The effects of hydrogen peroxide and lipopolysaccharide on rat alveolar L2 cells

Purpose: This study aimed to investigate differential cell responses of alveolar epithelial cells (AECs) after treatments with lipopolysaccharide (LPS) and hydrogen peroxide (H2O2) to mimic the exposure to inflammation and oxidative stress and the mechanisms of a double-hit model of apoptosis. Materials and Methods: AECs were cultured and treated with combinations of 1 μg/mL of LPS and 500 μM H2O2 as follows: LPS-only at 0 h, LPS at 0 h with H2O2 at 6 h (LPS + H2O2), H2O2-only at 0 h, H2O2 at 0 h with LPS at 6 h (H2O2 + LPS), and control. We investigated mRNA expression (TNF-α, Fas, Fas ligand, Bax, Bcl-2, Caspase-7), protein expression (Fas, Bax, Bcl-2, Caspase-7) and apoptosis (Caspase-3 activity, TUNEL assay) at 0, 3, 6, 9, 12, and 24 h. Results: In the H2O2 + LPS group, the Caspase-7, and Fas mRNA levels were significantly higher than the other groups at 9 h and 12 h, and Bax was higher at 12 h. The Bax/Bcl-2 protein expression ratio was significantly higher in the H2O2 + LPS group than that of the other groups at 12h and 24h. Apoptotic index was highest in the H2O2 + LPS group at 24 h. Conclusions: The sequence of stimulation may modify the cell response in rat AECs. The results suggest that previous oxidative stress and subsequent LPS-induced inflammation primarily influence apoptosis of L2 cells by up-regulation of cell signaling.