Lactobacillus reuteri 1 Enhances Intestinal Epithelial Barrier Function and Alleviates the Inflammatory Response Induced by Enterotoxigenic Escherichia coli K88 via Suppressing the MLCK Signaling Pathway in IPEC-J2 Cells

Abstract

Intestinal epithelial barrier injury disrupts immune homeostasis and leads to many intestinal disorders. Lactobacillus reuteri (L. reuteri) strains can influence immune system development and intestinal function. However, the underlying mechanisms of L. reuteri LR1 that regulate inflammatory response and intestinal integrity are still unknown. The present study aimed to determine the effects of LR1 on the ETEC K88-induced intestinal epithelial injury on the inflammatory response, intestinal epithelial barrier function, and the MLCK signal pathway and its underlying mechanism. Here, we showed that the 1 × 109 cfu/ml LR1 treatment for 4 h dramatically decreased interleukin-8 (IL-8) and IL-6 expression. Then, the data indicated that the 1 × 108 cfu/ml ETEC K88 treatment for 4 h dramatically enhanced IL-8, IL-6, and tumor necrosis factor-α (TNF-α) expression. Furthermore, scanning electron microscope (SEM) data indicated that pretreatment with LR1 inhibited the ETEC K88 that adhered on IPEC-J2 and alleviated the scratch injury of IPEC J2 cells. Moreover, LR1 pretreatment significantly reversed the declined transepithelial electrical resistance (TER) and tight junction protein level, and enhanced the induction by ETEC K88 treatment. Additionally, LR1 pretreatment dramatically declined IL-8, IL-17A, IL-6, and TNF-α levels compared with the ETEC K88 group. Then, ETEC K88-treated IPEC-J2 cells had a higher level of myosin light-chain kinase (MLCK), higher MLC levels, and a lower Rho-associated kinase (ROCK) level than the control group, while LR1 pretreatment significantly declined the MLCK and MLC expression and enhanced ROCK level in the ETEC K88-challenged IPEC-J2 cells. Mechanistically, depletion of MLCK significantly declined MLC expression in IPEC-J2 challenged with ETEC K88 compared to the si NC+ETEC K88 group. On the other hand, the TER of the si MLCK+ETEC K88 group was higher and the FD4 flux in the si MLCK+ETEC K88 group was lower compared with the si NC+ETEC K88 group. In addition, depletion of MLCK significantly enhanced Claudin-1 level and declined IL-8 and TNF-α levels in IPEC-J2 pretreated with LR1 followed by challenging with ETEC K88. In conclusion, our work indicated that L. reuteri LR1 can decline inflammatory response and improve intestinal epithelial barrier function through suppressing the MLCK signal pathway in the ETEC K88-challenged IPEC-J2.