Butyrate increases tight junction protein expression and enhances tight junction integrity in porcine IPEC-J2 cells stimulated with LPS

Abstract Text:

The intestinal mucosal barrier is maintained by tight junctions, which are intercellular adhesion complexes and prevent the passage of pathogens and toxins through the paracellular space. Dysfunction of tight junctions induced by endotoxin and mycotoxin is highly associated with a variety of gastrointestinal disorders in pigs. Butyrate has been shown to possess immunological and metabolic modulatory effects in various cells and tissues. Therefore, we investigated protective effect of butyrate on cell integrity and tight junction protein expressions during LPS stimulation in porcine IPEC-J2 cells. We found that butyrate (1mM) and LPS (10µg/ml) significantly induced TNFα, IL-1β, IL-6, IL-8 and MCP1 expression (P<0.05) as well as IL-8 secretion. However, although LPS upregulated TLR4 expression, butyrate downregulated it (P<0.01) indicating butyrate could inactivate LPS stimulation of TLR4 pathway. Barrier integrity was investigated with trans-epithelial electrical resistance (TEER) and fluorescein isothiocyanate-dextran (FITC-dextran) uptake based tests. Treatment with LPS for 24 h significantly decreased TEER (P=0.01) and increased cell permeability (P=0.02). On the contrary, butyrate (1mM) significantly increased TEER (P<0.01) and decreased cell permeability (P<0.01), indicating that butyrate could increase cell integrity and enhance epithelial barrier against LPS-induced damage. Butyrate also induced Claudin-1 (P=0.09), Claudin-3 (P<0.01) and Claudin-4 (P<0.01) mRNA expression, and Claudin-3 protein expression (P<0.05) in a dose-dependent manner, perhaps accounting for the increase in epithelial barrier integrity induced by butyrate. Butyrate also increased (P<0.01) activation of Akt by phosphorylation, whereas LPS exerted the opposite effect. Taken together, butyrate increased basal immune response and enhanced the integrity of the intestinal mucosal barrier against LPS-induced damage through an upregulation of cytokine expression and an increase in the synthesis of tight junction proteins.

Keywords: Akt, butyrate, epithelial barrier integrity, IPEC-J2 cells, tight junction protein