RESUMO
BACKGROUND: Imbalanced intestinal microbiota and damage to the intestinal barrier contribute to the development of necrotizing enterocolitis (NEC). Autoinducer-2 (AI-2) plays a crucial role in repairing intestinal damage and reducing inflammation. OBJECTIVE: This study aimed to investigate the impact of AI-2 on the expression of intestinal zonula occludens-1 (ZO-1) and occludin proteins in NEC. We evaluated its effects in vivo using NEC mice and in vitro using lipopolysaccharide (LPS)-stimulated intestinal cells. METHODS: Pathological changes in the intestines of neonatal mice were assessed using histological staining and scoring. Cell proliferation was measured using the cell counting kit-8 (CCK-8) assay to determine the optimal conditions for LPS and AI-2 interventions. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to analyze the mRNA levels of matrix metalloproteinase-3 (MMP3), protease activated receptor-2 (PAR2), interleukin-1ß (IL-1ß), and IL-6. Protein levels of MMP3, PAR2, ZO-1, and occludin were evaluated using western blot, immunohistochemistry, or immunofluorescence. RESULTS: AI-2 alleviated NEC-induced intestinal damage (P < 0.05) and enhanced the proliferation of damaged IEC-6 cells (P < 0.05). AI-2 intervention reduced the mRNA and protein expressions of MMP3 and PAR2 in intestinal tissue and cells (P < 0.05). Additionally, it increased the protein levels of ZO-1 and occludin (P < 0.05), while reducing IL-1ß and IL-6 mRNA expression (P < 0.05). CONCLUSION: AI-2 intervention enhances the expression of tight junction proteins (ZO-1 and occludin), mitigates intestinal damage in NEC neonatal mice and IEC-6 cells, potentially by modulating PAR2 and MMP3 signaling. AI-2 holds promise as a protective intervention for NEC. AI-2 plays a crucial role in repairing intestinal damage and reducing inflammation.
Assuntos
Enterocolite Necrosante , Metaloproteinase 3 da Matriz , Receptor PAR-2 , Transdução de Sinais , Animais , Humanos , Camundongos , Animais Recém-Nascidos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Enterocolite Necrosante/patologia , Enterocolite Necrosante/tratamento farmacológico , Enterocolite Necrosante/metabolismo , Homosserina/análogos & derivados , Homosserina/farmacologia , Mucosa Intestinal/patologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/imunologia , Intestinos/patologia , Intestinos/efeitos dos fármacos , Lactonas/farmacologia , Lipopolissacarídeos , Metaloproteinase 3 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/genética , Camundongos Endogâmicos C57BL , Ocludina/metabolismo , Ocludina/genética , Receptor PAR-2/metabolismo , Receptor PAR-2/genética , Transdução de Sinais/efeitos dos fármacos , Proteína da Zônula de Oclusão-1/metabolismo , Proteína da Zônula de Oclusão-1/genéticaRESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV) causes a highly contagious disease that threatens the global swine industry. Recent studies have focused on the damage that PRRSV causes to the reproductive system of male pigs, although pathological research is lacking. Therefore, we examined the pathogenic mechanisms in male piglets infected with PRRSV. Gross and histopathological changes indicated that PRRSV affected the entire reproductive system, as confirmed via immunohistochemical analysis. PRRSV infected Sertoli cells and spermatogonia. To test the new hypothesis that PRRSV infection in piglets impairs blood - testis barrier (BTB) development, we investigated the pathology of PRRSV damage in the BTB. PRRSV infection significantly decreased the quantity and proliferative capacity of Sertoli cells constituting the BTB. Zonula occludens-1 and ß-catenin were downregulated in cell - cell junctions. Transcriptome analysis revealed that several crucial genes and signalling pathways involved in the growth and development of Leydig cells, Sertoli cells, and tight junctions in the testes were downregulated. Apoptosis, necroptosis, inflammatory, and oxidative stress-related pathways were activated, whereas hormone secretion-related pathways were inhibited. Many Sertoli cells and spermatogonia underwent apoptosis during early differentiation. Infected piglets exhibited disrupted androgen secretion, leading to significantly reduced testosterone and anti-Müllerian hormone levels. A cytokine storm occurred, notably upregulating cytokines such as tumour necrosis factor-α and interleukin-6. Markers of oxidative-stress damage (i.e. H2O2, malondialdehyde, and glutathione) were upregulated, whereas antioxidant-enzyme activities (i.e. superoxide dismutase, total antioxidant capacity, and catalase) were downregulated. Our results demonstrated that PRRSV infected multiple organs in the male reproductive system, which impaired growth in the BTB.
Assuntos
Barreira Hematotesticular , Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína , Células de Sertoli , Testículo , Animais , Masculino , Suínos , Vírus da Síndrome Respiratória e Reprodutiva Suína/patogenicidade , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Síndrome Respiratória e Reprodutiva Suína/virologia , Síndrome Respiratória e Reprodutiva Suína/patologia , Células de Sertoli/virologia , Células de Sertoli/metabolismo , Barreira Hematotesticular/virologia , Testículo/virologia , Testículo/patologia , Espermatogônias/virologia , Apoptose , Células Intersticiais do Testículo/virologia , Citocinas/metabolismo , Testosterona/sangue , Proteína da Zônula de Oclusão-1/metabolismo , Proteína da Zônula de Oclusão-1/genéticaRESUMO
Research on plant and animal peptides has garnered significant attention, but there is a lack of studies on the functional properties of Tenebrio molitor peptides, particularly in relation to their potential mitigating effect on radiation damage and the underlying mechanisms. This study aims to explore the protective effects of Tenebrio molitor peptides against radiation-induced damage. Mice were divided into five groups: normal, radiation model, and low-, medium-, and high-dose Tenebrio molitor peptide (TMP) groups (0.15 g per kg BW, 0.30 g per kg BW, and 0.60 g per kg BW). Various parameters such as blood cell counts, bone marrow DNA content, immune organ indices, serum levels of D-lactic acid, diamine oxidase (DAO), endotoxin (LPS), and inflammatory factors were assessed at 3 and 15 days post gamma irradiation. Additionally, the intestinal tissue morphology was examined through H&E staining, RT-qPCR experiments were conducted to analyze the expression of inflammatory factors in the intestine, and immunohistochemistry was utilized to evaluate the expression of tight junction proteins ZO-1 and Occludin in the intestine. The findings revealed that high-dose TMP significantly enhanced the hematopoietic system function in mice post radiation exposure, leading to increased spleen index, thymus index, blood cell counts, and bone marrow DNA production (p < 0.05). Moreover, TMP improved the intestinal barrier integrity and reduced the intestinal permeability. Mechanistic insights suggested that these peptides may safeguard intestinal barrier function by downregulating the gene expression of inflammatory factors TNF-α, IL-1ß, and IL-6, while upregulating the expression of tight junction proteins ZO-1 and Occludin (p < 0.05). Overall, supplementation with TMP mitigates radiation-induced intestinal damage by enhancing the hematopoietic system and the intestinal barrier, offering valuable insights for further investigations into the mechanisms underlying the protective effects of these peptides against ionizing radiation.
Assuntos
Mucosa Intestinal , Peptídeos , Tenebrio , Animais , Camundongos , Peptídeos/farmacologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efeitos da radiação , Mucosa Intestinal/efeitos dos fármacos , Masculino , Sistema Hematopoético/efeitos dos fármacos , Sistema Hematopoético/efeitos da radiação , Protetores contra Radiação/farmacologia , Proteína da Zônula de Oclusão-1/metabolismo , Proteína da Zônula de Oclusão-1/genética , Raios gama/efeitos adversos , Ocludina/metabolismo , Ocludina/genética , Intestinos/efeitos dos fármacos , Intestinos/efeitos da radiaçãoRESUMO
Although bitter receptors, known as Tas2Rs, have been identified in the testes and mature sperm, their expression in testicular Sertoli cells (SCs) and their role in recognizing harmful substances to maintain the immune microenvironment remain unknown. To explore their potential function in spermatogenesis, this study utilized TM4 cells and discovered the high expression of the bitter receptor Tas2R143 in the cells. Interestingly, when the Tas2R143 gene was knocked down for 24 and 48 h, there was a significant downregulation (P < 0.05) in the expression of tight junction proteins (occludin and ZO-1) and NF-κB. Additionally, Western blot results demonstrated that the siRNA-133+NF-κB co-treatment group displayed a significant downregulation (P < 0.05) in the expression of occludin and ZO-1 compared to both the siRNA-133 transfection group and the NF-κB inhibitors treatment group. These findings suggest that Tas2R143 likely regulates the expression of occludin and ZO-1 through the NF-κB signaling pathway and provides a theoretical basis for studying the regulatory mechanism of bitter receptors in the reproductive system, aiming to attract attention to the chemical perception mechanism of spermatogenesis.
Assuntos
Barreira Hematotesticular , NF-kappa B , Transdução de Sinais , NF-kappa B/metabolismo , Barreira Hematotesticular/metabolismo , Masculino , Animais , Linhagem Celular , Camundongos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína da Zônula de Oclusão-1/metabolismo , Proteína da Zônula de Oclusão-1/genética , Ocludina/metabolismo , Ocludina/genética , Proteínas de Junções Íntimas/metabolismo , Proteínas de Junções Íntimas/genética , Células de Sertoli/metabolismoRESUMO
The activation of the vitamin D receptor (VDR) in the ileum has been shown to regulate Paneth cell-specific defensins, a large family of antimicrobial peptides; hence, this may serve as a potential mechanism to maintain intestinal homeostasis. Previously, we have demonstrated that a combination of vitamin D3 (VD) and fructooligosaccharides (FOSs) upregulates colonic Vdr in mice. Here, we aim to examine the effect of VD, alone or in combination with FOSs, on intestinal barrier integrity and the secretion of antimicrobial peptides, as well as the gut microbial community. Male and female C57BL/6J mice at 6 weeks old were randomized into three groups to receive the following dietary regimens (n = 10/sex/group) for 8 weeks: (1) standard AIN-93G control diet (CTR), (2) CTR + 5000 IU vitamin D3 (VD), and (3) VD + 5% fructooligosaccharides (VF). VD and VF differentially regulated the mRNA expressions of tight junction proteins in the colon and ileum. VF suppressed the upregulation of colonic ZO-1 and occludin, which was induced by VD supplementation alone. In the ileum, occludin but not ZO-1 was upregulated 20-fold in the VF-treated mice. While VD did not alter the mRNA expressions of Vdr and defensins in the ileum, these targets were downregulated by VF. Microbial analysis further reveals a shift of microbial beta diversity and a reduction in Romboutsia ilealis, a pathobiont, in VF-treated mice. Though the implications of these phenotypical and microbial changes remain to be determined, the administration of FOSs in the presence of VD may serve as an effective dietary intervention for maintaining intestinal homeostasis.
Assuntos
Colecalciferol , Defensinas , Suplementos Nutricionais , Microbioma Gastrointestinal , Oligossacarídeos , Animais , Feminino , Masculino , Camundongos , Colecalciferol/farmacologia , Colo/metabolismo , Colo/efeitos dos fármacos , Defensinas/metabolismo , Defensinas/genética , Regulação para Baixo/efeitos dos fármacos , Microbioma Gastrointestinal/efeitos dos fármacos , Íleo/metabolismo , Íleo/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Ocludina/metabolismo , Ocludina/genética , Oligossacarídeos/farmacologia , Oligossacarídeos/administração & dosagem , Celulas de Paneth/metabolismo , Celulas de Paneth/efeitos dos fármacos , Receptores de Calcitriol/metabolismo , Receptores de Calcitriol/genética , Proteína da Zônula de Oclusão-1/metabolismo , Proteína da Zônula de Oclusão-1/genéticaRESUMO
Many COVID-19 patients suffer from gastrointestinal symptoms and impaired intestinal barrier function is thought to play a key role in Long COVID. Despite its importance, the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on intestinal epithelia is poorly understood. To address this, we established an intestinal barrier model integrating epithelial Caco-2 cells, mucus-secreting HT29 cells and Raji cells. This gut epithelial model allows efficient differentiation of Caco-2 cells into microfold-like cells, faithfully mimics intestinal barrier function, and is highly permissive to SARS-CoV-2 infection. Early strains of SARS-CoV-2 and the Delta variant replicated with high efficiency, severely disrupted barrier function, and depleted tight junction proteins, such as claudin-1, occludin, and ZO-1. In comparison, Omicron subvariants also depleted ZO-1 from tight junctions but had fewer damaging effects on mucosal integrity and barrier function. Remdesivir, the fusion inhibitor EK1 and the transmembrane serine protease 2 inhibitor Camostat inhibited SARS-CoV-2 replication and thus epithelial barrier damage, while the Cathepsin inhibitor E64d was ineffective. Our results support that SARS-CoV-2 disrupts intestinal barrier function but further suggest that circulating Omicron variants are less damaging than earlier viral strains.
Assuntos
COVID-19 , Mucosa Intestinal , SARS-CoV-2 , Junções Íntimas , Replicação Viral , Humanos , SARS-CoV-2/patogenicidade , Células CACO-2 , COVID-19/virologia , COVID-19/patologia , Mucosa Intestinal/virologia , Mucosa Intestinal/patologia , Junções Íntimas/virologia , Alanina/análogos & derivados , Proteína da Zônula de Oclusão-1/metabolismo , Proteína da Zônula de Oclusão-1/genética , Antivirais/farmacologia , Células HT29 , Ocludina/metabolismo , Ocludina/genética , Monofosfato de Adenosina/análogos & derivadosRESUMO
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
BACKGROUND: Ulcerative colitis (UC) is a type of inflammatory bowel disease associated with persistent inflammation. Animal studies proved the efficacy of metformin in UC. AIM: To investigate the potential role of metformin and its protective pathways in patients with UC. METHODS: This is a randomized, controlled, and double-blinded clinical trial that included 60 participants with mild to moderate UC and was divided randomly into two groups (n = 30). For 6 months, the mesalamine group received 1 g of mesalamine three times daily (t.i.d.). For six months, the metformin group received mesalamine 1 g t.i.d. and metformin 500 mg twice daily. A gastroenterologist evaluated patients at baseline and 6 months after starting the treatment in order to measure serum levels of zonulin, sphingosine 1 phosphate (S1P), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α). Biopsies from the colon were used to measure gene expression of zonula occuldin-1 (ZO-1), signal transducer and activator of factor-3 (STAT-3), and intracellular adhesion molecule-1 (ICAM-1). The numeric pain rating scale (NRS) and partial Mayo score were also assessed for each patient. RESULTS: When compared to the mesalamine group, the metformin group demonstrated a statistical decrease in serum IL-6, zonulin, TNF-α, SIP, gene expression of ICAM-1 and STAT-3, and a significant increase in colonic ZO-1 when compared to the mesalamine group. The metformin group also showed a significant decrease in NRS and partial Mayo score index in comparison with the mesalamine group. CONCLUSION: Metformin may be a promising additional therapy for UC patients. Trial registration identifier: NCT05553704.
Assuntos
Colite Ulcerativa , Mesalamina , Metformina , Humanos , Metformina/uso terapêutico , Colite Ulcerativa/tratamento farmacológico , Mesalamina/uso terapêutico , Método Duplo-Cego , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT3/genética , Reposicionamento de Medicamentos , Haptoglobinas/metabolismo , Interleucina-6/sangue , Proteína da Zônula de Oclusão-1/metabolismo , Proteína da Zônula de Oclusão-1/genética , Toxina da Cólera , Molécula 1 de Adesão Intercelular/sangue , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Anti-Inflamatórios não Esteroides/uso terapêutico , Fator de Necrose Tumoral alfa/sangue , Colo/patologia , Colo/efeitos dos fármacos , Resultado do Tratamento , Adulto Jovem , Quimioterapia Combinada , Precursores de ProteínasRESUMO
The damage to the mechanical barrier of the intestinal mucosa is the initiating factor and the core link of the progression of ulcerative colitis (UC). Protecting the mechanical barrier of the intestinal mucosa is of great significance for improving the health status of UC patients. ZO-1 is a key scaffold protein of the mechanical barrier of the intestinal mucosa, and its fusion with the membrane of the intestinal epithelium is a necessary condition to maintain the integrity of the mechanical barrier of the intestinal mucosa. Enteric glial cells (EGCs) play an important role in the maintenance of intestinal homeostasis and have become a new target for regulating intestinal health in recent years. In this study, we found that glycyrol (GC), a representative coumarin compound isolated from Licorice (Glycyrrhiza uralensis Fisch, used for medicine and food), can alleviate UC by promoting the production of neurotrophic factor GDNF in mice EGCs. Specifically, we demonstrated that GC promotes the production of GDNF, then activates its receptor RET, promotes ZO-1 fusion with cell membranes, and protects the intestinal mucosal mechanical barrier. The results of this study can provide new ideas for the prevention and treatment of UC.
Assuntos
Colite Ulcerativa , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Mucosa Intestinal , Neuroglia , Proteína da Zônula de Oclusão-1 , Animais , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/metabolismo , Camundongos , Humanos , Proteína da Zônula de Oclusão-1/metabolismo , Proteína da Zônula de Oclusão-1/genética , Masculino , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-ret/metabolismo , Proteínas Proto-Oncogênicas c-ret/genética , Camundongos Endogâmicos C57BL , Cumarínicos/farmacologia , Cumarínicos/química , Transdução de Sinais/efeitos dos fármacos , Glycyrrhiza/químicaRESUMO
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
PURPOSE: This study aimed to elucidate the role of the Cdk1/p53/p21 feedback loop in the pathogenesis of interstitial cystitis (IC)/bladder pain syndrome (BPS). MATERIALS AND METHODS: An IC/BPS cell model was established. Cell viability was determined using the CCK-8 assay. Flow cytometry was adopted to assess cell apoptosis rates. ELISA was employed to measure secretion levels of inflammatory factors (IL-6, IL-8, and TNF-α). Gene expressions were assessed using PCR, while protein expressions were analyzed through Western blotting analysis. Epithelial permeability was demonstrated using the phenol red leakage experiment and FITC-dextran permeability assay. The interaction between proteins was determined using co-immunoprecipitation, and protein localization was investigated using immunofluorescence. RESULTS: The CCK-8 assay revealed a significantly reduced viability of IC/BPS cells compared to normal epithelial cells (p < 0.05). Elevated levels of IL-6, IL-8, and TNF-α were detected in IC/BPS cells. Changes in the expressions of E-cadherin and ZO-1 were evident, leading to increased epithelial permeability in IC/BPS cells. Furthermore, within IC/BPS cells, Cdk1 phosphorylated p53 in the nucleus. The Cdk1/p53/p21 feedback loop was established to influence urothelial permeability. Both p21 and Cdk1 inhibitors notably reduced the epithelial permeability in IC/BPS cells. CONCLUSION: The Cdk1/p53/p21 feedback loop was instrumental in IC/BPS, acting as a regulator of urothelial permeability. This discovery offered a novel therapeutic approach for IC/BPS management.
Assuntos
Proteína Quinase CDC2 , Cistite Intersticial , Proteína Supressora de Tumor p53 , Humanos , Cistite Intersticial/metabolismo , Cistite Intersticial/patologia , Cistite Intersticial/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Quinase CDC2/metabolismo , Proteína Quinase CDC2/genética , Retroalimentação Fisiológica , Apoptose , Sobrevivência Celular , Proteína da Zônula de Oclusão-1/metabolismo , Proteína da Zônula de Oclusão-1/genética , Linhagem Celular , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Caderinas/metabolismo , Caderinas/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologiaRESUMO
An exceptional expression of claudins (CLDNs), tight junction (TJ) proteins, is observed in various solid cancer tissues. However, the pathophysiological roles of CLDNs have not been clarified in detail. CLDN14 is highly expressed in human colorectal cancer (CRC) tissues and cultured cancer epithelial cells. We found CLDN14 silencing decreased cell viability without affecting spheroid size in the three-dimensional (3D) spheroid model of DLD-1 cells derived from human CRC. Mitochondria activity and oxidative stress level were reduced by CLDN14 silencing. Furthermore, CLDN14 silencing decreased the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and its target antioxidative genes. CLDN14 was colocalized with ZO-1, a scaffolding protein in the TJ. CLDN14 silencing induced the disruption of TJ barrier such as the reduction of transepithelial electrical resistance and elevation of fluxes of small molecules including glucose in two-dimensional (2D) cultured model,. The depletion of glucose induced the elevation of ROS generation, mitochondria activity, and Nrf2 expression. These results suggest that CLDN14 increases Nrf2 expression in spheroids mediated via the formation of paracellular barrier to glucose. The cytotoxicities of doxorubicin, an anthracycline anticancer drug, and oxaliplatin, a platinum-based agent, were augmented by an Nrf2 activator in 2D cultured cells. The anticancer drug-induced toxicity was enhanced by CLDN14 silencing in 3D spheroids. We suggest that CLDN14 may potentiate chemoresistance mediated by the suppression of paracellular glucose permeability and activation of the Nrf2 signaling pathway in CRC cells.
Assuntos
Claudinas , Neoplasias Colorretais , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos , Inativação Gênica , Fator 2 Relacionado a NF-E2 , Humanos , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Claudinas/metabolismo , Claudinas/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Esferoides Celulares/metabolismo , Esferoides Celulares/efeitos dos fármacos , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo , Proteína da Zônula de Oclusão-1/genéticaRESUMO
OBJECTIVE: Hepatic stellate cells (HSCs) play a crucial role in liver fibrosis. Early-stage liver fibrosis is reversible and intimately associated with the state of HSCs. Kruppel-like factor 4 (KLF4) plays a pivotal role in a wide array of physiological and pathological processes. This study aimed to investigate the effect of KLF4 on the proliferation, apoptosis and phenotype of quiescent HSCs METHODS: We designed a KLF4 lentiviral vector and a KLF4 siRNA lentiviral vector, to upregulate and silence KLF4 expression in human HSC LX-2 cells via transfection. Cell proliferation was assessed using the CCK-8 assay. Flow cytometry was used to detect the cell cycle distribution and apoptosis rate. Western blotting was used to determine the levels of some quiescence and activation markers of HSCs RESULTS: Overexpression of KLF4 significantly increased the levels of E-cadherin and ZO-1, which are quiescent HSC markers, while significantly decreased the levels of N-cadherin and a-SMA, known activated HSC markers. In contrast, cell proliferation and apoptosis rates were elevated in LX-2 cells in which KLF4 expression was silenced CONCLUSION: KLF4 inhibits the proliferation and activation of human LX-2 HSCs. It might be a key regulatory protein in the maintenance of HSC quiescence and may serve as a target for the inhibition of hepatic fibrosis.
Assuntos
Apoptose , Proliferação de Células , Células Estreladas do Fígado , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like , Humanos , Células Estreladas do Fígado/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Proliferação de Células/genética , Apoptose/genética , Caderinas/metabolismo , Caderinas/genética , Linhagem Celular , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Proteína da Zônula de Oclusão-1/metabolismo , Proteína da Zônula de Oclusão-1/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ciclo Celular/genética , Actinas/metabolismo , Actinas/genéticaRESUMO
Despite of yet unknown mechanism, microvascular deposition of oligomeric Tau (oTau) has been implicated in alteration of the Blood-Brain Barrier (BBB) function in Alzheimer's disease (AD) brains. In this study, we employed an in vitro BBB model using primary mouse cerebral endothelial cells (CECs) to investigate the mechanism underlying the effects of oTau on BBB function. We found that exposing CECs to oTau induced oxidative stress through NADPH oxidase, increased oxidative damage to proteins, decreased proteasome activity, and expressions of tight junction (TJ) proteins including occludin, zonula occludens-1 (ZO-1) and claudin-5. These effects were suppressed by the pretreatment with Fasudil, a RhoA/ROCK signaling inhibitor. Consistent with the biochemical alterations, we found that exposing the basolateral side of CECs to oTau in the BBB model disrupted the integrity of the BBB, as indicated by an increase in FITC-dextran transport across the model, and a decrease in trans endothelial electrical resistance (TEER). oTau also increased the transmigration of peripheral blood mononuclear cells (PBMCs) in the BBB model. These functional alterations in the BBB induced by oTau were also suppressed by Fasudil. Taken together, our findings suggest that targeting the RhoA/ROCK pathway can be a potential therapeutic strategy to maintain BBB function in AD.
Assuntos
Barreira Hematoencefálica , Células Endoteliais , Transdução de Sinais , Proteínas tau , Animais , Humanos , Camundongos , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Células Cultivadas , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Endoteliais/efeitos dos fármacos , Estresse Oxidativo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Proteínas tau/metabolismo , Proteínas tau/genética , Junções Íntimas/metabolismo , Junções Íntimas/efeitos dos fármacos , Proteína da Zônula de Oclusão-1/metabolismo , Proteína da Zônula de Oclusão-1/genéticaRESUMO
Adequate extravillous trophoblast (EVT) invasion into the maternal decidua is important for human placental development. We identified that E2F transcription factor 8 (E2F8) suppresses EVT invasion, and that tight junction protein-1 (TJP1) is a potential downstream target gene of E2F8. We investigated the role of TJP1 in the human placenta and regulation of TJP1 expression by E2F8. TJP1 expression decreased in E2F8 knockdown HTR-8/SVneo cells. TJP1 and E2F8 were co-expressed in villi in the first-trimester placenta and in EVTs and villi in the third-trimester placenta. TJP1 was significantly increased in the pre-eclamptic compared with control placenta. TJP1 knockdown increased the invasion of HTR-8/SVneo cells, while TJP1 overexpression inhibited cell invasion. Halo-E2F8 overexpression significantly increased TJP1 expression and TJP1 transcription compared with control placenta. Our findings suggest that E2F8 promotes TJP1 transcription, and that TJP1 expression by E2F8 inhibits EVT invasion. TJP1 and E2F8 may be related to pre-eclampsia pathogenesis.
Assuntos
Movimento Celular , Placenta , Pré-Eclâmpsia , Proteínas Repressoras , Trofoblastos , Proteína da Zônula de Oclusão-1 , Adulto , Feminino , Humanos , Gravidez , Linhagem Celular , Movimento Celular/genética , Técnicas de Silenciamento de Genes , Placenta/metabolismo , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/patologia , Trofoblastos/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo , Proteína da Zônula de Oclusão-1/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
Zonula occludens-1 (ZO-1) is involved in the regulation of cell-cell junctions between endothelial cells (ECs). Here we identify the ZO-1 protein interactome and uncover ZO-1 interactions with RNA-binding proteins that are part of stress granules (SGs). Downregulation of ZO-1 increased SG formation in response to stress and protected ECs from cellular insults. The ZO-1 interactome uncovered an association between ZO-1 and Y-box binding protein 1 (YB-1), a constituent of SGs. Arsenite treatment of ECs decreased the interaction between ZO-1 and YB-1, and drove SG assembly. YB-1 expression is essential for SG formation and for the cytoprotective effects induced by ZO-1 downregulation. In the developing retinal vascular plexus of newborn mice, ECs at the front of growing vessels express less ZO-1 but display more YB-1-positive granules than ECs located in the vascular plexus. Endothelial-specific deletion of ZO-1 in mice at post-natal day 7 markedly increased the presence of YB-1-positive granules in ECs of retinal blood vessels, altered tip EC morphology and vascular patterning, resulting in aberrant endothelial proliferation, and arrest in the expansion of the retinal vasculature. Our findings suggest that, through its interaction with YB-1, ZO-1 controls SG formation and the response of ECs to stress during angiogenesis.
Assuntos
Células Endoteliais , Proteína 1 de Ligação a Y-Box , Proteína da Zônula de Oclusão-1 , Animais , Proteína 1 de Ligação a Y-Box/metabolismo , Proteína 1 de Ligação a Y-Box/genética , Proteína da Zônula de Oclusão-1/metabolismo , Proteína da Zônula de Oclusão-1/genética , Camundongos , Humanos , Células Endoteliais/metabolismo , Grânulos de Estresse/metabolismo , Neovascularização Fisiológica , Vasos Retinianos/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Angiogênese , Fatores de TranscriçãoRESUMO
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
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
Heat-killed Lactiplantibacillus plantarum L-137 (HK L-137) has been suggested to enhance the intestinal barrier in obese mice, leading to improvement of metabolic abnormalities and adipose tissue inflammation, and in healthy humans with overweight, leading to improvement of systemic inflammation. However, its detailed mechanism of action has not been clarified. Therefore, this study investigated the effects of HK L-137 on the permeability of rat small intestinal epithelial IEC-6 cells, tight junction-related gene and protein expression and localization, and intracellular signaling pathways involved in barrier function. Treatment of IEC-6 cells with HK L-137 for 26 h significantly reduced the permeability to fluorescein isothiocyanate-dextran (FD-4). HK L-137 also increased gene and protein expression of zonula occludens-1 (ZO-1), an important tight junction protein, without affecting the localization. Furthermore, inhibition of the extracellular signal-regulated kinase (ERK)1/2 pathway in IEC-6 cells canceled the HK L-137-related reduction in permeability to FD-4. Phosphorylation of ERK in IEC-6 cells was induced 15 min after the addition of HK L-137. These results suggest that HK L-137 reduces intestinal permeability partly through activating the ERK pathway and increasing expression of the ZO-1 gene and protein. Enhancement of intestinal barrier function with HK L-137 might be effective in preventing and treating leaky gut, for which no specific therapeutic tool has been established.
Assuntos
Células Epiteliais , Mucosa Intestinal , Proteína da Zônula de Oclusão-1 , Animais , Ratos , Proteína da Zônula de Oclusão-1/metabolismo , Proteína da Zônula de Oclusão-1/genética , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Linhagem Celular , Intestino Delgado/metabolismo , Intestino Delgado/microbiologia , Probióticos/farmacologia , Permeabilidade , Lactobacillus plantarum/fisiologia , Junções Íntimas/metabolismo , Temperatura Alta , Sistema de Sinalização das MAP Quinases , Fosforilação , Função da Barreira IntestinalRESUMO
Adaptive metabolic responses and innate metabolites hold promising therapeutic potential for stroke, while targeted interventions require a thorough understanding of underlying mechanisms. Adiposity is a noted modifiable metabolic risk factor for stroke, and recent research suggests that it benefits neurological rehabilitation. During the early phase of experimental stroke, the lipidomic results showed that fat depots underwent pronounced lipolysis and released fatty acids (FAs) that feed into consequent hepatic FA oxidation and ketogenesis. Systemic supplementation with the predominant ketone beta-hydroxybutyrate (BHB) is found to exert discernible effects on preserving blood-brain barrier (BBB) integrity and facilitating neuroinflammation resolution. Meanwhile, blocking FAO-ketogenesis processes by administration of CPT1α antagonist or shRNA targeting HMGCS2 exacerbated endothelial damage and aggravated stroke severity, whereas BHB supplementation blunted these injuries. Mechanistically, it is unveiled that BHB infusion is taken up by monocarboxylic acid transporter 1 (MCT1) specifically expressed in cerebral endothelium and upregulated the expression of tight junction protein ZO-1 by enhancing local ß-hydroxybutyrylation of H3K9 at the promoter of TJP1 gene. Conclusively, an adaptive metabolic mechanism is elucidated by which acute lipolysis stimulates FAO-ketogenesis processes to restore BBB integrity after stroke. Ketogenesis functions as an early metabolic responder to restrain stroke progression, providing novel prospectives for clinical translation.