PKCα/HMGB1/NF-κB Pathway Involved In Vascular Endothelial Cell Injury Under Impinging Flow

Background and Purpose: Intracranial aneurysms(IA)are aneurysmal bulges in the walls of the arterial vessels of the brain,whose rupture causes subarachnoid haemorrhage and often devastating injuries to patients.Despite significant advances in diagnosis and treatment,the mechanism of formation of IA remains unknown.In recent years,it has been found that haemodynamic factors can lead to the development of an inflammatory response within the vessel wall,endothelial damage and abnormal remodelling of the vessel wall,which may be closely related to the development of IA formation.In this study,in vitro experiments were performed to simulate the mechanisms associated with the changes in haemodynamic stress to which vascular endothelial cells are subjected at the vascular bifurcation,resulting in inflammation and damage to the endothelium.Methods: In our study,we used a modified T-shaped hydrodynamic system developed by our group to simulate the hemodynamic changes at the intracranial vascular bifurcation and construct a model of vascular endothelial cell injury.Human umbilical vein endothelial cells(HUVEC)were shocked at a flow rate of 500 ml/min at 37℃ and 5% CO2 for different times(0.5h,1h,2h,3h,6h)to observe changes in cell density and morphology.q PCR was used to detect the expression and regulation of p-PKCα,HMGB1 and NF-κB proteins,IL-6 and IL-8 expression changes were detected by q PCR.HUVEC was then subjected to PKCα phosphorylation inhibitors as well as plasmid knockdown of HMGB1 to explore the regulatory linkage between p-PKCα,HMGB1 and NF-κB.Results: The cells in region I remained confluent at different times of shock flow,and no significant changes were observed in their density and morphology;in region II,the cell density decreased and the gap increased after 0.5h and 1h of shock flow,and the number of cells decreased progressively with increasing time of shock flow(2h,3h,6h),and the cell morphology showed diversification;the cells in region III did not change significantly after 0.5h and 1h of shock flow,and the cell density and morphology increased with increasing time of shock flow(2h,3h,6h).The cell density and morphology in region III did not change significantly after 0.5h and 1h of shock flow,while the cell pooling density increased and the connection was tightened with the increase of the duration of shock flow(2h,3h and 6h).Protein blotting revealed that endothelial cells in the three regions of the pool activated the expression of p-PKC α,HMGB1 and NF-κ B after 0.5h of shock flow,and the expression increased gradually with the increase of shock time(1h,2h,3h),reaching the highest level after 3h of shock(p < 0.01).q PCR detected that shock flow activated the expression levels of IL-1 β,IL-6,IL-The expression of IL-1 β,IL-6 and IL-8m RNA increased progressively with the duration of shock,and also reached a high level after 3h of shock(p<0.05),so 3h of shock was selected as the time point for subsequent intervention.Specific inhibition of p-PKC α activation reduced intracellular HMGB1 and NF-κB expression and inhibited IL-1β,IL-6 and IL-8levels.Knockdown of HMGB1 resulted in a decrease in NF-κ B expression and inhibition of IL-1β,IL-6 and IL-8 levels,but had a limited effect on p-PKCα.Conclusion: In the shock flow-induced vascular endothelial cell injury model,a progressive decrease in cell density,an increase in gap and significant morphological changes in the high wall shear stress region were seen with increasing shock flow time.Shock flow activated the expression of p-PKCα,HMGB1,NF-κB,IL-1β,IL-6and IL-8 in a time-dependent manner and promoted endothelial inflammation and participated in vascular endothelial injury through the PKCα/HMGB1/NF-κB signalling pathway.