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1.
J Agric Food Chem ; 67(49): 13758-13766, 2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31789514

RESUMO

Probiotics, such as Lactobacillus, have been proven to be effective in maintaining intestinal homeostasis. The modulatory effect of Lactobacillus on intestinal epithelial development in early life is still unclear. In this study, Lactobacillus isolates with good probiotic abilities were screened and orally administered to detect their regulatory effect on intestinal development in chickens. L. reuteri 22 was isolated from chickens and chosen for subsequent chicken experiments due to its strong acid and bile salt resistance and ability to adhere to epithelial cells. The 3-day-old chickens were orally administrated with 108 CFU L. reuteri 22 for consecutive 7 days. L. reuteri 22 increased Lgr5 mRNA expression (3.23 ± 0.40, P = 0.001) and activated the Wnt/ß-catenin signaling pathway, with increasing expression of proliferating cell nuclear antigen (PCNA) (49.27 ± 9.81, P = 0.021) to support the proliferation of chicken intestinal epithelial cells. Moreover, L. reuteri 22 also inhibited the Notch signaling pathway to induce intestinal stem cell differentiation into goblet cells with increased mucin 2 (Muc-2) expression (1.72 ± 0.34, P = 0.047). L. reuteri 22 significantly enhanced lysozyme mRNA expression (2.32 ± 0.55, P = 0.019) to improve intestinal innate mucosal immunity. This study demonstrated that L. reuteri administration could regulate chicken intestinal epithelium development to ensure the function of the intestinal mucosal barrier, which is beneficial for newborn animals.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/citologia , Células Caliciformes/efeitos dos fármacos , Lactobacillus reuteri/fisiologia , Probióticos/farmacologia , Animais , Galinhas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Caliciformes/citologia , Células Caliciformes/metabolismo , Intestinos/citologia , Intestinos/efeitos dos fármacos , Mucina-2/genética , Mucina-2/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Transdução de Sinais/efeitos dos fármacos , beta Catenina/genética , beta Catenina/metabolismo
2.
J Biochem Mol Toxicol ; 33(11): e22397, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31557363

RESUMO

Fumonisins (Fums) are mycotoxins widely distributed in crops and feed, and ingestion of Fums-contaminated crops is harmful to animal health. The purpose of this study is to explore the effect of Fum B1 (FB1 ) on barrier functions of porcine intestinal epithelial cells, IPEC-J2, to clarify the intestinal toxicity of Fums in pigs. The results showed that the persistent treatment of FB1 significantly decreased the viability of IPEC-J2. Moreover, the expressions of Claudin 1, Occludin, Zonula Occluden-1 (ZO-1) on the messenger RNA (mRNA), and protein levels and MUC1 on the mRNA level were significantly inhibited after FB1 treatment, while the mRNA relative expression level of MUC2 was clearly increased. FB1 also enhanced the monolayer cell permeability of IPEC-J2. Importantly, FB1 promoted the expression of phosphorylated extracellular regulated protein kinase (p-ERK1/2 ). These data suggest that long-term treatment of FB1 can suppress IPEC-J2 proliferation, damage tight junctions of IPEC-J2, and regulate expression of mucins to induce the damage of barrier functions of porcine intestinal epithelial cells, which may be associated with the ERK1/2 phosphorylation pathway.


Assuntos
Células Epiteliais/metabolismo , Fumonisinas/farmacologia , Mucosa Intestinal/citologia , Micotoxinas/farmacologia , Permeabilidade/efeitos dos fármacos , Junções Íntimas/efeitos dos fármacos , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Fusarium/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mucina-1/genética , Mucina-1/metabolismo , Mucina-2/genética , Mucina-2/metabolismo , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos , Suínos , Proteínas de Junções Íntimas/genética , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/metabolismo
3.
Nat Commun ; 10(1): 4306, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31541089

RESUMO

The mucus layer is the first line of innate host defense in the gut that protects the epithelium by spatially separating commensal bacteria. MUC2 mucin is produced and stored by goblet cells that is constitutively exocytosed or hyper secreted upon sensing a threat. How coordinated mucus exocytosis maintains homeostasis in the intestinal epithelium and modulates the immunological landscape remains elusive. Here we describe how the vesicle SNARE protein VAMP8 coordinates mucin exocytosis from goblet cells. Vamp8-/- exhibit a mild pro-inflammatory state basally due to an altered mucus layer and increased encounters with microbial antigens. Microbial diversity shifts to a detrimental microbiota with an increase abundance of pathogenic and mucolytic bacteria. To alleviate the heavy microbial burden and inflammatory state basally, Vamp8-/- skews towards tolerance. Despite this, Vamp8-/- is highly susceptible to both chemical and infectious colitis demonstrating the fragility of the intestinal mucosa without proper mucus exocytosis mechanisms.


Assuntos
Colo/metabolismo , Exocitose/fisiologia , Células Caliciformes/metabolismo , Mucosa Intestinal/metabolismo , Mucina-2/metabolismo , Proteínas R-SNARE/metabolismo , Animais , Biodiversidade , Colo/patologia , Citocinas/metabolismo , Células Caliciformes/patologia , Homeostase , Humanos , Intestinos/patologia , Camundongos Knockout , Microbiota , Mucina-2/genética , Muco/metabolismo , Fenótipo , Proteínas R-SNARE/genética , Proteínas SNARE/metabolismo
4.
J Agric Food Chem ; 67(35): 9831-9839, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31407897

RESUMO

Probiotic lactobacilli and their exopolysaccharides (EPS) are thought to modulate mucosal homeostasis; however, their mechanisms remain elusive. Thus, we tried to clarify the role of exopolysaccharides from Lactobacillus plantarum NCU116 (EPS116) in the intestinal mucosal homeostasis. Our results indicated that EPS116 regulated the colon mucosal healing and homeostasis, enhanced the goblet cell differentiation, and promoted the expression of Muc2 gene in vivo and in vitro. Further experiments showed that EPS116 promoted the expression and phosphorylation of transcription factor c-Jun and facilitated its binding to the promoter of Muc2. Moreover, knocking down c-Jun or inhibiting its function in LS 174T cells treated with EPS116 led to decreased expression of Muc2, implying that EPS116 promoted the colonic mucosal homeostasis and Muc2 expression via c-Jun. Therefore, our study uncovered a novel model where EPS116 enhanced colon mucosal homeostasis by controlling the epithelial cell differentiation and c-Jun/Muc2 signaling.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Colo/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Lactobacillus plantarum/química , Mucina-2/metabolismo , Polissacarídeos Bacterianos/farmacologia , Proteínas Proto-Oncogênicas c-jun/metabolismo , Animais , Linhagem Celular Tumoral , Colo/citologia , Colo/metabolismo , Colo/fisiopatologia , Homeostase/efeitos dos fármacos , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mucina-2/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-jun/genética , Transdução de Sinais/efeitos dos fármacos
5.
Redox Biol ; 26: 101269, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31330482

RESUMO

The gastrointestinal (GI) tract can play a critical role in the development of pathologies associated with overeating, overweight and obesity. We previously observed that supplementation with anthocyanins (AC) (particularly glycosides of cyanidin and delphinidin) mitigated high fat diet (HFD)-induced development of obesity, dyslipidemia, insulin resistance and steatosis in C57BL/6J mice. This paper investigated whether these beneficial effects could be related to AC capacity to sustain intestinal monolayer integrity, prevent endotoxemia, and HFD-associated dysbiosis. The involvement of redox-related mechanisms were further investigated in Caco-2 cell monolayers. Consumption of a HFD for 14 weeks caused intestinal permeabilization and endotoxemia, which were associated with a decreased ileum expression of tight junction (TJ) proteins (occludin, ZO-1 and claudin-1), increased expression of NADPH oxidase (NOX1 and NOX4) and NOS2 and oxidative stress, and activation of redox sensitive signals (NF-κB and ERK1/2) that regulate TJ dynamics. AC supplementation mitigated all these events and increased GLP-2 levels, the intestinal hormone that upregulates TJ protein expression. AC also prevented, in vitro, tumor necrosis factor alpha-induced Caco-2 monolayer permeabilization, NOX1/4 upregulation, oxidative stress, and NF-κB and ERK activation. HFD-induced obesity in mice caused dysbiosis and affected the levels and secretion of MUC2, a mucin that participates in intestinal cell barrier protection and immune response. AC supplementation restored microbiota composition and MUC2 levels and distribution in HFD-fed mice. Thus, AC, particularly delphinidin and cyanidin, can preserve GI physiology in HFD-induced obesity in part through redox-regulated mechanisms. This can in part explain AC capacity to mitigate pathologies, i.e. insulin resistance and steatosis, associated with HFD-associated obesity.


Assuntos
Antocianinas/farmacologia , Dieta Hiperlipídica/efeitos adversos , Microbioma Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/metabolismo , Oxirredução , Substâncias Protetoras/farmacologia , Células CACO-2 , Disbiose , Endotoxemia/tratamento farmacológico , Endotoxemia/etiologia , Endotoxemia/metabolismo , Células Caliciformes/metabolismo , Humanos , Mucina-2/genética , Mucina-2/metabolismo , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Permeabilidade/efeitos dos fármacos , Transdução de Sinais
6.
mBio ; 10(3)2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31213556

RESUMO

Much remains unknown about how the intestinal microbiome interfaces with the protective intestinal mucus layer. Bifidobacterium species colonize the intestinal mucus layer and can modulate mucus production by goblet cells. However, select Bifidobacterium strains can also degrade protective glycans on mucin proteins. We hypothesized that the human-derived species Bifidobacterium dentium would increase intestinal mucus synthesis and expulsion, without extensive degradation of mucin glycans. In silico data revealed that B. dentium lacked the enzymes necessary to extensively degrade mucin glycans. This finding was confirmed by demonstrating that B. dentium could not use naive mucin glycans as primary carbon sources in vitro To examine B. dentium mucus modulation in vivo, Swiss Webster germfree mice were monoassociated with live or heat-killed B. dentium Live B. dentium-monoassociated mice exhibited increased colonic expression of goblet cell markers Krüppel-like factor 4 (Klf4), Trefoil factor 3 (Tff3), Relm-ß, Muc2, and several glycosyltransferases compared to both heat-killed B. dentium and germfree counterparts. Likewise, live B. dentium-monoassociated colon had increased acidic mucin-filled goblet cells, as denoted by Periodic Acid-Schiff-Alcian Blue (PAS-AB) staining and MUC2 immunostaining. In vitro, B. dentium-secreted products, including acetate, were able to increase MUC2 levels in T84 cells. We also identified that B. dentium-secreted products, such as γ-aminobutyric acid (GABA), stimulated autophagy-mediated calcium signaling and MUC2 release. This work illustrates that B. dentium is capable of enhancing the intestinal mucus layer and goblet cell function via upregulation of gene expression and autophagy signaling pathways, with a net increase in mucin production.IMPORTANCE Microbe-host interactions in the intestine occur along the mucus-covered epithelium. In the gastrointestinal tract, mucus is composed of glycan-covered proteins, or mucins, which are secreted by goblet cells to form a protective gel-like structure above the epithelium. Low levels of mucin or alterations in mucin glycans are associated with inflammation and colitis in mice and humans. Although current literature links microbes to the modulation of goblet cells and mucins, the molecular pathways involved are not yet fully understood. Using a combination of gnotobiotic mice and mucus-secreting cell lines, we have identified a human-derived microbe, Bifidobacterium dentium, which adheres to intestinal mucus and secretes metabolites that upregulate the major mucin MUC2 and modulate goblet cell function. Unlike other Bifidobacterium species, B. dentium does not extensively degrade mucin glycans and cannot grow on mucin alone. This work points to the potential of using B. dentium and similar mucin-friendly microbes as therapeutic agents for intestinal disorders with disruptions in the mucus barrier.


Assuntos
Autofagia , Bifidobacterium/fisiologia , Sinalização do Cálcio , Interações entre Hospedeiro e Microrganismos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Animais , Bifidobacterium/enzimologia , Feminino , Microbioma Gastrointestinal , Vida Livre de Germes , Células Caliciformes/fisiologia , Masculino , Camundongos , Mucina-2/genética , Mucinas/metabolismo , Mucinas/farmacologia , Polissacarídeos/metabolismo
7.
Surg Today ; 49(11): 887-893, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30879148

RESUMO

Intraductal papillary mucinous neoplasm (IPMN) of the pancreas is characterized by cystic dilation of the pancreatic duct, caused by mucin hypersecretion, with slow progression via the adenoma-carcinoma sequence mechanism. Mutation of GNAS at codon 201 is found exclusively in IPMNs, occurring at a rate of 41-75%. Recent advances in molecular biological techniques have demonstrated that GNAS mutation might play a role in the transformation of IPMNs after the appearance of neoplastic cells, rather than in the tumorigenesis of IPMNs. GNAS mutation is observed frequently in the intestinal subtype of IPMNs with MUC2 expression, and less frequently in IPMNs with concomitant pancreatic ductal adenocarcinoma (PDAC). Research has focused on assessing GNAS mutation status in clinical practice using various samples. In this review, we discuss the clinical application of GNAS mutation assessment to differentiate invasive IPMNs from concomitant PDAC, examine the clonality of recurrent IPMNs in the remnant pancreas using resected specimens, and differentiate pancreatic cystic lesions using cystic fluid collected by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA), duodenal fluid, and serum liquid biopsy samples.


Assuntos
Cromograninas/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Mutação , Neoplasias Primárias Múltiplas , Neoplasias Intraductais Pancreáticas/genética , Neoplasias Pancreáticas/genética , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Transformação Celular Neoplásica/genética , Códon/genética , Diagnóstico Diferencial , Aspiração por Agulha Fina Guiada por Ultrassom Endoscópico , Expressão Gênica , Humanos , Mucina-2/genética , Mucina-2/metabolismo , Neoplasias Intraductais Pancreáticas/diagnóstico , Neoplasias Intraductais Pancreáticas/patologia , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)
8.
Cancer Chemother Pharmacol ; 83(5): 893-904, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30815720

RESUMO

Irinotecan-induced mucositis is a major oncological problem. Goblet cells secrete mucus, protecting the intestinal mucosa, with secretion altered during mucositis. The enteric nervous system is involved in regulating gut motility and secretion. The aim of this study was to determine whether enteric neural cells and goblet cells are altered following irinotecan treatment. Tumour-bearing Dark Agouti rats were administered a single dose of 175 mg/kg of irinotecan intraperitoneally and 0.01 mg/kg atropine subcutaneously. Experimental and untreated control rats were killed at times 6, 24, 48, 72, 96 and 120 h after treatment. Jejunum and colon samples were formalin fixed. Haematoxylin and eosin staining, Alcian Blue-PAS staining, and immunohistochemistry with S-100 antibody (neural cell marker) were carried out. Statistical analyses were carried out using Kruskal-Wallis test with Dunns post test, Mann Whitney U test, and nonlinear regression. Total goblet cells decreased at 72 h compared with controls in the colon (p < 0.05). The percentage of cavitated goblet cells decreased compared to all other time points at 120 h in the colon. The number of S-100-positive cells in the submucosal plexus decreased in the colon (p = 0.0046) and in the myenteric plexus of the jejunum and colon (p = 0.0058 and p = 0.0022, respectively), on comparing treated with control. Enteric ganglia in the myenteric plexus of the jejunum decreased at 24 h and 96 h. Irinotecan-induced mucositis is associated with increases in mucus secretion and enteric neural cell change. These changes may contribute to the pathophysiology of mucositis through the dysregulation of neural signalling.


Assuntos
Adenocarcinoma/tratamento farmacológico , Enterócitos/efeitos dos fármacos , Irinotecano/efeitos adversos , Neoplasias Mamárias Experimentais/tratamento farmacológico , Mucina-2/metabolismo , Mucina-4/metabolismo , Mucosite/induzido quimicamente , Animais , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Enterócitos/metabolismo , Enterócitos/patologia , Feminino , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/metabolismo , Células Caliciformes/patologia , Irinotecano/uso terapêutico , Jejuno/efeitos dos fármacos , Jejuno/metabolismo , Jejuno/patologia , Mucina-2/genética , Mucina-4/genética , Mucosite/metabolismo , Mucosite/patologia , Ratos Endogâmicos
9.
Toxicol Lett ; 309: 1-9, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30904571

RESUMO

Aflatoxin M1 (AFM1), ochratoxin A (OTA), and zearalenone (ZEA) are mycotoxins commonly found in milk. Mycotoxin contamination has caused food safety concerns worldwide since most of the toxic effects in humans are serious. The combined toxic effects of these mycotoxins on intestinal epithelial cells have not been reported. Herein, we investigated the combined effects of AFM1, OTA, and ZEA on intestinal integrity and define the underlying mechanisms(s) of their effects in Caco-2/HT29-MTX co-cultures. Our results showed that the mixtures of AFM1 + OTA, AFM1 + ZEA, and AFM1 + ZEA + OTA significantly increased epithelial permeability. Immunofluorescence analysis and transmission electron microscopy revealed that mycotoxins altered TJ proteins morphology and disrupted their structures. Also, the present study showed that mixtures of mycotoxins significantly modulated MUC5AC and MUC5B mRNA levels and protein secretion. This study demonstrated that the effects of mixtures of mycotoxins on intestinal barrier function were more significant than AFM1 alone. More importantly, the damage of intestinal integrity caused by mycotoxins was correlated to the change of the TJ proteins location and the decrease of mucin secretion. Mixtures of AFM1, OTA, and ZEA in food might pose a health risk to consumers, particularly in children, and toxin risks should be considered.


Assuntos
Aflatoxina M1/toxicidade , Mucosa Intestinal/efeitos dos fármacos , Mucinas/metabolismo , Ocratoxinas/toxicidade , Zearalenona/toxicidade , Células CACO-2 , Técnicas de Cocultura , Contaminação de Alimentos , Células HT29 , Humanos , Mucosa Intestinal/metabolismo , Mucina-5AC/análise , Mucina-5AC/genética , Mucina-2/análise , Mucina-2/genética , Mucina-5B/análise , Mucina-5B/genética , Mucinas/genética , Permeabilidade , RNA Mensageiro/análise , Proteínas de Junções Íntimas/análise
10.
Toxins (Basel) ; 11(2)2019 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-30813459

RESUMO

Aflatoxin M1 (AFM1) and ochratoxin A (OTA), which widely coexist in milk, may pose a serious threat to human health. Mucin is a major component of the intestinal mucus layer, which plays an important role in maintaining intestinal mucosal homeostasis. However, the effect of mycotoxins AFM1 and OTA on intestinal mucin production is still not clear. This study aimed to investigate individual and interactive effects of mycotoxins AFM1 and OTA on the intestinal barrier and the mRNA expression of intestinal mucin (MUC2, MUC5AC and MUC5B) and on protein production in Caco-2/HT29-MTX cultures after 48 h of exposure. Our results show that individual mycotoxins and their mixtures significantly reduced intestinal cell viability and transepithelial electrical resistance (TEER) values, as well as significantly altered intestinal mucin mRNA expression and protein abundance. Moreover, OTA showed toxicity similar to AFM1 in cell viability and TEER value at the same concentration. When the two mycotoxins acted in combination, the synergistic effects observed in the assessment of cell viability and protein abundance in all mono- and co-cultures. In general, this study provides evidence that AFM1 and OTA can damage the intestine, and it contributes to optimized maximum permissible limits of mycotoxins in milk.


Assuntos
Aflatoxina M1/toxicidade , Mucina-5AC/genética , Mucina-2/genética , Mucina-5B/genética , Ocratoxinas/toxicidade , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Células HT29 , Humanos , Mucina-5AC/metabolismo , Mucina-2/metabolismo , Mucina-5B/metabolismo , RNA Mensageiro/metabolismo
11.
J Med Food ; 22(3): 294-304, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30724689

RESUMO

Constipation is an acute or chronic illness attributed to various causes, ranging from lifestyle habits to side effects of a disease. To improve the laxative effects of some traditional medicines, herbal mixtures of Liriope platyphylla, Glycyrrhiza uralensis, and Cinnamomum cassia (LGC) were evaluated for their mechanism of action and therapeutic effects in loperamide (Lop)-induced constipated Sprague Dawley rats by examining alterations in excretion parameters, histological structure, mucin secretion, and related protein levels. Food intake and water consumption were constant for all animals. We observed that the Lop+LGC-treated group had significantly greater excretion of stool and urine than was observed in the Lop+Vehicle-treated group. Administration of LGC in the constipation model restored the intestinal transit ratio to normal levels, and increased the number of goblet cells, mucosal layer, and muscle thickness. Mucin secretion was greater in the Lop+LGC-treated group than in the Lop+Vehicle-treated group, and the expression of MUC2 and AQP8 genes were also increased. In addition, reverse transcription polymerase chain reaction and Western blot revealed an increase in the muscarinic acetylcholine receptors (mAChRs) in the Lop+LGC-treated group compared to the Lop+Vehicle-treated group. Furthermore, compared with the Lop+Vehicle-treated group, treatment with LGC reduced the phosphorylation of PKC and PI3K, and expression of Gα protein, but increased levels of IP3. Our results suggest that the traditional herbal mixture of LGC induces a potent laxative effect in Lop-induced constipation through mucosal tissue changes and mucin production. We also demonstrated that the laxative effect of LGC is closely related to the expression of mAChR and its downstream signals, suggesting the possibility of developing a constipation-laxative agent using LGC.


Assuntos
Cinnamomum aromaticum/química , Constipação Intestinal/tratamento farmacológico , Glycyrrhiza uralensis/química , Laxantes/administração & dosagem , Liriope (Planta)/química , Extratos Vegetais/administração & dosagem , Animais , Aquaporinas/genética , Aquaporinas/metabolismo , Constipação Intestinal/induzido quimicamente , Constipação Intestinal/genética , Constipação Intestinal/metabolismo , Sinergismo Farmacológico , Loperamida/efeitos adversos , Masculino , Mucina-2/genética , Mucina-2/metabolismo , Ratos , Ratos Sprague-Dawley
12.
Gene ; 696: 206-218, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30802540

RESUMO

Mounting evidence suggests that long noncoding RNAs (lncRNAs) play an important role in tumor biology. To date, some lncRNAs have been found to be involved in competitive binding of miRNAs, a major group of competitive endogenous RNAs (ceRNAs), through participation in a regulatory network of protein-coding gene expression. However, the functional roles of lncRNA-mediated ceRNAs in esophageal squamous cell carcinoma (ESCC) have rarely been reported. Here, we construct a hypothetical ceRNA network by analyzing differential expression of lncRNAs, miRNAs and mRNAs obtained from 96 ESCC tissues and 13 normal tissues in the Cancer Genome Atlas. Ultimately, 95 lncRNAs, 9 miRNAs, and 40 mRNAs were identified (fold change >1.5, P < .05) and included in the ceRNA network for ESCC. Moreover, three lncRNAs (IGF2-AS, MUC2 and SOX2-OT) were found to be significantly associated with overall survival (log-rank test, P < .05), and further experiments revealed that lncRNA DLX6-AS1 knockdown inhibited the proliferation and invasion of esophageal cancer cells by enhancing the endogenous function of mTOR. We believe that the identified ceRNA network can facilitate a better understanding of lncRNA-related mechanisms in ESCC.


Assuntos
Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago/genética , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , RNA Antissenso/metabolismo , Linhagem Celular Tumoral , Conjuntos de Dados como Assunto , Neoplasias Esofágicas/mortalidade , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/mortalidade , Carcinoma de Células Escamosas do Esôfago/patologia , Esôfago/patologia , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Mucina-2/genética , Invasividade Neoplásica/genética , Prognóstico , RNA Antissenso/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , Serina-Treonina Quinases TOR/genética
13.
Breast Cancer Res Treat ; 173(2): 289-299, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30317423

RESUMO

PURPOSE: Tumors that secrete large volumes of mucus are chemotherapy resistant, however, mechanisms underlying this resistance are unknown. One protein highly expressed in mucin secreting breast cancers is the secreted mucin, Mucin 2 (MUC2). While MUC2 is expressed in some breast cancers it is absent in normal breast tissue, implicating it in breast cancer. However, the effects of MUC2 on breast cancer are largely unknown. This study examined the role of MUC2 in modulating breast cancer proliferation, response to chemotherapy and metastasis. METHODS: Using patient derived xenografts we developed two novel cell lines, called BCK4 and PT12, which express high levels of MUC2. To modulate MUC2 levels, BCK4 and PT12 cells were engineered to express shRNA targeted to MUC2 (shMUC2, low MUC2) or a non-targeting control (shCONT, high MUC2) and proliferation and apoptosis were measured in vitro and in vivo. BCK4 cells with shCONT or shMUC2 were labeled with GFP-luciferase and examined in an experimental metastasis model; disease burden and site specific dissemination were monitored by intravital imaging and fluorescence guided dissection, respectively. RESULTS: Proliferation decreased in BCK4 and PT12 shMUC2 cells versus control cells both in vitro and in vivo. Chemotherapy induced minimal apoptosis in control cells expressing high MUC2 but increased apoptosis in shMUC2 cells containing low MUC2. An experimental metastasis model showed disease burden decreased when breast cancer cells contained low versus high MUC2. Treatment with Epidermal Growth Factor (EGF) increased MUC2 expression in BCK4 cells; this induction was abolished by the EGF-receptor inhibitor, Erlotinib. CONCLUSIONS: MUC2 plays an important role in mediating proliferation, apoptosis and metastasis of breast cancer cells. MUC2 may be important in guiding treatment and predicting outcomes in breast cancer patients.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Mucina-2/metabolismo , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Cloridrato de Erlotinib/farmacologia , Cloridrato de Erlotinib/uso terapêutico , Feminino , Humanos , Camundongos , Mucina-2/genética , RNA Interferente Pequeno/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
14.
J Immunol ; 202(2): 598-607, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30530480

RESUMO

Regulation of the intestinal mucus layer by goblet cells is important for preventing inflammation and controlling infection. IL-33, a cytokine upregulated in inflammatory bowel disease and helminth infection, induces intestinal goblet cells, but the mechanism remains unclear. Enteroids are three-dimensional structures of primary small intestinal epithelial cells that contain all differentiated intestinal epithelial cell types. We developed an enteroid-immune cell coculture model to determine the mechanism through which IL-33 affects intestinal goblet cell differentiation. We report that IL-33 does not directly induce goblet cell differentiation in murine enteroids; however, IL-13, a cytokine induced by IL-33, markedly induces goblet cells and gene expression consistent with goblet cell differentiation. When enteroids are cocultured with CD90+ mesenteric lymph node cells from IL-33-treated mice, IL-33 then induces IL-13 secretion by group 2 innate lymphoid cells and enteroid gene expression consistent with goblet cell differentiation. In cocultures, IL-33-induced Muc2 expression is dependent on enteroid Il4ra expression, demonstrating a requirement for IL-13 signaling in epithelial cells. In vivo, IL-33-induced intestinal goblet cell hyperplasia is dependent on IL-13. These studies demonstrate that IL-33 induces intestinal goblet cell differentiation not through direct action on epithelial cells but indirectly through IL-13 production by goup 2 innate lymphoid cells.


Assuntos
Diferenciação Celular , Células Caliciformes/imunologia , Imunidade Inata , Interleucina-13/imunologia , Interleucina-33/imunologia , Linfócitos/imunologia , Animais , Técnicas de Cocultura , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucina-2/genética , Mucina-2/imunologia , Receptores de Superfície Celular/genética , Transdução de Sinais
15.
Mol Nutr Food Res ; 63(3): e1800658, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30407734

RESUMO

SCOPE: Necrotizing enterocolitis (NEC) is a leading cause of morbidity and death in preterm infants, occurring more often in formula-fed than breastfed infants. Studies in both rats and humans show that human milk oligosaccharides (HMOs) lower the incidence of NEC, but the mechanism underlying such protection is currently unclear. METHODS AND RESULTS: By extracting HMOs from pooled human breastmilk, the impact of HMOs on the intestinal mucin levels in a murine model of NEC are investigated. To confirm the results, the findings are validated by exposing human intestinal epithelial cells and intestinal organoids to HMOs and evaluated for mucin expression. HMO-gavage to pups increases Muc2 levels and decreases intestinal permeability to macromolecular dextran. HMO-treated cells have increased Muc2 expression, decreased bacterial attachment and dextran permeability during challenge by enteric pathogens. To identify the mediators involved in HMO induction of mucins, it is demonstrated that HMOs directly induce the expression of chaperone proteins including protein disulfide isomerase (PDI). Suppression of PDI activity removes the protective effects of HMOs on barrier function in vitro as well as NEC protection in vivo. CONCLUSIONS: Taken together, the results provide insights to the possible mechanisms by which HMOs protect the neonatal intestine through upregulation of mucins.


Assuntos
Enterocolite Necrosante/prevenção & controle , Leite Humano/química , Mucina-2/genética , Oligossacarídeos/farmacologia , Animais , Animais Recém-Nascidos , Células CACO-2 , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Enterocolite Necrosante/metabolismo , Células Caliciformes/efeitos dos fármacos , Humanos , Mucosa Intestinal/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Mucina-2/análise , Isomerases de Dissulfetos de Proteínas/fisiologia
16.
J Biol Chem ; 294(3): 816-826, 2019 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-30482841

RESUMO

Regulated mucin secretion is essential for the formation of the mucus layer that protects the underlying epithelial cells from foreign particles. Alterations in the quantity or quality of secreted mucins are therefore detrimental to airway and colon physiology. Based on various biochemical assays in several human cell lines, we report here that Na+/Ca2+ exchanger 2 (NCX2) works in conjunction with transient receptor potential cation channel subfamily M member 4 (TRPM4), and perhaps TRPM5, Na+ channels to control Ca2+-mediated secretion of both mucin 2 (MUC2) and MUC5AC from HT29-18N2 colonic cancer cells. Differentiated normal bronchial epithelial (NHBE) cells and tracheal cells from patients with cystic fibrosis (CFT1-LC3) expressed only TRPM4 and all three isoforms of NCXs. Blocking the activity of TRPM4 or NCX proteins abrogated MUC5AC secretion from NHBE and CFT1-LC3 cells. Altogether, our findings reveal that NCX and TRPM4/TRPM5 are both required for mucin secretion. We therefore propose that these two proteins could be potential pharmacological targets to control mucus-related pathologies such as cystic fibrosis.


Assuntos
Cálcio/metabolismo , Células Caliciformes/metabolismo , Mucina-5AC/metabolismo , Mucina-2/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Canais de Cátion TRPM/metabolismo , Linhagem Celular , Fibrose Cística/genética , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Células Caliciformes/patologia , Humanos , Mucina-5AC/genética , Mucina-2/genética , Trocador de Sódio e Cálcio/genética , Canais de Cátion TRPM/genética
17.
Sci Rep ; 8(1): 17503, 2018 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-30504806

RESUMO

The DNA sequence of the two human mucin genes MUC2 and MUC6 have not been completely resolved due to the repetitive nature of their central exon coding for Proline, Threonine and Serine rich sequences. The exact nucleotide sequence of these exons has remained unknown for a long time due to limitations in traditional sequencing techniques. These are still very poorly covered in new whole genome sequencing projects with the corresponding protein sequences partly missing. We used a BAC clone containing both these genes and third generation sequencing technology, SMRT sequencing, to obtain the full-length contiguous MUC2 and MUC6 tandem repeat sequences. The new sequences span the entire repeat regions with good coverage revealing their length, variation in repeat sequences and their internal organization. The sequences obtained were used to compare with available sequences from whole genome sequencing projects indicating variation in number of repeats and their internal organization between individuals. The lack of these sequences has limited the association of genetic alterations with disease. The full sequences of these mucins will now allow such studies, which could be of importance for inflammatory bowel diseases for MUC2 and gastric ulcer diseases for MUC6 where deficient mucus protection is assumed to play an important role.


Assuntos
Éxons , Mucina-2/genética , Mucina-6/genética , Sequências Repetitivas de Ácido Nucleico , Sequência de Aminoácidos , Cromossomos Artificiais Bacterianos , Doenças Inflamatórias Intestinais/genética , Mucina-2/química , Mucina-6/química , Polimorfismo Genético , Recombinação Genética , Úlcera Gástrica/genética
18.
PLoS One ; 13(11): e0207196, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30408134

RESUMO

Due to their complex chemical and physical properties, the effects and mechanisms of action of natural sources of dietary fiber on the intestine are unclear. Pigs are commonly fed high-fiber diets to reduce production costs and non-starch polysaccharide (NSP)-degrading enzymes have been used to increase fiber digestibility. We evaluated the expression of mucin 2 (MUC2), presence of goblet cells, and ileal immune profile of pigs housed individually for 28 days and fed either a low fiber diet based on corn-soybean meal (CSB, n = 9), or two high fiber diets formulated adding 40% corn distillers' dried grains with solubles (DDGS, n = 9) or 30% wheat middlings (WM, n = 9) to CSB-based diet. Pigs were also fed those diets supplemented with a NSP enzymes mix (E) of xylanase, ß-glucanase, mannanase, and galactosidase (n = 8, 10, and 9 for CSB+E, DDGS+E and WM+E, respectively). Feeding DDGS and WM diets increased ileal MUC2 expression compared with CSB diet, and this effect was reversed by the addition of enzymes. There were no differences in abundance of goblet cells among treatments. In general, enzyme supplementation increased gene expression and concentrations of IL-1ß, and reduced the concentrations of IL-4, IL-17A and IL-11. The effects of diet-induced cytokines on modulating intestinal MUC2 were assessed in vitro by treating mouse and swine enteroids with 1 ng/ml of IL-4 and IL-1ß. In accordance with previous studies, treatment with Il-4 induced Muc2 and expansion of goblet cells in mouse enteroids. However, swine enteroids did not change MUC2 expression or number of goblet cells when treated with IL-4 or IL-1ß. Our results suggest that mucin and immune profile are regulated by diet in the swine intestine, but by mechanisms different to mouse, emphasizing the need for using appropriate models to study responses to dietary fiber in swine.


Assuntos
Fibras na Dieta/administração & dosagem , Glicosídeo Hidrolases/administração & dosagem , Glicosídeo Hidrolases/metabolismo , Íleo/imunologia , Íleo/metabolismo , Mucina-2/metabolismo , Polissacarídeos/metabolismo , Ração Animal/análise , Animais , Citocinas/administração & dosagem , Citocinas/metabolismo , Fibras na Dieta/análise , Suplementos Nutricionais , Grão Comestível , Feminino , Expressão Gênica , Células Caliciformes/citologia , Células Caliciformes/metabolismo , Íleo/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mucina-2/genética , Soja , Sus scrofa , Triticum , Zea mays
19.
World J Gastroenterol ; 24(36): 4164-4177, 2018 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-30271081

RESUMO

AIM: To determine tissue expression (mRNA, protein) of two types of mucins [mucin 1 (MUC1) and mucin 2 (MUC2)] in patients with colorectal cancer (CRC). METHODS: Expression of membrane-bound mucin (MUC1) and secretory mucin (MUC2) in CRC (mRNA, protein) were analyzed in tissue material including fragments of tumors obtained from CRC patients (n = 34), and fragments of normal colorectal tissue from the same patients (control). The analysis was conducted using real-time quantitative polymerase chain reaction (RT-qPCR) (transcripts), immunohistochemistry (IHC) (apomucins), and the modern approach for morphometric analysis of IHC reaction (HSV filter software). Results on tissue expression of both mucins (mRNA, protein) were compared to histological alterations in colorectal cancer samples and correlated with selected clinical data in the patients. The statistical analysis was conducted using Statistica PL v. 12.0 software. RESULTS: Significantly higher expression of the MUC1 mRNA in the CRC, compared with the control and the borderline correlation of mRNA expression with MUC1 protein levels in colorectal samples was observed. The expression of apomucins concerned cell membranes (MUC1) and cytoplasm (MUC2) and occurred both in control tissues and in most cancerous samples. There were no significant relationships between MUC1 (mRNA, protein) and the clinicopathological data of patients. MUC2 protein expression was significantly lower as compared to the control, while MUC2 mRNA expression was comparable in both groups. The MUC1/MUC2 ratio was significantly higher in CRC tissues than in the control. The higher expression of MUC2 was a feature of mucinous CRC subtypes, and characterized higher histological stage of tumors. Negative correlations have been obtained between MUC2 and the Ki-67 antigen, as well as between MUC2 and p53 protein expressions in CRC. CONCLUSION: A combination of tissue overexpression of MUC1, reduced MUC2 expression, and high ratio of MUC1/MUC2 is a factor of poor prognosis in CRC patients. MUC2 tissue expression allows to differentiate mucinous and nonmucinous CRC subtypes.


Assuntos
Adenocarcinoma Mucinoso/patologia , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/patologia , Mucina-1/metabolismo , Mucina-2/metabolismo , Adenocarcinoma Mucinoso/mortalidade , Adenocarcinoma Mucinoso/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/cirurgia , Feminino , Humanos , Intestino Grosso/patologia , Masculino , Pessoa de Meia-Idade , Mucina-1/análise , Mucina-1/genética , Mucina-2/análise , Mucina-2/genética , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/metabolismo , Taxa de Sobrevida
20.
Inflamm Res ; 67(11-12): 975-984, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30317465

RESUMO

OBJECTIVE AND DESIGN: The primary component in gut mucus is mucin 2 (MUC2) secreted by goblet cells. Fluctuations in MUC2 expression are considered a useful indicator for evaluating mucosal damage and protective effect of various agents using animal studies. However, there are few in vitro studies evaluating mucosal damage using MUC2 as the indicator. Hence, we attempted to establish a novel in vitro model with MUC2 as the indicator for evaluating drug-induced mucosal damage and protective effect using enterocytes derived from human iPS cells. METHODS: Compounds were added into enterocytes derived from human iPS cells, and MUC2 mRNA and protein expression levels were evaluated. Further, the effect of compounds on membrane permeability was investigated. RESULTS: Nonsteroidal anti-inflammatory drugs were found to decrease MUC2 mRNA expression in enterocytes, whereas mucosal protective agents increased mRNA levels. Changes in MUC2 protein expression were consistent with those of mRNA. Additionally, our results indicated that indomethacin caused mucosal damage, affecting membrane permeability of the drug. Moreover, we observed protective effect of rebamipide against the indomethacin-induced permeability increase. CONCLUSIONS: The developed model could facilitate evaluating drug-induced mucosal damage and protective effects of various agents and could impact drug development studies regarding pharmacological efficacy and safety.


Assuntos
Alanina/análogos & derivados , Anti-Inflamatórios não Esteroides/toxicidade , Antiulcerosos/farmacologia , Enterócitos/efeitos dos fármacos , Indometacina/toxicidade , Mucina-2/metabolismo , Quinolonas/farmacologia , Alanina/farmacologia , Alternativas aos Testes com Animais , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Enterócitos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Mucina-2/genética
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