Effect Of Blackcurrant On Vascular Endothelial Cells By Hydrogen Peroxide (H₂O₂) Injury

Objectives: To investigate the protective effects and mechanisms of blackcurrant on human umbilical vascular endothelial cells(HUVECs,ECV-304) following the damage of hydrogen peroxide(H₂O₂). Methods: 1. The oxidative injury model of ECV-304 in vitro: Human umbilical vascular endothelial cells (ECV-304) were cultured in vitro. They were divided into normal control group ,three oxidative groups which were injured by H₂O₂with different concentration (0.40mmol/L,2.00mmol/L,10.00mmo1/L, respectively). After 24,48,72h exposure , give the hydrogen peroxide. After 4 hours, the levels of MTT(OD) of cells in each group were measured. Moreover, the morphological changes of ECV-304 were observed by the light microscope, respectively.2. Protective effects of blackcurrant on ECV-304 following oxidative injury in vitro: ECV-304 were divided into seven groups. They were normal control, Negative control group :DMSO, damage group: H₂O₂ oxidative control ( 2.00mmol/L), blackcurrant group -L, M, H (3.13μg/mL,12.50μg/mL,50.00μg/mL)+ H₂O₂ (2.00mmol/L), Positive control group (VitaminE,100.00μmol/mL) + H₂O₂ (2.00mmol/L). ECV-304 were injuryed for 4 hours by 2.00mmol/L H₂O₂ after they were incubated with different dosages of blackcurrant,Vitamin E and their combination for 24 hours. The activity(MTT),MDA,NO,LDH,ET,6-keto- Fla were measured. 3. Anti-apoptosis effects of blackcurrant on ECV-304 induced by H₂O₂ in vitro: The condition and group were the same as 2. Apoptosis ratio of ECV-304 was detected by FCM. Results: 1. The oxidative injury model of ECV-304 in vitro:â‘ From the morphological changes of ECV-304, the H₂O₂ damage groups(0.40mmol/L , 2.00mmol/L , 10.00mmo1/L, respectively) were significantly different from the normal control, were distorted while the cell boundary was still clear. Along with the hydrogen peroxide concentration increase, the more obvious morphological changes in the more severe the injury.â‘¡The levels of MTT (OD) of cells in the H₂O₂ damage groups were significantly lower than those in the normal control group (P<0.01) . 2. Protective effects of blackcurrant on ECV-304 following oxidative injury in vitro: There were significant differences between damage group and blackcurrant, Positive control group in cell growth condition under light microscope.The contents of NO,PGI2 of cell in the damage group were significantly lower than those in the normal control. The contents of MDA,ET and LDH activity of cell in the damage group were significantly higher than those in the normal control. The level of MTT(OD), the content of NO,PGI2 of cell in the in the normal control, (blackcurrant group - M, H) +H₂O₂ groups and Positive control (VitaminE) + H₂O₂ group were significantly higher than those in the damage group :H₂O₂ oxidative control(P<0.05 or P<0.01). Besides the content of MDA,ET and LDH activity of cell in the normal control ,(blackcurrant group - M, H) + H₂O₂ groups and Positive control (VitaminE) + H₂O₂ group were significantly lower than those in the damage group : H₂O₂ oxidative control(P<0.05 or P<0.01) except Negative control group (P>0.05). 3. Anti-apoptosis effects of blackcurrant on ECV-304 induced by H₂O₂ in vitro: the morning apoptosis ratio and the total apoptosis ratio of cell in the normal control, Positive control (VitaminE) + H₂O₂ group and (blackcurrant group- L,M, H)+ H₂O₂ groups were significantly lower than those in the H₂O₂ oxidative control. Conclusion:1. ECV-304 can be injured by H₂O₂. The extent of cellular damage depends on the concentration and time of H₂O₂ 2.00mmol/L and 4 hours is a better condition that is suitable to study the protective effects on ECV-304 following oxidative injury in vitro.2.Blackcurrant group can improve the cell activity (MTT), increase the level of NO and PGI2, and decrease the content of MDA,ET and the activity of LDH depends on their concentration in some extent. 3. Blackcurrant group and Positive control group can decrease the apoptosis ratio induced by H₂O₂.4. The protective effect of blackcurrant and the Vitamin E may be related to antioxidant and apoptosis.