Research Paper Volume 12, Issue 11 pp 10370—10380

Keap1-Nrf2 signaling activation by Bardoxolone-methyl ameliorates high glucose-induced oxidative injury in human umbilical vein endothelial cells

Figure 2. BARD inhibits high glucose-induced oxidative injury in HUVECs. HUVECs were pretreated with Bardoxolone Methyl (BARD, at 50 nM) for 1h, followed by HG stimulation and cultured for applied time periods, the cellular superoxide contents (A), the GSH/GSSH ratio (B) and mitochondrial depolarization (JC-1 green intensity, C) were tested; Cell viability and death were tested by CCK-8 (D) and medium LDH release (E) assays, respectively, with cell apoptosis analyzed by caspase-3 activity (F), nuclear TUNEL staining (G) and Annexin V-FACS (H) assays. For TUNEL staining assays, at least 500 nuclei in five random views (1×200 magnification) for each condition were included to calculate the TUNEL/DAPI ratio (same for all Figures). Error bars stand for mean ± standard deviation (SD, n=5). “Ctrl” stands for cells-cultured in the normal glucose medium (same for all Figures). ** p<0.01 vs. “Ctrl” treatment. ##p<0.01. vs. HG only treatment (no BARD pretreatment). Each experiment was repeated five times to insure the consistency of experimental results.