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1.
Mol Biol Rep ; 51(1): 704, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38824233

RESUMO

BACKGROUND: Tumor modeling using organoids holds potential in studies of cancer development, enlightening both the intracellular and extracellular molecular mechanisms behind different cancer types, biobanking, and drug screening. Intestinal organoids can be generated in vitro using a unique type of adult stem cells which are found at the base of crypts and are characterized by their high Lgr5 expression levels. METHODS AND RESULTS: In this study, we successfully established intestinal cancer organoid models by using both the BALB/c derived and mouse embryonic stem cells (mESCs)-derived intestinal organoids. In both cases, carcinogenesis-like model was developed by using azoxymethane (AOM) treatment. Carcinogenesis-like model was verified by H&E staining, immunostaining, relative mRNA expression analysis, and LC/MS analysis. The morphologic analysis demonstrated that the number of generated organoids, the number of crypts, and the intensity of the organoids were significantly augmented in AOM-treated intestinal organoids compared to non-AOM-treated ones. Relative mRNA expression data revealed that there was a significant increase in both Wnt signaling pathway-related genes and pluripotency transcription factors in the AOM-induced intestinal organoids. CONCLUSION: We successfully developed simple carcinogenesis-like models using mESC-based and Lgr5 + stem cell-based intestinal organoids. Intestinal organoid based carcinogenesi models might be used for personalized cancer therapy in the future.


Assuntos
Azoximetano , Carcinogênese , Células-Tronco Embrionárias Murinas , Organoides , Via de Sinalização Wnt , Animais , Organoides/metabolismo , Organoides/patologia , Camundongos , Azoximetano/toxicidade , Carcinogênese/patologia , Carcinogênese/induzido quimicamente , Carcinogênese/genética , Células-Tronco Embrionárias Murinas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Camundongos Endogâmicos BALB C , Intestinos/patologia , Neoplasias Intestinais/patologia , Neoplasias Intestinais/induzido quimicamente , Neoplasias Intestinais/genética , Neoplasias Intestinais/metabolismo , Modelos Animais de Doenças , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia
2.
PLoS One ; 19(6): e0304526, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38857221

RESUMO

In vitro models, such as primary cells and continuous cell lines routinely used for evaluating drug candidates, have limitations in their translational relevance to human diseases. Organotypic cultures are increasingly being used to assess therapeutics for various cancers and infectious diseases. Monitoring drug cytotoxicity in cell cultures is crucial in drug development, and several commercially available kits for cytotoxicity assessment offer distinct advantages and limitations. Given the complexity of organoid cultures, including donor-driven variability, we investigated drug-treated, tissue stem cell-derived human intestinal organoid responses with commonly used cell cytotoxicity assay kits. Using seven different compounds, we compared the cytotoxicity assay performance of two different leaky membrane-based and two metabolism-based assays. Significant variability was seen in reported viability outcomes across assays and organoid lines. High baseline activity of lactate dehydrogenase (LDH) in four human intestinal organoid lines required modification of the standard LDH assay protocol. Additionally, the LDH assay reported unique resilience to damage in a genetically-modified line contrasting results compared to other assays. This study highlights factors that can impact the measurement of cell cytotoxicity in intestinal organoid models, which are emerging as valuable new tools for research and pre-clinical drug testing and suggest the need for using multiple assay types to ensure reliable cytotoxicity assessment.


Assuntos
L-Lactato Desidrogenase , Organoides , Humanos , Organoides/efeitos dos fármacos , Organoides/metabolismo , Organoides/citologia , L-Lactato Desidrogenase/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Intestinos/citologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo
3.
Int J Nanomedicine ; 19: 5273-5295, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38859952

RESUMO

Purpose: Reducing the first-pass hepatic effect via intestinal lymphatic transport is an effective way to increase the oral absorption of drugs. 2-Monoacylglycerol (2-MAG) as a primary digestive product of dietary lipids triglyceride, can be assembled in chylomicrons and then transported from the intestine into the lymphatic system. Herein, we propose a biomimetic strategy and report a 2-MAG mimetic nanocarrier to target the intestinal lymphatic system via the lipid absorption pathway and improve oral bioavailability. Methods: The 2-MAG mimetic liposomes were designed by covalently bonding serinol (SER) on the surface of liposomes named SER-LPs to simulate the structure of 2-MAG. Dihydroartemisinin (DHA) was chosen as the model drug because of its disadvantages such as poor solubility and high first-pass effect. The endocytosis and exocytosis mechanisms were investigated in Caco-2 cells and Caco-2 cell monolayers. The capacity of intestinal lymphatic transport was evaluated by ex vivo biodistribution and in vivo pharmacokinetic experiments. Results: DHA loaded SER-LPs (SER-LPs-DHA) had a particle size of 70 nm and a desirable entrapment efficiency of 93%. SER-LPs showed sustained release for DHA in the simulated gastrointestinal environment. In vitro cell studies demonstrated that the cellular uptake of SER-LPs primarily relied on the caveolae- rather than clathrin-mediated endocytosis pathway and preferred to integrate into the chylomicron assembly process through the endoplasmic reticulum/Golgi apparatus route. After oral administration, SER-LPs efficiently promoted drug accumulation in mesenteric lymphatic nodes. The oral bioavailability of DHA from SER-LPs was 10.40-fold and 1.17-fold larger than that of free DHA and unmodified liposomes at the same dose, respectively. Conclusion: SER-LPs improved oral bioavailability through efficient intestinal lymphatic transport. These findings of the current study provide a good alternative strategy for oral delivery of drugs with high first-pass hepatic metabolism.


Assuntos
Artemisininas , Disponibilidade Biológica , Lipossomos , Animais , Lipossomos/química , Lipossomos/farmacocinética , Células CACO-2 , Humanos , Administração Oral , Artemisininas/farmacocinética , Artemisininas/química , Artemisininas/administração & dosagem , Absorção Intestinal/efeitos dos fármacos , Masculino , Distribuição Tecidual , Tamanho da Partícula , Camundongos , Sistema Linfático/metabolismo , Sistema Linfático/efeitos dos fármacos , Ratos Sprague-Dawley , Ratos , Materiais Biomiméticos/farmacocinética , Materiais Biomiméticos/química , Mucosa Intestinal/metabolismo
4.
Proc Natl Acad Sci U S A ; 121(25): e2322588121, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38861598

RESUMO

The nematode intestine is the primary site for nutrient uptake and storage as well as the synthesis of biomolecules; lysosome-related organelles known as gut granules are important for many of these functions. Aspects of intestine biology are not well understood, including the export of the nutrients it imports and the molecules it synthesizes, as well as the complete functions and protein content of the gut granules. Here, we report a mass spectrometry (MS)-based proteomic analysis of the intestine of the Caenorhabditis elegans and of its gut granules. Overall, we identified approximately 5,000 proteins each in the intestine and the gonad and showed that most of these proteins can be detected in samples extracted from a single worm, suggesting the feasibility of individual-level genetic analysis using proteomes. Comparing proteomes and published transcriptomes of the intestine and the gonad, we identified proteins that appear to be synthesized in the intestine and then transferred to the gonad. To identify gut granule proteins, we compared the proteome of individual intestines deficient in gut granules to the wild type. The identified gut granule proteome includes proteins known to be exclusively localized to the granules and additional putative gut granule proteins. We selected two of these putative gut granule proteins for validation via immunohistochemistry, and our successful confirmation of both suggests that our strategy was effective in identifying the gut granule proteome. Our results demonstrate the practicability of single-tissue MS-based proteomic analysis in small organisms and in its future utility.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Lisossomos , Proteômica , Animais , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteômica/métodos , Lisossomos/metabolismo , Proteoma/metabolismo , Intestinos , Mucosa Intestinal/metabolismo , Gônadas/metabolismo , Espectrometria de Massas/métodos , Organelas/metabolismo
5.
Cell ; 187(12): 3039-3055.e14, 2024 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-38848677

RESUMO

In the prevailing model, Lgr5+ cells are the only intestinal stem cells (ISCs) that sustain homeostatic epithelial regeneration by upward migration of progeny through elusive upper crypt transit-amplifying (TA) intermediates. Here, we identify a proliferative upper crypt population marked by Fgfbp1, in the location of putative TA cells, that is transcriptionally distinct from Lgr5+ cells. Using a kinetic reporter for time-resolved fate mapping and Fgfbp1-CreERT2 lineage tracing, we establish that Fgfbp1+ cells are multi-potent and give rise to Lgr5+ cells, consistent with their ISC function. Fgfbp1+ cells also sustain epithelial regeneration following Lgr5+ cell depletion. We demonstrate that FGFBP1, produced by the upper crypt cells, is an essential factor for crypt proliferation and epithelial homeostasis. Our findings support a model in which tissue regeneration originates from upper crypt Fgfbp1+ cells that generate progeny propagating bi-directionally along the crypt-villus axis and serve as a source of Lgr5+ cells in the crypt base.


Assuntos
Mucosa Intestinal , Receptores Acoplados a Proteínas G , Receptores Acoplados a Proteínas G/metabolismo , Animais , Camundongos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/citologia , Células-Tronco/metabolismo , Células-Tronco/citologia , Linhagem da Célula , Regeneração , Proliferação de Células , Células Epiteliais/metabolismo , Células Epiteliais/citologia , Camundongos Endogâmicos C57BL , Homeostase
6.
Sci Rep ; 14(1): 13177, 2024 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-38849503

RESUMO

Overconsumption of dietary sugar can lead to many negative health effects including the development of Type 2 diabetes, metabolic syndrome, cardiovascular disease, and neurodegenerative disorders. Recently, the human intestinal microbiota, strongly associated with our overall health, has also been known to be affected by diet. However, mechanistic insight into the importance of the human intestinal microbiota and the effects of chronic sugar ingestion has not been possible largely due to the complexity of the human microbiome which contains hundreds of types of organisms. Here, we use an interspecies C. elegans/E. coli system, where E. coli are subjected to high sugar, then consumed by the bacterivore host C. elegans to become the microbiota. This glucose-fed microbiota results in a significant lifespan reduction accompanied by reduced healthspan (locomotion), reduced stress resistance, and changes in behavior and feeding. Lifespan reduction is also accompanied by two potential major contributors: increased intestinal bacterial density and increased concentration of reactive oxygen species. The glucose-fed microbiota accelerated the age-related development of intestinal cell permeability, intestinal distention, and dysregulation of immune effectors. Ultimately, the changes in the intestinal epithelium due to aging with the glucose-fed microbiota results in increased susceptibility to multiple bacterial pathogens. Taken together, our data reveal that chronic ingestion of sugar, such as a Western diet, has profound health effects on the host due to changes in the microbiota and may contribute to the current increased incidence of ailments including inflammatory bowel diseases as well as multiple age-related diseases.


Assuntos
Caenorhabditis elegans , Escherichia coli , Microbioma Gastrointestinal , Glucose , Mucosa Intestinal , Caenorhabditis elegans/microbiologia , Animais , Glucose/metabolismo , Mucosa Intestinal/microbiologia , Mucosa Intestinal/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Longevidade , Suscetibilidade a Doenças
7.
Proc Natl Acad Sci U S A ; 121(25): e2321228121, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38857399

RESUMO

Ciliary defects are linked to ciliopathies, but impairments in the sensory cilia of Caenorhabditis elegans neurons extend lifespan, a phenomenon with previously unclear mechanisms. Our study reveals that neuronal cilia defects trigger the unfolded protein response of the endoplasmic reticulum (UPRER) within intestinal cells, a process dependent on the insulin/insulin-like growth factor 1 (IGF-1) signaling transcription factor and the release of neuronal signaling molecules. While inhibiting UPRER doesn't alter the lifespan of wild-type worms, it normalizes the extended lifespan of ciliary mutants. Notably, deactivating the cyclic nucleotide-gated (CNG) channel TAX-4 on the ciliary membrane promotes lifespan extension through a UPRER-dependent mechanism. Conversely, constitutive activation of TAX-4 attenuates intestinal UPRER in ciliary mutants. Administering a CNG channel blocker to worm larvae activates intestinal UPRER and increases adult longevity. These findings suggest that ciliary dysfunction in sensory neurons triggers intestinal UPRER, contributing to lifespan extension and implying that transiently inhibiting ciliary channel activity may effectively prolong lifespan.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Cílios , Longevidade , Resposta a Proteínas não Dobradas , Animais , Caenorhabditis elegans/metabolismo , Cílios/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Intestinos/citologia , Transdução de Sinais , Neurônios/metabolismo , Retículo Endoplasmático/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Mucosa Intestinal/metabolismo
9.
J Vis Exp ; (207)2024 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-38856194

RESUMO

An advanced intestine-on-chip model recreating epithelial 3D organotypic villus-like and crypt-like structures has been developed. The immunocompetent model includes Human Umbilical Vein Endothelial Cells (HUVEC), Caco-2 intestinal epithelial cells, tissue-resident macrophages, and dendritic cells, which self-organize within the tissue, mirroring characteristics of the human intestinal mucosa. A unique aspect of this platform is its capacity to integrate circulating human primary immune cells, enhancing physiological relevance. The model is designed to investigate the intestinal immune system's response to bacterial and fungal colonization and infection. Due to its enlarged cavity size, the model offers diverse functional readouts such as permeation assays, cytokine release, and immune cell infiltration, and is compatible with immunofluorescence measurement of 3D structures formed by the epithelial cell layer. It hereby provides comprehensive insights into cell differentiation and function. The intestine-on-chip platform has demonstrated its potential in elucidating complex interactions between surrogates of a living microbiota and human host tissue within a microphysiological perfused biochip platform.


Assuntos
Mucosa Intestinal , Humanos , Mucosa Intestinal/imunologia , Mucosa Intestinal/citologia , Células CACO-2 , Células Endoteliais da Veia Umbilical Humana , Imunidade nas Mucosas/imunologia , Dispositivos Lab-On-A-Chip , Células Dendríticas/imunologia , Células Dendríticas/citologia , Macrófagos/imunologia , Macrófagos/citologia
10.
Nat Commun ; 15(1): 4775, 2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38839750

RESUMO

The metal ion transporter SLC39A8 is associated with physiological traits and diseases, including blood manganese (Mn) levels and inflammatory bowel diseases (IBD). The mechanisms by which SLC39A8 controls Mn homeostasis and epithelial integrity remain elusive. Here, we generate Slc39a8 intestinal epithelial cell-specific-knockout (Slc39a8-IEC KO) mice, which display markedly decreased Mn levels in blood and most organs. Radiotracer studies reveal impaired intestinal absorption of dietary Mn in Slc39a8-IEC KO mice. SLC39A8 is localized to the apical membrane and mediates 54Mn uptake in intestinal organoid monolayer cultures. Unbiased transcriptomic analysis identifies alkaline ceramidase 1 (ACER1), a key enzyme in sphingolipid metabolism, as a potential therapeutic target for SLC39A8-associated IBDs. Importantly, treatment with an ACER1 inhibitor attenuates colitis in Slc39a8-IEC KO mice by remedying barrier dysfunction. Our results highlight the essential roles of SLC39A8 in intestinal Mn absorption and epithelial integrity and offer a therapeutic target for IBD associated with impaired Mn homeostasis.


Assuntos
Ceramidase Alcalina , Proteínas de Transporte de Cátions , Doenças Inflamatórias Intestinais , Mucosa Intestinal , Manganês , Camundongos Knockout , Animais , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Transporte de Cátions/genética , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/patologia , Manganês/metabolismo , Camundongos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Ceramidase Alcalina/metabolismo , Ceramidase Alcalina/genética , Humanos , Camundongos Endogâmicos C57BL , Homeostase , Masculino , Colite/metabolismo , Colite/genética , Colite/patologia , Absorção Intestinal , Células Epiteliais/metabolismo
11.
Front Immunol ; 15: 1405622, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38827741

RESUMO

Background: Severe acute pancreatitis (SAP) is an inflammatory disorder affecting the gastrointestinal system. Intestinal injury plays an important role in the treatment of severe acute pancreatitis. In this study, we mainly investigated the role of S1PR2 in regulating macrophage pyroptosis in the intestinal injury of severe acute pancreatitis. Methods: The SAP model was constructed using cerulein and lipopolysaccharide, and the expression of S1PR2 was inhibited by JTE-013 to detect the degree of pancreatitis and intestinal tissue damage in mice. Meanwhile, the level of pyroptosis-related protein was detected by western blot, the level of related mRNA was detected by PCR, and the level of serum inflammatory factors was detected by ELISA. In vitro experiments, LPS+ATP was used to construct the pyroptosis model of THP-1. After knockdown and overexpression of S1PR2, the pyroptosis proteins level was detected by western blot, the related mRNA level was detected by PCR, and the level of cell supernatant inflammatory factors were detected by ELISA. A rescue experiment was used to verify the sufficient necessity of the RhoA/ROCK pathway in S1PR2-induced pyroptosis. Meanwhile, THP-1 and FHC were co-cultured to verify that cytokines released by THP-1 after damage could regulate FHC damage. Results: Our results demonstrated that JTE-013 effectively attenuated intestinal injury and inflammation in mice with SAP. Furthermore, we observed a significant reduction in the expression of pyroptosis-related proteins within the intestinal tissue of SAP mice upon treatment with JTE-013. We confirmed the involvement of S1PR2 in THP-1 cell pyroptosis in vitro. Specifically, activation of S1PR2 triggered pyroptosis in THP-1 cells through the RhoA/ROCK signaling pathway. Moreover, it was observed that inflammatory factors released during THP-1 cell pyroptosis exerted an impact on cohesin expression in FHC cells. Conclusion: The involvement of S1PR2 in SAP-induced intestinal mucosal injury may be attributed to its regulation of macrophage pyroptosis.


Assuntos
Modelos Animais de Doenças , Macrófagos , Pancreatite , Piroptose , Receptores de Esfingosina-1-Fosfato , Animais , Camundongos , Humanos , Macrófagos/metabolismo , Macrófagos/imunologia , Pancreatite/metabolismo , Pancreatite/imunologia , Pancreatite/patologia , Pancreatite/induzido quimicamente , Receptores de Esfingosina-1-Fosfato/metabolismo , Receptores de Esfingosina-1-Fosfato/genética , Masculino , Transdução de Sinais , Camundongos Endogâmicos C57BL , Proteína rhoA de Ligação ao GTP/metabolismo , Células THP-1 , Quinases Associadas a rho/metabolismo , Quinases Associadas a rho/genética , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Intestinos/patologia , Intestinos/imunologia , Citocinas/metabolismo , Lipopolissacarídeos , Pirazóis , Piridinas
13.
Sci Rep ; 14(1): 12960, 2024 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-38839819

RESUMO

The maintenance of intestinal integrity and barrier function under conditions of restricted oxygen availability is crucial to avoid bacterial translocation and local inflammation. Both lead to secondary diseases after hemorrhagic shock and might increase morbidity and mortality after surviving the initial event. Monitoring of the intestinal integrity especially in the early course of critical illness remains challenging. Since microcirculation and mitochondrial respiration are main components of the terminal stretch of tissue oxygenation, the evaluation of microcirculatory and mitochondrial variables could identify tissues at risk during hypoxic challenges, indicate an increase of intestinal injury, and improve our understanding of regional pathophysiology during acute hemorrhage. Furthermore, improving intestinal microcirculation or mitochondrial respiration, e.g. by remote ischemic preconditioning (RIPC) that was reported to exert a sufficient tissue protection in various tissues and was linked to mediators with vasoactive properties could maintain intestinal integrity. In this study, postcapillary oxygen saturation (µHbO2), microvascular flow index (MFI) and plasmatic D-lactate concentration revealed to be early markers of intestinal injury in a rodent model of experimental hemorrhagic shock. Mitochondrial function was not impaired in this experimental model of acute hemorrhage. Remote ischemic preconditioning (RIPC) failed to improve intestinal microcirculation and intestinal damage during hemorrhagic shock.


Assuntos
Biomarcadores , Intestinos , Precondicionamento Isquêmico , Microcirculação , Choque Hemorrágico , Animais , Precondicionamento Isquêmico/métodos , Ratos , Choque Hemorrágico/terapia , Intestinos/irrigação sanguínea , Masculino , Biomarcadores/sangue , Modelos Animais de Doenças , Mitocôndrias/metabolismo , Mucosa Intestinal/metabolismo , Ácido Láctico/sangue , Ácido Láctico/metabolismo
14.
Sci Rep ; 14(1): 12879, 2024 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-38839896

RESUMO

Paneth cells (PCs), a subset of intestinal epithelial cells (IECs) found at the base of small intestinal crypts, play an essential role in maintaining intestinal homeostasis. Altered PCs function is associated with diverse intestinal pathologies, including ileal Crohn's disease (CD). CD patients with ileal involvement have been previously demonstrated to display impairment in PCs and decreased levels of anti-microbial peptides. Although the immunosuppressive drug Azathioprine (AZA) is widely used in CD therapy, the impact of AZA on IEC differentiation remains largely elusive. In the present study, we hypothesized that the orally administered drug AZA also exerts its effect through modulation of the intestinal epithelium and specifically via modulation of PC function. AZA-treated CD patients exhibited an ileal upregulation of AMPs on both mRNA and protein levels compared to non-AZA treated patients. Upon in vitro AZA stimulation, intestinal epithelial cell line MODE-K exhibited heightened expression levels of PC marker in concert with diminished cell proliferation but boosted mitochondrial OXPHOS activity. Moreover, differentiation of IECs, including PCs differentiation, was boosted in AZA-treated murine small intestinal organoids and was associated with decreased D-glucose consumption and decreased growth rates. Of note, AZA treatment strongly decreased Lgr5 mRNA expression as well as Ki67 positive cells. Further, AZA restored dysregulated PCs associated with mitochondrial dysfunction. AZA-dependent inhibition of IEC proliferation is accompanied by boosted mitochondria function and IEC differentiation into PC.


Assuntos
Azatioprina , Diferenciação Celular , Doença de Crohn , Mucosa Intestinal , Celulas de Paneth , Doença de Crohn/tratamento farmacológico , Doença de Crohn/patologia , Doença de Crohn/metabolismo , Azatioprina/farmacologia , Celulas de Paneth/metabolismo , Celulas de Paneth/efeitos dos fármacos , Celulas de Paneth/patologia , Humanos , Diferenciação Celular/efeitos dos fármacos , Animais , Camundongos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Feminino , Masculino , Íleo/efeitos dos fármacos , Íleo/metabolismo , Íleo/patologia , Adulto , Organoides/efeitos dos fármacos , Organoides/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Proliferação de Células/efeitos dos fármacos , Pessoa de Meia-Idade , Linhagem Celular , Índice de Gravidade de Doença
15.
BMC Res Notes ; 17(1): 154, 2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38840260

RESUMO

OBJECTIVE: The IPEC-J2 cell line is used as an in vitro small intestine model for swine, but it is also used as a model for the human intestine, presenting a relatively unique setting. By combining electric cell-substrate impedance sensing, with next-generation-sequencing technology, we showed that mRNA gene expression profiles and related pathways can depend on the growth phase of IPEC-J2 cells. Our investigative approach welcomes scientists to reproduce or modify our protocols and endorses putting their gene expression data in the context of the respective growth phase of the cells. RESULTS: Three time points are presented: (TP1) 1 h after medium change (= 6 h after seeding of cells), (TP2) the time point of the first derivative maximum of the cell growth curve, and a third point at the beginning of the plateau phase (TP3). Significantly outstanding at TP1 compared to TP2 was upregulated PLEKHN1, further FOSB and DEGS2 were significantly downregulated at TP2 compared to TP3. Any provided data can be used to improve next-generation experiments with IPEC-J2 cells.


Assuntos
Proliferação de Células , Perfilação da Expressão Gênica , RNA Mensageiro , Animais , Linhagem Celular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Suínos , Perfilação da Expressão Gênica/métodos , Proliferação de Células/genética , Intestino Delgado/metabolismo , Intestino Delgado/citologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/citologia , Transcriptoma/genética
16.
Vet Q ; 44(1): 1-11, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38832661

RESUMO

Hemorrhagic bowel syndrome (HBS) is characterized by a dissecting intramucosal hematoma at the small bowel, causing obstruction and severe hemorrhage in dairy cattle. Recent investigation revealed the presence of early-stage lesions in cows affected by HBS. These are presumed to be the initial stage of the hematoma, as both share unique dissection of the lamina muscularis mucosae (LMM) as histological hallmark. Early-stage lesions of HBS have not been characterized in greater detail, and neither has the hypothesis of mucosal abrasion as etiology been explored. Therefore, the first objective of the present study was to characterize the morphology of early-stage lesions, by gross examination, histochemistry, immunohistochemistry and transmission electron microscopy. The second objective was to determine the effect of mucosal abrasion to the small intestine in an ex vivo model. A total of 86 early-stage lesions from 10 cows with HBS were characterized. No underlying alterations at the LMM were evident which could explain their occurrence. However, degeneration at the ultrastructural level of the LMM smooth muscle cells was present in 3 of 4 lesions, it is however unclear whether this is primary or secondary. Bacteriological examination did not reveal any association with a specific bacterium. Experimental-induced and early-stage lesions were gross and histologically evaluated and scored in three cows with HBS and seven controls. Experimentally induced lesions in both affected cows and controls, were histologically very similar to the naturally occurring early-stage lesions. Altogether, the results are suggestive for mucosal trauma to play a role in the pathogenesis of HBS.


Assuntos
Doenças dos Bovinos , Hemorragia Gastrointestinal , Mucosa Intestinal , Animais , Bovinos , Doenças dos Bovinos/patologia , Mucosa Intestinal/patologia , Mucosa Intestinal/ultraestrutura , Feminino , Hemorragia Gastrointestinal/veterinária , Hemorragia Gastrointestinal/patologia , Microscopia Eletrônica de Transmissão/veterinária , Intestino Delgado/patologia , Imuno-Histoquímica/veterinária , Enteropatias/veterinária , Enteropatias/patologia
17.
Nat Commun ; 15(1): 4764, 2024 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-38834561

RESUMO

Bacteriophage are sophisticated cellular parasites that can not only parasitize bacteria but are increasingly recognized for their direct interactions with mammalian hosts. Phage adherence to mucus is known to mediate enhanced antimicrobial effects in vitro. However, little is known about the therapeutic efficacy of mucus-adherent phages in vivo. Here, using a combination of in vitro gastrointestinal cell lines, a gut-on-a-chip microfluidic model, and an in vivo murine gut model, we demonstrated that a E. coli phage, øPNJ-6, provided enhanced gastrointestinal persistence and antimicrobial effects. øPNJ-6 bound fucose residues, of the gut secreted glycoprotein MUC2, through domain 1 of its Hoc protein, which led to increased intestinal mucus production that was suggestive of a positive feedback loop mediated by the mucus-adherent phage. These findings extend the Bacteriophage Adherence to Mucus model into phage therapy, demonstrating that øPNJ-6 displays enhanced persistence within the murine gut, leading to targeted depletion of intestinal pathogenic bacteria.


Assuntos
Infecções por Escherichia coli , Escherichia coli , Mucosa Intestinal , Mucina-2 , Animais , Escherichia coli/virologia , Camundongos , Mucosa Intestinal/microbiologia , Mucosa Intestinal/virologia , Mucina-2/metabolismo , Humanos , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/terapia , Terapia por Fagos/métodos , Aderência Bacteriana , Feminino , Muco/metabolismo , Muco/virologia , Colífagos/fisiologia , Fucose/metabolismo , Camundongos Endogâmicos C57BL
18.
Front Immunol ; 15: 1368545, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38835764

RESUMO

There is a rapidly growing interest in how the avian intestine is affected by dietary components and feed additives. The paucity of physiologically relevant models has limited research in this field of poultry gut health and led to an over-reliance on the use of live birds for experiments. The development of complex 3D intestinal organoids or "mini-guts" has created ample opportunities for poultry research in this field. A major advantage of the floating chicken intestinal organoids is the combination of a complex cell system with an easily accessible apical-out orientation grown in a simple culture medium without an extracellular matrix. The objective was to investigate the impact of a commercial proprietary blend of organic acids and essential oils (OA+EO) on the innate immune responses and kinome of chicken intestinal organoids in a Salmonella challenge model. To mimic the in vivo prolonged exposure of the intestine to the product, the intestinal organoids were treated for 2 days with 0.5 or 0.25 mg/mL OA+EO and either uninfected or infected with Salmonella and bacterial load in the organoids was quantified at 3 hours post infection. The bacteria were also treated with OA+EO for 1 day prior to challenge of the organoids to mimic intestinal exposure. The treatment of the organoids with OA+EO resulted in a significant decrease in the bacterial load compared to untreated infected organoids. The expression of 88 innate immune genes was investigated using a high throughput qPCR array, measuring the expression of 88 innate immune genes. Salmonella invasion of the untreated intestinal organoids resulted in a significant increase in the expression of inflammatory cytokine and chemokines as well as genes involved in intracellular signaling. In contrast, when the organoids were treated with OA+EO and challenged with Salmonella, the inflammatory responses were significantly downregulated. The kinome array data suggested decreased phosphorylation elicited by the OA+EO with Salmonella in agreement with the gene expression data sets. This study demonstrates that the in vitro chicken intestinal organoids are a new tool to measure the effect of the feed additives in a bacterial challenge model by measuring innate immune and protein kinases responses.


Assuntos
Ração Animal , Galinhas , Intestinos , Organoides , Animais , Intestinos/imunologia , Intestinos/efeitos dos fármacos , Intestinos/microbiologia , Imunidade Inata , Óleos Voláteis/farmacologia , Salmonelose Animal/imunologia , Salmonelose Animal/microbiologia , Doenças das Aves Domésticas/microbiologia , Doenças das Aves Domésticas/imunologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/efeitos dos fármacos
19.
PLoS One ; 19(6): e0304686, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38837998

RESUMO

Microplastics, which are tiny plastic particles less than 5 mm in diameter, are widely present in the environment, have become a serious threat to aquatic life and human health, potentially causing ecosystem disorders and health problems. The present study aimed to investigate the effects of microplastics, specifically microplastics-polystyrene (MPs-PS), on the structural integrity, gene expression related to tight junctions, and gut microbiota in mice. A total of 24 Kunming mice aged 30 days were randomly assigned into four groups: control male (CM), control female (CF), PS-exposed male (PSM), and PS-exposed female (PSF)(n = 6). There were significant differences in villus height, width, intestinal surface area, and villus height to crypt depth ratio (V/C) between the PS group and the control group(C) (p <0.05). Gene expression analysis demonstrated the downregulation of Claudin-1, Claudin-2, Claudin-15, and Occludin, in both duodenum and jejunum of the PS group (p < 0.05). Analysis of microbial species using 16S rRNA sequencing indicated decreased diversity in the PSF group, as well as reduced diversity in the PSM group at various taxonomic levels. Beta diversity analysis showed a significant difference in gut microbiota distribution between the PS-exposed and C groups (R2 = 0.113, p<0.01), with this difference being more pronounced among females exposed to MPs-PS. KEGG analysis revealed enrichment of differential microbiota mainly involved in seven signaling pathways, such as nucleotide metabolism(p<0.05). The relative abundance ratio of transcriptional pathways was significantly increased for the PSF group (p<0.01), while excretory system pathways were for PSM group(p<0.05). Overall findings suggest that MPs-PS exhibit a notable sex-dependent impact on mouse gut microbiota, with a stronger effect observed among females; reduced expression of tight junction genes may be associated with dysbiosis, particularly elevated levels of Prevotellaceae.


Assuntos
Microbioma Gastrointestinal , Microplásticos , Poliestirenos , Junções Íntimas , Animais , Microbioma Gastrointestinal/efeitos dos fármacos , Microplásticos/toxicidade , Poliestirenos/toxicidade , Camundongos , Masculino , Feminino , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , RNA Ribossômico 16S/genética , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Ocludina/metabolismo , Ocludina/genética , Claudinas/genética , Claudinas/metabolismo , Claudina-1/genética , Claudina-1/metabolismo , Proteínas de Junções Íntimas/metabolismo , Proteínas de Junções Íntimas/genética
20.
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue ; 36(5): 496-502, 2024 May.
Artigo em Chinês | MEDLINE | ID: mdl-38845496

RESUMO

OBJECTIVE: To analyze the impact of cecal ligation and puncture (CLP)-induced sepsis on the proliferation and differentiation of intestinal epithelial cells. METHODS: (1) Animal experiment: sixteen male C57BL/6 mice were divided into sham operation group (Sham group) and CLP-induced sepsis model group (CLP group) by random number table method, with 8 mice in each group. After 5 days of operation, the jejunal tissues were taken for determination of leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5) and intestinal alkaline phosphatase (IAP) by polymerase chain reaction (PCR). The translation of LGR5 was detected by Western blotting. The expression of proliferating cell nuclear antigen (Ki67) was analyzed by immunohistochemistry. IAP level was detected by modified calcium cobalt staining and colorimetry. Immunofluorescence staining was used to detect the expression of Paneth cell marker molecule lysozyme 1 (LYZ1) and goblet cell marker molecule mucin 2 (MUC2). (2) Cell experiment: IEC6 cells in logarithmic growth stage were divided into blank control group and lipopolysaccharide (LPS) group (LPS 5 µg/mL). Twenty-four hours after treatment, PCR and Western blotting were used to analyze the transcription and translation of LGR5. The proliferation of IEC6 cells were detected by 5-ethynyl-2'-deoxyuridine (EdU) staining. The transcription and translation of IAP were detected by PCR and colorimetric method respectively. RESULTS: (1) Animal experiment: the immunohistochemical results showed that the positive rate of Ki67 staining in the jejunal tissue of CLP group was lower than that of Sham group [(41.7±2.5)% vs. (48.7±1.4)%, P = 0.01]. PCR and Western blotting results showed that there were no statistical differences in the mRNA and protein expressions of LGR5 in the jejunal tissue between the CLP group and Sham group (Lgr5 mRNA: 0.7±0.1 vs. 1.0±0.2, P = 0.11; LGR5/ß-actin: 0.83±0.17 vs. 0.68±0.19, P = 0.24). The mRNA (0.4±0.1 vs. 1.0±0.1, P < 0.01) and protein (U/g: 47.3±6.0 vs. 73.1±15.3, P < 0.01) levels of IAP in the jejunal tissue were lower in CLP group. Immunofluorescence saining analysis showed that the expressions of LYZ1 and MUC2 in the CLP group were lower than those in the Sham group. (2) Cell experiment: PCR and Western blotting results showed that there was no significant difference in the expression of LGR5 between the LPS group and the blank control group (Lgr5 mRNA: 0.9±0.1 vs. 1.0±0.2, P = 0.33; LGR5/ß-actin: 0.71±0.18 vs. 0.69±0.04, P = 0.81). The proliferation rate of IEC6 cells in the LPS group was lower than that in the blank control group, but there was no significant difference [positivity rate of EdU: (40.5±3.8)% vs. (46.5±3.6)%, P = 0.11]. The mRNA (0.5±0.1 vs. 1.0±0.2, P < 0.01) and protein (U/g: 15.0±4.0 vs. 41.2±10.4, P < 0.01) of IAP in the LPS group were lower than those in the blank control group. CONCLUSIONS: CLP-induced sepsis inhibits the proliferation and differentiation of intestinal epithelial cells, impairing the self-renewal ability of intestinal epithelium.


Assuntos
Diferenciação Celular , Proliferação de Células , Camundongos Endogâmicos C57BL , Receptores Acoplados a Proteínas G , Sepse , Células-Tronco , Animais , Masculino , Sepse/metabolismo , Camundongos , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco/metabolismo , Células-Tronco/citologia , Ceco , Mucosa Intestinal/metabolismo , Ligadura , Mucina-2
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