| Objectives:In our study, we established oxidative damage model of human umbilical vascular endothelial cells (HUVECs) by using hydrogen peroxide (H2O2) to injure HUVEC, and investigated the protective effects and mechanisms of SSTF on HUVEC following oxidative injury.Methods:1. The foundation of oxidative injury model of HUVECHUVEC were cultured in vitro.They were divided into normal control group and nine oxidative groups which were injured by H2O2 with different concentrations(0.1,0.2,0.5,1,2,4,5,8,10mmo1/L) and different times(4 hours,8 hours). We observed morphological changes of HUVEC of each group by the light microscope.Moreover, we measured the cell vitality (OD value), LDH activity in normal control group and H2O2 groups (0.5, 1, 2, 4,5mmo1/L) when the inguryed time is 4 hours.2.Protective effects and mechanisms of SSTF on HUVEC following oxidative injurythe cultured HUVEC were divided into 6 groups:①normal control group;②H2O2 oxidative injury group(2mmol/L);③-⑤groups of SSTF with different dosages(low,middle,high):firstly they were incubated with different dosages of SSTF(which are 100mg/L,200mg/L,400mg/L) for 24 hours and then they were injured by 2mmol/L H2O2 for 4 hours;⑥VC group:firstly they were incubated with VC(20mg/L)for 24 hours and then they were injured by 2mmol/L H2O2 for 4 hours Finally, the cell vitality (OD value) by MTT,MDA content, LDH activity, SOD activity were measured;apoptosis ratio of control group, H2O2 oxidative injury group,SSTF(high)group,VC group,was check by FCM;Inhibition apoptosis protein Bcl-2 by western-blot of each group were measured.Result1. The foundation of oxidative injury model of HUVEC1.1 The effect of different concentration of H2O2 on Morphological changes of HUVEC Morphological changes of HUVEC by the light microscope of each group show that when the inguryed time is 8 hours, the cells in the lower concentration H2O2 groups were obviously injuryed, moreover, when the concentration was a little more bigger, the damage was serious, the cells became round and swelling,and the boundary was not clear.There were some or more of dead cells fall off from the bottom of wells.therefore,we chose 4 hours as injuryed time.Under this condition, the H2O2 groups(0.1,0.2mmol/L)were similar to the normal control group, the H2O2 groups(0.5,1mmol/L)were relatively different from the normal, and the H2O2 group(2mmol/L)were significantly different from the normal ,the cell shape were distorted while the cell boundary was still clear. The cell shape of H2O2 group (4mmol/L) was distorted apparently and the boundary was not clear. There were some or more of dead cells fall off from the bottom of wells.1.2 The effect of different concentration of H2O2 on oxidative injury of HUVEC①The cell vitality (OD value) in all the H2O2 groups were significantly lower than those in the normal control group(P<0.01);②The active of LDH in the H2O2 groups(0.5,1mmol/L) were relatively higher than those in the normal control group(P<0.05),while the active of LDH in the H2O2 groups(2,4mmol/L) were significantly higher than those in the normal control group(P<0.01). 2. Protective effects and mechanisms of SSTF on HUVEC following oxidative injury2.1 The effect of protection①The cell activity(OD value) in the SSTF groups(200 mg/L,400 mg/L) was significantly higher than those in H2O2 oxidative injury group( P<0.05 or P<0.01),while in the SSTF group(100 mg/L),there was no significance(P>0.05);②The active of LDH in all the SSTF groups was significantly lower than those in the H2O2 oxidative injury(P<0.05 or P<0.01).2.2 The mechanism of protection①The contents of MDA in all the SSTF groups was significantly lower than those in H2O2 oxidative injury group( P<0.05 or P<0.01);②The active of SOD in all the SSTF groups was significantly higher than those in H2O2 oxidative injury group (P<0.05 or P<0.01);③apoptosis ratios of SSTF(high)group were significantly lower than those in H2O2 oxidative injury group (P<0.01).④The level of Bcl-2 in H2O2 oxidative injury group were significantly lower than those in normal control group(P<0.01), while in all the SSTF groups,they were significantly higher than those in H2O2 oxidative injury group (P<0.01).Conclusion1. The foundation of oxidative injury model of HUVEC : the concentration (2mmol/L H2O2) and time (4 hours) is a best condition of our study.2. SSTF can fight H2O2 , improve the cell vitality, reduce the activity of LDH, and finally protect HUVEC following oxidative injury.3. The mechanism of the protection of SSTF on HUVEC following oxidative injury may concern that it can fight free radicals, reduce the contents of MDA,enhance the activity of SOD, improve the level of Bcl-2 to inhibit the apoptosis of HUVEC.
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