Glutathione supplementation attenuates lipopolysaccharide-induced mitochondrial dysfunction and apoptosis in a mouse model of acute lung injury

Abstract

Acute lung injury (ALI) is a life threatening condition associated with hypoxemia, diffuse alveolar damage, inflammation, and loss of lung function. Lipopolysaccharide (LPS; endotoxin) from the outer membrane of Gram-negative bacteria is a major virulence factor involved in the development of ALI. The depletion of glutathione (GSH), an essential intra- and extra-cellular protective antioxidant, by LPS is an important event that contributes to the elevation in reactive oxygen species. Whether restoring GSH homeostasis can effectively ameliorate mitochondrial dysfunction and cellular apoptosis in ALI is unknown and therefore, was the focus of this study. In peripheral lung tissue of LPS-treated mice, hydrogen peroxide and protein nitration levels were significantly increased. Pre-treatment with GSH-ethyl ester (GSH-EE) prevented this increase in oxidative stress. LPS also increased the lactate/pyruvate ratio, attenuated SOD2 protein levels, and decreased ATP levels in the mouse lung indicative of mitochondrial dysfunction. Again, GSH-EE treatment preserved the mitochondrial function. Finally, our studies showed that LPS induced an increase in the mitochondrial translocation of Bax, caspase 3 activation, and nuclear DNA fragmentation and these parameters were all prevented with GSH-EE. Thus, this study suggests that GSH-EE supplementation may reduce the mitochondrial dysfunction associated with ALI.

Keywords: acute lung injury; apoptosis; glutathione ethyl ester; lipopolysaccharide; mitochondrial dysfunction.

Figure 1

GSH-EE supplementation improves LPS-mediated decrease in GSH levels and attenuates LPS-induced oxidative and nitrosative stress. Lungs from vehicle, LPS, GSH-EE + LPS, and GSH-EE treated mice were used for the analysis of GSH, H₂O₂, and nitrated proteins levels. LPS significantly decreased GSH levels but GSH-EE pre-treatment prevented the loss of GSH in these animals (A). The Amplex Red assay measurement indicated a twofold increase in H₂O₂ levels in LPS-treated mice lungs as compared to controls (B). Total nitrated protein levels were measured by dot blot analysis (C). LPS caused significant increases in the formation of H₂O₂ and nitrated proteins, whereas GSH-EE pre-treatment prevented the LPS-induced increase in these oxidative and nitrosative stress parameters. Values are mean ± SEM; n = 6/group. *P < 0.05 vs. vehicle; ^†P < 0.05 vs. LPS alone.

Figure 2

GSH-EE supplementation prevents LPS-mediated mitochondrial dysfunction. Protein extracts prepared from peripheral lungs of vehicle, LPS, GSH-EE + LPS, and GSH-EE treated mice were analyzed by Western blot analysis using a specific antiserum raised against SOD1 (A) or SOD2 (B). Protein levels were normalized for loading using β-actin. A representative blot and normalized densitometric values are shown. There was a significant decrease in SOD1 and SOD2 protein levels in LPS-treated mice and this was prevented by GHS-EE pre-treatment. The lactate/pyruvate ratio was also determined in all four groups (C). LPS-exposed mice had a significantly higher lactate/pyruvate ratio (C). Pre-treatment with GSH-EE preserved the lactate/pyruvate ratio. There was a significant reduction in lung ATP levels after LPS exposure (D). However, the LPS-mediated decrease in ATP levels was not observed in mice pre-treated with GSH-EE (D). Values are mean ± SEM; n = 6/group. *P < 0.05 vs. vehicle; ^†P < 0.05 vs. LPS.

Figure 3

GSH supplementation prevents the LPS-mediated translocation of Bax and caspase 3 activation. Bax protein levels were measured in the mitochondria isolated from lung homogenates of vehicle, LPS, LPS + GSH-EE, and GSH-EE treated mice using Western blot analysis (A). Mitochondrial Bax protein levels from LPS-treated mouse lungs were significantly higher and GSH-EE prevented this translocation (A). Activated caspase 3 and 7 protein levels and activity were also determined in lung homogenates from all four groups. LPS-treated mice had significantly increased activated caspase 3 protein levels that was reduced by GSH-EE pre-treatment (B). The activated caspase 7 protein levels were also upregulated in LPS-treated mice, but did not change after GSH-EE pre-treatment (C). The increase in activated caspase 3 protein levels correlated with an increase in caspase 3/7 activity in lungs of mice exposed to LPS, which was significantly decreased in presence of GSH-EE (D). Values are mean ± SEM; n = 3–6/group. *P < 0.05 vs. vehicle; ^†P < 0.05 vs. LPS.

Figure 4

GSH supplementation inhibits the LPS-induced endothelial cell apoptosis in the mouse lung. Lung tissue sections from vehicle, LPS, LPS + GSH-EE, and GSH-EE treated mice were analyzed for the presence of apoptotic nuclei using the DeadEnd Fluorometric TUNEL System. The DNA breaks were labeled with fluorescein-12-dUTP (TUNEL; green), and the nuclei were stained using propidium iodide (PI; red). Vascular endothelial cells were labeled with anti-Von Willebrand Factor (VWF; blue) (A). The quantification of the TUNEL positive endothelial nuclei and total endothelial nuclei was processed by Image-Pro software and presented as a percentage. LPS treatment caused a significant increase in endothelial apoptosis and this was attenuated by GSH-EE pre-treatment (B). Values are mean ± SEM; n = 4/group. *P < 0.05 vs. vehicle; ^†P < 0.05 vs. LPS.