The effect of lipopolysaccharide and hydrogen peroxide on apoptosis in rat alveolar epithelial cells

Abstract

Background: Apoptotic cell death following lung inflammation has been increasingly considered to be an underlying pathophysiology in acute lung injury (ALI) or chronic lung disease such as bronchopulmonary dysplasia and emphysema. Although there have been many studies on lipopolysaccharide (LPS) or H2O2-induced apoptosis in alveolar epithelial cells (AECs), studies on apoptosis after sequential treatment with both LPS and H2O2 on AECs are limited. Objectives: To investigate whether there is a differential response in cell death/apoptosis of AECs after sequential treatment of LPS and H2O2 and its possible mechanisms. Methods: L2, type II rat alveolar epithelial cells (AECs), were cultured and treated with 1 μg/mL of LPS or 500 μM of H2O2 at 0 and 6 hour sequentially according to the group assignment (LPS only, LPS+ H2O2 (L+H), H2O2 only, H2O2+LPS (H+L), and the control group). The level of IL-6, cell viability assay (MTS assay and LDH activity), detection of apoptosis (colorimetric caspase-3 activity and TUNEL assay), and mRNA expression levels of apoptotic factors (TNF-α, Bax, Bcl-2, caspase-7, Fas, Fas ligand, P50, P65, IκB-β, IκK-β) were analyzed at 0, 3, 6, 9, 12, and 24 hours. The results were compared between 5 experimental groups. Results: LPS significantly induced the level of IL-6 expression from 3 h and H2O2 alone had no effect on IL-6 expression. There was a significant decrease in MTS activity in all groups at 9 h compared to the control (P<0.05). LDH release increased at 9 h in all groups compared to the control (P<0.05) and the highest level was shown in group H+L (P<0.05). TUNEL positive cells were significantly increased at all measured time points and in all groups compared to the control (P<0.05). At 24 h, TUNEL positive cells were at the highest level in the H+L group and significantly higher than in other groups (P<0.05). TNF-α, Bax, caspase-7 mRNA levels were highest at 12 h and Fas levels at 9 h in H+L group. TNF-α, caspase-7 and Fas mRNA level in H+L group at 9 or 12 h was significantly higher than that of LPS group at 3 h, respectively (P<0.05). P50 (NFκB 1), P65 (Rel A), IκB-β mRNA levels were affected more by LPS than by H2O2. Conclusions: The apoptotic response of AEC may be different according to the sequence of stimulation in vitro. The highest TUNEL positivity in H+L group at 24 h was associated with up-regulation of TNF-α, caspase-7, Fas, and possibly Bax. Hydrogen peroxide may potentiate apoptosis of the LPS pretreated L2 cells by up-regulating several apoptotic factors.