Role Of Slingshot-mediated Cofilin Activation In Barrier Disrution Of Hypoxic Intestinal Epithelial Cells

For a long time,the intestine has been considered to be the central organ of the surgical stress such as severe trauma and burn injury.It has been well recognized that intestinal barrier disruption plays very important role in pathophysiologica disorders following severe burns,but the mechanism is not yet fully clear.Our previous studies showed that intestinal epithelial barrier dysfunction associated with severe burns or ischemia/hypoxic damage is closely related to tight junction(TJ)proteins,filamentous actin(F-actin)and so on,but the molecular mechanism remains unclear.It has been reported that cofilin,a kind of actin binding protein,plays a critical role in the regulation of polymerization and depolymerization of cytoskeleton actin dynamics.The activity of cofilin is directly regulated by slingshot(SSH),an upstream signal molecule of cofilin.It has been well demonstrated that slingshot can cause the activation of cofilin by dephosphorylating cofilin,and then regulate the polymerization and depolymerization of cytoskeleton actin dynamics.It has been recognized that the essence of the early damage following severe burn is ischemic/hypoxic damage.Therefore,we assume that ischemia/hypoxia occurring at the early stage after severe burn injury may cause cofilin activation by inducing the expression of SSH protein,lead to the imbalance of polymerization and depolymerization of actin,and then cause the changes of tight junction proteins,resulting in the disruption of intestinal epithelial barrier function.Thus,in the current study we developed an in vitro intestinal epithelial barrier model to investigate the changes of the SSH/cofilin/F-actin pathway in intestinal epithelial cells exposed to hypoxia and its role in the intestinal epithelial barrier dysfunction,so as to further elucidate the molecular mechanisms of intestinal epithelial barrier disruption caused by severe burns.Methods1.The human intestinal epithelial cell line Caco-2 were cultured by regular method,and then planted on Transwell polycarbonate membrane inserted in culture plate to grow as cell monolayers.For hypoxia treatment,the Caco-2 cell monolayers were exposed to 1% O2 environmental condition for 0,1,2,6,12 and 24 h respectively.In addition,the Caco-2 cell monolayers were respectively treated with jasplakinolide and latruculin A(LatA)for 0,1,2,6,12 and 24 h.2.In order to evaluate the barrier integrity of intestinal epithelial cell monolayers,the transepithelial electrical resistance(TER)of Caco-2 cell monolayers was monitored with a volt-ohmmeter.The fluorescein isothiocyanate-labeled dextrane(FITC-dextran)probe was used to detect the permeability of Caco-2 cell monolayers.3.The changes of F-actin distribution and the ratio of F-actin and G-actin content were analyzed by fluorescence method.4.The distribution changes of tight junction proteins ZO-1 and occludin were respectively analyzed by the indirect immunofluorescence assay.In addition,the Western blot assay was used to detect the protein expression of ZO-1,occludin and claudin-1.5.The changes of protein expression of cofilin,p-cofilin,SSH-1,SSH-2 and SSH-3 were analyzed by the Western blot assay.Results1.Hypoxia treatment induced a significant decrease of TER in Caco-2 cell monolayers.2.Exposure of Caco-2 cell monolayers to hypoxia induced obviously morphological changes in F-action distribution,and a significant decrease in the ratio of F-actin and G-actin.3.Hypoxia had no significant effect on the protein expression of cofilin in each phase,but induced a significant decrease in the protein expression of p-cofilin in Caco-2 cell monolayers.4.Treatment of Caco-2 cell monolayers with hypoxia caused a significant decrease in the protein expression of tight junction proteins ZO-1,occludin and claudin-1,and also caused obvious changes in the distribution of ZO-1 and occludin.5.The protein expression of SSH-1 and SSH-3 had no significant change after treatment of Caco-2 cell monolayers to hypoxia.But hypoxia caused a significant increase in SSH-2 protein expression.6.Both jasplakinolide and Lat A caused significant decrease of TER and increase of permeability in Caco-2 cell monolayers.7.Both jasplakinolide and LatA induced a significant decrease in the protein expression of tight junction proteins ZO-1,occludin and claudin-1,and also caused obvious changes in the distribution of ZO-1 and occludin in Caco-2 cell monolayers.8.Jasplakinolide treatment increased F-actin/G-actin ratio in Caco-2 cell monolayers,especially significantly at 24 h,while LatA decreased F-actin/G-actin ratio in Caco-2 monolayers at 24 h.9.The protein expression of cofilin was insignificantly changed by both jasplakinolide and Lat A in Caco-2 cell monolayers in each phase.But both jasplakinolide and LatA induced a significant decrease in the protein expression of p-cofilin.ConclusionsThe results of this in vitro study suggest that hypoxia treatment can disrupt the intestinal epithelial barrier function.The underlying molecular mechanism involved in the intestinal epithelial barrier disruption induced by hypoxia is probably that hypoxia activates cofilin by inducing slingshot-2 protein expression,which in turn leads to the depolymerization of F-actin and the disruption of dynamic balance between F-actin and G-actin,resulting in the changed expression and distribution of tight junction proteins.