Role of 15-Deoxy12,14 Prostaglandin J2 and Nrf2 Pathways in Protection against Acute Lung Injury
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美国呼吸和危急护理医学 2005年第6期
Department of Respiratory Medicine
Institute of Basic Medical Sciences and Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba
Laboratory of Food and Biodynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan
ABSTRACT
Rationale: Acute lung injury (ALI) is a disease process that is characterized by diffuse inflammation in the lung parenchyma. Recent studies demonstrated that cyclooxygenase-2 (COX-2) induced at the late phase of inflammation aids in the resolution of inflammation by generating 15-deoxy-12,14-prostaglandin J2 (15d-PGJ2). Transcription factor Nrf2 is activated by electrophiles and exerts antiinflammatory effects by inducing the gene expression of antioxidant and detoxification enzymes. Objectives: Because 15d-PGJ2 is an endogenous electrophile, we hypothesized that it protects against ALI by activating Nrf2. Methods: To test this hypothesis, we generated a reversible ALI model by intratracheal injection of carrageenin, an inducer of acute inflammation, whose stimulation has been known to induce COX-2. Main Results: We found that ALI induced by carrageenin was markedly exacerbated in Nrf2-knockout mice, compared with wild-type mice. Analysis of bronchoalveolar lavage fluids also revealed that the magnitude and the duration of acute inflammation, indicated by albumin concentration and the number of neutrophils, were significantly enhanced in Nrf2-knockout mice. Treatment of wild-type mice with NS-398, a selective COX-2 inhibitor, significantly exacerbated ALI to the level of Nrf2-knockout mice. In the lungs of NS-398eCtreated wild-type mice, both the accumulation of 15d-PGJ2 and the induction of Nrf2 target antioxidant genes were significantly attenuated. Exogenous administration of 15d-PGJ2 reversed the exacerbating effects of NS-398 with the induction of antioxidant genes. Conclusions: These results demonstrated in vivo that 15d-PGJ2 plays a protective role against ALI by exploiting the Nrf2-mediated transcriptional pathway.
Key Words: alveolar macrophages cyclooxygenase-2 inflammation transcription factor
Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome, represent a clinical syndrome that results from numerous causes and is responsible for significant mortality. The most characteristic pathologic findings associated with ALI are alveolar edema, caused by endothelial and epithelial injury, and the infiltration of inflammatory cells, especially neutrophils, into airspaces (1eC3). Several xenobiotics, such as chemicals, toxins, and their electrophilic metabolites, can directly injure endothelial and epithelial cells and induce lung parenchymal inflammation. Moreover, reactive oxygen species and nitrogen species generated by activated inflammatory cells secondarily lead to cell and tissue injury via various mechanisms, including direct damage to DNA, lipid peroxidation, oxidation of proteins, and alteration of transcription factor activity (4eC8).
To protect against these toxic compounds and oxidative stress, animals posses several defense systems. Antioxidant responsive element has been shown to regulate the expression of a group of phase 2 detoxification enzymes and antioxidant enzymes (9, 10). Antioxidant responsive elementeCmediated induction of these enzymes cooperatively serves to reduce inflammation and injury by lowering xenobiotic and oxidative stress (9, 10). Nrf2 (NF-E2eCrelated factor 2) is a member of the "cap 'n collar" basic leucine zipper transcription factor family and has been identified as a pivotal factor in the coordinate induction of phase 2 detoxifying and antioxidant enzymes under the regulatory influence of antioxidant responsive element (11eC13). In mouse models, Nrf2 plays essential roles in the protection against acute lung inflammation and damage induced by a number of stimuli, including butylated hydroxytoluene (14) and hyperoxia (15); such protection is performed via the transcriptional activation of phase 2 detoxification enzymes and antioxidant enzymes. In a homeostatic condition, Keap1 (Kelch-like ECH-associated protein 1), a cytosolic actin-binding protein (16, 17), constitutively represses the Nrf2 activity. Keap1 sequesters Nrf2 in the cytoplasm and enhances proteasomal degradation of Nrf2. When cells encounter oxidative or xenobiotic stress, Nrf2 is released from Keap1 (and the actin cytoskeleton) and rapidly accumulates in the nucleus.
It is well known that cyclooxygenases (COXs), especially COX-2, are upregulated at sites of acute inflammation. In a rat carrageenin-induced pleurisy model, COX-2 is induced at early (2 hours) and late (48 hours) phases of pleurisy, and it is proposed that the late-phase induction of COX-2 may contribute to the resolution of inflammation by producing cyclopentenone prostaglandins, including 15-deoxy-12,14-prostaglandin J2 (15d-PGJ2) (18). It has been proposed that 15d-PGJ2 exerts its antiinflammatory activity through the activation of peroxisome proliferator-activated receptor- (PPAR-) (19, 20), and also via the inactivation of nuclear factoreCB by directly inhibiting the effect of IB kinase or the p50 subunit (21, 22). However, it remains unclear whether these molecules actually participate in the protection against or in the resolution of acute lung inflammation in vivo. We recently demonstrated that cyclopentenone prostaglandins, including 15d-PGJ2, activate Nrf2 by directly binding to Keap1 (23).
Carrageenin is a polysaccharide obtained from red seaweed, and it induces acute inflammation at the site of injection. Although carrageenin is often used as an inducer of pleurisy (24, 25) and paw edema (26), it has not been used as an inducer of ALI because of its viscosity. The present study established a novel carrageenin-induced ALI model in mice using a low concentration of carrageenin. Then, with the help of this new model, the protective role of the COX-2, 15d-PGJ2, and Nrf2 pathways were investigated.
METHODS
Animals
Nrf2-deficient (Nrf2eC/eC) mice with an ICR/129sv background (13) were backcrossed with BALB/c mice for nine generations. Wild-type balb/c mice were purchased from Charles River Japan (Kanagawa, Japan). All mice used in this study were 6 to 8 weeks old and were maintained in our animal facilities under specific pathogen-free conditions. All animal studies were approved by the Institutional Review Board at the University of Tsukuba.
Assessment of Pulmonary Inflammation
Mice were injected intratracheally with 0.25% carrageenin (Cayman Chemical, Ann Arbor, MI) solution in 0.1 ml saline, or saline as a vehicle control. One day after the injection, the lungs were removed, fixed, and embedded in paraffin, and 2-eeCthick sections were stained with hematoxylin and eosin. Pulmonary inflammation was also assessed using lung digests and bronchoalveolar lavage (BAL) fluids. At 1, 3, and 8 days after carrageenin injection, the lungs were removed, minced, and incubated at 37°C for 90 minutes with RPMI 1640 (Gibco BRL, Grand Island, NY) containing 10% fetal bovine serum and 75 U/ml collagenase (type 1; Sigma Chemical, St. Louis, MO). The cells were then filtered through 20-e nylon mesh. To obtain the BAL fluids, the lungs were lavaged five times with 1 ml of saline solution containing 0.5% heparin. The supernatant of the first BAL fluid was used for an analysis of the albumin concentration, on the basis of its color reaction with bromocresol green (Sigma Chemical), as previously described (27). The remaining pooled BAL was centrifuged and resuspended in phosphate-buffered saline (PBS). Cells were counted using a hemocytometer, and a differential cell count was performed by standard light microscopic techniques based on staining with Diff-Quik (American Scientific Products, MacGaw Park, IL).
Immunohistochemistry
Cells obtained by BAL were cytospined onto poly-L-lysineeCcoated slides. Endogenous peroxidases were quenched with 0.3% hydrogen peroxide in methanol for 30 minutes. The sections were reacted with antieC15d-PGJ2 monoclonal antibody (28), washed, and incubated for 30 minutes with Histofine Simple Stain MAX-PO (Nichirei, Tokyo, Japan); we used nonimmune mouse IgG as a negative control. Diaminobenzidine was used as a chromogen.
Detection of Antioxidant Genes
The expression of peroxiredoxin-I (PrxI), heme oxygenase-1 (HO-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was determined by reverse transcriptioneCpolymerase chain reaction using the following primers:
PrxI sense 5'-TGAACGATTTGGCCGTATTGG-3' and antisense 5'-GAAGGCCATGCCAGTGAGCTT-3'
HO-1 sense 5'-GAGCAGAACCAGCCTGAACTA-3' and antisense 5'-GGTACAAGGAAGCCATCACCA-3'
GAPDH sense 5'-ACCACAGTCCATGCCATCAC-3' and antisense 5'-TCCACCACCCTGTTGCTGTA-3'
Polymerase chain reaction products were analyzed by 1.5% agarose gel, and the results were quantified by densitometry analysis using National Institutes of Health image software (NIH Image, Bethesda, MD). The gene expression was normalized to respective GAPDH expression.
Determination of the Effects of NS-398 and 15d-PGJ2
NS-398 and 15d-PGJ2 (Cayman Chemical) were dissolved in dimethyl sulfoxide and then 50-fold diluted with PBS. Mice were intraperitoneally administered 100 e of the NS-398 solution (10 mg/kg), or carrier, every 24 hours beginning 1 hour before the injection of carrageenin. The mice were injected with 50 e of the 15d-PGJ2 solution (100 e/kg) intratracheally at the same time they received the carrageenin injections.
Statistics
Data were expressed as means ± SEM. Statistical analysis was done using an analysis of variance followed by a Bonferroni posttest. A level of p < 0.05 was accepted as statistically significant.
RESULTS
Effects of Nrf2 Disruption on Carrageenin-induced ALI in Mice
To evaluate the protective role of Nrf2 against ALI, we first examined the pulmonary responses to carrageenin in both wild-type and Nrf2eC/eC mice. We preliminarily tested the various concentrations of carrageenin to determine an adequate concentration for use in the present study, and we found that 0.25% carrageenin adequately provoked the acute inflammation and recovery.
To elucidate the role played by Nrf2 in the protection against carrageenin-induced ALI, we first evaluated the histopathology of the lungs 1 day after the injection of carrageenin. No pathologic changes were observed in either wild-type or Nrf2eC/eC mice after the injection of saline on microscopic examination (Figure 1, saline). After carrageenin injection, acute pulmonary inflammation characterized by alveolar edema and the infiltration of neutrophils into the airspaces was observed in both wild-type and Nrf2eC/eC mice (Figure 1, carrageenin). However, these pathologic changes were much more severe in Nrf2eC/eC mice than in wild-type mice.
We then quantified the degree of acute inflammation at the lung and the airway levels. The accumulation of neutrophils into the lung was evaluated by measuring the number of neutrophils in the whole lung digests. The number of neutrophils in the lung digests was significantly increased in both wild-type and Nrf2eC/eC mice 1 day after carrageenin injection (Figure 2A). However, the number of neutrophils was significantly higher in Nrf2eC/eC mice than in wild-type mice (Figure 2A). Furthermore, the number of neutrophils was still elevated 3 days after carrageenin injection in Nrf2eC/eC mice, whereas the number of neutrophils decreased to the control values in wild-type mice at that time (Figure 2A). The number of neutrophils reversed to the baseline level 8 days after carrageenin injection in both genotypes of mice (Figure 2A).
We then assessed the accumulation of neutrophils into the airspaces using BAL fluid. An increased number of neutrophils was observed in the BAL fluids of both wild-type and Nrf2eC/eC mice 1 and 3 days after carrageenin injection (Figure 2B). The number of neutrophils was significantly greater in Nrf2eC/eC mice than in wild-type mice at both time points (Figure 2B). Neutrophils remained in the airspaces until Day 8 in Nrf2eC/eC mice (Figure 2B).
We further quantified the lung permeability damage by the measuring the albumin concentration in the BAL fluids. The BAL albumin concentration showed a peak at 1 day after the injection in both wild-type and Nrf2eC/eC mice (Figure 2C). However, the concentration was significantly higher in Nrf2eC/eC mice than that in wild-type mice (Figure 2C). Although the albumin concentration had decreased to the control values in wild-type mice by Day 3, the level of albumin was still high in Nrf2eC/eC mice (Figure 2C). The albumin concentration recovered to the control values in both genotypes of mice by Day 8 (Figure 2C). These pathologic findings and the quantitative data indicate that both the magnitude and duration of carrageenin-induced ALI were significantly enhanced in Nrf2eC/eC mice.
15d-PGJ2 Accumulation in Macrophages
We previously reported data supporting the notion that 15d-PGJ2 binds to Keap1 and activates Nrf2 in macrophages (23). To clarify whether 15d-PGJ2 is generated in the present model, we determined the accumulation of 15d-PGJ2 in BAL-recovered cells using a specific monoclonal antibody against 15d-PGJ2. Demonstrating the specificity of the antieC15d-PGJ2 antibody, none of the cells recovered from the BAL fluid before carrageenin injection was positive for the antieC15d-PGJ2 antibody (Figure 3A, Before). By contrast, strong immunoreactivity to the antibody was emerged in alveolar macrophages, but not in neutrophils, on Days 1 and 3 after carrageenin injection in both wild-type and Nrf2eC/eC mice (Figure 3A). More than 70% of alveolar macrophages were positive for the antieC15d-PGJ2 antibody at these time points in both wild-type and Nrf2eC/eC mice, and the proportion of the 15d-PGJ2 antibodyeCpositive cells is comparable between the two genotype mice (Figure 3B). The number of 15d-PGJ2eCpositive cells decreased by Day 8 in both genotypes of mice (Figures 3A and 3B). No immunoreactivity was detected with nonimmune IgG at any time point. These observations thus indicate that 15d-PGJ2 accumulates in alveolar macrophages from the peak to the resolution period of carrageenin-induced ALI in both wild-type and Nrf2eC/eC mice.
Effect of NS-398 on ALI in Wild-Type Mice
In a carrageenin-induced pleurisy model, it was demonstrated that the late-phase induction of COX-2 aids in the resolution of inflammation by generating 15d-PGJ2 (18). To clarify the contribution of COX-2 to the resolution of ALI in the present model, we then treated mice with NS-398, a selective COX-2 inhibitor. Treatment with NS-398 markedly reduced the accumulation of 15d-PGJ2 in alveolar macrophages of wild-type mice, both 1 and 3 days after carrageenin injection (Figure 3A, Wild+NS398). The proportion of antieC15d-PGJ2 antibodyeCpositive cells in alveolar macrophages decreased significantly in wild-type mice treated with NS-398, as compared with the results obtained from mice not treated with NS-398 (Figure 3B). These results suggest that COX-2 contributes to the generation of 15d-PGJ2 in the present model, and that the amount of NS-398 used in this study was sufficient to inhibit COX-2 activity to generate 15d-PGJ2.
We then assessed the effects of NS-398 on carrageenin-induced ALI. In NS-398eCtreated wild-type mice, the pathologic changes in the lung 1 day after carrageenin injection were much more severe than those in the lung of mice not treated with NS-398 (Figure 1, NS398). The quantitative analysis also revealed that the number of neutrophils in the total lung digest had significantly increased 3 days after carrageenin injection in NS-398eCtreated wild-type mice to the level of Nrf2eC/eC mice (Figure 4A). The number of neutrophils and the albumin concentration in the BAL fluids increased significantly in NS-398eCtreated wild-type mice 1 day after carrageenin injection, as compared with the values in NS-398eCuntreated wild-type mice (Figures 4B and 4C). Treatment with NS-398 did not alter these inflammatory parameters in Nrf2eC/eC mice (Figure 1, NS398, and Figures 4DeC4F). Thus, the inhibition of COX-2 most likely caused an exacerbation of carrageenin-induced ALI in wild-type mice to the same level as that observed in Nrf2eC/eC mice.
Effect of Exogenous Administration of 15d-PGJ2 in Wild-Type Mice
We then assessed the therapeutic effects of exogenously administered 15d-PGJ2 on the exacerbation described previously in NS-398eCtreated animals. The dose of 15d-PGJ2 (100 e/kg) was determined according to the previous studies (18, 29). The intratracheal administration of 100 e/kg of 15d-PGJ2 was found to reverse the pathologic changes in the lungs of NS-398eCtreated wild-type mice (Figure 1, NS398+PGJ2). The administration of 15d-PGJ2 reduced the number of neutrophils in both the lung digests and the BAL fluids of NS-398eCtreated wild-type mice (Figures 4A and 4B). 15d-PGJ2 also reversed the albumin concentration in BAL fluids to the level observed in NS-398eCuntreated mice (Figure 4C). However, the administration of 15d-PGJ2 did not affect these inflammatory parameters in Nrf2eC/eC mice (Figures 1, NS398+PGJ2, and 4DeC4F).
Stimulation of Nrf2 Target Gene Expression by 15d-PGJ2
To clarify the effects of COX-2 and 15d-PGJ2 on the expression of antioxidative Nrf2 target genes, we examined the expression of PrxI and HO-1 genes in the lung tissues and BAL cells of both wild-type and Nrf2eC/eC mice 1 day after carrageenin injection. No expression of PrxI and HO-1 mRNA was observed in the lungs of wild-type and Nrf2eC/eC mice before the injection of carrageenin by reverse transcriptioneCpolymerase chain reaction analyses. Significant induction of PrxI and HO-1 mRNAs was observed in the lungs of wild-type mice 1 day after carrageenin injection (Figures 5A and 5B, Wild). However, the induction of these mRNAs was markedly reduced in the lungs of Nrf2eC/eC mice, compared with that observed in wild-type mice (Figures 5A and 5B, KO). Similarly, the induction of PrxI and HO-1 gene expression was significantly reduced in wild-type mice treated with NS-398 (Figures 5A and 5B, Wild+NS). The expression of PrxI and HO-1 mRNAs was significantly upregulated in the lungs of NS-398eCtreated wild-type mice by the exogenous administration of 15d-PGJ2 (Figures 5A and 5B, Wild+NS+PGJ2). However, the administration of 15d-PGJ2 did not affect the expression level of PrxI and HO-1 in Nrf2eC/eC mice (Figures 5A and 5B, KO+PGJ2). We further examined the expression of PrxI and HO-1 using BAL cells, and we obtained results similar to those obtained with lung tissue samples (Figures 5C and 5D). These results suggest that 15d-PGJ2 is protective against ALI, at least in part by the activation of an Nrf2 transcriptional pathway.
DISCUSSION
The present study established a novel animal model of reversible ALI by intratracheal inoculation with a low concentration of carrageenin, which appears to be useful to study time course and resolution of the lung inflammation. Using this model, we found that both the magnitude and the duration of neutrophilic infiltration and permeability damage were significantly enhanced in Nrf2eC/eC mice, compared with the results obtained with wild-type mice. The result, together with the other results obtained in this study, indicates that Nrf2 is critical for determining susceptibility to ALI induced by carrageenin.
Carrageenin is known as an inflammatory agent that causes acute inflammation at the site of injection. A recent study demonstrated that the late-phase induction of COX-2 exhibits antiinflammatory activity by producing cyclopentenone prostaglandins, including 15d-PGJ2, during rat carrageenin-induced inflammation (18). Therefore, in the present study, we determined the role played by the COX-2, 15d-PGJ2, and Nrf2 pathways in the resolution of ALI. The results of the present study unveiled the correlations between the accumulation of 15d-PGJ2 and the activation of Nrf2 during the resolution of carrageenin-induced ALI on the basis of the following observations. First, the magnitude and the duration of acute lung inflammation, as indicated by the number of neutrophils and albumin permeability, were significantly enhanced in Nrf2eC/eC mice. Second, the treatment of wild-type mice with a COX-2 inhibitor, NS-398, exacerbated the degree of acute pulmonary inflammation to a level matching that of Nrf2eC/eC mice, together with an attenuation of 15d-PGJ2 accumulation in alveolar macrophages. NS-398 at the same time repressed the expression of PrxI and HO-1 genes, which are known to be Nrf2 target genes in these cells. Third, exogenous administration of 15d-PGJ2 reversed the exacerbation of acute lung inflammation in NS-398eCtreated wild-type mice. The administration of 15d-PGJ2 also reversed the attenuation of PrxI and HO-1 expression in NS-398eCtreated wild-type mice. On the other hand, the administration of 15d-PGJ2 altered neither the degree of inflammation nor the expression of PrxI and HO-1 genes in Nrf2eC/eC mice. These results demonstrate that the lack of Nrf2 leads to an inability to mediate the antiinflammatory effects of 15d-PGJ2. Thus, Nrf2 is suggested to play important roles in the resolution of acute lung inflammation as the downstream molecule of 15d-PGJ2, as summarized in Figure 6.
15d-PGJ2 is a member of cyclopentenone prostaglandin family, and it seems to exert antiinflammatory activity via multiple mechanisms. Because 15d-PGJ2 possesses an electrophilic cyclopentenone ring and acts as an endogenous electrophile, it is likely that 15d-PGJ2 possesses the ability to activate Nrf2. Furthermore, our recent analyses demonstrate that 15d-PGJ2 directly binds to Keap1 and thereby releases Nrf2 from the Keap1 repression (23). We observed in the present study both the accumulation of 15d-PGJ2 and expression of HO-1 and PrxI genes in alveolar macrophages (Figure 3 and data not shown). This observation suggests that 15d-PGJ2 and the Nrf2 antiinflammatory pathway mainly work in macrophages. Because macrophages are the major source of reactive oxygen species and proteases, as well as inflammatory cytokines, it seems reasonable to hypothesize that the aberrant gene expression in Nrf2eC/eC macrophages may result in an overwhelming inflammatory response and thus induce lung injury. A defense system based on macrophages is therefore important in the protection against acute lung inflammation, and Nrf2 must be a key molecule in this defense system.
For the detection of 15d-PGJ2, we used antieC15d-PGJ2 monoclonal antibody (23, 28, 30). Specificity of this antibody against the prostaglandin has been tested previously (28). For instance, the antibody specifically recognized 15d-PGJ2; the binding affinity to PGJ2 and 12-PGJ2 was 50 times less than that to 15d-PGJ2. The antibody did not recognize other prostaglandins, such as PGA2, PGB2, PGD2, PGE2, PGF2, and PGI2. This study found that the immunoreactive substance to the antibody disappears with the treatment with COX-2 inhibitor (Figure 4A). The result strongly argues that the signal detected is not merely a cross-reaction of the antibody to certain cellular components, but that the antibody specifically recognizes 15d-PGJ2.
To inhibit the COX-2 activity, we used its selective inhibitor, NS-398, because the specificity of NS-398 to COX-2 is widely accepted. NS-398 inhibits the COX-2 activity in a concentration-dependent manner, with an IC50 value of 3.8 x 10eC6 M (31). To serve as a selective COX-2 inhibitor in vivo, administration of NS-398 at a dose of 0.3 to 5 mg/kg was required in rats (32). On the other hand, NS-398 did not affect COX-1 activity, at least until 10eC4 M (31). Taken together, the dose of NS-398 used in the present study is that which specifically inhibits COX-2. Furthermore, treatment with NS-398 almost completely blocked the accumulation of 15d-PGJ2 in macrophages; these findings thus suggest that the amount of NS-398 used in this study was sufficient to inhibit the COX-2 activity, thus inhibiting the generation of 15d-PGJ2.
Although it remains to be elucidated how Nrf2 regulates acute pulmonary inflammation, it is likely that Nrf2 target genes cooperatively function to repress the inflammatory reactions. HO-1 and PrxI are likely to influence the inflammatory process (12). It was recently demonstrated that 15d-PGJ2 could activate HO-1 gene expression via a stress-responsive element and an Nrf2-mediated mechanism (33). Carbon monoxide, generated as a consequence of HO-1 activation, can inhibit the expression of tumor necrosis factor (34). With respect to PrxI, its human counterpart, PAG, was reported to directly bind to and negatively regulate -tautomerase activity of macrophage migration inhibitory factor, which is one of the central regulators of inflammation (35). Although the physiologic significance of the -tautomerase activity of migration inhibitory factor is unclear at present, the repression of migration inhibitory factor activity by PrxI may play an important role in the regulation of inflammation. Furthermore, it has been suggested that the overexpression of PrxI represses tumor necrosis factoreC signaling by the removal of hydrogen peroxide (36). Besides antioxidant genes, a recent study has demonstrated that Nrf2 transcriptionally regulates CD36 molcules (37). CD36 is a scavenger receptor that mediates the phagocytosis of apoptotic neutrophils, which is an essential process for the resolution of inflammation. These broad observations suggest that Nrf2 regulates multiple downstream target genes that are functional in the acute inflammatory process.
This study demonstrates for the first time that Nrf2 exerts antiinflammatory activity in vivo as a downstream regulator of 15d-PGJ2. Considering the importance of Nrf2 in the protection against ALI and also the fact that COX-2 is readily inducible by a variety of stimuli, we envisage that the COX-2eCNrf2 pathway identified in this study exerts general roles in the protection of inflammation, especially in the lung. ALI, and its severe form, acute respiratory distress syndrome, are a lethal lung inflammatory disorder for which therapeutic options are not readily available. Thus, activation of Nrf2 might be a useful therapeutic approach for the protection and the resolution of this lethal disorder.
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Institute of Basic Medical Sciences and Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba
Laboratory of Food and Biodynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan
ABSTRACT
Rationale: Acute lung injury (ALI) is a disease process that is characterized by diffuse inflammation in the lung parenchyma. Recent studies demonstrated that cyclooxygenase-2 (COX-2) induced at the late phase of inflammation aids in the resolution of inflammation by generating 15-deoxy-12,14-prostaglandin J2 (15d-PGJ2). Transcription factor Nrf2 is activated by electrophiles and exerts antiinflammatory effects by inducing the gene expression of antioxidant and detoxification enzymes. Objectives: Because 15d-PGJ2 is an endogenous electrophile, we hypothesized that it protects against ALI by activating Nrf2. Methods: To test this hypothesis, we generated a reversible ALI model by intratracheal injection of carrageenin, an inducer of acute inflammation, whose stimulation has been known to induce COX-2. Main Results: We found that ALI induced by carrageenin was markedly exacerbated in Nrf2-knockout mice, compared with wild-type mice. Analysis of bronchoalveolar lavage fluids also revealed that the magnitude and the duration of acute inflammation, indicated by albumin concentration and the number of neutrophils, were significantly enhanced in Nrf2-knockout mice. Treatment of wild-type mice with NS-398, a selective COX-2 inhibitor, significantly exacerbated ALI to the level of Nrf2-knockout mice. In the lungs of NS-398eCtreated wild-type mice, both the accumulation of 15d-PGJ2 and the induction of Nrf2 target antioxidant genes were significantly attenuated. Exogenous administration of 15d-PGJ2 reversed the exacerbating effects of NS-398 with the induction of antioxidant genes. Conclusions: These results demonstrated in vivo that 15d-PGJ2 plays a protective role against ALI by exploiting the Nrf2-mediated transcriptional pathway.
Key Words: alveolar macrophages cyclooxygenase-2 inflammation transcription factor
Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome, represent a clinical syndrome that results from numerous causes and is responsible for significant mortality. The most characteristic pathologic findings associated with ALI are alveolar edema, caused by endothelial and epithelial injury, and the infiltration of inflammatory cells, especially neutrophils, into airspaces (1eC3). Several xenobiotics, such as chemicals, toxins, and their electrophilic metabolites, can directly injure endothelial and epithelial cells and induce lung parenchymal inflammation. Moreover, reactive oxygen species and nitrogen species generated by activated inflammatory cells secondarily lead to cell and tissue injury via various mechanisms, including direct damage to DNA, lipid peroxidation, oxidation of proteins, and alteration of transcription factor activity (4eC8).
To protect against these toxic compounds and oxidative stress, animals posses several defense systems. Antioxidant responsive element has been shown to regulate the expression of a group of phase 2 detoxification enzymes and antioxidant enzymes (9, 10). Antioxidant responsive elementeCmediated induction of these enzymes cooperatively serves to reduce inflammation and injury by lowering xenobiotic and oxidative stress (9, 10). Nrf2 (NF-E2eCrelated factor 2) is a member of the "cap 'n collar" basic leucine zipper transcription factor family and has been identified as a pivotal factor in the coordinate induction of phase 2 detoxifying and antioxidant enzymes under the regulatory influence of antioxidant responsive element (11eC13). In mouse models, Nrf2 plays essential roles in the protection against acute lung inflammation and damage induced by a number of stimuli, including butylated hydroxytoluene (14) and hyperoxia (15); such protection is performed via the transcriptional activation of phase 2 detoxification enzymes and antioxidant enzymes. In a homeostatic condition, Keap1 (Kelch-like ECH-associated protein 1), a cytosolic actin-binding protein (16, 17), constitutively represses the Nrf2 activity. Keap1 sequesters Nrf2 in the cytoplasm and enhances proteasomal degradation of Nrf2. When cells encounter oxidative or xenobiotic stress, Nrf2 is released from Keap1 (and the actin cytoskeleton) and rapidly accumulates in the nucleus.
It is well known that cyclooxygenases (COXs), especially COX-2, are upregulated at sites of acute inflammation. In a rat carrageenin-induced pleurisy model, COX-2 is induced at early (2 hours) and late (48 hours) phases of pleurisy, and it is proposed that the late-phase induction of COX-2 may contribute to the resolution of inflammation by producing cyclopentenone prostaglandins, including 15-deoxy-12,14-prostaglandin J2 (15d-PGJ2) (18). It has been proposed that 15d-PGJ2 exerts its antiinflammatory activity through the activation of peroxisome proliferator-activated receptor- (PPAR-) (19, 20), and also via the inactivation of nuclear factoreCB by directly inhibiting the effect of IB kinase or the p50 subunit (21, 22). However, it remains unclear whether these molecules actually participate in the protection against or in the resolution of acute lung inflammation in vivo. We recently demonstrated that cyclopentenone prostaglandins, including 15d-PGJ2, activate Nrf2 by directly binding to Keap1 (23).
Carrageenin is a polysaccharide obtained from red seaweed, and it induces acute inflammation at the site of injection. Although carrageenin is often used as an inducer of pleurisy (24, 25) and paw edema (26), it has not been used as an inducer of ALI because of its viscosity. The present study established a novel carrageenin-induced ALI model in mice using a low concentration of carrageenin. Then, with the help of this new model, the protective role of the COX-2, 15d-PGJ2, and Nrf2 pathways were investigated.
METHODS
Animals
Nrf2-deficient (Nrf2eC/eC) mice with an ICR/129sv background (13) were backcrossed with BALB/c mice for nine generations. Wild-type balb/c mice were purchased from Charles River Japan (Kanagawa, Japan). All mice used in this study were 6 to 8 weeks old and were maintained in our animal facilities under specific pathogen-free conditions. All animal studies were approved by the Institutional Review Board at the University of Tsukuba.
Assessment of Pulmonary Inflammation
Mice were injected intratracheally with 0.25% carrageenin (Cayman Chemical, Ann Arbor, MI) solution in 0.1 ml saline, or saline as a vehicle control. One day after the injection, the lungs were removed, fixed, and embedded in paraffin, and 2-eeCthick sections were stained with hematoxylin and eosin. Pulmonary inflammation was also assessed using lung digests and bronchoalveolar lavage (BAL) fluids. At 1, 3, and 8 days after carrageenin injection, the lungs were removed, minced, and incubated at 37°C for 90 minutes with RPMI 1640 (Gibco BRL, Grand Island, NY) containing 10% fetal bovine serum and 75 U/ml collagenase (type 1; Sigma Chemical, St. Louis, MO). The cells were then filtered through 20-e nylon mesh. To obtain the BAL fluids, the lungs were lavaged five times with 1 ml of saline solution containing 0.5% heparin. The supernatant of the first BAL fluid was used for an analysis of the albumin concentration, on the basis of its color reaction with bromocresol green (Sigma Chemical), as previously described (27). The remaining pooled BAL was centrifuged and resuspended in phosphate-buffered saline (PBS). Cells were counted using a hemocytometer, and a differential cell count was performed by standard light microscopic techniques based on staining with Diff-Quik (American Scientific Products, MacGaw Park, IL).
Immunohistochemistry
Cells obtained by BAL were cytospined onto poly-L-lysineeCcoated slides. Endogenous peroxidases were quenched with 0.3% hydrogen peroxide in methanol for 30 minutes. The sections were reacted with antieC15d-PGJ2 monoclonal antibody (28), washed, and incubated for 30 minutes with Histofine Simple Stain MAX-PO (Nichirei, Tokyo, Japan); we used nonimmune mouse IgG as a negative control. Diaminobenzidine was used as a chromogen.
Detection of Antioxidant Genes
The expression of peroxiredoxin-I (PrxI), heme oxygenase-1 (HO-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was determined by reverse transcriptioneCpolymerase chain reaction using the following primers:
PrxI sense 5'-TGAACGATTTGGCCGTATTGG-3' and antisense 5'-GAAGGCCATGCCAGTGAGCTT-3'
HO-1 sense 5'-GAGCAGAACCAGCCTGAACTA-3' and antisense 5'-GGTACAAGGAAGCCATCACCA-3'
GAPDH sense 5'-ACCACAGTCCATGCCATCAC-3' and antisense 5'-TCCACCACCCTGTTGCTGTA-3'
Polymerase chain reaction products were analyzed by 1.5% agarose gel, and the results were quantified by densitometry analysis using National Institutes of Health image software (NIH Image, Bethesda, MD). The gene expression was normalized to respective GAPDH expression.
Determination of the Effects of NS-398 and 15d-PGJ2
NS-398 and 15d-PGJ2 (Cayman Chemical) were dissolved in dimethyl sulfoxide and then 50-fold diluted with PBS. Mice were intraperitoneally administered 100 e of the NS-398 solution (10 mg/kg), or carrier, every 24 hours beginning 1 hour before the injection of carrageenin. The mice were injected with 50 e of the 15d-PGJ2 solution (100 e/kg) intratracheally at the same time they received the carrageenin injections.
Statistics
Data were expressed as means ± SEM. Statistical analysis was done using an analysis of variance followed by a Bonferroni posttest. A level of p < 0.05 was accepted as statistically significant.
RESULTS
Effects of Nrf2 Disruption on Carrageenin-induced ALI in Mice
To evaluate the protective role of Nrf2 against ALI, we first examined the pulmonary responses to carrageenin in both wild-type and Nrf2eC/eC mice. We preliminarily tested the various concentrations of carrageenin to determine an adequate concentration for use in the present study, and we found that 0.25% carrageenin adequately provoked the acute inflammation and recovery.
To elucidate the role played by Nrf2 in the protection against carrageenin-induced ALI, we first evaluated the histopathology of the lungs 1 day after the injection of carrageenin. No pathologic changes were observed in either wild-type or Nrf2eC/eC mice after the injection of saline on microscopic examination (Figure 1, saline). After carrageenin injection, acute pulmonary inflammation characterized by alveolar edema and the infiltration of neutrophils into the airspaces was observed in both wild-type and Nrf2eC/eC mice (Figure 1, carrageenin). However, these pathologic changes were much more severe in Nrf2eC/eC mice than in wild-type mice.
We then quantified the degree of acute inflammation at the lung and the airway levels. The accumulation of neutrophils into the lung was evaluated by measuring the number of neutrophils in the whole lung digests. The number of neutrophils in the lung digests was significantly increased in both wild-type and Nrf2eC/eC mice 1 day after carrageenin injection (Figure 2A). However, the number of neutrophils was significantly higher in Nrf2eC/eC mice than in wild-type mice (Figure 2A). Furthermore, the number of neutrophils was still elevated 3 days after carrageenin injection in Nrf2eC/eC mice, whereas the number of neutrophils decreased to the control values in wild-type mice at that time (Figure 2A). The number of neutrophils reversed to the baseline level 8 days after carrageenin injection in both genotypes of mice (Figure 2A).
We then assessed the accumulation of neutrophils into the airspaces using BAL fluid. An increased number of neutrophils was observed in the BAL fluids of both wild-type and Nrf2eC/eC mice 1 and 3 days after carrageenin injection (Figure 2B). The number of neutrophils was significantly greater in Nrf2eC/eC mice than in wild-type mice at both time points (Figure 2B). Neutrophils remained in the airspaces until Day 8 in Nrf2eC/eC mice (Figure 2B).
We further quantified the lung permeability damage by the measuring the albumin concentration in the BAL fluids. The BAL albumin concentration showed a peak at 1 day after the injection in both wild-type and Nrf2eC/eC mice (Figure 2C). However, the concentration was significantly higher in Nrf2eC/eC mice than that in wild-type mice (Figure 2C). Although the albumin concentration had decreased to the control values in wild-type mice by Day 3, the level of albumin was still high in Nrf2eC/eC mice (Figure 2C). The albumin concentration recovered to the control values in both genotypes of mice by Day 8 (Figure 2C). These pathologic findings and the quantitative data indicate that both the magnitude and duration of carrageenin-induced ALI were significantly enhanced in Nrf2eC/eC mice.
15d-PGJ2 Accumulation in Macrophages
We previously reported data supporting the notion that 15d-PGJ2 binds to Keap1 and activates Nrf2 in macrophages (23). To clarify whether 15d-PGJ2 is generated in the present model, we determined the accumulation of 15d-PGJ2 in BAL-recovered cells using a specific monoclonal antibody against 15d-PGJ2. Demonstrating the specificity of the antieC15d-PGJ2 antibody, none of the cells recovered from the BAL fluid before carrageenin injection was positive for the antieC15d-PGJ2 antibody (Figure 3A, Before). By contrast, strong immunoreactivity to the antibody was emerged in alveolar macrophages, but not in neutrophils, on Days 1 and 3 after carrageenin injection in both wild-type and Nrf2eC/eC mice (Figure 3A). More than 70% of alveolar macrophages were positive for the antieC15d-PGJ2 antibody at these time points in both wild-type and Nrf2eC/eC mice, and the proportion of the 15d-PGJ2 antibodyeCpositive cells is comparable between the two genotype mice (Figure 3B). The number of 15d-PGJ2eCpositive cells decreased by Day 8 in both genotypes of mice (Figures 3A and 3B). No immunoreactivity was detected with nonimmune IgG at any time point. These observations thus indicate that 15d-PGJ2 accumulates in alveolar macrophages from the peak to the resolution period of carrageenin-induced ALI in both wild-type and Nrf2eC/eC mice.
Effect of NS-398 on ALI in Wild-Type Mice
In a carrageenin-induced pleurisy model, it was demonstrated that the late-phase induction of COX-2 aids in the resolution of inflammation by generating 15d-PGJ2 (18). To clarify the contribution of COX-2 to the resolution of ALI in the present model, we then treated mice with NS-398, a selective COX-2 inhibitor. Treatment with NS-398 markedly reduced the accumulation of 15d-PGJ2 in alveolar macrophages of wild-type mice, both 1 and 3 days after carrageenin injection (Figure 3A, Wild+NS398). The proportion of antieC15d-PGJ2 antibodyeCpositive cells in alveolar macrophages decreased significantly in wild-type mice treated with NS-398, as compared with the results obtained from mice not treated with NS-398 (Figure 3B). These results suggest that COX-2 contributes to the generation of 15d-PGJ2 in the present model, and that the amount of NS-398 used in this study was sufficient to inhibit COX-2 activity to generate 15d-PGJ2.
We then assessed the effects of NS-398 on carrageenin-induced ALI. In NS-398eCtreated wild-type mice, the pathologic changes in the lung 1 day after carrageenin injection were much more severe than those in the lung of mice not treated with NS-398 (Figure 1, NS398). The quantitative analysis also revealed that the number of neutrophils in the total lung digest had significantly increased 3 days after carrageenin injection in NS-398eCtreated wild-type mice to the level of Nrf2eC/eC mice (Figure 4A). The number of neutrophils and the albumin concentration in the BAL fluids increased significantly in NS-398eCtreated wild-type mice 1 day after carrageenin injection, as compared with the values in NS-398eCuntreated wild-type mice (Figures 4B and 4C). Treatment with NS-398 did not alter these inflammatory parameters in Nrf2eC/eC mice (Figure 1, NS398, and Figures 4DeC4F). Thus, the inhibition of COX-2 most likely caused an exacerbation of carrageenin-induced ALI in wild-type mice to the same level as that observed in Nrf2eC/eC mice.
Effect of Exogenous Administration of 15d-PGJ2 in Wild-Type Mice
We then assessed the therapeutic effects of exogenously administered 15d-PGJ2 on the exacerbation described previously in NS-398eCtreated animals. The dose of 15d-PGJ2 (100 e/kg) was determined according to the previous studies (18, 29). The intratracheal administration of 100 e/kg of 15d-PGJ2 was found to reverse the pathologic changes in the lungs of NS-398eCtreated wild-type mice (Figure 1, NS398+PGJ2). The administration of 15d-PGJ2 reduced the number of neutrophils in both the lung digests and the BAL fluids of NS-398eCtreated wild-type mice (Figures 4A and 4B). 15d-PGJ2 also reversed the albumin concentration in BAL fluids to the level observed in NS-398eCuntreated mice (Figure 4C). However, the administration of 15d-PGJ2 did not affect these inflammatory parameters in Nrf2eC/eC mice (Figures 1, NS398+PGJ2, and 4DeC4F).
Stimulation of Nrf2 Target Gene Expression by 15d-PGJ2
To clarify the effects of COX-2 and 15d-PGJ2 on the expression of antioxidative Nrf2 target genes, we examined the expression of PrxI and HO-1 genes in the lung tissues and BAL cells of both wild-type and Nrf2eC/eC mice 1 day after carrageenin injection. No expression of PrxI and HO-1 mRNA was observed in the lungs of wild-type and Nrf2eC/eC mice before the injection of carrageenin by reverse transcriptioneCpolymerase chain reaction analyses. Significant induction of PrxI and HO-1 mRNAs was observed in the lungs of wild-type mice 1 day after carrageenin injection (Figures 5A and 5B, Wild). However, the induction of these mRNAs was markedly reduced in the lungs of Nrf2eC/eC mice, compared with that observed in wild-type mice (Figures 5A and 5B, KO). Similarly, the induction of PrxI and HO-1 gene expression was significantly reduced in wild-type mice treated with NS-398 (Figures 5A and 5B, Wild+NS). The expression of PrxI and HO-1 mRNAs was significantly upregulated in the lungs of NS-398eCtreated wild-type mice by the exogenous administration of 15d-PGJ2 (Figures 5A and 5B, Wild+NS+PGJ2). However, the administration of 15d-PGJ2 did not affect the expression level of PrxI and HO-1 in Nrf2eC/eC mice (Figures 5A and 5B, KO+PGJ2). We further examined the expression of PrxI and HO-1 using BAL cells, and we obtained results similar to those obtained with lung tissue samples (Figures 5C and 5D). These results suggest that 15d-PGJ2 is protective against ALI, at least in part by the activation of an Nrf2 transcriptional pathway.
DISCUSSION
The present study established a novel animal model of reversible ALI by intratracheal inoculation with a low concentration of carrageenin, which appears to be useful to study time course and resolution of the lung inflammation. Using this model, we found that both the magnitude and the duration of neutrophilic infiltration and permeability damage were significantly enhanced in Nrf2eC/eC mice, compared with the results obtained with wild-type mice. The result, together with the other results obtained in this study, indicates that Nrf2 is critical for determining susceptibility to ALI induced by carrageenin.
Carrageenin is known as an inflammatory agent that causes acute inflammation at the site of injection. A recent study demonstrated that the late-phase induction of COX-2 exhibits antiinflammatory activity by producing cyclopentenone prostaglandins, including 15d-PGJ2, during rat carrageenin-induced inflammation (18). Therefore, in the present study, we determined the role played by the COX-2, 15d-PGJ2, and Nrf2 pathways in the resolution of ALI. The results of the present study unveiled the correlations between the accumulation of 15d-PGJ2 and the activation of Nrf2 during the resolution of carrageenin-induced ALI on the basis of the following observations. First, the magnitude and the duration of acute lung inflammation, as indicated by the number of neutrophils and albumin permeability, were significantly enhanced in Nrf2eC/eC mice. Second, the treatment of wild-type mice with a COX-2 inhibitor, NS-398, exacerbated the degree of acute pulmonary inflammation to a level matching that of Nrf2eC/eC mice, together with an attenuation of 15d-PGJ2 accumulation in alveolar macrophages. NS-398 at the same time repressed the expression of PrxI and HO-1 genes, which are known to be Nrf2 target genes in these cells. Third, exogenous administration of 15d-PGJ2 reversed the exacerbation of acute lung inflammation in NS-398eCtreated wild-type mice. The administration of 15d-PGJ2 also reversed the attenuation of PrxI and HO-1 expression in NS-398eCtreated wild-type mice. On the other hand, the administration of 15d-PGJ2 altered neither the degree of inflammation nor the expression of PrxI and HO-1 genes in Nrf2eC/eC mice. These results demonstrate that the lack of Nrf2 leads to an inability to mediate the antiinflammatory effects of 15d-PGJ2. Thus, Nrf2 is suggested to play important roles in the resolution of acute lung inflammation as the downstream molecule of 15d-PGJ2, as summarized in Figure 6.
15d-PGJ2 is a member of cyclopentenone prostaglandin family, and it seems to exert antiinflammatory activity via multiple mechanisms. Because 15d-PGJ2 possesses an electrophilic cyclopentenone ring and acts as an endogenous electrophile, it is likely that 15d-PGJ2 possesses the ability to activate Nrf2. Furthermore, our recent analyses demonstrate that 15d-PGJ2 directly binds to Keap1 and thereby releases Nrf2 from the Keap1 repression (23). We observed in the present study both the accumulation of 15d-PGJ2 and expression of HO-1 and PrxI genes in alveolar macrophages (Figure 3 and data not shown). This observation suggests that 15d-PGJ2 and the Nrf2 antiinflammatory pathway mainly work in macrophages. Because macrophages are the major source of reactive oxygen species and proteases, as well as inflammatory cytokines, it seems reasonable to hypothesize that the aberrant gene expression in Nrf2eC/eC macrophages may result in an overwhelming inflammatory response and thus induce lung injury. A defense system based on macrophages is therefore important in the protection against acute lung inflammation, and Nrf2 must be a key molecule in this defense system.
For the detection of 15d-PGJ2, we used antieC15d-PGJ2 monoclonal antibody (23, 28, 30). Specificity of this antibody against the prostaglandin has been tested previously (28). For instance, the antibody specifically recognized 15d-PGJ2; the binding affinity to PGJ2 and 12-PGJ2 was 50 times less than that to 15d-PGJ2. The antibody did not recognize other prostaglandins, such as PGA2, PGB2, PGD2, PGE2, PGF2, and PGI2. This study found that the immunoreactive substance to the antibody disappears with the treatment with COX-2 inhibitor (Figure 4A). The result strongly argues that the signal detected is not merely a cross-reaction of the antibody to certain cellular components, but that the antibody specifically recognizes 15d-PGJ2.
To inhibit the COX-2 activity, we used its selective inhibitor, NS-398, because the specificity of NS-398 to COX-2 is widely accepted. NS-398 inhibits the COX-2 activity in a concentration-dependent manner, with an IC50 value of 3.8 x 10eC6 M (31). To serve as a selective COX-2 inhibitor in vivo, administration of NS-398 at a dose of 0.3 to 5 mg/kg was required in rats (32). On the other hand, NS-398 did not affect COX-1 activity, at least until 10eC4 M (31). Taken together, the dose of NS-398 used in the present study is that which specifically inhibits COX-2. Furthermore, treatment with NS-398 almost completely blocked the accumulation of 15d-PGJ2 in macrophages; these findings thus suggest that the amount of NS-398 used in this study was sufficient to inhibit the COX-2 activity, thus inhibiting the generation of 15d-PGJ2.
Although it remains to be elucidated how Nrf2 regulates acute pulmonary inflammation, it is likely that Nrf2 target genes cooperatively function to repress the inflammatory reactions. HO-1 and PrxI are likely to influence the inflammatory process (12). It was recently demonstrated that 15d-PGJ2 could activate HO-1 gene expression via a stress-responsive element and an Nrf2-mediated mechanism (33). Carbon monoxide, generated as a consequence of HO-1 activation, can inhibit the expression of tumor necrosis factor (34). With respect to PrxI, its human counterpart, PAG, was reported to directly bind to and negatively regulate -tautomerase activity of macrophage migration inhibitory factor, which is one of the central regulators of inflammation (35). Although the physiologic significance of the -tautomerase activity of migration inhibitory factor is unclear at present, the repression of migration inhibitory factor activity by PrxI may play an important role in the regulation of inflammation. Furthermore, it has been suggested that the overexpression of PrxI represses tumor necrosis factoreC signaling by the removal of hydrogen peroxide (36). Besides antioxidant genes, a recent study has demonstrated that Nrf2 transcriptionally regulates CD36 molcules (37). CD36 is a scavenger receptor that mediates the phagocytosis of apoptotic neutrophils, which is an essential process for the resolution of inflammation. These broad observations suggest that Nrf2 regulates multiple downstream target genes that are functional in the acute inflammatory process.
This study demonstrates for the first time that Nrf2 exerts antiinflammatory activity in vivo as a downstream regulator of 15d-PGJ2. Considering the importance of Nrf2 in the protection against ALI and also the fact that COX-2 is readily inducible by a variety of stimuli, we envisage that the COX-2eCNrf2 pathway identified in this study exerts general roles in the protection of inflammation, especially in the lung. ALI, and its severe form, acute respiratory distress syndrome, are a lethal lung inflammatory disorder for which therapeutic options are not readily available. Thus, activation of Nrf2 might be a useful therapeutic approach for the protection and the resolution of this lethal disorder.
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