Sepsis can increase cell permeability, and lead to capillary leakage syndrome (CLS), which causes severe hypoproteinemia, hypovolemia, tissue hypoperfusion, edema, shock and multiple organ dysfunction syndrome [22–24]. Our previous studies have shown that TH can improve the permeability of septic cells . In this study, we found that TH significantly mitigated the LPS-increased permeability in EA. Hy926 cells. Given that cellular permeability is crucial for the pathogenesis of CLS the decreased permeability by TH suggests that TH may be valuable for control of septic shock, like VEGF (vascular endothelial growth factor) antagonist , nitric oxide inducer , inhibition of MLC phosphorylation , protection of cell connection .
The Rap1/Rho/MLC signaling is a critical regulator of cell permeability. We found that compared with the control group, LPS stimulation significantly decreased Rap1 and Ve-cadherin expression, but increased RhoA expression and MLC phosphorylation in EA. hy926 cells, which were enhanced by Rap1 silencing. In contrast, TH significantly mitigated the effects of LPS by preserving Rap1 and Ve-cadherin expression and reducing the LPS-stimulated RhoA expression and MLC phosphorylation in EA. hy926 cells. Such inhibitory effects of TH were attenuated by Rap1 silencing. Such novel data demonstrated that TH mitigated the LPS-increased permeability by preserving Rap1 expression in EA. hy926 cells.
The available data indicated that Rap1 were crucial for cell-matrix adhesion and cell-cell adhesion. Rap1 inhibits the RhoA activity, which can activate ROCK to promote non-muscle myosin II activation and actin contraction, and stress fiber formation to enhance local adhesion . In addition, activated ROCK can promote MLC phosphorylation to increase permeability . Therefore, Rap1 can reduce vascular permeability under both resting and stress conditions and dynamically regulate the barrier function of endothelial cells . During the process of sepsis, Rap1 inhibits RhoA and Rac activities to preserve endothelial cell permeability . Actually, inhibition of Rap1 can increase vascular permeability to deteriorate ARDS (acute respiratory distress syndrome)  while enhancement of Rap1 activity can accelerate the recovery of LPS-induced lung injury and vascular endothelial cell function .
Previous studies have shown that many factors, such as histamine, bradykinin, platelet activating factor and thrombin, increase vascular permeability by regulating Ve-cadherin expression in endothelial cells [16, 17, 33, 34]. We found that LPS stimulation increased the MLC phosphorylation in EA. hy926 cells, which explained why LPS increased cell permeability , consistent with previous observations [36, 37]. However, TH mitigated the LPS-decreased Ve-cadherin expression such data indicated that TH preserved Rap1 expression to inhibit the LPS-increased RhoA expression and MLC phosphorylation and LPS-decreased Ve-cadherin expression in EA. hy926 cells [14, 20, 21, 38]. Thus, the Rap1/RhoA/MLC signaling may be valuable targets for intervention of LPS-induced high endothelial cell permeability. We are interested in further investigating the therapeutic effects of TH in vivo and the potential mechanisms underlying the action of TH during the process of septic shock.