Intermittent hypoxia simulating obstructive sleep apnea causes pulmonary inflammation and activates the Nrf2/HO-1 pathway

Abstract

Obstructive sleep apnea (OSA) is a disorder with high morbidity in adults. OSA damages multiple organs and tissues, including the cardiovascular and cerebrovascular systems, the metabolism system, the lungs, liver and heart. OSA-induced damage is earliest and greatest to the pulmonary tissue. The present study established a rat OSA model of differing severity by inducing intermittent hypoxia with different concentrations of O₂ and it was determined that OSA caused a severe oxidative stress response and pulmonary inflammation in a dose-dependent manner. OSA increased serum levels of C-reactive protein and 8-isoprostane and elevated the expression of malondialdehyde, tumor necrosis factor α, interleukin (IL)-1β and IL-6 in the pulmonary tissue. Furthermore, the expression of two important antioxidants, superoxide dismutase and glutathione, was downregulated following intermittent hypoxia. By contrast, levels of cylooxygenase 2 and inducible nitric oxide synthase, which are crucial in the antioxidative response, increased. In addition, OSA activates the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase (OH)-1 antioxidative signaling pathway. Finally, all increases and decreases in levels of inflammatory and antioxidative substances were dependent on oxygen concentrations. Therefore, the present study demonstrated that OSA, simulated by intermittent hypoxia, caused an oxidative stress response and pulmonary inflammation, and activated the canonical antioxidative Nrf2/HO-1 signaling pathway in a dose-dependent manner. These results may facilitate the development of clinical therapies to treat pulmonary diseases caused by OSA.

Keywords: inflammation; intermittent hypoxia; nuclear factor erythroid 2-related factor 2; obstructive sleep apnea; oxidative stress.

Figure 1.

Establishment of the rat obstructive sleep apnea model. (A) Protein content of BALF was detected by Bradford assay. (B) Leukocyte numbers in the BALF of rats at different concentrations of O₂. (C) Monocyte numbers in the BALF of rats at different concentrations of O₂. (D) Polykaryocyte numbers in the BALF of rats at different concentrations of O₂. (E) Hematoxylin and eosin staining of rat pulmonary tissues from each group. Scale bar=100 µm. *P<0.05, **P<0.01, ***P<0.001 vs. normoxia. Ns, no significance; BALF, bronchoalveolar lavage fluid.

Figure 2.

Obstructive sleep apnea causes an oxidative stress response and pulmonary inflammation. (A) CRP levels in the serum. (B) Levels of 8-isoprostane in the serum. (C) MDA content in rat pulmonary tissues. (D) SOD activity in the pulmonary tissues of rats. (E) GSH content in the pulmonary tissues of rats. (F) The expression of TNF-α in the pulmonary tissues of rats. (G) The expression IL-1β in the pulmonary tissues of rats. (H) The expression of IL-6 in the pulmonary tissues of rats. *P<0.05, **P<0.01, ***P<0.001 vs. normoxia. CRP, C reactive protein; MDA, malondialdehyde; SOD, superoxide dismutase; GSH, glutathione; TNF, tumor necrosis factor; IL, interleukin; ns, no significance.

Figure 3.

Obstructive sleep apnea increases expression of COX-2 and iNOS. (A) The expression of COX-2 in pulmonary tissue of rats detected by western blotting. (B) The expression of iNOS in the pulmonary tissue of rats detected by western blotting. (C) Immunohistochemical detection of anti-COX-2 in the pulmonary tissue of rats. (D) Immunohistochemical detection of anti-iNOS in the pulmonary tissue of rats. Scale bar=50 µm. ***P<0.001 vs. normoxia. iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase; ns, no significance.

Figure 4.

Obstructive sleep apnea activates the Nrf2/HO-1 signaling pathway. (A) The expression of HO-1 in the pulmonary tissue of rats as detected by western blotting. (B) The expression of Nrf2 in the pulmonary tissue nuclei of rats as detected by western blotting. **P<0.01, ***P<0.001 vs. normoxia. HO-1, heme oxygenase 1; Nrf2, nuclear factor erythroid 2-related factor 2; ns, no significance.