PKCα/ERK/PPARγ/NF-κB Signaling Pathway Is Involved In Vascular Endothelial Injury Under Impinging Flow

Background and Objective:The mechanism of the formation of intracranial aneurysms is unclear.At present,it is considered that vascular endothelial injury,vascular inflammation and vascular wall reconstruction caused by hemodynamic changes are the possible causes of intracranial aneurysm formation and rupture,and vascular endothelial injury is considered as the initial link of intracranial aneurysm formation.Therefore,it is very important for the etiology of intracranial aneurysms to study the mechanism of vascular endothelial injury induced by hemodynamics.PKCαis an important transmitter of intracellular signal transduction,which is closely related to vascular diseases.Studies have shown that PKCαcan activate NF-κB in vascular endothelial cells to trigger inflammatory reaction and participate in vascular endothelial injury.PKCαcan also enhance the expression of MMPs by activating ERK,which accelerate the degradation of extracellular matrix,and damages the vessel wall.PPARγis a kind of vascular protective factor,which can alleviate vascular injury by reducing inflammation of vascular wall and play an important role in inhibiting the formation and development of aneurysm.The purpose of this study is to explore the relationship between PKCαand PPARγ,and to verify whether PKCα/ERK/PPARγ/NF-κB signaling pathway is involved in vascular endothelial injury under the impin ging flow.Methods:In this study,we designed and modified the“T”chamber system to simulate the hemodynamic environment at the vascular bifurcation and construct the vascular endothelial injury model.Umbilical vein endothelial cells（HUVECs）were cultured in vitro and were transfected with RNAi PKCαrecombinant lentivirus.The above-mentioned cells were cultured on glass slides and placed in the“T”chamber system after reaching the experimental conditions.The density and morphological changes of vascular endothelial cells were observed under inverted microscope after being exposed to impin ging flow for 3h and 6h.Then PKCα,p-PKCα,ERK,p-ERK,PPARγ,NF-κB and MMP2 expression of vascular endothelial cells were detected by Western Blot.Result:1.Effects of imp inging flow on the density and morphology of endothelial cell:zone 1:the density and morphology of the cells had no obvious change,and the cells still maintained the state of fusion;zone 2:under the action of high wall shear stress,the cell density decreased obviously,the gap enlarged,the morphology was various and elongated,and changed obviously with the extension of action time;zone 3:the density of cells increased and the connection was tight.2.Effects of imping ing flow on the activation of PKCαand expression of PPARγ,NF-κB and MMP2 in vascular endothelial cells under:under impin ging flow,the expression of p-PKCα,NF-κB and MMP2 increased significantly（P<0.05）and PPARγdecreased significantly（P<0.05）,but the high expression of p-PKCαin endothelial cells can be blocked by Ca²⁺inhibitor.3.Effects of impin ging flow on the activation of ERK/NF-κB in vascular endothelial cells:under impin ging flow,the expression of p-ERK,NF-κB and MMP2 increased significantly（P<0.05）,NF-κB and MMP2 were blocked by ERK inhibitors,but NF-κB and MMP2 not by P38 inhibitor or PI3K/Akt inhibitor.4.Effects of PKCαon the expression of endothelial cells ERK,NF-κB and MMP2:under impin ging flow,the expression of p-ERK,NF-κB and MMP2 in normal endothelial cells increased significantly（P<0.05）,but no obvious change was observed（P>0.05）in PKCαknockdown endothelial cells.5.Effects of PPARγon the expression of endothelial cells NF-κB and MMP2:under impin ging flow,the expression of PPARγdecreased significantly（P<0.05）,NF-κB and MMP2 increased significantly（P<0.05）in normal endothelial cells,but NF-κB and MMP2 not significantly changed（P>0.05）after pretreatment with PPARγagonist.Conclusion:Under the impin ging flow,PKCαis activated by Ca²⁺and then further activates ERK.The activated ERK can increase the expression of NF-κB and MMP2 by directly or indirectly inhibit PPARγwhich promotes vascular endothelial inflammation and extracellular matrix degradation and vascular endothelial injury.Therefore,we believe that the activation of PKCα/ERK/PPARγ/NF-κB signaling pathway is involved in vascular endothelial injury induced by impin ging flow,and this pathway may be one of the formation mechanisms of intracranial aneurysms.