Impact of deoxynivalenol and kaempferol on expression of tight junction proteins at different stages of Caco-2 cell proliferation and differentiation

Abstract

The barrier function of intestinal tract is essential to gut health, and the expression of tight junction (TJ) proteins in human epithelial colorectal adenocarcinoma (Caco-2) cells, mimicking the intestinal stem cell proliferation and differentiation, was investigated after treatment by the mycotoxin of deoxynivalenol (DON) and phenolic compound of kaempferol (KAM). The results showed that DON (5 μM) significantly reduced the expression of claudin-4 while kaempferol (100 μM) increased the expression of claudin-3 during Caco-2 cell proliferation. After being cultured for 11 days, the DON treatment, unexpectedly, augmented the expression of claudin-4 with an increased TEER. For differentiated Caco-2 cells after a 21 day culture, both the TEER and claudin-4 levels were significantly reduced by DON while KAM pretreatment alleviated the damage caused by DON accompanying an increase of TEER, claudin-3, and ZO-1. Thus, kaempferol and DON differentially affected the expression of tight junction proteins at different stages of Caco-2 cell proliferation and differentiation, and an increase of the integrity of TJ after KAM pretreatment indicates KAM has the potential to protect the integrity of the barrier function of the intestinal epithelium for improved health.

Fig. 1. The TEER value (A) and FD-4 permeability (B) at different stages of Caco-2 cell proliferation and differentiation. The data were presented as mean ± SD of at least three replicates.

Fig. 2. Alkaline phosphatase activity at different stages of proliferation and differentiation of Caco-2 cells modeling. AP: apical side, BL: basolateral side. The data were presented as mean ± SD of at least three replicates.

Fig. 3. Effects of DON (A) and kaempferol (B) on proliferation inhibition rate of Caco-2 cells. The data were presented as mean ± SD of at least three replicates.

Fig. 4. Effect of DON and KAM on the permeability (normalized by control) of Caco-2 cell monolayer after treatment by DON (A and B) or KAM (C and D) for 24 h. Fold changes referred to the ratio with initial values measured before treatment. The data were presented as mean ± SD of at least three replicates. Different letters represent a significant difference at a level of p < 0.05.

Fig. 5. Effects of KAM pretreatment on the permeability (A and B) and tight junction protein expression (C and D) in Caco-2 cells during the proliferation stage (3 days after inoculation). The fold changes refer to the ratios relative to initial values before treatment. The data were presented as mean ± SD of at least three replicates. Different letters of the same parameter indicating significant differences at the 0.05 level.

Fig. 6. Effects of KAM pretreatment on the permeability (A and B) and tight junction protein expression (C and D) in Caco-2 cells during the intermediate stage of differentiation (11 days after inoculation). The fold changes refer to the ratios relative to initial values before treatment. The data were presented as mean ± SD of at least three replicates. Different letters of the same parameter indicating significant differences at the 0.05 level.

Fig. 7. Effects of KAM pretreatment on the permeability (A and B) and tight junction protein expression (C and D) in differentiated Caco-2 cell monolayer (21 days after inoculation). The fold changes refer to the ratios relative to initial values before treatment. The data were presented as mean ± SD of at least three replicates. Different letters of the same parameter indicating significant differences at the 0.05 level.

Fig. 8. The heatmap analysis of the TJ protein expression and the permeability of the Caco-2 cell models at stages of proliferation and differentiated state (left side), and including the intermediate stage (11th day). A: control, B: DON, C: KAM, D: KAM + DON. The number after the parameter represents different stages (1: 3rd day, 2: 11th day, 3: 21st day).