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CDNA microarray analysis of sulforaphane effect on mouse acute lung injury caused by hyperoxia

ABSTRACT: Protective roles of Nrf2, a key transcription factor for antioxidant and defense genes, have been determined in oxidative lung injury, and health benefits of Nrf2 agonists including sulforaphane have been demonstrated. The current study was designed to investigate the effect of sulforaphane on model acute lung injury and sulforaphane-mediated transcriptome changes in mouse lungs. Adult mice genetically deficient in Nrf2 (Nrf2-/-) and wild-type controls (Nrf2+/+, ICR) received oral sulforaphane (9 mmol/daily) or vehicle before (-5, -3, -1 days) hyperoxia or air exposure (3 days), and lung injury and gene expression changes were assessed. Sulforaphane significantly reduced hyperoxia-induced airway injury, inflammation, and mucus hypersecretion in Nrf2+/+ mice while relatively marginal treatment effect was found in Nrf2-/- mice. Sulforaphane significantly altered expression of lung genes associated with oxidative phosphorylation and mitochondrial dysfunction (Atp2a2, Cox7a1, Ndufa1) basally and cell function/cycle and protein metabolism (Actr1a, Wasf2, Ccne1, Gtpbp4) after hyperoxia in Nrf2+/+ mice. Nrf2-dependently modulated lung genes by sulforaphane and hyperoxia were associated with tissue development and hereditary disorders (Slc25a3, Pccb, Psmc3ip). Results demonstrate preventive roles of sulforaphane against oxidant lung injury in mice, and reveal potential downstream mechanisms. Our observations also suggest Nrf2-independent mechanisms of sulforaphane in prevention of acute lung injury.

ORGANISM(S): Mus musculus

PROVIDER: GSE58654 | GEO | 2015/06/01

SECONDARY ACCESSION(S): PRJNA253109

REPOSITORIES: GEO

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Project description:Background: Nrf2 is an essential cytoprotective transcription factor. However, association of Nrf2 in organ development and neonatal disease is rarely examined. Hyperoxia exposure to newborn rodents generates pulmonary phenotypes which resemble bronchopulmonary dysplasia (BPD) of prematurity. Methods: To investigate the role of Nrf2 in lung maturation and BPD pathogenesis, Nrf2-deficient (Nrf2-/-) and wild-type (Nrf2+/+) neonates were exposed to air or hyperoxia (O2). Transcriptome analysis determined Nrf2-directed mechanisms in premature lung. Lung injury was assessed by bronchoalveolar lavage analysis and histopathology. Results: In Nrf2-/- neonates, basal expression of cell cycle machinery, redox balance, and lipid/carbohydrate metabolism genes were suppressed while immunity genes were overexpressed compared to Nrf2+/+ pups. O2-induced mortality and pulmonary inflammation/injury were significantly higher in Nrf2-/- than in Nrf2+/+. Lung DNA lesion and oxidation were greater in Nrf2-/- than in Nrf2+/+, constitutively and after O2. Nrf2-dependent genes modulated cellular growth/proliferation, defense, immunity, and lipid metabolism against hyperoxia. Bioinformatic elucidation of Nrf2 binding motifs and augmented O2-induced inflammation in genetically deficient neonates validated Gpx2 and Marco as Nrf2 effectors. Conclusion: Overall, Nrf2 in underdeveloped lungs orchestrated cell cycle, morphogenesis, and immunity as well as cellular defense constitutively and under oxidant stress. Results provide putative molecular mechanisms of Nrf2-directed lung alveolarization and BPD of prematurity. PARALLEL study design with 42 samples comparing 14 groups of age (P1 to P4 corresponding to day 0 to day 3 animals), gene, and exposure: (4 groups Nrf+/+ wild type P1-P4 air exposure) (4 groups Nrf -/- knockout P1-P4 air exposure), (3 groups Nrf+/+ wild type P2-P4 with 100 percent O2 (hyperoxia exposure) and 3 groupsNrf -/- knockout P2-P4 with 100 percent O2 (hyperoxia exposure)) Biological replicates: 3 per group

Project description:RATIONALE: Mechanical ventilation (MV) is an indispensable therapy for critically ill patients with acute lung injury and the adult respiratory distress syndrome. However, the mechanisms by which conventional MV induces lung injury remain unclear. OBJECTIVES: We hypothesized that disruption of the gene encoding Nrf2, a transcription factor which regulates the induction of several antioxidant enzymes, enhances susceptibility to ventilator-induced lung injury (VILI), while antioxidant supplementation attenuates such effect. METHODS: To test our hypothesis and to examine the relevance of oxidative stress in VILI, here we have assessed lung injury and inflammatory responses in Nrf2-deficient (Nrf2(-/-)) mice and wildtype (Nrf2(+/+)) animals following acute (2 h) injurious model of MV with or without administration of antioxidant. MEASUREMENTS AND MAIN RESULTS: Nrf2(-/-) mice displayed greater levels of lung alveolar and vascular permeability and inflammatory responses to MV as compared to Nrf2(+/+) mice. Nrf2-deficieny enhances the levels of several pro-inflammatory cytokines implicated in the pathogenesis of VILI. We found diminished levels of critical antioxidant enzymes and redox imbalance by MV in the lungs of Nrf2(-/-) mice; however antioxidant supplementation to Nrf2(-/-) mice remarkably attenuated VILI. When subjected to clinically relevant prolong period of MV, Nrf2(-/-) mice displayed greater levels of VILI than Nrf2(+/+) mice. Expression profiling revealed lack of induction of several VILI genes, stress response and solute carrier proteins and phosphatases in Nrf2(-/-) mice. CONCLUSIONS: Collectively, our data demonstrate for the first time a critical role for Nrf2 in VILI, which confers protection against cellular responses induced by MV by modulating oxidative stress. Experiment Overall Design: The Nrf2 wildtype (Nrf2+/+) and Nrf2-deficient (Nrf2â/â) CD-1/ICR strains of mice were subjected to mechanical ventilation with high (HVT) amounts of tidal volumes (VT) at 30 ml/kg for 2 hours. The animals subjected to spontaneous ventilation (SpV) for 2 hours were used as controls. Lungs were immediately removed and processed for total RNA isolation using TRIzol reagent (LifeTechnologies, Grand Island, NY). The isolated RNA was applied to Murine Genome 430A GeneChip arrays (Affymetrix, Santa Clara, CA), which contain probes for detecting ~14,500 well-characterized genes and 4371 expressed sequence tags according to standard microarray protocol. Scanned output files were analyzed by using Affymetrix GeneChip Operating Software and were independently normalized to an average intensity of 500.

Project description:Background: Obesity has become a worldwide concern. Acute respiratory distress syndrome (ARDS) comprises 10.4% of total intensive care unit admissions and is associated with very high mortality. ARDS incidence is increased in obese patients. Exposure of rodents to hyperoxia mimics many of the clinical and pathologic features observed in patients with ARDS. The aim of this study was to determine the impact of high fat diet-induced obesity on the susceptibility to hyperoxic acute lung injury in mice. Methods: Male C57BL/6 mice received 60% fat versus ingredient matched 10% fat diet. Mice were exposed to >95% oxygen to induce lung damage. RNA was isolated from lung homogenates and by comparing RNA sequencing results with mouse Mitocarta, an inventory of genes encoding proteins with mitochondrial localization, we identified fatty acid synthase (FASN), an enzyme catalyzing de novo fatty acid synthesis, as one of the mitochondrial genes significantly changed with diet and with hyperoxia. We generated mice deficient in FASN in alveolar epithelial cells by using a tamoxifen inducible Cre recombinase construct (FASNflox/flox SPC Cre+/-) and subjected them to hyperoxia and high fat diet. Results: Mice receiving 60% fat diet had significantly higher weight, serum cholesterol and fasting glucose. High fat diet mice had significantly reduced survival and increased lung damage, as assessed by BAL protein and LDH, histology and TUNEL staining. By RNA sequencing of lung homogenates we identified FASN as one of the mitochondrial genes significantly reduced in mice receiving 60% compared to 10% fat diet and further reduced with hyperoxia. We confirmed that FASN protein levels in the lung of high fat diet mice were lower by immunoblotting and immunohistochemistry. After 48 hours of hyperoxia FASNflox/flox SPC Cre+/- mice displayed increased levels of BAL protein and LDH and more severe histologic lung injury. FASNflox/flox SPC Cre+/- mice remained more prone to lung injury after hyperoxic exposure even when they received 60% fat diet. Conclusions: These results demonstrate that obesity increases the severity of hyperoxia induced acute lung injury in mice by altering FASN levels in the lung of high fat diet fed rodents. To our knowledge, this is the first study to show that high fat diet leads to altered FASN expression in the lung and that both high fat diet and reduced FASN in alveolar epithelial cells lead to increased lung injury under hyperoxic conditions.

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CDNA microarray analysis of sulforaphane effect on mouse acute lung injury caused by hyperoxia

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