Effects Of NBP On HBMEC Cells Apoptosis Induced By A?1-42 Via Inhibiting TLR4/COX-2

Objective:?-amyloid protein?A??sedimentary lead to vascular endothelial dysfunction may be one of the pathogenesis of Alzheimer's disease?AD?.A?may participate in the vascular endothelial cell apoptosis,inflammation,and endothelial dysfunction.Toll Like Receptor 4?TLR4?mediated signaling pathway is one of the important pathways in the body involved in inflammation.A?induced endothelial cell apoptosis may be associated with TLR4/COX-2 signaling pathways.Butyl phthalide?NBP?have the effect of nerve protective,improve the cognitive behavior ability of AD patients.Whether the NBP have the protection effection on the vascular endothelial cell apoptosis induced by A?,and the mechanism are not clear at present.So this reaserch will investigate the protective effects of NBP on the apoptosis of HBMEC induced by A?1-42,and explore the protective effect related with the TLR4/cox-2 signal pathway.Methods:?1?To explore the concentration of A?1-42:HBMEC were cultivated in vitro with A?1-42 seven different concentrations,time of stimulate were 6 different time,cell viability were observed by CCK-8.?2?The effections of NBP on the viability of HBMEC induced by A?1-42:HBMEC were divided into NBP 160?g/L,80?g/L,40?g/L,model group,control group and TAK242 group.A?1-42stimulated dose is 50?mol/L,HBMEC viability were detected by CCK-8.?3?The effections of NBP on the apoptosis of HBMEC induced by A?1-42:HBMEC were divided into NBP 160?g/L,80?g/L,40?g/L,model group,control group and TAK242 group.HBMEC were stimulated with A?1-4250?mol/L.fluorescent dye were detected apoptosis,the nucleus changes were charged by the color of cells,cell morphological changes.Hoechst33342/PI dye were used to judge Whether the cell apoptosis.Flow cytometry were used to test the quantitative apoptotic cells.?4?The effections of NBP on the expression of TLR4,COX-2 of HBMEC induced by A?1-42:HBMEC were divided into NBP 160?g/L,80?g/L,40?g/L,model group,control group and TAK242group.HBMEC were stimulated with A?1-42 50?mol/L,expression of TLR4,COX-2 protein were detected by western blot.?5?The effections of NBP on the concentration of IL-1 of HBMEC induced by A?1-42:HBMEC were divided into NBP 160?g/L,80?g/L,40?g/L,model group,control group and TAK242 group.HBMEC were stimulated with A?1-42 50?mol/L,the concentration of IL-1 were detected by ELISA.?6?The effections of NBP on the concentration of IL-6 of HBMEC induced by A?1-42:HBMEC were divided into NBP 160?g/L,80?g/L,40?g/L,and TAK242 group.HBMEC were stimulated with A?1-42 50?mol/L?7?The effections of NBP on the concentration of TNF-?of HBMEC induced by A?1-42:HBMEC were divided into NBP 160?g/L,80?g/L,40?g/L,model group,control group and TAK242group.HBMEC were stimulated with A?1-42 50?mol/L,the concentration of TNF-?were detected by ELISA.Results:?1?Compared with control group,cell viability of groups with 5×10²?mol·L^-11 A?1-42 were changed different in different time,administrated 0.5 h and 2h.cell viability of groups with5×10¹?mol·L^-1A?1-42 were changed significantly in 24h and48h?P<0.05?.5?mol·L^-1A?1-42 were decreased significantly in 48h?P<0.05?.?2?The effects of NBP on the cell viability of HBMEC induced by A?_1-42:Compared with control group,NBP could increase HBMEC cell survival rate?P<0.05??3?The effects of NBP on the apoptosis of HBMEC induced by A?1-42:The apoptosis rate and cell death rate of HBMEC cells which administrated by NBP were less than model group obviously.Fluorescence dyeing results judgment mainly according to the nucleus dyeing.If the nuclei are blue,tips for normal cells.If the nucleus is light blue,tips for apoptotic cells.?4?The effects of NBP on the expression of TLR4/COX-2:Compared with model group,NBPcould decreased the expression of TLR4/COX-2?P<0.05?.?5?The effects of NBP on the expression of IL-1:Compared with model group,NBPcould decreased the concentration of IL-1?P<0.05?.?6?The effects of NBP on the expression ofIL-6:Compared with model group,NBPcould decreased the concentration of IL-6?P<0.05?.?7?The effects of NBP on the expression of TNF-?:Compared with model group,NBPcould decreased the concentration ofTNF-??P<0.05?.Conclusion:?1?NBP has a protective effect to HBMEC injury and decrease the HBMEC apoptosis induced by A?1-42.?2?mechanism of NBP protective effect may be related to inhibiting TLR4,cox-2 expression and reduce inflammation factor IL-1,IL-6 and TNF-?.