RESUMEN
Oxidative stress is a prominent causal factor in the premature senescence of microvascular endothelial cells and the ensuing blood-brain barrier (BBB) dysfunction. Through the exposure of an in vitro model of human BBB, composed of brain microvascular endothelial cells (BMECs), astrocytes, and pericytes to H2O2, this study examined whether a specific targeting of the p38MAPK/NF-κB pathway and/or senescent cells could delay oxidative stress-mediated EC senescence and protect the BBB. Enlarged BMECs, displaying higher ß-galactosidase activity, γH2AX staining, p16 expression, and impaired tubulogenic capacity, were regarded as senescent. The BBB established with senescent BMECs had reduced transendothelial electrical resistance and increased paracellular flux, which are markers of BBB integrity and function, respectively. Premature senescence disrupted plasma-membrane localization of the tight junction protein, zonula occludens-1, and elevated basement membrane-degrading matrix metalloproteinase-2 activity and pro-inflammatory cytokine release. Inhibition of p38MAPK by BIRB796 and NF-κB by QNZ and the elimination of senescent cells by a combination of dasatinib and quercetin attenuated the effects of H2O2 on senescence markers; suppressed release of the pro-inflammatory cytokines interleukin-8, monocyte chemoattractant protein-1, and intercellular adhesion molecule-1; restored tight junctional unity; and improved BBB function. In conclusion, therapeutic approaches that mitigate p38MAPK/NF-κB activity and senescent cell accumulation in the cerebrovasculature may successfully protect BBB from oxidative stress-induced BBB dysfunction.
Asunto(s)
Barrera Hematoencefálica , Senescencia Celular , Células Endoteliales , Peróxido de Hidrógeno , FN-kappa B , Estrés Oxidativo , Senoterapéuticos , Proteínas Quinasas p38 Activadas por Mitógenos , Estrés Oxidativo/efectos de los fármacos , Humanos , Senescencia Celular/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/efectos de los fármacos , FN-kappa B/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Senoterapéuticos/farmacología , Peróxido de Hidrógeno/farmacología , Transducción de Señal/efectos de los fármacos , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
Segmental grafts from living donors have advantages over grafts from deceased donors when used for small intestine transplantation. However, storage time for small intestine grafts can be extremely short and optimal graft preservation conditions for short-term storage remain undetermined. Secreted factors from mesenchymal stem cells (MSCs) that allow direct activation of preserved small intestine grafts. Freshly excised Luc-Tg LEW rat tissues were incubated in preservation solutions containing MSC-conditioned medium (MSC-CM). Preserved Luc-Tg rat-derived grafts were then transplanted to wild-type recipients, after which survival, injury score, and tight junction protein expression were examined. Luminance for each graft was determined using in vivo imaging. The findings indicated that 30-100 and 3-10 kDa fractions of MSC-CM have superior activating effects for small intestine preservation. Expression of the tight-junction proteins claudin-3, and zonula occludens-1 preserved for 24 h in University of Wisconsin (UW) solution containing MSC-CM with 50-100 kDa, as shown by immunostaining, also indicated effectiveness. Reflecting the improved graft preservation, MSC-CM preloading of grafts increased survival rate from 0% to 87%. This is the first report of successful transplantation of small intestine grafts preserved for more than 24 h using a rodent model to evaluate graft preservation conditions that mimic clinical conditions.
Asunto(s)
Intestino Delgado , Células Madre Mesenquimatosas , Preservación de Órganos , Ratas Endogámicas Lew , Animales , Intestino Delgado/trasplante , Ratas , Preservación de Órganos/métodos , Masculino , Soluciones Preservantes de Órganos , Supervivencia de Injerto , Medios de Cultivo Condicionados , Proteína de la Zonula Occludens-1/metabolismo , Claudina-3/metabolismo , Ratas Transgénicas , Glutatión , Rafinosa , Alopurinol , Insulina , AdenosinaRESUMEN
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.
Asunto(s)
COVID-19 , Mucosa Intestinal , SARS-CoV-2 , Uniones Estrechas , Replicación Viral , Humanos , SARS-CoV-2/patogenicidad , Células CACO-2 , COVID-19/virología , COVID-19/patología , Mucosa Intestinal/virología , Mucosa Intestinal/patología , Uniones Estrechas/virología , Alanina/análogos & derivados , Proteína de la Zonula Occludens-1/metabolismo , Proteína de la Zonula Occludens-1/genética , Antivirales/farmacología , Células HT29 , Ocludina/metabolismo , Ocludina/genética , Adenosina Monofosfato/análogos & derivadosRESUMEN
BACKGROUND: Intestinal barrier dysfunction and systemic inflammation are common in obstructive sleep apnoea (OSA). We aimed to investigate the role of melatonin, an anti-inflammatory mediator, in mediating the relationships between OSA, intestinal barrier dysfunction and systemic inflammation. METHODS: Two hundred and thirty-five male participants who complained with sleep problems and underwent whole night polysomnography at our sleep centre between 2017 and 2018 were enrolled. Polysomnographic data, anthropometric measurements and biochemical indicators were collected. Serum melatonin, intestinal barrier function biomarker zonula occludens-1 (ZO-1) and inflammatory biomarkers C-reactive protein (CRP) with lipopolysaccharide (LPS) were detected. Spearman's correlation analysis assessed the correlations between sleep parameters, melatonin and biomarkers (ZO-1, LPS and CRP). Mediation analysis explored the effect of OSA on intestinal barrier dysfunction and systemic inflammation in moderate-severe OSA patients. RESULTS: As OSA severity increased, serum melatonin decreased, whereas ZO-1, LPS and CRP increased. Spearman's correlation analysis showed that serum melatonin was significantly negatively correlated with ZO-1 (r = -0.19, p < .05) and LPS (r = -0.20, p < .05) in the moderate-OSA group; serum melatonin was significantly negatively correlated with ZO-1 (r = -0.46, p < .01), LPS (r = -0.35, p < .01) and CPR (r = -0.30, p < .05) in the severe-OSA group. Mediation analyses showed melatonin explain 36.12% and 35.38% of the effect of apnoea-hypopnea index (AHI) on ZO-1 and LPS in moderate to severe OSA patients. CONCLUSIONS: Our study revealed that melatonin may be involved in mediating intestinal barrier dysfunction and systemic inflammation in moderate-to-severe OSA patients.
Asunto(s)
Biomarcadores , Proteína C-Reactiva , Inflamación , Melatonina , Polisomnografía , Apnea Obstructiva del Sueño , Proteína de la Zonula Occludens-1 , Humanos , Melatonina/sangre , Masculino , Apnea Obstructiva del Sueño/sangre , Apnea Obstructiva del Sueño/fisiopatología , Apnea Obstructiva del Sueño/complicaciones , Persona de Mediana Edad , Proteína C-Reactiva/análisis , Proteína C-Reactiva/metabolismo , Inflamación/sangre , Adulto , Proteína de la Zonula Occludens-1/metabolismo , Proteína de la Zonula Occludens-1/sangre , Biomarcadores/sangre , Mucosa Intestinal/metabolismo , Índice de Severidad de la Enfermedad , LipopolisacáridosRESUMEN
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.
Asunto(s)
Hipoxia de la Célula , Proteínas de Uniones Estrechas , Uniones Estrechas , Urotelio , Humanos , Urotelio/metabolismo , Urotelio/patología , Uniones Estrechas/metabolismo , Proteínas de Uniones Estrechas/metabolismo , Proteínas de Uniones Estrechas/genética , Línea Celular , Proteína de la Zonula Occludens-1/metabolismo , Proteína de la Zonula Occludens-1/genética , Ocludina/metabolismo , Ocludina/genética , Claudina-1/metabolismo , Claudina-1/genética , Impedancia Eléctrica , Regulación de la Expresión GénicaRESUMEN
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.
Asunto(s)
Mucosa Intestinal , Péptidos , Tenebrio , Animales , Ratones , Péptidos/farmacología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efectos de la radiación , Mucosa Intestinal/efectos de los fármacos , Masculino , Sistema Hematopoyético/efectos de los fármacos , Sistema Hematopoyético/efectos de la radiación , Protectores contra Radiación/farmacología , Proteína de la Zonula Occludens-1/metabolismo , Proteína de la Zonula Occludens-1/genética , Rayos gamma/efectos adversos , Ocludina/metabolismo , Ocludina/genética , Intestinos/efectos de los fármacos , Intestinos/efectos de la radiaciónRESUMEN
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.
Asunto(s)
Colecalciferol , Defensinas , Suplementos Dietéticos , Microbioma Gastrointestinal , Oligosacáridos , Animales , Femenino , Masculino , Ratones , Colecalciferol/farmacología , Colon/metabolismo , Colon/efectos de los fármacos , Defensinas/metabolismo , Defensinas/genética , Regulación hacia Abajo/efectos de los fármacos , Microbioma Gastrointestinal/efectos de los fármacos , Íleon/metabolismo , Íleon/efectos de los fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efectos de los fármacos , Ratones Endogámicos C57BL , Ocludina/metabolismo , Ocludina/genética , Oligosacáridos/farmacología , Oligosacáridos/administración & dosificación , Células de Paneth/metabolismo , Células de Paneth/efectos de los fármacos , Receptores de Calcitriol/metabolismo , Receptores de Calcitriol/genética , Proteína de la Zonula Occludens-1/metabolismo , Proteína de la Zonula Occludens-1/genéticaRESUMEN
Epithelial-Mesenchymal Transition (EMT) of retinal pigment epithelial (RPE) cells is recognized as pivotal in various retinal diseases. Previous studies have suggested a reciprocal regulation between reactive oxygen species (ROS) and EMT, though the involvement of peroxidized lipids or the effects of reducing them has remained unclear. The present study disclosed that EMT of ARPE-19 cells induced by TGF-ß2 and TNF-α involves increased lipid peroxidation, and Ferrostatin-1 (Fer-1), a lipophilic antioxidative agent, successfully inhibited the increase in lipid peroxidation. Fer-1 suppressed the formation of EMT-associated fibrotic deposits, while EMT induction or Fer-1 treatment did not influence the cell viability or proliferation. Functionally, Fer-1 impeded EMT-driven cell migration and reduction in transepithelial electrical resistance. It demonstrated regulatory prowess by downregulating the mesenchymal marker fibronectin, upregulating the epithelial marker ZO-1, and inhibiting the EMT-associated transcriptional factor ZEB1. Additionally, VEGF, a major pathogenic cytokine in various retinal diseases, is also upregulated during EMT, and Fer-1 significantly mitigated the effect. The present study disclosed the involvement of lipid peroxidation in EMT of RPE cells, and suggests the suppression of lipid peroxidation may be a potential therapeutic target in retinal diseases in which EMT is implicated.
Asunto(s)
Transición Epitelial-Mesenquimal , Peroxidación de Lípido , Epitelio Pigmentado de la Retina , Transición Epitelial-Mesenquimal/efectos de los fármacos , Humanos , Epitelio Pigmentado de la Retina/metabolismo , Línea Celular , Movimiento Celular/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor de Crecimiento Transformador beta2/metabolismo , Células Epiteliales/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Supervivencia Celular/efectos de los fármacos , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Proliferación Celular , Proteína de la Zonula Occludens-1/metabolismo , Fibronectinas/metabolismoRESUMEN
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.
Asunto(s)
Barrera Hematotesticular , Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Células de Sertoli , Testículo , Animales , Masculino , Porcinos , Virus del Síndrome Respiratorio y Reproductivo Porcino/patogenicidad , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Síndrome Respiratorio y de la Reproducción Porcina/virología , Síndrome Respiratorio y de la Reproducción Porcina/patología , Células de Sertoli/virología , Células de Sertoli/metabolismo , Barrera Hematotesticular/virología , Testículo/virología , Testículo/patología , Espermatogonias/virología , Apoptosis , Células Intersticiales del Testículo/virología , Citocinas/metabolismo , Testosterona/sangre , Proteína de la Zonula Occludens-1/metabolismo , Proteína de la Zonula Occludens-1/genéticaRESUMEN
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.
Asunto(s)
Enterocolitis Necrotizante , Metaloproteinasa 3 de la Matriz , Receptor PAR-2 , Transducción de Señal , Animales , Humanos , Ratones , Animales Recién Nacidos , Línea Celular , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Enterocolitis Necrotizante/patología , Enterocolitis Necrotizante/tratamiento farmacológico , Enterocolitis Necrotizante/metabolismo , Homoserina/análogos & derivados , Homoserina/farmacología , Mucosa Intestinal/patología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/inmunología , Intestinos/patología , Intestinos/efectos de los fármacos , Lactonas/farmacología , Lipopolisacáridos , Metaloproteinasa 3 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/genética , Ratones Endogámicos C57BL , Ocludina/metabolismo , Ocludina/genética , Receptor PAR-2/metabolismo , Receptor PAR-2/genética , Transducción de Señal/efectos de los fármacos , Proteína de la Zonula Occludens-1/metabolismo , Proteína de la Zonula Occludens-1/genéticaRESUMEN
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.
Asunto(s)
Barrera Hematotesticular , FN-kappa B , Transducción de Señal , FN-kappa B/metabolismo , Barrera Hematotesticular/metabolismo , Masculino , Animales , Línea Celular , Ratones , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Proteína de la Zonula Occludens-1/metabolismo , Proteína de la Zonula Occludens-1/genética , Ocludina/metabolismo , Ocludina/genética , Proteínas de Uniones Estrechas/metabolismo , Proteínas de Uniones Estrechas/genética , Células de Sertoli/metabolismoRESUMEN
Background and Objectives: Irritable Bowel Syndrome (IBS) is a common gastrointestinal disorder often exacerbated by stress, influencing the brain-gut axis (BGA). BGA dysregulation, disrupted intestinal barrier function, altered visceral sensitivity and immune imbalance defects underlying IBS pathogenesis have been emphasized in recent investigations. Phosphoproteomics reveals unique phosphorylation details resulting from environmental stress. Here, we employ phosphoproteomics to explore the molecular mechanisms underlying IBS-like symptoms, mainly focusing on the role of ZO-1 and IL-1RAP phosphorylation. Materials and Methods: Morris water maze (MWM) was used to evaluate memory function for single prolonged stress (SPS). To assess visceral hypersensitivity of IBS-like symptoms, use the Abdominal withdrawal reflex (AWR). Colonic bead expulsion and defecation were used to determine fecal characteristics of the IBS-like symptoms. Then, we applied a phosphoproteomic approach to BGA research to discover the molecular mechanisms underlying the process of visceral hypersensitivity in IBS-like mice following SPS. ZO-1, p-S179-ZO1, IL-1RAP, p-S566-IL-1RAP and GFAP levels in BGA were measured by western blotting, immunofluorescence staining, and enzyme-linked immunosorbent assay to validate phosphorylation quantification. Fluorescein isothiocyanate-dextran 4000 and electron-microscopy were performed to observe the structure and function of the intestinal epithelial barrier. Results: The SPS group showed changes in learning and memory ability. SPS exposure affects visceral hypersensitivity, increased fecal water content, and significant diarrheal symptoms. Phosphoproteomic analysis displayed that p-S179-ZO1 and p-S566-IL-1RAP were significantly differentially expressed following SPS. In addition, p-S179-ZO1 was reduced in mice's DRG, colon, small intestine, spinal and hippocampus and intestinal epithelial permeability was increased. GFAP, IL-1ß and p-S566-IL-1RAP were also increased at the same levels in the BGA. And IL-1ß showed no significant difference was observed in serum. Our findings reveal substantial alterations in ZO-1 and IL-1RAP phosphorylation, correlating with increased epithelial permeability and immune imbalance. Conclusions: Overall, decreased p-S179-ZO1 and increased p-S566-IL-1RAP on the BGA result in changes to tight junction structure, compromising the structure and function of the intestinal epithelial barrier and exacerbating immune imbalance in IBS-like stressed mice.
Asunto(s)
Eje Cerebro-Intestino , Proteína Accesoria del Receptor de Interleucina-1 , Síndrome del Colon Irritable , Proteína de la Zonula Occludens-1 , Animales , Humanos , Masculino , Ratones , Modelos Animales de Enfermedad , Proteína Accesoria del Receptor de Interleucina-1/metabolismo , Síndrome del Colon Irritable/metabolismo , Síndrome del Colon Irritable/patología , Ratones Endogámicos C57BL , Fosforilación , Estrés Psicológico/metabolismo , Estrés Psicológico/inmunología , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
Podocyte injury or dysfunction can lead to proteinuria and glomerulosclerosis. Zonula occludens 1 (ZO-1) is a tight junction protein which connects slit diaphragm (SD) proteins to the actin cytoskeleton. Previous studies have shown that the expression of ZO-1 is decreased in chronic kidney disease (CKD). Thus, elucidation of the regulation mechanism of ZO-1 has considerable clinical importance. Triptolide (TP) has been reported to exert a strong antiproteinuric effect by inhibiting podocyte epithelial mesenchymal transition (EMT) and inflammatory response. However, the underlying mechanisms are still unclear. We found that TP upregulates ZO-1 expression and increases the fluorescence intensity of ZO-1 in a puromycin aminonucleoside (PAN)-induced podocyte injury model. Permeablity assay showed TP decreases podocyte permeability in PAN-treated podocyte. TP also upregulates the DNA demethylase TET2. Our results showed that treatment with the DNA methyltransferase inhibitors 5-azacytidine (5-AzaC) and RG108 significantly increased ZO-1 expression in PAN-treated podocytes. Methylated DNA immunoprecipitation (MeDIP) and hydroxymethylated DNA immunoprecipitation (hMeDIP) results showed that TP regulates the methylation status of the ZO-1 promoter. Knockdown of TET2 decreased ZO-1 expression and increased methylation of its promoter, resulting in the increase of podocyte permeability. Altogether, these results indicate that TP upregulates the expression of ZO-1 and decreases podocyte permeability through TET2-mediated 5 mC demethylation. These findings suggest that TP may alleviate podocyte permeability through TET2-mediated hydroxymethylation of ZO-1.
Asunto(s)
Dioxigenasas , Diterpenos , Compuestos Epoxi , Fenantrenos , Podocitos , Proteína de la Zonula Occludens-1 , Podocitos/metabolismo , Podocitos/efectos de los fármacos , Podocitos/patología , Proteína de la Zonula Occludens-1/metabolismo , Fenantrenos/farmacología , Diterpenos/farmacología , Compuestos Epoxi/farmacología , Dioxigenasas/metabolismo , Animales , Proteínas de Unión al ADN/metabolismo , Ratones , Proteínas Proto-Oncogénicas/metabolismo , Permeabilidad/efectos de los fármacos , Humanos , Metilación de ADN/efectos de los fármacosRESUMEN
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.
Asunto(s)
Colitis Ulcerosa , Factor Neurotrófico Derivado de la Línea Celular Glial , Mucosa Intestinal , Neuroglía , Proteína de la Zonula Occludens-1 , Animales , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/metabolismo , Ratones , Humanos , Proteína de la Zonula Occludens-1/metabolismo , Proteína de la Zonula Occludens-1/genética , Masculino , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efectos de los fármacos , Membrana Celular/metabolismo , Membrana Celular/efectos de los fármacos , Proteínas Proto-Oncogénicas c-ret/metabolismo , Proteínas Proto-Oncogénicas c-ret/genética , Ratones Endogámicos C57BL , Cumarinas/farmacología , Cumarinas/química , Transducción de Señal/efectos de los fármacos , Glycyrrhiza/químicaRESUMEN
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.
Asunto(s)
Mucosa Intestinal , Estrés Oxidativo , Permeabilidad , Extractos Vegetales , Rubus , Uniones Estrechas , Factor de Necrosis Tumoral alfa , Humanos , Rubus/química , Células CACO-2 , Estrés Oxidativo/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efectos de los fármacos , Extractos Vegetales/farmacología , Permeabilidad/efectos de los fármacos , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Ocludina/metabolismo , Ocludina/genética , Claudina-1/metabolismo , Claudina-1/genética , Proteína de la Zonula Occludens-1/metabolismo , Proteína de la Zonula Occludens-1/genética , Interferón gamma/metabolismo , Frutas/químicaRESUMEN
Obstructive sleep apnea (OSA) can lead to intestinal injury, endotoxemia, and disturbance of intestinal flora. Additionally, as a crucial component of the endocannabinoid system, some studies have demonstrated that cannabinoid 1 (CB1) receptors are closely linked to the multiple organ dysfunction triggered by OSA. However, the role of the CB1 receptor in alleviating OSA-induced colon injury remains unclear. Here, through the construction of the OSA classic model, we found that the colon tissue of chronic intermittent hypoxia (CIH)-induced mice exhibited an overexpression of the CB1 receptor. The results of hematoxylin-eosin staining and transmission electron microscopy revealed that inhibition of the CB1 receptor could decrease the gap between the mucosa and muscularis mucosae, alleviate mitochondrial swelling, reduce microvilli shedding, and promote the recovery of tight junctions of CIH-induced mice. Furthermore, CB1 receptor inhibition reduced the levels of metabolic endotoxemia and inflammatory responses, exhibiting significant protective effects on the colon injury caused by CIH. At the molecular level, through western blotting and real-time polymerase chain reaction techniques, we found that inhibiting the CB1 receptor can significantly increase the expression of ZO-1 and Occludin proteins, which are closely related to the maintenance of intestinal mucosal barrier function. Through 16S rRNA high-throughput sequencing and short-chain fatty acid (SCFA) determination, we found that inhibition of the CB1 receptor increased the diversity of the microbial flora and controlled the makeup of intestinal flora. Moreover, butyric acid concentration and the amount of SCFA-producing bacteria, such as Ruminococcaceae and Lachnospiraceae, were both markedly elevated by CB1 receptor inhibition. The results of the spearman correlation study indicated that Lachnospiraceae showed a positive association with both ZO-1 and Occludin but was negatively correlated with the colon CB1 receptor, IL-1ß, and TNF-α. According to this study, we found that inhibiting CB1 receptor can improve CIH-induced colon injury by regulating gut microbiota, reducing mucosal damage and promoting tight junction recovery. KEY POINTS: â¢CIH leads to overexpression of CB1 receptor in colon tissue. â¢CIH causes intestinal flora disorder, intestinal mucosal damage, and disruption of tight junctions. â¢Inhibition of CB1 receptor can alleviate the colon injury caused by CIH through regulating the gut microbiota, reducing mucosal injury, and promoting tight junction recovery.
Asunto(s)
Colon , Modelos Animales de Enfermedad , Mucosa Intestinal , Receptor Cannabinoide CB1 , Animales , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB1/genética , Ratones , Colon/patología , Colon/microbiología , Colon/metabolismo , Masculino , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Hipoxia/metabolismo , Ratones Endogámicos C57BL , Proteína de la Zonula Occludens-1/metabolismo , Ocludina/metabolismo , Ocludina/genética , Microbioma Gastrointestinal , Uniones Estrechas/metabolismoRESUMEN
Matrine (MT) possesses anti-inflammatory, anti-allergic and antioxidative properties. However, the impact and underlying mechanisms of matrine on colitis are unclear. The purpose of this research was to examine the protective impact and regulatory mechanism of matrine on dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) in mice. MT alleviated DSS-induced UC by inhibiting weight loss, relieving colon shortening and reducing the disease activity index (DAI). Moreover, DSS-induced intestinal injury and the number of goblet cells were reversed by MT, as were alterations in the expression of zonula occludens-1 (ZO-1) and occludin in colon. Simultaneously, matrine not only effectively restored DSS-induced oxidative stress in colonic tissues but also reduced the production of inflammatory cytokines. Furthermore, MT could treat colitis mice by regulating the regulatory T cell (Treg)/T helper 17 (Th17) cell imbalance. We observed further evidence that MT alleviated the decrease in intestinal flora diversity, reduced the proportion of Firmicutes and Bacteroidetes, decreased the proportion of Proteobacteria and increased the relative abundance of Lactobacillus and Akkermansia in colitis mice. In conclusion, these results suggest that MT may mitigate DSS-induced colitis by enhancing the colon barrier integrity, reducing the Treg/Th17 cell imbalance, inhibiting intestinal inflammation, modulating oxidative stress and regulating the gut microbiota. These findings provide strong evidence for the development and application of MT as a dietary treatment for UC.
Asunto(s)
Alcaloides , Sulfato de Dextran , Microbioma Gastrointestinal , Matrinas , Estrés Oxidativo , Quinolizinas , Linfocitos T Reguladores , Animales , Alcaloides/farmacología , Microbioma Gastrointestinal/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Quinolizinas/farmacología , Quinolizinas/uso terapéutico , Ratones , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Masculino , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Colitis/metabolismo , Colitis/microbiología , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Inflamación/patología , Proteína de la Zonula Occludens-1/metabolismo , Colon/patología , Colon/metabolismo , Colon/efectos de los fármacos , Colon/microbiología , Células Th17/efectos de los fármacos , Células Th17/metabolismo , Células Th17/inmunología , Modelos Animales de Enfermedad , Citocinas/metabolismo , Ratones Endogámicos C57BL , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/microbiología , Colitis Ulcerosa/metabolismo , Colitis Ulcerosa/patología , Ocludina/metabolismoRESUMEN
The COVID-19 pandemic caused by SARS-CoV-2 has highlighted the urgent need for innovative antiviral strategies to fight viral infections. Although a substantial part of the overall effort has been directed at the Spike protein to create an effective global vaccination strategy, other proteins have also been examined and identified as possible therapeutic targets. Among them, although initially underestimated, there is the SARS-CoV-2 E-protein, which turned out to be a key factor in viral pathogenesis due to its role in virus budding, assembly and spreading. The C-terminus of E-protein contains a PDZ-binding motif (PBM) that plays a key role in SARS-CoV-2 virulence as it is recognized and bound by the PDZ2 domain of the human tight junction protein ZO-1. The binding between the PDZ2 domain of ZO-1 and the C-terminal portion of SARS-CoV-2 E-protein has been extensively characterized. Our results prompted us to develop a possible adjuvant therapeutic strategy aimed at slowing down or inhibiting virus-mediated pathogenesis. Such innovation consists in the design and synthesis of externally PDZ2-ZO1 functionalized PLGA-based nanoparticles to be used as intracellular decoy. Contrary to conventional strategies, this innovative approach aims to capitalize on the E protein-PDZ2 interaction to prevent virus assembly and replication. In fact, the conjugation of the PDZ2 domain to polymeric nanoparticles increases the affinity toward the E protein effectively creating a "molecular sponge" able to sequester E proteins within the intracellular environment of infected cells. Our in vitro studies on selected cellular models, show that these nanodevices significantly reduce SARS-CoV-2-mediated virulence, emphasizing the importance of exploiting viral-host interactions for therapeutic benefit.
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Nanopartículas , Dominios PDZ , SARS-CoV-2 , Humanos , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/metabolismo , Nanopartículas/química , COVID-19/virología , COVID-19/metabolismo , Proteína de la Zonula Occludens-1/metabolismo , Proteínas de la Envoltura de Coronavirus/metabolismo , Proteínas de la Envoltura de Coronavirus/química , Antivirales/farmacología , Antivirales/química , Tratamiento Farmacológico de COVID-19 , Animales , Unión ProteicaRESUMEN
Decrease of human corneal endothelial cell (CEC) density leads to corneal edema, progressive corneal opacity, and reduced visual acuity. A reduction in CEC density may be related to elevated levels of inflammatory cytokines, such as tumor necrosis factor (TNF)-α and interferon (INF)-γ. PANoptosis, characterized by the activation of apoptosis, necroptosis, and pyroptosis, could be a factor in the loss of CECs driven by TNF-α and INF-γ. Cytokines also stimulate monocytes adhesion to endothelium. It has been shown in previous research that curcumin plays protective roles against numerous corneal inflammatory diseases. However, it is not determined whether curcumin acts as an anti-PANoptotic agent or if it mitigates monocyte adhesion to CECs. Therefore, this research aimed to explor the potential therapeutic effects of curcumin and its underlying mechanisms in the loss of CECs. CEC injury models were established, and curcumin was injected subconjunctivally. Clinical evaluation of the corneas was conducted using a scoring system and anterior segment photography. Corneal observation was performed with hematoxylin and eosin staining and immunostaining of zona occludens-1(ZO-1). Apoptotic cells within the corneal endothelium were observed using TUNEL staining. The detection of primary proteins expression was accomplished through Western blot analysis. Interleukin (IL)-1ß and macrophage chemotactic protein 1 (MCP-1) levels were determined via ELISA, while the expression of cleaved caspase-3, gasdermin-D (GSDMD), phosphor-mixed lineage kinase domain-like protein (p-MLKL) and intercellular cell adhesion molecule-1 were confirmed by immunofluorescence. Myeloperoxidase (MPO) activity was measured in aqueous humors. Curcumin treatment attenuated the loss of CECs and corneal edema caused by TNF-α and IFN-γ. Besides, it decreased the count of TUNEL-positive cells, and inhibited the upregulation of cleaved caspase-3, cleaved caspase-6, cleaved caspase-7, and cleaved poly (ADP-ribose) polymerase. Moreover, both the expression and phosphorylation of MLKL and receptor-interacting protein 3 were decreased in curcumin-treated rats. Furthermore, curcumin also lowered the expression of cleaved caspase-1, diminished the levels of IL1ß and MCP-1, and inhibited the activity of MPO. Besides, the expression of intercellular cell adhesion molecule-1, vascular cell adhesion molecule-1, as well as the number of CD11b-positive cells adhered to the CECs decreased for the administration of curcumin.
Asunto(s)
Adhesión Celular , Curcumina , Modelos Animales de Enfermedad , Endotelio Corneal , Interferón gamma , Monocitos , Ratas Sprague-Dawley , Factor de Necrosis Tumoral alfa , Curcumina/farmacología , Endotelio Corneal/efectos de los fármacos , Endotelio Corneal/patología , Endotelio Corneal/metabolismo , Ratas , Animales , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Interferón gamma/metabolismo , Adhesión Celular/efectos de los fármacos , Masculino , Necroptosis/efectos de los fármacos , Proteína de la Zonula Occludens-1/metabolismo , Western BlottingRESUMEN
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.