The Effections And The Mechanisms Of Asiaticoside On The HUVEC Cells Induced By A?_1-42

Objective:To explore the protective effections of asiaticoside on the endothelial cell injury induced by A?1-42,and explore that weateher the mechanism of the protective effects realated to the endothelial cell apoptosis inhibition,the percentage of the Bcl-2/Bax regulation,the expression of IL-6,IL-1 and TNF-alpha inhibition or not,these will provide scientific evidences for prevention and control alzheimer's disease.Methods:(1)To explore the optimal concentration and time of the apoptosis of Human umbilical vein endothelial cells(HUVEC)induced by A?1-42.HUVEC were cultivated in vitro,A?1-42 were divided into 5×10-4?5×10-3?5×10-2?5×10-1?5?5×10?5×102?mol·L-1 respectively,time of stimulate were 0.5,2,6,12,24,48 h,cell viability were observed by CCK-8.(2)To explore the optimal concentration of asiaticoside:asiaticoside were divided into 10-2,10-3,10-4,10-5,10-6,10-7,10-8 mol.ml-1 respectively,HUVEC with asiaticoside were cultivated in vitro,cell viability were detected by CCK-8.(3)The effections of asiaticoside on the viability of HUVEC induced by A?1-42:HUVEC were stimulated with A?1-42 and asiaticoside 10-5,10-6,10-7,10-8mol.ml-l,HUVEC viability were detected by CCK-8.(4)The effections of asiaticoside on the apoptosis of HUVEC induced by A?1-42 asiaticoside were divided into 10-5,10-6,10-7,10-8mol.ml-1 respectively,HUVEC were stimulated with A?1-42 and asiaticoside,apoptosis of HUVEC were detected by fluorescence microscope with Hochest33342/PI dye and flow cytometry with Annexin V-FITC,expression of Bcl-2/BAX protein were detected by Western blot.(5)The effections of asiaticoside on the concentration of IL-1,IL-6 and TNF-? of HUVEC induced by A?1-42:the concentration of IL-1,IL-6 and TNF-? were detected by ELISA Results:(1)Compared with control group,cell viability of groups with A?1-42 0.5 h and 2h were changed slightly(P>0.05),cell viability of groups with 5×102?mol·L-1A?1-42 6h,12h,24h,48h were decreased significantly(P<0.05),cell viability of groups with 5×10?mol·L-1A?1-4224h,48h,5?mol·L-1A?1-42 48h were decreased significantly(P<0.05),cell viability of groups with 5×10-4?5x10-3?5×10-2?5×10-1?mol·L-1A?1-42 were changed slightly(P>0.05);(2)Compared with control group,10-2,10-3 mol.ml-1 asiaticoside could inhibite the growth of HUVEC cell,(P<0.01);10-4,10-5,10-6,10-7,10-8mol.ml-1 asiaticoside have no toxic effect of HUVEC cell in survival(P>0.05),according to the literature and experimental results,this experiment selects 10-5,10-6,10-7,10-8mol.ml-1 asiaticoside;(3)10-5,10-6,10-7,10-8mol.ml-1 asiaticoside could increase HUVEC cell survival rate,reduced HUVEC cell damage by A?1-42 in dose manner.The apoptosis and dead of HUVEC cells or apoptosis rate and cell death rate of HUVEC cells which administrated by asiticoside were less than model group obviously,but HUVEC cells still have seen apoptosis,dead cells(red),the nucleus of HUVEC cells were seen bright blue,asiaticoside reduce the expression of BAX and promote the expression of Bcl-2 protein,increase the Bcl-2/BAX ratio in a dose manner.compared with model group,asiaticoside 10-5,10-6,10-7 mol.ml-1 were increased significantly(P<0.05),the Bcl-2/BAX ratio of asiaticoside 10-8mol.ml-1 were no difference(P>0.05);(4)Compared with model group,asiaticoside could inhibition the concentration of IL-1,IL-6 and TNF-a in HUVEC cell in a dose manner(P<0.05).Conclusion:(1)Asiaticoside has a protective effect of HUVEC cells inducing by A?1-42 in a dose manner.(2)Asiaticoside could reduce apoptosis of HUVEC,increased Bcl-2/BAX ratio in a dose manner.(3)Asiaticoside could relieve the concentration of IL-1,IL-6 and TNF-a of HUVEC cells inducing by A?1-42 in a dose manner.(4)The mechanism of asiaticoside protection of HUVEC may be related to inhibit apoptosis,reduce the release of IL-1,IL-6 and TNF-?.