Genetic Ablation of Nrf2 Enhances Susceptibility to Acute Lung Injury After Traumatic Brain Injury in Mice

Abstract

Previous studies have shown that nuclear factor erythroid 2-related factor 2 (Nrf2) plays a unique role in many physiological stress processes. The present study investigated the role of Nrf2 in the regulation of traumatic brain injury (TBI)-induced acute lung injury (ALI). Wild-type Nrf2 (+/+) and Nrf2 (−/−)-deficient mice were subjected to a moderately severe weight-drop impact head injury. Pulmonary capillary permeability (PCP), wet/dry weight ratio, apoptosis, inflammatory cytokines and antioxidant/detoxifying enzymes were measured at 24 h after TBI. Mice lacking Nrf2 were found to be more susceptible to TBI-induced ALI, as characterized by the higher increase in PCP, wet/dry weight ratio and alveolar cells apoptosis after TBI. This exacerbation of lung injury in Nrf2-deficient mice was associated with increased pulmonary mRNA and protein expression of inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6); and with decreased pulmonary mRNA expression and enzymatic activities of antioxidant and detoxifying enzymes including NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione S-transferase α1 (GST-α1)—as compared with their wild-type Nrf2 (+/+) counterparts after TBI. The results of the present study suggest that Nrf2 reduces TBI-induced acute lung injury, possibly by decreasing pulmonary inflammation and inducing antioxidant and detoxifying enzymes.

Introduction

The association between traumatic brain injury (TBI) and subsequent pulmonary dysfunction has been increasingly recognized (1, 2). Development of acute lung injury (ALI) may not only influence the lung epithelium itself, but also impair brain oxygenation and aggravate the neurogenic injury. Several pieces of evidence have shown that inflammation and oxidative stress are involved in the progression of TBI-induced lung injury (3, 4). In addition, it has been demonstrated that oxidative stress can modulate inflammatory responses during tissue injury, possibly through activation of nuclear factor erythroid 2-related factor 2 (Nrf2) (5).

Nrf2 is a basic leucine zipper redox sensitive transcription factor which has been reported to be a pleiotropic regulator in cell survival mechanisms (6). Under basal conditions, Nrf2 is sequestered in the cytoplasm by the cytosolic regulatory protein Keap1. In conditions of oxidative or xenobiotic stress, Nrf2 translocates from the cytoplasm to the nucleus, and sequentially binds to a promoter sequence called the antioxidant response element (ARE), resulting in a cytoprotective response which is characterized by upregulation of a group of antioxidant and detoxifying enzymes, and decreased sensitivity to inflammation and oxidative stress (7, 8).

Nrf2 is considered to be a protector for many organs, including the lung (9). Nrf2 (−/−)-deficient mice have been shown to exhibit increased hyperoxic lung injury (10), cigarette smoke-induced emphysema (11), allergen-mediated airway inflammation (12), butylated hydroxytoluene-induced acute respiratory distress syndrome (13), and bleomycin-mediated pulmonary fibrosis (14). Nrf2 has also been reported as a crucial regulator of the innate immune response and survival during experimental sepsis (15). Furthermore, activation of Nrf2 by resveratrol, a polyphenolic phytoalexin, is able to protect human lung epithelial cells against cigarette smoke-mediated oxidative stress (16). In light of the findings described above, we hypothesize that Nrf2 contributes to protection against TBI-induced ALI. To test this hypothesis, we subjected wild-type Nrf2 (+/+) and Nrf2 (−/−)-deficient mice to a TBI model. Then we compared the outcomes in terms of pulmonary capillary permeability (PCP), wet/dry weight ratio, number of apoptotic cells, expression of inflammatory cytokines and antioxidant/detoxifying enzymes.

Experimental Procedures

Mouse Model of TBI.

All procedures were approved by the Institutional Animal Care Committee and were in accordance with the guidelines of the National Institutes of Health on the care and use of animals. Breeding pairs of Nrf2-deficient ICR mice were kindly provided by Dr. Thomas W. Kensler (Johns Hopkins University, Baltimore, MD, USA). Homozygous wild-type Nrf2 (+/+) and Nrf2 (−/−)-deficient mice were generated from inbred heterozygous Nrf2 (+/−) mice (17). Genotypes of Nrf2 (−/−) and Nrf2 (+/+) mice were confirmed by polymerase chain reaction (PCR) amplification of genomic DNA isolated from the blood. PCR amplification was carried out by using three different primers: 5′-TGGACGGGACTATTGAAGGCTG-3′ (sense for both genotypes), 5′-CGCCTTTTCAGTAGATGGAGG-3′ (antisense for wild-type) and 5′-GCGGATTGACCGTAATGGGATAGG-3′ (antisense for LacZ). Age- and weight-matched adult male mice (6–8 weeks, 28–32 g) were separated into four groups: I, sham + wild-type (Nrf2 +/+); II, TBI + wild-type (Nrf2 +/+); III, sham + deficient (Nrf2 −/−); IV, TBI + deficient (Nrf2 −/−).

The mouse model of TBI was employed as described (18) with recent minor modification (19). Mice were anesthetized by intraperitoneal injection with sodium pentobarbital (50 mg/kg). A round, flat and 6 mm diameter Teflon impounder was centered between the ears and eyes. TBI was induced by a 100 g weight dropped from a 12 cm height along a stainless steel string, which translated into 1200 g/cm. Brain injury-induced apnea was then treated for 3 mins with 100% oxygen administration and chest compression to stimulate the respiration (20). This model is generally associated with 20% of mortality within the first 5 mins post-injury and no delayed mortality was observed thereafter. Sham mice were subjected to identical treatment with the exception that no injury was performed. After operation procedures, the mice were returned to their cages. Heart rate, arterial blood pressure and rectal temperature were monitored, and the rectal temperature was kept at 37 ± 0.5°C (physical cooling if required) throughout the experimental and recovery periods.

At 24 h following sham or injury, mice were sacrificed for sample collection. For terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analysis, mice (n =6 per group) were transcardially perfused with cold saline (4°C) followed by 4% neutral-buffered formalin. The lungs were harvested, stored overnight in 4% neutral-buffered formalin, and embedded in paraffin. For wet/dry weight ratio assay, RNA reverse transcriptase-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and enzyme activity assay, mice (n =6 per group) were exsanguinated by cardiac puncture. The lungs were excised and the wet/dry weight ratio was immediately determined. Specimens of lung tissue were stored in liquid nitrogen.

Capillary Permeability in Lung Tissue.

PCP was determined by Evans blue (EB) extravasation method as previously described (21). Evans blue (50 mg/kg; Sigma Chemical Co., St. Louis, MO) in 250 μl of 0.9% saline was injected intravenously via the left internal vein at 30 mins before sacrifice. The mice were exsanguinated by cardiac puncture. The lung tissue was excised and weighed. To each sample, 1.5 ml formamide was added and incubated at 37°C for 24 h. After filtration with glass filter, the absorbance of the filtrate was measured at 620 nm using a Beckman spectrophotometer. The total amount of dye can be calculated by means of a standard calibration curve. The capillary permeability in lung tissue was shown as the micrograms of Evans blue in every milligram of tissue (n =6 per group).

Wet/Dry Weight Ratio in Lung Tissue.

Pulmonary edema was determined using the wet/dry method as previously described (22). The lung tissue samples were weighed before and after drying in a desic oven for 72 h at 80°C.

TUNEL Staining and Quantitation of Apoptotic Cells.

The formalin-fixed, paraffin-embedded sections (4 μm) were detected for apoptotic cells by the TUNEL method. The procedures were according to the kit instructions (ISCDD, Boehringer Mannheim, Germany). Microscopy of the stained tissue sections was performed by a pathologist blinded to the experimental condition. The extent of lung damage was evaluated by the apoptotic index (AI) which was the percent of average number of TUNEL-positive cells in each section counted in 10 microscopic fields.

RNA Extraction and RT-PCR.

The pulmonary mRNA expression levels of inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6), and antioxidant/detoxifying enzymes, including NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione S-transferase α1 (GST-α1), were determined by RT-PCR. Total RNA was extracted with TriPure Reagent (Roche Diagnostics Corp., Indianapolis, IN, USA) according to the manufacturer’s instructions. The cDNA synthesis from the isolated RNA was performed using a reverse transcriptional system. Briefly, 5 μg of total lung RNA was reversely transcribed using 0.5 μg oligo(dT)15 U Avian Myeloblastosis Virus Reverse Transcriptase (AMV RT) (Promega, Madison, WI, USA). The cDNA was then amplified by PCR using various primer sets: TNF-α, 5′-ACGGCATGGATCTCAAAGAC-3′ and 5′-GGTCACTGTCCCAGCATCTT-3′; IL-1β, 5′-GAGTGTGGATCCCAAGCAAT-3′ and 5′-CTCAGTG-CAGGCTATGACCA-3′; IL-6, 5′-AGTTGCCTTCTTGG-GACTGA-3′ and 5′-GCCACTCCTTCTGTGACTCC-3′; NQO1, 5′-CATTCTGAAAGGCTGGTTTGA-3′ and 5′-CTAGCTTTGATCTGGTTGTCAG-3′; GST-α1, 5′-AAGCCAGGACTCTCACTA-3 ′ and 5 ′-CTGCTGATTCTGCTCTTG-3′; β-actin, 5′-AGTGTGACGTTGACATCCGTA-3′ and 5′-GCCAGAGCAGTAATCTCCTTCT-3′. PCR was started within 5 mins incubation at 94°C followed by a three-step temperature cycle: denaturation at 94°C for 30 s, annealing at 53–55°C for 50 s, and extension at 72°C for 1 min for 25 to ~30 cycles. A final extension step at 72°C for 8 mins was included after the final cycle to complete polymerization. PCR products were detected by agarose gel electrophoresis in 1.5% NuSieve agarose gels (FMC, USA) and visualized by ethidium bromide staining. The intensity of the bands was quantified using Glyko Bandscan software, and the ratios of each gene product to β-actin product were used as indices of inflammatory cytokines and antioxidant/detoxifying enzymes mRNA expression.

ELISA.

The pulmonary protein expression levels of inflammatory cytokines were quantified using ELISA kits specific for mouse cytokines according to the manufacturers’ instructions (TNF-α from Diaclone Research, France; IL-1β, IL-6 from Biosource Europe SA, Belgium). Values were expressed as pg/mg protein.

Enzyme Activity Assay for Lung Tissue.

Lung tissue was dissected and homogenized in ice-cold 10 mM Tris-HCl (pH 7.8). The homogenates were centrifuged at 12,000 × g for 15 mins at 4°C. Protein concentration of the resulting supernatant was determined using a bicinchoninic acid assay kit with bovine serum albumin as the standard (Pierce Biochemicals, Rockford, IL). NQO1 enzyme activity was determined by calculating the dicumarol-sensitive fraction of 2,6-dichlorophenol-indo-phenol reduction (23). Reactions consisting of 30 μg protein, 25 mM Tris-HCl (pH 7.4), 0.7 mg/ml crystalline bovine serum albumin, 5 μM FAD, 0.2 mM NADH, and 0 or 20 μM dicoumarol were preincubated for 10 mins at 25°C (final concentrations in 200 μl of reaction volume). To initiate the reaction, 40 μM of 2,6-dichlorophenol-indo-phenol was added, and the initial velocity of the reduction of dichlorophenol-indo-phenol was measured spectrophotometrically at 540 nm. The extinction coefficient for 2,6-dichlorophenol-indo-phenol was 2.1 × 10⁴ M/cm. The total GST enzyme activity assay consisted of 100 μg protein, 1 mM 1-chloro-2,4-dinitrobenzene, and 1 mM glutathione at 37°C in 100 mM potassium phosphate buffer (pH 6.5) (final concentrations in 150 μl of reaction volume) (24). The reaction was monitored at 340 nm and the non-enzymatic slope was subtracted from the total observed slope. The extinction coefficient for 1-chloro-2, 4-dinitrobenzene was 9600 M/cm. All values were expressed as nmol/min/mg protein.

Statistical Analysis.

Software SPSS 13.0 was used for the statistical analysis. All data were expressed as mean ± standard error (SE). Student’s t test was used to analyze the differences between the sham and TBI groups within a single genotype as well as between genotypes. Statistical significance was accepted at P < 0.05.

Results

Pulmonary Capillary Permeability.

As shown in Figure 1, the PCP level in both sham-operated Nrf2 (+/+) and Nrf2 (−/−) mice was similar. An increased level of PCP was detected in both Nrf2 (+/+) and Nrf2 (−/−) mice at 24 h after TBI. However, the pathological change was more severe in Nrf2 (−/−) mice than in Nrf2 (+/+) mice.

Pulmonary Edema.

As shown in Figure 2 , lung wet/dry weight ratio in both sham-operated Nrf2 (+/+) and Nrf2 (−/−) mice was similar. An increased wet/dry weight ratio was detected in both Nrf2 (+/+) and Nrf2 (−/−) mice at 24 h after TBI. However, the pathological change was more severe in Nrf2 (−/−) mice as compared with Nrf2 (+/+) mice.

Apoptosis.

As detected by TUNEL staining, a similar number of characteristic nuclear chromatin condensed TUNEL-positive alveolar epithelial and endothelial cells was found in Nrf2 (+/+) and Nrf2 (−/−) mice (Fig. 3A, C ). An increased number of TUNEL-apoptotic alveolar cells was found in both Nrf2 (+/+) and Nrf2 (−/−) mice at 24 h after TBI (Fig. 3B, D ). However, the pathological change was more severe in Nrf2 (−/−) mice as compared with Nrf2 (+/+) mice (Fig. 3E ).

Expression of Inflammatory Cytokines in the Lung.

The levels of protein expression of TNF-α, IL-1β and IL-6 in both sham-operated Nrf2 (+/+) and Nrf2 (−/−) mice were also similar. TBI induced increased protein expression levels of TNF-α, IL-1β and IL-6 in the lung samples of both Nrf2 (+/+) and Nrf2 (−/−) mice at 24 h after TBI. Interestingly, higher pulmonary protein expression levels of inflammatory cytokines were found in Nrf2 (−/−) mice than in Nrf2 (+/+) mice after TBI (Fig. 6 ).

Expression of Antioxidant and Detoxifying Enzymes in the Lung.

The mRNA expression levels of antioxidant and detoxifying enzymes NQO1 and GST-α1 detected in the lung samples of both sham and injured Nrf2 (−/−) mice were significantly lower than those measured in the corresponding Nrf2 (+/+) mice. TBI induced increased cortical mRNA expression levels of NQO1 and GST-α1 in Nrf2 (+/+) mice but not Nrf2 (−/−) mice (P > 0.05) (Fig. 5 ).

The NQO1 and total GST activity measured in the lung samples of both sham and injured Nrf2 (−/−) mice were significantly lower than those measured in the corresponding Nrf2 (+/+) mice. TBI induced increased NQO1 activity in the lung samples of Nrf2 (+/+) mice but not Nrf2 (−/−) mice (P > 0.05). No TBI-induced changes were observed in the total GST activity in the lung samples of both genotypes of mice (P > 0.05) (Fig. 7 ).

Discussion

This study revealed that Nrf2 (−/−)-deficient mice have significantly enhanced TBI-induced ALI characterized by increased PCP, wet/dry weight ratio and alveolar epithelial and endothelial cells apoptosis after TBI. Increased TBI-induced inflammatory cytokines TNF-α, IL-1β and IL-6 mRNA and protein expression were observed in the lung samples of Nrf2 (−/−) mice compared with Nrf2 (+/+) mice. Furthermore, decreased antioxidant and detoxifying enzymes NQO1 and GST-α1 mRNA expression and enzymatic activities were found in the lung samples of Nrf2 (−/−) mice after TBI. To our knowledge, these findings reported here suggest for the first time that Nrf2 reduces TBI-induced ALI, possibly by decreasing pulmonary inflammation and inducing antioxidant and detoxifying enzymes.

ALI and its more severe form, acute respiratory distress syndrome (ARDS), involve a disruption of the alveolar-capillary membranes, with local inflammation ultimately resulting in increased tissue microvascular permeability and extravasation of vascular fluid (25). The filling of alveolar spaces by edema fluid and inflammatory cells can in turn lead to severe hypoxemia and respiratory failure (26). Indeed, the PCP and pulmonary wet/dry weight ratio, as the indexes of ALI, were significantly increased at 24 h after TBI in the present study. However, higher PCP and wet/dry weight ratio were observed in Nrf2 (−/−) mice than in Nrf2 (+/+) mice. These findings illustrate the protective role of Nrf2 in the TBI-induced ALI.

Apoptosis of epithelial and endothelial cells, which is induced by a variety of stimuli, contributes to the impairment of the barrier function of pulmonary endothelium and epithelium, and the development of pulmonary edema (27). Using TUNEL staining as a marker for apoptosis, we observed a trend toward more alveolar epithelial and endothelial cells apoptosis in Nrf2 (−/−) mice than in Nrf2 (+/+) mice at 24 h after TBI. These findings also illustrated the protective role of Nrf2 in the TBI-induced ALI.

While the exact mechanisms underlying TBI-induced ALI have been proved to be complex, it is clear that inflammatory response contributes to disease progression. Many researchers have focused on aspects of the inflammatory cytokine network, which is believed to be central to the pathophysiology of the inflammation process in the lung (28). TNF-α is reported to be a major initiator of inflammation and is released early after an inflammatory stimulus (29). IL-1β is regarded as the prototypic “multi-functional” cytokine and is induced in a multitude of cell types (30). IL-6 is increased after TNF-α and is considered to be an important pro-inflammatory cytokine in contribution to both morbidity and mortality in conditions of “uncontrolled” inflammation (31). Excessive expression of these cytokines, during trauma or other stress, potentiates inflammatory response through the subsequent induction of other inflammatory mediators. The prevailing theory has been that the dysregulation of the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system resulting from the TBI may initiate the stimulation of cytokine expression and an inflammatory process in the lung (3). In this study, the presence of Nrf2 function significantly inhibited TBI-induced inflammatory cytokines TNF-α, IL-1β and IL-6 production in the lung, whereas the absence of Nrf2 function resulted in greatly increased pulmonary inflammatory cytokines production after TBI. Therefore, it appeared that Nrf2 plays an important role in the regulation of pulmonary inflammation after TBI, which might be a mechanism to explain the protective role of Nrf2 in the TBI-induced ALI as described previously.

Corresponding well with our results of experimental study, the anti-inflammatory effect of Nrf2 has also been demonstrated in a variety of experimental models, such as cigarette smoke-induced emphysema (11), allergen-mediated airway inflammation (12), dextran sulfate sodium (DSS)-mediated colitis (32), inflammation-mediated colonic tumorigenesis (33) and ICH-induced brain injury (34). Thimmulappa et al. reported that Nrf2 can effectively regulate the innate immune response and survival during experimental sepsis. Inflammation in Nrf2 (−/−) mice was greatly intensified after lipopolysaccharide (LPS) challenge (15). Another study investigated the role of Nrf2 in the regulation of cigarette smoke-induced emphysema and the results showed that disruption of the Nrf2 gene in mice led to earlier-onset and more extensive lung inflammation and alveolar cell apoptosis in response to challenge with tobacco smoke (11). Furthermore, Rangasamy et al. also demonstrated that disruption of the Nrf2 gene leads to ovalbumin sensitization and challenge-driven airway inflammation and hyperresponsiveness in mice (12). Collectively, these studies implicate an important role of Nrf2 in inhibiting inflammatory response in response to a variety of stimuli.

Oxidative stress has also been shown to contribute to the ALI following TBI (4). Because oxidative stress has not only direct injurious effects, but also overarching effects on several important mechanisms involved in TBI-induced ALI, particularly inflammation and apoptosis, Nrf2-mediated antioxidant and detoxifying enzymes expression, such as NQO1 and GST, may afford wide protection against the lung injury secondary to TBI (5, 10). Indeed, associating with the aggravated lung injury and the augmented pulmonary inflammation, the absence of Nrf2 function in mice resulted in decreased pulmonary expression of antioxidant and detoxifying enzymes NQO1 and GST-α1 mRNA after TBI. In addition, NQO1 and GST enzymes activities were lower in the lung samples of both sham-operated and injured Nrf2 (−/−) mice, relative to Nrf2 (+/+) mice. These data are in agreement with another study illustrating the protective role of Nrf2 in the pathogenesis of hyperoxic lung injury (10). In that study, mRNA expression and activity levels of antioxidant and detoxifying enzymes were lower in lungs of Nrf2 (−/−) mice as compared with Nrf2 (+/+) mice after hyperoxia. Therefore, it appeared that mice lacking Nrf2 function have significantly aggravated lung injury secondary to TBI because of decreased ability to induce antioxidant and detoxifying enzymes.

Increasing evidence has demonstrated the protective role of these Nrf2-regulated gene products in pulmonary disorders. NQO1 is a reductive enzyme that reduces quinine and nitrogen oxide functional groups (35), whereas GST helps conjugate xenobiotics to glutathione (36). NQO1 is considered to play a protective role in hyperoxic lung injury in mice (10, 37). NQO1 polymorphism was associated with the risk of lung cancer (38). Alteration of GST activity was found in human lung cancer (39) and asthma (40). GSTs polymorphism was also associated with the risk of lung cancer (41).

In conclusion, the results of the present study indicate that Nrf2 plays an important role in protecting TBI-induced ALI, possibly by decreasing pulmonary inflammation and inducing antioxidant and detoxifying enzymes. These findings raise the possibility that Nrf2 will be a new therapeutic target for the treatment of severe acute lung injury secondary to TBI.

Figure 1.
Pulmonary capillary permeability in sham and injured Nrf2 (+/+) and Nrf2 (−/−) mice. The capillary permeability level was significantly increased and was higher in Nrf2 (−/−) mice than in Nrf2 (+/+) mice at 24 h after TBI (n =6 per group). P < 0.01 versus genotype-matched sham-operated mice. # P < 0.05 versus treatment-matched Nrf2 (+/+) mice.

Figure 2.
Lung wet/dry weight ratio in sham and injured Nrf2 (+/+) and Nrf2 (−/−) mice. The lung wet/dry weight ratio was significantly increased and was higher in Nrf2 (−/−) mice than in Nrf2 (+/+) mice at 24 h after TBI (n = 6 per group). P < 0.01 versus genotype-matched sham-operated mice. # P < 0.05 versus treatment-matched Nrf2 (+/+) mice.

Figure 3.
Pulmonary apoptosis in sham and injured Nrf2 (+/+) and Nrf2 (−/−) mice. Sham-operated Nrf2 (+/+) and Nrf2 (−/−) mice showing few TUNEL-apoptotic alveolar cells (A and C); injured Nrf2 (+/+) mice showing increased level of TUNEL-apoptotic alveolar cells as brown (B); injured Nrf2 (−/−) mice showing much more TUNEL-apoptotic alveolar cells compared with injured Nrf2 (+/+) mice (D); quantitative analysis showed that the level of TUNEL-apoptotic alveolar cells was significantly increased and was greater in Nrf2 (−/−) mice than in Nrf2 (+/+) mice at 24 h after TBI (E) (n = 6 per group). ** P < 0.01 versus genotype-matched sham-operated mice. ## P < 0.01 versus treatment-matched Nrf2 (+/+) mice.

Figure 4.
Differential mRNA expression levels of inflammatory cytokines in the lung samples of sham and injured Nrf2 (+/+) and Nrf2 (−/−) mice. Representative agarose gel images for each gene product are shown on top of the graphs, and the order of individual mRNA bands corresponds to that of graph bars (n = 6 per group). * P < 0.05 and P < 0.01 versus genotype-matched sham-operated mice. # P < 0.05 and ## P < 0.01 versus treatment-matched Nrf2 (+/+) mice.

Figure 5.
Differential mRNA expression levels of antioxidant and detoxifying enzymes in the lung samples of sham and injured Nrf2 (+/+) and Nrf2 (−/−) mice. Representative agarose gel images for each gene product are shown on top of the graphs, and the order of individual mRNA bands corresponds to that of graph bars (n = 6 per group). P < 0.01 versus genotype-matched sham-operated mice. ## P < 0.01 versus treatment-matched Nrf2 (+/+) mice.

Figure 6.
Protein expression levels of inflammatory cytokines in the lung samples of sham and injured Nrf2 (+/+) and Nrf2 (−/−) mice. Pulmonary levels of TNF-α, IL-1β and IL-6 were significantly increased and were greater in Nrf2 (−/−) mice than in Nrf2 (+/+) mice at 24 h after TBI (n = 6 per group). P < 0.01 versus genotype-matched sham-operated mice. # P < 0.05 versus treatment-matched Nrf2 (+/+) mice.

Figure 7.
Antioxidant and detoxifying enzymes activity in the lung samples of sham and injured Nrf2 (+/+) and Nrf2 (−/−) mice. The figure indicates that pulmonary basal levels of NQO1 and total GST activity were higher in Nrf2 (+/+) mice than in Nrf2 (−/−) mice (n = 6 per group). P < 0.01 versus genotype-matched sham-operated mice. # P < 0.05 and ## P < 0.01 versus treatment-matched Nrf2 (+/+) mice. mice compared with Nrf2 (+/+)

Footnotes

This work was supported by grants from Jinling Hospital of China.

Acknowledgements

The authors would like to thank Dr. Lizhi Xu, Dr. Bo Wu and Dr. Gengbao Feng for technical assistance.

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