In vivo antioxidant treatment protects against bleomycin-induced lung damage in rats

Abstract

1. This study examines the activity of the antioxidant N-acetylcysteine on bleomycin-induced pulmonary fibrosis in rats with emphasis on the early inflammatory phase. 2. Rats receiving N-acetylcysteine (300 mg kg(-1) day(-1), intraperitoneal) had less augmented lung wet weight, and lower levels of proteins, lactate dehydrogenase, neutrophil and macrophage counts in bronchoalveolar lavage fluid and lung myeloperoxidase activity with a betterment of histological score at 3 days postbleomycin. 3. A diminished lung GSH/GSSG ratio and augmented lipid hydroperoxides were observed 3 days postbleomycin. These changes were attenuated by N-acetylcysteine. Alveolar macrophages from bleomycin-exposed rats released augmented amounts of superoxide anion and nitric oxide. N-Acetylcysteine did not modify superoxide anion generation but reduced the increased production of nitric oxide. 4. N-Acetylcysteine suppressed the bleomycin-induced increased activation of lung NF-kappaB (shift assay and immunohistochemistry), and decreased the augmented levels of the early inflammatory cytokines, tumour necrosis factor-alpha, interleukin-beta, interleukin-6 and macrophage inflammatory protein-2 observed in bronchoalveolar lavage fluid at 1 and 3 days postbleomycin exposure. 5. At 15 days postbleomycin, N-acetylcysteine decreased collagen deposition in bleomycin-exposed rats (hydroxyproline content: 6351+/-669 and 4626+/-288 micro g per lung in drug vehicle- and N-acetylcysteine-treated rats, respectively; P<0.05). Semiquantitative histological assessment at this stage showed less collagen deposition in N-acetylcysteine-treated rats compared to those receiving bleomycin alone. 6. These results indicate that N-acetylcysteine reduces the primary inflammatory events, thus preventing cellular damage and the subsequent development of pulmonary fibrosis in the bleomycin rat model.

Figure 1

Lung weight/body weight (panel a) and lung hydroxyproline levels (panel b) in experimental groups and time points as indicated. Treatment with N-acetylcysteine (NAC; 300 mg kg⁻¹ per day, i.p.) reduced the lung weight and the lung content of hydroxyproline. Data are means±s.e.m. of 10 animals per group; ^*P<0.05 from group A; +P<0.05 from group B.

Figure 2

Time-course changes in the levels of tumour necrosis factor (TNF)-α, IL-1β, IL-6 and MIP-2 in bronchoalveolar lavage fluid (BALF) at 1, 3 and 15 days postbleomycin or saline in experimental groups as indicated. Treatment with N-acetylcysteine (NAC; 300 mg kg⁻¹ per day, i.p.) reduced the augmented levels of inflammatory cytokines in bleomycin-exposed rats. Data are means±s.e.m. of four to seven animals; ^*P<0.05 from group A; +P<0.05 from group C.

Figure 3

Effect of N-acetylcysteine (NAC; 300 mg kg⁻¹ per day, i.p.) on alveolar macrophage release of superoxide anion and nitrite in the culture medium. NAC did not significantly change superoxide anion release but attenuated nitric oxide production. Data are means±s.e.m. of five independent observations in each group. ^*P<0.05 from group A; +P<0.05 from group C.

Figure 4

Effect of N-acetylcysteine (NAC; 300 mg kg⁻¹ per day, i.p.) on the bleomycin-induced increase of NF-κB-binding activity assessed at 1, 3 and 15 days postexposure. Panel (a) An electrophoretic gel mobility shift assay showing NF-κB-binding activity in rat lung cell nuclear proteins from individual animals at 1 day postbleomycin. Lane 1: Free probe. Lanes 2, 3, 4 and 5 correspond to experimental groups A (drug vehicle + saline), B (NAC + saline), C (drug vehicle + bleomycin) and D (NAC + bleomycin), respectively. The specificity of the binding was confirmed by adding excess unlabelled NF-κB oligonucleotide (lane 6). Panel (b) The densitometric scanning of the band shift data of NF-κB-binding activity is expressed as relative values compared to their corresponding values in group A taken as unity. There was a significant increase in NF-κB-binding activity that was suppressed in NAC-treated rats. Columns are means±s.e.m. of three to four independent experiments for each group. ^*P<0.05 compared to group A; ⁺P<0.05 from group B.

Figure 5

Immunohistochemical analysis of NF-κB in lung sections obtained at 3 days postbleomycin or saline exposure in vehicle- or N-acetylcysteine (NAC, 300 mg kg⁻¹ per day, i.p.)-treated rats. Lung sections were obtained from control rats (vehicle+vehicle; (a, b)), and from rats exposed to bleomycin and receiving drug vehicle (c, d) or NAC (e, f). Panels show sections stained with anti-p65 antibody followed by the avidin–biotin complex. The immunohistochemical localization of NF-κB appears as dark-brown positive nuclear staining in airway epithelial cells as well as macrophages and other inflammatory cells like polymorphonuclear leukocytes in interalveolar septa. There is a low staining level in control lungs. Note the high level of staining in the epithelium of bleomycin-exposed rats (panel (d) compared to the virtual absence in epithelial cells from control animals (panel (b). The increased expression of p65 in vehicle+bleomycin rats was markedly diminished in NAC+bleomycin samples (cf. (c,d) and (e,f)). Original magnification × 25 (a, c, e) and × 40 (b, d, f).

Figure 6

Representative photomicrographs of lung histopathology in groups B (vehicle + bleomycin) and C (N-acetylcysteine (NAC)+ bleomycin) at 3 days (a) and (b), respectively) and 15 days (c, d) and (e), respectively) following endotracheal bleomycin. Lung sections were stained with haematoxylin–eosin. NAC dose was of 300 mg kg⁻¹ per day given intraperitoneally. Normal lungs observed for group A (vehicle + vehicle) are not shown. Three days after intratracheal bleomycin, a marked peribronchial interstitial infiltration of inflammatory cells and alveolar oedema were patent in vehicle-treated animals (a). These pulmonary lesions were not improved by NAC (b). At 15 days postbleomycin, inflammation and fibrosis were present in vehicle-treated rats ((c,d)), but NAC ameliorated the pulmonary lesions (e). Panels (f) (group B) and (g) (group C) show lung sections stained with Masson-trichrome at 15 days postbleomycin. The presence of interstitial collagen was diminished by NAC. Original magnification of panels × 10 (except (d) × 20).