Naringenin enhances intestinal barrier function through the expression and cytoskeletal association of tight junction proteins in Caco-2 cells

Abstract

We have previously reported that naringenin promotes the tight junction (TJ) integrity in intestinal Caco-2 cells. This study investigated the naringenin-mediated effect in Caco-2 cells with a particular focus on the modulation of TJ structure and claudin-4 transcriptional regulation. Naringenin (10~100 μM) dose-dependently enhanced TJ barrier integrity of Caco-2 cells, indicated by transepithelial electrical resistance and FITC-dextran flux. Immunoblot analysis showed that naringenin increased the cytoskeletal association of TJ proteins, zonula occludens-2, occludin, claudin-1, and claudin-4, simultaneously with increased occludin phosphorylation. The total expression of claudin-4 was also increased by naringenin. Quantitative RT-PCR and luciferase reporter assay revealed that naringenin transcriptionally increased the claudin-4 expression with activation of claudin-4 promoter. The mutation of the binding site of the transcriptional factor Sp1 in the claudin-4 promoter sequence and the pharmacological inhibition of Sp1 partially suppressed the naringenin-mediated activation of the claudin-4 promoter. Further, naringenin induced the heat shock protein 70 expression in the cells. Naringenin enhances barrier integrity through the assembly and expression of TJ proteins in intestinal epithelial cells. Naringenin-mediated claudin-4 expression occurs, at least partially, through Sp1-dependent transcriptional regulation. The induction of heat shock protein 70 may be also involved in the increased claudin-4 expression.