Effect Of FKN/CX3CR1 Signal Axis On The Inflammatory Response Of Human Umbilical Vein Endothelial Cells

Objective: The purpose of this study was to investigate the effect of FKN/CX3CR1 signal axis on endothelial cell inflammatory response,and lay a foundation for the molecular mechanism of atherosclerosis.Methods: 1)Select EA.HY926 cell line as experimental subjects.Cells were cultured with complete medium(M199:fetal bovine serum:penicillin=100:10:1)Cells were treated with FKN(0,12.5,25,50,100,200ng/mL)for 24 h.The expression of CX3CR1 mRNA was detected by qRT-PCR.The expression of CX3CR1 protein was detected by Western-blotting and the optimal concentration was determined;2)The EA.HY926 cell line(0,2,4,8,16,12,24,48h)was treated with FKN(25ng/mL).Real-time fluorescence quantitative PCR and Western blotting were used to detect the expression of CX3CR1 from mRNA and protein levels,and then to determine the optimal expression time;3)The lentiviral packaging CX3CR1-RNAi transfected endothelial cells,qRT-PCR and Western blotting were used to detect the expression of CX3CR1 mRNA and protein levels in the cells,to determine the interference;4)After FKN(25ng/mL)was applied to the CX3CR1-RNAi(56338-1)transfected endothelial cells for 8h,The contents of IL-6 and TNF-? in the supernatant of the cultured cells were detected by ELISA.The role of CX3CR1 in FKN-induced endothelial inflammatory responses was therefore determined.Results: 1)Increased expression of CX3CR1 protein in endothelial cells stimulated by 25 ng/mL FKN for 24 h;2)FKN(25 ng/mL)stimulated endothelial cells at different times.qRT-PCR showed that the expression of CX3CR1 mRNA increased after FKN-stimulated cells for 8 h.Western-blotting assay showed that protein levels increased after FKN stimulation for 24 h;3)CX3CR1-RNAi transfected endothelial cells 72 h,qRT-PCR and Western-blotting assay showed decreased expression of CX3CR1 mRNA and protein in endothelial cells transfected with CX3CR1-RNAi(56338-1);4)After stimulation of EA.HY926 endothelial cells with FKN(25 ng/mL)for 8 h,endothelial cells secreted IL-6(1.3-fold,P<0.05)and TNF-?inflammatory cytokines(1.08-fold);5)FKN(25 ng/mL)stimulated the secretion ofinflammatory cytokines IL-6(3.53 fold,P<0.05)and TNF-?(1.25 fold,P<0.05)after 8 h stimulation of human umbilical vein endothelial cells treated with CX3CR1.Conclusion:Therefore,exogenous FKN can synthesize and secrete IL-6 and TNF-? and promote the development of endothelial inflammatory reaction.However,the role of CX3CR1 in FKN-induced endothelial inflammatory response needs further study.