| Objects:Despite the remarkable progress in critical care and wound management, burn shock caused by massive and severe burn injury still has a high rate of morbidity and mortality. Vascular hyperpermeablity plays a central role in the occurrence of syndrome in bum. Fluid resuscitation was the only current accessible clinical method in the therapy of burn shock. Previous studies have approved that MLC phosphorylation is involved in inflammatory mediators-induced alteration of endothelial barrier function, while the level of phosphorylated MLC is regulated by MLC kinase and MLC phosphatase. Activation of Protein kinase C pathway has been approved to contribute to the signal transduction in enhanced microvascular permeability. PKC is also regarded as an important upstream signal for MLC phosphorylation. The objects of this study are to elucidate the property of burn serum induced-endothelial cytoskeleton reorganization and to explore the effect and possible signal pathway of MLC phosphorylation involved in this process. Methods:The serum from Sprague-Dawley rats with II degree burn injury (30-40% TBSA) was used to stimulate ECV-304 cells to observe its time and dose-dependent effects on endothelial cytoskeleton using the specific fluorescent probe of F-actin. ML-7, the specific inhibitor of MLCK, and Y-27632, the specific inhibitor of Rho kinase which could inhibit the activity of MLC phosphatase, were used to reduce the level of phosphorylated MLC and to detect the roles of MLCK and Rho kinase during the process of bum serum-induced endothelial cytoskeltal reorganization. The expression of PKCwas down-regulated by long term (24 h)-PMA stimulation in endothelial cells in order to know whether PKC activation played a role in burn serum induced-cytoskeletal reorganization. PMA treatment for 15min was used to specifically activate PKC so as to clarify the effects and relationship between PKC and MLC phosphorylation in burn serum induced-cytoskeleton morphological changes. Results:1. Compared with normal serum, serum from burn rats could induce remarkable morphological changes of endothelial cytoskeleton in a dose-dependent manner. But even 100% normal serum could cause the contraction of endothelial cells and destruct the integrity of endothelial monolayer.2. 15% burn serum stimulation for 15 min caused the formation of stress fibers in endothelial cells in a time-dependent pattern.3. Reduction of pohsphorylated MLC level in ECs was induced by pretreating the cells with ML-7 or/and Y-27632 for 30min. Burn serum could not induced any changes in the organization of F-actin in these cells. In ECs incubated with ML-7 and/or Y-27632 30 min before the 30 min stimulation of burn serum, few stress fibers could be seen compared with those cells pretreated with vehicle and burn serum or only with burn serum for 30min.4. Down-regulation of PKC activation induced by long-term PMA incubation attenuated the burn serum induced-changes of endothelial cytoskeleton organization.5. Activation of PKC by short-term stimulation with PMA caused the formation of stress fibers in endothelial cells, which resembled the alteration induced by burn serum. In endothelial cells treated with ML-7 or Y-27632,PMA stimulation could not caused the formation of stress fiber. 6. The result of western blot indicated an increased of MLC phosphorylation level in ECs treated with 15% burn serum. Conclusion:1. Burn serum could induce a dose- and time-dependent formation of stress fibers in enthelial cells. 100% serum treatment should not be used in burn model in vitro.2. Burn serum could induced a shift of MLC species from nonphosphorylated form to phosphorylated form, which is very important during the process of endothelial cytoskeletal changes induced by serum from burned rats. The activation of MLCK and Rho kinase may be involved in burn serum-induced cytosketal reorganization in endothelial cells.3. The reduction of MLC phosphorylation level by ML-7 and/or Y-27632 could reverse the cytoskeletal reorga... |
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