Effect Of Salvia Miltiorrhiza Beg.F.Alba On Protection Of Vascular Endothelial Cells And Its Mechanisms

ObjectiveTo observe the effect of Salvia miltiorrhiza bge.f.alba (SMA)on a model of injured human umbilical vascular endothelial cell (HUVEC) in vitro by H2O2;To investigate the relationship between the mechanisms and apoptosis; And to explore influence of Salvia miltiorrhiza bge.f.alba on bcl-2 expression. provide informations for evaluation of parmarchological value of Salvia miltiorrhiza bge.f.alba.MethodIn vitro perfusion and enzyme digestion method is used to culture HUVEC, And the cells are identified by morphological observation andⅧ:Ag immunoreactivity. select the cells in the exponential phase of growth and Groups are divided on experiment as follow: (1) normal control; (2) H2O2 damage group: H2O2(eventually concentration 2mmol/L); (3) high dosage SMA group: SMA(0.10 g/ml) + H2O2; (4) middling dosage SMA group: SMA(0.03 g/ml) + H2O2; (5) low dosage SMA group: SMA(0.01 g/ml) + H2O2. After the cells are treated for 4 hours, The cells viability is measured by morphological observation and the MTT assay. The cell apoptosis is determined by flow cytometric analysis and staining with AO and EB. eventually, immunofluorescence is used to determine Bcl-2 expression.Results1 .Cell culture and identify(1) morphological observation :the most cultured cells are fused after inoculation for 5 to 7 days, appearing typical'slabstone'. (2)Ⅷ:Ag immunoreactivity: under confocal microscopy, around 95% cellular nucleus appeared flavovirens fluorescence.The cells cultured in this method are proved to be HUVEC and fit for our study.2. Effect of SMA on Activity of HUVEC(1)morphological observation: in H2O2 damage group HUVECs are damaged obviously, while in groups with SMA ,condition of HUVECs is improved and depend on dosage of SMA. (2)the MTT assay: the OD value of the cells in normal control group is the maximum value as 0.744±0.043; in H2O2 damage group the OD value of the cells is minimum value as 0.082±0.009; the OD values in high dosage SMA group , middling dosage SMA group and low dosage SMA group are 0.621±0.065,0.529±0.065 and 0.305±0.023 in turn, appearing significant difference vs H2O2 damage group(P<0.01).3 .Effect of SMA on Apoptosis of HUVEC(1) flow cytometric analysis :Apoptosis index in H2O2 damage group is significantly higher than normal control group(P <0.01), but Apoptosis indexs in other groups decrease significantly after interference by SMA with 0.10 g/ml, 0.03 g/ml and 0.01 g/ml concentrations(P <0.01 or P <0.05). The effect is dose dependant. (2) The result of cell staining with AO and EB is similar with FCM.4. Effect of SMA on expression of Bcl-2 of HUVEC Compared with normal control, Expression of Bcl-2 in H2O2 damage group is significantly decrease. While in groups of SMA 0.10 g/ml and 0.03 g/ml concentrations , Expression of Bcl-2 is significantly higher and the effect is dose dependant(P <0.01). But The 0.01 g/ml concentration group has no significant difference (P >0.05).Conclusion1. SMA could prevent H2O2-induced apoptosis of HUVEC and protect its activity .2. The inhibition of SMA on apoptosis of HUVEC is correlated with an increase Bcl-2 expression.