Therapeutic hypothermia mitigates the sepsis-increased permeability in EA. hy926 cells by preserving Rap1 expression

Abstract Background: To determine the effect and potential mechanisms of therapeutic hypothermia (TH) on the permeability of septic cells.Main methods: Human EA. hy926 cells were transfected with, or without, control or Rap1-specific siRNA and treated with 2 µg/ml of lipopolysaccharide (LPS), followed by cultured in normal temperature (NT) or a temporary therapeutic hypothermia (TH) for 10 h. The cellular permeability of each group of cells was determined by transwell permeability assay and the relative levels of ras-proximate-1 (Rap1), RhoA (a small GTP enzyme of the Rho family), Ve-cadherin expression and myosin light chain (MLC) phosphorylation were quantified by western blot and immunofluorescent assays.Results: Compared with the control group, LPS stimulation increased cellular permeability, which was enhanced by Rap1 silencing in EA. hy926 cells under a NT condition, but significantly mitigated by TH. Furthermore, LPS up-regulated RhoA expression and MLC phosphorylation, but reduced Rap1 and Ve-cadherin expression, which were also enhanced by Rap1 silencing, but significantly mitigated by TH. Immunofluorescent analyses indicated that LPS significantly increased phosphorylated MLC, but decreased Ve-cadherin expression, which were deteriorated by Rap1 silencing, but significantly mitigated by TH in EA. hy926 cells.Conclusions: TH significantly mitigated the sepsis-increased permeability of EA. hy926 cells by enhancing the Rap1 expression to attenuate the RhoA/MLC signaling.