Figure 3.

Sal B inhibits H₂O₂-induced apoptosis in HUVECs. (A) HUVECs were treated with H₂O₂ in the presence or absence Sal B, and the cells were stained with Hoechst 33258 (white arrows indicate apoptotic cells; magnification, ×200). (B) Flow cytometric analysis of HUVEC apoptosis was stained by Annexin Ⅴ-FITC/propidium iodide double staining; (a) control group; (b) H₂O₂ group; (c) H₂O₂ + 5 µg/ml Sal B group; (d), H₂O₂ + 10 µg/ml Sal B group; (e) H₂O₂ + 20 µg/ml Sal B group. (C) Rate of apoptosis quantified by Hoechst. (D) Rate of apoptosis quantified by flow cytometry; Sal B decreased apoptosis of the HUVECs induced by H₂O₂. Data are representative of three independent experiments (mean ± SEM); ^##P<0.01 compared with the control group; ^*P<0.05 and ^**P<0.01 compared with the H₂O₂ group. (E) Sal B decreased H₂O₂ -induced caspase-3 activity. ^##P<0.01 compared with the control group; ^**P<0.01 compared with the H₂O₂ group. (F) HUVECs were incubated with different concentrations (5, 10, 20 µg/ml) of Sal B for 24 h and then stimulated with H₂O₂ (800 µM) for 24 h. The protein levels of cytochrome c and cleaved caspase-3 or β-actin were determined by western blotting. Bar charts show the quantification of (G) cytochrome c and (H) cleaved caspase-3. Relative protein levels are presented as the mean ± SEM of the optical density from three separate experiments. ^##P<0.01 compared with control group; ^**P<0.01 compared with H₂O₂ group. HUVECs, human umbilical vein endothelial cells; Sal B, salvianolic acid B; H₂O₂, hydrogen peroxide.