RESUMO
Hypoxia plays an important role in the pathological process of bladder outlet obstruction. Previous research has mostly focused on the dysfunction of bladder smooth muscle cells, which are directly related to bladder contraction. This study delves into the barrier function changes of the urothelial cells under exposure to hypoxia. Results indicated that after a 5-day culture, SV-HUC-1 formed a monolayer and/or bilayer of cell sheets, with tight junction formation, but no asymmetrical unit membrane was observed. qPCR and western blotting revealed the expression of TJ-associated proteins (occludin, claudin1 and ZO-1) was significantly decreased in the hypoxia group in a time-dependent manner. No expression changes were observed in uroplakins. When compared to normoxic groups, immunofluorescent staining revealed a reduction in the expression of TJ-associated proteins in the hypoxia group. Transepithelial electrical resistance (TEER) revealed a statistically significant decrease in resistance in the hypoxia group. Fluorescein isothiocyanate-conjugated dextran assay was inversely proportional to the results of TEER. Taken together, hypoxia down-regulates the expression of TJ-associated proteins and breaks tight junctions, thus impairing the barrier function in human urothelial cells.
Assuntos
Hipóxia Celular , Proteínas de Junções Íntimas , Junções Íntimas , Urotélio , Humanos , Urotélio/metabolismo , Urotélio/patologia , Junções Íntimas/metabolismo , Proteínas de Junções Íntimas/metabolismo , Proteínas de Junções Íntimas/genética , Linhagem Celular , Proteína da Zônula de Oclusão-1/metabolismo , Proteína da Zônula de Oclusão-1/genética , Ocludina/metabolismo , Ocludina/genética , Claudina-1/metabolismo , Claudina-1/genética , Impedância Elétrica , Regulação da Expressão GênicaRESUMO
PURPOSE: This study aimed to investigate the impact of hepatocyte growth factor (HGF) on colonic morphology and gut microbiota in a rat model of short bowel syndrome (SBS). METHODS: SD rats underwent jugular vein catheterization for total parenteral nutrition (TPN) and 90% small bowel resection [TPN + SBS (control group) or TPN + SBS + intravenous HGF (0.3 mg/kg/day, HGF group)]. Rats were harvested on day 7. Colonic morphology, gut microflora, tight junction, and Toll-like receptor-4 (TLR4) were evaluated. RESULTS: No significant differences were observed in the colonic morphological assessment. No significant differences were observed in the expression of tight junction-related genes in the proximal colon. However, the claudin-1 expression tended to increase and the claudin-3 expression tended to decrease in the distal colon of the HGF group. The Verrucomicrobiota in the gut microflora of the colon tended to increase in the HGF group. The abundance of most LPS-producing microbiota was lower in the HGF group than in the control group. The gene expression of TLR4 was significantly downregulated in the distal colon of the HGF group. CONCLUSION: HGF may enhance the mucus barrier through the tight junctions or gut microbiome in the distal colon.
Assuntos
Colo , Modelos Animais de Doenças , Microbioma Gastrointestinal , Fator de Crescimento de Hepatócito , Ratos Sprague-Dawley , Síndrome do Intestino Curto , Animais , Ratos , Fator de Crescimento de Hepatócito/metabolismo , Fator de Crescimento de Hepatócito/genética , Microbioma Gastrointestinal/efeitos dos fármacos , Colo/microbiologia , Colo/patologia , Síndrome do Intestino Curto/metabolismo , Síndrome do Intestino Curto/microbiologia , Masculino , Receptor 4 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Claudina-1/metabolismo , Claudina-1/genéticaRESUMO
Growing evidence showed the capacity of (poly)phenols to exert a protective role on intestinal health. Nevertheless, the existing findings are still heterogeneous and the underlying mechanisms remain unclear. This study investigated the potential benefits of a red raspberry (Rubus idaeus) powder on the integrity of the intestinal barrier, focusing on its ability to mitigate the effects of tumor necrosis factor-α (TNF-α)-induced intestinal permeability. Human colorectal adenocarcinoma cells (i.e., Caco-2 cells) were used as a model to assess the impact of red raspberry on intestinal permeability, tight junction expression, and oxidative stress. The Caco-2 cells were differentiated into polarized monolayers and treated with interferon-γ (IFN-γ) (10 ng mL-1) for 24 hours, followed by exposure to TNF-α (10 ng mL-1) in the presence or absence of red raspberry extract (1-5 mg mL-1). The integrity of the intestinal monolayer was evaluated using transepithelial electrical resistance (TEER) and fluorescein isothiocyanate-dextran (FITC-D) efflux assay. Markers of intestinal permeability (claudin-1, occludin, and zonula occludens-1 (ZO-1)) and oxidative stress (8-hydroxy-2-deoxyguanosine (8-OHdG) and protein carbonyl) were assessed using ELISA kits. Treatment with red raspberry resulted in a significant counteraction of TEER value loss (41%; p < 0.01) and a notable reduction in the efflux of FITC-D (-2.5 times; p < 0.01). Additionally, red raspberry attenuated the levels of 8-OHdG (-48.8%; p < 0.01), mitigating the detrimental effects induced by TNF-α. Moreover, red raspberry positively influenced the expression of the integral membrane protein claudin-1 (+18%; p < 0.01), an essential component of tight junctions. These findings contribute to the growing understanding of the beneficial effects of red raspberry in the context of the intestinal barrier. The effect of red raspberry against TNF-α-induced intestinal permeability observed in our in vitro model suggests, for the first time, its potential as a dietary strategy to promote gastrointestinal health.
Assuntos
Mucosa Intestinal , Estresse Oxidativo , Permeabilidade , Extratos Vegetais , Rubus , Junções Íntimas , Fator de Necrose Tumoral alfa , Humanos , Rubus/química , Células CACO-2 , Estresse Oxidativo/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Extratos Vegetais/farmacologia , Permeabilidade/efeitos dos fármacos , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Ocludina/metabolismo , Ocludina/genética , Claudina-1/metabolismo , Claudina-1/genética , Proteína da Zônula de Oclusão-1/metabolismo , Proteína da Zônula de Oclusão-1/genética , Interferon gama/metabolismo , Frutas/químicaRESUMO
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/genéticaRESUMO
The transmembrane protein claudin-1 is critical for formation of the epidermal barrier structure called tight junctions (TJ) and has been shown to be important in multiple disease states. These include neonatal ichthyosis and sclerosing cholangitis syndrome, atopic dermatitis and various viral infections. To develop a model to investigate the role of claudin-1 in different disease settings, we used CRISPR/Cas9 to generate human immortalized keratinocyte (KC) lines lacking claudin-1 (CLDN1 KO). We then determined whether loss of claudin-1 expression affects epidermal barrier formation/function and KC differentiation/stratification. The absence of claudin-1 resulted in significantly reduced barrier function in both monolayer and organotypic cultures. CLDN1 KO cells demonstrated decreases in gene transcripts encoding the barrier protein filaggrin and the differentiation marker cytokeratin-10. Marked morphological differences were also observed in CLDN1 KO organotypic cultures including diminished stratification and reduced formation of the stratum granulosum. We also detected increased proliferative KC in the basale layer of CLDN1 KO organotypic cultures. These results further support the role of claudin-1 in epidermal barrier and suggest an additional role of this protein in appropriate stratification of the epidermis.
Assuntos
Diferenciação Celular , Claudina-1 , Epiderme , Proteínas Filagrinas , Queratinócitos , Queratinócitos/metabolismo , Claudina-1/metabolismo , Claudina-1/genética , Humanos , Proteínas Filagrinas/metabolismo , Epiderme/metabolismo , Epiderme/patologia , Dermatopatias/genética , Dermatopatias/metabolismo , Junções Íntimas/metabolismo , Queratina-10/metabolismo , Queratina-10/genética , Técnicas de Inativação de Genes , Proliferação de Células , Sistemas CRISPR-CasRESUMO
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
OBJECTIVES: To investigate the effect of Peitu Yimu(strengthening spleen and soothing liver) acupuncture on intestinal mucosal barrier function and corticotropin-releasing factor (CRF)/CRF receptor 1 (CRFR1) pathway in rats with diarrhea-predominant irritable bowel syndrome (IBS-D), so as to explore its underlying mechanism in alleviating IBS-D. METHODS: Forty female SD rats were randomly divided into blank, model, electroacupuncture (EA), and agonist groups, with 10 rats in each group. Except for the blank group, rats in the other groups were given folium sennae infusion by gavage combined with chronic unpredictable mild stress to establish IBS-D model. Rats in the EA group received acupuncture at "Tianshu"(ST25) and EA at "Zusanli"(ST36) and "Taichong"(LR3) (2 Hz/15 Hz) on one side for 20 min, with the side chosen alternately every other day, for 14 days after modeling. Rats in the agonist group received acupuncture 30 min after intravenous injection of CRFR1 agonist urocortin, with the same manipulation method and time as the EA group. Before and after intervention, visceral pain threshold and stool Bristol scores were measured. Elevated plus maze test and open field test were used to detect anxiety and depression like behavior of rats. ELISA was used to detect the contents of CRF and CRFR1 in rats serum. Immunohistochemistry was used to detect the positive expressions of CRF, CRFR1, zonula occludens protein 1(ZO-1), occlusal protein(Occludin), and closure protein 1 (Claudin-1) in colon tissue. RESULTS: Compared with the blank group, the visceral pain threshold, open arm time percentage (OT%), total distance of movement in the open field test, and positive expression of ZO-1, Occludin, and Claudin-1 in colon were decreased (P<0.01, P<0.05), while Bristol stool scores, serum CRF and CRFR1 contents, and positive expressions of CRF and CRFR1 in colon were increased (P<0.01) in the model group. After intervention and compared with the model group, the visceral pain threshold, OT%, total distance of movement in the open field test, and positive expressions of ZO-1, Occludin, and Claudin-1 in colon were increased (P<0.05, P<0.01), while Bristol stool scores, serum CRF and CRFR1 contents, and positive expressions of CRF and CRFR1 in colon were decreased (P<0.01) in the EA groupï¼the Bristol stool scores, serum CRF content, and CRF positive expression in colon were significantly decreased in the agonist group (P<0.01). CONCLUSIONS: Peitu Yimu acupuncture can significantly improve visceral hypersensitivity and anxiety-depression state in IBS-D rats. Its mechanism may be related to the inhibition of CRF/CRFR1 pathway and restoration of intestinal tight junction protein expressions.
Assuntos
Terapia por Acupuntura , Diarreia , Mucosa Intestinal , Síndrome do Intestino Irritável , Receptores de Hormônio Liberador da Corticotropina , Animais , Feminino , Humanos , Ratos , Pontos de Acupuntura , Claudina-1/metabolismo , Claudina-1/genética , Hormônio Liberador da Corticotropina/metabolismo , Hormônio Liberador da Corticotropina/genética , Diarreia/terapia , Diarreia/metabolismo , Diarreia/genética , Modelos Animais de Doenças , Mucosa Intestinal/metabolismo , Síndrome do Intestino Irritável/terapia , Síndrome do Intestino Irritável/metabolismo , Síndrome do Intestino Irritável/genética , Ratos Sprague-Dawley , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Receptores de Hormônio Liberador da Corticotropina/genética , Proteína da Zônula de Oclusão-1/metabolismo , Proteína da Zônula de Oclusão-1/genéticaRESUMO
OBJECTIVE: To investigate the role of the EGFR/MAPK signaling pathway in PM2.5 in promoting the MUC5AC hypersecretion in airway and exacerbating airway inflammation. METHODS: By establishing rat model exposed to PM2.5, overexpressing miR-133b-5p and Claudin1, the content of IL-1 and TNF-α in serum were detected by ELISA, the pathology of lung tissue was observed by HE staining, p-EGFR, Claudin1, MUC5AC, p-ERK1/2, p-JNK, p-p38 in rats lung tissue were detected by immunohistochemical and WB, the expression level of miR-133b-5p in rats lung tissue were detected by qPCR. RESULTS: After the rats were exposed to PM2.5, the content of inflammatory factors in serum increased, the inflammatory damage of lung tissues occurred, the expression of miR-133b-5p was down-regulated, and the expression of MUC5AC protein was increased. The ELISA test results showed that the expression of IL-1 and TNF-α in the model group was significantly higher than that in the control group, and the model +AG1478 treatment group was down-regulated compared with the model group, and the +miR-133b-5p agomir treatment group was significantly lower than that in the control group, the model group and the model +Claudin1 overexpression blank load group, and the model +Claudin1 overexpression group was down-regulated compared with the model group and the model +Claudin1 overexpression blank load group. The protein detection results showed that the expression of p-EGFR, MUC5AC, p-ERK1/2, p-JNK and p-p38 proteins was increased and the expression of Claudin1 protein was decreased in the model group compared with the control group. In the model + AG1478 treatment group, model + miR-133b-5p agomir treatment group and model + Claudin1 overexpression group, compared with the model group, p-EGFR, MUC5AC, p-ERK1/2, p-JNK, p-p38 protein expression was down-regulated, and Claudin1 protein expression was up-regulated. CONCLUSIONS: PM2.5 inhibited the expression of miR-133b-5p to activate the EGFR/MAPK signal pathway, induce the hypersecretion of MUC5AC, thus aggravating PM2.5-related airway inflammation in rats.
Assuntos
Claudina-1 , Receptores ErbB , MicroRNAs , Mucina-5AC , Material Particulado , Animais , MicroRNAs/metabolismo , MicroRNAs/genética , Mucina-5AC/metabolismo , Mucina-5AC/genética , Ratos , Receptores ErbB/metabolismo , Receptores ErbB/genética , Material Particulado/toxicidade , Claudina-1/metabolismo , Claudina-1/genética , Masculino , Ratos Sprague-Dawley , Pulmão/metabolismo , Pulmão/patologia , Muco/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Sistema de Sinalização das MAP QuinasesRESUMO
Bronchopulmonary dysplasia (BPD) remains a significant challenge in neonatal care, the pathogenesis of which potentially involves altered lipid metabolism. Given the critical role of lipids in lung development and the injury response, we hypothesized that specific lipid species could serve as therapeutic agents in BPD. This study aimed to investigate the role of the lipid Phosphatidylcholine (PC) (16:0/14:0) in modulating BPD pathology and to elucidate its underlying mechanisms of action. Our approach integrated in vitro and in vivo methodologies to assess the effects of PC (16:0/14:0) on the histopathology, cellular proliferation, apoptosis, and molecular markers in lung tissue. In a hyperoxia-induced BPD rat model, we observed a reduction in alveolar number and an enlargement in alveolar size, which were ameliorated by PC (16:0/14:0) treatment. Correspondingly, in BPD cell models, PC (16:0/14:0) intervention led to increased cell viability, enhanced proliferation, reduced apoptosis, and elevated surfactant protein C (SPC) expression. RNA sequencing revealed significant gene expression differences between BPD and PC (16:0/14:0) treated groups, with a particular focus on Cldn1 (encoding claudin 1), which was significantly enriched in our analysis. Our findings suggest that PC (16:0/14:0) might protect against hyperoxia-induced alveolar type II cell damage by upregulating CLDN1 expression, potentially serving as a novel therapeutic target for BPD. This study not only advances our understanding of the role of lipids in BPD pathogenesis, but also highlights the significance of PC (16:0/14:0) in the prevention and treatment of BPD, offering new avenues for future research and therapeutic development.
Assuntos
Células Epiteliais Alveolares , Displasia Broncopulmonar , Claudina-1 , Hiperóxia , Fosfatidilcolinas , Regulação para Cima , Animais , Displasia Broncopulmonar/metabolismo , Displasia Broncopulmonar/patologia , Displasia Broncopulmonar/etiologia , Hiperóxia/metabolismo , Hiperóxia/complicações , Hiperóxia/patologia , Ratos , Claudina-1/metabolismo , Claudina-1/genética , Fosfatidilcolinas/metabolismo , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Ratos Sprague-Dawley , Apoptose , Proliferação de Células , Humanos , Alvéolos Pulmonares/patologia , Alvéolos Pulmonares/metabolismo , Animais Recém-Nascidos , Modelos Animais de DoençasRESUMO
OBJECTIVES: To observe the effects of moxibustion on intestinal barrier function and Toll-like receptor 4 (TLR4)/nuclear factor-κB p65 (NF-κB p65) signaling pathway in obese rats and explore the mechanism of moxibustion in the intervention of obesity. METHODS: Fifty-five Wistar rats of SPF grade were randomly divided into a normal group (10 rats) and a modeling group (45 rats). In the modeling group, the obesity model was established by feeding high-fat diet. Thirty successfully-modeled rats were randomized into a model group, a moxibustion group, and a placebo-control group, with 10 rats in each one. In the moxibustion group, moxibustion was applied at the site 3 cm to 5 cm far from the surface of "Zhongwan" (CV 12), with the temperature maintained at (46±1 ) â. In the placebo-control group, moxibustion was applied at the site 8 cm to 10 cm far from "Zhongwan" (CV 12), with the temperature maintained at (38±1) â. The intervention was delivered once daily for 8 weeks in the above two groups. The body mass and food intake of the rats were observed before and after intervention in each group. Using ELISA methool, the levels of serum triacylglycerol (TG), total cholesterol (TC) and lipopolysaccharide (LPS) were detected and the insulin resistance index (HOMA-IR) was calculated. HE staining was used to observe the morphology of colon tissue. The mRNA expression of zonula occludens-1 (ZO-1), Occludin, Claudin-1, TLR4 and NF-κB p65 in the colon tissue was detected by quantitative real-time PCR; and the protein expression of ZO-1, Occludin, Claudin-1, TLR4 and NF-κB p65 was detected by Western blot in the rats of each group. RESULTS: Compared with the normal group, the body mass, food intake, the level of HOMA-IR, and the serum levels of TC, TG and LPS were increased in the rats of the model group (P<0.01); those indexes in the moxibustion group were all reduced when compared with the model group and the placebo-control group respectively (P<0.01, P<0.05). Compared with the normal group, a large number of epithelial cells in the mucosa of colon tissue was damaged, shed, and the inflammatory cells were infiltrated obviously in the interstitium in the rats of the model group. When compared with the model group, in the moxibustion group, the damage of the colon tissue was recovered to various degrees and there were few infiltrated inflammatory cells in the interstitium, while, the epithelial injury of the colon tissue was slightly recovered and the infiltrated inflammatory cells in the interstitium were still seen in the placebo-control group. The mRNA and protein expressions of ZO-1, Occludin and Caudin-1 were decreased in the model group compared with those in the normal group (P<0.01). When compared with the model group and the placebo-control group, the mRNA and protein expressions of these indexes were increased in the moxibustion group (P<0.01, P<0.05). In the model group, the mRNA and protein expressions of TLR4 and NF-κB p65 were increased when compared with those in the normal group (P<0.01), and the mRNA and protein expressions of these indexes were reduced in the moxibustion group when compared with those in the model group and the placebo-control group (P<0.01). CONCLUSIONS: Moxibustion can reduce the body mass and food intake, regulate the blood lipid and improve insulin resistance in the rats of obesity. It may be related to alleviating inflammatory response through improving intestinal barrier function and modulating the intestinal TLR4/NF-κB p65 signaling pathway.
Assuntos
Resistência à Insulina , Moxibustão , Ratos , Animais , NF-kappa B/genética , NF-kappa B/metabolismo , Ratos Wistar , Receptor 4 Toll-Like/genética , Lipopolissacarídeos/metabolismo , Função da Barreira Intestinal , Ocludina/metabolismo , Claudina-1/metabolismo , Transdução de Sinais , Obesidade/genética , Obesidade/terapia , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Different levels of EspP2 expression are seen in strains of Glaesserella parasuis with high and low pathogenicity. As a potential virulence factor for G. parasuis, the pathogenic mechanism of EspP2 in infection of host cells is not clear. To begin to elucidate the effect of EspP2 on virulence, we used G. parasuis SC1401 in its wild-type form and SC1401, which was made EspP2-deficient. We demonstrated that EspP2 causes up-regulation of claudin-1 and occludin expression, thereby promoting the adhesion of G. parasuis to host cells; EspP2-deficiency resulted in significantly reduced adhesion of G. parasuis to cells. Transcriptome sequencing analysis of EspP2-treated PK15 cells revealed that the Rap1 signaling pathway is stimulated by EspP2. Blocking this pathway diminished occludin expression and adhesion. These results indicated that EspP2 regulates the adhesion of Glaesserella parasuis via Rap1 signaling pathway.
Assuntos
Haemophilus parasuis , Transdução de Sinais , Proteínas rap1 de Ligação ao GTP , Animais , Haemophilus parasuis/patogenicidade , Haemophilus parasuis/genética , Proteínas rap1 de Ligação ao GTP/metabolismo , Proteínas rap1 de Ligação ao GTP/genética , Aderência Bacteriana , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Ocludina/metabolismo , Ocludina/genética , Claudina-1/metabolismo , Claudina-1/genética , Linhagem Celular , SuínosRESUMO
The time for diabetic nephropathy (DN) to progress from mild to severe is long. Thus, methods to continuously repress DN are required to exert long-lasting effects mediated through epigenetic regulation. In this study, we demonstrated the ability of nicotinamide adenine dinucleotide (NAD) and its metabolites to reduce albuminuria through Sirt1- or Nampt-dependent epigenetic regulation. We previously reported that proximal tubular Sirt1 was lowered before glomerular Sirt1. Repressed glomerular Sirt1 was found to epigenetically elevate Claudin-1. In addition, we reported that proximal tubular Nampt deficiency epigenetically augmented TIMP-1 levels in Sirt6-mediated pathways, leading to type-IV collagen deposition and diabetic fibrosis. Altogether, we propose that the Sirt1/Claudin-1 axis may be crucial in the onset of albuminuria at the early stages of DN and that the Nampt/Sirt6/TIMP-1 axis promotes diabetic fibrosis in the middle to late stages of DN. Finally, administration of NMN, an NAD precursor, epigenetically potentiates the regression of the onset of DN to maintain Sirt1 and repress Claudin-1 in podocytes, suggesting the potential use of NAD metabolites as epigenetic medications for DN.
Assuntos
Albuminúria , Claudina-1 , Nefropatias Diabéticas , Epigênese Genética , NAD , Sirtuína 1 , Inibidor Tecidual de Metaloproteinase-1 , Animais , Humanos , Albuminúria/genética , Claudina-1/genética , Claudina-1/metabolismo , Citocinas/metabolismo , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Fibrose , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NAD/metabolismo , Mononucleotídeo de Nicotinamida/farmacologia , Nicotinamida Fosforribosiltransferase/genética , Nicotinamida Fosforribosiltransferase/metabolismo , Podócitos/metabolismo , Sirtuína 1/metabolismo , Sirtuína 1/genética , Sirtuínas/genética , Sirtuínas/metabolismo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-1/genéticaRESUMO
INTRODUCTION: Progestin, commonly used in oral contraception and preventing preterm birth, elicits various off-target side effects on brain and gastrointestinal (GI) functions, yet the precise mechanisms remain elusive. This study aims to probe progestin's impact on GI function and anxiety-like behaviors in female mice. METHODS: Colon stem cells were utilized to explore the mechanism underlying progestin 17-hydroxyprogesterone caproate (17-OHPC)-mediated suppression of claudin-1 (CLDN1), crucial for epithelial integrity. Chromatin immunoprecipitation and luciferase assays identified potential progestin-response elements on the CLDN1 promoter, with subsequent assessment of oxidative stress and pro-inflammatory cytokine release. Manipulation of vitamin D receptor (VDR) or estrogen receptor ß (ERß) expression elucidated their roles in 17-OHPC-mediated effects. Intestine-specific VDR deficient mice were generated to evaluate 17-OHPC's impact on GI dysfunction and anxiety-like behaviors in female mice. Additionally, gene expression was analyzed in various brain regions, including the amygdala, hypothalamus, and hippocampus. RESULTS: Exposure to 17-OHPC suppressed CLDN1 expression via epigenetic modifications and VDR dissociation from the CLDN1 promoter. Furthermore, 17-OHPC intensified oxidative stress and pro-inflammatory cytokine release. VDR knockdown partly mimicked, while overexpression of either VDR or ERß partly restored 17-OHPC-mediated effects. Intestinal VDR deficiency partly mirrored 17-OHPC-induced GI dysfunction, with minimal impact on 17-OHPC-mediated anxiety-like behaviors. CONCLUSIONS: 17-OHPC suppresses CLDN1 expression through VDR, contributing to GI dysfunction in female mice, distinct from 17-OHPC-induced anxiety-like behaviors. This study reveals a new mechanism and potential negative impact of progestin exposure on the GI tract, alongside inducing anxiety-like behaviors in female mice.
Assuntos
Ansiedade , Claudina-1 , Receptores de Calcitriol , Animais , Feminino , Ansiedade/induzido quimicamente , Ansiedade/metabolismo , Camundongos , Receptores de Calcitriol/metabolismo , Claudina-1/metabolismo , Camundongos Endogâmicos C57BL , Comportamento Animal/efeitos dos fármacos , Receptor beta de Estrogênio/metabolismo , Progestinas/farmacologiaRESUMO
Inulin-type fructan CP-A, a predominant polysaccharide in Codonopsis pilosula, demonstrates regulatory effects on immune activity and anti-inflammation. The efficacy of CP-A in treating ulcerative colitis (UC) is, however, not well-established. This study employed an in vitro lipopolysaccharide (LPS)-induced colonic epithelial cell model (NCM460) and an in vivo dextran sulfate sodium (DSS)-induced colitis mouse model to explore CP-A's protective effects against experimental colitis and its underlying mechanisms. We monitored the clinical symptoms in mice using various parameters: body weight, disease activity index (DAI), colon length, spleen weight, and histopathological scores. Additionally, molecular markers were assessed through enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence (IF), immunohistochemistry (IHC), and Western blotting assays. Results showed that CP-A significantly reduced reactive oxygen species (ROS), tumor necrosis factor-alpha (TNF-α), and interleukins (IL-6, IL-1ß, IL-18) in LPS-induced cells while increasing IL-4 and IL-10 levels and enhancing the expression of Claudin-1, ZO-1, and occludin proteins in NCM460 cells. Correspondingly, in vivo findings revealed that CP-A administration markedly improved DAI, reduced colon shortening, and decreased the production of myeloperoxidase (MPO), malondialdehyde (MDA), ROS, IL-1ß, IL-18, and NOD-like receptor protein 3 (NLRP3) inflammasome-associated genes/proteins in UC mice. CP-A treatment also elevated glutathione (GSH) and superoxide dismutase (SOD) levels, stimulated autophagy (LC3B, P62, Beclin-1, and ATG5), and reinforced Claudin-1 and ZO-1 expression, thereby aiding in intestinal epithelial barrier repair in colitis mice. Notably, the inhibition of autophagy via chloroquine (CQ) diminished CP-A's protective impact against colitis in vivo. These findings elucidate that CP-A's therapeutic effect on experimental colitis possibly involves mitigating intestinal inflammation through autophagy-mediated NLRP3 inflammasome inactivation. Consequently, inulin-type fructan CP-A emerges as a promising drug candidate for UC treatment.
Assuntos
Codonopsis , Colite Ulcerativa , Colite , Camundongos , Animais , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Inulina/metabolismo , Inulina/farmacologia , Inulina/uso terapêutico , Interleucina-18 , Codonopsis/metabolismo , Proteínas NLR/metabolismo , Frutanos/metabolismo , Frutanos/farmacologia , Frutanos/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Lipopolissacarídeos/farmacologia , Claudina-1/metabolismo , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/patologia , Autofagia , Sulfato de Dextrana , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Colo/metabolismo , Colo/patologiaRESUMO
OBJECTIVE: To investigate the protective effect of resveratrol on intestinal barrier in 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced Parkinson's disease (PD) mouse models and its mechanism for regulating TLR4/MyD88/NF-κB signaling to protect dopaminergic neurons. METHODS: Fifty-two C57BL/6J mice were randomized into control group (n= 12), MPTP group (n=14), MPTP + resveratrol (30 mg/kg) group (n=13), and MPTP + resveratrol (90 mg/kg) group (n=13), and mouse models were established by intraperitoneal MPTP (30 mg/kg) injection for 7 days in the latter 3 groups. Behavioral tests were conducted to evaluate the effect of resveratrol on motor symptoms of the mice. Western blotting was used to detect the expression of TH, α-syn, ZO-1, Claudin-1, TLR4, MyD88, and NF-κB in the brain tissues of the mice. Immunohistochemistry, immunofluorescence, ELISA and transmission electron microscopy were used to verify the effect of resveratrol for suppressing inflammation and protecting the intestinal barrier. RESULTS: Compared with those in the normal control group, the mice in MPTP group showed significant changes in motor function, number of dopaminergic neurons, neuroinflammation, levels of LPS and LBP, and expressions of tight junction proteins in the intestinal barrier. Resveratrol treatment significantly improved motor function of the PD mice (P < 0.01), increased the number of neurons and TH protein expression (P < 0.05), down-regulated the expressions of GFAP, Iba-1, and TLR4, lowered fecal and plasma levels of LPS and LBP (P < 0.05), restored the expression levels of ZO-1 and Claudin-1 (P < 0.01), and down-regulated the expressions of TLR4, MyD88, and NF-κB in the colon tissue (P < 0.05). The mice with resveratrol treatment at 30 mg/kg showed normal morphology of the tight junction complex with neatly and tightly arranged intestinal villi. CONCLUSION: Resveratrol repairs the intestinal barrier by inhibiting TLR4/MyD88/NF-κB signaling pathway-mediated inflammatory response, thereby improving motor function and neuropathy in mouse models of MPTP-induced PD.
Assuntos
Doença de Parkinson , Animais , Camundongos , Doença de Parkinson/tratamento farmacológico , Neurônios Dopaminérgicos/metabolismo , Resveratrol/farmacologia , Receptor 4 Toll-Like/metabolismo , NF-kappa B/metabolismo , Eixo Encéfalo-Intestino , Lipopolissacarídeos/farmacologia , Claudina-1/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Fator 88 de Diferenciação Mieloide/farmacologia , Camundongos Endogâmicos C57BL , Transdução de Sinais , Modelos Animais de Doenças , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/metabolismo , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologiaRESUMO
Obstructive sleep apnea (OSA) is characterized by intermittent repeated episodes of hypoxia-reoxygenation. OSA is associated with cerebrovascular consequences. An enhanced blood-brain barrier (BBB) permeability has been proposed as a marker of those disorders. We studied in mice the effects of 1 day and 15 days intermittent hypoxia (IH) exposure on BBB function. We focused on the dorsal part of the hippocampus and attempted to identify the molecular mechanisms by combining in vivo BBB permeability (Evans blue tests) and mRNA expression of several junction proteins (zona occludens (ZO-1,2,3), VE-cadherin, claudins (1,5,12), cingulin) and of aquaporins (1,4,9) on hippocampal brain tissues. After 15 days of IH exposure we observed an increase in BBB permeability, associated with increased mRNA expressions of claudins 1 and 12, aquaporins 1 and 9. IH seemed to increase early for claudin-1 mRNA expression as it doubled with 1 day of exposure and returned near to its base level after 15 days. Claudin-1 overexpression may represent an immediate response to IH exposure. Then, after 15 days of exposure, an increase in functional BBB permeability was associated with enhanced expression of aquaporin. These BBB alterations are possibly associated with a vasogenic oedema that may affect brain functions and accelerate neurodegenerative processes.
Assuntos
Aquaporinas , Apneia Obstrutiva do Sono , Camundongos , Animais , Barreira Hematoencefálica/metabolismo , Claudina-1/metabolismo , Modelos Animais de Doenças , Hipóxia/metabolismo , Claudinas/metabolismo , Apneia Obstrutiva do Sono/metabolismo , Permeabilidade , Aquaporinas/metabolismo , RNA Mensageiro/metabolismo , Claudina-5/metabolismoRESUMO
BACKGROUND: Early weaning is prone to damage intestinal barrier function, resulting in diarrhea, whereas rutin, as a natural flavonoid with multiple biological functions, shows potential in piglets. Therefore, the effects of dietary rutin on growth, antidiarrheal, barrier function, antioxidant status and cecal microbiota of weaned piglets were investigated with the control group (CON) (basal diet) and Rutin (basal diet+500 mg kg-1 rutin) groups fed for 14 days. RESULTS: The results showed that dietary 500 mg kg-1 rutin significantly decreased diarrhea index, serum diamine oxidase activity and total aerobic bacterial population in mesenteric lymph nodes, whereas it significantly increased the gain-to-feed ratio (G:F) and serum growth hormone content, jejunal villus height and villus height to crypt depth ratio, and also enhanced jejunal claudin-1 and zonula occludens-1 mRNA and protein expression. Meanwhile, dietary rutin significantly decreased inflammation-associated mRNA expression, malondialdehyde (MDA) content, swollen mitochondrial number and mitochondrial area in the jejunum, whereas it increased the total superoxide dismutase (T-SOD) and glutathione peroxidase activities and activated the Nrf2 signaling pathway. Moreover, dietary rutin significantly increased Firmicutes abundance and decreased Campylobacterota abundance, which were closely associated with the decreased diarrhea index and MDA content or increased Claudin-1 expression and T-SOD activity. CONCLUSION: Dietary 500 mg kg-1 rutin increased G:F by improving intestinal morphology, and alleviated diarrhea by enhancing intestinal barrier, which might be associated with the enhanced antioxidant capacity via activating the Nrf2/Keap1 signaling pathway and the improved cecal microbial composition in weaned piglets. © 2024 Society of Chemical Industry.
Assuntos
Antidiarreicos , Antioxidantes , Ceco , Diarreia , Microbioma Gastrointestinal , Mucosa Intestinal , Rutina , Desmame , Animais , Suínos/metabolismo , Suínos/crescimento & desenvolvimento , Microbioma Gastrointestinal/efeitos dos fármacos , Antioxidantes/metabolismo , Ceco/microbiologia , Ceco/metabolismo , Mucosa Intestinal/metabolismo , Diarreia/microbiologia , Diarreia/dietoterapia , Diarreia/veterinária , Antidiarreicos/administração & dosagem , Bactérias/classificação , Bactérias/isolamento & purificação , Bactérias/genética , Bactérias/metabolismo , Doenças dos Suínos/microbiologia , Doenças dos Suínos/metabolismo , Claudina-1/metabolismo , Claudina-1/genética , Ração Animal/análise , Jejuno/metabolismo , Jejuno/microbiologia , Suplementos Nutricionais/análise , Masculino , Superóxido Dismutase/metabolismo , Malondialdeído/metabolismo , Função da Barreira IntestinalRESUMO
OBJECTIVE: Disruption of the gingival epithelial barrier is often mediated by aging or the pathogen Porphyromonas gingivalis. This study examined the combined effects of aging and P. gingivalis exposure on gingival epithelial barrier molecules. METHODS: In vitro experiments involved treating young- and senescence-induced primary human gingival epithelial progenitor cells (HGEPp) with P. gingivalis lipopolysaccharide (LPS). Transepithelial electrical resistance (TER) and paracellular permeability were measured. In vivo, male C57BL/6J mice aged 10 (young) and 80 (old) weeks were divided into four groups: young, old, young with P. gingivalis (Pg-Young) inoculation, and old with P. gingivalis (Pg-Old) inoculation. P. gingivalis was inoculated orally thrice a week for 5 weeks. The mice were sacrificed 30 days after the last inoculation, and samples were collected for further procedures. The junctional molecules (Claudin-1, Claudin-2, E-cadherin, and Connexin) were analyzed for mRNA expression using qRT-PCR and protein production using western blotting and immunohistochemistry. The alveolar bone loss and inflammatory cytokine levels in gingival tissues were also assessed. RESULTS: LPS-treated senescent cells exhibited a pronounced reduction in TER, increased permeability to albumin protein, significant upregulation of Claudin-1 and Claudin-2, and significant downregulation of E-cadherin and Connexin. Furthermore, the Pg-Old group showed identical results with aging in addition to an increase in alveolar bone loss, significantly higher than that in the other groups. CONCLUSION: In conclusion, the host susceptibility to periodontal pathogens increases with age through changes in the gingival epithelial barrier molecules.
Assuntos
Perda do Osso Alveolar , Porphyromonas gingivalis , Masculino , Humanos , Animais , Camundongos , Porphyromonas gingivalis/metabolismo , Claudina-1/metabolismo , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/metabolismo , Claudina-2/metabolismo , Camundongos Endogâmicos C57BL , Caderinas/metabolismo , Envelhecimento , Conexinas/metabolismoRESUMO
Tight junctions (TJs) are important factors constituting the physical barriers of the skin, and their suppression has been described in various conditions, such as aged skin and atopic dermatitis lesions. However, the methods for improving skin TJ function remain insufficient. Therefore, to obtain compounds that can improve TJ function, we developed a novel high-throughput screening system termed live-cell immunostaining to evaluate cell surface-localized claudin-1 (CLDN1) with high selectivity using normal human epidermal keratinocytes (NHEKs). Heparinoid and phospho-pyridoxal (p-Pyr), a metabolite of pyridoxine, were identified as hit compounds. In addition, heparinoid was strongly suggested to increase CLDN1 expression by inhibiting epidermal growth factor receptor signaling. By contrast, p-Pyr did not enhance CLDN1 expression, but it accelerated the translocation of CLDN1 to the cell surface. Finally, we confirmed that heparinoid and p-Pyr improved barrier function in NHEKs in a transepithelial electrical resistance assay. In conclusion, heparinoid and p-Pyr could potentially ameliorate skin conditions by improving TJ function.
Assuntos
Heparinoides , Junções Íntimas , Humanos , Idoso , Claudina-1/metabolismo , Junções Íntimas/metabolismo , Heparinoides/metabolismo , Ensaios de Triagem em Larga Escala , Queratinócitos/metabolismo , Claudina-4/metabolismoRESUMO
This study investigates the intricate composition and spatial distribution of tight junction complex proteins during early mouse neurulation. The analyses focused on the cranial neural tube, which gives rise to all head structures. Neurulation brings about significant changes in the neuronal and non-neuronal ectoderm at a cellular and tissue level. During this process, precise coordination of both epithelial integrity and epithelial dynamics is essential for accurate tissue morphogenesis. Tight junctions are pivotal for epithelial integrity, yet their complex composition in this context remains poorly understood. Our examination of various tight junction proteins in the forebrain region of mouse embryos revealed distinct patterns in the neuronal and non-neuronal ectoderm, as well as mesoderm-derived mesenchymal cells. While claudin-4 exhibited exclusive expression in the non-neuronal ectoderm, we demonstrated a neuronal ectoderm specific localization for claudin-12 in the developing cranial neural tube. Claudin-5 was uniquely present in mesenchymal cells. Regarding the subcellular localization, canonical tight junction localization in the apical junctions was predominant for most tight junction complex proteins. ZO-1 (zona occludens protein-1), claudin-1, claudin-4, claudin-12, and occludin were detected at the apical junction. However, claudin-1 and occludin also appeared in basolateral domains. Intriguingly, claudin-3 displayed a non-canonical localization, overlapping with a nuclear lamina marker. These findings highlight the diverse tissue and subcellular distribution of tight junction proteins and emphasize the need for their precise regulation during the dynamic processes of forebrain development. The study can thereby contribute to a better understanding of the role of tight junction complex proteins in forebrain development.