Myosin Light Chain Kinase Is Involved in Lipopolysaccharide-Induced Disruption of Colonic Epithelial Barrier and Bacterial Translocation in Rats

Abstract

Sepsis is associated with bacterial translocation (BT)and changes in colonic paracellular permeability (CPP), but the link between these effects is unknown. The present study aimed to identify whether changes in CPP after lipopolysaccharide (LPS) administration triggers BT, colonic inflammation, visceral pain, and sickness behavior and to evaluate the role of myosin light chain kinase (MLCK) in colonocyte cytoskeleton contraction. Rats received the MLCK inhibitor ML-7 alone or combined with LPS. CPP was measured for 6 hours after administration. Visceral pain, food intake, BT, electron microscopy of tight junctions of colonocytes, cytokine levels, and Western blotting of phosphorylated MLC from colonic mucosa were assessed in a time range of 0 to 3 hours after treatment. Sepsis increased CPP at 0 to 6 hours after LPS and associated with tight junction morphological changes, increased MLC phosphorylation, and mucosal release of proinflammatory cytokines. Massive BT, visceral hyperalgesia, and reduced food intake were also observed. Addition of ML-7 prevented all LPSinduced effects, except for changes in food intake. In conclusion, LPS-mediated effects on CPP include gut inflammation, BT, and visceral hyperalgesia. Inhibition of MLCK-dependent colonocyte cytoskeleton contraction by ML-7 prevents the LPS-induced alterations of CPP and its subsequent effects. The gastrointestinal mucosal barrier plays a pivotal role in the body's protection against luminal pathogens and antigenic molecules. The impairment of the gastrointestinal barrier function with massive bacterial translocation (BT) occurs during sepsis, both in human and in animal models. [1][2][3][4][5] In these circumstances, bacteria and their lysis products, such as lipopolysaccharide (LPS), gain access to portal and systemic circulations, producing critical deleterious effects. Sepsis is the most common cause of death for hospitalized patients. The gastrointestinal barrier includes secreted mucus and the epithelial cell layer itself, which prevent BT of intestinal flora through both transcellular and paracellular pathways.7 Tight junction (TJ) opening is the major limiting factor of the paracellular pathway. Examination of isolated human and rodent small intestine cells and cell lines have shown that TJ opening is driven by myosin light chain (MLC) phosphorylation, which depends on myosin light chain kinase (MLCK) activation. 8,9 MLC phosphorylation occurs within the perijunctional actomyosin ring and co-localizes with the TJ.9 Studies using an inducible active MLCK have shown that this activity is sufficient to activate the downstream events necessary for TJ regulation. 10Moreover MLCK-mediated regulation of TJ opening appears to be a common intermediate in a variety of physiological and pathophysiological pathways related to altered paracellular permeability both in vitro and in vivo. 10 -12 For example, enteropathogenic Escherichia coli infection causes diarrhea in children. An in vitro model has demonstrated that enteropathogenic E. coli dramatically ...

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