Protective Effect Of ACEI, Milkvetch Root And Their Combination On Human Umbilical Vascular Endothelial Cells Following Oxidative Injury In Vitro.

Objectives:To investigate the protective effects and mechanisms of ACEI (Captopril),Milkvetch root and their combination on human umbilical vascular endothelial cells (HUVECs) following the damage of hydrogen peroxide(H₂O₂).Methods1. The oxidative injury and apoptosis model of HUVEC in vitro: Human umbilical vascular endothelial cells (HUVECs) were cultured in vitro.They were divided into normal control group and seven oxidative groups which were injured by H₂O₂ with different concentration (0.02, 0.10, 0.20, 1.00, 2.00, 10.00, 20.00mmol/L, respectively). After 4 hours, the levels of MTT(OD) of cells in each group were measured. Moreover, the contents of cell protein and malondialdehycle(MDA), the apoptosis rate in normal control and H₂O₂ 1, 3, 5, 7 goups(0.02, 0.20, 2.00, 20.00mmol/L, respectively) were detected and the morphological changes of HUVEC were observed by the light microscope, respectively.2. Protective effects of Captopril,Milkvetch root and their combination on HUVEC following oxidative injury in vitro: HUVEC were divided into 11 groups. They were normal control, H₂O₂ oxidative control(2.00mmol/L), Captopril-L, M, H (0.5, 5.0, 50.0μg/mL)+ H₂O₂(2.00mmol/L), Milkvetch root-L, M, H (0.1, 1.0, 10.0mg/mL) + H₂O₂(2.00mmol/L), Captopril-L, M, H (0.5, 5.0, 50.0μg/mL)+ Milkvetch root-L, M, H (0.1, 1.0, 10.0mg/mL) + H₂O₂(2.00mmol/L). HUVEC were injuryed for 4 hours by 2.00mmol/L H₂O₂ after they were incubated with different dosages of Captopril,Milkvetch root and their combination for 24 hours. The activity(MTT), antioxidabt indicatrix (MDA,SOD,NO) and the content of VEGF of cells were measured.3. Anti-apoptosis effects of Captopril,Milkvetch root and their combination on HUVEC induced by H₂O₂ in vitro: The condition and group were the same as 2. AO/EB fluorescent staining observed morphology changing of apoptosis. Apoptosis ratio of HUVEC was detected by FCM. Important inhibition apoptosis proteinum Bcl-2 was detected by Western blot.Result1. The oxidative injury and apoptosis model of HUVEC in vitroâ‘ The levels of MTT (OD) of cells in the H₂O₂ 3⁷ groups (0.20, 1.00, 2.00, 10.00, 20.00mmol/L H₂O₂, respectively) were significantly lower than those in the normal control group (P<0.01) except the H₂O₂ 1 and 2 group (0.02, 0.10mmol/L H₂O₂, respectively) which showed no significant difference with normal control group (P>0.05).â‘¡From the morphological changes of HUVEC, the H₂O₂ 1, 2, 3 groups(0.02, 0.10, 0.20mmol/L, respectively) were similar to the normal control, the H₂O₂ 4, 5, 6, 7 groups(1.00, 2.00, 10.00, 20.00mmol/L, respectively) were significantly different from the normal. The cell shape of H₂O₂ 4 and 5 group, which concentration are 1.00 and 2.00 mmol/L, were distorted while the cell boundary was still clear. The cell shape of H₂O₂ 6 and 7 group , which concentration are 10.00 and 20.00 mmol/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.â‘¢The contents of cell protein in H₂O₂ 5, 7 groups were significantly lower than those in the normal control group(P<0.01), while in H₂O₂ 1, 3 groups there were no significantly difference(P>0.05). Furthermore, the contents of MDA in H₂O₂ 5 and 7 groups were significantly higher than those in the normal control group(P<0.01), while in H₂O₂ 1 and 3 groups there were no significance (P>0.05). And the contentd of cell MDA in the H₂O₂ 7 group were significantly higher than those in the H₂O₂ 5 group(P<0.01). The moring and total apoptosis ratio of cells in the H₂O₂ 3, 5, 7 group were significantly lower than those in the normal control group(P<0.05) except the H₂O₂ 1 group(P>0.05). Among the total, the highest moring apotosis ratio was H₂O₂ 5 group, and the highest total apotosis ratio was H₂O₂ 7 group(P<0.05, vs other group).2. Protective effects of Captopril,Milkvetch root and their combination on HUVEC following oxidative injury in vitroâ‘ Protective effects of Captopril and Milkvetch root on HUVEC following oxidative injury in vitro: The level of MTT(OD), the cativity of SOD, the content of NO and VEGF of cell in the normal control and (Captopril-L, M, H / Milkvetch root-M, H)+ H₂O₂ groups were significantly higher than those in the H₂O₂ oxidative control(P<0.05 or P<0.01) except Milkvetch root-L+H₂O₂ group(P>0.05). Besides the content of MDA of cell in the normal control and (Captopril-L, M, H / Milkvetch root-M, H)+ H₂O₂ groups were significantly lower than those in the H₂O₂ oxidative control(P<0.05 or P<0.01) except Milkvetch root-L+H₂O₂ group(P>0.05).â‘¡The comparison of the combined effect of Captopril and Milkvetch root and their single effects on HUVEC following oxidative injury in vitro correspondingly: The effect of (Captopril + Milkvetch root)-L, M, H + H₂O₂ groups were significantly better than those in the Captopril / Milkvetch root-L, M, H + H₂O₂ groups(P<0.05 or P<0.01).â‘¢The relationship between the concentration of Captopril, Milkvetch root and the indicatrix: there were positive correlation in the level of MTT(OD), the content of NO and VEGF, the activity of SOD and the level of Captopril or Milkvetch root, while the contents of MDA of cells were inversely related to the level of Captopril or Milkvetch root.3. Anti-apoptosis effects of Captopril,Milkvetch root and their combination on HUVEC induced by H₂O₂ in vitroâ‘ Anti-apoptosis effects of of Captopril and Milkvetch root on HUVEC induced by H₂O₂ in vitro: the morning apoptosis ratio of cell in the normal control and (Captopril-L, M, H / Milkvetch root-M, H)+ H₂O₂ groups were significantly lower than those in the H₂O₂ oxidative control(P<0.05 or P<0.01) except Milkvetch root-L+H₂O₂ group(P>0.05). The total apoptosis ratio of cell in the normal control and (Captopril / Milkvetch root-M, H)+ H₂O₂ groups were significantly lower than those in the H₂O₂ oxidative control(P<0.05 or P<0.01) except Captopril / Milkvetch root-L+H₂O₂ group(P>0.05). Besides the content of important inhibition apoptosis proteinum Bcl-2 of cell in the normal control and (Captopril / Milkvetch root-M, H)+ H₂O₂ groups were significantly lower than those in the H₂O₂ oxidative control (P<0.05 or P<0.01) except (Captopril / Milkvetch root -L)+H₂O₂ group (P>0.05).â‘¡The comparison of the combined effect of Captopril and Milkvetch root and their single effects on HUVEC following oxidative injury in vitro correspondingly: The effect of (Captopril + Milkvetch root)-L, M, H + H₂O₂ groups were significantly better than those in the Captopril / Milkvetch root-L, M, H + H₂O₂ groups (P<0.05 or P<0.01).â‘¢The relationship between the concentration of Captopril, Milkvetch root and the indicatrix: there were positive correlation in the he morning,total apoptosis ratio and the level of Captopril or Milkvetch root, while the content of proteinum Bcl-2 of cells were inversely related to the level of Captopril or Milkvetch root.Conclusion1. HUVEC can be injured by H₂O₂. The extent of cellular damage and apoptosis 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 HUVEC following oxidative injury in vitro.2. Captopril, Milkvetch root and their combination can improve the cell activity (MTT), enhance the activity of SOD, increase the level of NO and VEGF, and decrease the content of MDA depends on their concentration in some extent. The combined effect of Captopril and Milkvetch root is better than the single effect of Captopril and Milkvetch root.3. Captopril, Milkvetch root and their combination can decrease the morning and apoptosis ratio induced by H₂O₂, increase the level of proteinum Bcl-2 of cell depends on their concentration in some extent. The combined effect of Captopril and Milkvetch root is better than the single effect of Captopril and Milkvetch root.