Distinct interactions of proanthocyanidins of various degrees of polymerization with TLR4 receptor on the integrity of differentiating Caco-2 cell monolayers through tight junction protein expression

Many studies have demonstrated the potential of proanthocyanidins (PACs) to protect the intestinal epithelial barrier by regulating the cellular levels of tight junction (TJ) proteins. The effects of five PAC fractions with different mean degrees of polymerization (DP), namely, DT-PAC (dimers – trimers), OPAC-1 (oligomeric-PAC, pentamer – hexamer), OPAC-2 (octamer – dodecamer), PPAC-1 (polymeric-PAC, 13–15 subunits), and PPAC-2 (20 subunits), on the barrier integrity of differentiating Caco-2 cell monolayers were investigated. The effects of PAC fractions on the integrity of Caco-2 monolayers were evaluated by measuring the permeability of fluorescein isothiocyanate (FITC)-dextran and cellular levels of TJ proteins by western blotting. DT-PAC fraction favored the barrier integrity by significantly increasing the cellular protein levels of barrier-forming zonula occludens-1 (ZO-1) and claudin-3. The effects of OPAC fractions were controversial as the treatments simultaneously increased the cellular protein levels of barrier-forming claudin-1 and pore-forming claudin-2. The PPAC fractions significantly increased the permeation of FITC-dextran through Caco-2 monolayers, indicating a loss in barrier integrity. PPAC fractions disrupted the barrier integrity by reducing the cellular protein levels of barrier-forming ZO-1 and claudin-3 while increasing the pore-forming claudin-2. Molecular docking experiments revealed that PAC molecules with higher DP may disrupt Caco-2 monolayers by stimulating the cell surface toll-like receptor 4 (TLR4). Interestingly, PAC molecules with lower DP may inhibit the activation of TLR4 signaling. Highly polymerized PACs may disrupt the barrier integrity of intestinal epithelium undergoing differentiation by suppressing barrier-forming and upregulating the pore-forming TJ proteins, while less polymerized PACs protect barrier integrity by depicting opposing effects.