The Study Of Low Shear Stress Induced Inflammatory Response Via CX3CR1/NF-κB Pathway In Human Umbilical Vein Endothelial Cells

Objective:To explore the important role of CX3CR1/NF-κB in the inflammatory response of human umbilical vein endothelial cells(HUVECs)induced by low shear stress.Methods:1.EA.hy926 human umbilical vein endothelial cell line was cultured in complete medium(M199:fetal bovine serum:stellosterin=100:10:1).The morphology of the endothelial cell strain was observed by light microscopy and electron microscopy,and the endothelial cell strain was identified by immunohistochemistry and growth curve.2.Different treatments were applied to endothelial cells,which were control group(C),negative control group(NC),low shear stress group(LSS)(4.14dyn/cm~2,2h),CX3CR1gene silencing group(ShCX3CR1),low shear stress+CX3CR1 gene silencing group(LSS+ShCX3CR1),low shear stress+PDTC group(LSS+PDTC),qRT-PCR was used to detect the expression of CX3CR1 mRNA in endothelial cells,and Western blotting was used to detect the expression of CX3CR1,NF-κB P65,IκB,ICAM-1,VCAM-1,IL-6in endothelial cells.3.The nuclear localization of NF-κB P65 was detected by immunofluorescence.4.Monocyte adhesion assay was used to detect the effect of different treatments on the adhesion between endothelial cells and monocytes.5.Scratch experiments were used to observe the effects of different treatments on endothelial cell migration.Results:1.Cell lines growth can be covered with single layer in 2~3 d,a spindle cells,vortex pattern growth,extend the visible lumen after sample structure,can be grown into stratified by observed with optical microscopes.Found that the transverse and longitudinal cutting Webel-Palade corpuscle by the transmission electron microscopy.Observed cytoplasm of tan particles under immunohistochemical.2.Both gene and protein assay results showed that the transfection efficiency of ShCX3CR1(56338)were the best.3.the gene and protein detection results showed that compared with the control group,the expression of CX3CR1 was significantly increased in the LSS group;compared with the LSS group,the expression of CX3CR1 in the LSS+ShCX3CR1 group was significantly reduced,the expression of CX3CR1 in the LSS+PDTC group did not change significantly.4.The results of Western-blotting showed that the expression of nuclear P65 in the LSS group was significantly higher than that in the C group.The expression of cytoplasm P65and IκB protein showed a significant decrease trend,and these changes caused by low shearstresscouldbeinhibitedbyShCX3CR1orP65inhibitorPDTC.Immunofluorescence assay showed that under low shear stress,P65 nuclear localization increased compared with group C,and this increase was inhibited by ShCX3CR1 or P65inhibitor PDTC.5.Western-blotting results showed that compared with group C,the expression of VCAM-1,ICAM-1,IL-6 protein in LSS group was significantly increased,and these changes caused by low shear stress could be inhibited by ShCX3CR1 or P65inhibitor PDTC.6.Adhesion experiments showed that the adhesion between endothelial cells and monocytes increased under low shear stress compared with group C,and this increase was inhibited by the ShCX3CR1 or P65 inhibitor PDTC;Scratch test results show that after applying low shear stress to endothelial cells,the migration ability of cells is enhanced,and this enhancement can be inhibited by ShCX3CR1 or P65 inhibitor PDTC.Conclusion:Low shear stress can promote the expression of CX3CR1 in endothelial cells,and it can induce the inflammatory reaction of endothelial cells through the activation of the downstream factor NF-κB,thereby promoting the occurrence and development of inflammatory diseases(atherosclerosis,etc.).The theoretical basis for the study of the mechanisms of sexual disease and the selection of effective molecular targets for early prevention,diagnosis and drug therapy.