Figure 3.

Withaferin A reduced H₂O₂-induced apoptotic death of HRMECs. HRMECs were pretreated with withaferin A (50 nmol/mL for 15 min) followed by treatment with H₂O₂ (100 μmol/L) for 2 h. Pretreatment of HRMECs with z−VAD-−FMK (20 μM, 1 h) significantly inhibited the protective effect of withaferin A on cell apoptotic death. (A) LDH assay and apoptotic rate. (B,C) Immunostaining of HRMECs for Annexin V (green), PI (red), and DAPI (blue). It is a method for the identification of apoptotic cells (Annexin V⁺/PI⁻, the early of apoptosis; Annexin V⁺/PI⁺, the late stages of apoptosis). (D) Western blots and quantification of cleaved caspase−3 in HRMECs. Two strips as a group, withaferin A inhibited the H₂O₂-induced apoptotic caspase−3 cleaved fragments. Results are expressed as the mean ± SEM. ** p < 0.01, *** p < 0.001; NS, no significance. N = 6 repeated experiments.