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1.
Int J Mol Sci ; 22(19)2021 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-34638666

RESUMO

Once weak ultraviolet ray-B (UVB) irradiates the skin cells, the generation of reactive nitrogen species (RNS), but not reactive oxygen species (ROS), is stimulated for the mislocalization of claudin-1 (CLDN1), an essential protein for forming tight junctions (TJs). Since our skin is constantly exposed to sunlight throughout our lives, an effective protection strategy is needed to maintain the skin barrier against weak UVB. In the present study, we investigated whether an ethanol extract of Brazilian green propolis (EBGP) and flavonoids had a protective effect against weak UVB irradiation-induced barrier dysfunction in human keratinocyte-derived HaCaT cells. A pretreatment with EBGP suppressed TJ permeability, RNS production, and the nitration level of CLDN1 in the weak UVB-exposed cells. Among the propolis components, apigenin and apigenin-like flavonoids have potent protective effects against NO production and the mislocalization of CLDN1 induced by UVB. The analyses between structures and biological function revealed that the chemically and structurally characteristic flavonoids with a hydroxyl group at the 4' position on the B-ring might contribute to its protective effect on barrier dysfunction caused by weak UVB irradiation. In conclusion, EBGP and its component apigenin protect HaCaT cells from weak UVB irradiation-induced TJ barrier dysfunction mediated by suppressing NO production.


Assuntos
Apigenina/farmacologia , Claudina-1/metabolismo , Óxido Nítrico/metabolismo , Própole/farmacologia , Substâncias Protetoras/farmacologia , Brasil , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Etanol/química , Células HaCaT , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Extratos Vegetais/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Raios Ultravioleta
2.
Cells ; 10(9)2021 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-34571860

RESUMO

Identifying molecular characteristics that are associated with aggressive cancer phenotypes through gene expression profiling can help predict treatment responses and clinical outcomes. Claudins are deregulated in colorectal cancer (CRC). In CRC, increased claudin-1 expression results in epithelial-to-mesenchymal transition and metastasis, while claudin-7 functions as a tumor suppressor. In this study, we have developed a molecular signature based on claudin-1 and claudin-7 associated with poor patient survival and chemoresistance. This signature was validated using an integrated approach including publicly available datasets and CRC samples from patients who either responded or did not respond to standard-of-care treatment, CRC cell lines, and patient-derived rectal and colon tumoroids. Transcriptomic analysis from a patient dataset initially yielded 23 genes that were differentially expressed along with higher claudin-1 and decreased claudin-7. From this analysis, we selected a claudins-associated molecular signature including PIK3CA, SLC6A6, TMEM43, and ASAP-1 based on their importance in CRC. The upregulation of these genes and their protein products was validated using multiple CRC patient datasets, in vitro chemoresistant cell lines, and patient-derived tumoroid models. Additionally, blocking these genes improved 5-FU sensitivity in chemoresistant CRC cells. Our findings propose a new claudin-based molecular signature that associates with poor prognosis as well as characteristics of treatment-resistant CRC including chemoresistance, metastasis, and relapse.


Assuntos
Claudina-1/genética , Claudinas/genética , Resistencia a Medicamentos Antineoplásicos/genética , Animais , Linhagem Celular Tumoral , Claudina-1/metabolismo , Claudinas/metabolismo , Claudinas/fisiologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Transição Epitelial-Mesenquimal/genética , Feminino , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Genes Supressores de Tumor , Humanos , Masculino , Camundongos , Recidiva Local de Neoplasia/genética , Fatores de Risco , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Zhonghua Yi Xue Za Zhi ; 101(28): 2233-2237, 2021 Jul 27.
Artigo em Chinês | MEDLINE | ID: mdl-34333937

RESUMO

Objective: To explore the diagnostic cut-off value of Claudin-1 (Cla-1) in papillary thyroid carcinoma (PTC) and the efficacy of the multiple test. Methods: Clinical and pathological data were collected from 295 patients with PTC and 137 patients with benign thyroid lesions (BTL) admitted to two hospitals in Pingdingshan City, Henan Province from January 2015 to June 2020. The expression index of Cla-1, the positive expression rate of high molecular weight cytokeratin (HMW-CK) and the negative expression rate of leukocyte differentiation antigen 56 (CD56) were calculated according to the results of immunohistochemistry. Receiver operating characteristic (ROC) curve was used to determine the cut-off value of Cla-1 for diagnosing PTC. Cla-1+HMW-CK, Cla-1+CD56, HMW-CK+CD56 and Cla-1+HMW-CK+CD56 were tested in series, and the diagnostic efficacy was evaluated by sensitivity, specificity and accuracy. Results: The age of PTC patients M (P25, P75) was 48.0 (41.0, 55.0) years old, among which 230 cases were male (78.0%). The age of BTL patients M (P25, P75) was 53.0 (46.0, 61.0) years old, including 34 males (24.8%). Cla-1 detection was conducted in all PTC and BTL patients, whereas only 198 and 97 cases conducted HMW-CK detection, 242 and 136 cases conducted CD56 detection, respectively. The M (P25, P75) of Cla-1 expression index in PTC was 80.0% (45.0%, 90.0%), which was higher than that in BTL [0.0 (0.0, 2.0%)] (P<0.001). The positive expression rate of HMW-CK was 93.9% (186/198) in PTC, and was negative in all BTL. The negative expression rate of D56 was 89.7% (217/242) in PTC, higher than that of BTL [1.5% (2/136)] (P<0.001). When expression index 19.0% was set as the cut-off value of Cla-1 for diagnosing PTC, the area under the ROC curve (AUC) was the maximum [AUC (95%CI): 0.981 (0.969, 0.994)], and the sensitivity, specificity and accuracy were 92.2% (272/295), 98.5% (135/137) and 94.2% (407/432), respectively. In multiple tests for diagnosis of PTC, the sensitivity ranged from 89.3% to 94.7%, specificity were all 100.0% and accuracy ranged from 93.1% to 96.9%. Conclusion: Cla-1 expression index ≥19.0% could be set as a cut-off value for the diagnosis of PTC and serial test of Cla-1 combined with HMW-CK(CD56) improves diagnostic specificity.


Assuntos
Carcinoma Papilar , Neoplasias da Glândula Tireoide , Biomarcadores Tumorais , Carcinoma Papilar/diagnóstico , Claudina-1 , Humanos , Masculino , Pessoa de Meia-Idade , Câncer Papilífero da Tireoide , Neoplasias da Glândula Tireoide/diagnóstico
4.
Int J Mol Sci ; 22(16)2021 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-34445450

RESUMO

Tumor necrosis factor alpha (TNFα) has been shown to impair the intestinal barrier, inducing and maintaining inflammatory states of the intestine. The aim of the current study was to analyze functional, molecular and regulatory effects of TNFα in a newly established non-transformed jejunal enterocyte model, namely IPEC-J2 monolayers. Incubation with 1000 U/mL TNFα induced a marked decrease in transepithelial electrical resistance (TEER), and an increase in permeability for the paracellular flux marker [3H]-D-mannitol compared to controls. Immunoblots revealed a significant decrease in tight junction (TJ) proteins occludin, claudin-1 and claudin-3. Moreover, a dose-dependent increase in the TNF receptor (TNFR)-1 was detected, explaining the exponential nature of pro-inflammatory effects, while TNFR-2 remained unchanged. Recovery experiments revealed reversible effects after the removal of the cytokine, excluding apoptosis as a reason for the observed changes. Furthermore, TNFα signaling could be inhibited by the specific myosin light chain kinase (MLCK) blocker ML-7. Results of confocal laser scanning immunofluorescence microscopy were in accordance with all quantitative changes. This study explains the self-enhancing effects of TNFα mediated by MLCK, leading to a differential regulation of TJ proteins resulting in barrier impairment in the intestinal epithelium.


Assuntos
Mucosa Intestinal/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Proteínas de Junções Íntimas/genética , Junções Íntimas , Fator de Necrose Tumoral alfa/metabolismo , Animais , Linhagem Celular , Claudina-1/genética , Claudina-3/genética , Regulação da Expressão Gênica , Mucosa Intestinal/fisiologia , Jejuno/metabolismo , Jejuno/fisiologia , Manitol/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Ocludina/genética , Permeabilidade , Transdução de Sinais , Sus scrofa/metabolismo , Sus scrofa/fisiologia , Fator de Necrose Tumoral alfa/farmacologia
5.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445491

RESUMO

In this study we assessed the effects of antigen exposure in mice pre-sensitized with allergen following viral infection on changes in lung function, cellular responses and tight junction expression. Female BALB/c mice were sensitized to ovalbumin and infected with influenza A before receiving a second ovalbumin sensitization and challenge with saline, ovalbumin (OVA) or house dust mite (HDM). Fifteen days post-infection, bronchoalveolar inflammation, serum antibodies, responsiveness to methacholine and barrier integrity were assessed. There was no effect of infection alone on bronchoalveolar lavage cellular inflammation 15 days post-infection; however, OVA or HDM challenge resulted in increased bronchoalveolar inflammation dominated by eosinophils/neutrophils or neutrophils, respectively. Previously infected mice had higher serum OVA-specific IgE compared with uninfected mice. Mice previously infected, sensitized and challenged with OVA were most responsive to methacholine with respect to airway resistance, while HDM challenge caused significant increases in both tissue damping and tissue elastance regardless of previous infection status. Previous influenza infection was associated with decreased claudin-1 expression in all groups and decreased occludin expression in OVA or HDM-challenged mice. This study demonstrates the importance of the respiratory epithelium in pre-sensitized individuals, where influenza-infection-induced barrier disruption resulted in increased systemic OVA sensitization and downstream effects on lung function.


Assuntos
Hiper-Reatividade Brônquica/tratamento farmacológico , Cloreto de Metacolina/administração & dosagem , Infecções por Orthomyxoviridae/complicações , Ovalbumina/imunologia , Pyroglyphidae/imunologia , Resistência das Vias Respiratórias/efeitos dos fármacos , Animais , Hiper-Reatividade Brônquica/etiologia , Claudina-1/metabolismo , Regulação para Baixo , Feminino , Vírus da Influenza A/patogenicidade , Cloreto de Metacolina/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/imunologia , Ovalbumina/administração & dosagem , Resultado do Tratamento
6.
Biochem Biophys Res Commun ; 565: 36-42, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34090208

RESUMO

Aberrant expression of tight junction proteins has recently been focused on in the cancer research field. We previously showed that claudin-1 is aberrantly expressed from an early stage of uterine cervical adenocarcinoma and contributes to malignant potentials. To elucidate the molecular mechanisms underlying tumor-promoting roles of claudin-1, we established and analyzed claudin-1 knockout cells. Knockout of claudin-1 suppressed conventional tight junctional functions, barrier and fence functions, and expression of cell adhesion-associated proteins including E-cadherin. Comparative proteome analysis revealed that expression of claudin-1 affected expression of a wide range of proteins, especially proteins that are associated with cell adhesion and actin cytoskeleton remodeling. Interactome analysis of the identified proteins revealed that E-cadherin and focal adhesion kinase play central roles in the claudin-1-dependently affected protein network. Moreover, knockout of claudin-1 significantly suppressed microvilli formation and activity of Ezrin/Radixin/Moesin. Taken together, the results indicate that expression of claudin-1 affects not only conventional tight junction function but also expression and activity of a wide range of proteins, especially proteins that are associated with cell adhesion and actin cytoskeleton remodeling, to contribute to malignant potentials and microvilli formation in cervical adenocarcinoma cells.


Assuntos
Claudina-1/metabolismo , Microvilosidades/metabolismo , Adesão Celular , Claudina-1/deficiência , Claudina-1/genética , Humanos , Células Tumorais Cultivadas
7.
Microb Pathog ; 157: 105012, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34062228

RESUMO

Classical swine fever (CSF) is one of the most epidemic viral diseases in swine industry. The causative pathogen is CSF virus (CSFV), a small enveloped RNA virus of Flaviviridae family. Claudin-1 was reported to be involved in the infections of a number of viruses, including many from Flaviviridae family, but no studies have investigated the role of porcine claudin-1 during CSFV infection in PK-15 cells. In this study, on the one hand, we demonstrated that CSFV infection reduced the claudin-1 expression at both mRNA and protein levels; on the other hand, CSFV infection was enhanced after claudin-1 knockdown, but inhibited by claudin-1 overexpression in a dose-dependent manner. Furthermore, negative correlation was demonstrated between the claudin-1 expression and CSFV titer. In conclusion, claudin-1 might be a barrier for CSFV infection in PK-15 cells, while CSFV bypasses the barrier through lysosome mediated degradation of claudin-1, which could be repressed by bafilomycin A1. Although the elaborate mechanisms how claudin-1 plays its roles in CSFV infection require further investigations, this study may advance our understanding of the molecular host-pathogen interaction mechanisms underlying CSFV infection and suggests enhancement of porcine claudin-1 as a potential preventive or therapeutic strategy for CSF control.


Assuntos
Vírus da Febre Suína Clássica , Peste Suína Clássica , Animais , Linhagem Celular , Claudina-1/genética , Suínos , Replicação Viral
8.
Clinics (Sao Paulo) ; 76: e2167, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34008771

RESUMO

OBJECTIVES: Leukoaraiosis is described as white matter lesions that are associated with cognitive dysfunction, neurodegenerative disorders, etc. Myelin depletion is a salient pathological feature of, and the loss of oligodendrocytes is one of the most robust alterations evident in, white matter degeneration. Recent studies have revealed that claudin proteins are aberrantly expressed in leukoaraiosis and regulate oligodendrocyte activity. However, the roles of claudin-1 and claudin-3 in oligodendrocytes and leukoaraiosis are still not well-defined. METHODS: Quantitative polymerase chain reaction was used to measure the expression of claudin-1 (CLDN1), claudin-3 (CLDN3), and myelinogenesis-related genes such as myelin basic protein (MBP), proteolipid protein (PLP), oligodendrocyte transcription factor 2 (OLIG2), and SRY-box transcription factor 10 (SOX10) in leukoaraiosis patients (n=122) and healthy controls (n=122). The expression of claudin-1 and claudin-3 was either ectopically silenced or augmented in Oli-neu oligodendrocytes, and colony formation, apoptosis, and migration assays were performed. Finally, the expression of myelin proteins was evaluated by western blotting. RESULTS: Our results revealed that in addition to SOX10, the expression levels of claudin-1, claudin-3, and myelinogenesis-related proteins were prominently downregulated in leukoaraiosis patients, compared to those in healthy controls. Furthermore, the growth and migration of Oli-neu cells were downregulated upon silencing claudin-1 or claudin-3. However, the overexpression of claudin-1 or claudin-3 resulted in the reduction of the degree of apoptosis in Oli-neu cells. In addition, claudin-1 and claudin-3 promoted the expression of MBP, OLIG2, PLP, and SOX10 at the translational level. CONCLUSION: Our data has demonstrated that the abnormal expression of claudin-1 and claudin-3 regulates the pathological progression of leukoaraiosis by governing the viability and myelination of oligodendrocytes. These findings provide novel insights into the regulatory mechanisms underlying the roles of claudin-1 and claudin-3 in leukoaraiosis.


Assuntos
Leucoaraiose , Claudina-1 , Claudina-3/genética , Humanos , Bainha de Mielina , Oligodendroglia
9.
PLoS One ; 16(4): e0249814, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33852594

RESUMO

The blood-brain barrier (BBB) keeps pathogens and toxins out of the brain but also impedes the entry of pharmaceuticals. Human cerebral microvascular endothelial cells (hCMECs) and astrocytes are the main functional cell components of the BBB. Although available commercially as cryopreserved cells in suspension, improvements in their cryopreservation and distribution as cryopreserved monolayers could enhance BBB in vitro studies. Here, we examined the response to slow cooling and storage in liquid nitrogen of immortalized hCMEC/D3 cells and human primary astrocytes in suspension and in monolayers. HCMEC/D3 cells in suspension cryopreserved in 5% dimethyl sulfoxide (DMSO) and 95% fetal bovine serum or in 5% DMSO and 6% hydroxyethyl starch (HES) showed post-thaw membrane integrities above 90%, similar to unfrozen control. Cryopreservation did not affect the time-dependent ability of hCMEC/D3 cells to form tubes on Matrigel. Primary astrocytes in suspension cryopreserved in the presence of 5% DMSO and 6% HES had improved viability over those cryopreserved in 10% DMSO. Monolayers of single cultures or co-cultures of hCMEC/D3 cells and astrocytes on fibronectin-coated Rinzl coverslips retained membrane integrities and metabolic function, after freezing in 5% DMSO, 6% HES, and 2% chondroitin sulfate, that were comparable to those of unfrozen controls even after overnight incubation. Rinzl is better than glass or Thermanox as an underlying solid substrate for cryopreserving hCMEC/D3 monolayers. Cryopreserved hCMEC/D3 monolayers expressed the junction proteins ZO-1 and claudin-5 similar to their unfrozen counterparts. Hence, we describe improved cryopreservation protocols for hCMEC/D3 cells and astrocytes in suspension, and a novel protocol for the cryopreservation of monolayers of hCMEC/D3 cells and astrocytes as single cultures or co-cultures that could expand their distribution for research on disease modeling, drug screening, and targeted therapy pertaining to the BBB.


Assuntos
Astrócitos/citologia , Barreira Hematoencefálica/citologia , Criopreservação/métodos , Astrócitos/metabolismo , Células Cultivadas , Claudina-1/genética , Claudina-1/metabolismo , Dimetil Sulfóxido/química , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Microvasos/citologia , Nitrogênio/química , Soroalbumina Bovina/química , Amido/análogos & derivados , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismo
10.
J Am Soc Nephrol ; 32(6): 1355-1370, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33795425

RESUMO

BACKGROUND: The activation of NAD+-dependent deacetylase, Sirt1, by the administration of nicotinamide mononucleotide (NMN) ameliorates various aging-related diseases. METHODS: Diabetic db/db mice were treated with NMN transiently for 2 weeks and observed for effects on diabetic nephropathy (DN). RESULTS: At 14 weeks after the treatment period, NMN attenuated the increases in urinary albumin excretion in db/db mice without ameliorating hemoglobin A1c levels. Short-term NMN treatment mitigated mesangium expansion and foot process effacement, while ameliorating decreased Sirt1 expression and increased claudin-1 expression in the kidneys of db/db mice. This treatment also improved the decrease in the expression of H3K9me2 and DNMT1. Short-term NMN treatment also increased kidney concentrations of NAD+ and the expression of Sirt1 and nicotinamide phosphoribosyltransferase (Nampt), and it maintained nicotinamide mononucleotide adenyltransferase1 (Nmnat1) expression in the kidneys. In addition, survival rates improved after NMN treatment. CONCLUSIONS: Short-term NMN treatment in early-stage DN has remote renal protective effects through the upregulation of Sirt1 and activation of the NAD+ salvage pathway, both of which indicate NMN legacy effects on DN.


Assuntos
Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/metabolismo , NAD/metabolismo , Mononucleotídeo de Nicotinamida/uso terapêutico , Sirtuína 1/metabolismo , Albuminúria/etiologia , Albuminúria/urina , Animais , Claudina-1/metabolismo , Citocinas/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Nefropatias Diabéticas/complicações , Nefropatias Diabéticas/patologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Epigênese Genética/efeitos dos fármacos , Mesângio Glomerular/patologia , Hemoglobina A Glicada/metabolismo , Histonas/metabolismo , Masculino , Camundongos , Camundongos Knockout , Mononucleotídeo de Nicotinamida/administração & dosagem , Nicotinamida Fosforribosiltransferase/metabolismo , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Podócitos/patologia , Sirtuína 1/genética , Taxa de Sobrevida , Fatores de Tempo
11.
Oncol Rep ; 45(4)2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33649777

RESUMO

In human head and neck squamous cell carcinoma (HNSCC), the invasion and metastatic properties of cancer cells are promoted by junctional adhesion molecule­A (JAM­A) and claudin­1; these are epithelial tight junction molecules regulated by histone deacetylases (HDACs) and transcription factor p63. HDAC expression is reportedly upregulated in HNSCC, and HDAC inhibitors suppress cancer cell proliferation by initiating proliferative arrest or apoptosis. However, little is known of the anti­cancer mechanisms of HDAC inhibitors in HNSCC. Thus, in the present study, the HNSCC Detroit 562 cell line and primary cultured HNSCC cells were treated with HDAC inhibitors to investigate their effects in HNSCC. Higher expression of p63, HDAC1, JAM­A and claudin­1 was observed in HNSCC tissues compared with the adjacent dysplastic regions. In Detroit 562 cells, treatment with trichostatin A (TSA), an inhibitor of HDAC1 and 6, downregulated the expression of p63, JAM­A and claudin­1, and upregulated that of acetylated tubulin; conversely, p63 knockdown resulted in the downregulation of JAM­A and claudin­1. Collectively, inhibiting HDAC suppressed the migration and invasiveness of cancer cells. In addition, treatment with TSA suppressed cancer cell proliferation via G2/M arrest, as well as upregulating p21 and downregulating cyclin D1 expression. TSA also downregulated the expression of epidermal growth factor receptor (EGFR) and phospho­ERK1/2. p63 knockdown and treatment with an EGFR inhibitor induced G1 arrest and downregulated EGFR and phospho­ERK1/2 levels, respectively. HDAC inhibition also suppressed the migration and invasiveness of primary cultured HNSCC cells. Collectively, the results of the present study indicate that HDAC inhibitors suppress the proliferation, migration and invasiveness of HNSCC by downregulating the p63­mediated tight junction molecules JAM­A and claudin­1, and inducing p63 or p21­mediated growth arrest.


Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Junções Íntimas/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Idoso , Apoptose/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Claudina-1/metabolismo , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Inibidores de Histona Desacetilases/uso terapêutico , Humanos , Masculino , Pessoa de Meia-Idade , Receptores de Superfície Celular/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Junções Íntimas/metabolismo , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética
12.
Mediators Inflamm ; 2021: 1315797, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33642941

RESUMO

Stroke is the second leading cause of death worldwide. Patients who have a stroke are susceptible to many gastrointestinal (GI) complications, such as dysphagia, GI bleeding, and fecal incontinence. However, there are few studies focusing on the GI tract after stroke. The current study is to investigate the changes of intestinal structure and function in mice after ischemic stroke. Ischemic stroke was made as a disease model in mice, in which brain and ileal tissues were collected for experiments on the 1st and 7th day after stroke. Intestinal motility of mice was inhibited, and intestinal permeability was increased after stroke. Hematoxylin-eosin (HE) staining showed the accumulation of leucocytes in the intestinal mucosa. Myeloperoxidase (MPO) activity and inflammatory proteins (nuclear factor kappa-B (NF-κB), inducible nitric oxide synthase (iNOS)) in the small intestine were significantly increased in mice after stroke. The expression of tight junction (TJ) proteins (zonula occludens-1 (ZO-1), occludin, and claudin-1) was downregulated, and transmission electron microscopy (TEM) showed broken TJ of the intestinal mucosa after stroke. Glial fibrillary acidic protein (GFAP) and the apoptosis-associated proteins (tumor necrosis factor (TNF-α), caspase-3, and cleaved caspase-3) were notably upregulated as well. Ischemic stroke led to negative changes on intestinal structure and function. Inflammatory mediators and TNF-α-induced death receptor signaling pathways may be involved and disrupt the small intestinal barrier function. These results suggest that stroke patients should pay attention to GI protection.


Assuntos
Intestinos/fisiologia , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/fisiopatologia , Actinas/genética , Actinas/metabolismo , Animais , Apoptose/fisiologia , Western Blotting , Claudina-1/genética , Claudina-1/metabolismo , Inflamação/metabolismo , Inflamação/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Neuroglia/metabolismo , Neuroglia/fisiologia , Ocludina/genética , Ocludina/metabolismo , Peroxidase/genética , Peroxidase/metabolismo , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismo
13.
Mol Med Rep ; 23(2)2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33655320

RESUMO

Atezolizumab can reduce immunosuppression caused by T lymphocyte apoptosis in various cancer types. The current study aimed to investigate whether this drug can also alleviate immunosuppression during sepsis. For that purpose, a C57BL/6 mouse sepsis model was generated. Mice were randomly assigned to three groups: Sham, cecal ligation and puncture (CLP) and atezolizumab groups. Atezolizumab was administered in vivo by intraperitoneal injection. The expression of programmed death ligand­1 (PD­L1) on neutrophils and programmed death­1 (PD­1) on T lymphocytes was evaluated, and endotoxin concentration, intestinal permeability, ileum histopathological score and tight junction protein expression were assessed to determine the extent of disease in each group. The rate of T lymphocyte apoptosis was determined to assess the effects of atezolizumab on T lymphocyte apoptosis in vivo and in vitro. Survival times were also recorded to compare mouse prognosis during sepsis. In the CLP group, the proportion of PD­L1+ neutrophils was significantly higher at 48, 72 and 96 h in blood, and at 24, 48, 72 and 96 h in bone marrow, compared with those of the sham group (P<0.05). The proportion of PD­1+ T lymphocytes was also upregulated at 72 h in blood. In the atezolizumab group, endotoxin concentration, intestinal permeability and ileum histopathological score were lower compared with those in the CLP group (P<0.05), whereas the expression of claudin­1 and occludin proteins on ileum was higher compared with that in the CLP group (P<0.05). Both in vivo and in vitro experiments indicated that the rate of T lymphocyte apoptosis following atezolizumab treatment was lower compared with that in the CLP group (P<0.05). Survival analysis demonstrated that mice in the atezolizumab group survived longer compared with those in the CLP group (P<0.05). The current study demonstrated that treatment with atezolizumab may be an effective method for treating immunosuppression induced by sepsis.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Íleo/patologia , Neutrófilos/imunologia , Sepse/tratamento farmacológico , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/uso terapêutico , Antígeno B7-H1 , Ceco/lesões , Claudina-1/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica , Íleo/metabolismo , Injeções Intraperitoneais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ocludina/genética , Sepse/imunologia , Resultado do Tratamento
14.
Chin J Nat Med ; 19(3): 188-194, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33781452

RESUMO

Triptolide (TP), an active component of Tripterygium wilfordiiHook. f. (TWHF), has been widely used for centuries as a traditional Chinese medicine. However, the clinical application of TP has been restricted due to multitarget toxicity, such as hepatotoxicity. In this study, 28 days of oral TP administration (100, 200, or 400 µg·kg-1·d-1) induced the occurrence of cholestasis in female Wistar rats, as evidenced by increased serum levels of γ-glutamyl transpeptidase (γ-GGT), alkaline phosphatase (ALP) and hepatic total bile acids (TBAs). In addition, the heptocyte polarity associated with the strcture of tight junctions (TJs) was disrupted in both rats and sandwich-cultured primary hepatocytes. Immunoblotting revealed decreased expression of the TJ-associated proteins occludin, claudin-1, and zonula occludens protein (ZO-1), and downregulated mRNA levels of these TJs was also detected by real-time PCR. An immunofluorescence analysis showed abnormal subcellular localization of occludin, claudin-1 and ZO-1, which was also confirmed by transmission electron microscopy. Moreover, the concentration of FITC-dextran, a marker of paracellular penetration, was found to increase rapidly in bile increased rapidly (within 6 minutes) after treatment with TP, which indicated the functional impairment of TJs. Taken together, these results suggest that the administration of TP for 28 consecutive days to rats could induce cholestatic injury in the liver, and the increased paracellular permeability might play an important role in these pathological changes.


Assuntos
Colestase , Diterpenos/efeitos adversos , Fígado/efeitos dos fármacos , Fenantrenos/efeitos adversos , Junções Íntimas , Animais , Colestase/induzido quimicamente , Claudina-1 , Compostos de Epóxi/efeitos adversos , Feminino , Hepatócitos/efeitos dos fármacos , Ocludina , Ratos , Ratos Wistar , Junções Íntimas/patologia , Proteína da Zônula de Oclusão-1
15.
Histol Histopathol ; 36(4): 437-446, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33629735

RESUMO

BACKGROUND: A large body of scientific evidence points to the important roles of tight junction proteins in tumor development, progression and dissemination. The larynx has only a few studies, analyzing the role of this group of junctional proteins in its oncogenesis. In this study, the author sheds some light on the expression and possible role of claudin-1 in laryngeal squamous cell carcinomas. MATERIALS AND METHODS: This study analyzed the expression of claudin-1, using immunohistochemistry, in a tissue microarray of 80 cases of laryngeal squamous cell cancers. Clinicopathological parameters were analyzed according to claudin-1 expression in the tissue microarray. Furthermore, the expression of slug/snail1, an Epithelial-Mesenchymal Transition (EMT) linked protein, was analyzed by immunohistochemistry in the same microarray, and the expressions of the two proteins were assessed for correlation. RESULTS: A significant majority of laryngeal squamous cell cancers exhibited positive expression of claudin-1 proteins. The majority of those tumors expressed claudin-1 in their cytoplasm. The overall majority of those same tumors also exhibited a cytoplasmic shift of the slug-snail-1 protein from the nuclei to the cytoplasm. There was also evidence of correlation of the two proteins' expressions in the cytoplasm of laryngeal tumors. CONCLUSION: The above may suggest a role for claudin-1 in the development and progression of laryngeal squamous cell carcinoma. Overall, claudin-1's aberrant expression in laryngeal cancer is in line with evidence seen in other head and neck cancers. Its co-expression with slug/snail-1 in LSCC patients should be investigated further to understand the nature of the relationship of the two proteins in LSCC and their possible contribution to its development and progression.


Assuntos
Biomarcadores Tumorais/metabolismo , Claudina-1/metabolismo , Neoplasias Laríngeas , Carcinoma de Células Escamosas de Cabeça e Pescoço , Caderinas/metabolismo , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Imuno-Histoquímica , Neoplasias Laríngeas/metabolismo , Neoplasias Laríngeas/patologia , Fatores de Transcrição da Família Snail/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia
16.
Life Sci ; 275: 119254, 2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-33636174

RESUMO

AIMS: Defective tight junctions (TJs) can induce intestinal epithelial dysfunction, which participates in various diseases such as irritable bowel syndrome. However, the mechanisms of TJ defects remain unclear. Our study revealed the role of Piezo1 in regulating intestinal epithelial function and TJs. MATERIALS AND METHODS: The human colonic adenocarcinoma cell line Caco-2 were cultured on Transwell plate to form an epithelial barrier in vitro, and Piezo1 expression was manipulated using a lentivirus vector. Epithelial function was evaluated by measuring transepithelial electronic resistance (TEER) and 4-kDa FITC-dextran (FD4) transmission. TJ proteins (claudin-1, occludin, ZO-1) were evaluated by RT-PCR, western blot, and immunostaining analysis. Potential signal pathways, including the ROCK and Erk pathways, were detected. Moreover, to explore the regulatory effect of Piezo1 activity on epithelial function, inhibitors (ruthenium red, GsMTx4) and an agonist (Yoda1) were introduced both ex vivo and in vitro. KEY FINDINGS: Alteration of Piezo1 expression altered epithelial function and the expression of the tight junction protein claudin-1. Piezo1 expression regulated phosphorylated ROCK1/2 expression, whereas interference on ROCK1/2 prevented the regulation of claudin-1 by Piezo1. In both Caco-2 monolayer and mouse colon epithelium, Piezo1 activity directly modulated epithelial function and permeability. SIGNIFICANCE: Piezo1 negatively regulates epithelial barrier function by affecting the expression of claudin-1. Such regulation may be achieved partially via the ROCK1/2 pathway. Moreover, activating Piezo1 can induce epithelial dysfunction.


Assuntos
Claudina-1/fisiologia , Mucosa Intestinal/fisiologia , Canais Iônicos/fisiologia , Transdução de Sinais , Quinases Associadas a rho/metabolismo , Animais , Western Blotting , Células CACO-2 , Claudina-1/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ocludina/metabolismo , Ocludina/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/fisiologia , Proteína da Zônula de Oclusão-1/metabolismo , Proteína da Zônula de Oclusão-1/fisiologia
17.
J Radiat Res ; 62(2): 259-268, 2021 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-33592097

RESUMO

To assess the effects of exposure to extremely low-frequency magnetic fields (ELF-MFs) on MDCK cell lines, experiments were performed in a chamber under controlled conditions (temperature, humidity and CO2). Therefore, the measured physicochemical and electrical changes in the cells are due solely to the magnetic field exposure and not to external factors. A developed sinusoidal magnetic field generator produced the ELF-MFs with a uniform magnetic field and adjustable intensity and frequency. Three experimental indicators were used: (i) transepithelial electrical impedance (TEEI); (ii) cell migration and proliferation; and (iii) expression of the proteins of the tight junctions, and changes in the area and shape of the cell nuclei. No significant effects on TEEI values were observed when 10 and 50 G 60 Hz magnetic fields were applied to confluent cell monolayers. There were no significant differences in migration and proliferation of the cell monolayer exposed to 60 Hz magnetic fields10 and 50 G , but a contact inhibition factor was observed. The expression of the CLDN-1 protein decreased by 90% compared with the control, while ZO-1 protein expression increased by 120%. No significant effects were observed in the area and shape of the cell nuclei. Experimentation in a controlled environment, under physiological conditions, ensures that the observed effects were strictly due to exposure to magnetic fields. Different exposure conditions are necessary to determine the impact on TEEI and cell migration-proliferation indicators.


Assuntos
Ambiente Controlado , Células Epiteliais/fisiologia , Campos Magnéticos , Animais , Núcleo Celular/metabolismo , Claudina-1/metabolismo , Cães , Impedância Elétrica , Células Epiteliais/metabolismo , Fluorescência , Células Madin Darby de Rim Canino , Proteína da Zônula de Oclusão-1/metabolismo
18.
Gastroenterology ; 160(6): 2103-2118, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33465371

RESUMO

BACKGROUND & AIMS: Liver tight junctions (TJs) establish tissue barriers that isolate bile from the blood circulation. TJP2/ZO-2-inactivating mutations cause progressive cholestatic liver disease in humans. Because the underlying mechanisms remain elusive, we characterized mice with liver-specific inactivation of Tjp2. METHODS: Tjp2 was deleted in hepatocytes, cholangiocytes, or both. Effects on the liver were assessed by biochemical analyses of plasma, liver, and bile and by electron microscopy, histology, and immunostaining. TJ barrier permeability was evaluated using fluorescein isothiocyanate-dextran (4 kDa). Cholic acid (CA) diet was used to assess susceptibility to liver injury. RESULTS: Liver-specific deletion of Tjp2 resulted in lower Cldn1 protein levels, minor changes to the TJ, dilated canaliculi, lower microvilli density, and aberrant radixin and bile salt export pump (BSEP) distribution, without an overt increase in TJ permeability. Hepatic Tjp2-defcient mice presented with mild progressive cholestasis with lower expression levels of bile acid transporter Abcb11/Bsep and detoxification enzyme Cyp2b10. A CA diet tolerated by control mice caused severe cholestasis and liver necrosis in Tjp2-deficient animals. 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene ameliorated CA-induced injury by enhancing Cyp2b10 expression, and ursodeoxycholic acid provided partial improvement. Inactivating Tjp2 separately in hepatocytes or cholangiocytes showed only mild CA-induced liver injury. CONCLUSION: Tjp2 is required for normal cortical distribution of radixin, canalicular volume regulation, and microvilli density. Its inactivation deregulated expression of Cldn1 and key bile acid transporters and detoxification enzymes. The mice provide a novel animal model for cholestatic liver disease caused by TJP2-inactivating mutations in humans.


Assuntos
Canalículos Biliares/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/genética , Colestase/genética , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-2/genética , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/genética , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/metabolismo , Ácidos e Sais Biliares/metabolismo , Canalículos Biliares/patologia , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Colagogos e Coleréticos/uso terapêutico , Ácido Cólico , Claudina-1/metabolismo , Família 2 do Citocromo P450/metabolismo , Proteínas do Citoesqueleto/metabolismo , Células Epiteliais , Feminino , Fibrose , Predisposição Genética para Doença , Hepatócitos , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Mutação , Oxazóis/uso terapêutico , Permeabilidade , Fatores de Proteção , RNA Mensageiro/metabolismo , Esteroide Hidroxilases/metabolismo , Junções Íntimas/ultraestrutura , Ácido Ursodesoxicólico/uso terapêutico , Proteína da Zônula de Oclusão-2/deficiência
19.
Toxicol Appl Pharmacol ; 414: 115411, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33476678

RESUMO

Di-2-ethylhexyl phosphate (DEHP) and its main toxic metabolite mono-2-ethylhexyl phthalate (MEHP) are the typical endocrine disrupting chemicals (EDCs) and widely affect human health. Our previous research reported that synthetic nonionic dietary emulsifier polysorbate 80 (P80, E433) had the promotional effect on the oral absorption of DEHP in rats. The aim of this study was to explore its mechanism of promoting oral absorption, focusing on the mucus barrier and mucosal barrier of the small intestine. A small molecule fluorescent probe 5-aminofluorescein-MEHP (MEHP-AF) was used as a tracker of MEHP in vivo and in vitro. First of all, we verified that P80 promoted the bioavailability of MEHP-AF in the long-term and low-dose exposure of MEHP-AF with P80 as a result of increasing the intestinal absorption of MEHP-AF. Afterwards, experimental results from Western blot, qPCR, immunohistochemistry, and immunofluorescence showed that P80 decreased the expression of proteins (mucus protein mucin-2, tight junction proteins claudin-1 and occludin) related to mucus barrier and mucosal barrier in the intestine, changed the integrity of intestinal epithelial cell, and increased the permeability of intestinal epithelial mucosa. These results indicated that P80 promoted the oral absorption of MEHP-AF by altering the intestinal mucus barrier and mucosal barrier. These findings are of great importance for assessing the safety risks of some food emulsifiers and clarifying the absorption mechanism of chemical pollutants in food, especially for EDCs.


Assuntos
Dietilexilftalato/análogos & derivados , Emulsificantes/toxicidade , Células Epiteliais/efeitos dos fármacos , Absorção Intestinal/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Intestino Delgado/efeitos dos fármacos , Polissorbatos/toxicidade , Animais , Disponibilidade Biológica , Células CACO-2 , Claudina-1/metabolismo , Dietilexilftalato/farmacocinética , Dietilexilftalato/toxicidade , Células Epiteliais/metabolismo , Fluoresceínas/metabolismo , Corantes Fluorescentes/metabolismo , Células HT29 , Humanos , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Masculino , Camundongos Endogâmicos ICR , Mucina-2/metabolismo , Ocludina/metabolismo , Permeabilidade , Ratos Sprague-Dawley , Distribuição Tecidual , Toxicocinética
20.
Oncol Rep ; 45(1): 368-378, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33416138

RESUMO

TP53 is the most common gene mutated in human cancers, including in cholangiocarcinoma (CCA). The gain­of­function properties of p53 variants are often involved in cancer progression. The present study demonstrated that a truncated del p53 variant, del p53M213, exhibited gain­of­function properties and was highly expressed in the invasive liver fluke Opisthorchis viverrini­associated CCA cell line, KKU­M213. The del p53M213 variant lacked exons 7­9 and contained a V31I substitution (p53­p72­Δ225­331­V31I). Stably transfected p53­null human non­small cell lung H1299 cells exhibited a del p53M213 localization in both the cell cytosol and nucleus. Del p53M213 lacked anti­growth functions, and instead enhanced migration and invasiveness. In addition, this p53 variant downregulated claudin­1 expression and promoted Cdc42 activation, consistent with the roles of claudin­1 and Cdc42 in inhibiting cell­cell dissociation and promoting cell migration, respectively. On the whole, although del p53M213 is an important driver of cancer cell migration and invasiveness, other properties related to its novel gain­of­function properties require further investigation in order to develop effective treatment strategies for cancers bearing this truncated TP53 allele.


Assuntos
Neoplasias dos Ductos Biliares/genética , Colangiocarcinoma/genética , Claudina-1/genética , Proteína Supressora de Tumor p53/genética , Proteína cdc42 de Ligação ao GTP/genética , Alelos , Neoplasias dos Ductos Biliares/patologia , Carcinogênese/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Colangiocarcinoma/patologia , Regulação para Baixo , Mutação com Ganho de Função , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Variação Genética , Humanos , Invasividade Neoplásica/genética , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismo
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