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1.
Life Sci ; 258: 118136, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32726662

RESUMO

The endothelium is a critical regulator of vascular homeostasis, controlling vascular tone and permeability as well as interactions of leukocytes and platelets with blood vessel walls. Consequently, endothelial dysfunction featuring inflammation and reduced vasodilation are considered central to cardiovascular disease (CVD) pathogenesis and have become a therapeutic area of focus. Type II endothelial cell (EC) activation by stress-related stimuli such as tumor necrosis factor-α (TNF-α) initiates the nuclear factor-κB (NF-κB) signaling pathway, a master regulator of inflammatory responses. Because dysregulated NF-κB signaling has been tightly linked to several CVDs, EC-specific inhibition of NF-κB represents an attractive pharmacological strategy. As accumulating evidence highlights the clinical benefits of tea catechin for multiple diseases including CVDs, we sought to determine whether the tea catechin epigallocatechin gallate (EGCG) that displays antioxidative, anti-inflammatory, hypolipidemic, anti-thrombogenic, and anti-hypertensive properties offers protection against CVDs by suppressing the canonical NF-κB pathway. Our findings indicate that EGCG downregulates multiple components of the TNF-α-induced NF-κB signaling pathway and thereby reduces the consequent increase in inflammatory gene transcription and protein expression. Furthermore, EGCG blocked type II EC activation, evidenced by diminished EC leakage and monocyte adhesion in EGCG-treated cells. In summary, our study advances knowledge of EGCG's anti-inflammatory effects on the NF-κB pathway and hence its benefits on endothelial health, supporting its therapeutic potential for CVDs.


Assuntos
Catequina/análogos & derivados , Vasos Coronários/patologia , Células Endoteliais/patologia , Inflamação/tratamento farmacológico , Catequina/farmacologia , Catequina/uso terapêutico , Adesão Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Inflamação/genética , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/patologia , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
2.
Int J Nanomedicine ; 15: 4573-4589, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32606693

RESUMO

Background: Therapeutic efficiency of ceragenins against cancers may be limited by lack of their hemocompatibility when high concentrations of molecules are required to reach a desired result. Synergistic effects observed upon administration of anticancer agents and metal nanoparticles may provide an opportunity to limit toxicity of immobilized ceragenins on the surface of metal nanoparticles and to improve their therapeutic efficiency at the same time. The aim of present work is to investigate the anticancer activities and hemocompatibility of nanoformulations consisting of ceragenin CSA-131 united with aminosilane-modified iron oxide-based magnetic nanoparticles (MNP) and prepared by 1) covalent bonding (MNP@CSA-131) or 2) by combining CSA-131 with MNP in 1:1 ratio (CSA-131 + MNP). Possible synergistic interactions between CSA-131 and magnetic nanoparticles were also quantified. Methods: MNP@CSA-131 and CSA-131+MNP were tested in vitro against selected lung and colon cancer cells using colorimetric, fluorimetric and flow cytometry methods. Results: Performed analysis demonstrates that MNP-based nanosystems significantly improve the killing efficiency of tested ceragenin, decreasing the viability of extra 1.37±4.72% to 76.07±15.30% cancer cells when compared to free CSA-131. Quantification of synergistic effects indicates the favorable interactions between CSA-131 and magnetic nanoparticles (CI < 1 for all tested doses), revealing at the same time a reduction in effective doses of ceragenin from 1.17 ± 0.61 to 34.57 ± 12.78 times when combined with MNP. We demonstrate that both MNP@CSA-131 and CSA-131+MNP induce significantly apoptosis of cancer cells and prevent the division of colon cancer cells even at relatively low doses of the active compound (10 µg/mL). Importantly, combining CSA-131 with MNP decreases the hemolytic activity of free ceragenin 4.72 to 7.88 times, which indicates a considerable improvement of hemotoxicity profile. Conclusion: Comparative analyses have revealed that both developed CSA-containing nanoformulations due to the utility of synergistic interactions between MNP and CSA-131, which are effective against lung and colon cancer cells. This indicates the new directions in preparation of MNP-based therapeutics, which are relatively easy to synthetize, cost-effective and safe when intravenously administrated.


Assuntos
Compostos Férricos/uso terapêutico , Nanopartículas de Magnetita/uso terapêutico , Esteroides/farmacologia , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/patologia , Sinergismo Farmacológico , Humanos , Neoplasias Pulmonares/patologia , Nanopartículas de Magnetita/administração & dosagem , Nanopartículas de Magnetita/ultraestrutura , Teste de Materiais
3.
Nat Commun ; 11(1): 3184, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576824

RESUMO

Peptide antibiotics are an abundant and synthetically tractable source of molecular diversity, but they are often cationic and can be cytotoxic, nephrotoxic and/or ototoxic, which has limited their clinical development. Here we report structure-guided optimization of an amphipathic peptide, arenicin-3, originally isolated from the marine lugworm Arenicola marina. The peptide induces bacterial membrane permeability and ATP release, with serial passaging resulting in a mutation in mlaC, a phospholipid transport gene. Structure-based design led to AA139, an antibiotic with broad-spectrum in vitro activity against multidrug-resistant and extensively drug-resistant bacteria, including ESBL, carbapenem- and colistin-resistant clinical isolates. The antibiotic induces a 3-4 log reduction in bacterial burden in mouse models of peritonitis, pneumonia and urinary tract infection. Cytotoxicity and haemolysis of the progenitor peptide is ameliorated with AA139, and the 'no observable adverse effect level' (NOAEL) dose in mice is ~10-fold greater than the dose generally required for efficacy in the infection models.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Animais , Carbapenêmicos/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Colistina/farmacologia , Modelos Animais de Doenças , Descoberta de Drogas , Feminino , Proteínas de Helminto/química , Proteínas de Helminto/farmacologia , Humanos , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Peritonite/tratamento farmacológico , Peritonite/microbiologia , Pneumonia/tratamento farmacológico , Pneumonia/microbiologia , Infecções Urinárias/tratamento farmacológico , Infecções Urinárias/microbiologia
4.
Life Sci ; 256: 117884, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32502546

RESUMO

AIMS: Endothelial barrier dysfunction is associated with multiple diseases, and barrier repair may be a possible therapeutic target. Yes-associated protein and its pathway have been implicated in organ repair after injury. However, the mechanisms underlying barrier repair and any role YAP plays in the process are unclear. This study aimed to explore the role and mechanism of YAP in the repair of endothelial cell permeability after TNF-α-induced injury. MAIN METHODS: A trans-endothelial electrical resistance assay was performed to investigate changes in endothelial cell permeability. Lentivirus packaging by calcium phosphate transfection was used to construct endothelial cell lines with knocked down or overexpressed YAP. Western blotting, immunofluorescence, CO-IP, and real-time PCR were used to detect related protein and gene expression. KEY FINDINGS: YAP is involved in the repair process of TNF-α-induced endothelial cell permeability injury; its overexpression promotes repair of endothelial cell permeability, and knockdown weakens repair ability. Moreover, YAP may promote repair by down-regulating STAT3 activity, thereby inhibiting VEGF expression. SIGNIFICANCE: Elucidating the role of YAP in endothelial cell permeability repair process after injury might reveal mechanisms of endothelial barrier repair and provide therapeutic targets for treatment of vascular hyper-permeability disease.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia
5.
Nat Commun ; 11(1): 2992, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32532974

RESUMO

Activated protein C (APC) is a plasma serine protease with antithrombotic and cytoprotective functions. Based on the hypothesis that specific inhibition of APC's anticoagulant but not its cytoprotective activity can be beneficial for hemophilia therapy, 2 types of inhibitory monoclonal antibodies (mAbs) are tested: A type I active-site binding mAb and a type II mAb binding to an exosite on APC (required for anticoagulant activity) as shown by X-ray crystallography. Both mAbs increase thrombin generation and promote plasma clotting. Type I blocks all APC activities, whereas type II preserves APC's cytoprotective function. In normal monkeys, type I causes many adverse effects including animal death. In contrast, type II is well-tolerated in normal monkeys and shows both acute and prophylactic dose-dependent efficacy in hemophilic monkeys. Our data show that the type II mAb can specifically inhibit APC's anticoagulant function without compromising its cytoprotective function and offers superior therapeutic opportunities for hemophilia.


Assuntos
Anticorpos Monoclonais/farmacologia , Hemofilia A/prevenção & controle , Fragmentos Fab das Imunoglobulinas/imunologia , Inibidor da Proteína C/farmacologia , Proteína C/antagonistas & inibidores , Animais , Anticorpos Monoclonais/classificação , Anticorpos Monoclonais/imunologia , Tempo de Sangramento , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Cristalografia por Raios X , Hemofilia A/sangue , Hemorragia/prevenção & controle , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Macaca fascicularis , Masculino , Proteína C/química , Proteína C/imunologia , Proteína C/metabolismo , Inibidor da Proteína C/sangue , Inibidor da Proteína C/farmacocinética
6.
Toxicology ; 440: 152491, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32413421

RESUMO

Multi-walled carbon nanotubes (MWCNTs) have promising applications in neurology depending on their unique physicochemical properties. However, there is limited understanding of their impacts on brain microvascular endothelial cells, the cells lining the vessels and maintaining the low and selective permeability of the blood-brain barrier. In this study, we examined the influence of pristine MWCNT (p-MWCNT) and carboxylated MWCNT (c-MWCNT) on permeability and tight junction tightness of murine brain microvascular endothelial cells, and investigated the potential mechanisms in the sight of hemichannel activity. Treatment with p-MWCNT for 24 h at subtoxic concentration (20 µg/mL) decreased the protein expression of occludin, disrupted zonula occludens-1 continuity, and elevated monolayer permeability as quantified by transendothelial electrical resistance and paracellular flux of 4000 Da fluorescein isothiocyanate-dextran conjugates. Moreover, p-MWCNT exposure also increased hemichannel activity with upregulated protein expression and altered subcellular localization of connexin (Cx)43 and pannexin (Panx)1. p-MWCNT-induced elevation in endothelial permeability could be prevented by hemichannel inhibitor carbenoxolone and peptide blocker of Cx43 and Panx1, indicating the crucial role of activated Cx43 and Panx1 hemichannels. Furthermore, Cx43 and Panx1 hemichannel-mediated ATP release might be involved in p-MWCNT-induced rise in endothelial permeability. In contrast, the above effects caused by p-MWCNT were not observed in cells treated with c-MWCNT, the functionalized form with more stable dispersion and a lower tendency to aggregate. Our study contributes further understanding of the impact of MWCNTs on brain endothelial tightness and permeability, which may have important implications for the safety application of MWCNTs in nanomedicine.


Assuntos
Células Endoteliais/efeitos dos fármacos , Nanotubos de Carbono , Trifosfato de Adenosina/metabolismo , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Capilares/citologia , Capilares/efeitos dos fármacos , Capilares/metabolismo , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sobrevivência Celular , Conexina 43/antagonistas & inibidores , Conexinas/antagonistas & inibidores , Impedância Elétrica , Camundongos , Proteínas do Tecido Nervoso/antagonistas & inibidores , Ocludina/biossíntese , Junções Íntimas/efeitos dos fármacos
7.
Am J Physiol Cell Physiol ; 319(1): C129-C135, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32374677

RESUMO

The purpose of this study was to evaluate a new pharmacological strategy using a first-generation succinate prodrug, NV118, in peripheral blood mononuclear cells (PBMCs) obtained from subjects with carbon monoxide (CO) poisoning and healthy controls. We obtained human blood cells from subjects with CO poisoning and healthy control subjects. Intact PBMCs from subjects in the CO and Control group were analyzed with high-resolution respirometry measured in pmol O2 per second per 10-6 PBMCs. In addition to obtaining baseline respiration, NV118 (100 µM) was injected, and the same parameters of respiration were obtained for comparison in PBMCs. We measured mitochondrial dynamics with microscopy with the same conditions. We enrolled 37 patients (17 in the CO group and 20 in the Control group for comparison) in the study. PMBCs obtained from subjects in the CO group had overall significantly lower respiration compared with the Control group (P < 0.0001). There was a significant increase in respiration with NV118, specifically with an increase in maximum respiration and respiration from complex II and complex IV (P < 0.0001). The mitochondria in PBMCs demonstrated an overall increase in net movement compared with the Control group. Our results of this study suggest that the therapeutic compound, NV118, increases respiration at complex II and IV as well as restoration of mitochondrial movement in PBMCs obtained from subjects with CO poisoning. Mitochondrial-directed therapy offers a potential future strategy with further exploration in vivo.


Assuntos
Intoxicação por Monóxido de Carbono/metabolismo , Permeabilidade da Membrana Celular/fisiologia , Leucócitos Mononucleares/metabolismo , Mitocôndrias/metabolismo , Pró-Fármacos/metabolismo , Ácido Succínico/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Respiração Celular/efeitos dos fármacos , Respiração Celular/fisiologia , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Pró-Fármacos/administração & dosagem , Ácido Succínico/administração & dosagem
8.
Nat Commun ; 11(1): 1995, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32332732

RESUMO

Gut microbial dysbiosis is associated with the development of autoimmune disease, but the mechanisms by which microbial dysbiosis affects the transition from asymptomatic autoimmunity to inflammatory disease are incompletely characterized. Here, we identify intestinal barrier integrity as an important checkpoint in translating autoimmunity to inflammation. Zonulin family peptide (zonulin), a potent regulator for intestinal tight junctions, is highly expressed in autoimmune mice and humans and can be used to predict transition from autoimmunity to inflammatory arthritis. Increased serum zonulin levels are accompanied by a leaky intestinal barrier, dysbiosis and inflammation. Restoration of the intestinal barrier in the pre-phase of arthritis using butyrate or a cannabinoid type 1 receptor agonist inhibits the development of arthritis. Moreover, treatment with the zonulin antagonist larazotide acetate, which specifically increases intestinal barrier integrity, effectively reduces arthritis onset. These data identify a preventive approach for the onset of autoimmune disease by specifically targeting impaired intestinal barrier function.


Assuntos
Artrite Reumatoide/prevenção & controle , Permeabilidade da Membrana Celular/efeitos dos fármacos , Disbiose/complicações , Haptoglobinas/antagonistas & inibidores , Mucosa Intestinal/efeitos dos fármacos , Oligopeptídeos/administração & dosagem , Precursores de Proteínas/antagonistas & inibidores , Adulto , Animais , Artrite Experimental/sangue , Artrite Experimental/imunologia , Artrite Experimental/microbiologia , Artrite Experimental/prevenção & controle , Artrite Reumatoide/sangue , Artrite Reumatoide/imunologia , Artrite Reumatoide/microbiologia , Translocação Bacteriana/efeitos dos fármacos , Translocação Bacteriana/imunologia , Células CACO-2 , Permeabilidade da Membrana Celular/imunologia , Estudos de Coortes , Estudos Transversais , Disbiose/imunologia , Disbiose/microbiologia , Feminino , Microbioma Gastrointestinal/imunologia , Haptoglobinas/metabolismo , Voluntários Saudáveis , Humanos , Íleo/citologia , Íleo/efeitos dos fármacos , Íleo/microbiologia , Íleo/patologia , Mucosa Intestinal/citologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Precursores de Proteínas/sangue , Precursores de Proteínas/metabolismo , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo
9.
Biochim Biophys Acta Biomembr ; 1862(8): 183302, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32311341

RESUMO

All antibiotics have to engage bacterial amphiphilic barriers such as the lipopolysaccharide-rich outer membrane or the phospholipid-based inner membrane in some manner, either by disrupting them outright and/or permeating them and thereby allow the antibiotic to get into bacteria. There is a growing class of cyclic antibiotics, many of which are of bacterial origin, that exhibit activity against Gram-negative bacteria, which constitute an urgent problem in human health. We examine a diverse collection of these cyclic antibiotics, both natural and synthetic, which include bactenecin, polymyxin B, octapeptin, capreomycin, and Kirshenbaum peptoids, in order to identify what they have in common when they interact with bacterial lipid membranes. We find that they virtually all have the ability to induce negative Gaussian curvature (NGC) in bacterial membranes, the type of curvature geometrically required for permeation mechanisms such as pore formation, blebbing, and budding. This is interesting since permeation of membranes is a function usually ascribed to antimicrobial peptides (AMPs) from innate immunity. As prototypical test cases of cyclic antibiotics, we analyzed amino acid sequences of bactenecin, polymyxin B, and capreomycin using our recently developed machine-learning classifier trained on α-helical AMP sequences. Although the original classifier was not trained on cyclic antibiotics, a modified classifier approach correctly predicted that bactenecin and polymyxin B have the ability to induce NGC in membranes, while capreomycin does not. Moreover, the classifier was able to recapitulate empirical structure-activity relationships from alanine scans in polymyxin B surprisingly well. These results suggest that there exists some common ground in the sequence design of hybrid cyclic antibiotics and linear AMPs.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Permeabilidade da Membrana Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Membrana Celular/química , Bactérias Gram-Negativas/patogenicidade , Humanos , Aprendizado de Máquina , Testes de Sensibilidade Microbiana , Fosfolipídeos/química , Relação Estrutura-Atividade
10.
J Med Chem ; 63(8): 3996-4004, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32227886

RESUMO

Constitutive proteolytic activity of MALT1 is associated with highly aggressive B-cell lymphomas. Chemical tools that detect active MALT1 have been reported, but suffer from poor cell permeability and/or cross-reactivity with the cysteine protease cathepsin B. Here, we report that the non-natural amino acid pipecolinic acid in the P2 position of substrates and chemical probes leads to improved selectivity toward MALT1 and results in cell-permeable fluorescent probes.


Assuntos
Aminoácidos/síntese química , Aminoácidos/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , Aminoácidos/farmacologia , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/fisiologia , Desenho de Fármacos , Corantes Fluorescentes/farmacologia , Humanos , Células Jurkat , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
11.
Ecotoxicol Environ Saf ; 196: 110519, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32244116

RESUMO

On two rat cell lines, pheochromocytoma PC12 and ascites hepatoma AS-30D, and on rat liver mitochondria we studied action of paxilline (lipophilic mycotoxin from fungus Penicillium paxilli which is blocker of large-conductance potassium channels) against harmful effects of Cd(II) - one of the most dangerous toxic metals and environmental pollutants. We investigated an influence of paxilline on cell viability and mitochondrial function in the presence and in the absence of Cd2+. As found, paxilline protected partially from the Cd2+-induced cytotoxicity, namely taken in concentration of 1 µM it decreased the Cd2+-induced cell necrosis in average by 10-14 or 13-23% for AS-30D and PC12 cells, respectively. Nevertheless, paxilline did not affect the Cd2+-induced apoptosis of AS-30D cells. The alleviating concentration of paxilline reduced an intracellular production of reactive oxygen species (ROS) in PC12 cells intoxicated by Cd2+ and enhanced the ROS production in control AS-30D cells; however, it weakly affected mitochondrial membrane potential of the cells in the absence and in the presence of Cd2+. The ameliorative concentration of paxilline decreased the maximal respiration rates of control cells of both types after short-term (3-5 h) treatment with it while the rates reached their control levels after long-term (24-48 h) incubation with the drug. Paxilline was not protective against the Cd2+-induced membrane permeability and respiration rate changes in isolated rat liver mitochondria. As result, the mitochondrial electron transport chain was concluded to contribute in the mitigating effect of paxilline against the Cd2+-produced cell injury.


Assuntos
Apoptose/efeitos dos fármacos , Cádmio/toxicidade , Poluentes Ambientais/toxicidade , Indóis/farmacologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Animais , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Necrose , Células PC12 , Ratos , Espécies Reativas de Oxigênio/metabolismo
12.
PLoS One ; 15(4): e0231173, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32251487

RESUMO

Acetaminophen is one of the most common over-the-counter pain medications used worldwide and is considered safe at therapeutic dose. However, intentional and unintentional overdose accounts for up to 70% of acute liver failure cases in the western world. Extensive research has demonstrated that the induction of oxidative stress and mitochondrial dysfunction are central to the development of acetaminophen-induced liver injury. Despite the insight gained on the mechanism of acetaminophen toxicity, there still is only one clinically approved pharmacological treatment option, N-acetylcysteine. N-acetylcysteine increases the cell's antioxidant defense and protects liver cells from further acetaminophen-induced oxidative damage. Because it primarily protects healthy liver cells rather than rescuing the already injured cells alternative treatment strategies that target the latter cell population are warranted. In this study, we investigated mitochondria as therapeutic target for the development of novel treatment strategies for acetaminophen-induced liver injury. Characterization of the mitochondrial toxicity due to acute acetaminophen overdose in vitro in human cells using detailed respirometric analysis revealed that complex I-linked (NADH-dependent) but not complex II-linked (succinate-dependent) mitochondrial respiration is inhibited by acetaminophen. Treatment with a novel cell-permeable succinate prodrug rescues acetaminophen-induced impaired mitochondrial respiration. This suggests cell-permeable succinate prodrugs as a potential alternative treatment strategy to counteract acetaminophen-induced liver injury.


Assuntos
Acetaminofen/efeitos adversos , Analgésicos não Entorpecentes/efeitos adversos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Respiração Celular/efeitos dos fármacos , Overdose de Drogas/tratamento farmacológico , Mitocôndrias/metabolismo , Pró-Fármacos/farmacocinética , Ácido Succínico/farmacocinética , Acetaminofen/administração & dosagem , Acetaminofen/farmacologia , Idoso , Analgésicos não Entorpecentes/administração & dosagem , Analgésicos não Entorpecentes/farmacologia , Plaquetas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Overdose de Drogas/metabolismo , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Masculino , Mitocôndrias/efeitos dos fármacos
13.
Am J Chin Med ; 48(3): 679-702, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32329644

RESUMO

Autophagy is a process of active programmed cell death, where a dying cell induces autophagosomes and subsequently regulated by degradative machinery. The aim of this study was to investigate the mechanism behind induction of autophagic cell death by Naringin flavonoid in AGS cancer cells. Growth inhibition of AGS cells showed downregulation of PI3K/Akt/mTOR signaling by Naringin treatment. Transmission electron microscopy observation showed swollen mitochondria and lysosome near peri-nuclear zone fused with autophagic vacuoles. Rapamycin pre-treatment with Naringin showed significant decrease in mTOR phosphorylation and increase in LC3B activation in AGS cells. Decrease in mTOR phosphorylation is associated with lysosomal function activation was observed by time-dependent treatment of Naringin. Induction of lysosomal membrane permeabilization (LMP) was observed by LAMP1 activation leading lysosomal cell death by releasing Cathepsin D from lysosomal lumen to cytosol. Naringin treated AGS cells showed up-regulating BH3 domain Bad, down-regulating Bcl-xL, and Bad phosphorylation and significant mitochondrial fluorescence intensity expression. Significant localization of mitochondria and LC3B activation was examined by person coefficient correlation. Activation of ERK1/2-p38 MAPKs and production of intracellular ROS has been observed over Naringin treatment. It has also been elucidated that pre-treatment with NAC inhibited mitochondria-LC3B colocalization, where ROS acted as upstream of ERK1/2-p38 MAPKs activation. Lysosomal cell death involvement has been evaluated by BAF A1 pre-treatment, inhibiting LAMP1, Cathepsin D, ROS, and blocking autophagolysosome in AGS cell death. Taken together, these findings show that, Naringin induced autophagy cell death involves LMP mediated lysosomal damage and BH3 protein Bad activation in AGS cancer cells.


Assuntos
Autofagia/efeitos dos fármacos , Flavanonas/farmacologia , Lisossomos/patologia , Neoplasias Gástricas/patologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Humanos , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Células Tumorais Cultivadas
14.
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue ; 32(1): 83-87, 2020 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-32148237

RESUMO

OBJECTIVE: To investigate the effect and mechanism of paeoniflorin on the permeability of cardiac microvascular endothelial cells (CMECs) in sepsis. METHODS: Primary rat CMECs were isolated and cultured in vitro, and the cells in the logarithmic growth phase were used for experiments. Tetramethylazozolium colorimetry (MTT) was used to screen the safe and effective concentrations of paeoniflorin at 10, 20, and 40 µmol/L. The cells were divided into blank control group, lipopolysaccharide (LPS) group and low, medium and high concentration paeoniflorin pretreatment group. The cells in the blank control group were cultured in complete medium; the cells in the LPS group were challenged with LPS (1 mg/L) in complete medium; and the cells in the paeoniflorin pretreatment groups were pretreated with 10, 20, and 40 µmol/L paeoniflorin at 4 hours before LPS stimulation. The cells in each group were further cultured for 24 hours after LPS stimulation. The horseradish peroxidase (HRP) method was used to detect the permeability of rat CMECs. The enzyme-linked immunosorbent assay (ELISA) was used to detect the CXC chemokine ligand (CXCL1, CXCL2) levels in the cell supernatant. The real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect the mRNA expressions of CXCL1 and CXCL2 in the cells. Western Blot was used to detect phosphorylated Src (p-Src), vascular endothelial-cadherin (VE-cadherin) and phosphorylated mitogen activated protein kinase (p-MAPK). RESULTS: Compared with the blank control group, the permeability of rat CMECs in the LPS group was significantly increased. The cell permeability was improved to some extent after paeoniflorin pretreatment at different concentrations, and the improvement was most obvious in the 40 µmol/L paeoniflorin group, with statistically significant difference as compared with the LPS group (A value: 1.61±0.07 vs. 2.13±0.06, P < 0.01). ELISA results showed that there were moderate amounts of CXCL1 and CXCL2 in the cell supernatant of rat CMECs in the blank control group. However, the secretion of CXCL1 and CXCL2 in the cell supernatant was increased significantly under the induction of LPS. After pretreatment with paeoniflorin at different concentrations, the secretion of CXCL1 and CXCL2 in the cell supernatant was significantly reduced. The most obvious inhibitory effect on CXCL1 was 40 µmol/L paeoniflorin, and the most obvious inhibition on CXCL2 was 20 µmol/L paeoniflorin, the differences were statistically significant as compared with the LPS group [CXCL1 (ng/L): 337.51±68.04 vs. 829.86±65.06, CXCL2 (ng/L): 4.48±0.11 vs. 9.41±0.70, both P < 0.01]. RT-qPCR results showed that the mRNA expressions of CXCL1 and CXCL2 in the rat CMECs were consistent with the ELISA results. LPS could increase mRNA expressions of CXCL1 and CXCL2 in the rat CMECs, and pretreatment with different concentrations of paeoniflorin could significantly reduce the mRNA expressions of CXCL1 and CXCL2. The 40 µmol/L paeoniflorin had the best inhibitory effect on CXCL1 mRNA expression, and the 20 µmol/L paeoniflorin had the best inhibitory effect on CXCL2 mRNA expression, the differences were statistically significant as compared with the LPS group [CXCL1 mRNA (2-ΔΔCt): 0.543±0.004 vs. 0.812±0.089, CXCL2 mRNA (2-ΔΔCt): 10.52±0.71 vs. 17.68±1.09, both P < 0.01]. Western Blot results showed that moderate amounts of p-Src, VE-cadherin and p-MAPK proteins were expressed in the rat CMECs in the blank control group. After LPS stimulation, the expressions of p-Src and p-MAPK proteins were increased significantly, while the expression of VE-cadherin protein was decreased significantly. After pretreatment with different concentrations of paeoniflorin, the expressions of p-Src and p-MAPK proteins in the cells were decreased to varying degrees, while the expression of VE-cadherin protein was increased, and 40 µmol/L paeoniflorin had the most obvious effect, the differences were statistically significant as compared with the LPS group [p-Src protein (p-Src/GAPDH): 1.02±0.09 vs. 1.29±0.05, p-MAPK proteins (p-MAPK/GAPDH): 0.24±0.02 vs. 0.62±0.02, VE-cadherin protein (VE-cadherin/GAPDH): 0.64±0.03 vs. 0.31±0.02, all P < 0.01]. CONCLUSIONS: Paeoniflorin can regulate the Src/VE-cadherin pathway in CMECs, inhibit the expression and secretion of inflammation-related proteins and chemokines, and improve the cell permeability of CMECs induced by LPS.


Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Glucosídeos/farmacologia , Monoterpenos/farmacologia , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Transdução de Sinais , Animais , Células Cultivadas , Quimiocina CXCL1/análise , Quimiocina CXCL2/análise , Lipopolissacarídeos , Ratos
15.
Nat Commun ; 11(1): 1199, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32139686

RESUMO

To tackle the global antibiotic resistance crisis, antibiotic resistance acquired either vertically by chromosomal mutations or horizontally through antibiotic resistance genes (ARGs) have been studied. Yet, little is known about the interactions between the two, which may impact the evolution of antibiotic resistance. Here, we develop a multiplexed barcoded approach to assess the fitness of 144 mutant-ARG combinations in Escherichia coli subjected to eight different antibiotics at 11 different concentrations. While most interactions are neutral, we identify significant interactions for 12% of the mutant-ARG combinations. The ability of most ARGs to confer high-level resistance at a low fitness cost shields the selective dynamics of mutants at low drug concentrations. Therefore, high-fitness mutants are often selected regardless of their resistance level. Finally, we identify strong negative epistasis between two unrelated resistance mechanisms: the tetA tetracycline resistance gene and loss-of-function nuo mutations involved in aminoglycoside tolerance. Our study highlights important constraints that may allow better prediction and control of antibiotic resistance evolution.


Assuntos
Resistência Microbiana a Medicamentos/genética , Epistasia Genética , Mutação/genética , Aminoglicosídeos/farmacologia , Sequência de Bases , Permeabilidade da Membrana Celular/efeitos dos fármacos , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Epistasia Genética/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Membrana Transportadoras/metabolismo , Estreptomicina/farmacologia
16.
PLoS Biol ; 18(3): e3000651, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32191696

RESUMO

Rapid antibiotic susceptibility testing (AST) for Neisseria gonorrhoeae (Ng) is critically needed to counter widespread antibiotic resistance. Detection of nucleic acids in genotypic AST can be rapid, but it has not been successful for ß-lactams (the largest antibiotic class used to treat Ng). Rapid phenotypic AST for Ng is challenged by the pathogen's slow doubling time and the lack of methods to quickly quantify the pathogen's response to ß-lactams. Here, we asked two questions: (1) Is it possible to use nucleic acid quantification to measure the ß-lactam susceptibility phenotype of Ng very rapidly, using antibiotic-exposure times much shorter than the 1- to 2-h doubling time of Ng? (2) Would such short-term antibiotic exposures predict the antibiotic resistance profile of Ng measured by plate growth assays over multiple days? To answer these questions, we devised an innovative approach for performing a rapid phenotypic AST that measures DNA accessibility to exogenous nucleases after exposure to ß-lactams (termed nuclease-accessibility AST [nuc-aAST]). We showed that DNA in antibiotic-susceptible cells has increased accessibility upon exposure to ß-lactams and that a judiciously chosen surfactant permeabilized the outer membrane and enhanced this effect. We tested penicillin, cefixime, and ceftriaxone and found good agreement between the results of the nuc-aAST after 15-30 min of antibiotic exposure and the results of the gold-standard culture-based AST measured over days. These results provide a new pathway toward developing a critically needed phenotypic AST for Ng and additional global-health threats.


Assuntos
Antibacterianos/farmacologia , DNA Bacteriano/metabolismo , Desoxirribonuclease I/metabolismo , Neisseria gonorrhoeae/efeitos dos fármacos , Tensoativos/farmacologia , beta-Lactamas/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Gonorreia/microbiologia , Gonorreia/urina , Humanos , Testes de Sensibilidade Microbiana , Neisseria gonorrhoeae/crescimento & desenvolvimento , Neisseria gonorrhoeae/isolamento & purificação , Fenótipo , Reprodutibilidade dos Testes , Fatores de Tempo , Fluxo de Trabalho
17.
J Biotechnol ; 312: 56-62, 2020 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-32151642

RESUMO

Whole-cell biocatalysts have numerous advantages including ease of preparation and coenzyme recovery over purified industrially used enzymes. However, the cell membrane can occasionally hinder cytoplasmic diffusion of the substrate, resulting in reduced biotransformation efficiency. Psychrophiles can grow and reproduce at low temperatures; their cell membranes are highly flexible, and their permeability can be improved via heat treatment at a moderate temperature. The aim of this study was to generate a psychrophile-based simple biocatalyst (PSCats) using Shewanella livingstonensis Ac10. This biocatalyst contained two enzymes that were heterologously expressed and converted citric acid to itaconic acid, thereby serving as a potential platform replacing the petroleum-based counterparts. The efficiency of the biocatalyst was increased via heat treatment at 45 °C for 15 min, and itaconic acid productivity of the cells after heat treatment (1.41 g/L/h) was increased around 6-fold in comparison with those without heat treatment (0.22 g/L/h). A large part of the productivity remained (67.3 %) when the cells were reused for 5 times (10 h for each reaction). Therefore, the potential of this heat-permeabilized psychrophile host to increase the productivity of whole-cell biocatalyst was proved; however, further research is necessary to understand the underlying mechanism.


Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/fisiologia , Enzimas/farmacologia , Shewanella/metabolismo , Succinatos/metabolismo , Aconitato Hidratase , Ácido Cítrico/metabolismo , Temperatura Baixa , Citoplasma/metabolismo , Escherichia coli/genética , Temperatura Alta , Metaboloma , Shewanella/genética , Shewanella/crescimento & desenvolvimento
18.
Biochim Biophys Acta Biomembr ; 1862(8): 183241, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32126227

RESUMO

The presence of an asymmetric distribution of lipids in biological membranes was first described ca. 50 years ago. While various studies had reported the role of loss of lipid asymmetry on signaling processes, its effect on membrane physical properties and membrane-protein interactions lacks further understanding. The recent description of new technologies for the preparation of asymmetric model membranes has helped to fill part of this gap. However, the major effort so far has been on plasma membrane models. Here we describe the preparation of liposomes mimicking the mitochondria outer membrane (MOM) in regard to its lipid composition and asymmetry. By employing the methyl-ß-cyclodextrin-catalyzed lipid exchange technology and accurate quantification of lipid asymmetry with head group-specific probes we showed the successful preparation of a MOM model bearing a physiologically relevant lipid composition and asymmetry. In addition, by a direct comparison with its lipid symmetrical counterpart it is shown that asymmetric models were more resistant to tBid-promoted Bax-permeabilization, suggesting a role played by MOM lipid asymmetry on the mitochondria pathway of apoptosis. The barrier imposed by lipid asymmetry on membrane permeabilization was in part due to a decrease in the concentration of membrane-bound proteins, which was likely a consequence of the two mutually-dependent properties; i.e., the lower electrostatic surface potential and the higher molecular packing imposed by lipid asymmetry. It is proposed that MOM lipid asymmetry imparts different physical properties on the membrane and might add an additional component of regulation in intricate mitochondrial processes.


Assuntos
Lipídeos/química , Mitocôndrias/genética , Membranas Mitocondriais/química , Proteína X Associada a bcl-2/genética , Apoptose/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Humanos , Lipossomos/química , Lipossomos/ultraestrutura , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mitocôndrias/efeitos dos fármacos , Membranas Mitocondriais/ultraestrutura , Fosfolipídeos/química , Fosfolipídeos/genética
19.
Biochim Biophys Acta Biomembr ; 1862(8): 183275, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32173291

RESUMO

The search for novel compounds to combat multi-resistant bacterial infections includes exploring the potency of antimicrobial peptides and derivatives thereof. Complementary to high-throughput screening techniques, biophysical and biochemical studies of the biological activity of these compounds enable deep insight, which can be exploited in designing antimicrobial peptides with improved efficacy. This approach requires the combination of several techniques to study the effect of such peptides on both bacterial cells and simple mimics of their cell envelope, such as lipid-only vesicles. These efforts carry the challenge of bridging results across techniques and sample systems, including the proper choice of membrane mimics. This review describes some important concepts toward the development of potent antimicrobial peptides and how they translate to frequently applied experimental techniques, along with an outline of the biophysics pertaining to the killing mechanism of antimicrobial peptides.


Assuntos
Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Infecções Bacterianas/tratamento farmacológico , Membrana Celular/efeitos dos fármacos , Antibacterianos/uso terapêutico , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Bactérias/efeitos dos fármacos , Bactérias/patogenicidade , Infecções Bacterianas/microbiologia , Membrana Celular/microbiologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/patogenicidade , Humanos
20.
Biochim Biophys Acta Biomembr ; 1862(7): 183261, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32151610

RESUMO

Antimicrobial peptides are widely studied as an alternative to traditional antibiotics. However, they are difficult to develop, as multiple factors influence their potency and selectivity toward bacterial cells. In this paper, we investigate three simplified model peptides that bear crown ethers, and the effects of simple structural modifications (peptide length and crown ether ring size) on their secondary structures and their permeabilizing activity on living cells and model membranes made with egg yolk phosphatidylcholine or 1-palmitoyl-2-oleoylphosphatidylglycerol. Circular dichroism studies show that the peptide length and the crown ether ring size do influence the conformation, but no trend could be determined from the results. Permeabilization studies with model membranes and with red blood cells demonstrated that from 13 residues to 16 residues, there is a gradual increase in activity as the peptides get longer. However, the shortest tested analogs, with 12 residues, also exhibited an increase in activity caused by the removal of one amino acid that was bearing a crown ether. Permeabilization assays showed that larger ring size analogs showed higher hemolytic activities. Altogether, the results reported help design new and more selective antimicrobial peptides.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Membrana Celular/química , Éteres de Coroa/química , Fosfatidilcolinas/química , Sequência de Aminoácidos/genética , Antibacterianos/efeitos adversos , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Dicroísmo Circular , Éteres de Coroa/farmacologia , Gema de Ovo/química , Humanos , Bicamadas Lipídicas/química , Fosfatidilcolinas/farmacologia , Estrutura Secundária de Proteína , Relação Estrutura-Atividade
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