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1.
PLoS One ; 17(2): e0262855, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35108315

RESUMO

The incidence of nonalcoholic fatty liver disease (NAFLD) in postmenopausal women has increased significantly. Estrogen plays a very important role in NAFLD, but whether NAFLD in premenopausal women was caused by estrogen deficiency was unknown. Thus, it is of great clinical significance to explore the mechanism of NAFLD in premenopausal women. Gut microbiota and its metabolites short chain fatty acids (SCFA) have been shown to play important roles in the development of NAFLD. In this study, we investigated the impact of gut microbiota and SCFA in NAFLD patients and mice caused by estrogen deficiency. We showed that premenopause NAFLD patients had much lower estrogen levels. Estrogen deficient mice, due to ovariectomy (OVX), suffered more severe liver steatosis with an elevated body weight, abdominal fat weight, serum triglycerides and deterioration in hepatic steatosis. Altered gut microbiota composition and decreased butyrate content were found in NAFLD patients and in OVX mice. Furthermore, fecal microbiota transplantation (FMT) or supplementing with butyrate alleviated NAFLD in OVX mice. The production of antimicrobial peptides (AMP) Reg3É£, ß-defensins and the expression of intestinal epithelial tight junction, including ZO-1 and Occluding-5, were decreased in the OVX mice compared to control mice. Upregulation of PPAR-É£ and VLDLR, downregulation of PPAR-ɑ indicated that OVX mice suffered from abnormal lipid metabolism. These data indicate that changes in the gut microbiota and SCFA caused by estrogen reduction, together with a disorder in AMP production and lipid metabolism, promote NAFLD, thus provide SCFAs derived from microbiota as new therapeutic targets for the clinical prevention and treatment of NAFLD.


Assuntos
Butiratos/metabolismo , Estrogênios/metabolismo , Microbioma Gastrointestinal , Hepatopatia Gordurosa não Alcoólica/patologia , Adulto , Animais , Defensinas/genética , Defensinas/metabolismo , Modelos Animais de Doenças , Estrogênios/deficiência , Ácidos Graxos Voláteis/metabolismo , Transplante de Microbiota Fecal , Feminino , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/terapia , Ovariectomia , Pré-Menopausa , Triglicerídeos/sangue , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismo
2.
Commun Biol ; 5(1): 112, 2022 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-35132135

RESUMO

Thyroid hormone (T3) regulates adult intestine development through T3 receptors (TRs). It is difficult to study TR function during postembryonic intestinal maturation in mammals due to maternal influence. We chose intestinal remodeling during Xenopus tropicalis metamorphosis as a model to study TR function in adult organ development. By using ChIP (chromatin immunoprecipitation)-Seq, we identified over 3000 TR-bound genes in the intestine of premetamorphic wild type or TRα (the major TR expressed during premetamorphosis)-knockout tadpoles. Surprisingly, cell cycle-related GO (gene ontology) terms and biological pathways were highly enriched among TR target genes even though the first major event during intestinal metamorphosis is larval epithelial cell death, and TRα knockout drastically reduced this enrichment. More importantly, treatment of tadpoles with cell cycle inhibitors blocked T3-induced intestinal remodeling, especially larval epithelial cell death, suggesting that TRα-dependent activation of cell cycle is important for T3-induced apoptosis during intestinal remodeling.


Assuntos
Proteína Quinase CDC2/metabolismo , Morte Celular/fisiologia , Células Epiteliais/fisiologia , Mucosa Intestinal/citologia , Receptores alfa dos Hormônios Tireóideos/metabolismo , Hormônios Tireóideos/metabolismo , Animais , Proteína Quinase CDC2/genética , Morte Celular/genética , Deleção de Genes , Regulação da Expressão Gênica/fisiologia , Mucosa Intestinal/fisiologia , Larva/fisiologia , Receptores alfa dos Hormônios Tireóideos/genética , Hormônios Tireóideos/genética , Xenopus
3.
Nat Commun ; 13(1): 693, 2022 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-35121734

RESUMO

Intracellular pathogens are challenged with limited space and resources while replicating in a single host cell. Mechanisms for direct invasion of neighboring host cells have been discovered in cell culture, but we lack an understanding of how bacteria directly spread between host cells in vivo. Here, we describe the discovery of intracellular bacteria that use filamentation for spreading between the intestinal epithelial cells of a natural host, the rhabditid nematode Oscheius tipulae. The bacteria, which belong to the new species Bordetella atropi, can infect the nematodes following a fecal-oral route, and reduce host life span and fecundity. Filamentation requires UDP-glucose biosynthesis and sensing, a highly conserved pathway that is used by other bacteria to detect rich conditions and inhibit cell division. Our results indicate that B. atropi uses a pathway that normally regulates bacterial cell size to trigger filamentation inside host cells, thus facilitating cell-to-cell dissemination.


Assuntos
Bordetella/crescimento & desenvolvimento , Mucosa Intestinal/citologia , Rhabditoidea/citologia , Animais , Bordetella/classificação , Bordetella/patogenicidade , Divisão Celular/genética , Células Epiteliais/microbiologia , Células Epiteliais/ultraestrutura , Genoma Bacteriano/genética , Interações Hospedeiro-Patógeno , Hibridização in Situ Fluorescente , Mucosa Intestinal/microbiologia , Espaço Intracelular/microbiologia , Redes e Vias Metabólicas/genética , Microscopia Eletrônica de Transmissão , Filogenia , RNA Ribossômico 16S/genética , Rhabditoidea/genética , Rhabditoidea/microbiologia , Análise de Sequência de DNA , Virulência
4.
Nat Commun ; 13(1): 874, 2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-35169117

RESUMO

IL-18 is emerging as an IL-22-induced and epithelium-derived cytokine which contributes to host defence against intestinal infection and inflammation. In contrast to its known role in Goblet cells, regulation of barrier function at the molecular level by IL-18 is much less explored. Here we show that IL-18 is a bona fide IL-22-regulated gate keeper for intestinal epithelial barrier. IL-22 promotes crypt immunity both via induction of phospho-Stat3 binding to the Il-18 gene promoter and via Il-18 independent mechanisms. In organoid culture, while IL-22 primarily increases organoid size and inhibits expression of stem cell genes, IL-18 preferentially promotes organoid budding and induces signature genes of Lgr5+ stem cells via Akt-Tcf4 signalling. During adherent-invasive E. coli (AIEC) infection, systemic administration of IL-18 corrects compromised T-cell IFNγ production and restores Lysozyme+ Paneth cells in Il-22-/- mice, but IL-22 administration fails to restore these parameters in Il-18-/- mice, thereby placing IL-22-Stat3 signalling upstream of the IL-18-mediated barrier defence function. IL-18 in return regulates Stat3-mediated anti-microbial response in Paneth cells, Akt-Tcf4-triggered expansion of Lgr5+ stem cells to facilitate tissue repair, and AIEC clearance by promoting IFNγ+ T cells.


Assuntos
Infecções por Escherichia coli/imunologia , Imunidade nas Mucosas/imunologia , Interleucina-18/imunologia , Interleucinas/imunologia , Mucosa Intestinal/imunologia , Animais , Doença de Crohn/microbiologia , Doença de Crohn/patologia , Disbiose/microbiologia , Escherichia coli/imunologia , Interferon gama/imunologia , Interleucina-18/genética , Mucosa Intestinal/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Muramidase/metabolismo , Organoides , Celulas de Paneth/imunologia , Regiões Promotoras Genéticas/genética , Fator de Transcrição STAT3/metabolismo , Junções Íntimas/imunologia
5.
Bioengineered ; 13(3): 6490-6499, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35220895

RESUMO

Ulcerative colitis (UC) is a type of chronic disease of inflammation, and matrine has anti-inflammatory activity. However, it is unclear that whether matrine can alleviate UC. This study aimed to evaluate the effect of matrine on DSS-induced intestinal epithelial cell injury. Cell viability was performed by MTT assay. Then cell apoptosis was analyzed using the TUNEL assay and flow cytometry. The levels of interleukin (IL)-2, IL-6, TNF-α, and IL-1ß were evaluated using qRT-PCR. Myeloperoxidase (MPO) activity was detected using ELISA assay. Nitric oxide (NO) production was detected by the Griess reagent. Bax, cleaved caspase-3, Bcl-2, JAK2, p-JAK2, STAT3, p-STAT3, STAT5, p-STAT5 levels were measured by Western blot. Bax (6A7) was asses using immunoprecipitation and immunofluorescence assays. The results illustrated that cell viability was inhibited as the concentration of DSS increased. Matrine did not affect cell viability at the concentration of 0-2 mg/ml but inhibited cell viability in a time-independent manner. Matrine suppressed the levels of pro-inflammatory factors, MPO activity, NO production, and apoptosis of DSS-stimulated cells. Furthermore, we found that matrine inhibited the levels of p-JAK2/JAK2 and p-STAT3/STAT3 but did not affect p-STAT5/STAT5. AG490 treatment further enhanced the effect of matrine on the apoptosis and pro-inflammatory factor levels in DSS-induced cells. In summary, matrine protected NCM460 cell against injury by inactivating the JAK2/STAT3 pathway. These data suggested for the first time that matrine may effective in treating UC.


Assuntos
Alcaloides , Apoptose/efeitos dos fármacos , Colo , Mucosa Intestinal , Substâncias Protetoras , Quinolizinas , Alcaloides/química , Alcaloides/farmacologia , Linhagem Celular , Colite Ulcerativa , Colo/citologia , Colo/efeitos dos fármacos , Humanos , Inflamação/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Substâncias Protetoras/química , Substâncias Protetoras/farmacologia , Quinolizinas/química , Quinolizinas/farmacologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos
6.
Nat Commun ; 13(1): 715, 2022 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-35132078

RESUMO

Organs are anatomically compartmentalised to cater for specialised functions. In the small intestine (SI), regionalisation enables sequential processing of food and nutrient absorption. While several studies indicate the critical importance of non-epithelial cells during development and homeostasis, the extent to which these cells contribute to regionalisation during morphogenesis remains unexplored. Here, we identify a mesenchymal-epithelial crosstalk that shapes the developing SI during late morphogenesis. We find that subepithelial mesenchymal cells are characterised by gradients of factors supporting Wnt signalling and stimulate epithelial growth in vitro. Such a gradient impacts epithelial gene expression and regional villus formation along the anterior-posterior axis of the SI. Notably, we further provide evidence that Wnt signalling directly regulates epithelial expression of Sonic Hedgehog (SHH), which, in turn, acts on mesenchymal cells to drive villi formation. Taken together our results uncover a mechanistic link between Wnt and Hedgehog signalling across different cellular compartments that is central for anterior-posterior regionalisation and correct formation of the SI.


Assuntos
Proteínas Hedgehog/metabolismo , Mucosa Intestinal/metabolismo , Intestino Delgado/embriologia , Células-Tronco Mesenquimais/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Linhagem da Célula , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Mucosa Intestinal/citologia , Mucosa Intestinal/embriologia , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Células-Tronco Mesenquimais/citologia , Camundongos , Morfogênese , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Via de Sinalização Wnt/genética
7.
Science ; 375(6576): eaaw9021, 2022 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-34990240

RESUMO

Epithelial organoids are stem cell­derived tissues that approximate aspects of real organs, and thus they have potential as powerful tools in basic and translational research. By definition, they self-organize, but the structures formed are often heterogeneous and irreproducible, which limits their use in the lab and clinic. We describe methodologies for spatially and temporally controlling organoid formation, thereby rendering a stochastic process more deterministic. Bioengineered stem cell microenvironments are used to specify the initial geometry of intestinal organoids, which in turn controls their patterning and crypt formation. We leveraged the reproducibility and predictability of the culture to identify the underlying mechanisms of epithelial patterning, which may contribute to reinforcing intestinal regionalization in vivo. By controlling organoid culture, we demonstrate how these structures can be used to answer questions not readily addressable with the standard, more variable, organoid models.


Assuntos
Mucosa Intestinal/crescimento & desenvolvimento , Organogênese , Organoides/crescimento & desenvolvimento , Engenharia Tecidual , Animais , Diferenciação Celular , Forma Celular , Células Epiteliais/citologia , Hidrogéis , Mucosa Intestinal/anatomia & histologia , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Camundongos , Organoides/anatomia & histologia , Organoides/citologia , Organoides/metabolismo , Celulas de Paneth/citologia , Receptores Notch/metabolismo , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/fisiologia , Técnicas de Cultura de Tecidos , /metabolismo
8.
J Immunol ; 208(3): 745-752, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-35031577

RESUMO

Cystic fibrosis (CF) is an inherited life-threatening disease accompanied by repeated lung infections and multiorgan inflammation that affects tens of thousands of people worldwide. The causative gene, cystic fibrosis transmembrane conductance regulator (CFTR), is mutated in CF patients. CFTR functions in epithelial cells have traditionally been thought to cause the disease symptoms. Recent work has shown an additional defect: monocytes from CF patients show a deficiency in integrin activation and adhesion. Because monocytes play critical roles in controlling infections, defective monocyte function may contribute to CF progression. In this study, we demonstrate that monocytes from CFTRΔF508 mice (CF mice) show defective adhesion under flow. Transplanting CF mice with wild-type (WT) bone marrow after sublethal irradiation replaced most (60-80%) CF monocytes with WT monocytes, significantly improved survival, and reduced inflammation. WT/CF mixed bone marrow chimeras directly demonstrated defective CF monocyte recruitment to the bronchoalveolar lavage and the intestinal lamina propria in vivo. WT mice reconstituted with CF bone marrow also show lethality, suggesting that the CF defect in monocytes is not only necessary but also sufficient to cause disease. We also show that monocyte-specific knockout of CFTR retards weight gains and exacerbates dextran sulfate sodium-induced colitis. Our findings show that providing WT monocytes by bone marrow transfer rescues mortality in CF mice, suggesting that similar approaches may mitigate disease in CF patients.


Assuntos
Adesão Celular/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/terapia , Monócitos/imunologia , Monócitos/transplante , Animais , Transplante de Medula Óssea , Líquido da Lavagem Broncoalveolar/citologia , Colite/patologia , Fibrose Cística/patologia , Integrinas/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Camundongos , Camundongos Endogâmicos C57BL
9.
Biomed Mater ; 17(2)2022 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-35026740

RESUMO

The design of bone scaffolds is predominately aimed to well reproduce the natural bony environment by imitating the architecture/composition of host bone. Such biomimetic biomaterials are gaining increasing attention and acknowledged quite promising for bone tissue engineering. Herein, novel biomimetic bone scaffolds containing decellularized small intestinal submucosa matrix (SIS-ECM) and Sr2+/Fe3+co-doped hydroxyapatite (SrFeHA) are fabricated for the first time by the sophisticated self-assembled mineralization procedure, followed by cross-linking and lyophilization post-treatments. The results indicate the constructed SIS/SrFeHA scaffolds are characterized by highly porous structures, rough microsurface and improved mechanical strength, as well as efficient releasing of bioactive Sr2+/Fe3+and ECM components. These favorable physico-chemical properties endow SIS/SrFeHA scaffolds with an architectural/componential biomimetic bony environment which appears to be highly beneficial for inducing angiogenesis/osteogenesis bothin vitroandin vivo. In particular, the cellular functionality and bioactivity of endotheliocytes/osteoblasts are significantly enhanced by SIS/SrFeHA scaffolds, and the cranial defects model further verifies the potent ability of SIS/SrFeHA to acceleratein vivovascularization and bone regeneration following implantation. In this view these results highlight the considerable angiogenesis/osteogenesis potential of biomimetic porous SIS/SrFeHA scaffolds for inducing bone regeneration and thus may afford a new promising alternative for bone tissue engineering.


Assuntos
Regeneração Óssea/efeitos dos fármacos , Durapatita , Osteogênese/efeitos dos fármacos , Tecidos Suporte/química , Animais , Materiais Biomiméticos , Linhagem Celular , Células Cultivadas , /farmacologia , Durapatita/química , Durapatita/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Mucosa Intestinal/citologia , Intestino Delgado/citologia , Camundongos , Neovascularização Fisiológica/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Porosidade
10.
J Radiat Res ; 63(2): 149-157, 2022 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-35021216

RESUMO

Intestinal stem cells (ISCs) are essential for the regeneration of intestinal cells upon radiation or chemical agent damage. As for radiation-induced damage, the expression of AIM2, YAP, TLR3, PUMA or BVES can aggravate ISCs depletion, while the stimulation of TLR5, HGF/MET signaling, Ass1 gene, Slit/Robo signaling facilitate the radio-resistance of ISCs. Upon chemical agent treatment, the activation of TRAIL or p53/PUMA pathway exacerbate injury on ISCs, while the increased levels of IL-22, ß-arrestin1 can ease the damage. The transformation between reserve ISCs (rISCs) maintaining quiescent states and active ISCs (aISCs) that are highly proliferative has obtained much attention in recent years, in which ISCs expressing high levels of Hopx, Bmi1, mTert, Krt19 or Lrig1 are resistant to radiation injury, and SOX9, MSI2, clusterin, URI are essential for rISCs maintenance. The differentiated cells like Paneth cells and enteroendocrine cells can also obtain stemness driven by radiation injury mediated by Wnt or Notch signaling. Besides, Mex3a-expressed ISCs can survive and then proliferate into intestinal epithelial cells upon chemical agent damage. In addition, the modulation of symbiotic microbes harboring gastrointestinal (GI) tract is also a promising strategy to protect ISCs against radiation damage. Overall, the strategies targeting mechanisms modulating ISCs activities are conducive to alleviating GI injury of patients receiving chemoradiotherapy or victims of nuclear or chemical accident.


Assuntos
Mucosa Intestinal , Células-Tronco , Moléculas de Adesão Celular/metabolismo , Diferenciação Celular , Proliferação de Células , Humanos , Mucosa Intestinal/citologia , Intestinos/citologia , Proteínas Musculares/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Células-Tronco/efeitos dos fármacos , Células-Tronco/efeitos da radiação
11.
Cell Rep ; 38(1): 110179, 2022 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-34986353

RESUMO

G protein-coupled receptors (GPCRs) in intestinal enteroendocrine cells (EECs) respond to nutritional, neural, and microbial cues and modulate the release of gut hormones. Here we show that Gpr17, an orphan GPCR, is co-expressed in glucagon-like peptide-1 (GLP-1)-expressing EECs in human and rodent intestinal epithelium. Acute genetic ablation of Gpr17 in intestinal epithelium improves glucose tolerance and glucose-stimulated insulin secretion (GSIS). Importantly, inducible knockout (iKO) mice and Gpr17 null intestinal organoids respond to glucose or lipid ingestion with increased secretion of GLP-1, but not the other incretin glucose-dependent insulinotropic polypeptide (GIP). In an in vitro EEC model, overexpression or agonism of Gpr17 reduces voltage-gated calcium currents and decreases cyclic AMP (cAMP) production, and these are two critical factors regulating GLP-1 secretion. Together, our work shows that intestinal Gpr17 signaling functions as an inhibitory pathway for GLP-1 secretion in EECs, suggesting intestinal GPR17 is a potential target for diabetes and obesity intervention.


Assuntos
Células Enteroendócrinas/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Glucose/metabolismo , Mucosa Intestinal/metabolismo , Proteínas do Tecido Nervoso/genética , Receptores Acoplados a Proteínas G/genética , Animais , Glicemia/análise , Cálcio/metabolismo , Linhagem Celular , AMP Cíclico/metabolismo , Diabetes Mellitus/patologia , Feminino , Polipeptídeo Inibidor Gástrico/metabolismo , Teste de Tolerância a Glucose , Células HEK293 , Células HeLa , Humanos , Incretinas/metabolismo , Insulina/metabolismo , Secreção de Insulina/fisiologia , Mucosa Intestinal/citologia , Masculino , Camundongos , Camundongos Knockout , Obesidade/patologia , Receptores dos Hormônios Gastrointestinais/metabolismo
12.
Immunity ; 55(2): 237-253.e8, 2022 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-35081371

RESUMO

The Th17 cell-lineage-defining cytokine IL-17A contributes to host defense and inflammatory disease by coordinating multicellular immune responses. The IL-17 receptor (IL-17RA) is expressed by diverse intestinal cell types, and therapies targeting IL-17A induce adverse intestinal events, suggesting additional tissue-specific functions. Here, we used multiple conditional deletion models to identify a role for IL-17A in secretory epithelial cell differentiation in the gut. Paneth, tuft, goblet, and enteroendocrine cell numbers were dependent on IL-17A-mediated induction of the transcription factor ATOH1 in Lgr5+ intestinal epithelial stem cells. Although dispensable at steady state, IL-17RA signaling in ATOH1+ cells was required to regenerate secretory cells following injury. Finally, IL-17A stimulation of human-derived intestinal organoids that were locked into a cystic immature state induced ATOH1 expression and rescued secretory cell differentiation. Our data suggest that the cross talk between immune cells and stem cells regulates secretory cell lineage commitment and the integrity of the mucosa.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Mucosa Intestinal/citologia , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Interleucina-17/metabolismo , Células-Tronco/metabolismo , Animais , Comunicação Celular , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Colite/induzido quimicamente , Colite/metabolismo , Colite/patologia , Sulfato de Dextrana/efeitos adversos , Humanos , Interleucina-17/metabolismo , Interleucina-17/farmacologia , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Intestinos/metabolismo , Intestinos/patologia , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Receptores de Interleucina-17/deficiência , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais , Células-Tronco/citologia
13.
J Therm Biol ; 103: 103147, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-35027200

RESUMO

How temperature influences fish physiological systems, such as the intestinal barrier, is important for understanding and alleviating the impact of global warming on fish and aquaculture. Monolayers of the rainbow trout cell line, RTgutGC, with or without linear 500 µm wide gaps (wounds) were the in vitro models used to study the integrity and healing of intestinal epithelial sheets at different temperatures. Cultures at hypothermic (4 °C) or hyperthermic (≥ 26 °C) temperatures were compared to normothermic control cultures (18-22 °C). Monolayers remained intact for at least a week at temperatures from 4 to 28 °C, but had lost their integrity after 3 h at 32 °C as the cells pulled away from one another and from the plastic surface. F-actin appeared as prominent stress fibers in cells at 28 °C and as blobs in cells at 32 °C. At normothermia and at 26 °C, cells migrated as sheets into the gaps and closed (healed) the gaps within 5-6 days. By contrast, wounds took 14 days to heal at 4 °C. At 28 °C some cells migrated into the gap in the first few days but mainly as single cells rather than collectively and wounds never healed. When monolayers with wounds were challenged at 32 °C for 3 h and returned to 18-22 °C, cells lost their shape and actin organization and over the next 6 days detached and died. When monolayers were subjected to 26 °C for 24 h and challenged at 32 °C for 3 h prior to being placed at 18-22 °C, cell shape and actin cytoskeleton were maintained, and wounds were healed over 6 days. Thus, intestinal epithelial cells become thermostabilized for shape, cytoskeleton and migration by a prior heat exposure.


Assuntos
Citoesqueleto de Actina/metabolismo , Células Epiteliais/metabolismo , Temperatura , Cicatrização/fisiologia , Animais , Linhagem Celular , Sobrevivência Celular , Resposta ao Choque Térmico , Mucosa Intestinal/citologia , Oncorhynchus mykiss , Termotolerância
14.
Arch Toxicol ; 96(2): 499-510, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34654938

RESUMO

The small intestine plays a critical role in the absorption and metabolism of orally administered drugs. Therefore, a model capable of evaluating drug absorption and metabolism in the small intestine would be useful for drug discovery. Patients with genotype UGT1A1*6 (exon 1, 211G > A) treated with the antineoplastic drug SN-38 have been reported to exhibit decreased glucuronide conjugation and increased incidence of intestinal toxicity and its severe side effects, including severe diarrhea. To ensure the safety of drugs, we must develop a drug metabolism and toxicity evaluation model which considers UGT1A1*6. In this study, we generated CYP3A4·POR·UGT1A1 KI- and CYP3A4·POR·UGT1A1*6 KI-Caco-2 cells for pharmaceutical research using a PITCh system. The CYP3A4·POR·UGT1A1 KI-Caco-2 cells were shown to express functional CYP3A4 and UGT1A1. The CYP3A4·POR·UGT1A1*6 KI-Caco-2 cells were sensitive to SN-38-induced intestinal toxicity. We thus succeeded in generating CYP3A4·POR·UGT1A1 KI- and CYP3A4·POR·UGT1A1*6 KI-Caco-2 cells, which can be used in pharmaceutical research. We also developed an intestinal epithelial cell model of patients with UGT1A1*6 and showed that it was useful as a tool for drug discovery.


Assuntos
Citocromo P-450 CYP3A/genética , Glucuronosiltransferase/genética , Mucosa Intestinal/enzimologia , Intestino Delgado/enzimologia , Antineoplásicos/toxicidade , Células CACO-2/enzimologia , Descoberta de Drogas/métodos , Genótipo , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Intestino Delgado/citologia , Intestino Delgado/efeitos dos fármacos , Irinotecano/toxicidade
15.
Gastroenterology ; 162(2): 535-547.e13, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34688712

RESUMO

BACKGROUND AND AIMS: The gastrointestinal (GI) tract extracts nutrients from ingested meals while protecting the organism from infectious agents frequently present in meals. Consequently, most animals conduct the entire digestive process within the GI tract while keeping the luminal contents entirely outside the body, separated by the tightly sealed GI epithelium. Therefore, like the skin and oral cavity, the GI tract must sense the chemical and physical properties of the its external interface to optimize its function. Specialized sensory enteroendocrine cells (EECs) in GI epithelium interact intimately with luminal contents. A subpopulation of EECs express the mechanically gated ion channel Piezo2 and are developmentally and functionally like the skin's touch sensor- the Merkel cell. We hypothesized that Piezo2+ EECs endow the gut with intrinsic tactile sensitivity. METHODS: We generated transgenic mouse models with optogenetic activators in EECs and Piezo2 conditional knockouts. We used a range of reference standard and novel techniques from single cells to living animals, including single-cell RNA sequencing and opto-electrophysiology, opto-organ baths with luminal shear forces, and in vivo studies that assayed GI transit while manipulating the physical properties of luminal contents. RESULTS: Piezo2+ EECs have transcriptomic features of synaptically connected, mechanosensory epithelial cells. EEC activation by optogenetics and forces led to Piezo2-dependent alterations in colonic propagating contractions driven by intrinsic circuitry, with Piezo2+ EECs detecting the small luminal forces and physical properties of the luminal contents to regulate transit times in the small and large bowel. CONCLUSIONS: The GI tract has intrinsic tactile sensitivity that depends on Piezo2+ EECs and allows it to detect luminal forces and physical properties of luminal contents to modulate physiology.


Assuntos
Células Enteroendócrinas/metabolismo , Mucosa Intestinal/metabolismo , Canais Iônicos/genética , Tato/fisiologia , Animais , Células Enteroendócrinas/fisiologia , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Técnicas de Inativação de Genes , Mucosa Intestinal/citologia , Mucosa Intestinal/fisiologia , Canais Iônicos/metabolismo , Mecanorreceptores , Camundongos , Camundongos Transgênicos , Optogenética , Peristaltismo/fisiologia
16.
J Crohns Colitis ; 16(1): 109-121, 2022 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-34180971

RESUMO

BACKGROUND AND AIMS: Ulcerative colitis [UC] is a chronic inflammatory disease of the colon with frequent relapses. Telomere shortening in intestinal epithelial cells has been reported in severe or longstanding cases. However, its influence on UC pathogenesis remains unelucidated. To this end, we evaluated telomere shortening using a long-term organoid inflammation model that we had originally established. METHODS: A UC model using human colon organoids was established to assess telomere changes chronologically. MST-312 was used for the telomerase inhibition assay. The potential of telomerase activators as a novel UC treatment was evaluated with an in vitro model, including microarray analysis, and histological changes were assessed using xenotransplantation into mouse colonic mucosa. RESULTS: Our UC model reproduced telomere shortening in vitro, which was induced by the continuous suppression of telomerase activity via P53. MST-312-based analysis revealed that telomere shortening was involved in the pathogenesis of UC. Madecassoside [MD] improved the telomere length of the UC model and UC patient-derived organoids, which further promoted cell proliferation in vitro and improved the graft take-rate of xenotransplantation. Moreover, histological analysis revealed that MD induced normal crypt structure with abundant goblet cells. CONCLUSIONS: This study is the first to reveal the mechanism and importance of telomere shortening in the pathogenesis of UC. MD could be a novel candidate for UC treatment beyond endoscopic mucosal healing.


Assuntos
Colite Ulcerativa/patologia , Células Epiteliais/patologia , Mucosa Intestinal/citologia , Encurtamento do Telômero , Animais , Biópsia , Proliferação de Células , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Colonoscopia , Humanos , Camundongos , Organoides/metabolismo , Organoides/patologia , Organoides/transplante , Espécies Reativas de Oxigênio/metabolismo , Telomerase/metabolismo , Transplante Heterólogo
17.
J Crohns Colitis ; 16(1): 133-142, 2022 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-34309645

RESUMO

BACKGROUND: Shedding of intestinal epithelial cells [IECs] is a potent cause of barrier loss which plays an important role in the pathogenesis of inflammatory bowel disease [IBD]. TNFα can induce IEC shedding, but little is known about this process. METHODS: To investigate the molecular mechanism regulating IEC shedding, mice lacking interferon regulatory factor1 [IRF1], caspase-3, or gasdermin E [GSDME] and their control wild-type [WT] littermates were intravenously injected with tumour necrosis factor alpha [TNFα] to establish an IEC shedding model. A dual-luciferase reporter assay and a chromatin immunoprecipitation assay were used to determine the role of IRF1 in regulating caspase-3 expression. RESULTS: TNFα administration induced obvious IEC shedding in WT mice, but IRF1-/- and caspase-3-/-mice were completely protected from TNFα-induced IEC shedding. As a critical transcription factor, IRF1 was found to be required for caspase-3 expression in IECs by binding to IRF1-binding sites in the caspase-3 promoter. In WT mice, plasma membrane integrity was disrupted in shed IECs; these cells were swollen and contained GSDME-N terminal [NT] fragments which are responsible for the induction of pyroptosis. However, in GSDME-/- mice, plasma membrane integrity was not disrupted in shed IECs, which were not swollen and did not contain GSDME-NT, indicating that GSDME converted TNFα-induced IEC shedding into a pyroptotic cell death process. In addition, IRF1 deficiency resulted in decreases in mucosal inflammation and mucosal bacteria levels in TNFα-challenged colons. CONCLUSIONS: IRF1 deficiency maintains intestinal barrier integrity by restricting TNFα-induced IEC shedding.


Assuntos
Células Epiteliais/patologia , Fator Regulador 1 de Interferon/metabolismo , Mucosa Intestinal/citologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Caspase 3/metabolismo , Morte Celular , Células Cultivadas , Humanos , Camundongos , Transfecção
18.
Food Funct ; 13(1): 161-169, 2022 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-34874374

RESUMO

The public has gradually begun to regard inflammatory bowel disease (IBD) as a crucial health issue; however, its mode of action has not been fully elucidated. Sophorolipid (SPL), a glycolipid-type biosurfactant, could be used as a potential treatment in physical intestinal dystrophy. We conducted a 2 × 2 factorial experiment to investigate the protective effect of SPL in a dextran sulfate sodium (DSS)-induced colitis mouse model (first factor, presence of SPL in feed; second factor, presence of DSS in water). Forty C57BL/6 mice (8-week-old) were used, and they were allocated to treatments according to their initial body weight. After a 7 d adjustment period, the DSS treatment was initiated in specific groups. At day 14, DSS was withdrawn from mice, and half of the mice were randomly selected and euthanized to collect colon and colon content samples. Three days after the end of DSS treatment, the rest of the mice were euthanized to investigate the therapeutic effect of SPL. Dietary SPL improved the growth performance in 3 d after DSS treatment, and the histopathological score was lower in the DSS-treated SPL group than in the DSS-treated control group. Mucosal thickness and goblet cell numbers significantly increased in the SPL-supplemented groups compared to in the control group. Similarly, SPL supplementation upregulated the gene expression levels of mucin-2, interleukin-10, and transforming growth factor-ß, and increased the concentration of short chain fatty acid compared to the control groups. In conclusion, dietary supplementation with SPL attenuated the pathological response against acute and chronic inflammation by the maintenance of the mucosal barrier and wound healing capacity.


Assuntos
Colite/metabolismo , Intestino Grosso/efeitos dos fármacos , Ácidos Oleicos/farmacologia , Substâncias Protetoras/farmacologia , Animais , Colite/induzido quimicamente , Citocinas/metabolismo , Sulfato de Dextrana/efeitos adversos , Modelos Animais de Doenças , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Intestino Grosso/citologia , Intestino Grosso/patologia , Camundongos , Camundongos Endogâmicos C57BL
19.
Development ; 149(1)2022 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-34910127

RESUMO

Although Wnt signaling is clearly important for the intestinal epithelial homeostasis, the relevance of various sources of Wnt ligands themselves remains incompletely understood. Blocking the release of Wnt in distinct stromal cell types suggests obligatory functions of several stromal cell sources and yields different observations. The physiological contribution of epithelial Wnt to tissue homeostasis remains unclear. We show here that blocking epithelial Wnts affects colonic Reg4+ epithelial cell differentiation and impairs colonic epithelial regeneration after injury in mice. Single-cell RNA analysis of intestinal stroma showed that the majority of Wnt-producing cells were contained in transgelin (Tagln+) and smooth muscle actin α2 (Acta2+) expressing populations. We genetically attenuated Wnt production from these stromal cells using Tagln-Cre and Acta2-CreER drivers, and found that blockage of Wnt release from either epithelium or Tagln+ and Acta2+ stromal cells impaired colonic epithelial healing after chemical-induced injury. Aggregated blockage of Wnt release from both epithelium and Tagln+ or Acta2+ stromal cells drastically diminished epithelial repair, increasing morbidity and mortality. These results from two uncharacterized stromal populations suggested that colonic recovery from colitis-like injury depends on multiple Wnt-producing sources.


Assuntos
Actinas/metabolismo , Colite Ulcerativa/metabolismo , Mucosa Intestinal/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Proteína Wnt3A/metabolismo , Cicatrização , Actinas/genética , Animais , Células Cultivadas , Colo/citologia , Colo/metabolismo , Colo/fisiologia , Mucosa Intestinal/citologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/genética , Proteínas Musculares/genética , Proteínas Associadas a Pancreatite/genética , Proteínas Associadas a Pancreatite/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco/metabolismo , Proteína Wnt3A/genética
20.
J Ethnopharmacol ; 287: 114927, 2022 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-34954265

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Panax ginseng root has been used as tonic in traditional Chinese medicine (TCM) and traditional Japanese Kampo medicine. Steam processing of Panax ginseng root is carried out to enhance its nourishing effects on qi. AIM OF THE STUDY: In order to explore the mechanism of these beneficial effects behind the steam processing of the P. ginseng root, we evaluated effectiveness of processing on the granulocyte-colony stimulating factor (G-CSF) secretion in intestinal epithelial cell-like MCE301 cells. MATERIALS AND METHODS: We collected P. ginseng root samples in the markets of China and Japan. Fresh or dried samples were steamed for different time lengths and subsequently dried and extracted. MCE301 cells were incubated with the medium containing various P. ginseng root extracts, while the concentration of G-CSF in the medium was measured. We also investigated the active ingredients by size exclusion HPLC. RESULTS: The extracts of fresh P. ginseng hairy root samples steamed for more than 6 h significantly induced G-CSF secretion, and the maximum activity was recorded at a 9-h steaming. The same activity was noted when already dried P. ginseng hairy root samples were steamed. The extracts of fresh P. ginseng hairy root without steam processing and those of fresh P. ginseng root body samples with steam processing exhibited no activities. The active ingredients of steamed P. ginseng hairy root samples were high-molecular-weight compounds with an average molecular weight of 758 kDa, and the activity was mediated by the toll-like receptor (TLR) 9. CONCLUSIONS: Our results shed on more light on the mechanism underlying the appearance of immunostimulatory activity of the P. ginseng hairy root induced by steam processing.


Assuntos
Mucosa Intestinal/efeitos dos fármacos , Panax/química , Extratos Vegetais/farmacologia , Vapor , Animais , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fator Estimulador de Colônias de Granulócitos/metabolismo , Mucosa Intestinal/citologia , Camundongos , Extratos Vegetais/química , Raízes de Plantas
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