3,5,7,3′,4′-Pentamethoxyflavone Enhances the Barrier Function through Transcriptional Regulation of the Tight Junction in Human Intestinal Caco-2 Cells
Yunika Mayangsari, Natsumi Sugimachi, Wenxi Xu, Chinatsu Mano, Yuki Tanaka, Osamu Ueda, Tomohiro Sakuta, Yoshiharu Suzuki, Yoshinari Yamamoto, Takuya Suzuki
Abstract
The intestinal tight junction (TJ) barrier plays a pivotal role in the regulation of intestinal homeostasis. This study investigated the effects of 3,5,7,3′,4′-pentamethoxyflavone (PMF), a major polymethoxyflavone found in black ginger, on TJ barrier regulation using intestinal Caco-2 cells. PMF treatment enhanced the TJ barrier integrity in Caco-2 cells, indicated by increased transepithelial electrical resistance (control, 1261 ± 36 Ω·cm2; 100 μM PMF, 1383 ± 55 Ω·cm2 at 48 h, p < 0.05) and decreased permeability to fluorescein-conjugated dextran (control, 24.2 ± 1.8 pmol/(cm2 × h); 100 μM PMF, 18.6 ± 1.0 pmol/(cm2 × h), p < 0.05). Immunoblot analysis revealed that PMF increased the cytoskeletal association and cellular expression of the TJ proteins, zonula occludens-1, claudin-3, and claudin-4 (e.g., occludin; control, 1.00 ± 0.2; 100 μM PMF, 3.69 ± 0.86 at 48 h, p < 0.05). Quantitative reverse transcriptase-polymerase chain reaction analysis and a luciferase promoter assay showed that PMF enhanced the transcription of occludin, claudin-3, and claudin-4. The promoter assay with site-directed mutagenesis indicated that PMF-induced occludin and claudin-3 transcription was mediated by transcription factors, KLF5 and EGR1, respectively, while PMF activated claudin-4 transcription through GATA1 and AP1. Taken together, the transcriptional regulation of TJ proteins is involved in PMF-mediated promotion of the intestinal barrier in vitro.