RESUMO
The development of bioproducts able to accelerate wound healing is an important topic in biomedicine. In the current study, Pistacia lentiscus distilled leaves (PDL) extract and its two isolated glycosylated flavonoids, myricetin-3-O-rhamnoside (MM) and quercetin-3-O-rhamnoside (QM), were evaluated for their wound healing activity, including evaluation of wound closure, revascularization, wound re-epithelialization, fibroblast proliferation, and collagen deposition on rat skin samples. Moreover, hydroxyproline content, C-reactive protein (CRP) level, and immunohistochemistry study were evaluated on blood and tissues collected from rats on day 14 post-wounding. Results showed that the topical application of PDL (at a concentration of 20 mg/ml) (PDL 20), MM, and QM increased wound healing and decreased inflammatory cells infiltration compared to the negative control group. Moreover, the cutaneous wound tissues treated with PDL 20, MM, and QM exhibited significantly higher hydroxyproline content than the negative control group, which means a high collagen biosynthesis in wound tissues. Indeed, the level of the inflammatory protein CRP is significantly lower in groups treated with MM and QM than in the negative control group. Also, the expression of the pro-inflammatory factor TNF-α and the angiogenesis marker CD-31 in PDL 20, MM, and QM treated groups is lower than in the negative control group. Moreover, MM, and QM induced a good elastase inhibition at 100 µg/ml compared to the standard epigallocatechin gallate. Therefore, PDL 20, MM, and QM could be used as effective cutaneous wound healing agents.
Assuntos
Manosídeos/farmacologia , Quercetina/análogos & derivados , Cicatrização/efeitos dos fármacos , Administração Tópica , Animais , Pistacia , Extratos Vegetais , Folhas de Planta , Quercetina/farmacologia , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Exposure to UV radiation damages the skin and increases the risk of skin cancer. Sunscreen is used to protect the skin from the harmful effects of UV radiation. However, the chemical UV filters used in sunscreen can show toxicity and cause allergic reactions. A safe sunscreen that includes a lower content of chemical UV filters and exerts an excellent effect on UV protection needs to be developed. The objective of this study was to investigate whether the addition of afzelin to sunscreen could improve the sun protection factor (SPF). A synergistic effect between afzelin and organic sunscreen agents including padimate O and oxybenzone was confirmed. Interestingly, 100% in vitro SPF-boosting was observed when afzelin (0.05%) was applied with a standard SPF formulation containing organic sunscreens while afzelin alone had no contribution to the SPF. In vivo SPF analysis of the standard SPF formulation showed an SPF value of 13.3 that increased to 20.1 when supplemented with afzelin (0.05%). Additionally, afzelin showed no skin irritation in a human trial. These results suggest that afzelin is useful as a natural additive in sunscreen formulations and provides an SPF-boosting effect. Afzelin supplementation to the formulation showed the potential to reduce the use of synthetic photoprotectors, which could minimize the risk of synthetic agent toxicity.
Assuntos
Cosméticos/química , Manosídeos/química , Proantocianidinas/química , Fator de Proteção Solar/métodos , Protetores Solares/química , Adolescente , Adulto , Benzofenonas/farmacologia , Ensaios Clínicos como Assunto , Cosméticos/farmacologia , Composição de Medicamentos , Feminino , Humanos , Masculino , Manosídeos/farmacologia , Pessoa de Meia-Idade , Proantocianidinas/farmacologia , Pele , Protetores Solares/farmacologia , Raios Ultravioleta , para-Aminobenzoatos/farmacologiaRESUMO
BACKGROUND: Cervical cancer is the most common cancer and has the highest morbidity rate of gynaecological malignancies in women worldwide. So, the development of effective anti-cancer agents to treat this condition is vital. Considering the recent interest in free (unconjugated) curcuminoids delivery, the present study investigated the efficacy of a novel food-grade, free-curcuminoids (curcumin-galactomannoside complex; CGM) on cervical cancer cells (HeLa) of human origin. In this study, we examined the anticancer potential of CGM as well as its effects on the cell cycle and the apoptosis of HeLa cancer cell. METHODS: Determination of anti-proliferative and apoptosis validation of CGM on HeLa cells was performed by 3-(4,5-Dimethylthiazol-2-yl)-2, 5,-diphenyltetrazolium bromide (MTT), acridine orange/propidium iodide and annexin-V-fluorescein isothiocyanate assays. Measurement of Reactive Oxygen Species (ROS) production, Caspase activities and protein expression experiments were performed to investigate the potential mechanisms of action in the apoptotic process. RESULTS: The cytotoxic assays revealed that the CGM showed inhibition of cell survival and exhibited high cytotoxic activity against HeLa cells at 25 µg/mL. Further studies on morphological changes were done in CGM-treated cervical cancer cells contributing to apoptosis. Flow cytometry analysis with Annexin V-FITC and PI staining precisely indicated that CGM induced apoptosis in HeLa cell lines at 25 µg/mL. By the supplementation of CGM showed an increase in Bax and cleaved caspase-8 protein in HeLa cells after 48 h exposure. CONCLUSION: The evidence obtained from this study suggests that CGM is a potent and promising natural formulation against cervical cancer cells via induction of apoptosis through ROS mediated mitochondrial damage in HeLa cells. Hence, CGM could be further explored as a potential lead in treating cancer.
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Assuntos
Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Curcumina/farmacologia , Galactosídeos/farmacologia , Manosídeos/farmacologia , Neoplasias do Colo do Útero/tratamento farmacológico , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Células HeLa , Humanos , Mitocôndrias/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias do Colo do Útero/patologiaRESUMO
Copaifera langsdorffii Desf. is a plant found in South America, especially in Brazil. Oleoresin and the leaves of this plant is used as a popular medicinal agent. However, few studies on the chemical composition of aerial parts and related biological activities are known. This study aimed to examine the cytotoxic, genotoxic, and antigenotoxic potential of C. langsdorffii aerial parts hydroalcoholic extract (CLE) and two of its major compounds afzelin and quercitrin. The cytotoxic and antigenotoxic potential of CLE was determined as follows: 1) against genotoxicity induced by doxorubicin (DXR) or methyl methanesulfonate (MMS) in V79 cells; 2) by direct and indirect-acting mutagens in Salmonella typhimurium strains; and 3) by MMS in male Swiss mice. The protective effects of afzelin and quercitrin against DXR or MMS were also evaluated in V79 and HepG2 cells. CLE was cytotoxic as evidenced by clonogenic efficiency assay. Further, CLE did not induce a significant change in frequencies of chromosomal aberrations and micronuclei; as well as number of revertants in the Ames test demonstrating absence of genotoxicity. In contrast, CLE was found to be antigenotoxic in mammalian cells. The results also showed that CLE exerted inhibitory effect against indirect-acting mutagens in the Ames test. Afzelin and quercitrin did not reduce genotoxicity induced by DXR or MMS in V79 cells. However, treatments using afzelin and quercitrin decreased MMS-induced genotoxicity in HepG2 cells. The antigenotoxic effect of CLE observed in this study may be partially attributed to the antioxidant activity of the combination of major components afzelin and quercitrin.
Assuntos
Dano ao DNA/efeitos dos fármacos , Fabaceae/química , Manosídeos/farmacologia , Extratos Vegetais/farmacologia , Proantocianidinas/farmacologia , Substâncias Protetoras/farmacologia , Quercetina/análogos & derivados , Animais , Doxorrubicina/toxicidade , Células Hep G2 , Humanos , Masculino , Metanossulfonato de Metila/toxicidade , Camundongos , Mutagênicos/farmacologia , Mutagênicos/toxicidade , Extratos Vegetais/química , Folhas de Planta/química , Quercetina/farmacologia , Salmonella typhimurium/efeitos dos fármacosRESUMO
Immunomodulation, cytotoxicity and anti-cancer activity of selected amphiphilic non-ionic (thio)alkyl α-D-mannosides (with aglycone of C6-C12) were investigated in vitro in human cervix epitheloid carcinoma cell line HeLa, murine melanoma cancer cells B16, murine lymphocytic leukemia cell line L1210, murine fibroblast cell line NIH 3â¯T3 and murine macrophage cell line RAW 264.7. Toxicological studies revealed structure-dependent immunobiological effectivity based on a tight interaction with relevant cells. The results demonstrated diverse immunomodulation of macrophage cell-line RAW264.7 proliferation and production of Th1 and Th2 cytokines, and induction of pro-inflammatory interleukins IL-1α, TNFα, IL-6, IL-12 and IL-17 and anti-inflammatory IL-10 following (thio)alkyl α-D-mannosides 24 and 48â¯h exposure. Direct application of alkyl mannosides MOC10 and MOC12 and their thio analogues MSC10 and MSC12 in reconstructed human EpiDerm™ and MOC12 and MSC12 in EpiOcular™ model assays for dermal and ocular irritation together with quantification of human proinflammatory cytokines IL-1α, TNFα, IL-6 and IL-8 culture media release was used to ascertain toxicological safety.
Assuntos
Antineoplásicos/farmacologia , Fatores Imunológicos/farmacologia , Manosídeos/farmacologia , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Olho/efeitos dos fármacos , Humanos , Imunomodulação , Camundongos , Modelos Biológicos , Pele/efeitos dos fármacosRESUMO
N-ribosylation and N-mannosylation compounds have a great role in compounds activity as anticancer. The reaction of 2-thioxo-4-thiazolidinone (rhodanine) derivatives, as aglycon part, was done with ribofuranose and mannopyranose sugars (glycone part) followed by deacetylation without cleavage of the rhodanine under acidic medium. Conformational analysis has been studied using NMR methods (2D, DQF-COSY, HMQC and HMBC). All final the new deprotected nucleosides were screened against leukemia 1210, and were found to be considerably less potent (Ic50% 1.4-10.6 µM) than doxorubicin (Ic50% 0.02 µM). Compounds 10d and 10e which contain ribose moiety have better activity than those with mannose sugar. DFT calculations with B3LYP/6-31 + G (d) level were used to analyze the electronic and geometric characteristics deduced from the stable structure of the compounds. The principal quantum chemical descriptors showed a good correlation with the experimental observations. Rapid Overlay Comparison Similarity (ROCS) study was operated to explain the compounds similarity and to figure out the most important pharmacophoric features.
Assuntos
Antineoplásicos/farmacologia , Teoria da Densidade Funcional , Desenho de Fármacos , Manosídeos/farmacologia , Rodanina/farmacologia , Ribonucleosídeos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Manosídeos/química , Modelos Moleculares , Estrutura Molecular , Rodanina/síntese química , Rodanina/química , Ribonucleosídeos/química , Relação Estrutura-AtividadeRESUMO
Particulate matter (PM), a widespread airborne contaminant, is a complex mixture of solid and liquid particles suspended in the air. Recent studies have demonstrated that PM induces oxidative stress and inflammatory reactions, and may cause certain skin diseases. Afzelin is a flavonoid isolated from Thesium chinense Turcz, which has antiinflammatory, anticancer and antibacterial properties. Therefore, the present study aimed to investigate if afzelin affected inflammatory responses in human keratinocytes exposed to PM. HaCaT cells were treated with PM (25 µg/cm2) in the presence or absence of afzelin (200 µM). Here, standard reference material 1649b was used as PM. Cell viability was assessed using the watersoluble tetrazolium salt1 assay. The generation of reactive oxygen species (ROS) was measured using the dichlorodihydroâfluorescein diacetate assay. Gene and protein expression were investigated using reverse transcriptionquantitative polymerase chain reaction and western blot analysis, respectively. Levels of secreted inflammatory cytokines were measured using ELISA. The results suggested that afzelin inhibited PMinduced proinflammatory cytokine mRNA expression and protein secretion in HaCaT cells. In addition, afzelin suppressed PMinduced intracellular ROS generation, and p38 mitogenactivated protein kinase and transcription factor activator protein1 component cFos and cJun activation. The results indicated that afzelin exerts antiinflammatory and antioxidant effects in PMexposed HaCaT. Afzelin may have potential for preventing PMinduced inflammatory skin diseases.
Assuntos
Anti-Inflamatórios/farmacologia , Inflamação/tratamento farmacológico , Inflamação/etiologia , Queratinócitos/efeitos dos fármacos , Manosídeos/farmacologia , Material Particulado/efeitos adversos , Proantocianidinas/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citocinas/imunologia , Humanos , Inflamação/imunologia , Queratinócitos/imunologia , Espécies Reativas de Oxigênio/imunologiaRESUMO
Tumor-associated macrophages (TAMs) that exist in tumor microenvironment promote tumor progression and have been suggested as a promising therapeutic target for cancer therapy in preclinical studies. Development of theranostic systems capable of specific targeting, imaging, and ablation of TAMs will offer clinical benefits. Here we constructed a theranostic probe, namely, TPE-Man, by attaching mannose moieties to a red-emissive and AIE (aggregation-induced emission)-active photosensitizer. TPE-Man can specifically recognize a mannose receptor that is overexpressed on TAMs by the sugar-receptor interaction and enables fluorescent visualization of the mannose-receptor-positive TAMs in high contrast. The histologic study of mouse tumor sections further verifies TPE-Man's excellent targeting specificity being comparable with the commercial mannose-receptor antibody. TAMs can be effectively eradicated upon exposure to white light irradiation via a photodynamic therapy effect. To our knowledge, this is the first small molecular theranostic probe for TAMs that revealed combined advantages of low cost, high targeting specificity, fluorescent light-up imaging, and efficient photodynamic ablation.
Assuntos
Compostos de Benzilideno/farmacologia , Macrófagos/efeitos dos fármacos , Manosídeos/farmacologia , Fármacos Fotossensibilizantes/farmacologia , Animais , Compostos de Benzilideno/síntese química , Compostos de Benzilideno/efeitos da radiação , Compostos de Benzilideno/toxicidade , Manosídeos/síntese química , Manosídeos/efeitos da radiação , Manosídeos/toxicidade , Camundongos , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/síntese química , Fármacos Fotossensibilizantes/efeitos da radiação , Fármacos Fotossensibilizantes/toxicidade , Ratos Sprague-Dawley , Nanomedicina Teranóstica/métodosRESUMO
Preventing bacterial adhesion to host cells is a provocative and alternative approach to traditional antibiotic treatments given the increasing microbial resistance. A brief overview of common antibiotic treatments is described in light of their respective resistance and remaining susceptibility. This strategy has been seriously considered in the context of adherent-invasive infections in Crohn's disease and urinary tract infections in particular. The adhesions of various pathogenic Escherichia coli strains to host cells are primarily mediated through carbohydrate-protein interactions involving bacterial organelles called fimbriae that can recognize specific glycoconjugate receptors on host cells. Of particular interest are the FimH and PapG fimbriae, which bind to mannosylated glycoproteins and glycolipids of the galabiose series, respectively. Therefore, blocking FimH- and PapG-mediated bacterial adhesion to uroepithelial cells by high-affinity carbohydrate antagonists constitutes a challenging therapeutic target of high interest. This is of particular interest since bacterial adhesion to host cells is a parameter unlikely to be the subject of bacterial mutations without affecting the carbohydrate ligand binding interactions at the basis of the recognition and infection processes. To date, there have been several families of potent FimH antagonists that include natural O-linked as well as unnatural analogues of α-d-mannopyranosides. These observations led to a thorough understanding of the intimate binding site interactions that helped to reveal the so-called "tyrosine gate mechanism" at the origin of the strong necessary interactions with sugar-possessing hydrophobic aglycones. By modification of the aglycones of single monosaccharidic d-mannopyranosides, it was possible to replace the natural complex oligomannoside structure by simpler ones. An appealing and successful series of analogues have been disclosed, including nanomolecular architectures such as dendrimers, polymers, and liposomes. In addition, the data were compared to the above multivalent architectures and confirmed the possibility of working with small sugar candidates. This Account primarily concentrates on the most promising types of FimH inhibitors belonging to the family of α-C-linked mannopyranosides. However, one of the drawbacks associated with C-mannopyranosides has been that they were believed to be in the inverted chair conformation, which is obviously not recognized by the E. coli FimH. To decipher this situation, various synthetic approaches, conformational aspects, and restrictions are discussed using molecular modeling, high-field NMR spectroscopy, and X-ray analysis. These combined techniques pointed to the fact that several α-C-linked mannopyranosides do exist in the required 4C1 chair conformation. Ultimately, recent findings in this growing field of interest culminated in the identification of drug candidates that have reached clinical phase I.
Assuntos
Infecções por Escherichia coli/terapia , Manosídeos/química , Adesinas de Escherichia coli/metabolismo , Animais , Antibacterianos , Antígenos CD , Aderência Bacteriana/efeitos dos fármacos , Moléculas de Adesão Celular , Farmacorresistência Bacteriana , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Proteínas de Fímbrias/antagonistas & inibidores , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Proteínas Ligadas por GPI , Humanos , Manosídeos/farmacologia , Manosídeos/uso terapêutico , Infecções Urinárias/tratamento farmacológico , Infecções Urinárias/patologiaRESUMO
Oxidised low-density lipoprotein (ox-LDL) is a pro-atherogenic molecule, which induces inflammatory response and contributes to the pathogenesis of vascular dysfunction to atherosclerosis. The aim of the present study was to explore the anti-inflammatory effect of a novel bioavailable formulation of curcumin as 'curcumagalactomannosides' (CGM) against ox-LDL-induced inflammatory responses in human peripheral blood mononuclear cells (hPBMCs). Curcumagalactomannosides was made from natural curcumin using the soluble dietary fibre (galactomannans) derived from fenugreek seeds (Trigonella foenumgracum) and the hPBMCs were isolated from healthy human volunteers. The cells were cultured in collagen-coated plates at 37 °C and grouped as Group I (Control), Group II (ox-LDL treated) and Group III (ox-LDL + CGM treated). Further analysis of inflammatory markers, reactive oxygen species and mRNA expression levels indicated significantly increased expressions of iNOS, TNF-α, IL-6 and VCAM-1 in ox-LDL-treated group along with the nuclear translocation of NF-κB. Other inflammatory markers such as LOX, PGE2, total COX and lipid peroxidation level were also found to be significantly (p < 0.05) increased upon ox-LDL treatment. The treatment with CGM on the other hand was found to down-regulate and reverse the ox-LDL-induced alterations indicating its potential anti-inflammatory effect on hPBMCs via. NF-κB signalling pathway.
Assuntos
Curcumina/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Lipoproteínas LDL/toxicidade , Manosídeos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Galactosídeos/farmacologia , Humanos , Leucócitos Mononucleares/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , NF-kappa B/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/genética , Molécula 1 de Adesão de Célula Vascular/análise , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
Dengue virus (DENV), a mosquito-borne flavivirus, causes severe and potentially fatal symptoms in millions of infected individuals each year. Although dengue fever represents a major global public health problem, the vaccines or antiviral drugs proposed so far have not shown sufficient efficacy and safety, calling for new antiviral developments. Here we have shown that a mannoside glycolipid conjugate (MGC) bearing a trimannose head with a saturated lipid chain inhibited DENV productive infection. It showed remarkable cell promiscuity, being active in human skin dendritic cells, hepatoma cell lines and Vero cells, and was active against all four DENV serotypes, with an IC50 in the low micromolar range. Time-of-addition experiments and structure-activity analyses revealed the importance of the lipid chain to interfere with an early viral infection step. This, together with a correlation between antiviral activity and membrane polarization by the lipid moiety indicated that the inhibitor functions by blocking viral envelope fusion with the endosome membrane. These finding establish MGCs as a novel class of antivirals against the DENV.
Assuntos
Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Glicolipídeos/farmacologia , Manosídeos/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Chlorocebus aethiops , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/virologia , Vírus da Dengue/fisiologia , Descoberta de Drogas , Glicolipídeos/química , Células Hep G2 , Humanos , Concentração Inibidora 50 , Manosídeos/química , Sorogrupo , Células VeroRESUMO
BACKGROUND: Plant extracts are sources of valuable compounds with biological activity, especially for the anti-proliferative activity against pathogens or tumor cells. Myricetin is a flavonoid found in several plants that has been described as an inhibitor of Human immunodeficiency virus type 1 (HIV-1) through its action against the HIV reverse transcriptase, but myricetin derivatives have not been fully studied. The aim of this study was to evaluate the anti-HIV-1 activity of glycosylated metabolites obtained from Marcetia taxifolia and derived from myricetin: myricetin rhamnoside and myricetin 3-(6-rhamnosylgalactoside). METHODS: Compounds were obtained from organic extracts by maceration of aerial parts of M. taxifolia. All biological assays were performed in the MT4 cell line. Antiviral activity was measured as inhibition of p24 and reverse transcriptase with a fluorescent assay. RESULTS: Both flavonoids have antiviral activity in vitro, with an EC50 of 120 µM for myricetin 3-rhamnoside (MR) and 45 µM for myricetin 3-(6-rhamnosylgalactoside) (MRG), both significantly lower than the EC50 of myricetin (230 µM). Although both compounds inhibited the reverse transcriptase activity, with an IC50 of 10.6 µM for MR and 13.8 µM for MRG, myricetin was the most potent, with an IC50 of 7.6 µM, and an inhibition greater than 80%. Molecular docking approach showed correlation between the free energy of binding with the assays of enzyme inhibition. CONCLUSIONS: The results suggest that glycosylated moiety might enhance the anti-HIV-1 activity of myricetin, probably by favoring the internalization of the flavonoid into the cell. The inhibition of the HIV-1 reverse transcriptase is likely responsible for the antiviral activity.
Assuntos
Fármacos Anti-HIV/farmacologia , Flavonoides/farmacologia , Galactosídeos/farmacologia , Proteína do Núcleo p24 do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/antagonistas & inibidores , Manosídeos/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Linhagem Celular , Glicosilação , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Humanos , Simulação de Acoplamento Molecular , Replicação Viral/efeitos dos fármacosRESUMO
Fibrotic diseases are among the most serious health issues with severe burdens due to their chronic nature and a large number of patients suffering from the debilitating effects and long-term sequelae. Collagenase treatment is a nonsurgical option but has limited results. To date, there is no potent noninvasive solution for fibrosis. Part of the reason for this is the lack of appropriate in vitro live cell screening tools to assess the efficacy of new therapeutical agents. Here, we demonstrate the utility of a cell-based electrochemical impedance biosensor platform to screen the efficacy of potential antifibrotic compounds. The platform employs a label-free and noninvasive strategy to detect the progression of fibrosis and the potency of the antifibrotic molecules in real-time. The fundamental principle that governs this novel system is that dynamic changes in cell shape and adhesion during fibrosis can be measured accurately by monitoring the changes in the impedance. This is achieved by growing the cells on a transparent interdigitated indium tin oxide (ITO) electrodes. It was demonstrated by monitoring the efficacy of a model antifibrotic compound, PXS64, on cells collected from patients with Dupuytren's contracture. We confirmed the validity of the developed biochemical impedance biosensor as an tool for in vitro screening of antifibrotic compounds and provided quantitative information on subcellular influences of the examined chemical molecules using correlative microscopy analyses that monitor the average cell area, cell morphology, and the amount and directionality of the deposited extracellular matrix protein collagen and measurement of cytosolic Ca2+ changes.
Assuntos
Técnicas Biossensoriais/métodos , Contratura de Dupuytren/tratamento farmacológico , Impedância Elétrica , Manosídeos/farmacologia , Organofosfonatos/farmacologia , Células Cultivadas , Contratura de Dupuytren/metabolismo , Eletrodos , Humanos , Compostos de Estanho/químicaRESUMO
Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) affect 150 million people annually. Despite effective antibiotic therapy, 30-50% of patients experience recurrent UTIs. In addition, the growing prevalence of UPEC that are resistant to last-line antibiotic treatments, and more recently to carbapenems and colistin, make UTI a prime example of the antibiotic-resistance crisis and emphasize the need for new approaches to treat and prevent bacterial infections. UPEC strains establish reservoirs in the gut from which they are shed in the faeces, and can colonize the periurethral area or vagina and subsequently ascend through the urethra to the urinary tract, where they cause UTIs. UPEC isolates encode up to 16 distinct chaperone-usher pathway pili, and each pilus type may enable colonization of a habitat in the host or environment. For example, the type 1 pilus adhesin FimH binds mannose on the bladder surface, and mediates colonization of the bladder. However, little is known about the mechanisms underlying UPEC persistence in the gut. Here, using a mouse model, we show that F17-like and type 1 pili promote intestinal colonization and show distinct binding to epithelial cells distributed along colonic crypts. Phylogenomic and structural analyses reveal that F17-like pili are closely related to pilus types carried by intestinal pathogens, but are restricted to extra-intestinal pathogenic E. coli. Moreover, we show that targeting FimH with M4284, a high-affinity inhibitory mannoside, reduces intestinal colonization of genetically diverse UPEC isolates, while simultaneously treating UTI, without notably disrupting the structural configuration of the gut microbiota. By selectively depleting intestinal UPEC reservoirs, mannosides could markedly reduce the rate of UTIs and recurrent UTIs.
Assuntos
Proteínas de Fímbrias/antagonistas & inibidores , Intestinos/efeitos dos fármacos , Intestinos/microbiologia , Manosídeos/farmacologia , Ácidos Ftálicos/farmacologia , Infecções Urinárias/prevenção & controle , Escherichia coli Uropatogênica/efeitos dos fármacos , Escherichia coli Uropatogênica/isolamento & purificação , Adesinas de Escherichia coli/metabolismo , Sequência de Aminoácidos , Animais , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Fezes/microbiologia , Feminino , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/classificação , Fímbrias Bacterianas/efeitos dos fármacos , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Humanos , Intestinos/citologia , Manosídeos/uso terapêutico , Camundongos , Modelos Moleculares , Ácidos Ftálicos/uso terapêutico , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/microbiologia , Infecções Urinárias/tratamento farmacológico , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/classificação , Escherichia coli Uropatogênica/genéticaRESUMO
INTRODUCTION: The bacterial adhesin FimH is a virulence factor and an attractive therapeutic target for urinary tract infection (UTI) and Crohn's Disease (CD). Located on type 1 pili of uropathogenic E. coli (UPEC), the FimH adhesin plays an integral role in the pathogenesis of UPEC. Recent efforts have culminated in the development of small-molecule mannoside FimH antagonists that target the mannose-binding lectin domain of FimH, inhibiting its function and preventing UPEC from binding mannosylated host cells in the bladder, thereby circumventing infection. Areas covered: The authors describe the structure-guided design of mannoside ligands, and review the structural biology of the FimH lectin domain. Additionally, they discuss the lead optimization of mannosides for therapeutic application in UTI and CD, and describe various assays used to measure mannoside potency in vitro and mouse models used to determine efficacy in vivo. Expert opinion: To date, mannoside optimization has led to a diverse set of small-molecule FimH antagonists with oral bioavailability. With clinical trials already initiated in CD and on the horizon for UTI, it is the authors, opinion that mannosides will be a 'first-in-class' treatment strategy for UTI and CD, and will pave the way for treatment of other Gram-negative bacterial infections.
Assuntos
Doença de Crohn/tratamento farmacológico , Proteínas de Fímbrias/antagonistas & inibidores , Infecções Urinárias/tratamento farmacológico , Adesinas de Escherichia coli , Animais , Antibacterianos/administração & dosagem , Antibacterianos/farmacocinética , Antibacterianos/farmacologia , Disponibilidade Biológica , Doença de Crohn/microbiologia , Modelos Animais de Doenças , Desenho de Fármacos , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Humanos , Manosídeos/administração & dosagem , Manosídeos/farmacocinética , Manosídeos/farmacologia , Camundongos , Relação Estrutura-Atividade , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/isolamento & purificaçãoRESUMO
Melanogenesis refers to synthesis of the skin pigment melanin, which plays a critical role in the protection of skin against ultraviolet irradiation and oxidative stressors. We investigated the effects of afzelin on melanogenesis and its mechanisms of action in human epidermal melanocytes. In this study, we found that afzelin increased both melanin content and tyrosinase activity in a concentration-dependent manner. While the mRNA levels of microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related protein (TRP)-1 increased following afzelin treatment, the mRNA levels of TRP-2 were not affected by afzelin. Likewise, afzelin increased the protein levels of MITF, TRP-1, and tyrosinase but not TRP-2. Mechanistically, we found that afzelin regulated melanogenesis by upregulating MITF through phosphorylation of p38 mitogen-activated protein kinase (MAPK), independent of cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling. Taken together, these findings indicate that the promotion of melanogenesis by afzelin occurs through increased MITF gene expression, which is mediated by activation of p38 MAPK, and suggest that afzelin may be useful as a protective agent against ultraviolet irradiation.
Assuntos
Manosídeos/farmacologia , Pigmentação/efeitos dos fármacos , Proantocianidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Western Blotting , Células Cultivadas , Humanos , Imidazóis/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Melaninas/metabolismo , Melanócitos/citologia , Melanócitos/efeitos dos fármacos , Melanócitos/metabolismo , Fator de Transcrição Associado à Microftalmia/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Oxirredutases/metabolismo , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The anti-adhesive strategy, consisting of disrupting bacterial attachment to the host cells, is widely explored as an alternative to antibiotic therapies. Recently, thiazolylmannosides (TazMans) have been identified as strong anti-adhesives of E. coli strains implied in the gut inflammation of patients with Crohn's disease. In this work, we developed a second generation of TazMans with improved chemical stability. The anomeric nitrogen was substituted by short linkers and the compounds were assessed against the bacterial adhesin FimH and the clinically isolated LF82 E. coli strain in four in vitro assays. The results obtained on the FimH adhesin alone and the whole bacteria enabled the identification of a candidate for further in vivo evaluations.
Assuntos
Doença de Crohn/tratamento farmacológico , Escherichia coli/efeitos dos fármacos , Proteínas de Fímbrias/antagonistas & inibidores , Manosídeos/farmacologia , Tiazóis/farmacologia , Adesinas de Escherichia coli , Doença de Crohn/microbiologia , Ensaio de Imunoadsorção Enzimática , Escherichia coli/patogenicidade , Humanos , Espectroscopia de Ressonância Magnética , Manosídeos/química , Testes de Sensibilidade Microbiana , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tiazóis/químicaRESUMO
Blocking the adherence of bacteria to cells is an attractive complementary approach to current antibiotic treatments, which are faced with increasing resistance. This strategy has been particularly studied in the context of urinary tract infections (UTIs), in which the adhesion of pathogenic Escherichia coli strains to uroepithelial cells is prevented by blocking the FimH adhesin expressed at the tips of bacteria organelles called fimbriae. Recently, we extended the antiadhesive concept, showing that potent FimH antagonists can block the attachment of adherent-invasive E.â coli (AIEC) colonizing the intestinal mucosa of patients with Crohn's disease (CD). In this work, we designed a small library of analogues of heptyl mannoside (HM), a previously identified nanomolar FimH inhibitor, but one that displays poor antiadhesive effects in vivo. The anomeric oxygen atom was replaced by a sulfur or a methylene group to prevent hydrolysis by intestinal glycosidases, and chemical groups were attached at the end of the alkyl tail. Importantly, a lead compound was shown to reduce AIEC levels in the feces and in the colonic and ileal mucosa after oral administration (10â mg kg(-1) ) in a transgenic mouse model of CD. The compound showed a low bioavailability, preferable in this instance, thus suggesting the possibility of setting up an innovative antiadhesive therapy, based on the water-soluble and non-cytotoxic FimH antagonists developed here, for the CD subpopulation in which AIEC plays a key role.
Assuntos
Aderência Bacteriana/efeitos dos fármacos , Doença de Crohn/terapia , Escherichia coli/efeitos dos fármacos , Mucosa Intestinal/microbiologia , Manosídeos/farmacologia , Adesinas de Escherichia coli/metabolismo , Animais , Disponibilidade Biológica , Peso Corporal/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Doença de Crohn/metabolismo , Doença de Crohn/microbiologia , Doença de Crohn/patologia , Cristalografia por Raios X , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Proteínas de Fímbrias/antagonistas & inibidores , Proteínas de Fímbrias/metabolismo , Humanos , Manosídeos/química , Manosídeos/metabolismo , Camundongos , Camundongos Transgênicos , Ligação Proteica , Domínios ProteicosRESUMO
Solidago chilensis Meyenmost (Asteraceae), popularly known as "Brazilian arnica" or "arnica-do-campo," is widely used in the folk medicine to treat gastric disorders. Based on this, the gastroprotective activity of S. chilensis methanolic extract was investigated. Besides, a phytochemical study allowed isolation of two flavonoids (quercitrin and afzelin). The gastroprotective effects were investigated in acute gastric ulcer models, and the antisecretory activity was assessed in vivo and in vitro. The adhered mucus levels, reduced glutathione (GSH) content and myeloperoxidase (MPO) activity were quantified in ulcerated tissues. The contribution of isolated compounds in extract effects was evaluated, and its doses were calculated according to its yield. To evaluate the in vivo healing properties of S. chilensis methanolic extract, a chronic gastric ulcer was induced in mice by 10 % acetic acid. Evaluation of tumor necrosis factor (TNF) levels was also performed at the site of the acetic acid-induced gastric ulcer. In parallel, effects on cell viability and cell proliferation of fibroblasts (L929 cells) were determined by in vitro trials. Firstly, the S. chilensis methanolic extract (100 or 300 mg/kg) reduced the ulcer area induced by ethanol/HCl in mice when compared to the vehicle group. Moreover, the S. chilensis extract (300 mg/kg) prevented the mucus depletion, the increase in MPO activity and the decrease in the GSH levels in the ulcerated gastric tissue. The S. chilensis extract also was able to decrease the indomethacin-induced gastric ulcer in rats at a dose of 100 mg/kg. The antisecretory effect of the extract (100 mg/kg, intraduodenal (i.d.)) was confirmed by the reduction in the volume and acidity in parallel to an increase in the pH of gastric content. In addition, quercitrin (1.38 mg/kg, but not 0.46 mg/kg) and afzelin (0.026 and 0.078 mg/kg) decreased the ethanol/HCl-induced gastric ulcer. In this model, quercitrin (1.38 mg/kg) prevented the depletion of gastric GSH content and both quercitrin (1.38 mg/kg) and afzelin (0.078 mg/kg) reduced the MPO activity. These compounds also inhibited the H(+),K(+)-ATPase activity at a concentration of 1-100 µg/ml. In addition, the participation of quercitrin and afzelin in these effects also was confirmed. Furthermore, after 4 days of the treatment, an oral administration of S. chilensis methanolic extract (100 mg/kg) reduced the area of the gastric ulcer induced by acetic acid and the regeneration of the gastric mucosa was accompanied by a reduction in gastric TNF levels. The healing properties of the extract also were confirmed by enhancement of proliferation and coverage of scratched wounds in a fibroblast monolayer. Together, our results confirmed the gastroprotective effect of S. chilensis methanolic extract as well as its gastric healing potential and provided some support to the traditional use of S. chilensis for prevention and treatment of gastric lesions in complementation to its known anti-inflammatory properties.
Assuntos
Antiulcerosos/farmacologia , Mucosa Gástrica/efeitos dos fármacos , Manosídeos/farmacologia , Metanol/química , Extratos Vegetais/farmacologia , Proantocianidinas/farmacologia , Quercetina/análogos & derivados , Solidago/química , Solventes/química , Úlcera Gástrica/prevenção & controle , Cicatrização/efeitos dos fármacos , Animais , Anti-Inflamatórios/farmacologia , Antiulcerosos/química , Antiulcerosos/isolamento & purificação , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patologia , Glutationa/metabolismo , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Concentração de Íons de Hidrogênio , Manosídeos/química , Manosídeos/isolamento & purificação , Camundongos , Fitoterapia , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Folhas de Planta , Plantas Medicinais , Proantocianidinas/química , Proantocianidinas/isolamento & purificação , Inibidores da Bomba de Prótons/farmacologia , Quercetina/química , Quercetina/isolamento & purificação , Quercetina/farmacologia , Coelhos , Ratos Wistar , Úlcera Gástrica/induzido quimicamente , Úlcera Gástrica/metabolismo , Úlcera Gástrica/patologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
UNLABELLED: The ileal lesions of Crohn's disease (CD) patients are colonized by adherent-invasive Escherichia coli (AIEC) bacteria. These bacteria adhere to mannose residues expressed by CEACAM6 on host cells in a type 1 pilus-dependent manner. In this study, we investigated different antagonists of FimH, the adhesin of type 1 pili, for their ability to block AIEC adhesion to intestinal epithelial cells (IEC). Monovalent and multivalent derivatives of n-heptyl α-d-mannoside (HM), a nanomolar antagonist of FimH, were tested in vitro in IEC infected with the AIEC LF82 strain and in vivo by oral administration to CEACAM6-expressing mice infected with LF82 bacteria. In vitro, multivalent derivatives were more potent than the monovalent derivatives, with a gain of efficacy superior to their valencies, probably owing to their ability to form bacterial aggregates. Of note, HM and the multi-HM glycoconjugates exhibited lower efficacy in vivo in decreasing LF82 gut colonization. Interestingly, HM analogues functionalized with an isopropylamide (1A-HM) or ß-cyclodextrin pharmacophore at the end of the heptyl tail (1CD-HM) exerted beneficial effects in vivo. These two compounds strongly decreased the amount of LF82 bacteria in the feces of mice and that of bacteria associated with the gut mucosa when administered orally at a dose of 10 mg/kg of body weight after infection. Importantly, signs of colitis and intestinal inflammation induced by LF82 infection were also prevented. These results highlight the potential of the antiadhesive compounds to treat CD patients abnormally colonized by AIEC bacteria and point to an alternative to the current approach focusing on blocking proinflammatory mediators. IMPORTANCE: Current treatments for Crohn's disease (CD), including immunosuppressive agents, anti-tumor necrosis factor alpha (anti-TNF-α) and anti-integrin antibodies, focus on the symptoms but not on the cause of the disease. Adherent-invasive Escherichia coli (AIEC) bacteria abnormally colonize the ileal mucosa of CD patients via the interaction of the mannose-specific adhesin FimH of type 1 pili with CEACAM6 mannosylated proteins expressed on the epithelial cell surface. Thus, we decided to develop an antiadhesive strategy based on synthetic FimH antagonists specifically targeting AIEC bacteria that would decrease intestinal inflammation. Heptylmannoside (HM)-based glycocompounds strongly inhibit AIEC adhesion to intestinal epithelial cells in vitro. The antiadhesive effect of two of these compounds of relatively simple chemical structure was also observed in vivo in AIEC-infected CEACAM6-expressing mice and was associated with a reduction in the signs of colitis. These results suggest a new therapeutic approach for CD patients colonized by AIEC bacteria, based on the development of synthetic FimH antagonists.