RESUMO
We investigated the effect of theaflavins (TFs) on membrane barrier of Caco-2 cells. For fluorescein-transport experiments, the apparent permeability (Papp) of fluorescein in Caco-2 cells pretreated with 20 µM TFs were significantly decreased compared with that in untreated cells. Although the respective monomeric catechins did not show any Papp reduction, purpurogallin pretreatment resulted in a significant Papp reduction similar to that of TF-3'-O-gallate (TF3'G) pretreatment. This indicates that the benzotropolone moiety may play a crucial role in the Papp reduction or tight junction (TJ)-closing effect induced by TFs. In TF-3'-O-gallate-pretreated Caco-2 cells, fluorescein transport was completely restored by compound C (AMPK inhibitor). In addition, TF3'G significantly increased both the mRNA and protein expression of TJ-related proteins (occludin, claudin-1, and ZO-1) as well as the phosphorylation of AMPK. It was, thus, concluded that TFs could enhance intestinal barrier function by increasing the expression of TJ-related proteins through the activation of AMPK in Caco-2 cells.
Assuntos
Proteínas Quinases Ativadas por AMP/genética , Antioxidantes/farmacologia , Benzocicloeptenos/farmacologia , Biflavonoides/farmacologia , Catequina/farmacologia , Fluoresceína/metabolismo , Ácido Gálico/análogos & derivados , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Células CACO-2 , Permeabilidade da Membrana Celular/efeitos dos fármacos , Claudina-1/agonistas , Claudina-1/genética , Claudina-1/metabolismo , Relação Dose-Resposta a Droga , Ácido Gálico/farmacologia , Regulação da Expressão Gênica , Humanos , Ocludina/agonistas , Ocludina/genética , Ocludina/metabolismo , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-1/agonistas , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
We investigated whether tomatoside A (5α-furostane-3ß,22,26-triol-3-[O-ß-d-glucopyranosyl (1â2)-ß-d-glucopyranosyl (1â4)-ß-d-galactopyranoside] 26-O-ß-d-glucopyranoside), a tomato seed saponin, may play a role in the regulation of intestinal glucose transport in human intestinal Caco-2 cells. Tomatoside A could not penetrate through Caco-2 cell monolayers, as observed in the transport experiments using liquid chromatography-mass spectrometry. The treatment of cells with 10 µM tomatoside A for 3 h resulted in a 46.0% reduction in glucose transport as compared to untreated cells. Western blotting analyses revealed that tomatoside A significantly (p < 0.05) suppressed the expression of glucose transporter 2 (GLUT2) in Caco-2 cells, while no change in the expression of sodium-dependent glucose transporter 1 was observed. In glucose transport experiments, the reduced glucose transport by tomatoside A was ameliorated by a protein kinase C (PKC) inhibitor and a multidrug resistance-associated protein 2 (MRP2) inhibitor. The tomatoside A-induced reduction in glucose transport was restored in cells treated with apical sodium-dependent bile acid transporter (ASBT) siRNA or an ASBT antagonist. These findings demonstrated for the first time that the nontransportable tomato seed steroidal saponin, tomatoside A, suppressed GLUT2 expression via PKC signaling pathway during the ASBT-influx/MRP2-efflux process in Caco-2 cells.
Assuntos
Transportador de Glucose Tipo 2/metabolismo , Glucose/metabolismo , Glicosídeos/farmacologia , Extratos Vegetais/farmacologia , Solanum lycopersicum/química , Transporte Biológico/efeitos dos fármacos , Células CACO-2 , Transportador de Glucose Tipo 2/genética , Humanos , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Saponinas/farmacologia , Sementes/químicaRESUMO
The current study demonstrated that theasinensin A (TSA) had a potential to form the complex with hydrophobic Trp-containing dipeptides, and to reduce their membrane potential by artificial-lipid membrane taste sensor. At a 1:3 molar ratio of the 6 Trp-containing dipeptides together with TSA, we observed a significant chemical shift of the protons of the dipeptides (Δδ) to a high magnetic field, when analyzed using 1H-nuclear-magnetic resonance (NMR) spectroscopy. The Δδ values were correlated with the hydrophobicity (log P) of the dipeptides and significant correlations were obtained (P = 0.022, R2 = 0.77); e.g., Trp-Leu with the highest log P value of 1.623 among the tested dipeptides showed the highest Δδ value of 0.105 ppm for the H7 proton of Trp-Leu, while less chemical shifts were observed in theasinensin B and epigallocatechin-3-O-gallate. Diffusion-ordered NMR spectroscopy revealed that the diffusion coefficient of 3 mM of Trp-Leu (7.6 × 10-11 m2/s) at a pulse field gradient in the range 0.05-0.3 T/m decreased in the presence of 3 mM TSA (6.6 × 10-11 m2/s), suggesting that Trp-Leu forms a complex with TSA. Quantum mechanical calculations and rotating frame nuclear Overhauser effect-NMR spectroscopy provided configuration information on the geometry of the complex that Trp-Leu formed with TSA (1:1 complex) with a ΔG energy of -8.7 kJ/mol. A sensor analysis using artificial-lipid membranes demonstrated that the changes in membrane potential of 1 mM Trp-Leu (21.8 ± 1.3 mV) and Leu-Trp (5.3 ± 0.9 mV) were significantly (P < 0.001) reduced by 1 mM TSA (Trp-Leu, 13.1 ± 2.4 mV; Leu-Trp, 3.5 ± 0.5 mV; TSA alone, 0.2 ± 0.01 mV), indicating the effective suppression of hydrophobicity of dipeptides by TSA-formed complex.