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1.
Neuroreport ; 32(3): 244-251, 2021 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-33470765

RESUMO

OBJECTIVE: Parkinson's disease is a common neurodegenerative disease. Here, we investigated the protective effect and potential mechanisms of propionate on the intestinal epithelial barrier in mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease. METHODS: Gas chromatography was used to determine short-chain fatty acids (SCFA) concentrations in the fecal samples of Parkinson's disease patients and healthy controls. The stepping test was used to analyze forelimb akinesia, whisker test was used to analyze sensorimotor injury, cylinder test was used to analyze sensorimotor function, and Western blotting was used to analyze protein expression. RESULTS: The concentrations of SCFAs, including acetate, butyrate and propionate, were significantly downregulated in the fecal samples of Parkinson's disease patients, and among the SCFAs, propionate decreased the most. Propionate administration improved the stepping test score, whisker test score and cylinder test score of MPTP-induced Parkinson's disease mice. Additionally, propionate administration increased the protein expression of zonula occludens-1 and occludin. Moreover, the effects of propionate on motor behavior and the intestinal epithelial barrier were dependent on the proteirrserinc-threonine kinases (AKT) signaling pathway. More importantly, treatment with SC79, a specific AKT agonist, abolished the effects of propionate on the intestinal epithelial barrier and motor behavior. CONCLUSION: Our results demonstrated that propionate, which was decreased in the fecal samples of Parkinson's disease patients, exerted beneficial effects on intestinal epithelial barrier function and improved motor behavior in MPTP-induced Parkinson's disease mice through the AKT signaling pathway.


Assuntos
Microbioma Gastrointestinal , Mucosa Intestinal/efeitos dos fármacos , Atividade Motora/efeitos dos fármacos , Doença de Parkinson/microbiologia , Transtornos Parkinsonianos/metabolismo , Propionatos/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Acetatos/metabolismo , Acetatos/farmacologia , Idoso , Animais , Comportamento Animal/efeitos dos fármacos , Benzopiranos/farmacologia , Butiratos/metabolismo , Estudos de Casos e Controles , Modelos Animais de Doenças , Ácidos Graxos Voláteis/metabolismo , Fezes/química , Feminino , Humanos , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Ocludina/efeitos dos fármacos , Ocludina/metabolismo , Doença de Parkinson/metabolismo , Transtornos Parkinsonianos/microbiologia , Transtornos Parkinsonianos/fisiopatologia , Permeabilidade , Propionatos/metabolismo , Proteínas Proto-Oncogênicas c-akt/agonistas , Transdução de Sinais , Proteína da Zônula de Oclusão-1/efeitos dos fármacos , Proteína da Zônula de Oclusão-1/metabolismo
2.
Clin Exp Allergy ; 51(1): 87-98, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33090566

RESUMO

BACKGROUND: Staphylococcus aureus colonization and release of enterotoxin B (SEB) has been associated with severe chronic rhinosinusitis with nasal polyps (CRSwNP). The pathogenic mechanism of SEB on epithelial barriers, however, is largely unexplored. OBJECTIVE: We investigated the effect of SEB on nasal epithelial barrier function. METHODS: SEB was apically administered to air-liquid interface (ALI) cultures of primary polyp and nasal epithelial cells of CRSwNP patients and healthy controls, respectively. Epithelial cell integrity and tight junction expression were evaluated. The involvement of Toll-like receptor 2 (TLR2) activation was studied in vitro with TLR2 monoclonal antibodies and in vivo in tlr2-/- knockout mice. RESULTS: SEB applied to ALI cultures of polyp epithelial cells decreased epithelial cell integrity by diminishing occludin and zonula occludens (ZO)-1 protein expression. Antagonizing TLR2 prevented SEB-induced barrier disruption. SEB applied in the nose of control mice increased mucosal permeability and decreased mRNA expression of occludin and ZO-1, whereas mucosal integrity and tight junction expression remained unaltered in tlr2-/- mice. Furthermore, in vitro SEB stimulation resulted in epithelial production of IL-6 and IL-8, which was prevented by TLR2 antagonization. CONCLUSION & CLINICAL RELEVANCE: SEB damages nasal polyp epithelial cell integrity by triggering TLR2 in CRSwNP. Our results suggest that SEB might represent a driving factor of disease exacerbation, rather than a causal factor for epithelial defects in CRSwNP. Interfering with TLR2 triggering might provide a way to avoid the pathophysiological consequences of S. aureus on inflammation in CRSwNP.


Assuntos
Enterotoxinas/farmacologia , Mucosa Nasal/efeitos dos fármacos , Pólipos Nasais/metabolismo , Permeabilidade/efeitos dos fármacos , Rinite/metabolismo , Sinusite/metabolismo , Junções Íntimas/efeitos dos fármacos , Adolescente , Adulto , Idoso , Animais , Estudos de Casos e Controles , Linhagem Celular , Feminino , Humanos , Técnicas In Vitro , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Mucosa Nasal/metabolismo , Ocludina/efeitos dos fármacos , Ocludina/genética , Cultura Primária de Células , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Staphylococcus aureus/patogenicidade , Junções Íntimas/genética , Receptor 2 Toll-Like/antagonistas & inibidores , Receptor 2 Toll-Like/genética , Adulto Jovem , Proteína da Zônula de Oclusão-1/efeitos dos fármacos , Proteína da Zônula de Oclusão-1/genética
3.
Neuroreport ; 31(18): 1274-1282, 2020 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-33165199

RESUMO

The brain edema following intracerebral hemorrhage (ICH) plays a key role in the recovery process. Protocatechuic acid (PCA) has been proved possessing neuroprotection in ICH. Here we tried to explore its value in brain edema after ICH and reveal underlying mechanisms. ICH model was created in C57 mice using collagenase IV. PCA was injected intraperitoneally at 30 mg/kg every 24 h in PCA group. On day 3 after ICH, the water content of hemorrhagic ipsilateral hemisphere in PCA group was significantly reduced compared with vehicle group. AQP4, the main water channel, was remarkably decreased in PCA group. Additionally, ZO-1 and occludin expression were increased in PCA group. The bEnd.3 cells were cultured to understand the effect of PCA on the blood-brain barrier (BBB) integrity. Compare to hemin treated group, plus PCA enhanced the expression of HO-1 and Nrf2 nuclear translocation. Furtherly, the overexpression of HO-1, ZO-1, occludin, in the PCA treatment group was inhibited after knockdown of Nrf2. Taken together, our results proved PCA alleviated brain edema and BBB disruption in ICH by promoting the Nrf2/HO-1 signaling pathway.


Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Heme Oxigenase-1/efeitos dos fármacos , Hidroxibenzoatos/farmacologia , Proteínas de Membrana/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/efeitos dos fármacos , Animais , Aquaporina 4/efeitos dos fármacos , Aquaporina 4/metabolismo , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Edema Encefálico/metabolismo , Hemorragia Cerebral/metabolismo , Técnicas de Silenciamento de Genes , Heme Oxigenase-1/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Ocludina/efeitos dos fármacos , Ocludina/metabolismo , Transdução de Sinais , Proteína da Zônula de Oclusão-1/efeitos dos fármacos , Proteína da Zônula de Oclusão-1/metabolismo
4.
Toxicol Lett ; 332: 146-154, 2020 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-32683294

RESUMO

Occludin is an important tight junction (TJ) protein in pulmonary epithelial cells. In this study, we identified changes in occludin in arsenic-induced lung injury in vivo and in vitro. Upon intratracheal instillation with arsenic trioxide (As2O3) at a daily dose of 30 µg/kg for 1 week, levels of occludin mRNA and protein expression decreased significantly in mouse lung tissue. Levels of occludin mRNA and protein expression in BEAS-2B cells were reduced upon exposure to As2O3 in a concentration- and time-dependent manner. In addition, exposure to As2O3 significantly increased expression of p-p38, p-ERK1/2, p-ELK1, and MLCK in mouse lung tissue and BEAS-2B cells. Treatment with As2O3 induced oxidative stress in mouse lung tissue and BEAS-2B cells. In BEAS-2B cells, exposure to As2O3 reduced transepithelial resistance, which was partially restored with N-acetyl-cysteine (NAC) treatment. Reduced expression of occludin mRNA and protein induced by As2O3 was entirely restored with NAC and resveratrol. However, SB203580, PD98059, and ML-7 partially blocked As2O3-induced occludin reduction in BEAS-2B cells. These results indicate that As2O3 inhibits occludin expression in vivo and in vitro at least partially via the ROS/ERK/ELK1/MLCK and ROS/p38 MAPK signaling pathways.


Assuntos
Arsenitos/toxicidade , Pulmão/metabolismo , Ocludina/biossíntese , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular , Regulação para Baixo/efeitos dos fármacos , Glutationa/metabolismo , Humanos , Pulmão/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ocludina/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Peptídeos/efeitos dos fármacos , Peptídeos/metabolismo , Espécies Reativas de Oxigênio , Superóxido Dismutase/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacos
5.
Neuropharmacology ; 162: 107845, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31704276

RESUMO

Glibenclamide protects against ischemic injury in both preclinical and clinical studies, presumably by blocking the de novo assembled sulfonylurea receptor 1-transient receptor potential M4 (Sur1-Trpm4) channel induced by ischemia. However, glibenclamide may cause unexpected serious hypoglycemia. Here, we tested whether glimepiride, another sulfonylurea with better safety, has comparable efficacy with glibenclamide and whether gene deletion of Trpm4 (Trpm4-/-) exerts similar effect. Wild-type (WT) mice subjected to temporary middle cerebral artery occlusion (tMCAO) were randomized to receive glibenclamide (an initial dose of 10 µg/kg and additional doses of 1.2 µg every 8 h), three different doses of glimepiride (10 µg/kg, 100 µg/kg and 1 mg/kg) or vehicle after ischemia, while tMCAO-treated Trpm4-/- mice were randomized to receive vehicle or glimepiride. Neurological function, infarct volume, edema formation, the integrity of blood-brain barrier and inflammatory reaction were evaluated at 24 h after ischemia. In tMCAO-treated WT mice, 10 µg/kg and 100 µg/kg glimepiride had comparable efficacy with glibenclamide in improving longa score and grip test score, reducing infarct volume, mitigating brain edema, lessening extravasation of Evans blue dye and IgG, restoring tight junction protein expression as well as suppressing inflammatory cytokines. Compared with WT mice, Trpm4-/- mice showed less neurological deficit, smaller cerebral infarction, lighter brain edema and more integrity of blood-brain barrier. As expected, glimepiride did not provide additional neuroprotection compared with vehicle in the tMCAO-treated Trpm4-/- mice. Glimepiride shows comparable efficacy with glibenclamide in alleviating brain injury after ischemic stroke in mice, possibly via targeting the Sur1-Trpm4 channel.


Assuntos
Edema Encefálico/fisiopatologia , Encéfalo/efeitos dos fármacos , Glibureto/farmacologia , Hipoglicemiantes/farmacologia , Infarto da Artéria Cerebral Média/fisiopatologia , Inflamação/fisiopatologia , AVC Isquêmico/fisiopatologia , Compostos de Sulfonilureia/farmacologia , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/fisiopatologia , Edema Encefálico/metabolismo , Edema Encefálico/patologia , Claudina-5/efeitos dos fármacos , Claudina-5/genética , Ciclo-Oxigenase 2/efeitos dos fármacos , Ciclo-Oxigenase 2/genética , Perfilação da Expressão Gênica , Hipoglicemia/induzido quimicamente , Infarto da Artéria Cerebral Média/genética , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Inflamação/metabolismo , Inflamação/patologia , Interleucina-1beta/efeitos dos fármacos , Interleucina-1beta/genética , AVC Isquêmico/genética , AVC Isquêmico/metabolismo , AVC Isquêmico/patologia , Camundongos , Camundongos Knockout , Fármacos Neuroprotetores , Ocludina/efeitos dos fármacos , Ocludina/genética , Distribuição Aleatória , Receptores de Sulfonilureias/efeitos dos fármacos , Receptores de Sulfonilureias/genética , Receptores de Sulfonilureias/metabolismo , Canais de Cátion TRPM/efeitos dos fármacos , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética
6.
Stroke ; 49(9): 2211-2219, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30354988

RESUMO

Background and Purpose- tPA (tissue-type plasminogen activator) is the only recommended intravenous thrombolytic agent for ischemic stroke. However, its application is limited because of increased risk of hemorrhagic transformation beyond the time window. T541 is a Chinese compound medicine with potential to attenuate ischemia and reperfusion injury. This study was to explore whether T541-benefited subjects underwent tPA thrombolysis extending the time window. Methods- Male C57BL/6 N mice were subjected to carotid artery thrombosis by stimulation with 10% FeCl3 followed by 10 mg/kg tPA with/without 20 mg/kg T541 intervention at 4.5 hours. Thrombolysis and cerebral blood flow were observed dynamically until 24 hours after drug treatment. Neurological deficit scores, brain edema and hemorrhage, cerebral microvascular junctions and basement membrane proteins, and energy metabolism in cortex were assessed then. An in vitro hypoxia/reoxygenation model using human cerebral microvascular endothelial cells was used to evaluate effect of T541 on tight junctions and F-actin in the presence of tPA. Results- tPA administered at 4.5 hours after carotid thrombosis resulted in a decrease in thrombus area and survival rate, whereas no benefit on cerebral blood flow. Study at 24 hours after tPA administration revealed a significant angioedema and hemorrhage in the ischemia hemisphere, a decreased expression of junction proteins claudin-5, zonula occludens-1, occludin, junctional adhesion molecule-1 and vascular endothelial cadherin, and collagen IV and laminin. Meanwhile, ADP/ATP, AMP/ATP, and ATP5D (ATP synthase subunit) expression and activities of mitochondria complex I, II, and IV declined, whereas malondialdehyde and 8-Oxo-2'-deoxyguanosine increased and F-actin arrangement disordered. All the insults after tPA treatment were attenuated by addition of T541 dose dependently. Conclusions- The results suggest T541 as a potential remedy to attenuate delayed tPA-related angioedema and hemorrhage and extend time window for tPA treatment. The potential of T541 to upregulate energy metabolism and protect blood-brain barrier is likely attributable to its effects observed.


Assuntos
Alcenos/farmacologia , Edema Encefálico , Trombose das Artérias Carótidas , Circulação Cerebrovascular/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Hemorragias Intracranianas , Polifenóis/farmacologia , Traumatismo por Reperfusão , Saponinas/farmacologia , Animais , Antígenos CD/efeitos dos fármacos , Antígenos CD/metabolismo , Astrágalo , Encéfalo/irrigação sanguínea , Encéfalo/efeitos dos fármacos , Caderinas/efeitos dos fármacos , Caderinas/metabolismo , Moléculas de Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Claudina-5/efeitos dos fármacos , Claudina-5/metabolismo , Colágeno Tipo IV/efeitos dos fármacos , Colágeno Tipo IV/metabolismo , Modelos Animais de Doenças , Combinação de Medicamentos , Complexo I de Transporte de Elétrons , Complexo II de Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons , Laminina/efeitos dos fármacos , Laminina/metabolismo , Masculino , Camundongos , Ocludina/efeitos dos fármacos , Ocludina/metabolismo , Panax notoginseng , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/metabolismo , Ativador de Plasminogênio Tecidual/farmacologia , Proteína da Zônula de Oclusão-1/efeitos dos fármacos , Proteína da Zônula de Oclusão-1/metabolismo
7.
Artigo em Inglês | MEDLINE | ID: mdl-28861400

RESUMO

During intestinal invasion, Entamoeba histolytica opens tight junctions (TJs) reflected by transepithelial electrical resistance (TEER) dropping. To explore the molecular mechanisms underlying this, we studied in vitro and in vivo the damage produced by the recombinant E. histolytica cysteine protease (rEhCP112) on TJ functions and proteins. rEhCP112 reduced TEER in Caco-2 cells in a dose- and time-dependent manner; and EhCP112-overexpressing trophozoites provoked major epithelial injury compared to control trophozoites. rEhCP112 penetrated through the intercellular space, and consequently the ion flux increased and the TJs fence function was disturbed. However, macromolecular flux was not altered. Functional in vitro assays revealed specific association of rEhCP112 with claudin-1 and claudin-2, that are both involved in regulating ion flux and fence function. Of note, rEhCP112 did not interact with occludin that is responsible for regulating macromolecular flux. Moreover, rEhCP112 degraded and delocalized claudin-1, thus affecting interepithelial adhesion. Concomitantly, expression of the leaky claudin-2 at TJ, first increased and then it was degraded. In vivo, rEhCP112 increased intestinal epithelial permeability in the mouse colon, likely due to apical erosion and claudin-1 and claudin-2 degradation. In conclusion, we provide evidence that EhCP112 causes epithelial dysfunction by specifically altering claudins at TJ. Thus, EhCP112 could be a potential target for therapeutic approaches against amoebiasis.


Assuntos
Proteínas de Bactérias/farmacologia , Claudina-1/efeitos dos fármacos , Claudina-2/efeitos dos fármacos , Cisteína Endopeptidases/farmacologia , Entamoeba histolytica/metabolismo , Células Epiteliais/efeitos dos fármacos , Intestinos/efeitos dos fármacos , Junções Íntimas/efeitos dos fármacos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Claudina-1/metabolismo , Claudina-2/metabolismo , Claudina-4/efeitos dos fármacos , Colo/efeitos dos fármacos , Colo/parasitologia , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Modelos Animais de Doenças , Cães , Entamoeba histolytica/genética , Entamoeba histolytica/patogenicidade , Entamebíase/patologia , Regulação da Expressão Gênica , Humanos , Células Madin Darby de Rim Canino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ocludina/efeitos dos fármacos , Permeabilidade , Proteínas Recombinantes/farmacologia , Junções Íntimas/metabolismo , Trofozoítos/genética , Trofozoítos/metabolismo , Proteína da Zônula de Oclusão-1/efeitos dos fármacos
8.
Chest ; 151(3): 658-667, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27815150

RESUMO

BACKGROUND: The synthetic peptide solnatide is a novel pharmacologic agent that reduces extravascular lung water, blunts reactive oxygen species production, and improves lung function due to its ability to directly activate the epithelial sodium channel. The goal of this study was to investigate the effect of solnatide in pulmonary edema induced by acute hypobaric hypoxia and exercise in rats, which is considered a model for high-altitude pulmonary edema. METHODS: Sprague-Dawley rats were assigned to low-altitude control and eight treatment groups. Animals of all groups were subjected to exhaustive exercise in a hypobaric hypoxic environment simulating an altitude of 4,500 meters, followed by simulated ascent to 6,000 meters. After 48 h at 6,000 meters, rats were given sodium chloride, dexamethasone, aminophylline, p38 mitogen activated protein kinase inhibitor, and NOD-like receptor containing a pyrin domain 3 inhibitor, or one of three different doses of solnatide, once daily for 3 consecutive days. After 3 days, arterial blood gas, BAL fluid, lung water content, and histologic and ultra-microstructure analyses were performed. Tight junction protein occludin was assayed by using immunohistochemistry. RESULTS: Rats treated with solnatide had significantly lower BAL fluid protein and lung water content than high-altitude control rats. Lungs of solnatide-treated rats were intact and showed less hemorrhage and disruption of the alveolar-capillary barrier than those of high-altitude control animals. Occludin expression was significantly higher in solnatide-treated animals, compared with high-altitude control, dexamethasone-, and aminophylline-treated animals. CONCLUSIONS: Solnatide reduced pulmonary edema, increased occludin expression, and improved gas-blood barrier function during acute hypobaric hypoxia and exercise in rats. These results provide a rationale for the clinical application of solnatide to patients with pulmonary edema and exposure to a high-altitude hypoxic environment.


Assuntos
Doença da Altitude/metabolismo , Água Extravascular Pulmonar/efeitos dos fármacos , Hipertensão Pulmonar/metabolismo , Pulmão/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Condicionamento Físico Animal , Doença da Altitude/patologia , Aminofilina/farmacologia , Animais , Gasometria , Líquido da Lavagem Broncoalveolar , Broncodilatadores/farmacologia , Capilares/ultraestrutura , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Hemorragia , Hipertensão Pulmonar/patologia , Imuno-Histoquímica , Pulmão/metabolismo , Pulmão/patologia , Pulmão/ultraestrutura , Microscopia Eletrônica , Ocludina/efeitos dos fármacos , Ocludina/metabolismo , Alvéolos Pulmonares/ultraestrutura , Edema Pulmonar/metabolismo , Edema Pulmonar/patologia , Ratos , Ratos Sprague-Dawley
9.
Toxicology ; 373: 1-12, 2016 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-27818224

RESUMO

Perfluorooctane sulfonate (PFOS), an ubiquitous environmental pollutant, has been associated with male reproductive disorders. However, the underlying mechanisms are not yet fully understood. In this study, in vivo and in vitro models were used to explore the effects of PFOS on blood-testis barrier (BTB) and related molecular mechanisms. First, male ICR mice were orally administrated PFOS (0.5-10mg/kg/bw) for 4 weeks. Bodyweight, sperm count, BTB integrity and the expression of proteins including p38 mitogen-activated protein kinase (MAPK), activating transcription factor 2 (ATF2), matrix metalloproteinase 9 (MMP9), tissue inhibitor of metalloproteinase 1(TIMP1) and BTB related junction proteins were evaluated. Furthermore, mouse primary Sertoli cells were used to delineate the molecular mechanisms that mediate the effects of PFOS on BTB. Our results demonstrated that PFOS dose-dependently increased BTB permeability, p38/ATF2 phosphorylation and MMP9 expression, paralleled by decrease in BTB junction protein Occludin and Connexin43 expression. Additionally, similar to the in vivo results, treatment of PFOS time-dependently increased Sertoli cell-based BTB permeability, phosphorylated-p38/ATF2 level, translocation of ATF2 into the nucleus and MMP9 expression/activity, paralleled by decrease in Occludin and Connexin43 expression. Meanwhile, inhibition of p38 by SB203580, knockdown of ATF2, or inhibition of MMP9 was sufficient to reduce the effects of PFOS on the Sertoli cell BTB. As such, the present study highlights a role of the p38/ATF2/MMP9 signaling pathway in PFOS-induced BTB disruption, advancing our understanding of molecular mechanisms for PFOS-induced male reproductive disorders.


Assuntos
Ácidos Alcanossulfônicos/toxicidade , Barreira Hematotesticular/efeitos dos fármacos , Fluorocarbonos/toxicidade , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas de Junções Íntimas/efeitos dos fármacos , Fator 2 Ativador da Transcrição/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Conexina 43/efeitos dos fármacos , Relação Dose-Resposta a Droga , Técnicas de Silenciamento de Genes , Imuno-Histoquímica , Masculino , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos ICR , Ocludina/efeitos dos fármacos , Células de Sertoli/efeitos dos fármacos , Contagem de Espermatozoides , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacos
10.
Dig Dis Sci ; 61(9): 2522-34, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27146412

RESUMO

BACKGROUND/AIM: Obstructive jaundice (OJ) is frequently complicated by infections and has been associated with increased bacterial translocation, intestinal epithelial hyperpermeability, and oxidative stress, but the mechanism remains unclear. The potential effect of resveratrol (Res) on modifying intestinal epithelial dysfunction was evaluated both in vitro and in vivo. METHODS: Caco-2 cells (in vitro) and male Wistar rats (n = 60; in vivo) were used to evaluate the role of Res on intestinal epithelial dysfunction. Hydrogen peroxide was used to induce oxidative stress in the Caco-2 cells. In bile duct-ligated group, OJ was successfully established on Day 7 after bile duct ligation, whereas sham-operated and vehicle-treated rats served as controls. Western blot and RT-qPCR were performed to analyze TJ proteins expression in epithelium isolated from rat intestine. RESULTS: Intestinal hyperpermeability was associated with decreased expression and phosphorylation of occludin and zonula occluden (ZO-1), but increased oxidation in Caco-2 cells and the intestinal epithelium. Res treatment increased the epithelial expression and phosphorylation of occludin and ZO-1 in a concentration-dependent manner. Moreover, Res which protected Caco-2 cells from H2O2-induced oxidative damage clearly reduced malondialdehyde level and intracellular reactive oxygen species accumulation, but increased the expression levels of superoxide dismutase and heme oxygenase-1 (HO-1). Further studies showed that Res also inhibited H2O2-induced protein kinase C activity and p38 phosphorylation. Interestingly, these effects of Res were abolished by the HO-1 inhibitor zinc protoporphyrin or knockdown of HO-1 by siRNA. CONCLUSIONS: Res protected gut barrier function possibly by initiating HO-1-dependent signaling which is essential for common expression of key tight junction proteins. It also provides a rationale to develop Res clinical applications of intestinal disorders.


Assuntos
Antioxidantes/farmacologia , Heme Oxigenase-1/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Icterícia Obstrutiva/genética , Estresse Oxidativo/efeitos dos fármacos , Estilbenos/farmacologia , Junções Íntimas/efeitos dos fármacos , Animais , Ductos Biliares/cirurgia , Western Blotting , Células CACO-2 , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Técnicas In Vitro , Mucosa Intestinal/metabolismo , Icterícia Obstrutiva/metabolismo , Ligadura , Masculino , Malondialdeído/metabolismo , Ocludina/efeitos dos fármacos , Ocludina/metabolismo , Permeabilidade/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteína Quinase C/efeitos dos fármacos , Proteína Quinase C/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Resveratrol , Junções Íntimas/metabolismo , Regulação para Cima , Proteína da Zônula de Oclusão-1/efeitos dos fármacos , Proteína da Zônula de Oclusão-1/metabolismo
11.
Inflammation ; 39(3): 1099-107, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27059499

RESUMO

The aim of this study was to investigate the effects of interleukin (IL)-22 on proliferation function and inflammatory mediator production and barrier function of human umbilical vein endothelial cells (HUVECs). The expression of mRNA was detected by RT-PCR. The proliferation ability of cells was evaluated using a cell counting kit assay. Real-time quantitative PCR and Western blot were used to detect the expression of inflammatory mediators. The endothelial barrier permeability was assessed by measuring permeability to FITC-labeled dextran. The distribution of occludin was detected by immunofluorescence. IL-22R1 mRNA expression was noted in HUVECs. IL-22 could enhance the proliferation ability of HUVECs and suppress lipopolysaccharide (LPS)-induced proliferation inhibition in these cells. IL-22 also enhanced the production of CCL2 and CCL20 by HUVECs. Besides, IL-22 could improve barrier function and decrease LPS-induced increased cellular permeability and inhibit the LPS-induced destruction of occludin in HUVECs. IL-22 may play a protective role in the development of vasculitis.


Assuntos
Barreira Hematoaquosa/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Mediadores da Inflamação/análise , Interleucinas/farmacologia , Permeabilidade Capilar/efeitos dos fármacos , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Interleucinas/genética , Ocludina/efeitos dos fármacos , Interleucina 22
12.
Gastroenterology ; 148(7): 1417-26, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25747597

RESUMO

BACKGROUND & AIMS: Intraepithelial lymphocytes that express the γδ T-cell receptor (γδ IELs) limit pathogen translocation across the intestinal epithelium by unknown mechanisms. We investigated whether γδ IEL migration and interaction with epithelial cells promote mucosal barrier maintenance during enteric infection. METHODS: Salmonella typhimurium or Toxoplasma gondii were administered to knockout (KO) mice lacking either the T cell receptor δ chain (Tcrd) or CD103, or control TcrdEGFP C57BL/6 reporter mice. Intravital microscopy was used to visualize migration of green fluorescent protein (GFP)-tagged γδ T cells within the small intestinal mucosa of mice infected with DsRed-labeled S typhimurium. Mixed bone marrow chimeras were generated to assess the effects of γδ IEL migration on early pathogen invasion and chronic systemic infection. RESULTS: Morphometric analyses of intravital video microscopy data showed that γδ IELs rapidly localized to and remained near epithelial cells in direct contact with bacteria. Within 1 hour, greater numbers of T gondii or S typhimurium were present within mucosae of mice with migration-defective occludin KO γδ T cells, compared with controls. Pathogen invasion in Tcrd KO mice was quantitatively similar to that in mice with occludin-deficient γδ T cells, whereas invasion in CD103 KO mice, which have increased migration of γδ T cells into the lateral intercellular space, was reduced by 63%. Consistent with a role of γδ T-cell migration in early host defense, systemic salmonellosis developed more rapidly and with greater severity in mice with occludin-deficient γδ IELs, relative to those with wild-type or CD103 KO γδ IELs. CONCLUSIONS: In mice, intraepithelial migration to epithelial cells in contact with pathogens is essential to γδ IEL surveillance and immediate host defense. γδ IEL occludin is required for early surveillance that limits systemic disease.


Assuntos
Translocação Bacteriana , Quimiotaxia de Leucócito , Células Epiteliais/imunologia , Mucosa Intestinal/imunologia , Linfócitos/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Salmonelose Animal/imunologia , Salmonella typhimurium/patogenicidade , Toxoplasmose Animal/imunologia , Animais , Antígenos CD/genética , Transplante de Medula Óssea , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Células Epiteliais/parasitologia , Interações Hospedeiro-Patógeno , Imunidade Inata , Cadeias alfa de Integrinas/deficiência , Cadeias alfa de Integrinas/genética , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Mucosa Intestinal/parasitologia , Linfócitos/metabolismo , Linfócitos/microbiologia , Linfócitos/parasitologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ocludina/deficiência , Ocludina/efeitos dos fármacos , Permeabilidade , Receptores de Antígenos de Linfócitos T gama-delta/deficiência , Receptores de Antígenos de Linfócitos T gama-delta/efeitos dos fármacos , Salmonelose Animal/genética , Salmonelose Animal/metabolismo , Salmonelose Animal/microbiologia , Salmonella typhimurium/imunologia , Fatores de Tempo , Toxoplasmose Animal/genética , Toxoplasmose Animal/parasitologia , Quimeras de Transplante , Virulência
13.
J Biochem Mol Toxicol ; 29(7): 299-304, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25752684

RESUMO

Antimalarials chloroquine (CQ) and hydroxychloroquine (HCQ) are widely used as antiinflammatory drugs, but side effects include retinopathy and vision loss. The objective of this study was to examine the effect of CQ and HCQ on the barrier integrity of retinal pigment epithelial (RPE) cell monolayers in vitro. Permeability of ARPE-19 cell monolayers was determined using Fluorescein isothiocyanate (FITC)-labeled dextran. The influence of CQ and HCQ on cell death and the expression tight junction molecules was examined. CQ and HCQ significantly increased ARPE-19 monolayer permeability after 3 and 18 h, respectively, and enhanced mRNA levels for claudin-1 and occludin. Cytotoxicity was only observed after 18 h exposure. Thus, CQ and HCQ rapidly enhance RPE barrier permeability in vitro, independent of cytotoxicity or loss of zonula occludens-1, claudin-1, and occludin expression. Our findings suggest that CQ/HCQ-induced permeability of the RPE layer may contribute to blood-retinal barrier breakdown in case of CQ/HCQ-induced retinopathy.


Assuntos
Antimaláricos/farmacologia , Barreira Hematorretiniana/efeitos dos fármacos , Cloroquina/farmacologia , Hidroxicloroquina/farmacologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Junções Íntimas/efeitos dos fármacos , Barreira Hematorretiniana/metabolismo , Claudina-1/efeitos dos fármacos , Claudina-1/genética , Regulação da Expressão Gênica , Humanos , Ocludina/efeitos dos fármacos , Ocludina/genética , Permeabilidade/efeitos dos fármacos , RNA Mensageiro , Epitélio Pigmentado da Retina/metabolismo , Junções Íntimas/metabolismo
14.
BMC Gastroenterol ; 13: 151, 2013 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-24138544

RESUMO

BACKGROUND: This study aimed to investigate the mechanism of the probiotic VSL#3 in acute alcoholic intestinal injury, and evaluate the effect of VSL#3, glutamine,VSL#3+glutamine and heat-killed VSL#3 therapy in a rat model. METHODS: Six- to eight-week-old male wild-type rats were divided into seven groups. To establish the acute alcohol liver disease model, rats received three doses of corn starch dissolved in PBS/40% alcohol administered intra-gastrically every 12 hours. Treatment groups received an intra-gastric dose of VSL#3, Glutamine, heat-killed VSL#3, or VSL#3+Glutamine 30 minutes prior to alcohol administration. The placebo group was treated with PBS prior to alcohol administration. TNFα and endotoxin in plasma was measured by ELISA and Tachypleus Ameboctye Lysate assays, and electron microscopy, Western blotting, and reverse transcription polymerase chain reaction were used to identify the mechanisms of VSL#3 in the regulation of epithelial permeability. RESULTS: First, compared with control group, endotoxin and TNFα in alcohol group was obviously high. At the same time, in VSL#3 group,the expression of endotoxin and TNFα obviously lower than the alcohol group. And the trends of the expression of tight junction proteins in these groups were reversed with the change of endotoxin and TNFα. Second, compared the groups of VSL#3 with glutamine,VSL#3+glutamine and heat-killed VSL#3,we found that both VSL#3 and heat-killed VSL#3, glutamine were as effective as VSL#3+glutamine in the treatment of acute alcohol liver disease, the expression of endotoxin and TNFα were lower than the alcohol group, and tight junction proteins were higher than the alcohol group whereas the expression of tight junction proteins were higher in VSL#3 + glutamine group than either agent alone, but have no significant difference. CONCLUSION: We conclude that VSL#3 treatment can regulate the ecological balance of the gut microflora, preventing passage of endotoxin and other bacterial products from the gut lumen into the portal circulation and down-regulating the expression of TNFα, which could otherwise down-regulate the expression of tight junction proteins and increase epithelial permeability.


Assuntos
Depressores do Sistema Nervoso Central/intoxicação , Etanol/intoxicação , Glutamina/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Permeabilidade/efeitos dos fármacos , Probióticos/farmacologia , Junções Íntimas/efeitos dos fármacos , Animais , Bifidobacterium , Modelos Animais de Doenças , Endotoxinas/sangue , Mucosa Intestinal/metabolismo , Mucosa Intestinal/ultraestrutura , Intestinos/efeitos dos fármacos , Intestinos/lesões , Lactobacillus , Hepatopatias Alcoólicas/prevenção & controle , Masculino , Microscopia Eletrônica de Transmissão , Ocludina/efeitos dos fármacos , Ocludina/genética , Ratos , Streptococcus thermophilus , Junções Íntimas/ultraestrutura , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Proteína da Zônula de Oclusão-1/efeitos dos fármacos , Proteína da Zônula de Oclusão-1/metabolismo
15.
Invest Ophthalmol Vis Sci ; 54(10): 6518-25, 2013 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-24008412

RESUMO

PURPOSE: To investigate whether high glucose (HG)-induced downregulation of connexin 43 (Cx43), a gap junction protein, alters ZO-1 and occludin expression and cell monolayer permeability. METHODS: Rat retinal endothelial cells (RRECs) were grown in normal (N; 5 mM) medium, high glucose (HG; 30 mM) medium, N medium transfected with Cx43 siRNA, or N medium transfected with scrambled siRNA. To determine Cx43, occludin, and ZO-1 protein expression, Western blot (WB) analysis and immunostaining were performed. Gap junction intercellular communication (GJIC) was determined using scrape load dye transfer (SLDT) assay. In parallel, cell monolayer permeability was assessed in the four groups of cells, and in cells transfected with Cx43 plasmid or dominant negative Cx43 plasmid. RESULTS: Connexin 43 protein expression was significantly reduced in cells grown in HG (67 ± 15% of control), and a significant reduction in Cx43 was achieved when cells grown in N medium were transfected with Cx43 siRNA (76 ± 12% of control), with concomitant decrease in GJIC activity. Cells grown in HG showed significant reduction in occludin (77 ± 9% of control) and ZO-1 (80 ± 11% of control) protein level compared with cells grown in N media. Importantly, cells transfected with Cx43 siRNA and grown in N medium showed significant downregulation in occludin (78 ± 8% of control) and ZO-1 (81 ± 6% of control) expression, and exhibited increased cell monolayer permeability. Furthermore, Cx43 upregulation protected cells against HG-induced excess cell monolayer permeability. CONCLUSIONS: Our findings indicate that HG-induced downregulation of Cx43 expression and GJIC may contribute to the breakdown of endothelial barrier tight junctions associated with diabetic retinopathy.


Assuntos
Conexina 43/genética , Regulação para Baixo/efeitos dos fármacos , Glucose/farmacologia , Ocludina/genética , RNA Mensageiro/genética , Retina/metabolismo , Proteína da Zônula de Oclusão-1/genética , Animais , Western Blotting , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/genética , Células Cultivadas , Conexina 43/biossíntese , Conexina 43/efeitos dos fármacos , Diabetes Mellitus Experimental , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Ocludina/biossíntese , Ocludina/efeitos dos fármacos , Ratos , Retina/efeitos dos fármacos , Retina/patologia , Junções Íntimas/efeitos dos fármacos , Proteína da Zônula de Oclusão-1/biossíntese , Proteína da Zônula de Oclusão-1/efeitos dos fármacos
16.
Am J Nephrol ; 37(6): 518-25, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23689670

RESUMO

BACKGROUND: Chronic kidney disease (CKD) impairs intestinal barrier function which by allowing influx of noxious products causes systemic inflammation. We have recently shown that intestinal barrier dysfunction in CKD is due to degradation of epithelial tight junction (TJ) which is, in part, mediated by influx of urea and its conversion to ammonia by microbial urease. We hypothesized that by adsorbing urea and urea-derived ammonia, oral activated charcoal (AST-120) may ameliorate CKD-induced intestinal epithelial barrier disruption and systemic inflammation. METHODS: Rats were randomized to the CKD or control groups. The CKD group was fed a chow containing 0.7% adenine for 2 weeks. They were then randomized to receive a chow with or without AST-120 (4 g/kg/day) for 2 weeks. Rats consuming regular diet served as controls. Animals were then euthanized, colons were removed and processed for Western blot and immunohistology, and plasma was used to measure endotoxin and oxidative and inflammatory markers. RESULTS: Compared with the controls, the untreated CKD rats showed elevated plasma endotoxin, IL-6, TNF-α, MCP-1, CINC-3, L-selectin, ICAM-1, and malondialdehyde, and depletions of colonic epithelial TJ proteins, claudin-1, occludin, and ZO1. Administration of AST-120 resulted in partial restoration of the epithelial TJ proteins and reduction in plasma endotoxin and markers of oxidative stress and inflammation. CONCLUSIONS: CKD animals exhibited depletion of the key protein constituents of the colonic epithelial TJ which was associated with systemic inflammation, oxidative stress and endotoxemia. Administration of AST-120 attenuated uremia-induced disruption of colonic epithelial TJ and the associated endotoxemia, oxidative stress and inflammation.


Assuntos
Carbono/farmacologia , Fármacos Gastrointestinais/farmacologia , Enteropatias/etiologia , Mucosa Intestinal/efeitos dos fármacos , Óxidos/farmacologia , Insuficiência Renal Crônica/complicações , Junções Íntimas/efeitos dos fármacos , Administração Oral , Adsorção , Animais , Claudina-1/efeitos dos fármacos , Claudina-1/metabolismo , Endotoxemia/complicações , Endotoxinas/metabolismo , Inflamação , Mediadores da Inflamação/imunologia , Enteropatias/imunologia , Enteropatias/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Masculino , Ocludina/efeitos dos fármacos , Ocludina/metabolismo , Permeabilidade , Ratos , Ratos Sprague-Dawley , Junções Íntimas/imunologia , Junções Íntimas/metabolismo , Ureia/imunologia , Ureia/metabolismo , Proteína da Zônula de Oclusão-1/efeitos dos fármacos , Proteína da Zônula de Oclusão-1/metabolismo
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