FIGURE 7.

AICAR reduces apoptosis and necrosis following exposure of rat alveolar type II epithelial (L2) cells to Br₂ but not in those treated with small interfering RNA (siRNA) against heme oxygenase-1 (HO-1). L2 cells were treated with scrambled RNA (scRNA) or siRNA against HO-1 and then divided into three groups: Air, Br₂ and Br₂+AICAR. a) Representative flow cytometry analysis of Annexin V FITC-stained cells collected 24 h post Br₂ exposure showed a significant decrease in live cells (quadrant (Q) 3), an increase in late apoptotic/necrotic (Q2) cells and an increase in necrotic (Q1) cells. Q4 showed early apoptotic cells. b, c) Administration of a siRNA against HO-1 significantly reduced the levels of HO-1 protein 24 h post Br₂ and in non-exposed L2 cells. AICAR did not increase the protein levels of HO-1 in siRNA-treated cells. The control scRNA did not affect the levels of HO-1. d–g) Quantification of flow cytometry data. In AICAR-treated L2 cells that received the control scRNA, all indicators of cell injury were reduced compared to the vehicle-treated Br₂ groups. Administration of the siRNA against HO-1 did not increase the injury after Br₂ exposure in vehicle-treated cells, but prevented the protective effects of AICAR seen in the scRNA groups. Data are presented as individual data points and mean±sem. Experiments were conducted in triplicate. Significance was determined by one-way ANOVA followed by Tukey’s post hoc test.