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Increased intestinal inflammatory response and gut barrier dysfunction in Nrf2-deficient mice after traumatic brain injury
Cytokine 44 (2008) 135–140
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Cytokine journal homepage: www.elsevier.com/locate/issn/10434666
Increased intestinal inflammatory response and gut barrier dysfunction in Nrf2-deficient mice after traumatic brain injury Wei Jin a, Handong Wang a,*, Yan Ji b, Qingang Hu b, Wei Yan a, Gang Chen a, Hongxia Yin a a
Department of Neurosurgery, Jinling Hospital, School of Medicine, Nanjing University, 305 East Zhongshan Road, Nanjing 210002, Jiangsu Province, PR China Department of Oral and Maxillofacial Surgery, Affiliated Stomatology Hospital, School of Medicine, Nanjing University, 30 Zhongyang Road, Nanjing 210008, Jiangsu Province, PR China b
a r t i c l e
i n f o
Article history: Received 9 April 2008 Received in revised form 1 July 2008 Accepted 14 July 2008
Keywords: Nuclear factor E2-related factor 2 Traumatic brain injury Intestine Inflammation
a b s t r a c t Aim: To explore the role of nuclear factor erythroid 2-related factor 2 (Nrf2) in traumatic brain injury (TBI)-induced intestinal inflammatory response and gut barrier dysfunction in the mice. Methods: Wild-type Nrf2 (+/+) and Nrf2 (/)-deficient mice were subjected to a moderately severe weight-drop impact-acceleration head injury. We measured nuclear factor kappa B (NF-jB) by electrophoretic mobility shift assay (EMSA); tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b) and interleukin-6 (IL-6) by enzyme-linked immunosorbent assay (ELISA); intercellular adhesion molecule-1 (ICAM-1) by immunohistochemistry; intestinal permeability by lactulose/mannitol (L/M) test; plasma endotoxin by chromogenic limulus amebocyte lysate test. Results: Intestinal levels of NF-jB, pro-inflammatory cytokines and ICAM-1 in Nrf2 (/)-deficient mice were significantly higher compared with Nrf2 (+/+) mice at 24 h after TBI. Furthermore, higher intestinal permeability and plasma level of endotoxin were observed in the Nrf2 (/) mice compared with Nrf2 (+/+) mice. Conclusion: Nrf2 plays an important protective role in limiting intestinal inflammatory response and gut barrier dysfunction after TBI. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Gastrointestinal dysfunction is a known complication of traumatic brain injury (TBI) [1]. The relation between TBI and intestinal mucosa injury has been studied [2]. Major trauma and shock may initiate a cascade of intestinal events such as overproduction of intestinal cytokine [3], increased intestinal permeability [4], translocation of intestinal bacteria and endotoxins [5]. This pathologic course may not only influence the intestinal mucosa itself, but also impair the remote tissue and organs, leading to systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS). Previous studies of our laboratory have demonstrated that traumatic brain injury could lead to a concomitant and persistent up-regulation of inflammation-related factors such as nuclear factor kappa B (NF-jB), pro-inflammatory cytokines and intercellular adhesion molecule-1 (ICAM-1) in the intestine, which could then lead to significant gut structural alterations and barrier dysfunction [6,7]. Additional work is necessary to explore the underlying mechanism of the inflammatory response in the intestine after TBI. It has been reported that traumatic brain injury could trigger the intestinal accumulation of highly toxic reactive oxygen species (ROS) which leads to a state of oxidative stress [8]. Oxidative stress * Corresponding author. Fax: +86 25 84817581. E-mail address: [email protected] (H. Wang). 1043-4666/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2008.07.005
can not only directly cause tissue damage, but also modulate inflammatory response during tissue injury. Intracellular antioxidant defense systems play a crucial role in protection against oxidative damage by limiting ROS levels and thereby inhibit redoxmediate inflammatory response. Nuclear factor erythroid 2-related factor 2 (Nrf2) is reported to be the key transcription factor that, upon activation by oxidative stress, regulates the cellular antioxidant response which is critical for cellular protection. Activation of Nrf2 has been shown to protect cells from oxidant insults, thereby inhibiting redox-mediate inflammatory response [9–12]. Numerous studies have reported that Nrf2 plays a critical role in a variety of experimental models in counteracting inflammation. Nrf2 protects against allergen-mediated airway inflammation and cigarette smoke-induced emphysema [13,14]. In addition, Nrf2 plays essential roles in protection against inflammatory responses during skin wound healing [15]. Moreover, Nrf2 has been reported as a crucial regulator of the innate immune response and survival during experimental sepsis [16]. Furthermore, Nrf2 has also been shown to be a protector in dextran sulfate sodium (DSS)-mediated colitis and inflammation-mediated colonic tumorigenesis [17,18]. However, to our knowledge, the role of Nrf2 in the intestinal production of inflammatory agents which played crucial roles in the mechanisms of gut injury after TBI has not been studied to date. The purpose of this study was therefore to investigate the influence of Nrf2 genotype in modulating TBI-induced up-regulation of inflammatory agents in the intestine.
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2. Materials and methods 2.1. Animals All procedures in animals 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 (+/+) mice and Nrf2 (/)deficient mice were generated from inbred heterozygous Nrf2 (+/) mice [10]. 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, 50 -TGGACGGGACTATTGA AGGCTG-30 (sense for both genotypes), 50 -CGCCTTTTCAGTAGATGG AGG-30 (antisense for wild-type) and 50 -GCGGATTGACCGTAATGG GATAGG-30 (antisense for LacZ). Mice were housed at 23 ± 1 °C in humidity controlled animal quarters with 12-h light/dark cycle. All animals were allowed water and food ad libitum throughout the study. 2.2. Experiment protocol The mouse model of diffuse closed head injury was employed as described [19] with recent minor modification [20]. Age- and weight-matched adult male mice (6–8 weeks, 28–32 g) 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 min with 100% oxygen administration and chest compression to stimulate the respiration. This model is generally associated with 20% of mortality within the first 5 min post-injury 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. The sham mice were subjected to identical anesthetic without trauma. Mice were separated into four groups (n = 12 per group): group I, sham + wild-type Nrf2 (+/+); group II, TBI + wild-type Nrf2 (+/+); group III, sham + Nrf2 (/)-deficient; group IV, TBI + Nrf2 (/)deficient. Animals were decapitated at 24 h following sham or injury for sample collection. Six mice in each group were sacrificed for electrophoretic mobility shift assay (EMSA) and enzyme-linked immunosorbent assay (ELISA) and the others were for immunohistochemistry study. Blood samples were obtained from the dorsal vena cava and centrifuged with the plasma stored at 40 °C before the animals were killed. For EMSA and ELISA analysis, mice were exsanguinated by cardiac puncture, and a 3-cm segment of the jejunum 8 cm distal to Treitz ligament was rapidly removed and stored at liquid nitrogen immediately. For immunohistochemistry, mice were perfused via left ventricular puncture with cold saline (4 °C), followed by 4% neutral-buffered formalin. The 3-cm segment of the jejunum was taken, stored overnight in 4% neutral-buffered formalin, and then embedded in paraffin.
AGG C-30 ) was end-labeled with [c-32P]ATP (Free Biotech., Beijing, China) with T4-polynucleotide kinase. EMSA was performed according to previous study of our laboratory. Levels of NF-jB activity were quantified by computer-assisted densitometric analysis. 2.4. Detection of tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b) and interleukin-6 (IL-6) in intestinal tissue The intestinal levels of inflammatory cytokines were quantified using ELISA kits specific for mouse according to the manufacturers’ instructions (TNF-a from Diaclone Research, France; IL-1b, IL-6 from Biosource Europe SA, Belgium) and previous study of our laboratory [21]. The cytokine contents in the intestinal tissue were expressed as pg per milligram protein. 2.5. Detection of ICAM-1 expression in intestinal tissue The tissue sections (4 lm) were used for immunohistochemical assay, which was performed with an goat anti-mouse ICAM1(CD54) antibody (diluted 1:200, R&D Systems, Inc., MN, USA), according to previous studies of our laboratory [7,21]. Microscopy of the immunohistochemically stained tissue sections was performed by an experienced pathologist blinded to the experimental condition. Evaluation of sections was undertaken by assessing the intensity of staining (5 grades). ‘‘0” indicates no detectable positive cell; ‘‘1” indicates very low density of positive cells; ‘‘2” indicates a moderate density of positive cells; ‘‘3” indicates the higher, but not maximal density of positive cells and ‘‘4” indicates the highest density of positive cells. 2.6. Intestinal permeability Intestinal permeability was quantified using the lactulose/mannitol (L/M) test as described [22]. Six hours before the animals were sacrificed the mice with their bladders emptied were given the test solution of 200 ll by gastric tube feeding, containing 13.3 mg lactulose and 10.1 mg mannitol. All urine was collected for 6 h through the metabolic cage and stored at 40 °C for further analysis. Urinary lactulose and mannitol were measured using high-performance liquid chromatography (Waters Co., USA). Results were expressed as a ratio of percentage administered dose of lactulose excreted in the urine to the percentage administered dose of mannitol in the same urine collection (L/M ratio). 2.7. Plasma endotoxin determination The endotoxin content in plasma samples was assayed using limulus amebocyte lysate (LAL) kit (Sigma, USA) with a kinetic modification according to the test kit instruction and our laboratory methods [2]. Endotoxin concentrations were expressed as EU/ml. 2.8. Statistical analysis Software SPSS 13.0 was used for the statistical analysis. All data were expressed as means ± SEM, Student’s t-test was used to analysis 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.
2.3. Nuclear protein extract and EMSA
3. Results
Nuclear protein of intestinal tissue was extracted and quantified as described [6]. EMSA was performed using a commercial kit (Gel Shift Assay System; Promega, Madison, WI) as previously described. Consensus oligonucleotide probe (50 -AGT TGA GGG GAC TTT CCC
3.1. NF-jB binding activity in intestinal tissue NF-jB binding activity of intestinal nuclear protein was assessed by EMSA. As shown in Fig. 1, TBI-induced activation of
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3.2. Inflammatory cytokine concentrations in intestinal tissue Concentrations of TNF-a, IL-1b and IL-6 in the intestinal tissue were measured by ELISA. As shown in Fig. 2, TBI-induced up-regulation of inflammatory cytokines including TNF-a, IL-1b and IL-6 in the intestinal tissue of Nrf2 (+/+) and Nrf2 (/) mice (P < 0.05). Nrf2 (/) mice showed larger increase in levels of intestinal TNF-a, IL-1b and IL-6 compared with their wild-type Nrf2 (+/+) littermates after TBI (P < 0.05). 3.3. Expression of ICAM-1 in intestinal tissue As shown in Fig. 3, strong to moderate ICAM-1 immunoreactivity was observed in villous interstitium and lamina propria of Nrf2 (+/+) and Nrf2 (/) mice after TBI (P < 0.05). The immunoreactivity was almost undetectable in the sham-operated mice, and was found to be relatively weaker in Nrf2 (+/+) mice as compared with Nrf2 (/) mice at 24 h after TBI (P < 0.05). 3.4. Intestinal permeability in various groups As shown in Fig. 4, TBI caused a significant increase in the levels of intestinal permeability in Nrf2 (+/+) and Nrf2 (/) mice as compared with sham-operated mice (P < 0.05). Increased intestinal permeability was found in Nrf2 (/) mice as compared with their wild-type Nrf2 (+/+) counterparts at 24 h after TBI (P < 0.05). 3.5. Changes in plasma endotoxin level
Fig. 1. NF-jB activity in the intestine tissue of sham and injured Nrf2 (+/+) and Nrf2 (/) mice. (A) Nuclear protein of intestine tissue of Nrf2 (+/+) and Nrf2 (/) mice were assayed for NF-jB DNA binding activity by EMSA at 24 h after TBI. (B) Quantification of NF-jB DNA binding activity was performed by densitometric analysis. The figure indicates that intestinal NF-jB activity was significantly increased and was greater in Nrf2 (/) mice than in Nrf2 (+/+) mice after TBI (n = 6 per group). *P < 0.05 vs sham control of the same genotype. #P < 0.05 vs injured wild-type mice.
NF-jB in the intestinal tissue of Nrf2 (+/+) and Nrf2 (/) mice (P < 0.05). Nrf2 (/) mice showed an increased susceptibility to TBI-induced intestinal activation of NF-jB compared with their wild-type Nrf2 (+/+) counterparts (P < 0.05).
As shown in Fig. 5, at 24 h after TBI, plasma endotoxin levels in Nrf2 (+/+) and Nrf2 (/) mice were significantly higher than in sham-operated mice without TBI (P < 0.05). Plasma endotoxin level in Nrf2 (/) mice was significantly higher than in Nrf2 (+/+) mice at 24 h after TBI (P < 0.05). 4. Discussion This study revealed that disruption of Nrf2 in mice caused higher sensitivity to intestinal inflammatory response after experimental TBI. We found in this study that Nrf2 (/) mice have more inflammatory cytokine TNF-a, IL-1b and IL-6 production, ICAM-1 expression and their mediators NF-jB activation in the intestine after TBI compared with their wild-type Nrf2 (+/+) counterparts. Furthermore, higher intestinal permeability and plasma level of endotoxin were observed in the Nrf2 (/) mice compared with their wild-type Nrf2 (+/+) counterparts. These findings reported here suggest for the first time that Nrf2 plays a critical role in lim-
Fig. 2. Changes of inflammatory cytokines in the intestine tissue of sham and injured Nrf2 (+/+) and Nrf2 (/) mice. Concentrations of TNF-a, IL-1b and IL-6 were determined by ELISA in the intestinal tissue of Nrf2 (+/+) and Nrf2 (/) mice at 24 h after TBI. The figure indicates that intestinal levels of TNF-a, IL-1b and IL-6 were significantly increased and were greater in Nrf2 (/) mice than in Nrf2 (+/+) mice after TBI (n = 6 per group). *P < 0.05 vs sham control of the same genotype. #P < 0.05 vs injured wild-type mice.
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Fig. 3. Expression of ICAM-1 in the intestine tissue of sham and injured Nrf2 (+/+) and Nrf2 (/) mice. Immunohistochemical staining for ICAM-1 was performed in the intestinal tissue sections of Nrf2 (+/+) and Nrf2 (/) mice at 24 h after TBI. (A) Sham control Nrf2 (+/+) mice showing low ICAM-1 immunoreactivity. (B) Injured Nrf2 (+/+) mice showing increased ICAM-1 immunoreactivity. (C) Sham control Nrf2 (/) mice also showing low ICAM-1 immunoreactivity. (D) Injured Nrf2 (+/+) mice showing the strongest ICAM-1 immunoreactivity. (E) Intestinal expression of ICAM-1 was significantly increased and was greater in Nrf2 (/) mice than in Nrf2 (+/+) mice after TBI (n = 6 per group). *P < 0.05 vs sham control of the same genotype. #P < 0.05 vs injured wild-type mice.
iting the TBI-induced intestinal inflammatory response and gut barrier dysfunction in the mice TBI model. Inflammatory response in the intestine has been implicated in the pathogenesis of TBI-induced gastrointestinal dysfunction. It is generally accepted that the intestine may serve as an important organ in the development of severe complications under critical illness [23]. Indeed, the intestine has been proposed to be the ‘‘motor” of MODS in trauma [24], mainly through the inflammatory response mediated by NF-jB, pro-inflammatory cytokines and ICAM-1 [6,7]. NF-jB is one of the most important pro-inflammatory modulators, which can be activated by lesion-induced oxidative stress, bacterial endotoxin or cytokines and subsequently
transactivate the expression of many cytokines and adhesion molecules [25]. Pro-inflammatory cytokines including interleukins (ILs) and TNF-a released early after an inflammatory stimulus, can initiate the infiltration of inflammatory cells into the intestine by activating ICAM-1 and other adhesion molecules [26]. NF-jB activation enhances the transcription of pro-inflammatory cytokines, and the cytokines are known to in turn activate NF-jB [27]. The positive feedback is believed to serve to amplify inflammatory signals, inducing more tissue injury. The prevailing theory has been that the dysregulation of brain-gut axis and hypothalamic-pituitary-adrenal axis resulting from the TBI may initiate the inflammatory process in the intestine, but the potential mech-
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Fig. 4. Intestinal permeability in sham and injured Nrf2 (+/+) and Nrf2 (/) mice. Intestinal permeability of Nrf2 (+/+) and Nrf2 (/) mice were quantified using lactulose/mannitol (L/M) test at 24 h after TBI. The figure indicates that the level of intestinal permeability was significantly increased and was higher in Nrf2 (/) mice than in Nrf2 (+/+) mice after TBI (n = 6 per group). *P < 0.05 vs sham control of the same genotype. #P < 0.05 vs injured wild-type mice.
Fig. 5. Plasma endotoxin levels in sham and injured Nrf2 (+/+) and Nrf2 (/) mice. Plasma endotoxin levels of Nrf2 (+/+) and Nrf2 (/) mice were determined using limulus amebocyte lysate assay at 24 h after TBI. The figure indicates that plasma endotoxin level was significantly increased and was higher in Nrf2 (/) mice than in Nrf2 (+/+) mice after TBI (n = 6 per group). *P < 0.05 vs sham control of the same genotype. #P < 0.05 vs injured wild-type mice.
anism underlying this action is not clear [28,29]. In the present study, we found that at 24 h after TBI, up-regulation of inflammation-related factors including NF-jB, TNF-a, IL-1b, IL-6 and ICAM-1 was evident in the intestine and was greater in Nrf2 (/) mice than in Nrf2 (+/+) mice. These results support previous researches of our laboratory that TBI could provoke a significant intestinal inflammatory response and also suggest that Nrf2 deficiency could enhance the intestinal inflammatory response after TBI in the mice TBI model. The elevation of inflammation also contributes to the gut barrier dysfunction following TBI. Disruption of gut barrier occurs with increased intestinal permeability and high plasma level of endotoxin. It has been demonstrated that pro-inflammatory cyto-
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kines such as TNF-a, IL-1b and IL-6, which can be regulated by NF-jB, have cytotoxic effect that induced damages of microvilli, resulting in destruction of intercellular tight junctions and increased intestinal permeability [30]. Furthermore, increasing intestinal permeability permits bacterial and endotoxin translocation, which triggers systemic immunoinflammatory response to release pro-inflammatory cytokines and mediators, leading to aggravated SIRS and MODS [5,3]. The positive feedback is also believed to serve to amplify inflammatory tissue injury in the intestine following TBI. Previous study of our laboratory has shown that traumatic brain injury could lead to a significant increase in the intestinal permeability and plasma level of endotoxin, which implied that gut barrier function was disrupted [2]. Interestingly, we also observed in the present study that intestinal permeability and plasma level of endotoxin were increased in the mice of both genotypes after TBI. In addition, higher intestinal permeability and plasma level of endotoxin were found in Nrf2 (/) mice as compared with their Nrf2 (+/+) counterparts after TBI, which could be explained by evidence indicating that Nrf2 plays a protective role in TBI-induced gut barrier dysfunction in mice. Although numerous in vivo studies have reported in a variety of experimental models that Nrf2 plays a critical role in counteracting the inflammation [13–18], the findings which we have confirmed and extended in the model of TBI-induced gut injury in the present study, it is still not clear how Nrf2 regulates the expression of proinflammatory mediators and cytokines, several lines of evidence indicate that Nrf2-dependent antioxidant response provides protection against acute inflammation through modulation the NFjB signaling pathway. Previous studies have demonstrated that oxidative stress from reactive oxygen species (ROS) is accompanied by the up-regulation of inflammatory response at the site of inflammation [11]. ROS, such as superoxide, hydroperoxyl radicals, hydroxyl radicals and hydrogen peroxide, can not only directly cause tissue damage, DNA damage and lipid peroxidation, but also serve as important signaling molecules in the activation of redoxsensitive transcription factors NF-jB and regulation of gene expression for pro-inflammatory cytokines and adhesion molecules involved in the inflammatory response [9,31]. On the other hand, intracellular antioxidant defense systems play a crucial role in protection against oxidative damage by limiting ROS levels and thereby affect redox signaling pathways involved in the inflammation. Nrf2 has been reported to be a key antioxidant transcription factor which plays a central role in protecting cells and tissue from ROS and electrophiles. Under basal conditions, Nrf2 is sequestered in the cytoplasm by the cytosolic regulatory protein Keap1. In conditions of oxidative stress, Nrf2 translocates from the cytoplasm to the nucleus, and sequentially binds to a promoter sequence called the antioxidant response element (ARE), resulting in transactivating the expression of a group of antioxidant and detoxifying enzymes. The cytoprotective function of Nrf2 is mainly mediated by these Nrf2-regulated antioxidant and detoxifying enzymes [9– 12,32]. It is therefore implied that Nrf2 may play an important role in anti-inflammation by a mechanism of the augmentation of cellular antioxidative or detoxification systems via activation of Nrf2regulated enzymes resulting in decreased pro-inflammatory cytokines production and adhesion molecules expression via inactivation of NF-jB. We then in this study postulated that increased susceptibility of Nrf2 (/) mice to TBI-induced intestinal inflammatory damage may at least in part be due to the dysregulation of redox homeostasis and NF-jB signaling pathway. Additional work is necessary to elucidate the whole mechanisms involved in these complicated networks. In summary, this present study has shown that Nrf2 plays a protective role in TBI-induced intestinal inflammatory response and gut barrier dysfunction in mice. We found that Nrf2 (/) mice are more susceptible to TBI-induced intestinal NF-jB activation,
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inflammatory cytokine TNF-a, IL-1b and IL-6 production and ICAM-1 expression, which then contributed to augmented gut barrier dysfunction occurring with increased levels of intestinal permeability and plasma endotoxin. To the best of our knowledge, this is the first study that elucidates the role of Nrf2 in TBI-induced intestinal inflammatory damage. These findings raise the possibility that Nrf2 will be a new therapeutic target for the treatment of gut injury after TBI. Acknowledgments This work was supported by grants from Jinling Hospital of China. The authors thank Dr. Bo Wu and Dr. Geng-bao Feng for technical assistance. References [1] Pilitsis JG, Rengachary SS. Complications of head injury. Neurol Res 2001;23:227–36. [2] Hang CH, Shi JX, Li JS, Wu W, Yin HX. Alterations of intestinal mucosa structure and barrier function following traumatic brain injury in rats. World J Gastroenterol 2003;9:2776–81. [3] Grotz MR, Deitch EA, Ding J, Xu D, Huang Q, Regel G. Intestinal cytokine response after gut ischemia: role of gut barrier failure. Ann Surg 1999;229:478–86. [4] Faries PL, Simon RJ, Martella AT, Lee MJ, Machiedo GW. Intestinal permeability correlates with severity of injury in trauma patients. J Trauma 1998;44:1031–5. [5] Swank GM, Deitch EA. Role of the gut in multiple organ failure: bacterial translocation and permeability changes. World J Surg 1996;20:411–7. [6] Hang CH, Shi JX, Li JS, Li WQ, Wu W. Expressions of intestinal NF-kappaB, TNFalpha, and IL-6 following traumatic brain injury in rats. J Surg Res 2005;123:188–93. [7] Hang CH, Shi JX, Li JS, Li WQ, Yin HX. Up-regulation of intestinal nuclear factor kappa B and intercellular adhesion molecule-1 following traumatic brain injury in rats. World J Gastroenterol 2005;11:1149–54. [8] Shohami E, Gati I, Beit-Yannai E, Trembovler V, Kohen R. Closed head injury in the rat induces whole body oxidative stress: overall reducing antioxidant profile. J Neurotrauma 1999;16:365–76. [9] Chen XL, Kunsch C. Induction of cytoprotective genes through Nrf2/antioxidant response element pathway: a new therapeutic approach for the treatment of inflammatory diseases. Curr Pharm Des 2004;10:879–91. [10] Itoh K, Chiba T, Takahashi S, Ishii T, Igarashi K, Katoh Y, et al. An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochem Biophys Res Commun 1997;236:313–22. [11] Guo RF, Ward PA. Role of oxidants in lung injury during sepsis. Antioxid Redox Signal 2007;9:1991–2002. [12] Lee JM, Johnson JA. An important role of Nrf2-ARE pathway in the cellular defense mechanism. J Biochem Mol Biol 2004;37:139–43. [13] Rangasamy T, Guo J, Mitzner WA, Roman J, Singh A, Fryer AD, et al. Disruption of Nrf2 enhances susceptibility to severe airway inflammation and asthma in mice. J Exp Med 2005;202:47–59.
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Cytokine journal homepage: www.elsevier.com/locate/issn/10434666
Increased intestinal inflammatory response and gut barrier dysfunction in Nrf2-deficient mice after traumatic brain injury Wei Jin a, Handong Wang a,*, Yan Ji b, Qingang Hu b, Wei Yan a, Gang Chen a, Hongxia Yin a a
Department of Neurosurgery, Jinling Hospital, School of Medicine, Nanjing University, 305 East Zhongshan Road, Nanjing 210002, Jiangsu Province, PR China Department of Oral and Maxillofacial Surgery, Affiliated Stomatology Hospital, School of Medicine, Nanjing University, 30 Zhongyang Road, Nanjing 210008, Jiangsu Province, PR China b
a r t i c l e
i n f o
Article history: Received 9 April 2008 Received in revised form 1 July 2008 Accepted 14 July 2008
Keywords: Nuclear factor E2-related factor 2 Traumatic brain injury Intestine Inflammation
a b s t r a c t Aim: To explore the role of nuclear factor erythroid 2-related factor 2 (Nrf2) in traumatic brain injury (TBI)-induced intestinal inflammatory response and gut barrier dysfunction in the mice. Methods: Wild-type Nrf2 (+/+) and Nrf2 (/)-deficient mice were subjected to a moderately severe weight-drop impact-acceleration head injury. We measured nuclear factor kappa B (NF-jB) by electrophoretic mobility shift assay (EMSA); tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b) and interleukin-6 (IL-6) by enzyme-linked immunosorbent assay (ELISA); intercellular adhesion molecule-1 (ICAM-1) by immunohistochemistry; intestinal permeability by lactulose/mannitol (L/M) test; plasma endotoxin by chromogenic limulus amebocyte lysate test. Results: Intestinal levels of NF-jB, pro-inflammatory cytokines and ICAM-1 in Nrf2 (/)-deficient mice were significantly higher compared with Nrf2 (+/+) mice at 24 h after TBI. Furthermore, higher intestinal permeability and plasma level of endotoxin were observed in the Nrf2 (/) mice compared with Nrf2 (+/+) mice. Conclusion: Nrf2 plays an important protective role in limiting intestinal inflammatory response and gut barrier dysfunction after TBI. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Gastrointestinal dysfunction is a known complication of traumatic brain injury (TBI) [1]. The relation between TBI and intestinal mucosa injury has been studied [2]. Major trauma and shock may initiate a cascade of intestinal events such as overproduction of intestinal cytokine [3], increased intestinal permeability [4], translocation of intestinal bacteria and endotoxins [5]. This pathologic course may not only influence the intestinal mucosa itself, but also impair the remote tissue and organs, leading to systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS). Previous studies of our laboratory have demonstrated that traumatic brain injury could lead to a concomitant and persistent up-regulation of inflammation-related factors such as nuclear factor kappa B (NF-jB), pro-inflammatory cytokines and intercellular adhesion molecule-1 (ICAM-1) in the intestine, which could then lead to significant gut structural alterations and barrier dysfunction [6,7]. Additional work is necessary to explore the underlying mechanism of the inflammatory response in the intestine after TBI. It has been reported that traumatic brain injury could trigger the intestinal accumulation of highly toxic reactive oxygen species (ROS) which leads to a state of oxidative stress [8]. Oxidative stress * Corresponding author. Fax: +86 25 84817581. E-mail address: [email protected] (H. Wang). 1043-4666/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2008.07.005
can not only directly cause tissue damage, but also modulate inflammatory response during tissue injury. Intracellular antioxidant defense systems play a crucial role in protection against oxidative damage by limiting ROS levels and thereby inhibit redoxmediate inflammatory response. Nuclear factor erythroid 2-related factor 2 (Nrf2) is reported to be the key transcription factor that, upon activation by oxidative stress, regulates the cellular antioxidant response which is critical for cellular protection. Activation of Nrf2 has been shown to protect cells from oxidant insults, thereby inhibiting redox-mediate inflammatory response [9–12]. Numerous studies have reported that Nrf2 plays a critical role in a variety of experimental models in counteracting inflammation. Nrf2 protects against allergen-mediated airway inflammation and cigarette smoke-induced emphysema [13,14]. In addition, Nrf2 plays essential roles in protection against inflammatory responses during skin wound healing [15]. Moreover, Nrf2 has been reported as a crucial regulator of the innate immune response and survival during experimental sepsis [16]. Furthermore, Nrf2 has also been shown to be a protector in dextran sulfate sodium (DSS)-mediated colitis and inflammation-mediated colonic tumorigenesis [17,18]. However, to our knowledge, the role of Nrf2 in the intestinal production of inflammatory agents which played crucial roles in the mechanisms of gut injury after TBI has not been studied to date. The purpose of this study was therefore to investigate the influence of Nrf2 genotype in modulating TBI-induced up-regulation of inflammatory agents in the intestine.
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2. Materials and methods 2.1. Animals All procedures in animals 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 (+/+) mice and Nrf2 (/)deficient mice were generated from inbred heterozygous Nrf2 (+/) mice [10]. 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, 50 -TGGACGGGACTATTGA AGGCTG-30 (sense for both genotypes), 50 -CGCCTTTTCAGTAGATGG AGG-30 (antisense for wild-type) and 50 -GCGGATTGACCGTAATGG GATAGG-30 (antisense for LacZ). Mice were housed at 23 ± 1 °C in humidity controlled animal quarters with 12-h light/dark cycle. All animals were allowed water and food ad libitum throughout the study. 2.2. Experiment protocol The mouse model of diffuse closed head injury was employed as described [19] with recent minor modification [20]. Age- and weight-matched adult male mice (6–8 weeks, 28–32 g) 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 min with 100% oxygen administration and chest compression to stimulate the respiration. This model is generally associated with 20% of mortality within the first 5 min post-injury 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. The sham mice were subjected to identical anesthetic without trauma. Mice were separated into four groups (n = 12 per group): group I, sham + wild-type Nrf2 (+/+); group II, TBI + wild-type Nrf2 (+/+); group III, sham + Nrf2 (/)-deficient; group IV, TBI + Nrf2 (/)deficient. Animals were decapitated at 24 h following sham or injury for sample collection. Six mice in each group were sacrificed for electrophoretic mobility shift assay (EMSA) and enzyme-linked immunosorbent assay (ELISA) and the others were for immunohistochemistry study. Blood samples were obtained from the dorsal vena cava and centrifuged with the plasma stored at 40 °C before the animals were killed. For EMSA and ELISA analysis, mice were exsanguinated by cardiac puncture, and a 3-cm segment of the jejunum 8 cm distal to Treitz ligament was rapidly removed and stored at liquid nitrogen immediately. For immunohistochemistry, mice were perfused via left ventricular puncture with cold saline (4 °C), followed by 4% neutral-buffered formalin. The 3-cm segment of the jejunum was taken, stored overnight in 4% neutral-buffered formalin, and then embedded in paraffin.
AGG C-30 ) was end-labeled with [c-32P]ATP (Free Biotech., Beijing, China) with T4-polynucleotide kinase. EMSA was performed according to previous study of our laboratory. Levels of NF-jB activity were quantified by computer-assisted densitometric analysis. 2.4. Detection of tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b) and interleukin-6 (IL-6) in intestinal tissue The intestinal levels of inflammatory cytokines were quantified using ELISA kits specific for mouse according to the manufacturers’ instructions (TNF-a from Diaclone Research, France; IL-1b, IL-6 from Biosource Europe SA, Belgium) and previous study of our laboratory [21]. The cytokine contents in the intestinal tissue were expressed as pg per milligram protein. 2.5. Detection of ICAM-1 expression in intestinal tissue The tissue sections (4 lm) were used for immunohistochemical assay, which was performed with an goat anti-mouse ICAM1(CD54) antibody (diluted 1:200, R&D Systems, Inc., MN, USA), according to previous studies of our laboratory [7,21]. Microscopy of the immunohistochemically stained tissue sections was performed by an experienced pathologist blinded to the experimental condition. Evaluation of sections was undertaken by assessing the intensity of staining (5 grades). ‘‘0” indicates no detectable positive cell; ‘‘1” indicates very low density of positive cells; ‘‘2” indicates a moderate density of positive cells; ‘‘3” indicates the higher, but not maximal density of positive cells and ‘‘4” indicates the highest density of positive cells. 2.6. Intestinal permeability Intestinal permeability was quantified using the lactulose/mannitol (L/M) test as described [22]. Six hours before the animals were sacrificed the mice with their bladders emptied were given the test solution of 200 ll by gastric tube feeding, containing 13.3 mg lactulose and 10.1 mg mannitol. All urine was collected for 6 h through the metabolic cage and stored at 40 °C for further analysis. Urinary lactulose and mannitol were measured using high-performance liquid chromatography (Waters Co., USA). Results were expressed as a ratio of percentage administered dose of lactulose excreted in the urine to the percentage administered dose of mannitol in the same urine collection (L/M ratio). 2.7. Plasma endotoxin determination The endotoxin content in plasma samples was assayed using limulus amebocyte lysate (LAL) kit (Sigma, USA) with a kinetic modification according to the test kit instruction and our laboratory methods [2]. Endotoxin concentrations were expressed as EU/ml. 2.8. Statistical analysis Software SPSS 13.0 was used for the statistical analysis. All data were expressed as means ± SEM, Student’s t-test was used to analysis 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.
2.3. Nuclear protein extract and EMSA
3. Results
Nuclear protein of intestinal tissue was extracted and quantified as described [6]. EMSA was performed using a commercial kit (Gel Shift Assay System; Promega, Madison, WI) as previously described. Consensus oligonucleotide probe (50 -AGT TGA GGG GAC TTT CCC
3.1. NF-jB binding activity in intestinal tissue NF-jB binding activity of intestinal nuclear protein was assessed by EMSA. As shown in Fig. 1, TBI-induced activation of
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3.2. Inflammatory cytokine concentrations in intestinal tissue Concentrations of TNF-a, IL-1b and IL-6 in the intestinal tissue were measured by ELISA. As shown in Fig. 2, TBI-induced up-regulation of inflammatory cytokines including TNF-a, IL-1b and IL-6 in the intestinal tissue of Nrf2 (+/+) and Nrf2 (/) mice (P < 0.05). Nrf2 (/) mice showed larger increase in levels of intestinal TNF-a, IL-1b and IL-6 compared with their wild-type Nrf2 (+/+) littermates after TBI (P < 0.05). 3.3. Expression of ICAM-1 in intestinal tissue As shown in Fig. 3, strong to moderate ICAM-1 immunoreactivity was observed in villous interstitium and lamina propria of Nrf2 (+/+) and Nrf2 (/) mice after TBI (P < 0.05). The immunoreactivity was almost undetectable in the sham-operated mice, and was found to be relatively weaker in Nrf2 (+/+) mice as compared with Nrf2 (/) mice at 24 h after TBI (P < 0.05). 3.4. Intestinal permeability in various groups As shown in Fig. 4, TBI caused a significant increase in the levels of intestinal permeability in Nrf2 (+/+) and Nrf2 (/) mice as compared with sham-operated mice (P < 0.05). Increased intestinal permeability was found in Nrf2 (/) mice as compared with their wild-type Nrf2 (+/+) counterparts at 24 h after TBI (P < 0.05). 3.5. Changes in plasma endotoxin level
Fig. 1. NF-jB activity in the intestine tissue of sham and injured Nrf2 (+/+) and Nrf2 (/) mice. (A) Nuclear protein of intestine tissue of Nrf2 (+/+) and Nrf2 (/) mice were assayed for NF-jB DNA binding activity by EMSA at 24 h after TBI. (B) Quantification of NF-jB DNA binding activity was performed by densitometric analysis. The figure indicates that intestinal NF-jB activity was significantly increased and was greater in Nrf2 (/) mice than in Nrf2 (+/+) mice after TBI (n = 6 per group). *P < 0.05 vs sham control of the same genotype. #P < 0.05 vs injured wild-type mice.
NF-jB in the intestinal tissue of Nrf2 (+/+) and Nrf2 (/) mice (P < 0.05). Nrf2 (/) mice showed an increased susceptibility to TBI-induced intestinal activation of NF-jB compared with their wild-type Nrf2 (+/+) counterparts (P < 0.05).
As shown in Fig. 5, at 24 h after TBI, plasma endotoxin levels in Nrf2 (+/+) and Nrf2 (/) mice were significantly higher than in sham-operated mice without TBI (P < 0.05). Plasma endotoxin level in Nrf2 (/) mice was significantly higher than in Nrf2 (+/+) mice at 24 h after TBI (P < 0.05). 4. Discussion This study revealed that disruption of Nrf2 in mice caused higher sensitivity to intestinal inflammatory response after experimental TBI. We found in this study that Nrf2 (/) mice have more inflammatory cytokine TNF-a, IL-1b and IL-6 production, ICAM-1 expression and their mediators NF-jB activation in the intestine after TBI compared with their wild-type Nrf2 (+/+) counterparts. Furthermore, higher intestinal permeability and plasma level of endotoxin were observed in the Nrf2 (/) mice compared with their wild-type Nrf2 (+/+) counterparts. These findings reported here suggest for the first time that Nrf2 plays a critical role in lim-
Fig. 2. Changes of inflammatory cytokines in the intestine tissue of sham and injured Nrf2 (+/+) and Nrf2 (/) mice. Concentrations of TNF-a, IL-1b and IL-6 were determined by ELISA in the intestinal tissue of Nrf2 (+/+) and Nrf2 (/) mice at 24 h after TBI. The figure indicates that intestinal levels of TNF-a, IL-1b and IL-6 were significantly increased and were greater in Nrf2 (/) mice than in Nrf2 (+/+) mice after TBI (n = 6 per group). *P < 0.05 vs sham control of the same genotype. #P < 0.05 vs injured wild-type mice.
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Fig. 3. Expression of ICAM-1 in the intestine tissue of sham and injured Nrf2 (+/+) and Nrf2 (/) mice. Immunohistochemical staining for ICAM-1 was performed in the intestinal tissue sections of Nrf2 (+/+) and Nrf2 (/) mice at 24 h after TBI. (A) Sham control Nrf2 (+/+) mice showing low ICAM-1 immunoreactivity. (B) Injured Nrf2 (+/+) mice showing increased ICAM-1 immunoreactivity. (C) Sham control Nrf2 (/) mice also showing low ICAM-1 immunoreactivity. (D) Injured Nrf2 (+/+) mice showing the strongest ICAM-1 immunoreactivity. (E) Intestinal expression of ICAM-1 was significantly increased and was greater in Nrf2 (/) mice than in Nrf2 (+/+) mice after TBI (n = 6 per group). *P < 0.05 vs sham control of the same genotype. #P < 0.05 vs injured wild-type mice.
iting the TBI-induced intestinal inflammatory response and gut barrier dysfunction in the mice TBI model. Inflammatory response in the intestine has been implicated in the pathogenesis of TBI-induced gastrointestinal dysfunction. It is generally accepted that the intestine may serve as an important organ in the development of severe complications under critical illness [23]. Indeed, the intestine has been proposed to be the ‘‘motor” of MODS in trauma [24], mainly through the inflammatory response mediated by NF-jB, pro-inflammatory cytokines and ICAM-1 [6,7]. NF-jB is one of the most important pro-inflammatory modulators, which can be activated by lesion-induced oxidative stress, bacterial endotoxin or cytokines and subsequently
transactivate the expression of many cytokines and adhesion molecules [25]. Pro-inflammatory cytokines including interleukins (ILs) and TNF-a released early after an inflammatory stimulus, can initiate the infiltration of inflammatory cells into the intestine by activating ICAM-1 and other adhesion molecules [26]. NF-jB activation enhances the transcription of pro-inflammatory cytokines, and the cytokines are known to in turn activate NF-jB [27]. The positive feedback is believed to serve to amplify inflammatory signals, inducing more tissue injury. The prevailing theory has been that the dysregulation of brain-gut axis and hypothalamic-pituitary-adrenal axis resulting from the TBI may initiate the inflammatory process in the intestine, but the potential mech-
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Fig. 4. Intestinal permeability in sham and injured Nrf2 (+/+) and Nrf2 (/) mice. Intestinal permeability of Nrf2 (+/+) and Nrf2 (/) mice were quantified using lactulose/mannitol (L/M) test at 24 h after TBI. The figure indicates that the level of intestinal permeability was significantly increased and was higher in Nrf2 (/) mice than in Nrf2 (+/+) mice after TBI (n = 6 per group). *P < 0.05 vs sham control of the same genotype. #P < 0.05 vs injured wild-type mice.
Fig. 5. Plasma endotoxin levels in sham and injured Nrf2 (+/+) and Nrf2 (/) mice. Plasma endotoxin levels of Nrf2 (+/+) and Nrf2 (/) mice were determined using limulus amebocyte lysate assay at 24 h after TBI. The figure indicates that plasma endotoxin level was significantly increased and was higher in Nrf2 (/) mice than in Nrf2 (+/+) mice after TBI (n = 6 per group). *P < 0.05 vs sham control of the same genotype. #P < 0.05 vs injured wild-type mice.
anism underlying this action is not clear [28,29]. In the present study, we found that at 24 h after TBI, up-regulation of inflammation-related factors including NF-jB, TNF-a, IL-1b, IL-6 and ICAM-1 was evident in the intestine and was greater in Nrf2 (/) mice than in Nrf2 (+/+) mice. These results support previous researches of our laboratory that TBI could provoke a significant intestinal inflammatory response and also suggest that Nrf2 deficiency could enhance the intestinal inflammatory response after TBI in the mice TBI model. The elevation of inflammation also contributes to the gut barrier dysfunction following TBI. Disruption of gut barrier occurs with increased intestinal permeability and high plasma level of endotoxin. It has been demonstrated that pro-inflammatory cyto-
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kines such as TNF-a, IL-1b and IL-6, which can be regulated by NF-jB, have cytotoxic effect that induced damages of microvilli, resulting in destruction of intercellular tight junctions and increased intestinal permeability [30]. Furthermore, increasing intestinal permeability permits bacterial and endotoxin translocation, which triggers systemic immunoinflammatory response to release pro-inflammatory cytokines and mediators, leading to aggravated SIRS and MODS [5,3]. The positive feedback is also believed to serve to amplify inflammatory tissue injury in the intestine following TBI. Previous study of our laboratory has shown that traumatic brain injury could lead to a significant increase in the intestinal permeability and plasma level of endotoxin, which implied that gut barrier function was disrupted [2]. Interestingly, we also observed in the present study that intestinal permeability and plasma level of endotoxin were increased in the mice of both genotypes after TBI. In addition, higher intestinal permeability and plasma level of endotoxin were found in Nrf2 (/) mice as compared with their Nrf2 (+/+) counterparts after TBI, which could be explained by evidence indicating that Nrf2 plays a protective role in TBI-induced gut barrier dysfunction in mice. Although numerous in vivo studies have reported in a variety of experimental models that Nrf2 plays a critical role in counteracting the inflammation [13–18], the findings which we have confirmed and extended in the model of TBI-induced gut injury in the present study, it is still not clear how Nrf2 regulates the expression of proinflammatory mediators and cytokines, several lines of evidence indicate that Nrf2-dependent antioxidant response provides protection against acute inflammation through modulation the NFjB signaling pathway. Previous studies have demonstrated that oxidative stress from reactive oxygen species (ROS) is accompanied by the up-regulation of inflammatory response at the site of inflammation [11]. ROS, such as superoxide, hydroperoxyl radicals, hydroxyl radicals and hydrogen peroxide, can not only directly cause tissue damage, DNA damage and lipid peroxidation, but also serve as important signaling molecules in the activation of redoxsensitive transcription factors NF-jB and regulation of gene expression for pro-inflammatory cytokines and adhesion molecules involved in the inflammatory response [9,31]. On the other hand, intracellular antioxidant defense systems play a crucial role in protection against oxidative damage by limiting ROS levels and thereby affect redox signaling pathways involved in the inflammation. Nrf2 has been reported to be a key antioxidant transcription factor which plays a central role in protecting cells and tissue from ROS and electrophiles. Under basal conditions, Nrf2 is sequestered in the cytoplasm by the cytosolic regulatory protein Keap1. In conditions of oxidative stress, Nrf2 translocates from the cytoplasm to the nucleus, and sequentially binds to a promoter sequence called the antioxidant response element (ARE), resulting in transactivating the expression of a group of antioxidant and detoxifying enzymes. The cytoprotective function of Nrf2 is mainly mediated by these Nrf2-regulated antioxidant and detoxifying enzymes [9– 12,32]. It is therefore implied that Nrf2 may play an important role in anti-inflammation by a mechanism of the augmentation of cellular antioxidative or detoxification systems via activation of Nrf2regulated enzymes resulting in decreased pro-inflammatory cytokines production and adhesion molecules expression via inactivation of NF-jB. We then in this study postulated that increased susceptibility of Nrf2 (/) mice to TBI-induced intestinal inflammatory damage may at least in part be due to the dysregulation of redox homeostasis and NF-jB signaling pathway. Additional work is necessary to elucidate the whole mechanisms involved in these complicated networks. In summary, this present study has shown that Nrf2 plays a protective role in TBI-induced intestinal inflammatory response and gut barrier dysfunction in mice. We found that Nrf2 (/) mice are more susceptible to TBI-induced intestinal NF-jB activation,
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inflammatory cytokine TNF-a, IL-1b and IL-6 production and ICAM-1 expression, which then contributed to augmented gut barrier dysfunction occurring with increased levels of intestinal permeability and plasma endotoxin. To the best of our knowledge, this is the first study that elucidates the role of Nrf2 in TBI-induced intestinal inflammatory damage. These findings raise the possibility that Nrf2 will be a new therapeutic target for the treatment of gut injury after TBI. Acknowledgments This work was supported by grants from Jinling Hospital of China. The authors thank Dr. Bo Wu and Dr. Geng-bao Feng for technical assistance. References [1] Pilitsis JG, Rengachary SS. Complications of head injury. Neurol Res 2001;23:227–36. [2] Hang CH, Shi JX, Li JS, Wu W, Yin HX. Alterations of intestinal mucosa structure and barrier function following traumatic brain injury in rats. World J Gastroenterol 2003;9:2776–81. [3] Grotz MR, Deitch EA, Ding J, Xu D, Huang Q, Regel G. Intestinal cytokine response after gut ischemia: role of gut barrier failure. Ann Surg 1999;229:478–86. [4] Faries PL, Simon RJ, Martella AT, Lee MJ, Machiedo GW. Intestinal permeability correlates with severity of injury in trauma patients. J Trauma 1998;44:1031–5. [5] Swank GM, Deitch EA. Role of the gut in multiple organ failure: bacterial translocation and permeability changes. World J Surg 1996;20:411–7. [6] Hang CH, Shi JX, Li JS, Li WQ, Wu W. Expressions of intestinal NF-kappaB, TNFalpha, and IL-6 following traumatic brain injury in rats. J Surg Res 2005;123:188–93. [7] Hang CH, Shi JX, Li JS, Li WQ, Yin HX. Up-regulation of intestinal nuclear factor kappa B and intercellular adhesion molecule-1 following traumatic brain injury in rats. World J Gastroenterol 2005;11:1149–54. [8] Shohami E, Gati I, Beit-Yannai E, Trembovler V, Kohen R. Closed head injury in the rat induces whole body oxidative stress: overall reducing antioxidant profile. J Neurotrauma 1999;16:365–76. [9] Chen XL, Kunsch C. Induction of cytoprotective genes through Nrf2/antioxidant response element pathway: a new therapeutic approach for the treatment of inflammatory diseases. Curr Pharm Des 2004;10:879–91. [10] Itoh K, Chiba T, Takahashi S, Ishii T, Igarashi K, Katoh Y, et al. An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochem Biophys Res Commun 1997;236:313–22. [11] Guo RF, Ward PA. Role of oxidants in lung injury during sepsis. Antioxid Redox Signal 2007;9:1991–2002. [12] Lee JM, Johnson JA. An important role of Nrf2-ARE pathway in the cellular defense mechanism. J Biochem Mol Biol 2004;37:139–43. [13] Rangasamy T, Guo J, Mitzner WA, Roman J, Singh A, Fryer AD, et al. Disruption of Nrf2 enhances susceptibility to severe airway inflammation and asthma in mice. J Exp Med 2005;202:47–59.
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