In this study, it is demonstrated that severe burn injury causes the increase of intestinal permeability, and that the increased intestinal permeability induced by burn injury is accompanied by the histological damage of intestinal mucosa, redistribution of tight junction protein ZO-1 as well as upregulation of both MLCK protein and MLC phosphorylation. Our present study also reveals that MLCK inhibition with specific inhibitor ML-9 attenuates the burn-induced intestinal barrier disruption in vivo. These findings provide a new insight into the mechanisms involved in the intestinal barrier breakdown caused by severe burn injury.
It has been well documented that many critical surgical diseases, such as shock, trauma and burn injury, causes the disruption of intestinal epithelial barrier function, leading to the leakage of bacteria, microbial products or other antigens from the intestinal lumen into the mucosa or systemic circulation to initiate or exacerbate an inflammatory response 
. Here, we demonstrate that intestinal epithelial permeability to 4.4 kDa FITC-dextran starts to increase at 1 hour, peaks at 6 hours, and is still higher than sham burn at 24 hours following burn injury. This time-course pattern of postburn intestinal permeability is similar to a recently published study which reveals intestinal permeability to 4 kDa FITC-dextran peaks at 4 hours and returns to baseline at 24 hours following severe burn 
. In addition, our present data show that the burn-caused increase of intestinal permeability is accompanied by the histological damage of intestinal mucosa, which is consistent with other previous studies 
. Taken together, an intact intestinal epithelial barrier function is disrupted after severe thermal injury.
Given that the intestinal barrier disruption is induced following severe burn, the underlying mechanisms are still incompletely elucidated. Actually, the early systemic and intestinal damage caused by thermal injury is orchestrated by a series of pathophysiological events such as ischemia, hypoxia, and inflammation, which have been demonstrated to contribute to intestinal barrier dysfunction. We and others have previously shown that hypoxia or ischemia, which occurs rapidly following severe burns, is capable of causing barrier disruption 
. Furthermore, it has been well documented that some proinflammatory cytokines such as TNF-α, IL-1β, IL-6 and IFN-γ are significantly up-regulated in severely burned mice, rats and patients 
. These up-regulated proinflammatory cytokines may contribute to the burn-induced intestinal barrier disruption. We, along with other investigators, have previously revealed that proinflammatory cytokines, including IFN-γ, TNF-α, IL-1β, IL-6, IL-13 and TNF superfamily member LIGHT, are able to induce intestinal epithelial barrier defects 
. Hence, it is reasonable to suggest that ischemia, hypoxia and inflammation contribute to the intestinal epithelial barrier breakdown induced by severe burn injury.
Intestinal barrier function disruption is characterized by the increased paracellular permeability as well as the changes of tight junction protein expression and organization 
. Previously published data have demonstrated that hypoxia and proinflammatory cytokines are able to induce the relocalization of tight junction proteins 
. Here, we show that the intestinal barrier defects following severe burn injury is accompanied by the reorganization of tight junction proteins ZO-1, occludin and claudin-1. This is similar to the previous study revealing that both decreased expression and reorganization of intestinal tight junction proteins ZO-1 and occludin are induced in balb/c mice undergoing a 30% TBSA steam burn 
. However, it has been reported that both mRNA and protein expression of occludin are up-regulated in Wistar rats inflicted with 30% TBSA scald injury 
. Thus, it is suggested that the reorganization of tight junction proteins is involved in the burn-induced intestinal barrier disruption, whereas the role of altered expression of tight junction proteins is still controversial, and need to be defined.
MLCK activation, which directly leads to the phosphorylation of MLC, has been viewed as a common final pathway of acute tight junction regulation in response to a broad range of stimuli 
. It has been reported that MLCK activation alone is sufficient to increase tight junction permeability, which is associated with biological and morphological reorganization of the tight junction proteins ZO-1 and occludin 
. Our previous in vitro
studies have shown that increased MLC phosphorylation mediated by upregulated MLCK protein expression is critical to barrier breakdown induced by hypoxia or proinflammatory cytokines 
, and that the barrier dysfunction is prevented by pharmacological MLCK inhibition 
. In this study, we demonstrate that after severe burn injury, intestinal barrier breakdown is accompanied by the upregulation of both MLCK protein expression and MLC phosphorylation in ileal mucosa. In addition, MLCK inhibition with ML-9, an MLCK inhibitor that is known to block MLCK activity 
, not only abolishes the burn-induced increase of MLC phosphorylation in ileal mucosa, but also attenuates the increased intestinal permeability, histological damage of mucosa, as well as reorganization of tight junction protein ZO-1 following severe thermal injury. Thus, it is indicated that MLCK-dependent MLC phosphorylation signaling pathway is involved in the intestinal barrier disruption induced by severe burn injury.
It should be noted that MLCK inhibition with ML-9 just ameliorates, but not corrects, the burn-caused intestinal barrier breakdown. Thus, other signaling pathways should be considered in the pathogenesis of intestinal barrier disruption following severe burn injury. For example, the accumulating published data have shown that the percentage of apoptotic intestinal epithelial cells is significantly increased in mice suffering from 30% TBSA cutaneous full-thickness burn 
. Our previous studies have also shown that the increase of apoptosis is accompanied by the increased Caspase-3 activity and the decreased Bcl-2 expression in intestinal epithelial cells after 30% TBSA burn injury 
. Although the relevance of single-cell apoptosis to intestinal barrier dysfunction remains controversial owing to differing results in diverse experimental systems 
, extensive apoptosis of epithelial cells may lead to intestinal barrier dysruption. Therefore, the burn-induced increase in epithelial cell apoptosis may contribute to intestinal barrier dysfunction following thermal injury. In addition, we have previously demonstrated that Rho-associated kinase is involved in the intestinal barrier dysfunction in rats undergoing 30% TBSA thermal injury, and that pharmacological inhibition of Rho-associated kinase attenuates the burn-induced intestinal barrier dysfunction 
. Moreover, it has been reported that Toll-like receptor 4 and p38 mitogen-activated protein kinase signaling pathways also play critical roles in intestinal epithelial barrier disruption following severe burn injury 
In summary, our present data demonstrate the involvement of MLCK-dependent MLC phosphorylation signaling pathway in intestinal barrier dysfunction following severe burn injury. To the best of our knowledge, this is the first in vivo experimental study providing direct evidence to show the role of MLCK-dependent MLC phosphorylation in burn-induced intestinal epithelial barrier disruption. It is suggested that MLCK-dependent MLC phosphorylation may be a critical target for the therapeutic treatment of intestinal epithelial barrier disruption after severe burn injury.