Figure 2.

X22 attenuated PA‐induced myocardial oxidative stress in H9C2 cells. H9c2 cells were pretreated with X22 (20 μM) for 1 hr and then incubated with PA (500 μM) for 10 hrs. (A) DHE and DCFH‐DA probes were loaded, respectively, and the ROS positive cells were detected using fluorescence microscope. The images represent three independent experiments. (B) After being loaded with the probes, cells were processed by flow cytometry analysis for H₂O₂ levels, and the data are represented by mean fluorescence intensity (MFI) value. (C) After treatment, cells were lysed and the 30 μl of collected proteins were performed to GSH/GSSG assay. (D) X22 activates Nrf2. H9c2 cells were pretreated with X22 (20 μM) for 1 hr and then incubated with PA (500 μM) for 6 hrs. Then, the nuclear translocation of Nrf‐2 was detected using fluorescence microscope. (E) Cells were collected and the total RNA was extracted and processed for real‐time RT‐qPCR assay for Nrf2 downstream genes including HO‐1, GCLC and GCLM (n = 3 for all in vitro experiment; ^# and *P < 0.05).