| BackgroundTraumatic brain injury (TBI) specifically refers to the brain tissue damage caused by trauma, with high incidence, high morbidity and mortality characteristics, and has become the leading cause of human health. Globally, the incidence of TBI is generally reported as approximately 200/100.000 individuals with a mortality rate of about 20/100,000. Patients surviving severe TBI present different degree neurological and psychological disabilities, thus representing a significant social and economic burden. At present, however, there is no proven therapy for TBI other than supportive care. Therefore, there is a compelling need to understand the detailed cellular and biochemical mechanisms that contribute to dysfunction and death following TBI, so that we may devise novel treatment approaches to improve patient outcome.The pathophysiology of TBI is understood to consist of two main phases:a primary phase of injury, and a secondary injury phase. Primary damage occurs at the time of insult and is not reversible. Compared to the primary injury, secondary injures represents consecutive pathological processes initiated at the moment of injury with delayed clinical presentation. These events amplify the effect of the primary injury and may ultimately be the deciding factors in the patient's recovery. The pathological mechanisms of secondary brain injury are complex, including excitatory amino acid toxicity, oxygen free radicals, inflammation and calcium overload, etc. At present, the mechanisms of neuronal damage caused by TBI are considered to be similar to that of cerebral ischemia and reperfusion injury, which are the result of mutual interaction of various pathogenic mechanisms. Among these, reactive oxygen species (ROS) are considered as the core of pathological link. Trauma-induced excessive production of ROS can cause peroxidation of lipids, protein oxidation and cleavage of DNA. Additionally, ROS also can act as a proinflammatory stimulus and is responsible for initiation of the cascade of reactions that lead to immune inflammation. Furthermore, ROS also can cause apoptosis by inhibiting mitochondrial function and altering signal transduction. These alterations are associated with each other, which ultimately contribute neuronal death after TBI. Thus, the relationship between oxidative stress and TBI has generated considerable interest in the development of antioxidant therapies for neuroprotection. However, despite promising results in the treatment of TBI in animal models, evidence on successful antioxidant therapy in TBI patients is limited. In addition, there are a number of drawbacks to the use of exogenous antioxidants for treatment, as many antioxidants do not efficiently cross the BBB, are rather unstable in the body, have short therapeutic time windows, and should be given in very narrow range of therapeutic dosages owing to their toxicity at high doses. Hence, alternative therapeutic approach to inhibit the detrimental effects of ROS, for instance through the induction of endogenous enzymatic antioxidants, are desirable.Present studies suggest that the endogenous antioxidant systems are activated during the production of excessive ROS, which inhibiting the damage caused by oxidative stress so as to maintain the state of equilibrium in body. Nrf2-ARE pathway plays an important role in the endogenous antioxidant systems. In the human body, many kinds of antioxidases, the disintoxicating enzymes and calcium ion stable state protein have a common promoter subsequence-antioxidant response element (ARE). Among the transcription factors which regular the interaction of ARE, the nuclear factor erythroid 2-related factor 2 (Nrf2) was considered as the crucial one, and may induce by the extraneous factor. Nrf2 belongs to the family of the cap "n" collar basic region-leucine zipper (CNC bZip) transcription factors. Under physiological conditions, Nrf2 is localized within the cytoplasm by binding to its negative regulator Kelch-like ECH associating protein 1 (Keap1), which promotes Nrf2 ubiquitination by the Cu13-Rbkl complex and subsequent degradation by the proteasome. However, upon exposure to ROS, Nrf2 is liberated from the Keapl-Nrf2 complex and translocates from the cytoplasm to the nucleus, and sequentially binds to the ARE, a regulatory enhancer region within gene promoters, thus inducing the production of many phase?detoxifying and antioxidant enzyme genes such as heme oxygenase 1 (HO-1), NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione peroxidase (GSH-Px), which protect cells against oxidative stress as well as a wide range of other toxins.Recently, many lines of evidence have demonstrated that Nrf2 plays a critical role in the induction of endogenous antioxidant enzymes against oxidative damage in a variety of experimental models. Nrf2 protects cardiac fibroblasts and cardiomyocytes against oxidative stress by increasing detoxification pathways and antioxidant potentials. In addition, Nrf2 protects the lungs from oxidative injury caused by bleomycin and environmental oxidants including hyperoxia. diesel exhaust particles, and cigarette smoke. Moreover, it has been reported that Nrf2 plays a critical role for cytoprotection and mitigates oxidative stress by activating antioxidant genes and preventing the pathogenesis of liver and gastrointestinal diseases such as liver fibrosis, gallstone development, drug-induced hepatotoxicity, acute gastric mucosal lesions, and inflammatory bowel diseases. Furthermore, the protective effects of the Nrf2-ARE axis against oxidative insults in the central nervous system (CNS) have also been reported. It is found the Nrf2/ARE pathway has been activated in mouse brain after focal cerebral ischemia, which has increased glutathione content in brain tissue to alleviate ischemic brain injury. Additionally, activation of this pathway has been shown to protect the brain against oxidative stress, inflammation and apoptosis produced by intracerebral hemorrhage. However, the precise role of Nrf2 in limiting oxidative damage in TBI remained obscure.Sulforaphane (SFN) is an isothioeyanat ederived from natural product which is present in cruciferous vegetables such as broccoli and has been used as antioxidants and anti-tumor agents, which drawn many researchers attention. In vitro study has demonstrated that SFN can disrupt the Nrf2/Keapl interaction leading to Nrf2 stabilization and nuclear localization and to expression of ARE-containing phase II genes, which plays a major role in the detoxification of ROS produced by xenobiotics. However, the direct evidence of whether the antioxidative effect of SFN on TBI is medicated by Nrf2-ARE pathway and whether SFN is capable of inducing nuclear translocation and activation of Nrf2 under the TBI experimental condition is absence.The present study was designed to evaluate the antioxidative role of Nrf2 in experimental TBI. Controlled cortical impact (CCI) injury was performed in Sprague-Dawley rats and Nrf2-knockout or control mice. Sulforaphane (SFN), a potent Nrf2 activator found in cruciferous vegetables, was used to activate Nrf2. We determined:(1) whether Nrf2-ARE pathway plays an important role against TBI-induced oxidative damage and (2) whether the antioxidative role of SFN is mediated by activating the Nrf2 signaling pathway. Our work may pave the way to assess the therapeutic role of Nrf2 activators in patients with TBI. Part I The expression and significance of Nrf2-ARE signaling pathway in traumatic brain injuryObjectiveTo observe the dynamic expression and distribution of the Nrf2-ARE signal pathway after traumatic brain injury, and explore its relationship between Nrf2-ARE pathway and traumatic brain injury, so as to provide a new therapeutic target for traumatic brain injury treatment.Method1. Experimental animal and dividing into groups:choose 112 healthy grow up Sprague-Dawley male rats, the weight is 250-300 g, raise under the diet condition of freedom (offered by animal'scentre of academy of medical sciences of Zhejiang Province). Divide animal at random into sham group, trauma group. Controlled cortical impact (CCI) injury was performed in trauma group. The trauma group is divided into 6 groups according to different time:1 hour, 6 hour, 12 hour, 24 hour, 48 hour, and 72 hour. The animal of each group is 16.2. Western blot analysis of Nrf2 and Nrf2-dependent antioxidative and detoxifying enzymes:HO-1 and NQO1 after TBI:according to different time after TBI, rats (n=8 per group) were decapitated, the brains quickly removed, and the ipsilateral peri-core parietal cortex (injured) and the equivalent area in the sham-operates rapidly dissected and frozen in dry ice. The protein level of Nrf2, HO-1, and NQO1 were analyzed by western blot.3. RT-PCR analysis of Nrf2 and Nrf2-dependent antioxidative and detoxifying enzymes:HO-1 and NQO1 after TBI:according to different time after TBI, rats (n=8 per group) were decapitated, the brains quickly removed, and the ipsilateral peri-core parietal cortex (injured) and the equivalent area in the sham-operates rapidly dissected and frozen in dry ice. The level of Nrf2, HO-1, and NQO1 mRNA were analyzed by RT-PCR.4. Immunohistochemical Staining:For immunohistochemical examination, the tissue sections were incubated with an anti-Nrf2 polyclonal antibody. The expression and distribution of the Nrf2 after TBI was examined by immunofluorescence.Result1. The Western blot shows that the protein levels of nuclear Nrf2, HO-1, and NQO1 increased from 1 hour to 24 hour after TBI, and had begun to decrease again by 48 hour. There was a statistically significant difference between the sham group and each TBI group (P<0.01, respectively).2. RT-PCR shows that the mRNA levels of HO-1 and NQO1 increased from 1 hour to 24 hour after TBI, and had begun to decrease again by 48 hour. There was a statistically significant difference between the sham group and each TBI group (P<0.01, respectively). Meanwhile, there was no significant difference in Nrf2 mRNA levels after TBI (P>0.05).3. Immunofluorescence staining:After TBI, Nrf2 was induced in the ipsilateral cortex, which was the most vulnerable region to TBI. Little reactivity was observed in the sham group, whereas strong Nrf2 immunostaining was detected in these regions at 24 hour after TBI. Both nuclear and cytoplasmic stainings were observed in glial cells and neurons.Conclusion1. Nrf2-ARE pathway is activated in the brain in the early state after TBI. The regulation of Nrf2 expression after TBI was mediated by post-transcriptional mechanisms but not transcription.2. The distribution of Nrf2 immunostaining was detected in the ipsilateral cortex, which is vulnerable to TBI.3. Activation of Nrf2 after TBI resulted in upregulation of a battery of Nrf2-dependent antioxidative and detoxifying enzymes, which means the Nrf2/ARE signaling pathway may be an endogenous antioxidant mechanism in the cellular defense against oxidative stress after TBI.Part 2 Effect of Nrf2-ARE signaling pathway on the protection of traumatic brain injury in miceObjectiveTo investigate the protective role of Nrf2-ARE signaling pathway in modulating traumatic brain injury (TBI)-induced secondary brain injury through intervening on the expressions of Nrf2 and Nrf2-dependent antioxidative and detoxifying enzymes and the effects on oxidative stress, and to provide a novel target for prevention and treatment on TBI.Method1. TBI model and experimental groups:Controlled cortical impact (CCI) injury was performed in Nrf2-knockout or control mice. The experimental groups consisted of sham Nrf2+/+, sham Nrf2-/-, injured Nrf2T+/+, and injured Nrf2-/-2. Evaluation of neurological deficits:To examine the neurological deficits of animals after TBI, the neurological functions of each experimented group (n=8) were evaluated at 7 days after injury by an experimenter blinded to the treatment status of the groups, with the Modified Neurological Severity Score (mNSS). which included motor, sensory, reflex, and balance tests. One point was given for failure to perform a task, with a maximum of 18 points.3. Measurement of brain edema:Brain edema at 24 hour after TBI was determined using the wet:dry weight method.4. Calculation of cortical lesion volume:Each brain was sectioned coronally (20?m-thick slices at an interval of 500?m from 1 mm to-3.25 mm with respect to the bregma, covering the cortical lesion according to the rat brain atlas) and the sections were stained with 1% toluidine blue (Sigma-Aldrich) to more easily visualize the injury cavity. Images of all sections were photographed with a stereomicroscope using brightfield illumination. The periphery of the contralateral and ipsilateral hemispheres was traced on each image by an evaluator blinded to the injury and treatment status of the animals and the area of each hemisphere was calculated using the Image J software.5. Fluoro Jade B histochemistry:To assess the extent of neural injury, we used Fluoro-Jade B. a polyanionic fluorescein derivative that sensitively and specifically binds to degenerating and dying neurons.6. Biochemical assays of oxidative stress:Animals (n=8 per group) were sacrificed by decapitation at 24h after TBI. The brains were quickly removed, and the ipsilateral peri-core parietal cortex (injured) and the equivalent area in the sham-operates was dissected out in ice immediately and stored at-80?for posterior oxidative stress analyses. The levels of oxidation-mediated changes in proteins (protein carbonyls). lipids (4-HNE) and DNA (8-OHdG) in the injured cortex 24 h after TBI are analyzed by ELISA.7. Western blot and RT-PCR analysis for the nuclear Nrf2. HO-1, and NQO-1 protein and mRNA levels following TBI:Animals (n=8 per group) were sacrificed by decapitation at 24h after TBI. The brains were quickly removed, and the ipsilateral peri-core parietal cortex (injured) and the equivalent area in the sham-operates was dissected out in ice immediately and stored at -80?for posterior analyses. The protein and mRNA levels of nuclear Nrf2, HO-1, and NQO-1 in the injured cortex 24 h after TBI are analyzed by Western blot and RT-PCR respectively.Result1. The mNSS scores in both sham-operated Nrf2+/+and Nrf2-/-mice were similar. At 24 h after TBI, increased mNSS score, which reflected an impairment of sensory and motor function, was found in both injured Nrf2+/+and Nrf2-/-mice compared with their respective sham-operated mice. However, the neurological deficit was significantly greater in Nrf2-/-mice than in Nrf2+/+mice.2. Similar brain water content was detected in both sham-operated Nrf2+/+and Nrf2-/-mice. At 24h after TBI, brain water content significantly increased in both injured Nrf2-/- and Nrf2-/-mice compared with their respective sham-operated mice. However, the pathological change was more severe in Nrf2-/- mice than in Nrf2+/+mice.3. TBI induced a significant loss of cortical tissue in the ipsilateral parietal cortex in both injured Nrf2+/+and Nrf2 -/-mice, as reflected by gross reduction in toluidine blue staining intensity. However, the contusion volume was bigger in Nrf2-/-mice than in Nrf2+/+mice.4. In sham-operated Nrf2+/+and Nrf2-/-mice, there were no FJB-positive cells detected. At 24 h after TBI, FJB-positive cells with neuronal morphology were evident in the cortical contusion margin in the ipsilateral hemisphere in both injured Nrf2+/+and Nrf2-/-mice. However, the pathological change was more severe in Nrf2-/- mice than in Nrf2+/+mice.5. The levels of oxidation-mediated changes in proteins (protein carbonyls), lipids (4-HNE) and DNA (8-OHdG) were similar in the brain samples of both sham-operated Nrf2+/+and Nrf2-/-mice. At 24 h after TBI, increased cortical protein expression levels of indicators of oxidative stress were detected in both injured Nrf2+/+and Nrf2-/-mice compared with their respective sham-operated mice. Higher protein expression levels of these indicators of oxidative stress were found in Nrf2-/- mice than in Nrf2+/+mice.6. The mRNA and protein expression levels of the antioxidant and detoxifying enzymes HO-1 and NQO1 detected in the brain samples of both sham and injured Nrf2-/-mice were significantly lower than those measured in the corresponding Nrf2+/+mice. TBI induced increased cortical mRNA and protein expression levels of HO-1 and NQO1 in Nrf2+/+mice but not Nrf2-/-mice.Conclusion1. Nrf2-ARE signaling pathway plays an important role in protecting TBI-induced secondary brain injury.2. Its neuroprotective effect may be mediated through increased expression of antioxidant/detoxification enzyme which inhibits TBI-induced oxidative stress.3. Nrf2-ARE pathway may become a new target for drug treatment of TBI.Part 3 The study of neuroprotective effects of sulforaphane in traumatic brain injury and its potential mechanisms ObjectiveTo explore the effect of sulforaphane on the regulation of Nrf2-ARE pathway and its impact on oxidative stress following traumatic brain injury. And to further discuss its specific molecular mechanisms so as to provide a new strategy for the clinical treatment of TBI and also to provide a theoretical and experimental basis for SFN clinical application. Method1. TBI model and experimental groups:Controlled cortical impact (CCI) injury was performed in Sprague-Dawley rats and Nrf2-knockout or control mice. SD rats were randomly assigned to three experimental groups:(a) sham group:animals were subjected to sham surgery; (b) SFN-treated TBI group:animals were subjected to TBI, and SFN (LKT Laboratories, St. Paul, MN) at 5 mg/kg in 1% DMSO was administered ip 15 min later; (c) TBI-vehicle group:animals were subjected to TBI and received equal volumes of 1% DMSO with the same schedule. Mice were assigned to four groups:(a) injured Nrf2+/+group:Nrf2+/+mice were subjected to TBI; (b) injured Nrf2-/- group:Nrf2-/- mice were subjected to TBI; (c) SFN-treated Nrf2+/+TBI group:animals were subjected to TBI, and SFN at 5 mg/kg in 1% DMSO was administered ip 15 min later; (d) SFN-treated Nrf2-/- TBI group:animals were subjected to TBI, and SFN at 5 mg/kg in 1% DMSO was administered ip 15 min later.2. Evaluation of neurological deficits:To examine the effects of the SFN on the neurological deficits of animals after TBI, the neurological functions of each experimented group (n=8) were evaluated at 7 days after injury by an experimenter blinded to the treatment status of the groups, with the Modified Neurological Severity Score (mNSS), which included motor, sensory, reflex, and balance tests. One point was given for failure to perform a task, with a maximum of 18 points.3. Measurement of brain edema: Brain edema at 24 hour after TBI was determined using the wet:dry weight method in all experimental groups.4. Calculation of cortical lesion volume:Each brain was sectioned coronally (20?m-thick slices at an interval of 500?m) and the sections were stained with 1% toluidine blue (Sigma-Aldrich) to more easily visualize the injury cavity. Images of all sections were photographed with a stereomicroscope using brightfield illumination. The periphery of the contralateral and ipsilateral hemispheres was traced on each image by an evaluator blinded to the injury and treatment status of the animals and the area of each hemisphere was calculated using the Image J software.5. Fluoro Jade B histochemistry:To assess the extent of neural injury, we used Fluoro-Jade B, a polyanionic fluorescein derivative that sensitively and specifically binds to degenerating and dying neurons.6. Biochemical assays of oxidative stress:Three groups of rats and four groups of mice (n=8 per group) were sacrificed by decapitation at 24h after TBI. The brains were quickly removed, and the ipsilateral peri-core parietal cortex (injured) and the equivalent area in the sham-operates was dissected out in ice immediately and stored at -80?for posterior oxidative stress analyses. The levels of oxidation-mediated changes in proteins (protein carbonyls), lipids (4-HNE) and DNA (8-OHdG) in the injured cortex 24 h after TBI are analyzed by ELISA.7. Nrf2 protein expression and localization:To examine Nrf2 expression, the protein level of nuclear Nrf2 in the injured cortex 24 h in rat after TBI was analyzed by Western blot. To examine Nrf2 protein localization, we performed double-labeling immunofluorescence on 20?m coronal brain sections. The stained sections were examined with a Leica confocal fluorescence microscope8. Western blot and RT-PCR analysis for HO-1 and NQO-1 protein and mRNA levels following TBI:Rats (n=8 per group) were sacrificed by decapitation at 24h after TBI. The brains were quickly removed, and the ipsilateral peri-core parietal cortex (injured) and the equivalent area in the sham-operates was dissected out in ice immediately and stored at-80?for posterior analyses. The protein and mRNA levels of HO-1 and NQO-1 in the injured cortex 24 h after TBI are analyzed by Western blot and RT-PCR respectively. Result1. Using a rat TBI model, we found that TBI induced significant histologic and neurologic deficits, as evidenced by brain edema, contusion volume and neurological dysfunction. As compared with vehicle-treatment group, the SFN-treatment resulted in a significantly greater reduction of brain edema, contusion volume and neurologic deficits after TBI (P<0.01).2. In sham operation group, there were no FJB-positive cells detected. At 24h after TBI, FJB-positive cells with neuronal morphology were evident in the cortical contusion margin in the ipsilateral hemisphere, which indicated that CCI resulted in significant cortical neuronal damage in the ipsilateral cortex. SFN treatment significantly reduced the number of FJB-positive cells as compared with vehicle treatment (P<0.01).3. Basal protein level of protein carbonyls,4-HNE,8-OHdG were low in the cortex of sham-injured animals. After injury, protein carbonyls, 4-HNE, 8-OHdG protein levels in the ipsilateral cortex of vehicle-treated TBI rats were significantly increased compared with sham rats. Treatment with SFN significantly reduced the injury-induced increase in cortical tissue levels of all three oxidative stress markers.4. Western blotting showed that the basal protein level of Nrf2 in nuclear was low in the cortex of sham-injured animals. The protein level of Nrf2 in nuclear was upregulated at 24 h after TBI. Furthermore, after treatment with SFN, the protein expression of Nrf2 in the nuclei was significantly increased compared with vehicle control. Immunofluorescence staining showed that little reactivity of Nrf0 was observed in the sham group. After TBI, the number of cells stained by Nrf2 increased in the ischemic cortex, and Nrf2 expressed both at cytoplasm and nucleus in glial cells and neurons. Nrf2 immunostaining was detected in nuclear and cytoplasm. Furthermore, treatment with SFN significantly increased the number of cells labeled with Nrf2 when compared with vehicle control group, and a lot of cells labeled with Nrf2 in the nucleus increased.5. The expression of Nrf2 dependent antioxidative and detoxifying enzymes after SFN treatment under TBI was also tested. In sham group, the levels of mRNA and protein expressions of HO-1 and NQO1 were low. Additionally, the we found that both the mRNA and protein expressions of Nrf2-regulated genes encoding important antioxidative enzymes, including HO-1 and NQO1, were upregulated in the ipsilateral cortex in vehicle control and significantly induced by treatment with SFN.6. To further verify the special role of Nrf2 in oxidative stress after TBI, we analyzed markers of oxidative stress, neurologic function, and brain edema of both Nrf2-knockout mice and wild-type mice subjected to TBI in the presence or absence of SFN. As expected, Nrf2-/- mice had more oxidative markers such as protein carbonyls.4-HNE, and 8-OHdG production in the injured area and more severe neurologic dysfunction and brain edema after TBI compared with their wild-type Nrf2+/+counterparts. Furthermore, pharamacological activation of Nrf2 with SFN significantly reduced oxidative damage, neurologic deficient and brain edema produced by TBI in Nrf2+/+mice but not in mice lacking the Nrf2 gene, confirming both the neuroprotective action and Nrf2 dependence of SFN in vivo.Conclusion1. SFN exerts neuroprotective effects to protect the brain from secondary damage caused by TBI.2. The protective effect of SFN may be through activation of Nrf2 and upregulation of a battery of Nrf2-dependent antioxidative and detoxifying enzymes, which consequently resulted in reduction of oxidative damage around the damaged area after TBI.3. Pharmacological activation Nrf2-ARE pathway by small molecule inducers such an SFN might be a practical preventative treatment for TBI patients.In conclusion, Nrf2-ARE pathway can be activated in the brain in the early state after TBI, which plays a pivotal role in cell defenses against oxidative stress in the TBI. Furthermore, pharmacological activation Nrf2-ARE pathway may be one of the strategic targets for TBI therapies. |
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