Pharmacological investigation on the anti-oxidant and anti-inflammatory activity of N-acetylcysteine in an ex vivo model of COPD exacerbation

Abstract

Background: Oxidative stress is recognized to be one of predisposing factor in the pathogenesis of COPD. The oxidant/antioxidant imbalance is significantly pronounced in patients with COPD exacerbation. N-acetylcysteine (NAC) seems to be able to reduce COPD exacerbations by modulating the oxidative stress in addition to its well-known mucolytic activity, but there are discordant findings on the actual anti-oxidant activity of NAC.

Methods: The anti-oxidant effect of NAC and its impact on the inflammatory response have been pharmacologically characterized on a human ex vivo model of COPD exacerbation induced by lipopolysaccharide (LPS).

Results: NAC prevented the desensitization induced by LPS incubation on the contractile tone in linear concentration-response manner. Concentrations of NAC ≥1 μM reduced the pro-oxidant response (peroxidase activity, hydrogen peroxide, malondialdehyde, nitric oxide), and improved the anti-oxidant response (total anti-oxidant capacity, glutathione, superoxide dismutase) induced by LPS. Lower concentrations of NAC (<1 μM) did not modulate the bronchial oxidative imbalance. Concentrations of NAC ≥300 μM inhibited the inflammatory response (release of IL-1β, IL-8, and TNF-α) of human airways induced by the overnight stimulation with LPS, whereas lower concentrations of NAC (≥1 μM) were sufficient to reduce the release of IL-6 elicited by LPS. Both the anti-oxidant effect and the anti-inflammatory effect of NAC were inversely correlated with the release of NKA.

Conclusions: The findings of this study suggest that NAC may have a role in modulating the detrimental effect induced by LPS in course of COPD exacerbation. It may elicit both anti-oxidant and anti-inflammatory effects when administered at high concentrations.

Keywords: Anti-inflammatory effect; Anti-oxidant effect; COPD; Lipopolysaccharide; N-acetylcysteine.

Fig. 1

Frequency-response curves produced by EFS ranging from 1 Hz to 50 Hz in control bronchi and after overnight incubation with LPS (100 ng/ml), alone or together with GSH (100 μM) (a). Influence of increasing concentrations of NAC (b 10 nM to 10 μM; c 30 μM to 10 mM) on the desensitizing effect induced by LPS (100 ng/ml). Data are expressed as the mean ± SEM of n = 4 segmental bronchi from different subjects. # P < 0.05 vs. control bronchi: * P < 0.05 and **P < 0.01 vs. LPS-incubated bronchi (statistical significance assessed by two-way ANOVA for comparison among control, LPS, LPS + GSH and LPS + NAC treatments)

Fig. 2

Linear concentration-response curves by NAC on the desensitization induced by overnight incubation of human isolated bronchi with LPS (100 ng/ml). Data are expressed as the mean ± SEM contractile response to EFS delivered at 25 Hz (a) and 50 Hz (b) in n = 4 segmental bronchi from different subjects

Fig. 3

Influence of NAC on the pro-oxidant response (a peroxidase activity, b H₂O₂ concentrations, c malondialdehyde [MDA] concentrations and d nitric oxide concentrations) induced by overnight incubation of human isolated bronchi with LPS (100 ng/ml). Horizontal dotted lines indicate the pro-oxidant response in control bronchi (c), in bronchi treated with LPS alone or in the presence of GSH. Data are expressed as the mean ± SEM of experiments repeated in triplicate. * P < 0.05 and ***P < 0.001 vs. LPS-incubated bronchi (statistical significance assessed by two-way ANOVA)

Fig. 4

Influence of NAC on the anti-oxidant response (a total anti-oxidant capacity [TAC], b GSH concentrations and c superoxide dismutase [SOD] activity) induced by overnight incubation of human isolated bronchi with LPS (100 ng/ml). Horizontal dotted lines indicate the anti-oxidant response in control bronchi (c), in bronchi treated with LPS alone or in the presence of GSH. In B the LPS + GSH treatment has been not carried out in order to not influence the GSH quantification. Data are expressed as the mean ± SEM of experiments repeated in triplicate. ***P < 0.001 vs. LPS-incubated bronchi (statistical significance assessed by two-way ANOVA). ND: not detectable

Fig. 5

Influence of NAC on the cytokines release (a IL-1β concentrations, b IL-6 concentrations, c IL-8 concentrations and d TNF-α concentrations) induced by overnight incubation of human isolated bronchi with LPS (100 ng/ml). Horizontal dotted lines indicate the cytokines concentrations in control bronchi (c), in bronchi treated with LPS alone or in the presence of GSH. Data are expressed as the mean ± SEM of experiments repeated in triplicate. **P < 0.01 and ***P < 0.001 vs. LPS-incubated bronchi (statistical significance assessed by two-way ANOVA)

Fig. 6

Influence of NAC on the release of neurokinin A (NKA) induced by overnight incubation of human isolated bronchi with LPS. Data are expressed as the mean ± SEM of experiments repeated in triplicate. ***P < 0.001 vs. LPS-incubated bronchi (statistical significance assessed by two-way ANOVA)