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1.
Methods Mol Biol ; 2291: 285-296, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33704759

RESUMO

Human intestinal organoid cultures established from crypt-derived stem cells truly revolutionized our approach to study intestinal epithelial physiology and pathologies as they can be propagated indefinitely and preserve the genetic signature of the donor and the gut segment specificity in culture. Here we describe human stem cell-derived colonoid monolayers as a reliable and reproducible model to study Shiga toxin-producing Escherichia coli (STEC) infection and STEC-caused pathologies of the whole colonic epithelium comprising a mixture of colonocytes, goblet, enteroendocrine, and other rare cells present in human colonic epithelial tissue.


Assuntos
Colo , Células Epiteliais , Infecções por Escherichia coli/metabolismo , Interações Hospedeiro-Patógeno , Mucosa Intestinal , Modelos Biológicos , Escherichia coli Shiga Toxigênica/fisiologia , Colo/metabolismo , Colo/microbiologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia
2.
Life Sci ; 272: 119194, 2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-33609541

RESUMO

AIM: The aim of the present study was to investigate the anti-inflammatory response mediated of the M1 muscarinic acetylcholine receptor (mAChR) during experimental colitis. MATERIAL AND METHODS: After the induction of 6% acetic acid colitis, mice were treated with McN-A-343 0.5, 1.0, and 1.5 mg/kg or dexamethasone (DEXA, 2.0 mg/kg) or pirenzepine (PIR, 10 mg/kg; M1 mAChR antagonist). Colonic inflammation was assessed by macroscopic and microscopic lesion scores, colonic wet weight, myeloperoxidase (MPO) activity, interleukin-1 beta (IL1-ß) levels and tumor necrosis factor alpha (TNF-α), glutathione (GSH), malondialdehyde (MDA) and nitrate and nitrite (NO3/NO2), mRNA expression of IKKα, nuclear factor kappa beta (NF-kB) and cyclooxygenase-2 (COX-2), as well protein expression of NF-kB and COX-2. RESULTS: Treatment with McN-A-343 at a concentration of 1.5 mg/kg showed a significant reduction in intestinal damage as well as a decrease in wet weight, MPO activity, pro-inflammatory cytokine concentration, markers of oxidative stress and expression of inflammatory mediators. The action of the M1 agonist by the administration of pirenzepine, which promoted the blocking of the mAChR M1-mediated anti-inflammatory response, has also been proven. CONCLUSION: The results suggest that peripheral colonic M1 mAChR is involved in reversing the pro-inflammatory effect of experimentally induced colitis, which may represent a promising therapeutic alternative for patients with ulcerative colitis.


Assuntos
Cloreto de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamônio/farmacologia , Colite Ulcerativa/tratamento farmacológico , Cloreto de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamônio/metabolismo , Animais , Colite/tratamento farmacológico , Colite/metabolismo , Colite Ulcerativa/metabolismo , Colo/metabolismo , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Dexametasona/farmacologia , Modelos Animais de Doenças , Glutationa/metabolismo , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Interleucina-1beta/metabolismo , Masculino , Malondialdeído/metabolismo , Camundongos , Agonistas Muscarínicos/farmacologia , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Receptor Muscarínico M1 , Fator de Necrose Tumoral alfa/metabolismo
3.
Nat Rev Gastroenterol Hepatol ; 18(4): 269-283, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33589829

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread to more than 200 countries and regions globally. SARS-CoV-2 is thought to spread mainly through respiratory droplets and close contact. However, reports have shown that a notable proportion of patients with coronavirus disease 2019 (COVID-19) develop gastrointestinal symptoms and nearly half of patients confirmed to have COVID-19 have shown detectable SARS-CoV-2 RNA in their faecal samples. Moreover, SARS-CoV-2 infection reportedly alters intestinal microbiota, which correlated with the expression of inflammatory factors. Furthermore, multiple in vitro and in vivo animal studies have provided direct evidence of intestinal infection by SARS-CoV-2. These lines of evidence highlight the nature of SARS-CoV-2 gastrointestinal infection and its potential faecal-oral transmission. Here, we summarize the current findings on the gastrointestinal manifestations of COVID-19 and its possible mechanisms. We also discuss how SARS-CoV-2 gastrointestinal infection might occur and the current evidence and future studies needed to establish the occurrence of faecal-oral transmission.


Assuntos
/fisiopatologia , Diarreia/fisiopatologia , Disbiose/fisiopatologia , Gastroenterite/fisiopatologia , Microbioma Gastrointestinal , Náusea/fisiopatologia , Vômito/fisiopatologia , Dor Abdominal/fisiopatologia , Animais , Anorexia/fisiopatologia , Linhagem Celular , Colo/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Fezes/química , Gastroenterite/virologia , Humanos , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Complexo Antígeno L1 Leucocitário/metabolismo , Organoides , RNA Viral , /metabolismo , Serina Endopeptidases/metabolismo , Carga Viral , Eliminação de Partículas Virais
4.
Nat Rev Cancer ; 21(4): 239-256, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33627798

RESUMO

Cancer is a clonal disorder derived from a single ancestor cell and its progenies that are positively selected by acquisition of 'driver mutations'. However, the evolution of positively selected clones does not necessarily imply the presence of cancer. On the contrary, it has become clear that expansion of these clones in phenotypically normal or non-cancer tissues is commonly seen in association with ageing and/or in response to environmental insults and chronic inflammation. Recent studies have reported expansion of clones harbouring mutations in cancer driver genes in the blood, skin, oesophagus, bronchus, liver, endometrium and bladder, where the expansion could be so extensive that tissues undergo remodelling of an almost entire tissue. The presence of common cancer driver mutations in normal tissues suggests a strong link to cancer development, providing an opportunity to understand early carcinogenic processes. Nevertheless, some driver mutations are unique to normal tissues or have a mutation frequency that is much higher in normal tissue than in cancer, indicating that the respective clones may not necessarily be destined for evolution to cancer but even negatively selected for carcinogenesis depending on the mutated gene. Moreover, tissues that are remodelled by genetically altered clones might define functionalities of aged tissues or modified inflammatory processes. In this Review, we provide an overview of major findings on clonal expansion in phenotypically normal or non-cancer tissues and discuss their biological significance not only in cancer development but also in ageing and inflammatory diseases.


Assuntos
Envelhecimento/genética , Proliferação de Células/genética , Evolução Clonal , Células Clonais/citologia , Neoplasias/genética , Envelhecimento/patologia , Anemia Aplástica/genética , Anemia Aplástica/patologia , Esôfago de Barrett/genética , Esôfago de Barrett/patologia , Brônquios/citologia , Brônquios/metabolismo , Carcinogênese , Colite Ulcerativa/genética , Colite Ulcerativa/patologia , Colo/citologia , Colo/metabolismo , Endométrio/citologia , Endométrio/metabolismo , Esôfago/citologia , Esôfago/metabolismo , Feminino , Mucosa Gástrica/metabolismo , Humanos , Inflamação/genética , Inflamação/patologia , Fígado/citologia , Fígado/metabolismo , Cirrose Hepática/genética , Cirrose Hepática/patologia , Masculino , Metaplasia , Mutação , Neoplasias/patologia , Oncogenes/genética , Pele/citologia , Pele/metabolismo , Estômago/patologia , Urotélio/citologia , Urotélio/metabolismo
5.
Nat Commun ; 12(1): 836, 2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33547321

RESUMO

Dynamic regulation of intestinal cell differentiation is crucial for both homeostasis and the response to injury or inflammation. Sprouty2, an intracellular signaling regulator, controls pathways including PI3K and MAPKs that are implicated in differentiation and are dysregulated in inflammatory bowel disease. Here, we ask whether Sprouty2 controls secretory cell differentiation and the response to colitis. We report that colonic epithelial Sprouty2 deletion leads to expanded tuft and goblet cell populations. Sprouty2 loss induces PI3K/Akt signaling, leading to GSK3ß inhibition and epithelial interleukin (IL)-33 expression. In vivo, this results in increased stromal IL-13+ cells. IL-13 in turn induces tuft and goblet cell expansion in vitro and in vivo. Sprouty2 is downregulated by acute inflammation; this appears to be a protective response, as VillinCre;Sprouty2F/F mice are resistant to DSS colitis. In contrast, Sprouty2 is elevated in chronic colitis and in colons of inflammatory bowel disease patients, suggesting that this protective epithelial-stromal signaling mechanism is lost in disease.


Assuntos
Colite/genética , Glicogênio Sintase Quinase 3 beta/genética , Homeostase/genética , Interleucina-33/genética , Proteínas de Membrana/genética , Proteínas Serina-Treonina Quinases/genética , Animais , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Criança , Colite/induzido quimicamente , Colite/metabolismo , Colite/patologia , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Feminino , Regulação da Expressão Gênica , Glicogênio Sintase Quinase 3 beta/metabolismo , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/metabolismo , Células Caliciformes/patologia , Células HT29 , Homeostase/efeitos dos fármacos , Humanos , Interleucina-33/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Dodecilsulfato de Sódio/administração & dosagem
6.
BMC Bioinformatics ; 22(1): 3, 2021 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-33407079

RESUMO

BACKGROUND: Hydrogen cross-feeding microbes form a functionally important subset of the human colonic microbiota. The three major hydrogenotrophic functional groups of the colon: sulphate-reducing bacteria (SRB), methanogens and reductive acetogens, have been linked to wide ranging impacts on host physiology, health and wellbeing. RESULTS: An existing mathematical model for microbial community growth and metabolism was combined with models for each of the three hydrogenotrophic functional groups. The model was further developed for application to the colonic environment via inclusion of responsive pH, host metabolite absorption and the inclusion of host mucins. Predictions of the model, using two existing metabolic parameter sets, were compared to experimental faecal culture datasets. Model accuracy varied between experiments and measured variables and was most successful in predicting the growth of high relative abundance functional groups, such as the Bacteroides, and short chain fatty acid (SCFA) production. Two versions of the colonic model were developed: one representing the colon with sequential compartments and one utilising a continuous spatial representation. When applied to the colonic environment, the model predicted pH dynamics within the ranges measured in vivo and SCFA ratios comparable to those in the literature. The continuous version of the model simulated relative abundances of microbial functional groups comparable to measured values, but predictions were sensitive to the metabolic parameter values used for each functional group. Sulphate availability was found to strongly influence hydrogenotroph activity in the continuous version of the model, correlating positively with SRB and sulphide concentration and negatively with methanogen concentration, but had no effect in the compartmentalised model version. CONCLUSIONS: Although the model predictions compared well to only some experimental measurements, the important features of the colon environment included make it a novel and useful contribution to modelling the colonic microbiota.


Assuntos
Bactérias/metabolismo , Colo , Microbioma Gastrointestinal , Hidrogênio/metabolismo , Colo/metabolismo , Colo/microbiologia , Humanos , Modelos Biológicos , Sulfetos/metabolismo
7.
Int J Mol Sci ; 22(3)2021 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-33498392

RESUMO

Four drugs are currently approved for the treatment of Alzheimer's disease (AD) by the FDA. Three of these drugs-donepezil, rivastigmine, and galantamine-belong to the class of acetylcholine esterase inhibitors. Memantine, a NMDA receptor antagonist, represents the fourth and a combination of donepezil and memantine the fifth treatment option. Recently, the gut and its habitants, its microbiome, came into focus of AD research and added another important factor to therapeutic considerations. While the first data provide evidence that AD patients might carry an altered microbiome, the influence of administered drugs on gut properties and commensals have been largely ignored so far. However, the occurrence of digestive side effects with these drugs and the knowledge that cholinergic transmission is crucial for several gut functions enforces the question if, and how, this medication influences the gastrointestinal system and its microbial stocking. Here, we investigated aspects such as microbial viability, colonic propulsion, and properties of enteric neurons, affected by assumed intestinal concentration of the four drugs using the mouse as a model organism. All ex vivo administered drugs revealed no direct effect on fecal bacteria viability and only a high dosage of memantine resulted in reduced biofilm formation of E. coli. Memantine was additionally the only compound that elevated calcium influx in enteric neurons, while all acetylcholine esterase inhibitors significantly reduced esterase activity in colonic tissue specimen and prolonged propulsion time. Both, acetylcholine esterase inhibitors and memantine, had no effect on general viability and neurite outgrowth of enteric neurons. In sum, our findings indicate that all AD symptomatic drugs have the potential to affect distinct intestinal functions and with this-directly or indirectly-microbial commensals.


Assuntos
Inibidores da Colinesterase/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Memantina/farmacologia , Fármacos Neuroprotetores/farmacologia , Animais , Sinalização do Cálcio , Células Cultivadas , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/microbiologia , Colo/fisiologia , Sistema Nervoso Entérico/efeitos dos fármacos , Sistema Nervoso Entérico/metabolismo , Sistema Nervoso Entérico/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Crescimento Neuronal
8.
Food Chem ; 348: 129071, 2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-33493843

RESUMO

Dietary fibers (DFs) consumption promotes a healthier gut through colonic fermentation and the modulation of different types of gut bacteria. The aim of this study is to evaluate the production of short-chain fatty acids (SCFA), metabolization of polysaccharides, and changes in the bacterial profile related to DFs extracted from the pulp of unripe and ripe papayas, using a batch colonic in vitro fermentation model. Our results show that fermentation of DFs from papayas induce the production of SCFAs and are utilized in different ways by intestinal microbiota. DFs from ripe papayas showed faster degradation by human gut microorganisms due to higher level of water-soluble polysaccharides. The fermentation of unripe papaya fibers increased the abundance of microorganisms belonging to family Clostridiaceae and genera Coprobacillus, Bulleidia, and Slackia, whereas both fibers increased Clostridium and Bacteroides, showing fruit ripeness affects the fermentation pattern of fruit fibers and their probable beneficial health aspects.


Assuntos
Carica/química , Colo/metabolismo , Colo/microbiologia , Fibras na Dieta/análise , Fermentação , Microbioma Gastrointestinal/efeitos dos fármacos , Polissacarídeos/metabolismo , Colo/efeitos dos fármacos , Humanos , Polissacarídeos/farmacologia
9.
Nat Immunol ; 22(2): 216-228, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33462454

RESUMO

CD4+ effector lymphocytes (Teff) are traditionally classified by the cytokines they produce. To determine the states that Teff cells actually adopt in frontline tissues in vivo, we applied single-cell transcriptome and chromatin analyses to colonic Teff cells in germ-free or conventional mice or in mice after challenge with a range of phenotypically biasing microbes. Unexpected subsets were marked by the expression of the interferon (IFN) signature or myeloid-specific transcripts, but transcriptome or chromatin structure could not resolve discrete clusters fitting classic helper T cell (TH) subsets. At baseline or at different times of infection, transcripts encoding cytokines or proteins commonly used as TH markers were distributed in a polarized continuum, which was functionally validated. Clones derived from single progenitors gave rise to both IFN-γ- and interleukin (IL)-17-producing cells. Most of the transcriptional variance was tied to the infecting agent, independent of the cytokines produced, and chromatin variance primarily reflected activities of activator protein (AP)-1 and IFN-regulatory factor (IRF) transcription factor (TF) families, not the canonical subset master regulators T-bet, GATA3 or RORγ.


Assuntos
Bactérias/patogenicidade , Infecções Bacterianas/microbiologia , Linfócitos T CD4-Positivos/microbiologia , Linfócitos T CD4-Positivos/parasitologia , Colo/microbiologia , Colo/parasitologia , Microbioma Gastrointestinal , Heligmosomatoidea/patogenicidade , Enteropatias Parasitárias/parasitologia , Animais , Bactérias/imunologia , Infecções Bacterianas/genética , Infecções Bacterianas/imunologia , Infecções Bacterianas/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Cromatina/genética , Cromatina/metabolismo , Citrobacter rodentium/imunologia , Citrobacter rodentium/patogenicidade , Colo/imunologia , Colo/metabolismo , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Heligmosomatoidea/imunologia , Interações Hospedeiro-Patógeno , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Enteropatias Parasitárias/genética , Enteropatias Parasitárias/imunologia , Enteropatias Parasitárias/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Nematospiroides dubius/imunologia , Nematospiroides dubius/patogenicidade , Nippostrongylus/imunologia , Nippostrongylus/patogenicidade , Fenótipo , Salmonella enterica/imunologia , Salmonella enterica/patogenicidade , Análise de Célula Única , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Transcriptoma
10.
J Agric Food Chem ; 69(3): 992-1002, 2021 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-33428422

RESUMO

Anthocyanins have been known for their health benefits. However, the in vivo digestion and absorption of anthocyanins through the gastrointestinal tract have not been fully clarified, creating challenges for understanding why anthocyanins have high biological activities and purported low bioavailability in vivo. Twenty-seven male rats were intubated with a 500 mg/kg dose of cyanidin-3-glucoside (C3G). Samples from rats' stomach, duodenum, jejunum, ileum, colon, and serum were collected at 0.5, 1, 2, 3, 4, 5, 6, 12, and 24 h after intubation. Three rats without C3G were used as the control with samples collected at 0 h. C3G and its metabolites in each sample were analyzed using high-performance liquid chromatography-PDA-electrospray ionization-MS/MS. These in vivo studies' results unequivocally demonstrated that cyanidin and phenolic acids were the primary C3G metabolites absorbed, mainly in the jejunum and ileum, between 1 and 5 h post-ingestion. We speculate that C3G uses phloroglucinaldehyde and protocatechuic acid metabolic pathways in its metabolism in vivo.


Assuntos
Antocianinas/metabolismo , Hidroxibenzoatos/metabolismo , Íleo/metabolismo , Jejuno/metabolismo , Animais , Antocianinas/química , Disponibilidade Biológica , Cromatografia Líquida de Alta Pressão , Colo/metabolismo , Hidroxibenzoatos/química , Masculino , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas em Tandem
11.
Carbohydr Polym ; 255: 117367, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33436200

RESUMO

The aim of this work was to develop a comprehensive workflow to elucidate molecular features of artichoke pectic oligosaccharides (POS) contributing to high potential prebiotic activity. First, obtainment of artichoke POS by Pectinex® Ultra-Olio was optimised using an artificial neural network. Under optimal conditions (pH 6.86; 1.5 h; enzyme dose 520.5 U/g pectin) POS yield was 624 mg/g pectin. Oligosaccharide structures (Mw < 1.3 kDa) were characterised by MALDI-TOF-MS. Then, conformational analysis of glycosidic bonds was performed by replica exchange molecular dynamics simulations and interaction mechanisms between POS and several microbial glycosidases were proposed by molecular modelling. Chemical information was integrated in virtual simulations of colonic fermentation. Highest hydrolysis rate was obtained for GalA-Rha-GalA trisaccharide, while the presence of partial negative charges and high radius of gyration enhance short chain fatty acid formation in distal colon. Established structure-activity relationships could help the rational design of prebiotics and clinical trials.


Assuntos
Colo/metabolismo , Cynara scolymus/química , Ácidos Graxos Voláteis/biossíntese , Oligossacarídeos/metabolismo , Pectinas/metabolismo , Prebióticos/análise , Análise Fatorial , Ácidos Graxos Voláteis/química , Fermentação , Concentração de Íons de Hidrogênio , Hidrólise , Simulação de Dinâmica Molecular , Redes Neurais de Computação , Oligossacarídeos/química , Oligossacarídeos/isolamento & purificação , Pectinas/química , Pectinas/isolamento & purificação , Extratos Vegetais/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Relação Estrutura-Atividade , Interface Usuário-Computador
12.
Phytomedicine ; 80: 153372, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33113505

RESUMO

BACKGROUND: Feiyangchangweiyan capsule (FYC) is a traditional Chinese medicine formulation used in the clinical treatment of acute and chronic gastroenteritis and bacterial dysentery. However, the effect of FYC on ulcerative colitis (UC) and the mechanism thereof remains unknown. PURPOSE: To investigate the protective effect of FYC on UC mice induced by dextran sulfate sodium and illustrate the potential mechanism of this effect. METHODS: Here, we established a model of UC mice by dextran sulfate sodium and administered with FYC. The disease activity index (DAI), colon length, myeloperoxidase (MPO) content in serum, pathological structure and ultrastructural changes, and inflammatory cell infiltration of colon tissue were evaluated. Transcriptome and 16S rDNA sequencing were employed to illuminate the mechanism of FYC in the protection of UC mice. RESULTS: FYC significantly alleviates the pathological damage and the infiltration of inflammatory cells in colon tissue of dextran sulfate sodium induced UC mice, rescues shortened colon length, reduces DAI score, MPO content in serum, and pro-inflammatory factors including IL-1ß, IL-6, CCL11, MCP-1 and MIP-2, and increases anti-inflammatory factors such as IL-10. Transcriptomics revealed that Oncostatin M (OSM) and its receptor (OSMR) are the critical pathway for UC treatment by FYC. OSM and OSMR increased in UC mice compared to control mice, and decreased with FYC, which was verified via measurement of OSM and OSMR mRNA and protein levels. Furthermore, we observed that FYC modulates intestinal microbiome composition (e.g., the proportion of Barnesiella/Proteobacteria) by affecting the inflammatory factors. CONCLUSION: FYC exerts an effect on UC by inhibiting the OSM/OSMR pathway and regulating inflammatory factors to improve the intestinal flora.


Assuntos
Colite Ulcerativa/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Subunidade beta de Receptor de Oncostatina M/metabolismo , Oncostatina M/metabolismo , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Cápsulas , Quimiocinas/sangue , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/microbiologia , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Colo/ultraestrutura , Citocinas/sangue , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Microbioma Gastrointestinal/genética , Masculino , Camundongos Endogâmicos C57BL , Oncostatina M/genética , Subunidade beta de Receptor de Oncostatina M/genética , Substâncias Protetoras/farmacologia
14.
Anal Chem ; 93(3): 1393-1400, 2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33373197

RESUMO

In quantitative mass spectrometry imaging (MSI), the gold standard adds a single structural homologue of the target compound at a known concentration to the sample. This internal standard enables to map the detected intensity of the target molecule against an external calibration curve. This approach, however, ignores local noise levels and disproportional ion suppression effects, which might depend on the concentration of the target compound. To overcome these issues, we propose a novel approach that applies several isotopically labeled versions, each at a different concentration, to the sample. This allows creating individual internal calibration curves for every MSI pixel. As proof of principle, we have quantified an endogenous peptide of histone H4 by matrix-assisted laser desorption/ionization-Q-MSI (MALDI-Q-MSI), using a mixture of three isotopically labeled versions. The usage of a fourth label allowed us to compare the gold standard to our multilabel approach. We observed substantial heterogeneity in ion suppression across the tissue, which disclosed itself as varying slopes in the per-pixel regression analyses. These slopes were histology-dependent and differed from each other by up to a factor of 4. The results were validated by liquid chromatography-mass spectrometry (LC-MS), exhibiting a high agreement between LC-MS and MALDI-Q-MSI (Pearson correlation r = 0.87). A comparison between the multilabel and single-label approaches revealed a higher accuracy for the multilabel method when the local target compound concentration differed too much from the concentration of the single label. In conclusion, we show that the multilabel approach provides superior quantitation compared to a single-label approach, in case the target compound is inhomogeneously distributed at a wide concentration range in the tissue.


Assuntos
Histonas/química , Peptídeos/análise , Animais , Colo/química , Colo/metabolismo , Espectrometria de Massas , Suínos
15.
Biomed Pharmacother ; 134: 111129, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33348308

RESUMO

Ulcerative colitis (UC) is an inflammatory bowel disease with complex pathogenesis, which is affected by genetic factors, intestinal immune status and intestinal microbial homeostasis. Intestinal epithelial barrier defect is crucial to the development of UC. Berberine, extracted from Chinese medicine, can identify bitter taste receptor on intestinal Tuft cells and activate IL-25-ILC2-IL-13 immune pathway to impair damaged intestinal tract by promoting differentiation of intestinal stem cells, which might be a potential approach for the treatment of UC.


Assuntos
Anti-Inflamatórios/uso terapêutico , Berberina/uso terapêutico , Colite Ulcerativa/tratamento farmacológico , Colo/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Animais , Colite Ulcerativa/imunologia , Colite Ulcerativa/metabolismo , Colite Ulcerativa/patologia , Colo/imunologia , Colo/metabolismo , Colo/patologia , Citocinas/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Receptores Acoplados a Proteínas-G/agonistas , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais , Células-Tronco/imunologia , Células-Tronco/metabolismo , Células-Tronco/patologia
16.
Biomed Pharmacother ; 134: 111140, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33360052

RESUMO

Theanine and theobromine are abundantly present in tea and cocoa, respectively. This study was performed to assess the chemopreventive effects of these phytochemicals, alone or together, on dimethylhydrazine (DMH)-induced colon cancer. Thirty male Wistar rats were divided into five groups and subcutaneously injected with saline (negative control group) or 30 mg/kg DMH (the other groups) two times/week for 12 weeks. The negative and positive control animals were orally treated with drinking water, and the other groups were gavaged with theanine (400 mg/kg), theobromine (100 mg/kg), or their mixture for two weeks before and throughout the injection period. At the end of the study, the morphological and histopathological features, Ki-67 proliferation marker, and the expression of Akt/mTOR, JAK2/STAT3, MAPK/ERK, and TGF-ß/Smad pathways were investigated. Theanine and theobromine, alone or together, reduced the number of cancerous and precancerous lesions, the volume of tumors, the Ki-67 immunostaining, and the expression of Akt/mTOR and JAK2/STAT3 oncogenic pathways. The simultaneous treatment was more effective in the down-regulation of Akt and mTOR compared to either theanine or theobromine alone. Theobromine administration also caused more inhibitory effects on the Ki-67 and Akt/mTOR expression than theanine. Besides, all dietary interventions increased the mRNA and protein expression of Smad2. In conclusion, theanine and theobromine, alone and in combination, inhibited tumorigenesis through down-regulation of the Akt/mTOR and JAK2/STAT3 pathways and an increment of the Smad2 tumor suppressor. The inhibition of the Akt/mTOR pathway was more pronounced by simultaneous treatment.


Assuntos
Anticarcinógenos/farmacologia , Colo/efeitos dos fármacos , Neoplasias do Colo/prevenção & controle , Glutamatos/farmacologia , Teobromina/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Dimetilidrazinas , Modelos Animais de Doenças , Janus Quinase 2/metabolismo , Antígeno Ki-67/metabolismo , Masculino , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Wistar , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo
17.
Biomed Pharmacother ; 133: 111028, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33378943

RESUMO

Aspirin is one of the most commonly prescribed medications. Evidence shows that it can even treat and prevent intestinal tumors. However, it has also caused a great deal of controversy due to its intestinal side effects. We therefore explored whether aspirin was beneficial or harmful to the intestines. We used aspirin continuously interfered with C57BL/6 J mice for 48 weeks, examining their intestinal tissues at 13, 26 and 48 weeks to determine the drug's effect on the intestines. In addition, we used flow cytometry (FCM) used to detect T cells and expression of T-cell immunoreceptor with immunoglobulin (Ig)- and tyrosine-based inhibitory motif (ITIM) domain (TIGIT) on their surfaces to determine aspirin's immunomodulatory effects. The results showed that long-term aspirin intervention could reverse damage to the intestines, an effect related to the drug's significant inhibitory effect on TIGIT. The change in TIGIT level could regulate T-cell subsets, so that counts of Cluster of Differentiation 4 (CD4)+/chemokine (C-X3-C motif) receptor 3 (CXCR3)+ T-helper 1 (Th1) cells and CD4+/interleukin-4 (IL-4)+ Th2 cells increased, while those of CD4+/C-C chemokine receptor type 6 (CCR6)+ Th17 cells and CD4+/CD25+ regulatory T cells (Tregs) decreased. In summary, we demonstrated that long-term aspirin intervention could inhibit TIGIT, regulating T cells to reverse damage to the intestines. Furthermore, aspirin is a potential therapy for diseases related to an increase in TIGIT.


Assuntos
Aspirina/toxicidade , Colo/efeitos dos fármacos , Receptores Imunológicos/metabolismo , Reto/efeitos dos fármacos , Subpopulações de Linfócitos T/efeitos dos fármacos , Animais , Colo/imunologia , Colo/metabolismo , Colo/patologia , Regulação para Baixo , Masculino , Camundongos Endogâmicos C57BL , Fenótipo , Reto/imunologia , Reto/metabolismo , Reto/patologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th1/metabolismo , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Células Th17/metabolismo , Células Th2/efeitos dos fármacos , Células Th2/imunologia , Células Th2/metabolismo , Fatores de Tempo
18.
PLoS One ; 15(12): e0241148, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33332369

RESUMO

BACKGROUND: Intra-tumor heterogeneity (ITH) of colorectal cancer (CRC) complicates molecular tumor classification, such as transcriptional subtyping. Differences in cellular states, biopsy cell composition, and tumor microenvironment may all lead to ITH. Here we analyze ITH at the transcriptomic and proteomic levels to ascertain whether subtype discordance between multiregional biopsies reflects relevant biological ITH or lack of classifier robustness. Further, we study the impact of tumor location on ITH. METHODS: Multiregional biopsies from stage II and III CRC tumors were analyzed by RNA sequencing (41 biopsies, 14 tumors) and multiplex immune protein analysis (89 biopsies, 29 tumors). CRC subtyping was performed using consensus molecular subtypes (CMS), CRC intrinsic subtypes (CRIS), and TUMOR types. ITH-scores and network maps were defined to determine the origin of heterogeneity. A validation cohort was used with one biopsy per tumor (162 tumors). RESULTS: Overall, inter-tumor transcriptional variation exceeded ITH, and subtyping calls were frequently concordant between multiregional biopsies. Still, some tumors had high transcriptional ITH and were classified discordantly. Subtyping of proximal MSS tumors were discordant for 50% of the tumors, this ITH was related to differences in the microenvironment. Subtyping of distal MSS tumors were less discordant, here the ITH was more cancer-cell related. The subtype discordancy reflected actual molecular ITH within the tumors. The relevance of the subtypes was reflected at protein level where several inflammation markers were significantly increased in immune related transcriptional subtypes, which was verified in an independent cohort (Wilcoxon rank sum test; p<0.05). Unsupervised hierarchical clustering of the protein data identified large ITH at protein level; as the multiregional biopsies clustered together for only 9 out of 29 tumors. CONCLUSION: Our transcriptomic and proteomic analyses show that the tumor location along the colorectum influence the ITH of CRC, which again influence the concordance of subtyping.


Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Proteoma/metabolismo , Transcriptoma/genética , Idoso , Idoso de 80 Anos ou mais , Colo/metabolismo , Colo/patologia , Neoplasias Colorretais/patologia , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Masculino , Pessoa de Meia-Idade , Mapas de Interação de Proteínas , Proteômica , RNA-Seq , Reto/metabolismo , Reto/patologia , Distribuição Tecidual
19.
Molecules ; 25(24)2020 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-33371436

RESUMO

In the present study, the synthesis of gold nanoparticles (AuNPs) loaded with methotrexate (MTX) has been carried out in order to obtain controlled size and monodispersed nanocarriers of around 20 nm. The characterization study shows metallic AuNPs with MTX polydispersed on the surface. MTX is linked by the replacement of citrate by the MTX carboxyl group. The drug release profiles show faster MTX release when it is conjugated, which leads to the best control of plasma concentration. Moreover, the enhanced release observed at pH 5 could take advantage of the pH gradients that exist in tumor microenvironments to achieve high local drug concentrations. AuNP-MTX conjugates were tested by flow cytometry against lung (A-549) and colon (HTC-116) cancer cell lines. Results for A-549 showed a weaker dose-response effect than for colon cancer ones. This could be related to the presence of folate receptors in line HTC-116 in comparison to line A-549, supporting the specific uptake of folate-conjugated AuNP-MTX by folate receptor positive tumor cells. Conjugates exhibited considerably higher cytotoxic effects compared with the effects of equal doses of free MTX. Annexin V-PI tests sustained the cell death mechanism of apoptosis, which is normally disabled in cancer cells.


Assuntos
Antineoplásicos/farmacologia , Neoplasias do Colo/tratamento farmacológico , Ouro/química , Neoplasias Pulmonares/tratamento farmacológico , Nanopartículas Metálicas/química , Metotrexato/farmacologia , Células A549 , Anexina A5/metabolismo , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Colo/efeitos dos fármacos , Colo/metabolismo , Neoplasias do Colo/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Liberação Controlada de Fármacos/fisiologia , Ácido Fólico/farmacologia , Células HCT116 , Humanos , Concentração de Íons de Hidrogênio , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Neoplasias Pulmonares/metabolismo , Metotrexato/química , Microambiente Tumoral/efeitos dos fármacos
20.
Int J Mol Sci ; 21(24)2020 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-33322729

RESUMO

(1) Background: We characterized a novel animal model with obesity-induced constipation because constipation is rarely known in genetically engineered mice (GEM); (2) Methods: The changes in the constipation parameters and mechanisms were analyzed in CRISPR-Cas9-mediated leptin (Lep) knockout (KO) mice from eight to 24 weeks; (3) Results: Significant constipation phenotypes were observed in the Lep KO mice since 16 weeks old. These mice showed a significant decrease in the gastrointestinal motility, mucosal layer thickness and ability for mucin secretion as well as the abnormal ultrastructure of Lieberkühn crypts in the transverse colon. The density or function of the enteric neurons, intestinal Cajal cells (ICC), smooth muscle cells, and the concentration of gastrointestinal (GI) hormones for the GI motility were remarkably changed in Lep KO mice. The downstream signaling pathway of muscarinic acetylcholine receptors (mAChRs) were activated in Lep KO mice, while the expression of adipogenesis-regulating genes were alternatively reduced in the transverse colon of the same mice; (4) Conclusions: These results provide the first strong evidence that Lep KO mice can represent constipation successfully through dysregulation of the GI motility mediated by myenteric neurons, ICC, and smooth muscle cells in the transverse colon during an abnormal function of the lipid metabolism.


Assuntos
Colo/metabolismo , Constipação Intestinal/metabolismo , Motilidade Gastrointestinal , Leptina/metabolismo , Miócitos de Músculo Liso/metabolismo , Receptores Muscarínicos/metabolismo , Adipogenia/genética , Animais , Aquaporina 3/metabolismo , Aquaporinas/metabolismo , Sistemas CRISPR-Cas , Colo/citologia , Colo/patologia , Colo/ultraestrutura , Constipação Intestinal/complicações , Constipação Intestinal/genética , Constipação Intestinal/patologia , Modelos Animais de Doenças , Feminino , Hormônios Gastrointestinais/metabolismo , Motilidade Gastrointestinal/genética , Motilidade Gastrointestinal/fisiologia , Células Intersticiais de Cajal/metabolismo , Leptina/genética , Metabolismo dos Lipídeos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Mucinas/metabolismo , Neurônios/metabolismo , Obesidade/complicações , Obesidade/genética , Transdução de Sinais/genética
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