Role Of MLCK-mediated MLC Phosphorylation In Intestinal Epithelial Barrier Dysfunction After Severe Burn Injury

Background & AimsAn intact intestinal barrier is critical to normal physiological function and prevention of disease, and plays an important role in defending the translocation of opportunistic bacteria and toxins from the gut. It is well recognized that severe burn injury often induces the loss of intestinal barrier function, increased intestinal permeability, and the translocation of bacteria and/or endotoxin from the gut, which can lead to the development of distant organ damages and multiple organ failure. However, the mechanism and the signalling cascade of the intestinal barrier dysfunction after severe burn injury are not fully elucidated.The structure fundament of maintaining the intestinal barrier function is tight junction (TJ) and its associated proteins. TJ is the most apical component of the intercellular junctional complex, which is composed of multiple proteins interactionally, and plays an important role in regulating the intestinal permeability and cell polarity. There are at least three different types of transmembrane proteins, i.e., ZO-1, occludin and claudin. ZO-1 protein, an actin-and-myosin-binding protein, can communicate with the exterior signals of the cell. The relevant stimulating signal can alter the structure of linking proteins and impair the barrier function via skeleton protein activity and cell retraction, which forms intercellular space and increases the permeability.It has been shown that the changes and regulation of barrier permeability involve cytoskeleton rearrangement or redistribution, tight junction, adheren junction structure and so on. Cell retraction is the common pathway for the increase of barrier permeability, and mainly regulated by actin and myosin via a myosin light chain (MLC) phosphorylation-dependent mechanism.Although MLC phosphorylation is involved in the regulation of biological barrier function, the role of MLC phosphorylation in the pathogenesis of intestinal barrier dysfunction induced by severe burn injury is poorly understood. The aim of this study was to determine the role of MLC kinase (MLCK)-mediated MLC phosphorylation in intestinal epithelial barrier dysfunction after severe burn injury, hoping to better understand the mechanism of postburn intestinal barrier dysfunction.Methods1. Healthy adult Sprague Dawley rats were randomly divided into control, 1, 2, 6, 12 and 24h postburn groups. The intestinal permeability of rats was measured by fluorescein-isothiocyanate-dextran (FITC-Dextran) method. The changes of tight junction protein ZO-1, cytoskeleton F-actin, and the protein expression of phosphorylated MLC (p-MLC) in intestinal epithelia of the rats were analyzed by immunofluorescence.2. Healthy adult BALB/c mice were randomly divided into four groups, i.e., (1) sham burn mice given vehicle, (2) sham burn mice given ML-9 (2mg/kg peritoneal injection, immediately after sham burn), (3) mice given a 30% total body surface area (TBSA) full-thickness burn, and (4) burn rats given ML-9 (2mg/kg peritoneal injection, immediately after burn injury). The intestinal permeability of mice was measured by FITC-dextran method. The histological changes of the intestinal mucosa were observed by microscope and electromicroscope. The changes of F-actin and tight junction protein ZO-1 in intestinal mucosa were analyzed by immunofluorescence. The expressions of MLCK and p-MLC in intestinal mucosa were analyzed by Western blot.3. The cultured Caco-2 cell monolayers were randomly divided into control, 2, 6, 8, 12 and 24h posthypoxia groups. In some monolayers, the Caco-2 cells were treated with ML-9 (100μmol/L) prior to normoxia or hypoxia. Hypoxia was induced by 1%O2. The transepithelial electrical resistance (TER) of the monolayers was monitored with a volt/ohmmeter. The protein expressions of MLCK, MLC and p-MLC in Caco-2 cells were detected by Western blot analysis.Results1. After severe burn injury, the intestinal permeability of the rats increased significantly when compared with that of control, and peaked at 6h with 2.41 times higher than that of control. The increased intestinal permeability was accompanied by the striking reorganization of F-actin and the tight junction protein ZO-1, and the significant increase of p-MLC in intestinal epithelia of the burned rats.2. When compared with that of the control mice, the intestinal permeability of the burned mice increased significantly at 6h. Meanwhile, the intestinal mucosa was damaged, and the intercellular space of intestinal mucosa was increased in burned mice. The protein expressions of both MLCK and p-MLC were increased significantly. However, treatment of the mice with ML-9, a specific inhibitor of MLCK, can lessen the burn-induced changes mentioned above.3. Hypoxia treatment caused a significant decrease of TER in Caco-2 cell monolayers. Hypoxia also induced the increased expressions of MLCK and p-MLC protein in Caco-2 cells, whereas the expression of MLC protein was not significantly affected by hypoxia treatment. However, treatment the Caco-2 cell monolayers with ML-9, a specific inhibitor of MLCK, prevented the TER decrease and p-MLC increase, but not the MLCK increase induced by hypoxia.Conclusions1. The intestinal epithelial permeability increase significantly following severe brun injury, and peaks at 6h postburn. The rearragement or redistribution of F-actin and tight junction protein ZO-1 may contribute to the intestinal epithelial barrier dysfunction following severe burn injury.2. The increased intestinal epithelial permeability induced by severe burn injury is accompanied by the increased protein expressions of both MLCK and p-MLC. Specific inhibition of MLC phosphorylation mediated by MLCK can attenuate the permeability increase, the mucosa damage and the changes of F-actin and tight junction protein ZO-1, suggesting that the MLCK-mediated MLC phosphorylation is involved in the pathogenesis of intestinal epithelial barrier dysfunction following severe burn injury.3. The intestinal barrier dysfunction induced by hypoxia is also accompanied by the increase of both MLCK protein expression and MLC phosphorylation. Inhibiting MLCK activity prevents both the increased MLC phosphorylation and the damaged barrier function induced by hypoxia. It is suggested that the MLCK-mediated MLC phosphorylation may also play an critical role in the intestinal barrier dysfunction induced by hypoxia.