Attenuation of Hyperoxic Lung Injury in Newborn Thioredoxin-1-Overexpressing Mice through the Suppression of Proinflammatory Cytokine mRNA Expression

Abstract

The role of thioredoxin-1 (TRX), a small redox-active protein with antioxidant effects, during hyperoxic lung injury in newborns remains undetermined. We investigated TRX impact on hyperoxic lung injury in newborn TRX transgenic (TRX-Tg) and wildtype (WT) mice exposed to 21% or 95% O₂ for four days, after which some mice were allowed to recover in room air for up to 14 days. Lung morphology was assessed by hematoxylin/eosin and elastin staining, as well as immunostaining for macrophages. The gene expression levels of proinflammatory cytokines were evaluated using quantitative real-time polymerase chain reaction. During recovery from hyperoxia, TRX-Tg mice exhibited an improved mean linear intercept length and increased number of secondary septa in lungs compared with the WT mice. Neonatal hyperoxia enhanced the mRNA expression levels of proinflammatory cytokines in the lungs of both TRX-Tg and WT mice. However, interleukin-6, monocyte chemoattractant protein-1, and chemokine (C-X-C motif) ligand 2 mRNA expression levels were reduced in the lungs of TRX-Tg mice compared with the WT mice during recovery from hyperoxia. Furthermore, TRX-Tg mice exhibited reduced macrophage infiltration in lungs during recovery. These results suggest that in newborn mice TRX ameliorates hyperoxic lung injury during recovery likely through the suppression of proinflammatory cytokines.

Keywords: antioxidant effects; hyperoxic lung injury; newborn mice; proinflammatory cytokine gene expression; thioredoxin-1.

Figure 1

Changes in body weight after neonatal hyperoxic exposure. The changes in body weight were evaluated in 4 and 14-day-old WT and TRX-Tg mice following normoxic or hyperoxic exposure (n = 6 per group). Data are shown as means ± SEM. Comparisons between groups were performed using one-way ANOVA followed by Tukey’s test. ** p < 0.01. WT: wildtype; TRX: Thioredoxin-1; Tg: Transgenic; Air: Normoxia; O₂: Hyperoxia; SEM: Standard error of the mean; ANOV: Analysis of variance.

Figure 2

Alveolar development on day four after neonatal hyperoxic exposure. (A) H&E-stained histological sections. Scale bar = 100 µm (magnification × 100). (B) Lm (n = 6 per group). Lm was assessed in six nonoverlapping fields of lung parenchyma in one tissue section per animal. Animals were exposed to air (open bars) or O₂ (filled bars). (C) Elastin-stained histological sections. The black arrows indicate elastin-positive secondary septa. Scale bar = 50 µm (magnification × 200). (D) Number of secondary septa (n = 6 per group). The number of secondary septa was assessed in six nonoverlapping fields of lung parenchyma in one tissue section per animal. Animals were exposed to air (open bars) or O₂ (filled bars). Data are shown as the mean ± SEM. Comparisons between groups were performed using one-way ANOVA followed by Tukey’s test. ** p < 0.01. Air: Normoxia; O₂: Hyperoxia; WT: Wildtype; TRX: Thioredoxin-1; Tg: Transgenic; Lm: Mean linear intercept length; H&E: Hematoxylin and eosin; SEM: Standard error of the mean; ANOVA: Analysis of variance.

Figure 3

Alveolar development during recovery from neonatal hyperoxic exposure on day 14. (A) H&E-stained histological sections. Scale bar = 100 µm (magnification ×100). (B) Lm (n = 6, per group). Lm was assessed in six nonoverlapping fields of lung parenchyma in one tissue section per animal. Animals were exposed to air (open bars) or O₂ (filled bars). (C) Elastin-stained histological sections. The black arrows indicate elastin-positive secondary septa. Scale bar = 50 µm (magnification × 200). (D) Number of secondary septa (n = 6 per group). The number of secondary septa was assessed in six nonoverlapping fields of lung parenchyma in one tissue section per animal. Animals were exposed to air (open bars) or O₂ (filled bars). Data are shown as means ± SEM. Comparisons between groups were performed using one-way ANOVA followed by Tukey’s test. * p < 0.05; ** p < 0.01. Air: Normoxia; O₂: Hyperoxia; WT: Wildtype; TRX: Thioredoxin-1; Tg: Transgenic; Lm: Mean linear intercept length; H&E: Hematoxylin and eosin; SEM: Standard error of the mean; ANOVA: Analysis of variance.

Figure 4

Changes in mRNA expression levels of Il-6, Ho-1, Mcp-1, Il-1β, Tnf, Cxcl1, and Cxcl2 in lungs. Quantitative RT-PCR was performed on (A) day 4 and (B) day 14 (n = 6 per group). Animals were exposed to air (open bars) or O₂ (filled bars). Data are shown as means ± SEM. Comparison between groups were performed using one-way ANOVA followed by Tukey’s test. * p < 0.05; ** p < 0.01. IL: Interleukin; HO: Heme oxygenase; MCP-1: Monocyte chemotactic protein-1; TNF: Tumor necrosis factor; CXCL: Chemokine (C-X-C motif) ligand; Air: Normoxia; O₂: Hyperoxia; WT: Wildtype; TRX: Thioredoxin-1; Tg: Transgenic; SEM: Standard error of the mean; ANOVA: Analysis of variance.

Figure 5

Immunohistochemistry for macrophages. Immunohistochemistry using the macrophage marker F4/80 was performed on lung sections on (A) day 4 and (C) day 14. The black arrows indicate F4/80-positive macrophages. Scale bar = 25 μm (magnification × 400). Number of macrophages on (B) day 4 and (D) day 14 (n = 6 per group). The number of macrophages was assessed in six nonoverlapping fields of lung parenchyma in one tissue section per animal. Animals were exposed to air (open bars) or O₂ (filled bars). Data are shown as means ± SEM. Comparisons between groups were performed using one-way ANOVA followed by Tukey’s test. * p < 0.05; ** p < 0.01. Air: Normoxia; O₂: Hyperoxia; WT: Wildtype; TRX: Thioredoxin-1; Tg: Transgenic; SEM: Standard error of the mean; ANOVA: Analysis of variance.

Figure 6

(A,B) Mif mRNA expression levels. Quantitative RT-PCR for MIF was performed on (A) day 4 and (B) day 14 (n = 6 per group). (C,D) MIF protein expression levels. Western blot analysis for MIF was performed on (C) day 4 and (D) day 14. Animals were exposed to air (open bars) or O₂ (filled bars). Data are shown as means ± SEM. Comparisons between groups were performed using one-way ANOVA followed by Tukey’s test. * p < 0.05. Air: Normoxia; O₂: Hyperoxia; MIF: Macrophage migration inhibitory factor; WT: Wildtype; TRX: Thioredoxin-1; Tg: Transgenic; SEM: Standard error of the mean; ANOVA: Analysis of variance. (E) Immunoprecipitation of TRX with MIF. Immunoprecipitation using normal IgG and anti-TRX antibody was analyzed by Western blot analysis. The antibodies for Western blotting were anti-MIF, anti-hTRX, and anti-β-actin. hTRX: Human thioredoxin; MIF: Macrophage migration inhibitory factor.