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Research
Therapeutic hypothermia mitigates the sepsis-increased permeability in EA. hy926 cells by preserving Rap1 expression
Wu Ding
Zhejiang University School of Medicine Second Affiliated Hospital
Corresponding Author
ORCiD: https://orcid.org/0000-0001-5835-4002
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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.
Keywords
Therapeutic hypothermia, Rap1, Rho, VE-cadherin, Cell permeability
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Research
Therapeutic hypothermia mitigates the sepsis-increased permeability in EA. hy926 cells by preserving Rap1 expression
Wu Ding
Zhejiang University School of Medicine Second Affiliated Hospital
Corresponding Author
ORCiD: https://orcid.org/0000-0001-5835-4002
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 15 Apr, 2020
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
General Cell Biology & Physiology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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.
Figure 1
Figure 2
Figure 3