Effects of Lactobacillus Reuteri LR1 on Tight Junction Proteins Expression in Ipec-1 Cells during Enterotoxigenic Escherichia coli K88 Infection and Its Underlying Mechanisms

Intestinal epithelial barrier damages disrupt immune homeostasis and lead to many intestinal diseases. Lactobacilli reuteri strains have probiotic functions in modulation of microbiota and immune system in intestines. In this study, we investigated effects of L. reuteri LR1, a new strain isolated from the feces of weaning piglets, on intestinal epithelial barrier damages in IPEC-1 cells caused by enterotoxigenic Escherichia coli (ETEC) K88 infection. IPEC-1 cells were cultured in transwell dishes for 21 d until their transepithelial electrical resistance was stable. Monolayers of IPEC-1 cells were incubated for 6 h with serum and antibiotics-free medium, ETEC K88 (1×10⁷ CFU), L. reuteri LR1 (1×10⁸ CFU), or ETEC K88 (1×10⁷ CFU) plus L. reuteri LR1 (1×10⁸ CFU) in the upper layer. The FITC-dextran was used to determine the permeability of IPEC-1 cells monolayers. IPEC-1 cells were collected for colony counting, Real-time PCR and Western blotting analysis. Statistical analysis was performed using one-way ANOVA with SPSS 16.0 software. Our findings showed that L. reuteri LR1 effectively attenuated ETEC K88-induced increases of permeability of IPEC-1 cell monolayers (P<0.05), and decreased the adhesion and invasion of ETEC K88 in IPEC-1 cells (P<0.05). In addition, we demonstrated that L. reuteri LR1 increased the mRNA and protein levels of tight junction proteins zonula occluden-1 (ZO-1) and occludin in ETEC K88-infected IPEC-1 cells (P<0.05), whereas it had no effects on claudin-1 and F-actin expression (P>0.05). Using Colloidal gold immunoelectron microscopy, we confirmed that L. reuteri LR1 visualizedly increases ZO-1 and occludin expression in IPEC-1 cells. To explore the regulatory mechanisms of tight junction proteins by L. reuteri LR1, we investigate the effects of L. reuteri LR1 on myosin light chain kinase (MLCK) and phosphorylated MLCK in ETEC K88-infected IPEC-1 cells, and selected an inhibitor of MLCK (ML-7). We found L. reuteri LR1 treatment effectively attenuated ETEC K88-induced decreases of protein levels of MLCK in IPEC-1 cells (P<0.05). Furthermore, we found that the improvements of ZO-1 and occludin expression by L. reuteri LR1 were inhibited by ML-7 in ETEC K88-infected IPEC-1 cells (P<0.05). These data demonstrated that L. reuteri LR1’s improvements of ZO-1 and occludin could be dependent of MLCK pathway. In conclusion, L. reuteri LR1 improved ZO-1 and occludin expression via a MLCK-dependent manner in IPEC-1 cells during ETEC-K88 infection.