# Disruption of Nrf2 Enhances Upregulation of Nuclear Factor-κB Activity, Proinflammatory Cytokines, and Intercellular Adhesion Molecule-1 in the Brain after Traumatic Brain Injury

Authors: Wei Jin; Handong Wang; Wei Yan; Lizhi Xu; Xiaoliang Wang; Xiaoning Zhao; Xiaohe Yang; Gang Chen; Yan Ji
Journal: Mediators of Inflammation
 (2008)

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## Abstract

Inflammatory response plays an important role in the pathogenesis of secondary brain injury after traumatic brain injury (TBI). Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor that plays a crucial role in cytoprotection against inflammation. The present study investigated the role of Nrf2 in the cerebral upregulation of NF-κB activity, proinflammatory cytokine, and ICAM-1 after TBI. Wild-type Nrf2 (+/+) and Nrf2 (−/−)-deficient mice were subjected to a moderately severe weight-drop impact head injury. Electrophoretic mobility shift assays (EMSAs) were performed to analyze the activation of nuclear factor kappa B (NF-κB). Enzyme-linked immunosorbent assays were performed to quantify the production of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). Immunohistochemistry staining experiments were performed to detect the expression of intercellular adhesion molecule-1 (ICAM-1). Nrf2 (−/−) mice were shown to have more NF-κB activation, inflammatory cytokines TNF-α, IL-1β and IL-6 production, and ICAM-1 expression in brain after TBI compared with their wild-type Nrf2 (+/+) counterparts. The results suggest that Nrf2 plays an important protective role in limiting the cerebral upregulation of NF-κB activity, proinflammatory cytokine, and ICAM-1 after TBI.


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## Body

## 1. Introduction

Cerebral inflammation plays an
important role in the pathogenesis of secondary brain injury following
traumatic brain injury (TBI) [1, 2]. Proinflammatory nuclear factor kappa B (NF-κB)
signaling pathway has been well documented in previous studies of our
laboratory [3, 4]. Increased levels of inflammatory agents with the injured
brain, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β),
interleukin-6 (IL-6), and intercellular adhesion molecule 1 (ICAM-1), are
believed to contribute to the cerebral damage [5]. Their mediator NF-κB activation enhances the transcription of proinflammatory
cytokines [6], and the cytokines are known to in turn activate NF-κB [7]. The
positive feedback is believed to serve to amplify inflammatory signals and exacerbate
brain injury after TBI.Recent researches have demonstrated that nuclear factor
erythroid 2-related factor 2 (Nrf2), a key transcription factor that regulates
the cellular antioxidant response, plays a broader role in modulating acute
inflammatory response [8, 9]. 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 enzymes and decreased
sensitivity to oxidative damage [10–12]. These antioxidant enzymes have also
been shown to protect cells against acute inflammatory response [13].Numerous studies have reported that Nrf2 plays a critical
role in counteracting inflammation in a variety of experimental models. Nrf2
protects against allergen-mediated airway inflammation [14], cigarette
smoke-induced emphysema [15], dextran sulfate sodium (DSS)-mediated colitis
[16], inflammation-mediated colonic tumorigenesis [17], and inflammatory
responses during skin wound healing [18]. Furthermore, Nrf2 has also been
reported as a crucial regulator of the innate immune response and survival
during experimental sepsis [19]. In one of our previous studies, we have
demonstrated that TBI could induce Nrf2-ARE pathway activation in brain [20].Therefore, it may be reasonable to postulate that Nrf2 plays
an important role in limiting the cerebral inflammatory response after TBI. In
our study, we evaluated the influence of Nrf2 genotype on the cerebral upregulation
of NF-κB activity, proinflammatory cytokine, and ICAM-1 after TBI.


## 2. Materials and Methods

### 2.1. Animals

Our experiments were conformed to
Guide for the Care and Use of Laboratory Animals from National Institutes of
Health and approved by the Animal Care and Use Committee of Nanjing University.
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 [10]. Genotypes of Nrf2 (+/+) and Nrf2 (−/−) mice were confirmed by 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 (n=10 per group): group I, sham
wild-type (Nrf2 +/+); group II, injured
wild-type (Nrf2 +/+); group III, sham-deficient
(Nrf2 −/−); group IV, injured-deficient
(Nrf2 −/−). The mice of sham and injured groups were subjected to identical anesthetic alone or experimental TBI, respectively. Animals
were decapitated at 24 hours following sham or injury. Five mice
in each group were sacrificed for electrophoretic
mobility shift assay (EMSA) and enzyme-linked immunosorbent assay (ELISA)
analysis and the others were for immunohistochemistry study.


### 2.2. Induction of Experimental TBI

The mouse
model of TBI was employed as described [21] with recent minor modification [22]. The 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 minutes with 100% oxygen administration and chest compression to
stimulate the respiration. This model is generally associated with 20% of
mortality within the first 5 minutes postinjury and no delayed mortality was
observed thereafter. 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 experiments.At the 24 hours following sham or
injury, mice were sacrificed for sample
collection. For EMSA and ELISA analyses, mice were exsanguinated by cardiac
puncture. Cortex tissue was rapidly taken from the fresh brain at the site of
lesion (Figure1), and stored in liquid nitrogen immediately. For
immunohistochemistry, mice were perfused with cold saline (4°C),
followed by 4% neutral-buffered formalin. The cortex tissue was taken, stored
overnight in 4% neutral-buffered formalin, and then embedded in paraffin.Figure 1
Schematic representation of the area taken for assay.


### 2.3. Nuclear Protein Extract and EMSA

Nuclear protein was extracted and quantified as described [23]. Briefly,
frozen brain samples were homogenized in 0.8 mL ice-cold buffer A composed of 10 mmol/L HEPES pH 7.9, 10 mmol/L KCl, 2 mmol/L
MgCl2, 0.1 mmol/L EDTA, 1 mmol/L dithiothreitol (DTT), and 0.5 mmol/L phenylmethylsulfonyl
fluoride (PMSF) (all from Sigma Chemical Co., St. Louis, Mo, USA). The homogenates
were incubated on ice for 30 minutes and vortexed for 30 seconds after addition
of 50 μL 10% NP-40 (Sigma Chemical Co., Mo, USA). The mixture was then
centrifuged for 10 minutes (5000 × g, 4°C). The pellet was suspended in 100 μL ice-cold buffer B composed of 50 mmol/L
HEPES pH 7.9, 50 mmol/L KCl, 300 mM NaCl, 0.1 mmol/L EDTA, 1 mmol/LDTT, and 0.5 mmol/L PMSF, and 10% (v/v) glycerol
and incubated on ice 30 minutes with frequent mixing. After centrifugation
(12000 × g, 4°C) for 15 minutes, the supernatants were collected as nuclear extracts and
stored at −70°C for further use. Protein concentration was determined using a
bicinchoninic acid assay kit with bovine serum albumin as the standard (Pierce
Biochemicals, Rockford, Ill, USA).EMSA was performed using a
commercial kit (Gel Shift Assay System; Promega, Madison, Wis, USA)
following the methods in our laboratory [23]. Consensus oligonucleotide probe (5′-AGT TGA GGG GAC TTT CCC AGG C-3′) was end-labeled with [γ-P32]ATP
(Free Biotech., Beijing, China) with T4-polynucleotide
kinase. Nuclear protein (10 μg) was preincubated in a total volume of 9 μL in a
binding buffer, consisting of 10 mmol/L Tris-HCl (pH 7.5), 4% glycerol, 1 mmol/L
MgCl2, 0.5 mmol/L M EDTA, 0.5 mmol/L DTT, 0.5 mmol/L NaCl, and 0.05 g/L poly-(deoxyinosinic-deoxycytidylic
acid) for 15 minutes at room temperature. After addition of the 1 μL P32-labled
oligonucleotide probe, the incubation was continued for 20 minutes at room temperature.
Reaction was stopped by adding 1 μL of gel loading buffer and the mixture was
subjected to nondenaturing 4% polyacrylamide gel electrophoresis in 0.5 × TBE
buffer (Tris-borate-EDTA). After electrophoresis was conducted at 390 V for 1 hour,
the gel was vacuum-dried and exposed to X-ray film (Fuji Hyperfilm, Tokyo, Japan)
at −70°C with an intensifying screed. Levels of NF-κB DNA binding activity were
quantified by computer-assisted densitometric analysis.


### 2.4. ELISA Analysis

Frozen brain samples were homogenized in 1 mL of buffer containing 1 mmol/L of PMSF, 1 mg/L of pepstatin A, 1 mg/L of aprotinin, and 1 mg/L of leupeptin in PBS
solution (pH 7.2) with a glass homogenizer and then centrifuged at 12000 g for 20
minutes at 4°C. The supernatant was then collected and total
protein was determined by the Bradford method.
The levels of inflammatory cytokines were quantified using enzyme-linked
immunosorbent assay (ELISA) kits specific for mouse according to the
manufacturers’ instructions (TNF-α from Diaclone
Research, France; IL-1β, IL-6 from Biosource Europe SA, Belgium) and previous study of our laboratory [24].
The cytokine contents in the brain samples were expressed as pg per milligram protein.


### 2.5. Immunohistochemical Staining

The paraffin-embedded sections (4μm) were used for Immunohistochemical
assay, which was performed with a goat antimouse ICAM-1(CD54) antibody (diluted
1:200, R&D Systems, Inc.,
Minn, USA),
according to previous studies of our laboratory [7]. The sections were
incubated with the diluted antibody overnight at 4°C in a humid chamber, washed, and blocked with 1.6%  H2O2 in phosphate-buffered saline
(PBS) for 10 minutes. After washing with PBS again, sections were then
incubated with biotinylated second antibodies for 1 hour at room temperature.
Diaminobenzidine (DAB) was used as chromogen and counterstaining was done with
hematoxylin. The number of positive microvessels in each section was counted in
10 microscopic fields (at 100 × magnifications) and averaged for the positively
immunostained vessel number of per visual field.


### 2.6. Statistical Analysis

Software SPSS 13.0 was used for the statistical analysis. All data were
expressed as mean ± SEM, Student’st-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<.05.


## 2.1. Animals

Our experiments were conformed to
Guide for the Care and Use of Laboratory Animals from National Institutes of
Health and approved by the Animal Care and Use Committee of Nanjing University.
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 [10]. Genotypes of Nrf2 (+/+) and Nrf2 (−/−) mice were confirmed by 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 (n=10 per group): group I, sham
wild-type (Nrf2 +/+); group II, injured
wild-type (Nrf2 +/+); group III, sham-deficient
(Nrf2 −/−); group IV, injured-deficient
(Nrf2 −/−). The mice of sham and injured groups were subjected to identical anesthetic alone or experimental TBI, respectively. Animals
were decapitated at 24 hours following sham or injury. Five mice
in each group were sacrificed for electrophoretic
mobility shift assay (EMSA) and enzyme-linked immunosorbent assay (ELISA)
analysis and the others were for immunohistochemistry study.


## 2.2. Induction of Experimental TBI

The mouse
model of TBI was employed as described [21] with recent minor modification [22]. The 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 minutes with 100% oxygen administration and chest compression to
stimulate the respiration. This model is generally associated with 20% of
mortality within the first 5 minutes postinjury and no delayed mortality was
observed thereafter. 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 experiments.At the 24 hours following sham or
injury, mice were sacrificed for sample
collection. For EMSA and ELISA analyses, mice were exsanguinated by cardiac
puncture. Cortex tissue was rapidly taken from the fresh brain at the site of
lesion (Figure1), and stored in liquid nitrogen immediately. For
immunohistochemistry, mice were perfused with cold saline (4°C),
followed by 4% neutral-buffered formalin. The cortex tissue was taken, stored
overnight in 4% neutral-buffered formalin, and then embedded in paraffin.Figure 1
Schematic representation of the area taken for assay.


## 2.3. Nuclear Protein Extract and EMSA

Nuclear protein was extracted and quantified as described [23]. Briefly,
frozen brain samples were homogenized in 0.8 mL ice-cold buffer A composed of 10 mmol/L HEPES pH 7.9, 10 mmol/L KCl, 2 mmol/L
MgCl2, 0.1 mmol/L EDTA, 1 mmol/L dithiothreitol (DTT), and 0.5 mmol/L phenylmethylsulfonyl
fluoride (PMSF) (all from Sigma Chemical Co., St. Louis, Mo, USA). The homogenates
were incubated on ice for 30 minutes and vortexed for 30 seconds after addition
of 50 μL 10% NP-40 (Sigma Chemical Co., Mo, USA). The mixture was then
centrifuged for 10 minutes (5000 × g, 4°C). The pellet was suspended in 100 μL ice-cold buffer B composed of 50 mmol/L
HEPES pH 7.9, 50 mmol/L KCl, 300 mM NaCl, 0.1 mmol/L EDTA, 1 mmol/LDTT, and 0.5 mmol/L PMSF, and 10% (v/v) glycerol
and incubated on ice 30 minutes with frequent mixing. After centrifugation
(12000 × g, 4°C) for 15 minutes, the supernatants were collected as nuclear extracts and
stored at −70°C for further use. Protein concentration was determined using a
bicinchoninic acid assay kit with bovine serum albumin as the standard (Pierce
Biochemicals, Rockford, Ill, USA).EMSA was performed using a
commercial kit (Gel Shift Assay System; Promega, Madison, Wis, USA)
following the methods in our laboratory [23]. Consensus oligonucleotide probe (5′-AGT TGA GGG GAC TTT CCC AGG C-3′) was end-labeled with [γ-P32]ATP
(Free Biotech., Beijing, China) with T4-polynucleotide
kinase. Nuclear protein (10 μg) was preincubated in a total volume of 9 μL in a
binding buffer, consisting of 10 mmol/L Tris-HCl (pH 7.5), 4% glycerol, 1 mmol/L
MgCl2, 0.5 mmol/L M EDTA, 0.5 mmol/L DTT, 0.5 mmol/L NaCl, and 0.05 g/L poly-(deoxyinosinic-deoxycytidylic
acid) for 15 minutes at room temperature. After addition of the 1 μL P32-labled
oligonucleotide probe, the incubation was continued for 20 minutes at room temperature.
Reaction was stopped by adding 1 μL of gel loading buffer and the mixture was
subjected to nondenaturing 4% polyacrylamide gel electrophoresis in 0.5 × TBE
buffer (Tris-borate-EDTA). After electrophoresis was conducted at 390 V for 1 hour,
the gel was vacuum-dried and exposed to X-ray film (Fuji Hyperfilm, Tokyo, Japan)
at −70°C with an intensifying screed. Levels of NF-κB DNA binding activity were
quantified by computer-assisted densitometric analysis.


## 2.4. ELISA Analysis

Frozen brain samples were homogenized in 1 mL of buffer containing 1 mmol/L of PMSF, 1 mg/L of pepstatin A, 1 mg/L of aprotinin, and 1 mg/L of leupeptin in PBS
solution (pH 7.2) with a glass homogenizer and then centrifuged at 12000 g for 20
minutes at 4°C. The supernatant was then collected and total
protein was determined by the Bradford method.
The levels of inflammatory cytokines were quantified using enzyme-linked
immunosorbent assay (ELISA) kits specific for mouse according to the
manufacturers’ instructions (TNF-α from Diaclone
Research, France; IL-1β, IL-6 from Biosource Europe SA, Belgium) and previous study of our laboratory [24].
The cytokine contents in the brain samples were expressed as pg per milligram protein.


## 2.5. Immunohistochemical Staining

The paraffin-embedded sections (4μm) were used for Immunohistochemical
assay, which was performed with a goat antimouse ICAM-1(CD54) antibody (diluted
1:200, R&D Systems, Inc.,
Minn, USA),
according to previous studies of our laboratory [7]. The sections were
incubated with the diluted antibody overnight at 4°C in a humid chamber, washed, and blocked with 1.6%  H2O2 in phosphate-buffered saline
(PBS) for 10 minutes. After washing with PBS again, sections were then
incubated with biotinylated second antibodies for 1 hour at room temperature.
Diaminobenzidine (DAB) was used as chromogen and counterstaining was done with
hematoxylin. The number of positive microvessels in each section was counted in
10 microscopic fields (at 100 × magnifications) and averaged for the positively
immunostained vessel number of per visual field.


## 2.6. Statistical Analysis

Software SPSS 13.0 was used for the statistical analysis. All data were
expressed as mean ± SEM, Student’st-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<.05.


## 3. Results

### 3.1. EMSA for NF-κB

NF-κB activation in the nuclear
extracts was assessed by EMSA. As shown in
Figure 2, low NF-κB banding activity
(weak EMSA autoradiography) was observed in sham-operated mice of both
genotypes. TBI induced activation of NF-κB in the cortex
of both Nrf2 (+/+) and Nrf2 (−/−) mice. Nrf2 (−/−) mice showed an increased
susceptibility to TBI-induced activation of NF-κB than their wild-type Nrf2 (+/+) counterparts.NF-κB activity in the cortex of sham and injured Nrf2 (+/+)
and Nrf2 (−/−) mice. (a) Nuclear proteins of brain
samples of Nrf2 (+/+) and Nrf2 (−/−) mice were assayed for NF-κB DNA binding
activity by EMSA 24 hours after TBI. (b) Quantification of NF-κB DNA binding
activity was performed by densitometric analysis. The figure indicates that
cerebral NF-κB activity was significantly increased after TBI and was greater
in Nrf2 (−/−) mice than in Nrf2 (+/+) mice. Data
represents mean ± SEM (n=5 per group). P<.01** versus sham control of the
same genotype. ##P<.01 versus injured wild-type
mice.
(a)(b)


### 3.2. ELISA for Inflammatory Cytokines

Concentrations of TNF-α, IL-1β, and IL-6 in the brain samples were measured by
ELISA. As shown in Figure 3, low concentrations of TNF-α, IL-1β, and IL-6 were observed in sham-operated mice of both genotypes. TBI
induced upregulation of TNF-α, IL-1β, and IL-6 in the cortex of both Nrf2 (+/+) and Nrf2
(−/−) mice. Nrf2 (−/−) mice showed larger increase in cortical levels of TNF-α, IL-1β, and IL-6 than their wild-type
Nrf2 (+/+) littermates after TBI.Concentrations of inflammatory cytokines in the cortex of sham and injured Nrf2 (+/+) and Nrf2
(−/−) mice. Concentrations of (a) TNF-α, (b) IL-1β, and (c) IL-6 were determined by
ELISA in the brain samples of Nrf2 (+/+) and Nrf2 (−/−) mice 24 hours
after TBI. The figure indicates that concentrations of TNF-α, IL-1β, and IL-6
in brain were significantly increased after TBI and were
greater in Nrf2 (−/−) mice than in Nrf2 (+/+) mice. Data
represents mean ± SEM (n=5 per group). **P<.01 versus sham control of the
same genotype. #P<.05 and ##P<.01 versus
injured wild-type mice.
(a)(b)(c)


### 3.3. Immunohistochemistry for ICAM-1

For assessment of the expression of ICAM-A in the brain after TBI,
immunohistochemical study for ICAM-1 was performed. As shown in Figure4, few
ICAM-1-immunostained cerebral microvessels were observed in sham-operated mice
of both genotypes. At the 24 hours after TBI, the number of ICAM-1 positive
vessels was significantly increased in the cortex of both Nrf2 (+/+) and Nrf2
(−/−) mice. Nrf2 (−/−) mice showed larger increase in the number of ICAM-1
positive vessels than their wild-type
Nrf2 (+/+) littermates after TBI.Expression of ICAM-1 in the cortex of sham and injured Nrf2 (+/+) and Nrf2 (−/−) mice.
Immunohistochemical staining for ICAM-1 was performed in the cortex tissue sections of Nrf2 (+/+) and Nrf2 (−/−) mice 24 hours
after TBI. (a), (c) Sham-operated Nrf2 (+/+) and Nrf2 (−/−) mice showing few ICAM-1-immunostained cerebral microvessels. (b)
Injured Nrf2 (+/+) mice showing increased number of
ICAM-1 positive vessels. (d) Injured Nrf2 (−/−) mice showing larger increment in the number of ICAM-1 positive vessels compared
with injured Nrf2 (+/+) mice. (e) Quantitative analysis showed that the number of ICAM-1
positive vessels in brain was significantly increased after TBI and was greater
in Nrf2 (−/−) mice than in Nrf2 (+/+) mice. Data
represents mean ± SEM (n=5 per group). **P<.01 versus sham control of the same genotype. #P<.05 versus
injured wild-type mice.
(a)(b)(c)(d)(e)


## 3.1. EMSA for NF-κB

NF-κB activation in the nuclear
extracts was assessed by EMSA. As shown in
Figure 2, low NF-κB banding activity
(weak EMSA autoradiography) was observed in sham-operated mice of both
genotypes. TBI induced activation of NF-κB in the cortex
of both Nrf2 (+/+) and Nrf2 (−/−) mice. Nrf2 (−/−) mice showed an increased
susceptibility to TBI-induced activation of NF-κB than their wild-type Nrf2 (+/+) counterparts.NF-κB activity in the cortex of sham and injured Nrf2 (+/+)
and Nrf2 (−/−) mice. (a) Nuclear proteins of brain
samples of Nrf2 (+/+) and Nrf2 (−/−) mice were assayed for NF-κB DNA binding
activity by EMSA 24 hours after TBI. (b) Quantification of NF-κB DNA binding
activity was performed by densitometric analysis. The figure indicates that
cerebral NF-κB activity was significantly increased after TBI and was greater
in Nrf2 (−/−) mice than in Nrf2 (+/+) mice. Data
represents mean ± SEM (n=5 per group). P<.01** versus sham control of the
same genotype. ##P<.01 versus injured wild-type
mice.
(a)(b)


## 3.2. ELISA for Inflammatory Cytokines

Concentrations of TNF-α, IL-1β, and IL-6 in the brain samples were measured by
ELISA. As shown in Figure 3, low concentrations of TNF-α, IL-1β, and IL-6 were observed in sham-operated mice of both genotypes. TBI
induced upregulation of TNF-α, IL-1β, and IL-6 in the cortex of both Nrf2 (+/+) and Nrf2
(−/−) mice. Nrf2 (−/−) mice showed larger increase in cortical levels of TNF-α, IL-1β, and IL-6 than their wild-type
Nrf2 (+/+) littermates after TBI.Concentrations of inflammatory cytokines in the cortex of sham and injured Nrf2 (+/+) and Nrf2
(−/−) mice. Concentrations of (a) TNF-α, (b) IL-1β, and (c) IL-6 were determined by
ELISA in the brain samples of Nrf2 (+/+) and Nrf2 (−/−) mice 24 hours
after TBI. The figure indicates that concentrations of TNF-α, IL-1β, and IL-6
in brain were significantly increased after TBI and were
greater in Nrf2 (−/−) mice than in Nrf2 (+/+) mice. Data
represents mean ± SEM (n=5 per group). **P<.01 versus sham control of the
same genotype. #P<.05 and ##P<.01 versus
injured wild-type mice.
(a)(b)(c)


## 3.3. Immunohistochemistry for ICAM-1

For assessment of the expression of ICAM-A in the brain after TBI,
immunohistochemical study for ICAM-1 was performed. As shown in Figure4, few
ICAM-1-immunostained cerebral microvessels were observed in sham-operated mice
of both genotypes. At the 24 hours after TBI, the number of ICAM-1 positive
vessels was significantly increased in the cortex of both Nrf2 (+/+) and Nrf2
(−/−) mice. Nrf2 (−/−) mice showed larger increase in the number of ICAM-1
positive vessels than their wild-type
Nrf2 (+/+) littermates after TBI.Expression of ICAM-1 in the cortex of sham and injured Nrf2 (+/+) and Nrf2 (−/−) mice.
Immunohistochemical staining for ICAM-1 was performed in the cortex tissue sections of Nrf2 (+/+) and Nrf2 (−/−) mice 24 hours
after TBI. (a), (c) Sham-operated Nrf2 (+/+) and Nrf2 (−/−) mice showing few ICAM-1-immunostained cerebral microvessels. (b)
Injured Nrf2 (+/+) mice showing increased number of
ICAM-1 positive vessels. (d) Injured Nrf2 (−/−) mice showing larger increment in the number of ICAM-1 positive vessels compared
with injured Nrf2 (+/+) mice. (e) Quantitative analysis showed that the number of ICAM-1
positive vessels in brain was significantly increased after TBI and was greater
in Nrf2 (−/−) mice than in Nrf2 (+/+) mice. Data
represents mean ± SEM (n=5 per group). **P<.01 versus sham control of the same genotype. #P<.05 versus
injured wild-type mice.
(a)(b)(c)(d)(e)


## 4. Discussion

The most important finding of this
study is that Nrf2 (−/−) mice had more inflammatory cytokines TNF-α, IL-1β and IL-6
production, ICAM-1 expression, and their mediator NF-κB activation in brain after TBI compared
with their wild-type Nrf2 (+/+) counterparts. These findings reported here
suggest for the first time that Nrf2 may play an important role in limiting the
cerebral inflammatory response after TBI through modulating the proinflammatory
nuclear factor kappa B (NF-κB) signaling pathway.Activation of NF-κB signaling pathway has been shown to be
central to the pathophysiology of cerebral inflammatory response induced by TBI
[6, 7]. NF-κB can be activated by lesion-induced oxidative stress, bacterial
endotoxin, and cytokines [25]. The functional importance of NF-κB in
inflammation is based on its ability to regulate the promoters of multiple inflammatory
genes, including TNF-α, IL-1β, IL-6, and ICAM-1 [4]. TNF-α
is reported to be a major initiator of inflammation and is released early after
an inflammatory stimulus [26]. IL-1β is regarded as the prototypic “multifunctional”
cytokine and is induced in a multitude of consequences of cell types [27]. IL-6
is increased after TNF-α and is considered to be an important proinflammatory
cytokine in contribution to both morbidity and mortality in condition of
“uncontrolled” inflammation [28]. ICAM-1, a member of the immunoglobulin
superfamily which can be profound induced after cytokine challenge, is
important in the recruitment of leukocytes during the inflammatory process [29].
This inflammatory agent network is believed to be important in the generation
of acute inflammatory response. NF-κB activation enhances the transcription of
proinflammatory cytokines, and the cytokines are known to in turn activate
NF-κB [5]. The positive feedback is believed to serve to amplify inflammatory
signals and exacerbate brain injury after TBI. In the present study, we
evaluated the influence of Nrf2 genotype in the TBI-induced activation of proinflammatory
NF-κB signaling pathway in the brain. The results showed that disruption of Nrf2 in mice caused a greater activation of
NF-κB signaling pathway which played a critical role in the pathophysiology of
cerebral inflammatory response induced by TBI. The observed interplay between
Nrf2 and NF-κB signaling corresponds well to the results of study on experimental sepsis,
which have demonstrated that Nrf2-deficient mice displayed increased NF-κB
activation in response to lipopolysaccharide (LPS) [19].Although numerous in vivo studies
have reported that Nrf2 plays a critical role in counteracting the inflammation
in a variety of experimental models [14–19], the findings
which we have confirmed and extended in the model of TBI in the present study, the
precise mechanism underlying this network is still unclear. Several lines of
evidence suggest that Nrf2 regulates the inflammatory response by inhibiting
proinflammatory NF-κB activation through maintenance of redox homeostasis.
Oxidative stress from reactive oxygen species (ROS) is believed to be involved
in the progression of secondary brain injury following TBI [30]. Activation of
the NF-κB signaling pathway has been shown to be responsive to excess ROS and
is important in the generation of inflammation [19]. Nrf2, as a key antioxidant
transcription factor involved in the intracellular antioxidant defense systems,
has been shown to play an important role in limiting ROS levels and thereby
affect redox-sensitive NF-κB signaling pathway involved in the inflammation [8, 10–12]. The
protective function of Nrf2 is mainly mediated by a group of Nrf2-regulated
antioxidant and detoxifying enzymes. Therefore, the augmentation of cellular
antioxidative or detoxification systems via activation of Nrf2-regulated
enzymes resulting in decreased proinflammatory cytokines production and
adhesion molecules expression via inactivation of NF-κB represents a possible
anti-inflammatory mechanism for the attenuated inflammatory response seen in
brains from Nrf2 (+/+) mice but not Nrf2 (−/−) mice after TBI. We then in this
study postulated that Nrf2 regulates the TBI-induced cerebral inflammatory
response may at least in part through modulating the cerebral redox status and proinflammatory
NF-κB signaling pathway. Additional work is necessary to
elucidate the whole mechanisms involved in these complicated
networks.In summary, this present study showed that Nrf2 plays a
protective role in TBI-induced cerebral upregulation of inflammatory agents in
mice. We found that Nrf2 (−/−) mice are more susceptible to TBI-induced
cerebral NF-κB activation, inflammatory cytokine
TNF-α, IL-1β and IL-6 production, and ICAM-1 expression, which then contributed to exacerbated brain injury after TBI. To the
best of our knowledge, this is the first study that elucidates the interplay between Nrf2 and proinflammatory NF-κB signaling pathway in the brain following TBI. These
findings raise the possibility that Nrf2 will be a new therapeutic target for
the treatment TBI.



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*Source: 725174-2009-01-25.xml*