Aspirin Attenuates Hyperoxia-Induced Acute Respiratory Distress Syndrome (ARDS) by Suppressing Pulmonary Inflammation via the NF-κB Signaling Pathway

Abstract

Acute respiratory distress syndrome (ARDS) is a common destructive syndrome with high morbidity and mortality rates. Currently, few effective therapeutic interventions for ARDS are available. Clinical trials have shown that the effectiveness of aspirin is inconsistent. The contribution of platelets to the inflammatory response leading to the development of ARDS is increasingly recognized. The antiplatelet agent aspirin reportedly exerts a protective effect on acid- and hyperoxia-induced lung injury in murine models. Our previous study showed that pretreatment with aspirin exerts protective effects on hyperoxia-induced lung injury in mice. However, the mechanisms and therapeutic efficacy of aspirin in the posttreatment of hyperoxia-induced acute lung injury (ALI) remain unclear. In this study, we used a homozygous NF-κB-luciferase^+/+ transgenic mouse model and treated mice with low-dose (25 μg/g) or high-dose (50 μg/g) aspirin at 0, 24, and 48 h after exposure to hyperoxia (inspired oxygen fraction (FiO₂) > 95%). Hyperoxia-induced lung injury significantly increased the activation of NF-κB in the lung and increased the levels of macrophages infiltrating the lung and reactive oxygen species (ROS), increased the HO-1, NF-κB, TNF-α, IL-1β, and IL-4 protein levels, and reduced the CC10, SPC, eNOS, Nrp-1, and IκBα protein levels in the lung tissue. Pulmonary edema and alveolar infiltration of neutrophils were also observed in the lung tissue of mice exposed to hyperoxia. However, in vivo imaging revealed that posttreatment with aspirin reduced luciferase expression, suggesting that aspirin might reduce NF-κB activation. Posttreatment with aspirin also reduced hyperoxia-induced increases in the numbers of lung macrophages, intracellular ROS levels, and the expression of TNF-α, IL-1β, and IL-4; it also increased CC10, SPC and Nrp-1 levels compared with hyperoxia exposure alone. Lung histopathology also indicated that the aspirin posttreatment significantly reduced neutrophil infiltration and lung edema compared with hyperoxia exposure alone. Aspirin effectively induces an anti-inflammatory response in a model of hyperoxia-induced lung injury. Thus, aspirin may have potential as a novel treatment for hyperoxia-induced ALI.

Keywords: acute lung injury; acute respiratory distress syndrome; aspirin; hyperoxia; therapeutic efficacy.

FIGURE 1

Bioluminescence imaging indicating the therapeutic efficacy of aspirin in the lung tissues of NF-κB-luciferase^+/+ transgenic mice exposed to hyperoxia. NF-κB-luciferase^+/+ transgenic mice were assigned to four groups (n = 6 mice per group): N.C, treatment with PBS at 0, 24, and 48 h and exposure to normoxia; Mock, treatment with PBS at 0, 24, and 48 h and exposure to 72 h of hyperoxia; A25, treatment with 25 μg/g aspirin at 0, 24, and 48 h and exposure to 72 h of hyperoxia; and A50, treatment with 50 μg/g aspirin at 0, 24, and 48 h and exposure to 72 h of hyperoxia.

FIGURE 2

Therapeutic efficacy of aspirin against lung inflammation in mice (A) Gross appearance of the lungs from NF-κB-luciferase^+/+ transgenic mice exposed to hyperoxia. Scale bar: 1 cm (B) Histological changes in the lungs of NF-κB-luciferase^+/+ transgenic mice exposed to hyperoxia. Scale bars for the upper panel represent 200 µm and lower panel represent 50 µm (C) Lung-to-body weight ratio of NF-κB-luciferase^+/+ transgenic mice exposed to hyperoxia. The values are reported as the means ± SEM (n = 6 mice per group). ^## p < 0.01 compared with the N.C. group; *p < 0.05 and **p < 0.01 compared with the Mock group. N.C, treatment with PBS at 0, 24, and 48 h and exposure to normoxia; Mock, treatment with PBS at 0, 24, and 48 h and exposure to hyperoxia for 72 h; A25, treatment with 25 μg/g aspirin at 0, 24, and 48 h and exposure to hyperoxia for 72 h; A50, treatment with 50 μg/g aspirin at 0, 24, and 48 h and exposure to hyperoxia for 72 h. Arrow: red blood cells in the intra-alveolar space, consistent with hemorrhage. Triangle: intrapulmonary hemorrhage with some histiocyte aggregation.

FIGURE 3

Therapeutic efficacy of aspirin against acute lung injury in mice (A) Total cells in the bronchoalveolar lavage fluid (BALF) from NF-κB-luciferase^+/+ transgenic mice exposed to hyperoxia (B) The percentage of macrophages among total cells (C) The percentage of lymphocytes among total cells (D) The generation of intracellular reactive oxygen species (ROS) in bronchoalveolar lavage fluid (BALF) from NF-κB-luciferase^+/+ transgenic mice exposed to hyperoxia (E) The generation of extracellular ROS in bronchoalveolar lavage fluid (BALF) from NF-κB-luciferase^+/+ transgenic mice exposed to hyperoxia. The values are reported as the means ± SEM (n = 6 mice per group). ^# p < 0.05 and ^### p < 0.001 compared with the N.C. group; *p < 0.05 compared with the Mock group. N.C, treatment with PBS at 0, 24, and 48 h and exposure to normoxia; Mock, treatment with PBS at 0, 24, and 48 h and exposure to hyperoxia for 72 h; A25, treatment with 25 μg/g aspirin at 0, 24, and 48 h and exposure to hyperoxia for 72 h; A50, treatment with 50 μg/g aspirin at 0, 24, and 48 h and exposure to hyperoxia for 72 h.

FIGURE 4

Therapeutic efficacy of aspirin in improving survival and ameliorating the stress response in the lung tissue of NF-κB-luciferase^+/+ transgenic mice exposed to hyperoxia. The bands were quantified relative to β-actin bands using ImageJ software. The values are reported as the means ± SEM (n = 6 mice per group). ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 compared with the N.C. group; *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the Mock group. N.C, treatment with PBS at 0, 24, and 48 h and exposure to normoxia; Mock, treatment with PBS at 0, 24, and 48 h and exposure to hyperoxia for 72 h; A25, treatment with 25 μg/g aspirin at 0, 24, and 48 h and exposure to hyperoxia for 72 h; A50, treatment with 50 μg/g aspirin at 0, 24, and 48 h and exposure to hyperoxia for 72 h.

FIGURE 5

Therapeutic efficacy of aspirin in inhibiting hyperoxia-induced inflammation in the lung tissue of NF-κB-luciferase^+/+ transgenic mice. The bands were quantified relative to β-actin bands using ImageJ software. The values are reported as the means ± SEM (n = 6 mice per group). ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 compared with the N.C. group; *p < 0.05 and **p < 0.05 compared with the Mock group. N.C, treatment with PBS at 0, 24, and 48 h and exposure to normoxia; Mock, treatment with PBS at 0, 24, and 48 h and exposure to hyperoxia for 72 h; A25, treatment with 25 μg/g aspirin at 0, 24, and 48 h and exposure to hyperoxia for 72 h; A50, treatment with 50 μg/g aspirin at 0, 24, and 48 h and exposure to hyperoxia for 72 h.

FIGURE 6

Therapeutic efficacy of aspirin in altering levels of the CXCL4 protein induced by hyperoxia in NF-κB-luciferase^+/+ transgenic mice, as detected using immunohistochemical (IHC) staining (A) Images of alveoli; scale bars for the upper panel represent 200 µm and middle panel represent 50 µm (B) Images of bronchi; scale bars for the upper panel represent 200 µm and middle panel represent 50 µm (C) Images of blood vessels. Scale bars for the upper panel represent 200 µm and middle panel represent 50 µm. DAB-specific threshold selection (red selection) was performed using ImageJ software. NF-κB-luciferase^+/+ transgenic mice were assigned to four groups (n = 6 mice per group): Mock group, treatment with PBS at 0, 24 and 48 h, and exposure to hyperoxia for 72 h. A25 group, treatment with 25 μg/g aspirin at 0, 24 and 48 h, and exposure to hyperoxia for 72 h. A50 group: treatment with 50 μg/g aspirin at 0, 24 and 48 h, and exposure to hyperoxia for 72 h.

FIGURE 7

Immunohistochemical staining indicating the therapeutic efficacy of aspirin at altering levels of the CC10 protein in NF-κB-luciferase^+/+ transgenic mice exposed to hyperoxia. Scale bars for the upper panel represent 200 µm and lower panel represent 50 µm. NF-κB-luciferase^+/+ transgenic mice were assigned to four groups (n = 6 mice per group): Mock group, treatment with PBS at 0, 24, and 48 h and exposure to hyperoxia for 72 h; A25 group, treatment with 25 μg/g aspirin at 0, 24, and 48 h and exposure to hyperoxia for 72 h; A50 group, treatment with 50 μg/g aspirin at 0, 24, and 48 h and exposure to hyperoxia for 72 h.

FIGURE 8

Schematic of the pathway by which the aspirin posttreatment regulates hyperoxia-induced acute respiratory distress syndrome (ARDS) by suppressing pulmonary inflammation via NF-κB signaling.