The Effects of Chronic Intermittent Hypoxia in Bleomycin-Induced Lung Injury on Pulmonary Fibrosis via Regulating the NF-κB/Nrf2 Signaling Pathway

Abstract

Background: Obstructive sleep apnea (OSA) is associated with pulmonary fibrosis. Chronic intermittent hypoxia (CIH) is considered to be a surrogate of OSA. However, its exact role in pulmonary fibrosis remains uncertain. Therefore, we investigated the mechanism underlying CIH-induced pulmonary fibrosis and the role of the anti-fibrotic agent in bleomycin (BLE) induced lung injury.

Methods: Mice were divided into eight groups: the normoxia (NOR), CIH, NOR plus BLE, CIH plus BLE, NOR plus pirfenidone (PF), CIH plus PF, NOR plus BLE and PF, and CIH plus BLE and PF groups. BLE was administered intratracheally on day 14 following CIH or NOR exposure. Subsequently, the mice were exposed to CIH or NOR for an additional 4 weeks. PF was administered orally on day 5 after BLE instillation once daily for 3 weeks.

Results: In the BLE-treated groups, CIH-induced more collagen deposition in lung tissues than NOR, and significantly increased hydroxyproline and transforming growth factor-β expression. The CIH and BLE-treated groups showed increased lung inflammation compared to NOR or CIH groups. Following CIH with BLE treatment, nuclear factor-κB (NF-κB) protein expression was significantly increased, whereas nuclear factor-erythroid-related factor 2 (Nrf2) and heme oxygenase-1 protein levels were decreased. After PF treatment, NF-κB and Kelch-like ECH-associated protein 1 expression were suppressed, and Nrf2 expression was increased.

Conclusion: CIH accelerated lung fibrosis in BLE-induced lung injury in mice, potentially by regulating the NF-κB/Nrf2 signaling pathway. Our results implicate PF as a potential therapeutic agent for treating pulmonary fibrosis in individuals with OSA and idiopathic pulmonary fibrosis.

Keywords: Bleomycin; Pulmonary Fibrosis; Sleep Apnea, Obstructive.

Figure 1.

Schematic representation of the animal experimental protocol. Mice were exposed to normoxia (NOR) or chronic intermittent hypoxia (CIH) for 2 weeks, and then administered an intratracheal instillation of bleomycin (BLE) or saline on day 14. After BLE instillation, the mice were exposed to CIH or NOR for an additional 4 weeks. Pirfenidone (PF) was administered on day 5 after the BLE challenge by oral gavage (400 mg/kg), once daily, until the mice were sacrificed. IH: intermittent hypoxia.

Figure 2.

The effects of bleomycin (BLE), chronic intermittent hypoxia (CIH), and pirfenidone (PF) on body weights (BWs) and weight gain. (A) The BWs of mice exposed to normoxia (NOR) or CIH, with or without PF administration. BWs were recorded weekly. (B) Comparison of weight gains in mice (ΔBW=BW at 6 weeks–BW at 1 week). The data shown are expressed as the mean±SD. *p<0.05, **p<0.01 vs. the NOR group.

Figure 3.

The effects of bleomycin (BLE), chronic intermittent hypoxia (CIH), and pirfenidone (PF) on lung morphology. NOR: normoxia. Representative photomicrographs of lung tissues represent stained tissues with hematoxylin and eosin. Vascular morphology (upper panels) or peribronchial morphology (bottom panels) were observed as a measure of lobular inflammation (×200).

Figure 4.

The effect of bleomycin (BLE), chronic intermittent hypoxia (CIH), and pirfenidone (PF) on lung inflammation. (A) Bronchoalveolar lavage (BAL) cells were isolated, after which total cell numbers and the numbers of different inflammatory cell types were counted. (B) Activity of the inflammatory enzyme myeloperoxidase (MPO) was measured in total lung homogenates from mice. (C–F) The levels of albumin (C) and the proinflammatory cytokines interleukin (IL)-6 (D), IL-1β (E), and tumor necrosis factor α (TNF-α) (F) in BAL fluid were measured by enzyme-linked immunosorbent assay. The data shown are expressed as the mean±SD. *p<0.05, **p<0.01, ***p<0.001 vs. the CIH+BLE group. ^†p<0.05, ^††p<0.01, ^†††p<0.001 vs. the normoxia (NOR)+BLE group. ^‡p<0.05, ^‡‡p<0.01, ^‡‡‡p<0.001 vs. the CIH+BLE+PF group. ^§p<0.05, ^§§p<0.01, ^§§§p<0.001 vs. the NOR+BLE+PF group.

Figure 5.

The effects of bleomycin (BLE), chronic intermittent hypoxia (CIH), and pirfenidone (PF) on lung fibrosis. (A) Representative photomicrographs of lung tissues were stained with Masson’s trichrome stain as a measure of collagen accumulation. The upper panels show images of whole lung sections, and the bottom panels represent the enlarged images. Scale bars=2,000 μm, middle panels (×10); 100 μm, bottom panels (×200). (B) Sirius red staining (×200). (C) Hydroxyproline content was measured in lung tissues. (D) Serum transforming growth factor β (TGF-β) levels were measured by performing enzyme-linked immunosorbent assays. The data shown are expressed as the mean±SD. *p<0.05, **p<0.01, ***p<0.001 vs. the CIH+BLE group. ^†p<0.05, ^††p<0.01 vs. the normoxia (NOR)+BLE group.

Figure 6.

The effects of bleomycin (BLE), chronic intermittent hypoxia (CIH), and pirfenidone (PF) on oxidative stress. (A, B) The levels of malondialdehyde (MDA; as a lipid peroxidation marker) (A) and superoxide dismutase (SOD; as an indicator of oxidative stress) (B) were measured in the total lung homogenates from the mice in each group. NOR: normoxia. The data shown are expressed as the mean±SD. *p<0.05, **p<0.01 vs. the CIH+BLE group.

Figure 7.

The effects of bleomycin (BLE), chronic intermittent hypoxia (CIH), and pirfenidone (PF) on the nuclear factor-κB (NFκB)/nuclear factor-erythroid-related factor 2 (Nrf2) signaling pathway. Protein-expression levels in the total and nuclear fractions of lung tissues were analyzed by Western blotting. β-Actin or histone H3 were detected as loading controls for the total and nuclear fractions, respectively. Keap1: Kelch-like ECH-associated protein 1; HO1: heme oxygenase-1.

Figure 8.

Summary of the lung fibrosisrelated intracellular signaling pathways induced by bleomycin, chronic intermittent hypoxia, and anti-fibrotic drug mechanisms. PF: pirfenidone; Keap1: Kelch-like ECH-associated protein 1; Nrf2: nuclear factor-erythroid-related factor 2; HO-1: heme oxygenase-1.