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. 2009 Apr;296(4):L565-73.
doi: 10.1152/ajplung.90487.2008. Epub 2009 Jan 16.

Nrf2 increases survival and attenuates alveolar growth inhibition in neonatal mice exposed to hyperoxia

Sharon McGrath-Morrow  1 , Thomas Lauer, Min Yee, Enid Neptune, Megan Podowski, Rajesh K Thimmulappa, Michael O'Reilly, Shyam Biswal
Affiliations

Nrf2 increases survival and attenuates alveolar growth inhibition in neonatal mice exposed to hyperoxia

Sharon McGrath-Morrow et al. Am J Physiol Lung Cell Mol Physiol. 2009 Apr.

Abstract

Increased oxidative stress is associated with perinatal asphyxia and respiratory distress in the newborn period. Induction of nuclear factor erythroid 2 p45-related factor (Nrf2) has been shown to decrease oxidative stress through the regulation of specific gene pathways. We hypothesized that Nrf2 attenuates mortality and alveolar growth inhibition in newborn mice exposed to hyperoxia. Nrf2(+/+) and Nrf2(-/-) newborn mice were exposed to hyperoxia at 24 h. Survival was significantly less in Nrf2(-/-) mice exposed to 72 h of hyperoxia and returned to room air (P < 0.0001) and in Nrf2(-/-) mice exposed to hyperoxia for 8 continuous days (P < 0.005). To determine the response of Nrf2 target genes to hyperoxia, glutathione peroxidase 2 (Gpx2) and NAD(P)H:quinone oxidoreductase (NQO1) expression was measured from lung of newborn mice using real-time PCR. In the Nrf2(+/+) mice, significant induction of lung Gpx2 and NQO1 above room air controls was found with hyperoxia. In contrast, Nrf2(-/-) mice had minimal induction of lung Gpx2 and NQO1 with hyperoxia. Expression of p21 and IL-6, genes not regulated by Nrf2, were also measured. IL-6 expression in Nrf2(-/-) lung was markedly induced by 72 h of hyperoxia in contrast to the Nrf2(+/+) mice. p21 was induced in both Nrf2(+/+) and Nrf2(-/-) lung by hyperoxia. Mean linear intercept (MLI) and mean chord length (MCL) were significantly increased in 14-day-old Nrf2(-/-) mice previously exposed to hyperoxia compared with Nrf2(+/+) mice. The percentage of surfactant protein C (Sp-c(+)) type 2 alveolar cells in 14-day-old Nrf2(-/-) mice exposed to neonatal hyperoxia was also significantly less than Nrf2(+/+) mice (P < 0.02). In summary, these findings indicate that Nrf2 increases survival in newborn mice exposed to hyperoxia and that Nrf2 may help attenuate alveolar growth inhibition caused by hyperoxia exposure.

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Figures

Fig. 1.
Fig. 1.
Decreased survival of nuclear erythroid-related factor 2-deficient (Nrf2−/−) newborn mice in hyperoxia. Top: newborn Nrf2+/+ and Nrf2−/− mice were placed in hyperoxia at 24 h of age, exposed to hyperoxia for 72 h, and returned to room air. Mortality was significantly increased in the Nrf2−/− mice within the first 24 h of room air recovery compared with Nrf2+/+ mice (P < 0.0001). Nrf2+/+ mice, n = 17; Nrf2−/− mice, n = 19 (4 litters per group). Bottom: 24-h-old Nrf2+/+ and Nrf2−/− mice were placed in hyperoxia for 8 continuous days. Mortality was significantly increased in the Nrf2−/− mice at 5 days of continuous oxygen exposure compared with Nrf2+/+ mice (P < 0.003). Nrf2+/+ mice, n = 12; Nrf2−/− mice, n = 19 (2 litters per group). *Significant difference by P value using the Student's t-test.
Fig. 2.
Fig. 2.
Increased nitrotyrosine staining in lung of newborn Nrf2−/− mice exposed to hyperoxia. Top: representative example of nitrotyrosine staining from lung of 4-day-old Nrf2+/+ and 4-day-old Nrf2−/− mice exposed to 72 h of hyperoxia. Arrows point to nitrotyrosine staining. Bottom: significant increase in nitrotyrosine staining in lung of Nrf2−/− oxygen-exposed mice compared with Nrf2+/+ oxygen-exposed mice (P < 0.002) and Nrf2−/− room air mice (P < 0.004), n = 3 for each group. *Significant difference by P value using Student's t-test. RA, room air.
Fig. 3.
Fig. 3.
Increased terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) staining in lung of newborn Nrf2−/− mice exposed to hyperoxia. Top: representative example of TUNEL staining from lung of 4-day-old Nrf2+/+ and Nrf2−/− mice exposed to 72 h of hyperoxia. Arrows point to TUNEL staining. Bottom: significant increase in TUNEL staining in lung of Nrf2−/− mice exposed to hyperoxia compared with lung of Nrf2+/+ hyperoxia exposed mice (P < 0.05) and lung of Nrf2−/− mice that were born and raised in room air (P < 0.05), n = 3 for each group. *Significant difference by P value using Student's t-test.
Fig. 4.
Fig. 4.
Increased expression of lung glutathione peroxidase 2 (Gpx2) in Nrf2+/+ newborn mice exposed to hyperoxia. A: increased expression of lung Gpx2 above room air was found in newborn Nrf2+/+ mice exposed to hyperoxia for 24, 48, and 72 h. Minimal induction of lung Gpx2 expression was found in Nrf2−/− mice exposed to hyperoxia. Nrf2+/+ lung Gpx2 expression was significantly greater than Nrf2−/− at 48 and 72 h of hyperoxia (*P < 0.003 and **P < 0.0001, respectively), n = 3–7 (per group). B: Gpx2 protein was significantly decreased in lung from 4-day-old Nrf2−/− mice exposed to 72 h of hyperoxia (P < 0.004). C: representative example of Gpx2 Western blot. D: decreased Gpx2 staining in 4-day-old Nrf2−/− mice exposed to 72 h of hyperoxia by immunohistochemistry (arrows point to Gpx2 staining). n = 5–6 (per group).
Fig. 5.
Fig. 5.
Expression of lung NAD(P)H:quinone oxidoreductase (NQO1), p21, and IL-6 in Nrf2+/+ and Nrf2−/− newborn mice exposed to hyperoxia. A: lung NQO1 was significantly induced in Nrf2+/+ above room air controls but not in lung of Nrf2−/− mice exposed to hyperoxia. Nrf2+/+ lung NQO1 expression was significantly greater than Nrf2−/− at 48 and 72 h of hyperoxia (*P < 0.01 and **P < 0.0001). B: p21 was induced in lung of both Nrf2+/+ and Nrf2−/− mice in response to hyperoxia. At 72 h of Nrf2+/+ lung p21 expression was significantly greater than Nrf2−/− mice in hyperoxia (*P < 0.01). C: IL-6 was significantly induced in Nrf2−/− mice at 72 h of hyperoxia compared with Nrf2+/+ mice in hyperoxia (*P < 0.0001), n = 3–7 (per group).
Fig. 6.
Fig. 6.
Higher basal Gpx2 expression in lung of adult Nrf2+/+ mice compared with 1-day-old Nrf2+/+ newborn mice. Gpx2 expression at baseline was higher in adult Nrf2+/+ lung compared with newborn Nrf2+/+ lung. No difference in basal NQO1, p21, or IL-6 expression was found between adult Nrf2+/+ lung and newborn Nrf2+/+ lung, n = 3–7; error bars reflect SE of the mean (per group).
Fig. 7.
Fig. 7.
Similar postnatal alveolar growth in Nrf2+/+ and Nrf2−/− mice raised in room air at 7 and 14 days of life. There were no differences in mean linear intercept (MLI) measurements at 7 or 14 days of life between Nrf2+/+ and Nrf2−/− mice born and raised in room air conditions, n = 4–5 (per group).
Fig. 8.
Fig. 8.
Impaired alveolar growth in 2-wk-old Nrf2−/− mice exposed to hyperoxia in the newborn period. A representative example of lung from 2-wk-old Nrf2−/− and Nrf2+/+ mice placed in hyperoxia for 3 days starting at 48 h of life and then recovered in room is shown.
Fig. 9.
Fig. 9.
Decreased percentage of surfactant protein C (Sp-c+) type 2 alveolar cells in lung of 14-day-old Nrf2−/− mice exposed to hyperoxia in the newborn period. A: representative example of Sp-c (red), T1α staining (green) and 4′,6′-diamidino-2-phenylindole (DAPI; blue) in lung from 14-day-old Nrf2+/+ and Nrf2−/− mice raised in room air (top) or exposed to hyperoxia for 72 h in the newborn period (bottom). B: percentage of Sp-c staining cells over total cells was significantly decreased in both Nrf2+/+ and Nrf2−/− mice exposed to hyperoxia in the newborn period (*P < 0.03 and *P < 0.004, respectively). Percentage of Sp-c staining cells over total cells was significantly less in the Nrf2−/− lung exposed to hyperoxia compared with the Nrf2+/+ lung exposed to hyperoxia (P < 0.02), n = 3 (per group). The thin white arrows in the top point to Sp-c+ staining and the thick arrows in the bottom point to T1α staining.
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