Effect Of Water Extract Propolis On Cholesteryl Ester Accumulation And Apoptosis In Huasmc Induced By Ox-ldl

[Objective]The aim of this study to investigate the effect of water extract propolis on cholesteryl ester accumulation and apoptosis in human umbilical artery smooth muscle cell induced by oxidized low density lipoprotein, and explore the mechanism of water extract propolis on inhibition of atherosclerosis and it's implications.[Mothods]1.Preparation of WEP:Extract propolis by ultrasonic waves,chose the composition which can dissolved in water for the experiment.2.Culture human umbilical artery smooth muscle cell and identificate them:get umbilical cord and dissecte umbilical artery,culture HUASMC by explant method. Identificate them by immunocytochemistry technique.3.Preparation of oxidized low density lipoprotein and determination of the extent of oxidized modification:ox-LDL was produced from LDL after being incubated with CuSO4. in vitro;The extent of lipid peroxidation was estimated as for thiobarbituric acid-reactive substances. Tetramethoxypropane was used as a standard, and results are expressed as nanomoles of malondialdehyde equivalents per mg protein.4.Preparation of the smooth muscel cell derived foam cell model:coculture HUASMC with ox-LDL in concentration of 25mg/L,50mg/L,75mg/L respectively, the intracellular total cholesterol (TC)and free cholesterol(FC) at 12h,24h,36h,48h,60h were measured by high performance liquid chromatogram,the content of cholesterol ester was obtained by subtracting the FC from TC.The ratio of CE/TC>50% is the mark of foam cell,so coculture HUASMC with ox-LDL in concentration of 50mg/L for 48h is the optimum condition for foam cell formation.5.Experiment groups: Cultured HUASMCs were randomly divided into 5 groups:Control:without any drugModel:with 50mg/L ox-LDL for 48hPropolis groups:with 50mg/L ox-LDL and 50mg/L,100mg/L,200mg/LWEP respectively for 48h6.0il red O staining was used to stain lipids in the cultured HUASMC of various groups.7.Intracellular total cholesterol and free chelosterol were measured by high performance liquid chromatogram, the content of cholesterol ester was obtained by subtracting the FC from TC.8.Cellular apoptosis index was tested by flow cytometry.[Result]l.Estabilishment of smooth muscle cell derived foam cell:HUASMC were Cocultured with 50mg/Lox-LDL for 48h,the ratio of intracellular CE/TC is more than 50%,and the cells were transformed into foam cell.2.The morphology observation of the effect of WEP on cholesteryl ester accumulation in HUASMC induced by ox-LDLThe amount of intracellular red lipid granules in the model group are more than that in the control group;The amount of intracellular red lipid granules in 50 mg/L,100mg/L,200mg/L WEP groups are less than that in the model group,and with the increase of the concentration of WEP the amount of intracellular red lipid granules has the tendency of decreasing.3.Effect of WEP on the content of intracellular CE:The content of intracelluar CE in model group is more than that in control group (P<0.01); The content of intracellular CE in 50 mg/L,100mg/L,200mg/L WEP groups are less than that in the model group(P<0.01), and with the increase of the concentration of WEP the content of intracellular CE has the tendency of decreasing.4.The effect of WEP on cell apoptosis index:The cell apoptosis index in model group is more than that in control group (P<0.01); The cell apoptosis index in 50 mg/L,100mg/L,200mg/L WEP groups are less than that in the model group(P<0.01),and with and with the increase of the concentration of WEP the cell apoptosis index has the tendency of decreasing.5.The relationship between the content of intracellular CE and cell apoptosis:The content of intracellular CE was correlated with the cell apoptosis (rs=0.964,P<0.01).【Conclusion】1.ox-LDL can induces the cholestreyl ester accumulation in cultured HUASMC in vitro,and transforms it into foam cell, simultaneously it can raise the apoptosis index of HUASMC.2.WEP can reduce the extent of intracellular CE accumulation in HUASMC induced by ox-LDL.3.WEP can reduce the apoptosis index of HUASMC induced by ox-LDL which maybe concerned with the effect of reducing the content of intracellular CE.4.WEP reduce the extent of intracellular CE accumulation and cell apoptosis maybe one of the mechanisms of it's anti-atherosclerosis effect.