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Activation of Nrf2-ARE signal pathway in hippocampus of amygdala kindling rats
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Activation of Nrf2-ARE signal pathway in hippocampus of amygdala kindling rats
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Wei Wang a,b , Wei-ping Wang a,∗ , Guo-liang Zhang c , Yan-fen Wu b , Tao Xie a , Min-chen Kan a , Hai-bo Fang a , Hong-chao Wang a
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Key Laboratory of Neurology of Hebei Province, the Second Hospital of Hebei Medical University, Shijiazhuang, Hebei 050071, PR China Department of Neurology, Handan First Hospital, Handan, Hebei 056002, PR China Department of Neurobiology, Hebei Medical University, Shijiazhuang, Hebei 050017, PR China
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Oxidative stress was induced in hippocampus of amygdala kindling rats. Nrf2 and Nrf2-dependent gene products were significantly increased after seizure. Nrf2-dependent gene products can protect the cells against oxidative damage. To activate Nrf2-ARE signal pathway may be a new direction for treatment of epilepsy.
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Article history: Received 29 November 2012 Received in revised form 6 March 2013 Accepted 10 March 2013
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Oxidative stress resulting from excessive free-radical release is likely implicated in the initiation and progression of epilepsy. Therefore, antioxidant therapies have received considerable attention in epilepsy treatment. It is well known that the transcription factor NF-E2-related factor (Nrf2) binds to antioxidant response element (ARE) to induce antioxidant and phase II detoxification enzymes under conditions of oxidative stress, which reduces oxidative stress and accumulation of toxic metabolites. However, whether Nrf2-ARE pathway is activated after seizure has not been studied. In the present study, Wistar rats were rapidly kindled in the amygdala. Twenty-four hours after the last seizure, the hippocampus of control, sham and kindled rats were examined for oxidative stress parameters (malondialdehyde and glutathione) by spectrophotometry, the expression of Nrf2, heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase-1 (NQO1) were determined using immunohistochemistry, Western blot and real-time fluorescence quantitative polymerase chain reaction (PCR). The results showed that the kindled seizures induced oxidative stress, the expression of Nrf2, HO-1 and NQO1 at protein or gene levels significantly increased in hippocampus after seizure. According to these results, it could be postulated that Nrf2-ARE signal pathway was activated in the hippocampus after seizure. © 2013 Published by Elsevier Ireland Ltd.
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Keywords: Nrf2-ARE Heme oxygenase-1 NAD(P)H:quinone oxidoreductase-1 Epilepsy Oxidative stress
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1. Introduction
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Epilepsy, one of the most common neurological disorders, is a chronic disorder of abnormal electrical activity in the brain, characterized by recurrent unprovoked seizures [20]. The mechanisms underlying epileptogenic pathogenesis have been proved to be complex, including oxidative stress, glutamate excitotoxicity, calcium overload and so on [4]. Given that pathological processes involved in epileptogenesis are complex and interrelated, to find a target, which can interrupt multi-mechanisms underlying seizure, is desirable. Oxidative stress has been reported as an underlying mechanism in the initiation and progression of epilepsy, what’s
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∗ Corresponding author. Tel.: +86 031166002915; fax: +86 0311 66002915. E-mail address: ldh wwp [email protected] (W.-p. Wang).
more, excessive oxidative stress contributes to neuronal degeneration in the epileptic focus [21]. Increases in reactive oxygen species occur in response to sustained neuronal electrical activity and seizures [10]. Therefore, antioxidants have been suggested as therapeutic strategies for the treatment and modulation of epilepsy. The nuclear factor erythroid 2-related factor 2 (Nrf2) is essential for the induction of a battery of phase II detoxification genes through the antioxidant response element (ARE) that lies in their promoter region [27]. Genes driven by the ARE are up-regulated in response to various stressors of the cellular environment. Under numerous stimuli, Nrf2 translocates from the cytoplasm to the nucleus, and sequentially binds to ARE [13]. Nrf2-ARE signal pathway regulates the expression of a group of cytoprotective enzymes, such as heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase-1 (NQO1). These Nrf2-dependent gene products go on to protect the cells from oxidative or xenobiotic damage [28].
0304-3940/$ – see front matter © 2013 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.neulet.2013.03.038
Please cite this article in press as: W. Wang, et al., Activation of Nrf2-ARE signal pathway in hippocampus of amygdala kindling rats, Neurosci. Lett. (2013), http://dx.doi.org/10.1016/j.neulet.2013.03.038
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Previous studies have shown that up-regulation of HO-1 [1] or NQO1 [8] in neurons can protect them against oxidative and excitotoxic insults. HO-1 is a ubiquitous and redox-sensitive inducible stress protein that degrades heme to carbon monoxide, iron and biliverdin [13]. Reduced HO-1 protein expression is associated with more severe neurodegeneration after transient ischemia [11]. In contrast, induction of HO-1 could protect neurons from oxidative stress [6]. Another Nrf2-regulated gene product, NQO1, is considered to play a protective role in the pathogenesis of Parkinson’s disease [23]. Up-regulation of NQO1 by 4-hydroxybenzyl alcohol also reduces cerebral infarct size and improves neurological functions in rats [26]. However, to our knowledge, the Nrf2-ARE signal pathway and two Nrf2-regulated gene products (HO-1 and NQO1) have not been studied after seizure. Thus, the present study was aimed to examine oxidative stress parameters (malondialdehyde and glutathione) and determine the expression of Nrf2, HO-1 and NQO1 at protein or gene levels in hippocampus of amygdala kindling rats.
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2. Materials and methods
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2.1. Animals
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Adult male Wistar rats weighing 250–300 g (n = 50) were obtained from the Hebei Medial University. Rats were housed in a room with constant temperature (25 ± 1 ◦ C) and humidity (40–60%), and were kept on a 12 h light/dark cycle, with lights on at 8:00 AM and with free access to food and water. Animal experiments were performed according to the regulations of laboratory animal management promulgated by the Ministry of Science and Technology of the People’s Republic of China [1988] No.134, which coincides with internationally recognized NIH guidance. The rats were randomly assigned to three groups. Control group (n = 16) did not receive any treatment. Electrodes were implanted in the remaining 34 rats. Two rats could not be included in the experiments because of death during anesthesia or misplacement of the electrode. The remaining 32 rats were randomly distributed to the following groups: sham group (n = 16) did not receive any electrical current administration; kindling group (n = 16) were subjected to experimental kindling procedure as follow. Twenty-four hours after the last stimulation, all rats were sacrificed. Eight rats in each group were prepared for spectrophotometry, Western blot and real-time fluorescence quantitative PCR analysis, and the other eight were for immunohistochemical study.
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2.2. Electrode implantation
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For implantation, animals were anesthetized with chloral hydrate (300 mg/kg, i.p.), the sham and kindling groups were stereotactically implanted with twisted stainless steel wire bipolar stimulating electrodes in the left basolateral amygdale complex (Bregma: anterior: −2.8 mm; lateral: 4.8 mm; ventral: 8.8 mm from skull surface) [16]. Three screws positioned over the right frontal cortex, left and right occipital cortices served as recording, reference and ground electrodes. Dental cement bound the electrodes and screws to the skull. ABS connectors coupled the electrodes and screws to stimulating and recording equipments. After surgery, the rats were treated with antibiotics for 1 week to prevent infection.
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2.3. Kindling procedure
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The initial afterdischarge threshold (ADT) was determined using an electric stimulator (SEN-7203; Nihon Kohden) 1 week after electrode implantation. The stimulation parameters were performed by constant current stimulations (monophasic square-wave pulses,
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60 Hz for 1 s). The stimulus intensity started at 100 A, and thereafter increased in 40 A increments at 5 min intervals until an afterdischarge of at least 3 s duration was elicited (140–340 A). The next day animals of kindling group were kindled at 10 min intervals stimulation at 120% of their individual ADT. The kindled seizures were classified according to Racine’s five stages: stage 1, immobility and facial automatisms (eye closure, facial clonus); stage 2, head nodding accompanying mastication; stage 3, clonic forelimb convulsion; stage 4, rearing to a kangaroo-like posture or rearing with clonic forelimb clonus; and stage 5, generalized convulsion, including falling down. In the course of kindling, stimulation was delivered 40 times for the kindling group, with 20 times every day. In our study, all the kindling rats reached stage 5 at least three times.
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2.4. Experimental procedure
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The animals were sacrificed and the hippocampus was removed and cleaned with ice-cold (4 ◦ C) saline. Hippocampus tissue samples were homogenized with 10 times (w/v) ice-cold 0.1 M phosphate buffer (PB), pH 7.4. The homogenates were used to assess lipid peroxidation product and reduced glutathione. Malondialdehyde (MDA), an index of lipid peroxidation, was measured spectrophotometrically with the method described by Okhawa [14]; reduced glutathione (GSH) was estimated spectrophotometrically by the method described by Ellman [3]. MDA detection kit and GSH detection kit were obtained from Nanjing Jiancheng Bioengineering Institute (China). The rats used for immunohistochemical analysis were anesthetized with 4% chloral hydrate (300 mg/kg, i.p.) and perfused transcardially with saline, followed by 4% paraformaldehyde in 0.1 M PB, pH 7.4. Brains were carefully removed and postfixed in 4% paraformaldehyde in PB for 4 h at 4 ◦ C, and then embedded in paraffin. Brain sections (5 m thick) were blocked in 3% H2 O2 , 3% normal goat serum, and incubated with Nrf2 rabbit polyclonal antibody (1:100, Abcam Cambridge, USA), and HO-1 rabbit polyclonal antibody (1:100, Abcam Cambridge, USA) in 0.01 mol/L phosphate-buffered saline over night. The secondary antibodies (1:100, goat anti-rabbit IgG), secondary biotinylated conjugates and diaminobezidine were from the Vect ABC kit (Zhongshan Biology Technology Company, China). A computer-assisted image analysis system (Olympus, BX 61, Olympus DP71, U-TV0.5XC-3, Japan; Image-Pro Plus 6.0) was used for the average optical density (AOD) measurements of Nrf2 immunoreactive (Nrf2-ir) and HO-1 immunoreactive (HO-1-ir) intensity. After calibration of the system to eliminate saturation of gray levels, images were acquired for an accurate determination of optical density. Brain sections processed without primary antibody were used to determine the level of nonspecific staining for the entire experiment. After subtraction of the nonspecific staining, the AOD of Nrf2-ir and HO-1-ir cells in CA3 of hippocampus were measured. Measures were performed on both left and right sides and averaged for each section. To prevent differences arising from variations in the conditions of tissue processing and densitometric analysis, all sections were simultaneously processed. All microscopic and computer parameters were kept constant throughout the study. Ten sections of the hippocampus, with the interval of 5, were selected from each rat. The average of the AOD of Nrf2-ir or HO-1-ir cells in CA3 of hippocampus was presented for each rat in the results. Protein extraction for Nrf2 and HO-1 was performed as follows. The hippocampus was homogenized in ice-cold lysis buffer (10 mmol/L HEPES, pH 7.9, 10 mmol/L KCl, 0.1 mmol/L EDTA, 1 mmol/L DTT, 0.1 mmol/L EGTA) for 15 min. After adding NP-40, the homogenate was centrifuged 10,000 rpm at 4 ◦ C for 3 min and the supernatant was collected as cytoplasmic protein for HO-1.
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Fig. 1. The MDA (A) and GSH (B) levels express in hippocampus of rats. The ‘Con’, ‘sham’ and ‘EP’ in abscissa represent control, sham and kindling groups, respectively. Values are expressed as mean ± SD. *P < 0.01.
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The pellets were homogenized in ice-cold lysis buffer (20 mmol/L HEPES, pH 7.9, 400 mmol/L NaCl, 1 mmol/L EDTA, 0.1 mmol/L EGTA) for 15 min. Then the pellets were centrifuged 12,000 rpm at 4 ◦ C for 10 min, and the supernatant was collected. PMSF was then added to the supernatant with the final concentration 1 mmol/L as the nuclear protein for Nrf2. The protein concentration of the supernatant was determined using a BCA Protein Assay reagent kit (Novagen, Madison, WI, USA). Sample (50 g) was separated by SDS/PAGE, transferred on to PVDF membranes. Membranes were blocked with 5% skimmed milk 1 h at room temperature, and then were probed with polyclonal rabbit anti-Nrf2 antibody (1:500, Abcam Cambridge, USA) and polyclonal rabbit anti-HO-1 antibody (1:200, Abcam Cambridge, USA) overnight at 4 ◦ C. After washing three times with TPBS, IRDye® 800-conjugated goat antirabbit second antibody (1:3000, Rockland, Gilbertsville) was incubated with membranes for 1 h at room temperature. The relative density of bands was analyzed on an Odyssey infrared scanner (LI-COR Bioscience). The densitometric values were normalized with respect to the values of -actin immunoreactivity to correct for any loading and transfer differences between samples. Total RNA from the hippocampus tissue was obtained using Trizol reagent (Invitrogen, USA) following the manufacturer’s instruction. RNA concentration was determined by measuring the absorbance (A) of a diluted sample at the 260 nm wavelength in a UV spectrometer. A total of 2 g of total RNA was subjected to reverse transcription using random primer to obtain the firststrand cDNA template. Real-time fluorescence quantitative PCR was performed with 0.8 l cDNA (diluted 1:10), specific primers 2 l, and 2*GoTaq® Green Master Mix (Promega, USA) with a final volume of 20 l. PCR was performed as follows: an initial cycle at 95 ◦ C for 10 min, followed by 40 cycles at 95 ◦ C for 15 s, 58 ◦ C for 20 s and 72 ◦ C for 27 s. Then PCR products were analyzed by melting curve to confirm the specificity of amplification. Expression of Nrf2, HO-1 and NQO1 genes were analyzed with GAPDH used as an internal control. The sets of primers that we used were as follows: Nrf2(5 -GACCTAAAGCACAGCCAACACAT-3 and 5 CTCAATCGGCTTGAATGTTTGTC-3 ),HO-1(5 -TGTCCCAGGATTTGTCCGAG-3 and 5 -ACTGGGTTCTGCTTGTTTCGCT-3 ), NQO1(5 -GGGGACATGAACGTCATTCTCT-3 and 5 -AGTGGTGACTCCTCCCAGACAG-3 ), GAPDH(5 -TGAACGGGAAGCTCACTG3 and 5 -GCTTCACCACCTTCTTGATG-3 ). All data was analyzed by one-way ANOVA. Quantitative data were expressed as mean ± SD. All statistical analysis was performed with SPSS 13.0 software and P < 0.05 was considered statistically significant.
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As shown in Fig. 1, the level of GSH in kindling group was significantly lower compared to sham group (P < 0.01) and the level of MDA was much higher than that of sham group (P < 0.01). There was no difference between control and sham groups (P > 0.05). To localize Nrf2 and HO-1 expression, immunohistochemical study was performed. As shown in Fig. 2, in kindling group, the immunoreactive intensity of Nrf2 and HO-1 significantly increased in hippocampus, and Nrf2 expressed both at cytoplasm and nucleus. In addition, similar localization was found between Nrf2 and HO-1 in neurons and glial cells. The differences were shown between sham and kindling groups by AOD measurement in the expression of Nrf2 (P < 0.01) and HO1 (P < 0.01). There was no difference between control and sham groups (P > 0.05). As shown in Fig. 3, Western blot analysis was used to measure in protein level of Nrf2 in nuclear and HO-1 in cytoplasm extracted from hippocampus. Group differences were found between sham and kindling groups with the expression of Nrf2 (P < 0.01) and HO1 (P < 0.01). There was no difference between control and sham groups (P > 0.05). The expression of Nrf2, HO-1 and NQO1 mRNA in hippocampus was studied by real-time fluorescence quantitative PCR. As shown in Fig. 4, group differences were found between sham and kindling groups in the expression of Nrf2 mRNA (P < 0.01), HO-1 mRNA (P < 0.01) and NQO1 mRNA (P < 0.01). There was no difference between control and sham groups (P > 0.05).
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4. Discussion
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In this study we found that the seizure induced an increase in MDA levels and a decrease in GSH levels in hippocampus of rats. The protein level of Nrf2 in nuclear and HO-1 in cytoplasm were significantly increased, and the mRNA levels of Nrf2 and two Nrf2-regulated gene products (HO-1 and NQO1) were also up-regulated in hippocampus after seizure. It could be postulated that the seizure can induce oxidative stress in hippocampus of rats, which activates Nrf2-ARE signal pathway to result in up-regulation of antioxidative and detoxifying enzymes. Recent studies showed that oxidative stress plays an important role in epileptic pathogenesis [18,20]. MDA is an end product of free radical generation [9], and GSH is an endogenous antioxidant which plays a vital role as a free radical scavenger to protect cells against oxidative damage [15]. In the present study, the amygdala
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Fig. 2. Immunohistochemical study of Nrf2 (A) and HO-1 (B) expression in hippocampus. Bar graphs illustrate the AOD expression of Nrf2 (C) and HO-1 (D). Values are expressed as mean ± SD. *P < 0.01. Arrowhead () and arrow (→) respectively indicate neurons and glial cells. A1-3 and B1-3 Scale bars = 200 m. A4-6 and B4-6 Scale bars = 50 m. 266 267 268 269 270 271 272 273
kindled seizures caused a marked increase in MDA levels and a decrease in GSH levels in hippocampus, as compared to the control and sham groups. The imbalance between antioxidant and oxidant defense system may be at least partially responsible for seizures. In agreement with our findings, the seizure-induced oxidative stress has been reported in previous studies [25]. Thus, antioxidants have been suggested for therapeutic strategies in the treatment and modification of epilepsy.
Numerous studies have addressed a pivotal role of Nrf2 in protecting cells from oxidative stress [24]. One unique feature about the Nrf2-ARE signal pathway is that it coordinately up-regulates many protective detoxification and antioxidant genes, which can synergistically increase the efficiency of cellular defense system [7]. Many chronic neurodegenerative diseases (i.e. Parkinson’s disease and amyotrophic lateral sclerosis) are thought to involve oxidative stress as a component contributing to the progression of the
Please cite this article in press as: W. Wang, et al., Activation of Nrf2-ARE signal pathway in hippocampus of amygdala kindling rats, Neurosci. Lett. (2013), http://dx.doi.org/10.1016/j.neulet.2013.03.038
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Fig. 3. Western blot analysis for Nrf2 (A), and HO-1 (B) protein levels in hippocampus of rats. Bar graphs illustrate the protein expression of Nrf2 (C) and HO-1 (D), values are expressed as mean ± SD. *P < 0.01.
Fig. 4. The graphs show Nrf2 (A), HO-1 (B) and NQO1 (C) mRNA levels in control, sham and kindling groups. Values are expressed as mean ± SD. *P < 0.01.
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disease, and oxidative stress can be relieved by the Nrf2-ARE signal pathway as demonstrated previously [2,17]. But there is few data to show the relationship between the Nrf2-ARE signal pathway and epilepsy. In our study, we found that the expression of Nrf2, HO-1 and NQO1 were up-regulated at gene and protein level after seizure. Immunohistochemistry and real-time fluorescence quantitative PCR revealed that total Nrf2 was up-regulated, Western blot analysis illustrated that nuclear Nrf2 was induced after seizure, which provided the evidence that seizure could increase Nrf2 nuclear translocation. We also observed that there was a positive relationship between nuclear Nrf2 protein content and HO-1 or NQO1 mRNA levels. It could be postulated that Nrf2-ARE signal pathway is activated in hippocampus after seizure. Activation of Nrf2-ARE signal pathway after seizure results in up-regulation of antioxidative and detoxifying enzymes, which may reduce oxidative damage. It has been shown that mice lacking Nrf2 are more sensitive to kainic acid, and there is substantial evidence to support the notion that activation of Nrf2 is neuroprotective [5]. Therefore, activation of Nrf2 may represent a potential target for combating oxidative damage from seizure. Increasing evidence has demonstrated that the protective role of the Nrf2-ARE pathway in central nervous disease is mediated by HO-1 and NQO1 [24]. In our study, up-regulation of HO-1 and NQO1 mRNA levels was also observed. In addition, similar cellspecific localization was found between Nrf2 and HO-1. HO-1 activity reduced reactive oxygen species production by generating biliverdin, and further by biliverdin reductase to bilirubin, a potent antioxidant [13]. Systemically administered sulforaphane, a well-known Nrf2-ARE signal pathway activator, could increase
mRNA and protein levels of HO-1, and significantly decreased cerebral infarct volume following focal ischemia [27]. NQO1 is a flavoprotein that catalyzes the two-electron reduction and detoxification of quinines and their derivatives [19]. Some of NQO1 (−/−) mice have seizures, which is characterized by bending of the front legs in a praying motion, tension of the tail, salivation and occasionally spasm [22]. Thus, there was reason to suspect that NQO1 has an important role in central nervous system function. Agents that activate Nrf2-ARE signal pathway have been sought as chemoprotectants for a wide variety of conditions including neurodegeneration and epilepsy. As to this aspect, more studies should be conducted. In conclusion, the results of the present study indicate that the seizure can induce oxidative stress in hippocampus of rats, which activate Nrf2-ARE signal pathway to result in up-regulation of antioxidative and detoxifying enzymes. Therefore, to activate Nrf2-ARE signal pathway may be one of the strategic targets for epilepsy therapies.
Uncited reference [12].
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This project was financially supported by the Natural Science Foundation of Hebei Province, PR China (C2010000548).
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Activation of Nrf2-ARE signal pathway in hippocampus of amygdala kindling rats
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Wei Wang a,b , Wei-ping Wang a,∗ , Guo-liang Zhang c , Yan-fen Wu b , Tao Xie a , Min-chen Kan a , Hai-bo Fang a , Hong-chao Wang a
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Key Laboratory of Neurology of Hebei Province, the Second Hospital of Hebei Medical University, Shijiazhuang, Hebei 050071, PR China Department of Neurology, Handan First Hospital, Handan, Hebei 056002, PR China Department of Neurobiology, Hebei Medical University, Shijiazhuang, Hebei 050017, PR China
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Oxidative stress was induced in hippocampus of amygdala kindling rats. Nrf2 and Nrf2-dependent gene products were significantly increased after seizure. Nrf2-dependent gene products can protect the cells against oxidative damage. To activate Nrf2-ARE signal pathway may be a new direction for treatment of epilepsy.
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Article history: Received 29 November 2012 Received in revised form 6 March 2013 Accepted 10 March 2013
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Oxidative stress resulting from excessive free-radical release is likely implicated in the initiation and progression of epilepsy. Therefore, antioxidant therapies have received considerable attention in epilepsy treatment. It is well known that the transcription factor NF-E2-related factor (Nrf2) binds to antioxidant response element (ARE) to induce antioxidant and phase II detoxification enzymes under conditions of oxidative stress, which reduces oxidative stress and accumulation of toxic metabolites. However, whether Nrf2-ARE pathway is activated after seizure has not been studied. In the present study, Wistar rats were rapidly kindled in the amygdala. Twenty-four hours after the last seizure, the hippocampus of control, sham and kindled rats were examined for oxidative stress parameters (malondialdehyde and glutathione) by spectrophotometry, the expression of Nrf2, heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase-1 (NQO1) were determined using immunohistochemistry, Western blot and real-time fluorescence quantitative polymerase chain reaction (PCR). The results showed that the kindled seizures induced oxidative stress, the expression of Nrf2, HO-1 and NQO1 at protein or gene levels significantly increased in hippocampus after seizure. According to these results, it could be postulated that Nrf2-ARE signal pathway was activated in the hippocampus after seizure. © 2013 Published by Elsevier Ireland Ltd.
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Keywords: Nrf2-ARE Heme oxygenase-1 NAD(P)H:quinone oxidoreductase-1 Epilepsy Oxidative stress
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1. Introduction
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Epilepsy, one of the most common neurological disorders, is a chronic disorder of abnormal electrical activity in the brain, characterized by recurrent unprovoked seizures [20]. The mechanisms underlying epileptogenic pathogenesis have been proved to be complex, including oxidative stress, glutamate excitotoxicity, calcium overload and so on [4]. Given that pathological processes involved in epileptogenesis are complex and interrelated, to find a target, which can interrupt multi-mechanisms underlying seizure, is desirable. Oxidative stress has been reported as an underlying mechanism in the initiation and progression of epilepsy, what’s
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∗ Corresponding author. Tel.: +86 031166002915; fax: +86 0311 66002915. E-mail address: ldh wwp [email protected] (W.-p. Wang).
more, excessive oxidative stress contributes to neuronal degeneration in the epileptic focus [21]. Increases in reactive oxygen species occur in response to sustained neuronal electrical activity and seizures [10]. Therefore, antioxidants have been suggested as therapeutic strategies for the treatment and modulation of epilepsy. The nuclear factor erythroid 2-related factor 2 (Nrf2) is essential for the induction of a battery of phase II detoxification genes through the antioxidant response element (ARE) that lies in their promoter region [27]. Genes driven by the ARE are up-regulated in response to various stressors of the cellular environment. Under numerous stimuli, Nrf2 translocates from the cytoplasm to the nucleus, and sequentially binds to ARE [13]. Nrf2-ARE signal pathway regulates the expression of a group of cytoprotective enzymes, such as heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase-1 (NQO1). These Nrf2-dependent gene products go on to protect the cells from oxidative or xenobiotic damage [28].
0304-3940/$ – see front matter © 2013 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.neulet.2013.03.038
Please cite this article in press as: W. Wang, et al., Activation of Nrf2-ARE signal pathway in hippocampus of amygdala kindling rats, Neurosci. Lett. (2013), http://dx.doi.org/10.1016/j.neulet.2013.03.038
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Previous studies have shown that up-regulation of HO-1 [1] or NQO1 [8] in neurons can protect them against oxidative and excitotoxic insults. HO-1 is a ubiquitous and redox-sensitive inducible stress protein that degrades heme to carbon monoxide, iron and biliverdin [13]. Reduced HO-1 protein expression is associated with more severe neurodegeneration after transient ischemia [11]. In contrast, induction of HO-1 could protect neurons from oxidative stress [6]. Another Nrf2-regulated gene product, NQO1, is considered to play a protective role in the pathogenesis of Parkinson’s disease [23]. Up-regulation of NQO1 by 4-hydroxybenzyl alcohol also reduces cerebral infarct size and improves neurological functions in rats [26]. However, to our knowledge, the Nrf2-ARE signal pathway and two Nrf2-regulated gene products (HO-1 and NQO1) have not been studied after seizure. Thus, the present study was aimed to examine oxidative stress parameters (malondialdehyde and glutathione) and determine the expression of Nrf2, HO-1 and NQO1 at protein or gene levels in hippocampus of amygdala kindling rats.
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2. Materials and methods
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2.1. Animals
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Adult male Wistar rats weighing 250–300 g (n = 50) were obtained from the Hebei Medial University. Rats were housed in a room with constant temperature (25 ± 1 ◦ C) and humidity (40–60%), and were kept on a 12 h light/dark cycle, with lights on at 8:00 AM and with free access to food and water. Animal experiments were performed according to the regulations of laboratory animal management promulgated by the Ministry of Science and Technology of the People’s Republic of China [1988] No.134, which coincides with internationally recognized NIH guidance. The rats were randomly assigned to three groups. Control group (n = 16) did not receive any treatment. Electrodes were implanted in the remaining 34 rats. Two rats could not be included in the experiments because of death during anesthesia or misplacement of the electrode. The remaining 32 rats were randomly distributed to the following groups: sham group (n = 16) did not receive any electrical current administration; kindling group (n = 16) were subjected to experimental kindling procedure as follow. Twenty-four hours after the last stimulation, all rats were sacrificed. Eight rats in each group were prepared for spectrophotometry, Western blot and real-time fluorescence quantitative PCR analysis, and the other eight were for immunohistochemical study.
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For implantation, animals were anesthetized with chloral hydrate (300 mg/kg, i.p.), the sham and kindling groups were stereotactically implanted with twisted stainless steel wire bipolar stimulating electrodes in the left basolateral amygdale complex (Bregma: anterior: −2.8 mm; lateral: 4.8 mm; ventral: 8.8 mm from skull surface) [16]. Three screws positioned over the right frontal cortex, left and right occipital cortices served as recording, reference and ground electrodes. Dental cement bound the electrodes and screws to the skull. ABS connectors coupled the electrodes and screws to stimulating and recording equipments. After surgery, the rats were treated with antibiotics for 1 week to prevent infection.
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The initial afterdischarge threshold (ADT) was determined using an electric stimulator (SEN-7203; Nihon Kohden) 1 week after electrode implantation. The stimulation parameters were performed by constant current stimulations (monophasic square-wave pulses,
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60 Hz for 1 s). The stimulus intensity started at 100 A, and thereafter increased in 40 A increments at 5 min intervals until an afterdischarge of at least 3 s duration was elicited (140–340 A). The next day animals of kindling group were kindled at 10 min intervals stimulation at 120% of their individual ADT. The kindled seizures were classified according to Racine’s five stages: stage 1, immobility and facial automatisms (eye closure, facial clonus); stage 2, head nodding accompanying mastication; stage 3, clonic forelimb convulsion; stage 4, rearing to a kangaroo-like posture or rearing with clonic forelimb clonus; and stage 5, generalized convulsion, including falling down. In the course of kindling, stimulation was delivered 40 times for the kindling group, with 20 times every day. In our study, all the kindling rats reached stage 5 at least three times.
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2.4. Experimental procedure
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The animals were sacrificed and the hippocampus was removed and cleaned with ice-cold (4 ◦ C) saline. Hippocampus tissue samples were homogenized with 10 times (w/v) ice-cold 0.1 M phosphate buffer (PB), pH 7.4. The homogenates were used to assess lipid peroxidation product and reduced glutathione. Malondialdehyde (MDA), an index of lipid peroxidation, was measured spectrophotometrically with the method described by Okhawa [14]; reduced glutathione (GSH) was estimated spectrophotometrically by the method described by Ellman [3]. MDA detection kit and GSH detection kit were obtained from Nanjing Jiancheng Bioengineering Institute (China). The rats used for immunohistochemical analysis were anesthetized with 4% chloral hydrate (300 mg/kg, i.p.) and perfused transcardially with saline, followed by 4% paraformaldehyde in 0.1 M PB, pH 7.4. Brains were carefully removed and postfixed in 4% paraformaldehyde in PB for 4 h at 4 ◦ C, and then embedded in paraffin. Brain sections (5 m thick) were blocked in 3% H2 O2 , 3% normal goat serum, and incubated with Nrf2 rabbit polyclonal antibody (1:100, Abcam Cambridge, USA), and HO-1 rabbit polyclonal antibody (1:100, Abcam Cambridge, USA) in 0.01 mol/L phosphate-buffered saline over night. The secondary antibodies (1:100, goat anti-rabbit IgG), secondary biotinylated conjugates and diaminobezidine were from the Vect ABC kit (Zhongshan Biology Technology Company, China). A computer-assisted image analysis system (Olympus, BX 61, Olympus DP71, U-TV0.5XC-3, Japan; Image-Pro Plus 6.0) was used for the average optical density (AOD) measurements of Nrf2 immunoreactive (Nrf2-ir) and HO-1 immunoreactive (HO-1-ir) intensity. After calibration of the system to eliminate saturation of gray levels, images were acquired for an accurate determination of optical density. Brain sections processed without primary antibody were used to determine the level of nonspecific staining for the entire experiment. After subtraction of the nonspecific staining, the AOD of Nrf2-ir and HO-1-ir cells in CA3 of hippocampus were measured. Measures were performed on both left and right sides and averaged for each section. To prevent differences arising from variations in the conditions of tissue processing and densitometric analysis, all sections were simultaneously processed. All microscopic and computer parameters were kept constant throughout the study. Ten sections of the hippocampus, with the interval of 5, were selected from each rat. The average of the AOD of Nrf2-ir or HO-1-ir cells in CA3 of hippocampus was presented for each rat in the results. Protein extraction for Nrf2 and HO-1 was performed as follows. The hippocampus was homogenized in ice-cold lysis buffer (10 mmol/L HEPES, pH 7.9, 10 mmol/L KCl, 0.1 mmol/L EDTA, 1 mmol/L DTT, 0.1 mmol/L EGTA) for 15 min. After adding NP-40, the homogenate was centrifuged 10,000 rpm at 4 ◦ C for 3 min and the supernatant was collected as cytoplasmic protein for HO-1.
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Fig. 1. The MDA (A) and GSH (B) levels express in hippocampus of rats. The ‘Con’, ‘sham’ and ‘EP’ in abscissa represent control, sham and kindling groups, respectively. Values are expressed as mean ± SD. *P < 0.01.
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The pellets were homogenized in ice-cold lysis buffer (20 mmol/L HEPES, pH 7.9, 400 mmol/L NaCl, 1 mmol/L EDTA, 0.1 mmol/L EGTA) for 15 min. Then the pellets were centrifuged 12,000 rpm at 4 ◦ C for 10 min, and the supernatant was collected. PMSF was then added to the supernatant with the final concentration 1 mmol/L as the nuclear protein for Nrf2. The protein concentration of the supernatant was determined using a BCA Protein Assay reagent kit (Novagen, Madison, WI, USA). Sample (50 g) was separated by SDS/PAGE, transferred on to PVDF membranes. Membranes were blocked with 5% skimmed milk 1 h at room temperature, and then were probed with polyclonal rabbit anti-Nrf2 antibody (1:500, Abcam Cambridge, USA) and polyclonal rabbit anti-HO-1 antibody (1:200, Abcam Cambridge, USA) overnight at 4 ◦ C. After washing three times with TPBS, IRDye® 800-conjugated goat antirabbit second antibody (1:3000, Rockland, Gilbertsville) was incubated with membranes for 1 h at room temperature. The relative density of bands was analyzed on an Odyssey infrared scanner (LI-COR Bioscience). The densitometric values were normalized with respect to the values of -actin immunoreactivity to correct for any loading and transfer differences between samples. Total RNA from the hippocampus tissue was obtained using Trizol reagent (Invitrogen, USA) following the manufacturer’s instruction. RNA concentration was determined by measuring the absorbance (A) of a diluted sample at the 260 nm wavelength in a UV spectrometer. A total of 2 g of total RNA was subjected to reverse transcription using random primer to obtain the firststrand cDNA template. Real-time fluorescence quantitative PCR was performed with 0.8 l cDNA (diluted 1:10), specific primers 2 l, and 2*GoTaq® Green Master Mix (Promega, USA) with a final volume of 20 l. PCR was performed as follows: an initial cycle at 95 ◦ C for 10 min, followed by 40 cycles at 95 ◦ C for 15 s, 58 ◦ C for 20 s and 72 ◦ C for 27 s. Then PCR products were analyzed by melting curve to confirm the specificity of amplification. Expression of Nrf2, HO-1 and NQO1 genes were analyzed with GAPDH used as an internal control. The sets of primers that we used were as follows: Nrf2(5 -GACCTAAAGCACAGCCAACACAT-3 and 5 CTCAATCGGCTTGAATGTTTGTC-3 ),HO-1(5 -TGTCCCAGGATTTGTCCGAG-3 and 5 -ACTGGGTTCTGCTTGTTTCGCT-3 ), NQO1(5 -GGGGACATGAACGTCATTCTCT-3 and 5 -AGTGGTGACTCCTCCCAGACAG-3 ), GAPDH(5 -TGAACGGGAAGCTCACTG3 and 5 -GCTTCACCACCTTCTTGATG-3 ). All data was analyzed by one-way ANOVA. Quantitative data were expressed as mean ± SD. All statistical analysis was performed with SPSS 13.0 software and P < 0.05 was considered statistically significant.
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As shown in Fig. 1, the level of GSH in kindling group was significantly lower compared to sham group (P < 0.01) and the level of MDA was much higher than that of sham group (P < 0.01). There was no difference between control and sham groups (P > 0.05). To localize Nrf2 and HO-1 expression, immunohistochemical study was performed. As shown in Fig. 2, in kindling group, the immunoreactive intensity of Nrf2 and HO-1 significantly increased in hippocampus, and Nrf2 expressed both at cytoplasm and nucleus. In addition, similar localization was found between Nrf2 and HO-1 in neurons and glial cells. The differences were shown between sham and kindling groups by AOD measurement in the expression of Nrf2 (P < 0.01) and HO1 (P < 0.01). There was no difference between control and sham groups (P > 0.05). As shown in Fig. 3, Western blot analysis was used to measure in protein level of Nrf2 in nuclear and HO-1 in cytoplasm extracted from hippocampus. Group differences were found between sham and kindling groups with the expression of Nrf2 (P < 0.01) and HO1 (P < 0.01). There was no difference between control and sham groups (P > 0.05). The expression of Nrf2, HO-1 and NQO1 mRNA in hippocampus was studied by real-time fluorescence quantitative PCR. As shown in Fig. 4, group differences were found between sham and kindling groups in the expression of Nrf2 mRNA (P < 0.01), HO-1 mRNA (P < 0.01) and NQO1 mRNA (P < 0.01). There was no difference between control and sham groups (P > 0.05).
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In this study we found that the seizure induced an increase in MDA levels and a decrease in GSH levels in hippocampus of rats. The protein level of Nrf2 in nuclear and HO-1 in cytoplasm were significantly increased, and the mRNA levels of Nrf2 and two Nrf2-regulated gene products (HO-1 and NQO1) were also up-regulated in hippocampus after seizure. It could be postulated that the seizure can induce oxidative stress in hippocampus of rats, which activates Nrf2-ARE signal pathway to result in up-regulation of antioxidative and detoxifying enzymes. Recent studies showed that oxidative stress plays an important role in epileptic pathogenesis [18,20]. MDA is an end product of free radical generation [9], and GSH is an endogenous antioxidant which plays a vital role as a free radical scavenger to protect cells against oxidative damage [15]. In the present study, the amygdala
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Fig. 2. Immunohistochemical study of Nrf2 (A) and HO-1 (B) expression in hippocampus. Bar graphs illustrate the AOD expression of Nrf2 (C) and HO-1 (D). Values are expressed as mean ± SD. *P < 0.01. Arrowhead () and arrow (→) respectively indicate neurons and glial cells. A1-3 and B1-3 Scale bars = 200 m. A4-6 and B4-6 Scale bars = 50 m. 266 267 268 269 270 271 272 273
kindled seizures caused a marked increase in MDA levels and a decrease in GSH levels in hippocampus, as compared to the control and sham groups. The imbalance between antioxidant and oxidant defense system may be at least partially responsible for seizures. In agreement with our findings, the seizure-induced oxidative stress has been reported in previous studies [25]. Thus, antioxidants have been suggested for therapeutic strategies in the treatment and modification of epilepsy.
Numerous studies have addressed a pivotal role of Nrf2 in protecting cells from oxidative stress [24]. One unique feature about the Nrf2-ARE signal pathway is that it coordinately up-regulates many protective detoxification and antioxidant genes, which can synergistically increase the efficiency of cellular defense system [7]. Many chronic neurodegenerative diseases (i.e. Parkinson’s disease and amyotrophic lateral sclerosis) are thought to involve oxidative stress as a component contributing to the progression of the
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Fig. 3. Western blot analysis for Nrf2 (A), and HO-1 (B) protein levels in hippocampus of rats. Bar graphs illustrate the protein expression of Nrf2 (C) and HO-1 (D), values are expressed as mean ± SD. *P < 0.01.
Fig. 4. The graphs show Nrf2 (A), HO-1 (B) and NQO1 (C) mRNA levels in control, sham and kindling groups. Values are expressed as mean ± SD. *P < 0.01.
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disease, and oxidative stress can be relieved by the Nrf2-ARE signal pathway as demonstrated previously [2,17]. But there is few data to show the relationship between the Nrf2-ARE signal pathway and epilepsy. In our study, we found that the expression of Nrf2, HO-1 and NQO1 were up-regulated at gene and protein level after seizure. Immunohistochemistry and real-time fluorescence quantitative PCR revealed that total Nrf2 was up-regulated, Western blot analysis illustrated that nuclear Nrf2 was induced after seizure, which provided the evidence that seizure could increase Nrf2 nuclear translocation. We also observed that there was a positive relationship between nuclear Nrf2 protein content and HO-1 or NQO1 mRNA levels. It could be postulated that Nrf2-ARE signal pathway is activated in hippocampus after seizure. Activation of Nrf2-ARE signal pathway after seizure results in up-regulation of antioxidative and detoxifying enzymes, which may reduce oxidative damage. It has been shown that mice lacking Nrf2 are more sensitive to kainic acid, and there is substantial evidence to support the notion that activation of Nrf2 is neuroprotective [5]. Therefore, activation of Nrf2 may represent a potential target for combating oxidative damage from seizure. Increasing evidence has demonstrated that the protective role of the Nrf2-ARE pathway in central nervous disease is mediated by HO-1 and NQO1 [24]. In our study, up-regulation of HO-1 and NQO1 mRNA levels was also observed. In addition, similar cellspecific localization was found between Nrf2 and HO-1. HO-1 activity reduced reactive oxygen species production by generating biliverdin, and further by biliverdin reductase to bilirubin, a potent antioxidant [13]. Systemically administered sulforaphane, a well-known Nrf2-ARE signal pathway activator, could increase
mRNA and protein levels of HO-1, and significantly decreased cerebral infarct volume following focal ischemia [27]. NQO1 is a flavoprotein that catalyzes the two-electron reduction and detoxification of quinines and their derivatives [19]. Some of NQO1 (−/−) mice have seizures, which is characterized by bending of the front legs in a praying motion, tension of the tail, salivation and occasionally spasm [22]. Thus, there was reason to suspect that NQO1 has an important role in central nervous system function. Agents that activate Nrf2-ARE signal pathway have been sought as chemoprotectants for a wide variety of conditions including neurodegeneration and epilepsy. As to this aspect, more studies should be conducted. In conclusion, the results of the present study indicate that the seizure can induce oxidative stress in hippocampus of rats, which activate Nrf2-ARE signal pathway to result in up-regulation of antioxidative and detoxifying enzymes. Therefore, to activate Nrf2-ARE signal pathway may be one of the strategic targets for epilepsy therapies.
Uncited reference [12].
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This project was financially supported by the Natural Science Foundation of Hebei Province, PR China (C2010000548).
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Please cite this article in press as: W. Wang, et al., Activation of Nrf2-ARE signal pathway in hippocampus of amygdala kindling rats, Neurosci. Lett. (2013), http://dx.doi.org/10.1016/j.neulet.2013.03.038
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