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 3. Effects of PHC on rI/R serum-induced inflammatory reactions in NR8383 cells. (A, B) NR8383 cells were stimulated with PHC (5 μg/mL) for 1 h and then treated with serum from rI/R rats for 24 h. Western blotting was used to measure the protein levels of COX-2 and iNOS (A), as well as the nuclear concentration of Nrf2 and the protein levels of AMPK and p-AMPK (B). (C) NR8383 cells were stimulated with brusatol (an antagonist of Nrf2, 300 nM) for 1 h and then exposed to PHC (5 μg/mL) for 1 h before being treated with serum from rI/R rats for 24 h. Western blotting was used to measure Nrf2 levels. (D) NR8383 cells were stimulated with brusatol (300 nM) for 1 h, and then exposed to PHC (5 μg/mL) for 1 h before being treated with serum from rI/R rats for 24 h. Western blotting was used to measure the protein levels of NLRP3. (E) NR8383 cells were stimulated with brusatol (300 nM) for 1 h and then exposed to PHC (5 μg/mL) for 1 h before being treated with serum from rI/R rats for 24 h. Western blotting was used to measure the cytoplasmic and nuclear concentrations of p65, as well as the protein levels of IκBα and p-IκBα. 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 rI/R serum group. +P < 0.05, ++P < 0.01 vs. the PHC alone group.