RESUMO
Intestinal homeostasis involves the collaboration of gut barrier components, such as goblet cells and IgA-microbiota complexes, that are under the control of stress that promotes inflammatory responses addressed primarily in the colon. The aim of this study was to evaluate the effect of stress on mucins, goblet cells, and proinflammatory parameters in the proximal and distal regions of the small intestine. A group (n = 6) of female 8-week-old BALB/c mice underwent board immobilization stress (2 h per day for 4 days) and were sacrificed with isoflurane. Samples from proximal and distal small segments were collected to analyze the following: 1) goblet cells stained with periodic acid-Schiff (PAS) and with alcian blue (AB) to visualize histologically neutral and acidic mucins, respectively; 2) IgA-microbiota complexes identified by flow cytometry in intestinal lavages; and 3) MUC2, MUC5AC, and IL-18 mRNA levels in whole mucosal scrapings by reverse transcription-qPCR. Regarding the unstressed group, in the proximal region of small intestine both PAS+ and AB+ goblet cells were unchanged; however, MUC5AC and IL-18 mRNA levels were increased, and the percentage of IgA-microbiota complexes was reduced. In the distal segment, the number of PAS+ goblet cells was increased, whereas the number of AB+ goblet cells was reduced and did not affect the remaining parameters. The data suggest that stress induces inflammation in the proximal small intestine; these findings may provide an experimental reference for human diseases that may affect the proximal small intestine, such as Crohn's disease, in which stress contributes to the progression of intestinal inflammation or relapse.
Assuntos
Células Caliciformes , Intestino Delgado , Camundongos Endogâmicos BALB C , Mucinas , Animais , Intestino Delgado/metabolismo , Intestino Delgado/microbiologia , Intestino Delgado/patologia , Feminino , Camundongos , Células Caliciformes/metabolismo , Células Caliciformes/patologia , Mucinas/metabolismo , Estresse Psicológico/metabolismo , Estresse Psicológico/imunologia , Interleucina-18/metabolismo , Mucina-5AC/metabolismo , Estresse Fisiológico , Imunoglobulina A/metabolismo , Mucina-2/metabolismo , Mucina-2/genéticaRESUMO
Maintaining the mucus layer is crucial for the innate immune system. Urolithin A (Uro A) is a gut microbiota-derived metabolite; however, its effect on mucin production as a physical barrier remains unclear. This study aimed to elucidate the protective effects of Uro A on mucin production in the colon. In vivo experiments employing wild-type mice, NF-E2-related factor 2 (Nrf2)-deficient mice, and wild-type mice treated with an aryl hydrocarbon receptor (AhR) antagonist were conducted to investigate the physiological role of Uro A. Additionally, in vitro assays using mucin-producing cells (LS174T) were conducted to assess mucus production following Uro A treatment. We found that Uro A thickened murine colonic mucus via enhanced mucin 2 expression facilitated by Nrf2 and AhR signaling without altering tight junctions. Uro A reduced mucosal permeability in fluorescein isothiocyanate-dextran experiments and alleviated dextran sulfate sodium-induced colitis. Uro A treatment increased short-chain fatty acid-producing bacteria and propionic acid concentration. LS174T cell studies confirmed that Uro A promotes mucus production through the AhR and Nrf2 pathways. In conclusion, the enhanced intestinal mucus secretion induced by Uro A is mediated through the actions of Nrf-2 and AhR, which help maintain intestinal barrier function.
Assuntos
Colite , Cumarínicos , Mucosa Intestinal , Fator 2 Relacionado a NF-E2 , Receptores de Hidrocarboneto Arílico , Animais , Fator 2 Relacionado a NF-E2/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Camundongos , Mucosa Intestinal/metabolismo , Cumarínicos/farmacologia , Colite/metabolismo , Colite/induzido quimicamente , Mucina-2/metabolismo , Mucina-2/genética , Humanos , Colo/metabolismo , Camundongos Endogâmicos C57BL , Transdução de Sinais/efeitos dos fármacos , Masculino , Microbioma Gastrointestinal , Camundongos Knockout , Sulfato de Dextrana , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Função da Barreira IntestinalRESUMO
BACKGROUND: Intestinal barrier dysfunction is a significant contributor to the recurrence and refractory of ulcerative colitis (UC). Promoting the interaction between group 3 innate lymphoid cells (ILC3s) and gut flora is a valuable strategy for mucosal repair. Paeoniae decoction (PD) is a compound commonly used in clinical treatment of UC, but its exact mechanism remains unclear. PURPOSE: We aimed to investigate the protective effect of PD on intestinal mucosal injury induced by dextran sulfate sodium (DSS) in chronic colitis, as well as to elucidate its potential mechanism. METHODS: C57BL/6 mice were induced with chronic colitis by 2 % DSS and divided into four groups: control group, model group, PD low dose (4 g/kg), and high dose (8 g/kg) group. The effectiveness of PD in treating chronic colitis mice was evaluated based on changes in body weight, colon length, colon pathological tissue scores, and the mRNA levels of inflammatory factors IL-6 and IL-1ß. The expressions of intestinal epithelial tight junction proteins (ZO-1 and Occludin), IL-22, and MUC2 were observed using immunofluorescence and RT-PCR. Additionally, the proportion of ILC3 and natural cytotoxicity receptor (NCR)+ ILC3 in the colon were detected using flow cytometry. Furthermore, UHPLC-QE-MS was utilized to identify chemical components of PD and network pharmacology was employed to predict potential pathways for PD intervention in UC. Subsequently, MNK-3 cells (ILC3 in vitro cell line) and NCM460 cells were used to verify the network pharmacology results. Finally, the effects of PD on UC gut flora have been explored using in vitro fermentation and 16S rDNA techniques. RESULTS: The results showed that PD significantly restored body weight and colon length in mice with chronic colitis, while also reducing colon inflammatory cell infiltration and the expression of IL-6 and IL-1ß. Additionally, PD notably promoted the expression of MUC2, ZO-1, Occludin, and IL-22, as well as increasing the ratio of ILC3 and NCR+ILC3. UHPLC-QE-MS analysis identified 443 components of PD, and network pharmacology suggested that PD could target the aryl hydrocarbon receptor (AHR) signaling pathway, which was confirmed by MNK-3 cells and in vitro fermentation experiments. Furthermore, MNK-3-conditioned medium (CM) increased the expression of ZO-1 and Occludin in NCM460 cells. In addition, 16S rDNA results indicated that PD promoted the abundance of Lactobacillales, thus contributing to mucosal damage repair by activating the AHR signal in ILC3s. CONCLUSION: In summary, our study demonstrates that PD repairs intestinal mucosal damage in chronic colitis by regulating the interaction of gut flora with ILC3, and the specific mechanism is related to the activation of AHR signaling pathway.
Assuntos
Sulfato de Dextrana , Microbioma Gastrointestinal , Interleucina 22 , Mucosa Intestinal , Camundongos Endogâmicos C57BL , Paeonia , Animais , Microbioma Gastrointestinal/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Camundongos , Masculino , Paeonia/química , Linfócitos/efeitos dos fármacos , Proteína da Zônula de Oclusão-1/metabolismo , Interleucinas/metabolismo , Modelos Animais de Doenças , Interleucina-1beta/metabolismo , Ocludina/metabolismo , Interleucina-6/metabolismo , Colo/efeitos dos fármacos , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/induzido quimicamente , Medicamentos de Ervas Chinesas/farmacologia , Colite/tratamento farmacológico , Colite/induzido quimicamente , Mucina-2Assuntos
Adenocarcinoma , Neoplasias do Ânus , Fístula Retal , Humanos , Masculino , Adulto , Neoplasias do Ânus/patologia , Neoplasias do Ânus/genética , Neoplasias do Ânus/diagnóstico , Adenocarcinoma/patologia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Fístula Retal/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Mucina-2/metabolismoRESUMO
Mucosal-delivered drugs have to pass through the mucus layer before absorption through the epithelial cell membrane. Although there has been increasing interest in polymeric mucins, a major structural component of mucus, potentially acting as important physiological regulators of mucosal drug absorption, there are no reports that have systematically evaluated the interaction between mucins and drugs. In this study, we assessed the potential interaction between human polymeric mucins (MUC2, MUC5B, and MUC5AC) and various drugs with different chemical profiles by simple centrifugal method and fluorescence analysis. We found that paclitaxel, rifampicin, and theophylline likely induce the aggregation of MUC5B and/or MUC2. In addition, we showed that the binding affinity of drugs for polymeric mucins varied, not only between individual drugs but also among mucin subtypes. Furthermore, we demonstrated that deletion of MUC5AC and MUC5B in A549 cells increased the cytotoxic effects of cyclosporin A and paclitaxel, likely due to loss of mucin-drug interaction. In conclusion, our results indicate the necessity to determine the binding of drugs to mucins and their potential impact on the mucin network property.
Assuntos
Mucina-5AC , Paclitaxel , Humanos , Paclitaxel/farmacologia , Paclitaxel/metabolismo , Mucina-5AC/metabolismo , Mucina-5AC/genética , Células A549 , Interações Medicamentosas , Mucina-5B/metabolismo , Mucina-5B/genética , Mucinas/metabolismo , Mucina-2/metabolismo , Mucina-2/genética , Rifampina/farmacologia , Ciclosporina/farmacologia , Ligação ProteicaRESUMO
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-2RESUMO
OBJECTIVE: To explore the potential pathogenic genes of intestinal metaplasia. METHODS: Twenty-one patients with intestinal metaplasia admitted to the Department of Gastroenterology at the Second Affiliated Hospital of Anhui University of Chinese Medicine from January, 2022 to June, 2022, and 21 healthy subjects undergoing gastroscopic examination during the same period were enrolled in this study. All the participants underwent gastroscopy and pathological examination, and gastric tissue samples were collected for transcriptome sequencing to screen for differentially expressed genes (DEGs). The biological functions of the DEGs were analyzed using bioinformatics analysis, and qRT-PCR was used to validate the results. RESULTS: Transcriptomic sequencing identified a total of 1373 DEGs, including 827 upregulated and 546 downregulated ones. The top 6 upregulated genes (AGMAT, CCL25, FABP1, CDX1, SPINK4, and MUC2), ranked based on their significance and average expression level, were selected for validation, and qRT-PCR showed significant upregulation of their mRNAs in the gastric tissues of patients with intestinal metaplasia (P < 0.05). CONCLUSION: AGMAT, CCL25, FABP1, CDX1, SPINK4, and MUC2 participate in the occurrence and development of intestinal metaplasia, and may serve as potential biomarkers for diagnosing intestinal metaplasia.
Assuntos
Biologia Computacional , Metaplasia , Humanos , Metaplasia/genética , Biologia Computacional/métodos , Proteínas de Ligação a Ácido Graxo/genética , Transcriptoma , Mucina-2/genética , Mucina-2/metabolismo , Proteínas de Homeodomínio/genética , Perfilação da Expressão Gênica , Masculino , Mucosa Gástrica/patologia , Mucosa Gástrica/metabolismo , Intestinos/patologia , Feminino , RNA Mensageiro/genéticaRESUMO
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 C57BLRESUMO
Heavy metals interact with each other in a coexisting manner to produce complex combined toxicity to organisms. At present, the toxic effects of chronic co-exposure to heavy metals hexavalent chromium [Cr(VI)] and divalent nickel [Ni(II)] on organisms are seldom studied and the related mechanisms are poorly understood. In this study, we explored the mechanism of the colon injury in mice caused by chronic exposure to Cr or/and Ni. The results showed that, compared with the control group, Cr or/and Ni chronic exposure affected the body weight of mice, and led to infiltration of inflammatory cells in the colon, decreased the number of goblet cells, fusion of intracellular mucus particles and damaged cell structure of intestinal epithelial. In the Cr or/and Ni exposure group, the activity of nitric oxide synthase (iNOS) increased, the expression levels of MUC2 were significantly down-regulated, and those of ZO-1 and Occludin were significantly up-regulated. Interestingly, factorial analysis revealed an interaction between Cr and Ni, which was manifested as antagonistic effects on iNOS activity, ZO-1 and MUC2 mRNA expression levels. Transcriptome sequencing further revealed that the expression of genes-related to inflammation, intestinal mucus and tight junctions changed obviously. Moreover, the relative contents of Cr(VI) and Ni(II) in the Cr, Ni and Cr+Ni groups all changed with in-vitro gastrointestinal ï¼IVGï¼digestion, especially in the Cr+Ni group. Our results indicated that the chronic exposure to Cr or/and Ni can lead to damage to the mice colon, and the relative content changes of Cr(VI) and Ni(II) might be the main reason for the antagonistic effect of Cr+Ni exposure on the colon damage.
Assuntos
Cromo , Colo , Mucina-2 , Níquel , Animais , Cromo/toxicidade , Níquel/toxicidade , Camundongos , Colo/efeitos dos fármacos , Colo/patologia , Mucina-2/genética , Mucina-2/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Perfilação da Expressão Gênica , Masculino , Digestão/efeitos dos fármacos , Proteína da Zônula de Oclusão-1/metabolismo , Proteína da Zônula de Oclusão-1/genética , Transcriptoma/efeitos dos fármacos , Ocludina/metabolismo , Ocludina/genética , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologiaRESUMO
Human breast milk promotes maturation of the infant gastrointestinal barrier, including the promotion of mucus production. In the quest to produce next generation infant milk formula (IMF), we have produced IMF by membrane filtration (MEM-IMF). With a higher quantity of native whey protein, MEM-IMF more closely mimics human breast milk than IMF produced using conventional heat treatment (HT-IMF). After a 4-week dietary intervention in young pigs, animals fed a MEM-IMF diet had a higher number of goblet cells, acidic mucus and mucin-2 in the jejunum compared to pigs fed HT-IMF (P < 0.05). In the duodenum, MEM-IMF fed pigs had increased trypsin activity in the gut lumen, increased mRNA transcript levels of claudin 1 in the mucosal scrapings and increased lactase activity in brush border membrane vesicles than those pigs fed HT-IMF (P < 0.05). In conclusion, MEM-IMF is superior to HT-IMF in the promotion of mucus production in the young gut.
Assuntos
Filtração , Fórmulas Infantis , Muco , Animais , Fórmulas Infantis/química , Muco/metabolismo , Suínos , Proteínas do Soro do Leite/metabolismo , Intestino Delgado/metabolismo , Tripsina/metabolismo , Humanos , Células Caliciformes/metabolismo , Claudina-1/metabolismo , Claudina-1/genética , Lactase/metabolismo , Lactase/genética , Mucina-2/metabolismo , Mucina-2/genética , Mucosa Intestinal/metabolismo , Duodeno/metabolismo , Jejuno/metabolismo , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Proteínas do Leite/metabolismo , Proteínas do Leite/análiseRESUMO
Inflammatory bowel disease (IBD) is a multifactorial disease involving the interaction of the gut microbiota, genes, host immunity, and environmental factors. Dysbiosis in IBD is associated with pathobiont proliferation, so targeted antibiotic therapy is a rational strategy. When restoring the microbiota with probiotics, it is necessary to take into account the mutual influence of co-cultivated microorganisms, as the microbiota is a dynamic community of species that mediates homeostasis and physiological processes in the intestine. The aim of our study was to investigate the recovery efficacy of two potential probiotic bacteria, L. johnsonii and E. faecalis, in Muc2-/- mice with impaired mucosal layer. Two approaches were used to determine the efficacy of probiotic supplementation in mice with dysbiosis caused by mucin-2 deficiency: bacterial seeding on selective media and real-time PCR analysis. The recovery time and the type of probiotic bacteria relocated affected only the number of E. faecalis. A significant positive correlation was found between colony-forming unit (CFU) and the amount of E. faecalis DNA in the group that was replanted with probiotic E. faecalis. As for L. johnsonii, it could be restored to its original level even without any additional bacteria supplementation after two weeks. Interestingly, the treatment of mice with L. johnsonii caused a decrease in the amount of E. faecalis. Furthermore, either L. johnsonii or E. faecalis treatment eliminated protozoan overgrowth caused by antibiotic administration.
Assuntos
Antibacterianos , Disbiose , Enterococcus faecalis , Lactobacillus johnsonii , Probióticos , Animais , Enterococcus faecalis/efeitos dos fármacos , Camundongos , Antibacterianos/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Modelos Animais de Doenças , Mucina-2/metabolismo , Mucina-2/genética , Doenças Inflamatórias Intestinais/microbiologia , Camundongos KnockoutRESUMO
This study aimed to explore the ameliorative effects and potential mechanisms of Huangshan Umbilicaria esculenta polysaccharide (UEP) in dextran sulfate sodium-induced acute ulcerative colitis (UC) and UC secondary liver injury (SLI). Results showed that UEP could ameliorate both colon and liver pathologic injuries, upregulate mouse intestinal tight junction proteins (TJs) and MUC2 expression, and reduce LPS exposure, thereby attenuating the effects of the gut-liver axis. Importantly, UEP significantly downregulated the secretion levels of TNF-α, IL-1ß, and IL-6 through inhibition of the NF-κB pathway and activated the Nrf2 signaling pathway to increase the expression levels of SOD and GSH-Px. In vitro, UEP inhibited the LPS-induced phosphorylation of NF-κB P65 and promoted nuclear translocation of Nrf2 in RAW264.7 cells. These results revealed that UEP ameliorated UC and SLI through NF-κB and Nrf2-mediated inflammation and oxidative stress. The study first investigated the anticolitis effect of UEP, suggesting its potential for the treatment of colitis and colitis-associated liver disease.
Assuntos
Colite , Sulfato de Dextrana , Fator 2 Relacionado a NF-E2 , NF-kappa B , Polissacarídeos , Animais , Camundongos , Polissacarídeos/farmacologia , Polissacarídeos/química , Polissacarídeos/administração & dosagem , Sulfato de Dextrana/efeitos adversos , Masculino , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , Humanos , Colite/tratamento farmacológico , Colite/induzido quimicamente , Colite/metabolismo , Células RAW 264.7 , NF-kappa B/metabolismo , NF-kappa B/genética , Camundongos Endogâmicos C57BL , Substâncias Protetoras/farmacologia , Substâncias Protetoras/administração & dosagem , Substâncias Protetoras/química , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Estresse Oxidativo/efeitos dos fármacos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-1beta/imunologia , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-6/imunologia , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/metabolismo , Colite Ulcerativa/induzido quimicamente , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Mucina-2/genética , Mucina-2/metabolismoRESUMO
The MUC2 mucin protects the colonic epithelium by a two-layered mucus with an inner attached bacteria-free layer and an outer layer harboring commensal bacteria. CysD domains are 100 amino-acid-long sequences containing 10 cysteines that separate highly O-glycosylated proline, threonine, serine (PTS) regions in mucins. The structure of the second CysD, CysD2, of MUC2 is now solved by nuclear magnetic resonance. CysD2 shows a stable stalk region predicted to be partly covered by adjacent O-glycans attached to neighboring PTS sequences, whereas the CysD2 tip with three flexible loops is suggested to be well exposed. It shows transient dimer interactions at acidic pH, weakened at physiological pH. This transient interaction can be stabilized in vitro and in vivo by transglutaminase 3-catalyzed isopeptide bonds, preferring a specific glutamine residue on one flexible loop. This covalent dimer is modeled suggesting that CysD domains act as connecting hubs for covalent stabilization of mucins to form a protective mucus.
Assuntos
Mucina-2 , Domínios Proteicos , Transglutaminases , Mucina-2/metabolismo , Mucina-2/química , Humanos , Transglutaminases/metabolismo , Transglutaminases/química , Modelos Moleculares , Cisteína/metabolismo , Cisteína/química , Sequência de Aminoácidos , Multimerização Proteica , Reagentes de Ligações Cruzadas/química , Reagentes de Ligações Cruzadas/metabolismoRESUMO
OBJECTIVES: Ulcerative colitis (UC) is a colonic immune system disorder, manifested with long duration and easy relapse. Genistein has been reported to possess various biological activities. However, it remains unclear whether genistein can ameliorate UC by modulating the homeostasis of the intestinal bacterial community. METHODS: The dextran sodium sulfate (DSS)-induced UC mice were administrated with genistein (20 mg/kg/day) or genistein (40 mg/kg/day) for ten days. The general physical condition of the mice was monitored. After sacrifice, the changes in colon length and colonic pathological morphology were observed. The expression of intestinal barrier proteins, inflammatory cytokines, and macrophage markers in the colon was detected. The composition and metabolic products of the intestinal microbiota were analyzed. RESULTS: Genistein treatment visibly improved body weight change and disease activity index in DSS-induced mice. Genistein treatment ameliorated colonic pathological alterations and promoted the expression of mucin-2 and tight junction proteins. Genistein administration inhibited myeloperoxidase activity and colonic inflammatory cytokines. Furthermore, genistein administration improved the structure of the intestinal microbial community, promoted the production of short-chain fatty acids, and modulated macrophage polarization. CONCLUSIONS: These results revealed that genistein mediated macrophage polarization balance by improving intestinal microbiota and its metabolites, thereby alleviating DSS-induced colitis.
Assuntos
Sulfato de Dextrana , Microbioma Gastrointestinal , Genisteína , Macrófagos , Camundongos Endogâmicos C57BL , Animais , Genisteína/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Camundongos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Modelos Animais de Doenças , Colo/efeitos dos fármacos , Colo/metabolismo , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/induzido quimicamente , Citocinas/metabolismo , Colite/tratamento farmacológico , Colite/induzido quimicamente , Mucina-2/metabolismoRESUMO
This study investigated the impact of in vivo available colon-mango (poly)phenols on stress-induced impairment of intestinal barrier function. Caco-2/HT29-MTX cells were incubated with six extracts of ileal fluid collected pre- and 4-8 h post-mango consumption before being subjected to inflammatory stress. (Poly)phenols in ileal fluids were analysed by UHPLC-HR-MS. Epithelial barrier function was monitored by measurement of trans-epithelial electrical resistance (TEER) and the production of selected inflammatory markers (interleukin-8 (IL-8) and nitric oxide (NO)) and the major mucin of the mucosal layer (MUC2). Post-mango intake ileal fluids contained principally benzoic acids, hydroxybenzenes and galloyl derivatives. There was a high interindividual variability in the levels of these compounds, which was reflected by the degree of variability in the protective effects of individual ileal extracts on inflammatory changes in the treated cell cultures. The 24 h treatment with non-cytotoxic doses of extracts of 4-8 h post-mango intake ileal fluid significantly reduced the TEER decrease in monolayers treated with the inflammatory cytomix. This effect was not associated with changes in IL-8 expression and secretion or claudine-7 expression. The mango derived-ileal fluid extract (IFE) also mitigated cytomix-dependent nitrite secretion, as a proxy of NO production, and the MUC2 reduction observed upon the inflammatory challenge. These insights shed light on the potential protective effect of mango (poly)phenols on the intestinal barrier exposed to inflammatory conditions.
Assuntos
Interleucina-8 , Mucosa Intestinal , Mangifera , Mucina-2 , Humanos , Mangifera/química , Células CACO-2 , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Interleucina-8/metabolismo , Mucina-2/metabolismo , Células HT29 , Polifenóis/farmacologia , Colo/efeitos dos fármacos , Colo/metabolismo , Óxido Nítrico/metabolismo , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Inflamação/tratamento farmacológico , Função da Barreira IntestinalRESUMO
Sleep deprivation (SD) leads to impaired intestinal barrier function and intestinal flora disorder, especially a reduction in the abundance of the next generation of probiotic Faecalibacterium prausnitzii (F. prausnitzii). However, it remains largely unclear whether F. prausnitzii can ameliorate SD-induced intestinal barrier damage. A 72 h SD mouse model was used in this research, with or without the addition of F. prausnitzii. The findings indicated that pre-colonization with F. prausnitzii could protect against tissue damage from SD, enhance goblet cell count and MUC2 levels in the colon, boost tight-junction protein expression, decrease macrophage infiltration, suppress pro-inflammatory cytokine expression, and reduce apoptosis. We found that the presence of F. prausnitzii helped to balance the gut microbiota in SD mice by reducing harmful bacteria like Klebsiella and Staphylococcus, while increasing beneficial bacteria such as Akkermansia. Ion chromatography analysis revealed that F. prausnitzii pretreatment increased the fecal butyrate level in SD mice. Overall, these results suggested that incorporating F. prausnitzii could help reduce gut damage caused by SD, potentially by enhancing the intestinal barrier and balancing gut microflora. This provides a foundation for utilizing probiotics to protect against intestinal illnesses.
Assuntos
Disbiose , Faecalibacterium prausnitzii , Microbioma Gastrointestinal , Mucosa Intestinal , Probióticos , Privação do Sono , Animais , Privação do Sono/complicações , Camundongos , Probióticos/farmacologia , Probióticos/administração & dosagem , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Masculino , Fezes/microbiologia , Camundongos Endogâmicos C57BL , Suplementos Nutricionais , Modelos Animais de Doenças , Mucina-2/metabolismo , Butiratos/metabolismo , Colo/microbiologia , Colo/metabolismoRESUMO
Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a commonly encountered foodborne pathogen that can cause hemorrhagic enteritis and lead to hemolytic uremic syndrome (HUS) in severe cases. Bifidobacterium is a beneficial bacterium that naturally exists in the human gut and plays a vital role in maintaining a healthy balance in the gut microbiota. This study investigated the protective effects of B. longum K5 in a mouse model of EHEC O157:H7 infection. The results indicated that pretreatment with B. longum K5 mitigated the clinical symptoms of EHEC O157:H7 infection and attenuated the increase in myeloperoxidase (MPO) activity in the colon of the mice. In comparison to the model group, elevated serum D-lactic acid concentrations and diamine oxidase (DAO) levels were prevented in the K5-EHEC group of mice. The reduced mRNA expression of tight junction proteins (ZO-1, Occludin, and Claudin-1) and mucin MUC2, as well as the elevated expression of virulence factors Stx1A and Stx2A, was alleviated in the colon of both the K5-PBS and K5-EHEC groups. Additionally, the increase in the inflammatory cytokine levels of TNF-α and IL-1ß was inhibited and the production of IL-4 and IL-10 was promoted in the K5-EHEC group compared with the model group. B. longum K5 significantly prevented the reduction in the abundance and diversity of mouse gut microorganisms induced by EHEC O157:H7 infection, including blocking the decrease in the relative abundance of Roseburia, Lactobacillus, and Oscillibacter. Meanwhile, the intervention with B. longum K5 promoted the production of acetic acid and butyric acid in the gut. This study provides insights into the use of B. longum K5 for developing probiotic formulations to prevent intestinal diseases caused by pathogenic bacterial infections.
Assuntos
Bifidobacterium longum , Colo , Infecções por Escherichia coli , Escherichia coli O157 , Microbioma Gastrointestinal , Probióticos , Animais , Camundongos , Probióticos/farmacologia , Infecções por Escherichia coli/prevenção & controle , Infecções por Escherichia coli/microbiologia , Colo/microbiologia , Colo/metabolismo , Modelos Animais de Doenças , Mucina-2/metabolismo , Citocinas/metabolismo , Peroxidase/metabolismo , Amina Oxidase (contendo Cobre)/metabolismoRESUMO
The gastrointestinal (GI) tract's mucus layer serves as a critical barrier and a mediator in drug nanoparticle delivery. The mucus layer's diverse molecular structures and spatial complexity complicates the mechanistic study of the diffusion dynamics of particulate materials. In response, we developed a bi-component coarse-grained mucus model, specifically tailored for the colorectal cancer environment, that contained the two most abundant glycoproteins in GI mucus: Muc2 and Muc5AC. This model demonstrated the effects of molecular composition and concentration on mucus pore size, a key determinant in the permeability of nanoparticles. Using this computational model, we investigated the diffusion rate of polyethylene glycol (PEG) coated nanoparticles, a widely used muco-penetrating nanoparticle. We validated our model with experimentally characterized mucus pore sizes and the diffusional coefficients of PEG-coated nanoparticles in the mucus collected from cultured human colorectal goblet cells. Machine learning fingerprints were then employed to provide a mechanistic understanding of nanoparticle diffusional behavior. We found that larger nanoparticles tended to be trapped in mucus over longer durations but exhibited more ballistic diffusion over shorter time spans. Through these discoveries, our model provides a promising platform to study pharmacokinetics in the GI mucus layer.
Assuntos
Muco , Nanopartículas , Polietilenoglicóis , Humanos , Nanopartículas/química , Difusão , Polietilenoglicóis/química , Muco/metabolismo , Muco/química , Mucina-2/metabolismo , Mucina-2/química , Mucina-5AC/metabolismo , Mucina-5AC/química , Mucosa Intestinal/metabolismo , Trato Gastrointestinal/metabolismo , Células Caliciformes/metabolismo , Modelos BiológicosRESUMO
There is an increasing appreciation that colonic barrier function is closely related to the development and progression of colitis. The mucus layer is a crucial component of the colonic barrier, responsible for preventing harmful bacteria from invading the intestinal epithelium and causing inflammation. Furthermore, a defective mucus barrier is also a significant characteristic of ulcerative colitis (UC). Biochanin A (BCA), an isoflavonoid, has garnered increasing interest due to its significant biological activities. However, the impact of BCA on UC has not been reported yet. In this study, we used a dextran sodium sulfate (DSS)-induced ulcerative colitis model and the Muc2 deficient (Muc2-/-) mice spontaneous colitis model to explore the mechanisms of BCA in the treatment of UC. Here, we verified that DSS-induced UC was observably attenuated and spontaneous colitis in Muc2-/- mice was relieved by BCA. Treatment with BCA improved colitis-related symptoms and reduced intestinal permeability by upregulating the levels of goblet cells and tight junction (TJ) proteins. In addition, we confirmed that BCA promotes autophagy through the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)/Unc-51-like kinase 1 (ULK1) pathway, thereby alleviating DSS-induced UC. In addition, the administration of BCA was able to reduce apoptosis and promote proliferation by suppressing Cleaved Caspase-3 (Cleaved Cas-3) expression, and increasing PCNA and Ki67 levels. Further research revealed that BCA treatment ameliorated spontaneous colitis and alleviated epithelial damage in Muc2-/- mice by restoring the intestinal barrier and promoting autophagy. Our results demonstrated that BCA alleviated UC by enhancing intestinal barrier function and promoting autophagy. These findings indicate that BCA may be a novel treatment alternative for UC.
Assuntos
Colite Ulcerativa , Colo , Sulfato de Dextrana , Genisteína , Mucina-2 , Animais , Mucina-2/metabolismo , Mucina-2/genética , Sulfato de Dextrana/toxicidade , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/patologia , Colite Ulcerativa/metabolismo , Genisteína/farmacologia , Genisteína/uso terapêutico , Camundongos , Colo/patologia , Colo/efeitos dos fármacos , Colo/metabolismo , Autofagia/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Camundongos Knockout , Apoptose/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Proteínas Quinases Ativadas por AMP/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
BACKGROUND: Diarrheal irritable bowel syndrome (IBS-D), characterized primarily by the presence of diarrhea and abdominal pain, is a clinical manifestation resulting from a multitude of causative factors. Furthermore, Sishen Wan (SSW) has demonstrated efficacy in treating IBS-D. Nevertheless, its mechanism of action remains unclear. METHODS: A model of IBS-D was induced by a diet containing 45 % lactose and chronic unpredictable mild stress. Additionally, the impact of SSW was assessed by measuring body weight, visceral sensitivity, defecation parameters, intestinal transport velocity, intestinal neurotransmitter levels, immunohistochemistry, and transmission electron microscopy analysis. Immunofluorescent staining was used to detect the expression of Mucin 2 (MUC2) and Occludin in the colon. Western blotting was used to detect changes in proteins related to tight junction (TJ), autophagy, and endoplasmic reticulum (ER) stress in the colon. Finally, 16S rRNA amplicon sequencing was used to monitor the alteration of gut microbiota after SSW treatment. RESULTS: Our study revealed that SSW administration resulted in reduced visceral sensitivity, improved defecation parameters, decreased intestinal transport velocity, and reduced intestinal permeability in IBS-D mice. Furthermore, SSW promotes the secretion of colonic mucus by enhancing autophagy and inhibiting ER stress. SSW treatment caused remodeling of the gut microbiome by increasing the abundance of Blautia, Muribaculum and Ruminococcus torques group. CONCLUSION: SSW can improve intestinal barrier function by promoting autophagy and inhibiting ER stress, thus exerting a therapeutic effect on IBS-D.