Research Paper Volume 12, Issue 13 pp 13400—13421

Penehyclidine hydrochloride inhibits renal ischemia/reperfusion-induced acute lung injury by activating the Nrf2 pathway

Figure 2. Effects of PHC on Nrf2/AMPK signaling in NR8383 cells. (A, B) A dual-luciferase reporter analysis system was used to measure the effects of PHC on ARE luciferase activity at different time points (4, 8, 16 or 24 h) or PHC concentrations (1, 2.5 or 5 μg/mL) in NR8383 cells. (C) NR8383 cells were stimulated with PHC (5 μg/mL) for different durations (4, 8 or 16 h). Western blotting was used to determine the total, nuclear and cytoplasmic concentrations of Nrf2. (D) NR8383 cells were stimulated with PHC (5 μg/mL) for different durations (4, 8, 16 or 24 h). Western blotting was used to measure the protein levels of NQO1, HO-1, GCLM and GCLC. (E) NR8383 cells were incubated with PHC (5 μg/mL) for different durations (2, 4 or 8 h). Western blotting was used to measure the protein levels of p-AMPK and AMPK. (F) NR8383 cells were stimulated with CC (an antagonist of AMPK, 5 μM) for 24 h, and then were exposed to PHC for 3 h. Western blotting was used to measure the nuclear concentration of Nrf2 and the protein levels of p-AMPK and AMPK. GAPDH was used as an internal control for total and cytoplasmic proteins, while histone H3.1 was used as an internal control for nuclear proteins. Data are presented as the mean ± S.D. (n = 8). *P < 0.05, **P < 0.01 vs. the control group. +P < 0.05, ++P < 0.01 vs. the PHC alone group.