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

ABSTRACT

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.

KEYWORDS:

• alveolar epithelial cells
• apoptosis
• hydrogen peroxide
• lipopolysaccharide

Acknowledgments

The authors acknowledge Kyeung-Yeup Lee of the Department of Pediatrics of Seoul National University Children's Hospital, for performing experiments.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Funding

This work was supported in part by the Dongguk University Research Fund of 2016.