In Vivo Protective Effects of Nootkatone against Particles-Induced Lung Injury Caused by Diesel Exhaust Is Mediated via the NF-κB Pathway

Abstract

Numerous studies have shown that acute particulate air pollution exposure is linked with pulmonary adverse effects, including alterations of pulmonary function, inflammation, and oxidative stress. Nootkatone, a constituent of grapefruit, has antioxidant and anti-inflammatory effects. However, the effect of nootkatone on lung toxicity has not been reported so far. In this study we evaluated the possible protective effects of nootkatone on diesel exhaust particles (DEP)-induced lung toxicity, and the possible mechanisms underlying these effects. Mice were intratracheally (i.t.) instilled with either DEP (30 µg/mouse) or saline (control). Nootkatone was given to mice by gavage, 1 h before i.t. instillation, with either DEP or saline. Twenty-four hours following DEP exposure, several physiological and biochemical endpoints were assessed. Nootkatone pretreatment significantly prevented the DEP-induced increase in airway resistance in vivo, decreased neutrophil infiltration in bronchoalveolar lavage fluid, and abated macrophage and neutrophil infiltration in the lung interstitium, assessed by histolopathology. Moreover, DEP caused a significant increase in lung concentrations of 8-isoprostane and tumor necrosis factor α, and decreased the reduced glutathione concentration and total nitric oxide activity. These actions were all significantly alleviated by nootkatone pretreatment. Similarly, nootkatone prevented DEP-induced DNA damage and prevented the proteolytic cleavage of caspase-3. Moreover, nootkatone inhibited nuclear factor-kappaB (NF-κB) induced by DEP. We conclude that nootkatone prevented the DEP-induced increase in airway resistance, lung inflammation, oxidative stress, and the subsequent DNA damage and apoptosis through a mechanism involving inhibition of NF-κB activation. Nootkatone could possibly be considered a beneficial protective agent against air pollution-induced respiratory adverse effects.

Keywords: Lung; NF-κB; airway resistance; diesel exhaust particles; inflammation; nootkatone; oxidative stress.

Figure 1

Airway hyper-responsiveness. The airway resistance (R), after increasing concentrations of methacholine (0–40 mg/mL), was measured via the forced oscillation technique (FlexiVent), 24 h after intratracheal instillation of either saline or diesel exhaust particles (DEP, 30 µg/animal), with or without nootkatone (NK) pretreatment. There was a dose–response relationship of total respiratory system resistance to increasing doses of MCh (A). From the resistance MCh dose–response curve in (A), an index of airway responsiveness was calculated as the slope of the linear regression, using 0–40 mg/mL concentrations (B). Data are mean ± SEM (n = 6–8).

Figure 2

Representative light microscopy sections of lung tissues of mice, 24 h after administration of saline (A), nootkatone (NK)+saline (B), diesel exhaust particles (DEP; 30 µg/animal; C,D), and NK+DEP (E,F). (A,B) Both saline and NK+saline groups show normal lung tissue with unremarkable changes. (C,D) DEP-exposed lungs show particles within alveolar macrophages (thin arrows). There is severe expansion of the alveolar interstitial space with many neutrophil polymorphs (arrow head), and many macrophages (curved arrow). (E,F) The NK+DEP group shows DEP particles within alveolar macrophages (thin arrows). There is mild expansion of the alveolar interstitial space with a few neutrophil polymorphs (arrow head), and a few macrophages (curved arrow).

Figure 3

Number of cells (A) and polymorphonuclear neutrophils (PMN) (B) in bronchoalveolar lavage, 24 h after intratracheal instillation of either saline or diesel exhaust particles (DEP, 30 µg/animal), with or without nootkatone (NK) pretreatment. Data are mean ± SEM (n = 6–8 in each group).

Figure 4

Lung homogenate levels of 8-isoprostane (A); reduced glutathione (GSH, B); total nitric oxide (NO, C) and tumor necrosis factor α (TNFα, D); 24 h after intratracheal instillation of either saline or diesel exhaust particles (DEP, 30 µg/animal) with or without nootkatone (NK) pretreatment. Data are mean ± SEM (n = 5–8 in each group).

Figure 5

DNA migration (mm) in the lung tissues (A) evaluated by Comet assay, 24 h after intratracheal instillation of either saline or diesel exhaust particles (DEP, 30 µg/animal), with or without nootkatone (NK) pretreatment. Data are mean ± SEM (n = 5 in each group). Representative images, illustrating the quantification of the DNA migration by the Comet assay, under alkaline conditions, in control (B); DEP (C); NK+saline (D) and NK+DEP (E).

Figure 6

Cleaved caspase-3 levels in the lung tissues determined by Western blotting, 24 h after intratracheal instillation of either saline or diesel exhaust particles (DEP, 30 µg/animal), with or without nootkatone (NK) pretreatment. Data are mean ± SEM (n = 4 in each group).

Figure 7

Nuclear factor-kappaB (NF-κB) levels in the lung tissues determined by Western blotting, 24 h after intratracheal instillation of either saline or diesel exhaust particles (DEP, 30 µg/animal), with or without nootkatone (NK) pretreatment. Data are mean ± SEM (n = 4 in each group).