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NAC attenuates LPS-induced toxicity in aspirin-sensitized mouse macrophages via suppression of oxidative stress and mitochondrial dysfunction.

Raza H, John A, Shafarin J - PLoS ONE (2014)

Bottom Line: When ASA was added to LPS-treated macrophages, the increase in oxidative stress was significantly higher than that with LPS alone.These effects are presumed, at least in part, to be associated with alterations in NF-κB/Nrf-2 mediated cell signaling.These results suggest that macrophages are more sensitive to LPS when challenged with ASA and that NAC pre-treatment protects the macrophages from these deleterious effects.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates.

ABSTRACT
Bacterial endotoxin lipopolysaccharide (LPS) induces the production of inflammatory cytokines and reactive oxygen species (ROS) under in vivo and in vitro conditions. Acetylsalicylic acid (ASA, aspirin) is a commonly used anti-inflammatory drug. Our aim was to study the effects of N-acetyl cysteine (NAC), an antioxidant precursor of GSH synthesis, on aspirin-sensitized macrophages treated with LPS. We investigated the effects of LPS alone and in conjunction with a sub-toxic concentration of ASA, on metabolic and oxidative stress, apoptosis, and mitochondrial function using J774.2 mouse macrophage cell line. Protection from LPS-induced toxicity by NAC was also studied. LPS alone markedly induced ROS production and oxidative stress in macrophage cells. When ASA was added to LPS-treated macrophages, the increase in oxidative stress was significantly higher than that with LPS alone. Similarly, alteration in glutathione-dependent redox metabolism was also observed in macrophages after treatment with LPS and ASA. The combination of LPS and ASA selectively altered the CYP 3A4, CYP 2E1 and CYP 1A1 catalytic activities. Mitochondrial respiratory complexes and ATP production were also inhibited by LPS-ASA treatment. Furthermore a higher apoptotic cell death was also observed in LPS-ASA treated macrophages. NAC pre-treatment showed protection against oxidative stress induced apoptosis and mitochondrial dysfunction. These effects are presumed, at least in part, to be associated with alterations in NF-κB/Nrf-2 mediated cell signaling. These results suggest that macrophages are more sensitive to LPS when challenged with ASA and that NAC pre-treatment protects the macrophages from these deleterious effects.

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A–C: LPS- and ASA-induced alterations in mitochondrial respiratory functions.J774.2 macrophage cells were cultured to 80% confluence and treated with LPS alone or in combination with ASA with or without NAC as described above. Freshly isolated mitochondria from control and treated cells were used to assay Complex I and Complex IV activities (7A–7B) and ATP content (7C) as described before [13]. Results are expressed as mean +/−SEM of at least three experiments. Asterisks indicate significant difference (*p≤0.05, **p≤0.001) from control (C), ≠p≤0.05 compared to LPS-treated cells and δ p≤0.05 compared to LPS and ASA treated cells.
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pone-0103379-g007: A–C: LPS- and ASA-induced alterations in mitochondrial respiratory functions.J774.2 macrophage cells were cultured to 80% confluence and treated with LPS alone or in combination with ASA with or without NAC as described above. Freshly isolated mitochondria from control and treated cells were used to assay Complex I and Complex IV activities (7A–7B) and ATP content (7C) as described before [13]. Results are expressed as mean +/−SEM of at least three experiments. Asterisks indicate significant difference (*p≤0.05, **p≤0.001) from control (C), ≠p≤0.05 compared to LPS-treated cells and δ p≤0.05 compared to LPS and ASA treated cells.

Mentions: Our studies showed a significant loss in mitochondrial membrane potential (3–4 fold) after LPS treatment or after ASA treatment (about 2-fold) (Figure 6). A similar loss was observed when a combination of LPS and ASA was used. This was the basis to further study the effects of NAC on mitochondrial functions. As shown in figure 7A, Complex I activity was markedly inhibited in cells treated with LPS alone or combination with ASA. NAC pre-treatment only had a marginal effect on the recovery of this enzyme activity. Cytochrome c oxidase (Complex IV) activity (Figure 7B), on the other hand, was moderately inhibited by LPS alone but significantly by a combination of LPS and ASA treatment. NAC pre-treatment exhibited almost a complete recovery of enzyme activity in these cells.


NAC attenuates LPS-induced toxicity in aspirin-sensitized mouse macrophages via suppression of oxidative stress and mitochondrial dysfunction.

Raza H, John A, Shafarin J - PLoS ONE (2014)

A–C: LPS- and ASA-induced alterations in mitochondrial respiratory functions.J774.2 macrophage cells were cultured to 80% confluence and treated with LPS alone or in combination with ASA with or without NAC as described above. Freshly isolated mitochondria from control and treated cells were used to assay Complex I and Complex IV activities (7A–7B) and ATP content (7C) as described before [13]. Results are expressed as mean +/−SEM of at least three experiments. Asterisks indicate significant difference (*p≤0.05, **p≤0.001) from control (C), ≠p≤0.05 compared to LPS-treated cells and δ p≤0.05 compared to LPS and ASA treated cells.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4116207&req=5

pone-0103379-g007: A–C: LPS- and ASA-induced alterations in mitochondrial respiratory functions.J774.2 macrophage cells were cultured to 80% confluence and treated with LPS alone or in combination with ASA with or without NAC as described above. Freshly isolated mitochondria from control and treated cells were used to assay Complex I and Complex IV activities (7A–7B) and ATP content (7C) as described before [13]. Results are expressed as mean +/−SEM of at least three experiments. Asterisks indicate significant difference (*p≤0.05, **p≤0.001) from control (C), ≠p≤0.05 compared to LPS-treated cells and δ p≤0.05 compared to LPS and ASA treated cells.
Mentions: Our studies showed a significant loss in mitochondrial membrane potential (3–4 fold) after LPS treatment or after ASA treatment (about 2-fold) (Figure 6). A similar loss was observed when a combination of LPS and ASA was used. This was the basis to further study the effects of NAC on mitochondrial functions. As shown in figure 7A, Complex I activity was markedly inhibited in cells treated with LPS alone or combination with ASA. NAC pre-treatment only had a marginal effect on the recovery of this enzyme activity. Cytochrome c oxidase (Complex IV) activity (Figure 7B), on the other hand, was moderately inhibited by LPS alone but significantly by a combination of LPS and ASA treatment. NAC pre-treatment exhibited almost a complete recovery of enzyme activity in these cells.

Bottom Line: When ASA was added to LPS-treated macrophages, the increase in oxidative stress was significantly higher than that with LPS alone.These effects are presumed, at least in part, to be associated with alterations in NF-κB/Nrf-2 mediated cell signaling.These results suggest that macrophages are more sensitive to LPS when challenged with ASA and that NAC pre-treatment protects the macrophages from these deleterious effects.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates.

ABSTRACT
Bacterial endotoxin lipopolysaccharide (LPS) induces the production of inflammatory cytokines and reactive oxygen species (ROS) under in vivo and in vitro conditions. Acetylsalicylic acid (ASA, aspirin) is a commonly used anti-inflammatory drug. Our aim was to study the effects of N-acetyl cysteine (NAC), an antioxidant precursor of GSH synthesis, on aspirin-sensitized macrophages treated with LPS. We investigated the effects of LPS alone and in conjunction with a sub-toxic concentration of ASA, on metabolic and oxidative stress, apoptosis, and mitochondrial function using J774.2 mouse macrophage cell line. Protection from LPS-induced toxicity by NAC was also studied. LPS alone markedly induced ROS production and oxidative stress in macrophage cells. When ASA was added to LPS-treated macrophages, the increase in oxidative stress was significantly higher than that with LPS alone. Similarly, alteration in glutathione-dependent redox metabolism was also observed in macrophages after treatment with LPS and ASA. The combination of LPS and ASA selectively altered the CYP 3A4, CYP 2E1 and CYP 1A1 catalytic activities. Mitochondrial respiratory complexes and ATP production were also inhibited by LPS-ASA treatment. Furthermore a higher apoptotic cell death was also observed in LPS-ASA treated macrophages. NAC pre-treatment showed protection against oxidative stress induced apoptosis and mitochondrial dysfunction. These effects are presumed, at least in part, to be associated with alterations in NF-κB/Nrf-2 mediated cell signaling. These results suggest that macrophages are more sensitive to LPS when challenged with ASA and that NAC pre-treatment protects the macrophages from these deleterious effects.

Show MeSH
Related in: MedlinePlus