIP-2 were up-regulated. After BAEB treatment, the hyphae of the treatment group was decreased, the fungal load was decreased, the impaired mucosa showed different degrees of improvement, the inflammatory factors were decreased to varying degrees, and the protein and mRNA levels of chemokine IL-8 and MIP-2 were down-regulated. In conclusion, BAEB may be used to treat VVC by inhibiting vulvovaginal candidiasis via blocking neutrophils recruitment into vagina.


Assuntos
Candidíase Vulvovaginal/tratamento farmacológico , Quimiotaxia/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Neutrófilos/efeitos dos fármacos , 1-Butanol , Animais , Candida albicans , Feminino , Camundongos , Membrana Mucosa/citologia , Membrana Mucosa/efeitos dos fármacos , Neutrófilos/citologia , Vagina/citologia , Vagina/diagnóstico por imagem
15.
Curr Med Sci ; 40(1): 130-137, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32166675

RESUMO

Sinomenine (SN) has been used in the clinical treatment of systemic lupus erythematosus and rheumatoid arthritis for many years. Studies showed that SN held protective effects such as anti-inflammation, scavenging free radicals and suppressing immune response in many autoimmune diseases. The purpose of the present study is to explore the mechanism of anti-inflammation of SN on lipopolysaccharide (LPS)-induced macrophages activation and investigate whether the TLR4/NF-κB signaling pathway participated in. Macrophages isolated from mouse peritoneal cavity were stimulated by 1 µg/mL LPS for 24 h. And then the cells were treated with various concentrations of SN, TLR4 inhibitor respectively for additional 48 h. Drug toxicity was detected by MTT assay and Transwell experiment was used to assess chemotaxis. Furthermore, TLR4 and MyD88 mRNA levels were detected by real-time PCR. Western blotting was used to examine TLR4, MyD88 and phosphorylated IκB protein expression in macrophages. Immunofluorescence assay was applied to observe p65 NF-κB protein expression in macrophage nucleus. We extracted macrophages with high purity and activity from the abdominal cavity of mice. SN remarkably inhibited the chemotaxis and secretion function of LPS-stimulated macrophages. It also down-regulated both the protein levels of inflammatory cytokines (TNF-α, IL-1ß and IL-6) and the RNA and protein levels of the key factors (TLR4, MyD88, P-IκB) in TLR4 pathway. The expression of p65 NF-κB protein in nuclei was down-regulated, which was correlated with a similar decrease in P-IκB protein level. In conclusion, SN can inhibit the LPS induced immune responses in macrophages by blocking the activated TLR4/NF-κB signaling pathway. These results may provide a therapeutic approach to regulate inflammatory responses.


Assuntos
Anti-Inflamatórios/farmacologia , Lipopolissacarídeos/efeitos adversos , Macrófagos/citologia , Morfinanos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Camundongos , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo
16.
J Vis Exp ; (156)2020 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-32116294

RESUMO

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically been studied in vivo in animal models showing characteristic patterns of cell migration. However, in vivo models have several limitations, including intercellular mediators that are difficult to access and analyze, as well as the inability to directly analyze human neutrophils. Because of these limitations, there is a need for an in vitro platform that studies swarming with human neutrophils and provides easy access to the molecular signals generated during swarming. Here, a multistep microstamping process is used to generate a bioparticle microarray that stimulates swarming by mimicking an in vivo infection. The bioparticle microarray induces neutrophils to swarm in a controlled and stable manner. On the microarray, neutrophils increase in speed and form stable swarms around bioparticle clusters. Additionally, supernatant generated by the neutrophils was analyzed and 16 proteins were discovered to have been differentially expressed over the course of swarming. This in vitro swarming platform facilitates direct analysis of neutrophil migration and protein release in a reproducible, spatially controlled manner.


Assuntos
Quimiotaxia , Análise em Microsséries , Neutrófilos/citologia , Animais , Quimiotaxia/efeitos dos fármacos , Humanos , Processamento de Imagem Assistida por Computador , Microtecnologia , Neutrófilos/efeitos dos fármacos , Zimosan/farmacologia
17.
Molecules ; 25(4)2020 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-32085423

RESUMO

Several anti-inflammatory agents based on pyrazole and imidazopyrazole scaffolds and a large library of substituted catechol PDE4D inhibitors were reported by us in the recent past. To obtain new molecules potentially able to act on different targets involved in inflammation onset we designed and synthesized a series of hybrid compounds by linking pyrazole and imidazo-pyrazole scaffolds to differently decorated catechol moieties through an acylhydrazone chain. Some compounds showed antioxidant activity, inhibiting reactive oxygen species (ROS) elevation in neutrophils, and a good inhibition of phosphodiesterases type 4D and, particularly, type 4B, the isoform most involved in inflammation. In addition, most compounds inhibited ROS production also in platelets, confirming their ability to exert an antiinflammatory response by two independent mechanism. Structure-activity relationship (SAR) analyses evidenced that both heterocyclic scaffolds (pyrazole and imidazopyrazole) and the substituted catechol moiety were determinant for the pharmacodynamic properties, even if hybrid molecules bearing to the pyrazole series were more active than the imidazopyrazole ones. In addition, the pivotal role of the catechol substituents has been analyzed. In conclusion the hybridization approach gave a new serie of multitarget antiinflammatory compounds, characterized by a strong antioxidant activity in different biological targets.


Assuntos
Anti-Inflamatórios/farmacologia , Pirazóis/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/química , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/farmacologia , Humanos , Masculino , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Oxirredução , Inibidores da Fosfodiesterase 4/síntese química , Inibidores da Fosfodiesterase 4/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Pirazóis/síntese química , Pirazóis/química , Relação Estrutura-Atividade
18.
J Neurosci ; 40(17): 3320-3331, 2020 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-32060170

RESUMO

Microglial cells are considered as sensors of brain pathology by detecting any sign of brain lesions, infections, or dysfunction and can influence the onset and progression of neurological diseases. They are capable of sensing their neuronal environment via many different signaling molecules, such as neurotransmitters, neurohormones and neuropeptides. The neuropeptide VGF has been associated with many metabolic and neurological disorders. TLQP21 is a VGF-derived peptide and has been shown to signal via C3aR1 and C1qBP receptors. The effect of TLQP21 on microglial functions in health or disease is not known. Studying microglial cells in acute brain slices, we found that TLQP21 impaired metabotropic purinergic signaling. Specifically, it attenuated the ATP-induced activation of a K+ conductance, the UDP-stimulated phagocytic activity, and the ATP-dependent laser lesion-induced process outgrowth. These impairments were reversed by blocking C1qBP, but not C3aR1 receptors. While microglia in brain slices from male mice lack C3aR1 receptors, both receptors are expressed in primary cultured microglia. In addition to the negative impact on purinergic signaling, we found stimulating effects of TLQP21 in cultured microglia, which were mediated by C3aR1 receptors: it directly evoked membrane currents, stimulated basal phagocytic activity, evoked intracellular Ca2+ transient elevations, and served as a chemotactic signal. We conclude that TLQP21 has differential effects on microglia depending on C3aR1 activation or C1qBP-dependent attenuation of purinergic signaling. Thus, TLQP21 can modulate the functional phenotype of microglia, which may have an impact on their function in health and disease.SIGNIFICANCE STATEMENT The neuropeptide VGF and its peptides have been associated with many metabolic and neurological disorders. TLQP21 is a VGF-derived peptide that activates C1qBP receptors, which are expressed by microglia. We show here, for the first time, that TLQP21 impairs P2Y-mediated purinergic signaling and related functions. These include modulation of phagocytic activity and responses to injury. As purinergic signaling is central for microglial actions in the brain, this TLQP21-mediated mechanism might regulate microglial activity in health and disease. We furthermore show that, in addition to C1qBP, functional C3aR1 responses contribute to TLQP21 action on microglia. However, C3aR1 responses were only present in primary cultures but not in situ, suggesting that the expression of these receptors might vary between different microglial activation states.


Assuntos
Quimiotaxia/efeitos dos fármacos , Microglia/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Fagocitose/efeitos dos fármacos , Receptores Purinérgicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Cálcio/metabolismo , Células Cultivadas , Quimiotaxia/fisiologia , Feminino , Masculino , Camundongos , Microglia/metabolismo , Fagocitose/fisiologia , Transdução de Sinais/fisiologia
19.
Biochem Biophys Res Commun ; 524(4): 916-922, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32057367

RESUMO

Macrophage-mediated inflammation is a key pathophysiological component of cardiovascular diseases, but the underlying mechanisms by which the macrophage regulates inflammation have been unclear. In our study, we, for the first time, showed an endogenous sulfur dioxide (SO2) production in RAW267.4 macrophages by using HPLC and SO2-specific fluorescent probe assays. Moreover, the endogenous SO2 generating enzyme aspartate aminotransferase (AAT) was found to be expressed by the macrophages. Furthermore, we showed that AAT2 knockdown triggered spontaneous macrophage-mediated inflammation, as represented by the increased TNF-α and IL-6 levels and the enhanced macrophage chemotaxis; these effects could be reversed by the treatment with a SO2 donor. Mechanistically, AAT2 knockdown activated the NF-κB signaling pathway in macrophages, while SO2 successfully rescued NF-κB activation. In contrast, forced AAT2 expression reversed AngII-induced NF-κB activation and subsequent macrophage inflammation. Moreover, treatment with a SO2 donor also alleviated macrophage infiltration in AngII-treated mouse hearts. Collectively, our data suggest that macrophage-derived SO2 is an important regulator of macrophage activation and it acts as an endogenous "on-off switch" in the control of macrophage activation. This knowledge might enable a new therapeutic strategy for cardiovascular diseases.


Assuntos
Aspartato Aminotransferases/genética , Miócitos Cardíacos/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , NF-kappa B/genética , Dióxido de Enxofre/farmacologia , Angiotensina II/farmacologia , Animais , Aspartato Aminotransferases/antagonistas & inibidores , Aspartato Aminotransferases/imunologia , Linhagem Celular , Quimiotaxia/efeitos dos fármacos , Regulação da Expressão Gênica , Inflamação , Interleucina-6/genética , Interleucina-6/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/citologia , Miócitos Cardíacos/imunologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/imunologia , NF-kappa B/imunologia , Células RAW 264.7 , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Transdução de Sinais , Sulfitos/química , Sulfitos/farmacologia , Dióxido de Enxofre/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
20.
Molecules ; 25(3)2020 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-32050419

RESUMO

Root-knot nematode diseases cause severe yield and economic losses each year in global agricultural production. Virgibacillus dokdonensis MCCC 1A00493, a deep-sea bacterium, shows a significant nematicidal activity against Meloidogyne incognita in vitro. However, information about the active substances of V. dokdonensis MCCC 1A00493 is limited. In this study, volatile organic compounds (VOCs) from V. dokdonensis MCCC 1A00493 were isolated and analyzed through solid-phase microextraction and gas chromatography-mass spectrometry. Four VOCs, namely, acetaldehyde, dimethyl disulfide, ethylbenzene, and 2-butanone, were identified, and their nematicidal activities were evaluated. The four VOCs had a variety of active modes on M. incognita juveniles. Acetaldehyde had direct contact killing, fumigation, and attraction activities; dimethyl disulfide had direct contact killing and attraction activities; ethylbenzene had an attraction activity; and 2-butanone had a repellent activity. Only acetaldehyde had a fumigant activity to inhibit egg hatching. Combining this fumigant activity against eggs and juveniles could be an effective strategy to control the different developmental stages of M. incognita. The combination of direct contact and attraction activities could also establish trapping and killing strategies against root-knot nematodes. Considering all nematicidal modes or strategies, we could use V. dokdonensis MCCC 1A00493 to set up an integrated strategy to control root-knot nematodes.


Assuntos
Antinematódeos/isolamento & purificação , Doenças das Plantas/prevenção & controle , Tylenchoidea/efeitos dos fármacos , Virgibacillus/química , Compostos Orgânicos Voláteis/isolamento & purificação , Acetaldeído/isolamento & purificação , Acetaldeído/farmacologia , Animais , Antinematódeos/farmacologia , Organismos Aquáticos , Derivados de Benzeno/isolamento & purificação , Derivados de Benzeno/farmacologia , Butanonas/isolamento & purificação , Butanonas/farmacologia , Quimiotaxia/efeitos dos fármacos , Dissulfetos/isolamento & purificação , Dissulfetos/farmacologia , Cromatografia Gasosa-Espectrometria de Massas , Lycopersicon esculentum/efeitos dos fármacos , Lycopersicon esculentum/parasitologia , Contagem de Ovos de Parasitas , Doenças das Plantas/parasitologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/parasitologia , Microextração em Fase Sólida , Tylenchoidea/crescimento & desenvolvimento , Compostos Orgânicos Voláteis/farmacologia
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