- Email: [email protected]
reperfusion in rats
Neuroscience 284 (2015) 815–823
TARGETING THIOREDOXIN-1 WITH SIRNA EXACERBATES OXIDATIVE STRESS INJURY AFTER CEREBRAL ISCHEMIA/REPERFUSION IN RATS L. LI, a,c K. ZHU, a,c Y. LIU, b,c X. WU, a,c J. WU, b,c Y. ZHAO b,c AND J. ZHAO a,c*
Key words: thioredoxin-1, oxidative stress, peroxiredoxin, nuclear factor erythroid 2-related factor 2.
a
Department of Pathophysiology, Chongqing Medical University, Chongqing, People’s Republic of China b Department of Pathology, Chongqing Medical University, Chongqing, People’s Republic of China
INTRODUCTION
c Institute of Neuroscience, Chongqing Medical University, Chongqing, People’s Republic of China
Stroke is a leading cause of death and long-term disability in developing countries. Oxidative stress caused by reactive oxygen species (ROS) is a critical component of cerebral ischemic injury (Tewari et al., 2014). Oxidative damage can induce neuronal injury and necrosis in brain tissue by oxidizing intracellular molecules such as lipids, proteins, and DNA (Heo et al., 2005; Oztanir et al., 2014). Therefore, it was thought that antioxidant agents, which have the ability to scavenge ROS, could attenuate neurological damage. Thioredoxin-1 (Trx-1) is a 12-kDa ubiquitous protein present in all living cells. It has a wide range of physiological functions, including DNA synthesis, oxidation damage repair, and regulation apoptosis (Lu and Holmgren, 2012; Chang et al., 2013; Sengupta and Holmgren, 2013). Recent study has demonstrated that exogenous rhTrx-1 attenuates post-ischemic brain damage and neuronal apoptosis by reducing oxidative/nitrative stress (Ma et al., 2012). With thiol-reducing activity at its conserved active site, Trx-1 exhibits cytoprotective effects against oxidative stress by scavenging ROS and cooperating with peroxiredoxin (Prdx) (Das and Das, 2000; Lu and Holmgren, 2014). Prdx is a general term that refers to a family of small (22–27 kDa) non-seleno peroxidases, representing a class of important antioxidants in mammals (Park et al., 2014). In fact, Prdx, thioredoxin, and thioredoxin reductase form the mammalian thioredoxin system that plays crucial roles in defending oxidative stress injury, especially typical 2-Cys Prdxs (Prdx1–4) (Zhu et al., 2012). Therefore, elucidating the relationship between Trx-1 and Prdx1–4 in cerebral I/R injury is particularly important. It is well acknowledged that enhanced ROS and electrophiles can evoke a series of antioxidant genes by activating nuclear factor erythroid 2-related factor 2 (Nrf2), a critical transcription factor that regulates the expression of major antioxidant enzymes and phase II detoxification enzymes (Motohashi and Yamamoto, 2004; Wang et al., 2014). Hemin has been shown to induce activation of the thioredoxin gene by regulating Nrf2 through the antioxidant responsive element (ARE) in K562 cells (Kim et al., 2001). Moreover, the expression of Nrf2 has been shown to be significantly up-regulated in the peri-infarct region and to subsequently induce Trx
Abstract—Reactive oxygen species and their detrimental effects on the brain after transient ischemia/reperfusion (I/R) have been implicated in the pathogenesis of ischemic reperfusion injury. Thioredoxin-1 (Trx-1) is an endogenous antioxidant protein that has neuroprotective effects. We hypothesized that Trx-1 plays a crucial role in regulating cerebral I/R injury. To be able to test this, 190 Sprague– Dawley rats were subjected to transient middle cerebral artery occlusion (tMCAO) with Trx-1 siRNA (small interference RNA) injected 24 h prior to ischemia. At 24 h after tMCAO, we measured neurological deficits, infarct volume, and brain water content, and found that neurological dysfunction, brain infarct size, and brain edema were worse in the Trx-1 siRNA group than in the control group. Oxidative stress was evaluated by measuring superoxide dismutase activity and malondialdehyde level. The levels of Trx-1 and its cofactor, peroxiredoxin (Prdx), were significantly decreased after Trx-1 down-regulated. However, there is no significant difference in the Prdx mRNA level after administration of Trx-1 siRNA. In contrast, Prdx-SO3 protein levels were significantly increased in the Trx-1 siRNA group. We also investigated the specific role of nuclear factor erythroid 2-related factor 2 (Nrf2) in Trx-1 induction by knocking down Nrf2. Nrf2 siRNA injection decreased Trx-1 mRNA and protein expression. Our results suggest that the exacerbation of brain damage was associated with enhanced cerebral peroxidation in brain tissues. Moreover, these results revealed that Trx-1, which is more likely regulated by Nrf2, exerts a neuroprotective role probably through maintaining the reduction activity of Prdx1–4. Ó 2014 Published by Elsevier Ltd. on behalf of IBRO.
*Correspondence to: J. Zhao, Department of Pathophysiology, Chongqing Medical University, Yixueyuan Road 1, Chongqing 400016, People’s Republic of China. Tel/fax: +86-23-6848-5868. E-mail address: [email protected] (J. Zhao). Abbreviations: ANOVA, analysis of variance; ARE, antioxidant responsive element; CV, Cresyl Violet; I/R, ischemia/reperfusion; MCA, middle cerebral artery; MCAO, middle cerebral artery occlusion; MDA, malondialdehyde; MEFs, mouse embryo fibroblasts; Nrf2, nuclear factor erythroid 2-related factor 2; Prdx, peroxiredoxin; qPCR, quantitative PCR; ROS, reactive oxygen species; SOD, superoxide dismutase; Trx-1, thioredoxin-1. http://dx.doi.org/10.1016/j.neuroscience.2014.10.066 0306-4522/Ó 2014 Published by Elsevier Ltd. on behalf of IBRO. 815
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expression in male ICR mice following middle cerebral artery (MCA) occlusion (MCAO) (Tanaka et al., 2011). However, in spite of these various observations, the role of Nrf2 in regulating Trx-1 gene expression in the context of ischemic brain injury has yet to be fully investigated. The purpose of this study was to evaluate the role of Trx-1 in ischemic injury using an experimental MCAO model with Trx-1 knock-down. We evaluated whether the Trx1 down-regulation could exacerbate brain damage. Furthermore, we elucidated the relationship between Trx-1 and Prdx1–4 in cerebral I/R injury. Because our results highlight the importance of Trx-1 in modulating the extent of brain damage, we have also investigated the influence of Nrf2 on Trx-1 expression level by interfering Nrf2 gene expression.
EXPERIMENTAL PROCEDURES Animals and groups Adult male Sprague–Dawley rats, weighed 270–310 g (n = 190, 36 died within 24 h) and aged 90 ± 4 d were bred and held at the Experimental Animal Center of Chongqing Medical University. All rats were allowed free access to food and water before the operation under optimal conditions (12/12-h light/dark with humidity 60 ± 5%, 22 ± 3 °C). All experimental animals were randomly allocated to the following groups: sham surgery group (n = 28, no died), untreated controls with MCAO (n = 51, 9 died), scramble siRNA of Trx-1siRNA-injected group (n = 37, 7 died), Trx-1 siRNA group (n = 41, 11 died), scramble siRNA of Nrf2siRNA-injected group (n = 15, 3 died) and Nrf2 siRNA group (n = 18, 6 died). All experimental procedures were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Chongqing Medical University, China. Administration of Trx-1 siRNA and Nrf2 siRNA Trx-1 siRNA (The sense primer 5-AAGCUCGAAGCCAC UAUUATT-3 and antisense primer 5-UAAUAGUGGCUU CGAGCUUTT-3), Nrf2 siRNA (The sense primer 5-CCC UGUGUAAAGCUUUCAATT-3 and antisense primer 5-UUGAAAGCUUUACACAGGGTT-3) were designed and chemically synthesized by GenePharma Corporation, Shanghai, China. Scramble siRNA, which has the same nucleotide composition of the target gene siRNA with no sequence homology to any known rat genes was used as the control. All siRNAs were dissolved in RNase-free water, making the final concentration 2 lg/ll. Rats were anesthetized with 3.5% chloral hydrate (350 mg/kg, ip) and placed in a stereotaxic apparatus (Taimeng Software, Chengdu, China). 10 ll of siRNA or scramble siRNA was injected ipsilaterally into the left lateral cerebral ventricle at 24 h prior to the induction of MCAO, respectively. siRNA or scramble siRNA was slowly injected into the left lateral ventricle over a 20-min duration using a Hamilton microsyringe with the coordinates of 1.0 mm posterior to the bregma, 2.0 mm lateral to the midline, and 4.0 mm ventral to the surface
of the skull under the guidance of a stereotaxic instrument. The injection rate was 0.5 ll/min. After injection, the needle was held in place for 5 min and then removed slowly over 2 min. Cerebral ischemia/reperfusion (I/R) model Transient focal cerebral ischemia was introduced into rats by left MCA occlusion technique according to our previous methods (Chen et al., 2012). In brief, a 4–0 monofilament nylon suture (Beijing Sunbio Biotech Co Ltd, Beijing, China) with a rounded tip was inserted into the left internal carotid artery through the common carotid artery stump and gently advanced to occlude the MCA. After 60 min of MCAO, the suture was removed to restore blood flow. The reperfusion was confirmed by laser Doppler (Periflux System 5000, Perimed AB, Stockholm, Sweden). The same procedure was performed on sham-operated rats, but the MCA was not occluded. Rats that did not show neurological deficits after reperfusion (neurological score < 1) were excluded from the study, as well as animals that died after ischemia induction. Rats that showed neurological deficits immediately after reperfusion (neurological score > 0) but were found to be experiencing skull base or subarachnoid hemorrhage were also excluded from the study. Core body temperatures were monitored with a rectal probe and maintained at 37 °C during the whole procedure. Sample processing All rats were sacrificed at 24 h after reperfusion, and the brains were quickly removed to collect the cerebral cortex for an analysis of oxidative stress assay, western blotting, and real-time quantitative PCR (qPCR). A 4-mm coronal section was taken from the area perfused by the MCA starting at 5 mm from the frontal pole. Fresh cortical tissue was collected from the MCA territory of the ischemic left hemisphere, frozen immediately in liquid nitrogen, and stored at 80 °C until needed for further processing. Tissue samples from all animals were analyzed individually. Evaluation of neurological deficit Neurological deficit scores were evaluated by an examiner blinded to the experimental groups after 24 h of reperfusion. The deficits were scored on a modified scoring system developed by Longa et al. (Longa et al., 1989), as follows: 0, no neurological deficits; 1, failure to extend right forepaw fully; 2, circling to right; 3, falling to right; 4, did not walk spontaneously and has depressed levels of consciousness. The higher the neurological deficit score, the more severe the impairment is of motor motion injury. Brains from these rats were analyzed for water content, infarct volume, oxidative stress analysis, western blot, and real-time qPCR. Measurement of brain water content Brain water content was measured using the standard wet– dry method. Six rats in each group were anesthetized with chloral hydrate and killed by decapitation at 24 h after
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MCAO. The brains were rapidly removed and dissected into the ipsilateral and contralateral hemispheres. The two hemispheres were immediately weighed on an electronic balance to obtain the wet weight and then dried in an oven at 100 °C for 24 h to obtain the dry weight. Brain water content was calculated as follows: Brain water content (%) = (wet weight dry weight)/wet weight 100%. Infarct volume analysis Ischemic infarct volume was measured as described previously (Tureyen et al., 2004). For Cresyl Violet (CV) staining, the slides were immersed in xylene for 30 min, and then in 100%, 95%, and 70% ethanol and doubledistilled water for 5 min each. Next, the slides were stained in 0.1% CV solution for 5 min and then briefly rinsed in double-distilled water. These were then dehydrated again in 70%, 95%, and 100% ethanol for 1 min each. The slices were placed in xylene for another 10 min and then coverslipped. The normal tissue was stained blue whereas the infarct tissue did not stain and appeared white. All the CV-stained sections were scanned using a flatbed scanner and the images were stored as TIFFs. The non-ischemic and ischemic hemisphere infarct areas were measured using ImageJ (ver1.37c, NIH) software. All four infarct area measurements were calculated with a 2-mm distance between the slices. Using these measurements, the total infarct volume was calculated for each brain. To compensate for the effect of brain edema, the corrected volume was calculated using the following equation: Percentage hemisphere lesion volume (% HLV) = {[total infarct volume (left hemisphere volume right hemisphere volume)]/right hemisphere volume} 100%. Determination of superoxide dismutase (SOD) and malondialdehyde (MDA) levels Cortical tissues from 6 rats of each group were collected at 24 h after MCAO. The samples were rinsed, weighed, and then homogenized in 9 volumes of 9 g/l ice-cold saline. Supernatant homogenate was collected after centrifugation at 4000 rpm/min for 10 min at 4 °C. SOD and MDA levels were measured using commercially available assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturers’ instructions. SOD activity was measured by the hydroxylamine method. Absorbance was determined at 550 nm by spectrometry. The MDA level was measured by the thiobarbituric acid method. Absorbance was measured at 532 nm by spectrometry. The content of protein in the supernatant was determined by the Enhanced BCA Protein Assay Kit (Beyotime, Jiangsu, China). Western blot analysis Left cortical samples were recovered and homogenized in the RIPA buffer. Protein extracts were resolved by SDS– PAGE and transferred onto nitro-cellulose membranes. The membranes were blocked in 5% non-fat milk
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containing 20 mM Tris–HCl, pH 7.6, 137 mM NaCl, 0.1% Tween-20 (TBS-T) and then incubated at 4 °C overnight with primary antibodies. The primary antibodies used and their respective dilutions were as follows: rabbit polyclonal antibody to Trx-1 (1:2000, AB9328, Millipore, Boston, USA), rabbit polyclonal to Prdx1 (1:2000, ab59538, Abcam, Cambridge, MA, USA), rabbit polyclonal to Prdx2 (1:2000, ab59539, Abcam, Cambridge, MA, USA), rabbit polyclonal to Prdx3 (1:2000, ab73349, Abcam, Cambridge, MA, USA), mouse monoclonal to Prdx4 (1:2000, ab16943, Abcam, Cambridge, MA, USA), rabbit polyclonal to Prdx-SO3 (1:2000, ab16830, Abcam, Cambridge, MA, USA), rabbit polyclonal to Nrf2 (1:1000, ab31163, Abcam, Cambridge, MA, USA), and mouse monoclonal to GAPDH (1:500, TA-08, ZSGB-BIO, Beijing, China). This step was followed by 2-h incubation with respective HRP-conjugated secondary antibodies (ZSGB-BIO, Beijing, China; dilution 1:3000). Chemiluminescence of protein bands was developed using the ECL kit (Millipore, Temecula, California, USA). The relative density of bands was scanned with imaging densitometer (Bio-Rad, Foster City, CA, USA), and the results were quantified using Quantity One 1-D analysis software. GAPDH was used as an internal loading control. RNA isolation and cDNA synthesis Total RNA from left cortical samples was isolated using the RNAiso Plus reagent (TaKaRa Biotechnology, Dalian, China) according to the manufacturer’s instructions. The RNA content was assessed by A260/A280 ratio and agarose gel electrophoresis prior to performing qPCR. Equivalent amounts of RNA extracted from each sample served as template for cDNA synthesis using the PrimeScriptÒ RT reagent Kit With gDNA Eraser (Perfect Real Time) (TaKaRa Biotechnology, Dalian, China) following the manufacturer’s instructions. Real-time qPCR analysis qPCR was performed on a Bio-Rad CFX-96 Connect Real-Time System (Bio-Rad, Foster City, CA, USA) using TaKaRa SYBRÒ Premix Ex Taqä II (Tli RNaseH Plus) (TaKaRa Biotechnology, Dalian, China). The primer sequences and amplicon sizes are listed in Table 1. The data were analyzed using Bio-Rad CFX Manager software (version 2.0). The expression levels of the genes were normalized to GAPDH. The optimized qPCR assays were carried out with 12.5 ll of SYBRÒ Premix Ex Taqä II (Tli RNaseH Plus), 2 ll of cDNA, 1-ll forward primer (10 lM), 1-ll reverse primer (10 lM), and 8.5 ll of RNase-free water in a total volume of 25 ll, making the final primer concentration 0.4 lM. Amplification began with an initial incubation at 95 °C for 3 min followed by 40 cycles of denaturation at 95 °C for 10 s and annealing and extension at 58 °C for 30 s. A melting curve procedure was added to the Bio-Rad CFX96 Connect Real-Time System to analyze the specificity of the products. Non-template controls containing all reagents except cDNA was included in each PCR run. These controls generated C(t) > 40 in all experiments. To determine
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Table 1. Informatiwon on the qPCR Primers Gene symbol
Gen Bank accession no.
Primer sequence (50 ? 30 )
Product size (b.p.)
Trx-1
NM_053800.3
143
Prdx1
NM_057114.1
Prdx2
NM_017169.1
Prdx3
NM_022540.1
Prdx4
NM_053512.2
Nrf2
NM_031789.2
GAPDH
NM_017008.4
F cct tct ttc att ccc tct gtg a R ccc aac ctt ttg acc ctt ttt a F cat tgc tca gga tta tgg agt c R cat tta ttg tta tct ggc gaa gg F cgt ggt cct ctt ttt cta tcc a R ctt tta gtc aca tca gca agc a F tgc ttt tct tct acc ctt tgg a R cat tct ttc ttg gcg tgt tga t F cct ctg ctg ctg ttc ctg tta c R aaa tct tgg ctt tgc tta ggt g F ctg gtc atc aaa aag tcc cat t R tta ttc ttc cct ctc ctg cgt a F aca gca aca ggg tgg tgg ac R ttt gag ggt gca gcg aac tt
the amplification efficiency of the corresponding gene, a relative standard curve was constructed.
104 219 165 175 223 252
RESULTS
Statistical analysis
Trx-1 siRNA treatment aggravated neurologic deficit scores and cerebral tissue outcomes following transient MCAO
All data are expressed as mean ± S.E.M. The differences among various groups were analyzed by a one-way analysis of variance (ANOVA) followed by Student–Newman–Keuls for multiple comparisons. The neurological deficit scores were analyzed by Mann– Whitney U test. A probability level of less than 0.05 was considered to be statistically significant. The statistical software package SPSS18.0 was used.
Treatment with Trx-1 siRNA significantly increased neurological scores (Fig. 1A) and brain water content (Fig. 1B) compared to MCAO group and scrambled siRNA-treated animals. No significant difference in brain water content was observed in contralateral hemispheres. In addition, treatment with Trx-1 siRNA also significantly increased infarct volume compared to MCAO group and scrambled siRNA-treated animals (Fig. 1C, D).
Fig. 1. Effects of Trx-1 siRNA on neurologic deficit, brain water content and brain infarct volume. (A) Neurologic deficit scores in the MCAO group were significantly higher than those in the Sham group, but the scores after injection with Trx-1 siRNA were higher than those in the MCAO group. (B) The differences in the brain water content of ipsilateral hemispheres among groups followed the same trend as that of the neurological scores. No statistical difference was observed in contralateral hemispheres (p > 0.05). (C) No ischemic lesion was found in the sham group. The infarct volume in Trx-1 siRNA group was larger than that of the MCAO group. Quantification of C is shown in D. The error bars represent mean ± standard error of the mean (⁄p < 0.01 vs. Sham; #p < 0.01 vs. MCAO, ANOVA). Panel A: n = 20 in Sham group, n = 30 in other groups; Panel B and D: n = 4 in Sham group, n = 6 in other groups.
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Trx-1 siRNA aggravated oxidative stress in rats after MCAO The effects of Trx-1 siRNA on the cortex SOD and MDA levels of subjects are shown in Fig. 2. SOD activity in the cortex was significantly decreased in rats subjected to I/R (Fig. 2A). Animals administered with Trx-1 siRNA showed more reduction of SOD activity than the MCAO group (25.31 ± 2.04 vs. 53.52 ± 5.11, p < 0.05). Likewise, the cortex MDA level (Fig. 2B) was greater in Trx-1 siRNA animals than that in the MCAO group (9.58 ± 0.24 vs. 7.03 ± 0.73, p < 0.05). Trx-1 down-regulation modulates the expression of Prdx1–4 and Prdx-SO3 To explore the impact of Trx-1 on the expression level of Prdx1–4, Western blot and qPCR were performed. Trx-1 expression increased about 3 times at the protein level after I/R (Fig. 3A) and twice at the mRNA level (Fig. 4A). In the Trx-1 siRNA group, the level of Trx-1 was reduced to almost basal level. In parallel, Prdx1–4 levels were significantly increased in the MCAO group (Fig. 3E), however, these increases were prevented by Trx-1 siRNA. In contrast, Western blot analysis using a Prdx-SO3-specific antibody revealed that the level of
Fig. 2. Trx-1 siRNA exacerbated oxidative stress 24 h after focal cerebral I/R injury in rats. (A) SOD activity in the MCAO group was lower than that in the Sham group. The SOD activity was even lower in the Trx-1 siRNA group. (B) MDA level in the Trx-1 siRNA group was significantly higher than that in the MCAO group. The error bars represent mean ± standard error of the mean (⁄p < 0.05 vs. Sham; # p < 0.05 vs. MCAO, ANOVA). Panels A and B: n = 4 in Sham group, n = 6 in other groups.
Prdx1–4 hyperoxidation was higher in the Trx-1 siRNA group than in the MCAO group (Fig. 3C). Interestingly, there was no statistically significant difference in the mRNA level of Prdx1–4 between the siRNA group and MCAO group (Fig. 4B–E). The expression of Trx-1 was suppressed upon knockdown of Nrf2 Both western blot and qPCR results demonstrated that with I/R, Nrf2, and Trx-1 levels are increased after 24-h reperfusion (Figs. 5 and 6). In the MCAO group, Trx-1 was up-regulated to about 3.5 times (Fig. 5C). The expression of Trx-1 was suppressed even less than normal level following Nrf2 knockdown. This was not observed when the scramble siRNA was injected. The variation in the mRNA level of Trx-1 was similar to that of its protein level (Fig. 6B).
DISCUSSION In the present study, we have demonstrated elevation in the expression of Trx-1 in the cortex after cerebral I/R. Trx-1 silencing, on the contrary, effectively weakened this I/R-induced Trx-1 elevation in the brain tissues. Trx-1 down-regulation was associated with reduction of Prdx1–4 while it elevated Prdx-SO3 level in the brains of siRNA-treated rats. Trx-1 silencing resulted in the exacerbation of neurologic deficits and increase in cerebral infarction and brain water content. The concomitant change in the antioxidant activity of Trx-1 was indicated by changes in the representative endogenous antioxidant markers (SOD and MDA). In addition to these observations, we also found a reduction in Trx-1 level after Nrf2 knockdown. This work reveals the role of Trx-1 through Prdx1–4 in maintaining the balance of cellular redox homeostasis after cerebral I/R. To our knowledge, this is the first study demonstrating such involvement in the experimental MCAO model. The significance of the present work is further supported by demonstrating that Trx-1 downregulation targets mechanisms of over-oxidation in brain tissues during focal ischemic reperfusion. Oxygen-derived free radicals have been implicated in the pathogenesis of cerebral infarction after cerebral ischemia (Chan, 1994; Jia et al., 2014). The Trx-1 system, composed of NADPH, Trx reductase, Trx-1, and Prdxs, is ubiquitous in all cells and involved in many redox-dependent signaling pathways. Trx-1 reduces protein disulfide groups in cooperation with Trx reductase and NADPH. It has reported that Trx-1 has radical scavenging activity against singlet oxygen and H2O2-induced cytotoxicity in in vitro assays (Cai et al., 2012; Goy et al., 2014). Takagi observed an increase in both protein and mRNA levels of Trx-1 in the penumbra during permanent MCAO in rats (Takagi et al., 1998). Treatment with recombinant human Trx-1 (rhTrx-1) attenuated ONOO and superoxide anion formation in focal transient cerebral ischemia, thereby diminishing oxidative/nitrative stress-induced neuronal apoptotic cell death in the ischemic brain (Ma et al., 2012). Moreover, C57BL/6 transgenic mice overexpressing human Trx1 are more resistant to various types of
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Fig. 3. Western blot analysis of Trx-1, Prdx1–4, and Prdx-SO3 levels in the ischemic cortex after 24 h of reperfusion in rats. After I/R, Trx-1, and Prdx1–4 levels were up-regulated. However, Trx-1 was effectively down-regulated following administration of Trx-1 siRNA (panel B). Upon Trx-1 knockdown, Prdx1–4 protein levels decreased (panel F, G, H, I). In contrast, the level of Prdx-SO3, an oxidized form of Prdxs, in the group treated with Trx-1 siRNA was higher than that in the MCAO group (panel D). Quantification of A, C, and E is shown in B, D, F, G, H, and I, respectively. The error bars represent mean ± standard error of the mean (⁄p < 0.05 vs. Sham; #p < 0.05 vs. MCAO, ANOVA, n = 4 in Sham group, n = 6 in other groups).
oxidative stress than control mice (Mitsui et al., 2002). These results demonstrate the antioxidant capability of Trx-1. In our experiment, neurologic deficits were aggravated and cerebral infarction and brain water content were increased after administration of Trx-1 siRNA. Moreover, Trx-1 down-regulation increased the level of MDA product and decreased SOD activity, two typical markers reflecting oxidative stress, after I/R. There is a possibility that this exacerbation of oxidative stress injury in the brain could be in part due to the aggravation of acute injury of I/R brought about by Trx-1 siRNA injection prior to injury. The antioxidant effect of Trx-1 is determined by its thiol-reducing activity at its conserved active site: CysGly-Pro-Cys (Lu and Holmgren, 2014). Trx-1 can directly and independently quench singlet oxygen and scavenge hydroxyl radicals; its most widely studied antioxidant capacity has thus far centered on its co-operation with the peroxide scavengers Prdxs (Bell and Hardingham, 2011). Prdxs is a group of enzymes that protect against oxidation using a thiol-containing system. Overexpression of Prdx enzymes in cultured neurons attenuates cell injury caused by hypoxia and reoxygenation (Ichimiya et al.,
1997; Boulos et al., 2007). Moreover, intraventricular administration of recombinant Prdx3 significantly decreased lipid peroxidation and attenuated neuronal apoptosis in ischemia brain (Hwang et al., 2010). Our present study also revealed an increase in the expression level of Prdx after cerebral I/R. Previous studies have demonstrated that typical 2-Cys Prdxs (Prdx1–4) use Trx-1 as a donor of electrons to reduce the disulfide form (Manevich and Fisher, 2005; Bell and Hardingham, 2011). Chae et al. have shown in yeast that thioredoxin peroxidase (TPx) reduces peroxides with the use of electrons provided by the Trx system (Trx, TR, and NADPH). This work has also revealed that Prdx1, Prdx2, and Prdx3 can reduce peroxides in the presence of thioredoxin-1 in rat tissues and cultured cells (Chae et al., 1994, 1999). In accordance with the notion that typical 2-Cys Prdx (Prdx1–4) exhibits antioxidant activity through Trx-1, we observed a significant reduction in Prdx1–4 after treatment with Trx-1 siRNA in this study. However, without Trx-1, the expression of Prdx-SO3 was remarkably up-regulated indicating hyperoxidation of Prdx1–4 cysteine. This finding also reveals a crucial role of Trx-1 in
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Fig. 4. Trx-1 and Prdx1–4 mRNA levels as determined by real-time qPCR. The variation in Trx-1 gene expression was similar to that of its protein expression (panel A). Interestingly, there was no statistically significant difference in the transcriptional level of Prdx1–4 between siRNA group and MCAO group (B, C, D, and E, p > 0.05). The error bars represent mean ± standard error of the mean (⁄p < 0.01 vs. Sham; #p < 0.05 vs. MCAO, ANOVA, n = 4 in Sham group, n = 6 in other groups).
Fig. 5. Effects of Nrf2 siRNA on the expression of Trx-1 in rats. Nrf2 expression level was elevated by about twofold at 24 h after reperfusion (panel B). In the Nrf2 siRNA group, Nrf2 was significantly decreased to less than its basal level. The expression of Trx-1 was elevated by about 3 times in the MCAO group (panel D) but remarkably suppressed after Nrf2 knockdown. This change in the expression level in Trx-1 was similar to that of Nrf2. Quantification of A and C is shown in B and D, respectively. The error bars represent mean ± standard error of the mean (⁄p < 0.01 vs. Sham; # p < 0.01 vs. MCAO, ANOVA, n = 4 in Sham group, n = 6 in other groups).
maintaining the reduction activity of Prdx1–4. Interestingly, no statistically significant difference in the expression levels of Prdx1–4 genes was detected after knockdown of Trx-1. This is probably because Trx-1 regulates Prdx1–4 at primarily at the protein level rather than at the transcription level. The transcriptional regulation of Trx-1 was further explored in our experiment. Recent studies have reported that Trx-1 is regulated by Nrf2-ARE in in vitro trials. Moreover, Trx-1 expression can be transcriptionally induced by hemin in K562 cells by regulating Nrf2/small Maf complex to ARE, and Trx-1 is activated by tBHQ through the Nrf2–ARE pathway (Kim et al., 2001, 2003). Trx is not detectable in Nrf2-deficient mouse embryo
fibroblasts (MEFs) but constitutively expressed at a high level in Keap1-deficient MEFs and in SH-SY5Y cells treated with sulforaphane (Niso-Santano et al., 2010). However, there has been no study examining the relationship between Trx-1 and Nrf2 in a cerebral I/R model. In line with all the previous data, Nrf2 and Trx-1 levels were elevated after cerebral I/R but decreased in Nrf2 knockdown rats. This means that Nrf2 deficiency blocked the effects of I/R to induce Trx-1. It is possible to speculate that Trx-1 gene expression is partly regulated via the Nrf2 pathway. This current study examined the neuroprotective capability of Trx-1 against I/R injury. Our findings suggest that Trx-1 is worth being considered in designing therapeutic strategies against stroke.
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Fig. 6. Effects of Nrf2 siRNA on the transcriptional level of Trx-1. In the MCAO group, Nrf2 and Trx-1 levels were up-regulated. Following knockdown of Nrf2, the expression of Trx-1 was suppressed, which was not observed in the scramble siRNA group. The error bars represent mean ± standard error of the mean (⁄p < 0.01 vs. Sham; # p < 0.01 vs. MCAO, ANOVA, n = 4 in Sham group, n = 6 in other groups).
CONCLUSIONS Trx-1 silencing increased the production of ROS after cerebral I/R which then worsened the extent of brain damage. The increase in ROS levels may involve a decrease in activated Prdx1–4. The suppression of Prdx1–4 more likely stems from the knockdown of Trx-1, in which its expression level is probably in part regulated by Nrf2.
DISCLAIMER STATEMENT No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication. Acknowledgments—This work was supported by The National Natural Science Foundation of China (Nos. 81171090 and 81271460), Natural Science Youth Foundation of China (No. 81301125) and Natural Science Foundation of Chongqing Education Committee, China (No. KJ110313). We would like to thank Dr. Leonard L. Seelig, Jr. for the excellent editorial support.
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(Accepted 28 October 2014) (Available online 8 November 2014)
TARGETING THIOREDOXIN-1 WITH SIRNA EXACERBATES OXIDATIVE STRESS INJURY AFTER CEREBRAL ISCHEMIA/REPERFUSION IN RATS L. LI, a,c K. ZHU, a,c Y. LIU, b,c X. WU, a,c J. WU, b,c Y. ZHAO b,c AND J. ZHAO a,c*
Key words: thioredoxin-1, oxidative stress, peroxiredoxin, nuclear factor erythroid 2-related factor 2.
a
Department of Pathophysiology, Chongqing Medical University, Chongqing, People’s Republic of China b Department of Pathology, Chongqing Medical University, Chongqing, People’s Republic of China
INTRODUCTION
c Institute of Neuroscience, Chongqing Medical University, Chongqing, People’s Republic of China
Stroke is a leading cause of death and long-term disability in developing countries. Oxidative stress caused by reactive oxygen species (ROS) is a critical component of cerebral ischemic injury (Tewari et al., 2014). Oxidative damage can induce neuronal injury and necrosis in brain tissue by oxidizing intracellular molecules such as lipids, proteins, and DNA (Heo et al., 2005; Oztanir et al., 2014). Therefore, it was thought that antioxidant agents, which have the ability to scavenge ROS, could attenuate neurological damage. Thioredoxin-1 (Trx-1) is a 12-kDa ubiquitous protein present in all living cells. It has a wide range of physiological functions, including DNA synthesis, oxidation damage repair, and regulation apoptosis (Lu and Holmgren, 2012; Chang et al., 2013; Sengupta and Holmgren, 2013). Recent study has demonstrated that exogenous rhTrx-1 attenuates post-ischemic brain damage and neuronal apoptosis by reducing oxidative/nitrative stress (Ma et al., 2012). With thiol-reducing activity at its conserved active site, Trx-1 exhibits cytoprotective effects against oxidative stress by scavenging ROS and cooperating with peroxiredoxin (Prdx) (Das and Das, 2000; Lu and Holmgren, 2014). Prdx is a general term that refers to a family of small (22–27 kDa) non-seleno peroxidases, representing a class of important antioxidants in mammals (Park et al., 2014). In fact, Prdx, thioredoxin, and thioredoxin reductase form the mammalian thioredoxin system that plays crucial roles in defending oxidative stress injury, especially typical 2-Cys Prdxs (Prdx1–4) (Zhu et al., 2012). Therefore, elucidating the relationship between Trx-1 and Prdx1–4 in cerebral I/R injury is particularly important. It is well acknowledged that enhanced ROS and electrophiles can evoke a series of antioxidant genes by activating nuclear factor erythroid 2-related factor 2 (Nrf2), a critical transcription factor that regulates the expression of major antioxidant enzymes and phase II detoxification enzymes (Motohashi and Yamamoto, 2004; Wang et al., 2014). Hemin has been shown to induce activation of the thioredoxin gene by regulating Nrf2 through the antioxidant responsive element (ARE) in K562 cells (Kim et al., 2001). Moreover, the expression of Nrf2 has been shown to be significantly up-regulated in the peri-infarct region and to subsequently induce Trx
Abstract—Reactive oxygen species and their detrimental effects on the brain after transient ischemia/reperfusion (I/R) have been implicated in the pathogenesis of ischemic reperfusion injury. Thioredoxin-1 (Trx-1) is an endogenous antioxidant protein that has neuroprotective effects. We hypothesized that Trx-1 plays a crucial role in regulating cerebral I/R injury. To be able to test this, 190 Sprague– Dawley rats were subjected to transient middle cerebral artery occlusion (tMCAO) with Trx-1 siRNA (small interference RNA) injected 24 h prior to ischemia. At 24 h after tMCAO, we measured neurological deficits, infarct volume, and brain water content, and found that neurological dysfunction, brain infarct size, and brain edema were worse in the Trx-1 siRNA group than in the control group. Oxidative stress was evaluated by measuring superoxide dismutase activity and malondialdehyde level. The levels of Trx-1 and its cofactor, peroxiredoxin (Prdx), were significantly decreased after Trx-1 down-regulated. However, there is no significant difference in the Prdx mRNA level after administration of Trx-1 siRNA. In contrast, Prdx-SO3 protein levels were significantly increased in the Trx-1 siRNA group. We also investigated the specific role of nuclear factor erythroid 2-related factor 2 (Nrf2) in Trx-1 induction by knocking down Nrf2. Nrf2 siRNA injection decreased Trx-1 mRNA and protein expression. Our results suggest that the exacerbation of brain damage was associated with enhanced cerebral peroxidation in brain tissues. Moreover, these results revealed that Trx-1, which is more likely regulated by Nrf2, exerts a neuroprotective role probably through maintaining the reduction activity of Prdx1–4. Ó 2014 Published by Elsevier Ltd. on behalf of IBRO.
*Correspondence to: J. Zhao, Department of Pathophysiology, Chongqing Medical University, Yixueyuan Road 1, Chongqing 400016, People’s Republic of China. Tel/fax: +86-23-6848-5868. E-mail address: [email protected] (J. Zhao). Abbreviations: ANOVA, analysis of variance; ARE, antioxidant responsive element; CV, Cresyl Violet; I/R, ischemia/reperfusion; MCA, middle cerebral artery; MCAO, middle cerebral artery occlusion; MDA, malondialdehyde; MEFs, mouse embryo fibroblasts; Nrf2, nuclear factor erythroid 2-related factor 2; Prdx, peroxiredoxin; qPCR, quantitative PCR; ROS, reactive oxygen species; SOD, superoxide dismutase; Trx-1, thioredoxin-1. http://dx.doi.org/10.1016/j.neuroscience.2014.10.066 0306-4522/Ó 2014 Published by Elsevier Ltd. on behalf of IBRO. 815
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expression in male ICR mice following middle cerebral artery (MCA) occlusion (MCAO) (Tanaka et al., 2011). However, in spite of these various observations, the role of Nrf2 in regulating Trx-1 gene expression in the context of ischemic brain injury has yet to be fully investigated. The purpose of this study was to evaluate the role of Trx-1 in ischemic injury using an experimental MCAO model with Trx-1 knock-down. We evaluated whether the Trx1 down-regulation could exacerbate brain damage. Furthermore, we elucidated the relationship between Trx-1 and Prdx1–4 in cerebral I/R injury. Because our results highlight the importance of Trx-1 in modulating the extent of brain damage, we have also investigated the influence of Nrf2 on Trx-1 expression level by interfering Nrf2 gene expression.
EXPERIMENTAL PROCEDURES Animals and groups Adult male Sprague–Dawley rats, weighed 270–310 g (n = 190, 36 died within 24 h) and aged 90 ± 4 d were bred and held at the Experimental Animal Center of Chongqing Medical University. All rats were allowed free access to food and water before the operation under optimal conditions (12/12-h light/dark with humidity 60 ± 5%, 22 ± 3 °C). All experimental animals were randomly allocated to the following groups: sham surgery group (n = 28, no died), untreated controls with MCAO (n = 51, 9 died), scramble siRNA of Trx-1siRNA-injected group (n = 37, 7 died), Trx-1 siRNA group (n = 41, 11 died), scramble siRNA of Nrf2siRNA-injected group (n = 15, 3 died) and Nrf2 siRNA group (n = 18, 6 died). All experimental procedures were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Chongqing Medical University, China. Administration of Trx-1 siRNA and Nrf2 siRNA Trx-1 siRNA (The sense primer 5-AAGCUCGAAGCCAC UAUUATT-3 and antisense primer 5-UAAUAGUGGCUU CGAGCUUTT-3), Nrf2 siRNA (The sense primer 5-CCC UGUGUAAAGCUUUCAATT-3 and antisense primer 5-UUGAAAGCUUUACACAGGGTT-3) were designed and chemically synthesized by GenePharma Corporation, Shanghai, China. Scramble siRNA, which has the same nucleotide composition of the target gene siRNA with no sequence homology to any known rat genes was used as the control. All siRNAs were dissolved in RNase-free water, making the final concentration 2 lg/ll. Rats were anesthetized with 3.5% chloral hydrate (350 mg/kg, ip) and placed in a stereotaxic apparatus (Taimeng Software, Chengdu, China). 10 ll of siRNA or scramble siRNA was injected ipsilaterally into the left lateral cerebral ventricle at 24 h prior to the induction of MCAO, respectively. siRNA or scramble siRNA was slowly injected into the left lateral ventricle over a 20-min duration using a Hamilton microsyringe with the coordinates of 1.0 mm posterior to the bregma, 2.0 mm lateral to the midline, and 4.0 mm ventral to the surface
of the skull under the guidance of a stereotaxic instrument. The injection rate was 0.5 ll/min. After injection, the needle was held in place for 5 min and then removed slowly over 2 min. Cerebral ischemia/reperfusion (I/R) model Transient focal cerebral ischemia was introduced into rats by left MCA occlusion technique according to our previous methods (Chen et al., 2012). In brief, a 4–0 monofilament nylon suture (Beijing Sunbio Biotech Co Ltd, Beijing, China) with a rounded tip was inserted into the left internal carotid artery through the common carotid artery stump and gently advanced to occlude the MCA. After 60 min of MCAO, the suture was removed to restore blood flow. The reperfusion was confirmed by laser Doppler (Periflux System 5000, Perimed AB, Stockholm, Sweden). The same procedure was performed on sham-operated rats, but the MCA was not occluded. Rats that did not show neurological deficits after reperfusion (neurological score < 1) were excluded from the study, as well as animals that died after ischemia induction. Rats that showed neurological deficits immediately after reperfusion (neurological score > 0) but were found to be experiencing skull base or subarachnoid hemorrhage were also excluded from the study. Core body temperatures were monitored with a rectal probe and maintained at 37 °C during the whole procedure. Sample processing All rats were sacrificed at 24 h after reperfusion, and the brains were quickly removed to collect the cerebral cortex for an analysis of oxidative stress assay, western blotting, and real-time quantitative PCR (qPCR). A 4-mm coronal section was taken from the area perfused by the MCA starting at 5 mm from the frontal pole. Fresh cortical tissue was collected from the MCA territory of the ischemic left hemisphere, frozen immediately in liquid nitrogen, and stored at 80 °C until needed for further processing. Tissue samples from all animals were analyzed individually. Evaluation of neurological deficit Neurological deficit scores were evaluated by an examiner blinded to the experimental groups after 24 h of reperfusion. The deficits were scored on a modified scoring system developed by Longa et al. (Longa et al., 1989), as follows: 0, no neurological deficits; 1, failure to extend right forepaw fully; 2, circling to right; 3, falling to right; 4, did not walk spontaneously and has depressed levels of consciousness. The higher the neurological deficit score, the more severe the impairment is of motor motion injury. Brains from these rats were analyzed for water content, infarct volume, oxidative stress analysis, western blot, and real-time qPCR. Measurement of brain water content Brain water content was measured using the standard wet– dry method. Six rats in each group were anesthetized with chloral hydrate and killed by decapitation at 24 h after
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MCAO. The brains were rapidly removed and dissected into the ipsilateral and contralateral hemispheres. The two hemispheres were immediately weighed on an electronic balance to obtain the wet weight and then dried in an oven at 100 °C for 24 h to obtain the dry weight. Brain water content was calculated as follows: Brain water content (%) = (wet weight dry weight)/wet weight 100%. Infarct volume analysis Ischemic infarct volume was measured as described previously (Tureyen et al., 2004). For Cresyl Violet (CV) staining, the slides were immersed in xylene for 30 min, and then in 100%, 95%, and 70% ethanol and doubledistilled water for 5 min each. Next, the slides were stained in 0.1% CV solution for 5 min and then briefly rinsed in double-distilled water. These were then dehydrated again in 70%, 95%, and 100% ethanol for 1 min each. The slices were placed in xylene for another 10 min and then coverslipped. The normal tissue was stained blue whereas the infarct tissue did not stain and appeared white. All the CV-stained sections were scanned using a flatbed scanner and the images were stored as TIFFs. The non-ischemic and ischemic hemisphere infarct areas were measured using ImageJ (ver1.37c, NIH) software. All four infarct area measurements were calculated with a 2-mm distance between the slices. Using these measurements, the total infarct volume was calculated for each brain. To compensate for the effect of brain edema, the corrected volume was calculated using the following equation: Percentage hemisphere lesion volume (% HLV) = {[total infarct volume (left hemisphere volume right hemisphere volume)]/right hemisphere volume} 100%. Determination of superoxide dismutase (SOD) and malondialdehyde (MDA) levels Cortical tissues from 6 rats of each group were collected at 24 h after MCAO. The samples were rinsed, weighed, and then homogenized in 9 volumes of 9 g/l ice-cold saline. Supernatant homogenate was collected after centrifugation at 4000 rpm/min for 10 min at 4 °C. SOD and MDA levels were measured using commercially available assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturers’ instructions. SOD activity was measured by the hydroxylamine method. Absorbance was determined at 550 nm by spectrometry. The MDA level was measured by the thiobarbituric acid method. Absorbance was measured at 532 nm by spectrometry. The content of protein in the supernatant was determined by the Enhanced BCA Protein Assay Kit (Beyotime, Jiangsu, China). Western blot analysis Left cortical samples were recovered and homogenized in the RIPA buffer. Protein extracts were resolved by SDS– PAGE and transferred onto nitro-cellulose membranes. The membranes were blocked in 5% non-fat milk
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containing 20 mM Tris–HCl, pH 7.6, 137 mM NaCl, 0.1% Tween-20 (TBS-T) and then incubated at 4 °C overnight with primary antibodies. The primary antibodies used and their respective dilutions were as follows: rabbit polyclonal antibody to Trx-1 (1:2000, AB9328, Millipore, Boston, USA), rabbit polyclonal to Prdx1 (1:2000, ab59538, Abcam, Cambridge, MA, USA), rabbit polyclonal to Prdx2 (1:2000, ab59539, Abcam, Cambridge, MA, USA), rabbit polyclonal to Prdx3 (1:2000, ab73349, Abcam, Cambridge, MA, USA), mouse monoclonal to Prdx4 (1:2000, ab16943, Abcam, Cambridge, MA, USA), rabbit polyclonal to Prdx-SO3 (1:2000, ab16830, Abcam, Cambridge, MA, USA), rabbit polyclonal to Nrf2 (1:1000, ab31163, Abcam, Cambridge, MA, USA), and mouse monoclonal to GAPDH (1:500, TA-08, ZSGB-BIO, Beijing, China). This step was followed by 2-h incubation with respective HRP-conjugated secondary antibodies (ZSGB-BIO, Beijing, China; dilution 1:3000). Chemiluminescence of protein bands was developed using the ECL kit (Millipore, Temecula, California, USA). The relative density of bands was scanned with imaging densitometer (Bio-Rad, Foster City, CA, USA), and the results were quantified using Quantity One 1-D analysis software. GAPDH was used as an internal loading control. RNA isolation and cDNA synthesis Total RNA from left cortical samples was isolated using the RNAiso Plus reagent (TaKaRa Biotechnology, Dalian, China) according to the manufacturer’s instructions. The RNA content was assessed by A260/A280 ratio and agarose gel electrophoresis prior to performing qPCR. Equivalent amounts of RNA extracted from each sample served as template for cDNA synthesis using the PrimeScriptÒ RT reagent Kit With gDNA Eraser (Perfect Real Time) (TaKaRa Biotechnology, Dalian, China) following the manufacturer’s instructions. Real-time qPCR analysis qPCR was performed on a Bio-Rad CFX-96 Connect Real-Time System (Bio-Rad, Foster City, CA, USA) using TaKaRa SYBRÒ Premix Ex Taqä II (Tli RNaseH Plus) (TaKaRa Biotechnology, Dalian, China). The primer sequences and amplicon sizes are listed in Table 1. The data were analyzed using Bio-Rad CFX Manager software (version 2.0). The expression levels of the genes were normalized to GAPDH. The optimized qPCR assays were carried out with 12.5 ll of SYBRÒ Premix Ex Taqä II (Tli RNaseH Plus), 2 ll of cDNA, 1-ll forward primer (10 lM), 1-ll reverse primer (10 lM), and 8.5 ll of RNase-free water in a total volume of 25 ll, making the final primer concentration 0.4 lM. Amplification began with an initial incubation at 95 °C for 3 min followed by 40 cycles of denaturation at 95 °C for 10 s and annealing and extension at 58 °C for 30 s. A melting curve procedure was added to the Bio-Rad CFX96 Connect Real-Time System to analyze the specificity of the products. Non-template controls containing all reagents except cDNA was included in each PCR run. These controls generated C(t) > 40 in all experiments. To determine
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Table 1. Informatiwon on the qPCR Primers Gene symbol
Gen Bank accession no.
Primer sequence (50 ? 30 )
Product size (b.p.)
Trx-1
NM_053800.3
143
Prdx1
NM_057114.1
Prdx2
NM_017169.1
Prdx3
NM_022540.1
Prdx4
NM_053512.2
Nrf2
NM_031789.2
GAPDH
NM_017008.4
F cct tct ttc att ccc tct gtg a R ccc aac ctt ttg acc ctt ttt a F cat tgc tca gga tta tgg agt c R cat tta ttg tta tct ggc gaa gg F cgt ggt cct ctt ttt cta tcc a R ctt tta gtc aca tca gca agc a F tgc ttt tct tct acc ctt tgg a R cat tct ttc ttg gcg tgt tga t F cct ctg ctg ctg ttc ctg tta c R aaa tct tgg ctt tgc tta ggt g F ctg gtc atc aaa aag tcc cat t R tta ttc ttc cct ctc ctg cgt a F aca gca aca ggg tgg tgg ac R ttt gag ggt gca gcg aac tt
the amplification efficiency of the corresponding gene, a relative standard curve was constructed.
104 219 165 175 223 252
RESULTS
Statistical analysis
Trx-1 siRNA treatment aggravated neurologic deficit scores and cerebral tissue outcomes following transient MCAO
All data are expressed as mean ± S.E.M. The differences among various groups were analyzed by a one-way analysis of variance (ANOVA) followed by Student–Newman–Keuls for multiple comparisons. The neurological deficit scores were analyzed by Mann– Whitney U test. A probability level of less than 0.05 was considered to be statistically significant. The statistical software package SPSS18.0 was used.
Treatment with Trx-1 siRNA significantly increased neurological scores (Fig. 1A) and brain water content (Fig. 1B) compared to MCAO group and scrambled siRNA-treated animals. No significant difference in brain water content was observed in contralateral hemispheres. In addition, treatment with Trx-1 siRNA also significantly increased infarct volume compared to MCAO group and scrambled siRNA-treated animals (Fig. 1C, D).
Fig. 1. Effects of Trx-1 siRNA on neurologic deficit, brain water content and brain infarct volume. (A) Neurologic deficit scores in the MCAO group were significantly higher than those in the Sham group, but the scores after injection with Trx-1 siRNA were higher than those in the MCAO group. (B) The differences in the brain water content of ipsilateral hemispheres among groups followed the same trend as that of the neurological scores. No statistical difference was observed in contralateral hemispheres (p > 0.05). (C) No ischemic lesion was found in the sham group. The infarct volume in Trx-1 siRNA group was larger than that of the MCAO group. Quantification of C is shown in D. The error bars represent mean ± standard error of the mean (⁄p < 0.01 vs. Sham; #p < 0.01 vs. MCAO, ANOVA). Panel A: n = 20 in Sham group, n = 30 in other groups; Panel B and D: n = 4 in Sham group, n = 6 in other groups.
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Trx-1 siRNA aggravated oxidative stress in rats after MCAO The effects of Trx-1 siRNA on the cortex SOD and MDA levels of subjects are shown in Fig. 2. SOD activity in the cortex was significantly decreased in rats subjected to I/R (Fig. 2A). Animals administered with Trx-1 siRNA showed more reduction of SOD activity than the MCAO group (25.31 ± 2.04 vs. 53.52 ± 5.11, p < 0.05). Likewise, the cortex MDA level (Fig. 2B) was greater in Trx-1 siRNA animals than that in the MCAO group (9.58 ± 0.24 vs. 7.03 ± 0.73, p < 0.05). Trx-1 down-regulation modulates the expression of Prdx1–4 and Prdx-SO3 To explore the impact of Trx-1 on the expression level of Prdx1–4, Western blot and qPCR were performed. Trx-1 expression increased about 3 times at the protein level after I/R (Fig. 3A) and twice at the mRNA level (Fig. 4A). In the Trx-1 siRNA group, the level of Trx-1 was reduced to almost basal level. In parallel, Prdx1–4 levels were significantly increased in the MCAO group (Fig. 3E), however, these increases were prevented by Trx-1 siRNA. In contrast, Western blot analysis using a Prdx-SO3-specific antibody revealed that the level of
Fig. 2. Trx-1 siRNA exacerbated oxidative stress 24 h after focal cerebral I/R injury in rats. (A) SOD activity in the MCAO group was lower than that in the Sham group. The SOD activity was even lower in the Trx-1 siRNA group. (B) MDA level in the Trx-1 siRNA group was significantly higher than that in the MCAO group. The error bars represent mean ± standard error of the mean (⁄p < 0.05 vs. Sham; # p < 0.05 vs. MCAO, ANOVA). Panels A and B: n = 4 in Sham group, n = 6 in other groups.
Prdx1–4 hyperoxidation was higher in the Trx-1 siRNA group than in the MCAO group (Fig. 3C). Interestingly, there was no statistically significant difference in the mRNA level of Prdx1–4 between the siRNA group and MCAO group (Fig. 4B–E). The expression of Trx-1 was suppressed upon knockdown of Nrf2 Both western blot and qPCR results demonstrated that with I/R, Nrf2, and Trx-1 levels are increased after 24-h reperfusion (Figs. 5 and 6). In the MCAO group, Trx-1 was up-regulated to about 3.5 times (Fig. 5C). The expression of Trx-1 was suppressed even less than normal level following Nrf2 knockdown. This was not observed when the scramble siRNA was injected. The variation in the mRNA level of Trx-1 was similar to that of its protein level (Fig. 6B).
DISCUSSION In the present study, we have demonstrated elevation in the expression of Trx-1 in the cortex after cerebral I/R. Trx-1 silencing, on the contrary, effectively weakened this I/R-induced Trx-1 elevation in the brain tissues. Trx-1 down-regulation was associated with reduction of Prdx1–4 while it elevated Prdx-SO3 level in the brains of siRNA-treated rats. Trx-1 silencing resulted in the exacerbation of neurologic deficits and increase in cerebral infarction and brain water content. The concomitant change in the antioxidant activity of Trx-1 was indicated by changes in the representative endogenous antioxidant markers (SOD and MDA). In addition to these observations, we also found a reduction in Trx-1 level after Nrf2 knockdown. This work reveals the role of Trx-1 through Prdx1–4 in maintaining the balance of cellular redox homeostasis after cerebral I/R. To our knowledge, this is the first study demonstrating such involvement in the experimental MCAO model. The significance of the present work is further supported by demonstrating that Trx-1 downregulation targets mechanisms of over-oxidation in brain tissues during focal ischemic reperfusion. Oxygen-derived free radicals have been implicated in the pathogenesis of cerebral infarction after cerebral ischemia (Chan, 1994; Jia et al., 2014). The Trx-1 system, composed of NADPH, Trx reductase, Trx-1, and Prdxs, is ubiquitous in all cells and involved in many redox-dependent signaling pathways. Trx-1 reduces protein disulfide groups in cooperation with Trx reductase and NADPH. It has reported that Trx-1 has radical scavenging activity against singlet oxygen and H2O2-induced cytotoxicity in in vitro assays (Cai et al., 2012; Goy et al., 2014). Takagi observed an increase in both protein and mRNA levels of Trx-1 in the penumbra during permanent MCAO in rats (Takagi et al., 1998). Treatment with recombinant human Trx-1 (rhTrx-1) attenuated ONOO and superoxide anion formation in focal transient cerebral ischemia, thereby diminishing oxidative/nitrative stress-induced neuronal apoptotic cell death in the ischemic brain (Ma et al., 2012). Moreover, C57BL/6 transgenic mice overexpressing human Trx1 are more resistant to various types of
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Fig. 3. Western blot analysis of Trx-1, Prdx1–4, and Prdx-SO3 levels in the ischemic cortex after 24 h of reperfusion in rats. After I/R, Trx-1, and Prdx1–4 levels were up-regulated. However, Trx-1 was effectively down-regulated following administration of Trx-1 siRNA (panel B). Upon Trx-1 knockdown, Prdx1–4 protein levels decreased (panel F, G, H, I). In contrast, the level of Prdx-SO3, an oxidized form of Prdxs, in the group treated with Trx-1 siRNA was higher than that in the MCAO group (panel D). Quantification of A, C, and E is shown in B, D, F, G, H, and I, respectively. The error bars represent mean ± standard error of the mean (⁄p < 0.05 vs. Sham; #p < 0.05 vs. MCAO, ANOVA, n = 4 in Sham group, n = 6 in other groups).
oxidative stress than control mice (Mitsui et al., 2002). These results demonstrate the antioxidant capability of Trx-1. In our experiment, neurologic deficits were aggravated and cerebral infarction and brain water content were increased after administration of Trx-1 siRNA. Moreover, Trx-1 down-regulation increased the level of MDA product and decreased SOD activity, two typical markers reflecting oxidative stress, after I/R. There is a possibility that this exacerbation of oxidative stress injury in the brain could be in part due to the aggravation of acute injury of I/R brought about by Trx-1 siRNA injection prior to injury. The antioxidant effect of Trx-1 is determined by its thiol-reducing activity at its conserved active site: CysGly-Pro-Cys (Lu and Holmgren, 2014). Trx-1 can directly and independently quench singlet oxygen and scavenge hydroxyl radicals; its most widely studied antioxidant capacity has thus far centered on its co-operation with the peroxide scavengers Prdxs (Bell and Hardingham, 2011). Prdxs is a group of enzymes that protect against oxidation using a thiol-containing system. Overexpression of Prdx enzymes in cultured neurons attenuates cell injury caused by hypoxia and reoxygenation (Ichimiya et al.,
1997; Boulos et al., 2007). Moreover, intraventricular administration of recombinant Prdx3 significantly decreased lipid peroxidation and attenuated neuronal apoptosis in ischemia brain (Hwang et al., 2010). Our present study also revealed an increase in the expression level of Prdx after cerebral I/R. Previous studies have demonstrated that typical 2-Cys Prdxs (Prdx1–4) use Trx-1 as a donor of electrons to reduce the disulfide form (Manevich and Fisher, 2005; Bell and Hardingham, 2011). Chae et al. have shown in yeast that thioredoxin peroxidase (TPx) reduces peroxides with the use of electrons provided by the Trx system (Trx, TR, and NADPH). This work has also revealed that Prdx1, Prdx2, and Prdx3 can reduce peroxides in the presence of thioredoxin-1 in rat tissues and cultured cells (Chae et al., 1994, 1999). In accordance with the notion that typical 2-Cys Prdx (Prdx1–4) exhibits antioxidant activity through Trx-1, we observed a significant reduction in Prdx1–4 after treatment with Trx-1 siRNA in this study. However, without Trx-1, the expression of Prdx-SO3 was remarkably up-regulated indicating hyperoxidation of Prdx1–4 cysteine. This finding also reveals a crucial role of Trx-1 in
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Fig. 4. Trx-1 and Prdx1–4 mRNA levels as determined by real-time qPCR. The variation in Trx-1 gene expression was similar to that of its protein expression (panel A). Interestingly, there was no statistically significant difference in the transcriptional level of Prdx1–4 between siRNA group and MCAO group (B, C, D, and E, p > 0.05). The error bars represent mean ± standard error of the mean (⁄p < 0.01 vs. Sham; #p < 0.05 vs. MCAO, ANOVA, n = 4 in Sham group, n = 6 in other groups).
Fig. 5. Effects of Nrf2 siRNA on the expression of Trx-1 in rats. Nrf2 expression level was elevated by about twofold at 24 h after reperfusion (panel B). In the Nrf2 siRNA group, Nrf2 was significantly decreased to less than its basal level. The expression of Trx-1 was elevated by about 3 times in the MCAO group (panel D) but remarkably suppressed after Nrf2 knockdown. This change in the expression level in Trx-1 was similar to that of Nrf2. Quantification of A and C is shown in B and D, respectively. The error bars represent mean ± standard error of the mean (⁄p < 0.01 vs. Sham; # p < 0.01 vs. MCAO, ANOVA, n = 4 in Sham group, n = 6 in other groups).
maintaining the reduction activity of Prdx1–4. Interestingly, no statistically significant difference in the expression levels of Prdx1–4 genes was detected after knockdown of Trx-1. This is probably because Trx-1 regulates Prdx1–4 at primarily at the protein level rather than at the transcription level. The transcriptional regulation of Trx-1 was further explored in our experiment. Recent studies have reported that Trx-1 is regulated by Nrf2-ARE in in vitro trials. Moreover, Trx-1 expression can be transcriptionally induced by hemin in K562 cells by regulating Nrf2/small Maf complex to ARE, and Trx-1 is activated by tBHQ through the Nrf2–ARE pathway (Kim et al., 2001, 2003). Trx is not detectable in Nrf2-deficient mouse embryo
fibroblasts (MEFs) but constitutively expressed at a high level in Keap1-deficient MEFs and in SH-SY5Y cells treated with sulforaphane (Niso-Santano et al., 2010). However, there has been no study examining the relationship between Trx-1 and Nrf2 in a cerebral I/R model. In line with all the previous data, Nrf2 and Trx-1 levels were elevated after cerebral I/R but decreased in Nrf2 knockdown rats. This means that Nrf2 deficiency blocked the effects of I/R to induce Trx-1. It is possible to speculate that Trx-1 gene expression is partly regulated via the Nrf2 pathway. This current study examined the neuroprotective capability of Trx-1 against I/R injury. Our findings suggest that Trx-1 is worth being considered in designing therapeutic strategies against stroke.
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Fig. 6. Effects of Nrf2 siRNA on the transcriptional level of Trx-1. In the MCAO group, Nrf2 and Trx-1 levels were up-regulated. Following knockdown of Nrf2, the expression of Trx-1 was suppressed, which was not observed in the scramble siRNA group. The error bars represent mean ± standard error of the mean (⁄p < 0.01 vs. Sham; # p < 0.01 vs. MCAO, ANOVA, n = 4 in Sham group, n = 6 in other groups).
CONCLUSIONS Trx-1 silencing increased the production of ROS after cerebral I/R which then worsened the extent of brain damage. The increase in ROS levels may involve a decrease in activated Prdx1–4. The suppression of Prdx1–4 more likely stems from the knockdown of Trx-1, in which its expression level is probably in part regulated by Nrf2.
DISCLAIMER STATEMENT No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication. Acknowledgments—This work was supported by The National Natural Science Foundation of China (Nos. 81171090 and 81271460), Natural Science Youth Foundation of China (No. 81301125) and Natural Science Foundation of Chongqing Education Committee, China (No. KJ110313). We would like to thank Dr. Leonard L. Seelig, Jr. for the excellent editorial support.
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(Accepted 28 October 2014) (Available online 8 November 2014)