P38 Pathway Regulated The High Permeability Changes Of HUVECs Induced By Heatstroke Through The Translocation Of VE-Cadherin

Because of the injury of systemic inflammatory response syndrome(SIRS),heatstroke causes widespread vascular endothelial damage,resulting in vascular leakage,bleeding,tissue edema and other pathological damage,and finally leads to clinical outcomes of shock,microcirculatory disturbance,multiple organ dysfunction syndrome(MODS)and its like.It is well known that vascular permeability is closely related to the endothelial adherens junctions(AJ)and VE-Cadherin.However,the changes in vascular permeability of heatstroke has not yet been studied systematically,and its in-depth molecular mechanism and signal conditioning mechanism has not been explored.Objection:1.To study the changes of permeability of human umbilical venous endothelial cells(HUVECs)induced by heatstroke.2.To investigate the regulatory mechanism of p38 MAPK pathway in the permeability changes of HUVECs induced by heatstroke.Method:1.Heatstroke cause endothelial cell permeability changes and its molecular mechanisms.1.1 Constructed the HUVECs heatstroke model in vitro.According to the different heatstroke temperature,the cells was divided into 4 groups:Control group,39 ℃ group,41 ℃ group and 43 ℃ group.Transwell technique was used to detect the transendothelial electrical resistance(TEER)during different heatstroke situation,to describe the permeability of single layer HUVECs.Transmembrane fluorescent protein leakage test implemented,permeability coefficient(Pa)was calculated to describe the effect of different heatstroke temperature on the permeability of single layer HUVECs.The lower the TEER is,the higher the permeability is.The higher the Pa is,the higher the permeability is.1.2.Constructed the HUVECs heatstroke model in vitro.According to the different heatstroke time,the cells was divided into 4 groups:Control group,1h group,2h group and 3h group.Detect the TEER and Pa to describe the permeability of the HUVECs.1.3.Constructed the HUVECs heatstroke model in vitro,the cells were divided into 5 groups:Oh group,0.5h group,1h group,1.5h group and 2h group,according to different heatstroke time.The effect of different heatstroke time(temperature 43 ℃,time gradient)on the expression of VE-Cadherin total protein,membrane protein and cytoplasm protein was detected by Western blot.2.The role of p38 MAPK signal pathway in endothelial cell permeability changes under heatstroke2.1.The HUVECs heatstroke model was constructed.According to the different heatstroke time,the cells were divided into 5 groups:Control hgroup,0.5h group,1h group,1.5h group and 2h group.Western blot was used to detect the effect on the phosphorylation level of p38 MAPK in HUVECs under different heatstroke time.2.2.The HUVECs heatstroke model was constructed,and set the control group,SB group,HS group and SB + HS group.The p38 inhibitor SB203580(concentration of 10uM)was incubated with the HUVECs in the cell incubation for 2 hours beforehand.After 2 hours ’ heatstriking(43 ℃),the cells were rewarmed.Examining the TEER and calculating the Pa to study the role of MAPK signaling pathway in HUVECs permeability changes caused by heatstroke.2.3.The HUVECs heatstroke model was constructed,and set the control group,SB group,HS group and SB + HS group.The expression of VE-Cadherin total protein,membrane protein and plasma protein were tested respectively by Western Blot,and observe by the Immunofluorescence technology.Results:1.Heatstroke cause endothelial cell permeability changes and its molecular mechanisms.1.1.Permeability changes of HUVECs under different heatstroke temperature:In the process of the heatstriking,the TEER of HUVECs decreased and the Pa increased,meaning that the cell permeability increased.The higher the heatstroke temperature was,the more obviously the permeability increased,the more obviously the Pa increased.1.2.Permeability changes of HUVECs under different heatstroke time:The longer the heatstroke time was,the more obviously theTEER decreased,the more obviously the Pa increased.1.3.VE-Cadherin changes of HUVECs under heatstroke:1.3.1.The total amount of VE-Cadherin in HUVECs showed little change in the short-term heatstroke.1.3.2.In the process of heatstroke,the distribution of VE-Cadherin in HUVECs decreased in the membrane,while increasing in the cytoplasm.2.The role of p38 MAPK signal pathway in endothelial cell permeability changes under heatstroke2.1.Short-term heatstroke could increase the phosphorylation level of p38 in HUVECs.2.2.After pretreatment with p38 pathway inhibitor SB203580,the TEER decline of HUVECs caused by heatstroke was reduced,while the increasing Pa of HUVECs induced by heatstroke was reduced,with the permeability tabilized.2.3.After the pretreatment with SB203580,with the permeability stabilized.2.4.After pretreatment with SB203580,western blot and immunofluorescence showed that the internalization of VE-Cadherin in HUVECs induced by heatstroke was weakened.Conclusion:HUVECs permeability increased in a temperature-dependent and time-dependent manner,while the VE-Cadherin was redistributed from the membrane to the cytoplasm.Inchoate heatstroke could cause p38 phosphorylation activation,which would cause VE-Cadherin translocation from membrane to cytoplasm,resulting in damage of the intercellular adherent junction and high cell permeability through the p38 MAPK pathway.