Functional research and molecular mechanism of Kainic acid-induced denitrosylation of thioredoxin-1 in rat hippocampus

Accepted Manuscript Functional research and molecular mechanism of Kainic acid-induced denitrosylation of thioredoxin-1 in rat hippocampus Hongning Yang, Ningjun Zhao, Lanxin Lv, Xianliang Yan, Shuqun Hu, Tie Xu PII:

S0197-0186(17)30015-3

DOI:

10.1016/j.neuint.2017.06.004

Reference:

NCI 4091

To appear in:

Neurochemistry International

Received Date: 16 January 2017 Revised Date:

31 May 2017

Accepted Date: 7 June 2017

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ACCEPTED MANUSCRIPT Title:

Functional

Research

and

Molecular

Mechanism

of

Kainic

Acid-induced

Denitrosylation of Thioredoxin-1 in Rat Hippocampus Authors: Hongning Yanga,c,#, Ningjun Zhaoa,b,#, Lanxin Lva, Xianliang Yana,b,*, Shuqun Hua*, Tie Xua,b,* Institute of Emergency Rescue Medicine, Xuzhou Medical University, Xuzhou 221002, China

b

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a

Emergency Center of the Affiliated Hospital of Xuzhou Medical University, Xuzhou, 221002,

China c

Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical University, Xuzhou,

#

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221004, China These authors contributed equally to this work.

*

Name: Tie Xu Tel: +86-(0516)85802051; Fax: +86-(0516)85802051; E-mail: [email protected]

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Name: Shuqun Hu

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Corresponding author:

Tel: +86-(0516)85802187;

Fax: +86-(0516)85802187;

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E-mail: [email protected] Name: Xianliang Yan

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Tel: +86-(0516)85802105; Fax: +86-(0516)85802105;

E-mail: [email protected]

Author Contributions statement: All authors reviewed the manuscript. Tie Xu and Shuqun Hu designed the research, Hongning Yang and Xianliang Yan wrote the main manuscript text, Hongning Yang, Ningjun Zhao and Lanxin Lv Performed the experiments, Hongning Yang and Shuqun Hu analyzed data and prepared figures. Additional Information 1

ACCEPTED MANUSCRIPT Competing financial interests: The authors declare no competing financial interests. Abstract Thioredoxin-1 (Trx1) has long been recognized as a redox regulator, and is implicated in the inhibition of cell apoptosis. Trx1 is essential for the maintenance of the S-nitrosylation of

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molecules in cells. The S-nitrosylation of Trx1 is essential for the physiological function such as preservation of the redox regulatory activity. The mechanisms underlying Trx1 denitrosylation induced by kainate acid (KA) injection still remain uncharacterized. Our results showed that the S-nitrosylation levels of Trx1 were decreased subsequent to KA injection and that the glutamate

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receptor 6 (GluR6) antagonist NS102 could inhibit the denitrosylation of Trx1. Moreover, the denitrosylation of Trx1 following KA treatment could be suppressed by the Fas ligand (FasL) oligodeoxynucleotides

(AS-ODNs),

the

Trx

reductase

(TrxR)

inhibitor

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antisense

dinitrochlorobenzene (DNCB), or the Nitric oxide (NO) donors sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO). Subsequently, these mechanisms were morphologically validated by cresyl violet staining, in situ TUNEL staining to detect the survival of CA1 and CA3/DG pyramidal neurons. NS102, FasL AS-ODNs, GSNO and SNP could provide neuroprotection of the

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pyramidal neurons of CA1 and CA3/dentate gyrus (DG) regions by attenuating Trx1 denitrosylation. Our results also showed that the denitrosylation of Trx1 induced by KA injection can active the caspase-3 which results in apoptosis.

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Keywords: Thioredoxin-1; Denitrosylation; Kainite Acid; Glutamate receptor 6 1. Introduction

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NO as an easily diffusible and highly reactive molecule has been linked to numerous physiological and pathophysiological events (Tuteja, Chandra et al. 2004). Earlier studies showed that NO was an important signaling molecule, and exerted its effect mainly through the NO/cGMP channels, hence inducing cGMP production for signal transduction (Schmidt, Lohmann et al. 1993). Nowadays, more and more attention is being focused on the cGMP-independent NO signaling pathways involving S-nitrosation and denitrosylation (Tannenbaum and Kim 2005, Chvanov, Gerasimenko et al. 2006). Protein S-nitrosylation and denitrosylation has been postulated to be the principal post-translational modification by which NO exerts a myriad of biological effects. Wherein, S-nitrosylation is the covalent attachment of an NO group to a Cys thiol side chain, a fundamental mechanism in cellular signal transduction. The stability of proteins, 2

ACCEPTED MANUSCRIPT cleavage of zymogens and modification of active proteins can be controlled by this post-translational modification process (Benhar, Forrester et al. 2009, Foster, Hess et al. 2009). More recently, nitrosylation has been found to be reversible. Nitrosylated Cys thiol groups can be reduced to free thiols under different conditions, which is defined as denitrosylation and plays an

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important role in signal regulation innumerous diseases (Holmgren 2008, Benhar, Forrester et al. 2009).

KA is a potent exogenous agonist of the KAR, and the KA injection induces epilepsy in rats with attendant neuronal damage, particularly in the limbic structures such as the hippocampal

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pyramidal neurons. The seizures in rodents which induced by KA have been widely used as a model of human temporal lobe epilepsy (Fisher and Alger 1984).

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With a molecular weight of 12 kDa, Human Trx1 is a 104-amino acid protein that exists primarily in the cytosol, but is also found in the nucleus and the plasma. Trx2 is found primarily in the mitochondria (Watson, Yang et al. 2004, Lee, Park et al. 2016). Trx has only two Cysteine residues in the conserved active site, but mammalian cytosolic Trx1 have additional structural cysteines, which are located in positions 62, 69, and 73 (Wu, Liu et al. 2010, Zhang, Zhao et al.

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2016). Trx1 is S-nitrosylated at Cys 69, which is required for the scavenging of reactive oxygen species and the preservation of the redox regulatory activity of Trx1 (Haendeler, Hoffmann et al. 2002, Li and Wan 2013). The Trx system, which comprises Trx proteins, TrxR proteins and

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NADPH, is a major protein disulphide reductase system present in all living organisms. Trx systems have been discovered as a protein disulfide reductase which mediates Cys denitrosylation

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in cell (Hashemy and Holmgren 2008, Wu, Liu et al. 2010, Sengupta and Holmgren 2012). Trx1 per se can denitrosylate the S-nitrosylated proteins (SNO proteins) by the conversion of its free cysteine-thiol in active-site Cys32 and Cys35 (Trx-(SH)2) to dithiol moiety, resulting in a reduced protein (protein-SH) and oxidized Trx1 (Trx-S2). Oxidized Trx1 is reduced (Trx1 reactivation) by NADPH and the seleno-flavoprotein TrxR (Hashemy and Holmgren 2008). The denitrosylation of Trx1 can reduce the S-nitrosylation of procaspase3 at Cys 163, resulting in Caspase3 activation and leading to apoptosis (Mitchell, Morton et al. 2007, Sun, Hao et al. 2013). DNCB, the inhibitors of TrxR, can block the reduction of S-nitrosylated Trx1, therefore attenuating denitrosylation of other proteins. Our study confirmed that the KA-induced Trx1 denitrosylation is via GluR6-containing 3

ACCEPTED MANUSCRIPT kainate receptor (GluR6-KAR) pathway. Previous studies have suggested that KA injection can enhance the assembly of the GluR6-PSD95-MLK3 module and facilitate the c-Jun N-terminal Kinase (JNK) activation, which may up-regulate the expression of the FasL via c-Jun/AP-1-mediated transcriptional regulation and ultimately contribute to Fas receptor-mediated

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apoptosis (Tian, Zhang et al. 2005, Li, Xu et al. 2010, Wu, Li et al. 2015). Fas induces the denitrosylation of Trx1 and then active Caspase-3 and damage of the neuronal cells (Mannick, Hausladen et al. 1999, Sun, Hao et al. 2013). 2. Materials and methods

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2.1 Antibodies and reagents

The following primary antibodies and reagents were used. Antibodies anti-Trx1 (sc-20146),

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anti-Fas (sc-726) and anti-FasL (sc-6237), as well as the reagents DNCB (sc-434) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-procaspase-3 (9662) and anti-caspase-3 (9661) were purchased from Cell Signaling Technology, Inc. (Cell Signaling, MA, USA). 6, 7, 8, 9-Tetrahydro-5-nitro-1H–benz[g]indole-2, 3-dione 3-oxime (NS102, N179), S-nitrosoglutathione (GSNO, N4148), methyl methylthiomethyl sulfoxide (MMTS, 177954),

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(+)-Sodium L-ascorbate (Ascorbate, A7631), and neocuproine (N1501), were purchased from Sigma-Aldrich Co (St. Louis, MO, USA). Kainic acid (EA-123) was purchased from Enzo Life Sciences, Inc. (Plymouth Meeting, PA, USA) (N-(6-(Biotinamido)hexyl)-3'-(2'-pyridyldithio)

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-propionamide (Biotin-HPDP, 21341) was purchased from Thermo Fisher Scientific Inc. (Rockford, IL, USA). BCIP and NBT were obtained from Promega Biotech Co., Ltd. (Beijing,

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China). Guava TUNEL Kit (4500-0121) was purchased from Millipore Co. (Bedford, MA, USA). BCA Kit (P0012) and Methyl Green Staining Solution (C0115) were purchased from Beyotime Co. (Beyotime, Jiangsu, China). Sodium Nitroprusside (SNP) was purchased from a local hospital. FasL antisense oligodeoxynucleotides (FasL AS-ODNs), FasL sense oligodeoxynucleotides (FasL S-ODNs), FasL primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). All the other chemicals used in our experment were obtained from Sigma unless indicated otherwise. 2.2 Drug treatments Unilateral intracerebroventricular injection of 10 µl KA dissolved in 0.9% NaCl (normal saline) was administered at a dose of 0.06 µg/µl in the rats at the coordinates (1.5 mm lateral, 0.8 mm posterior, 3.5 mm deep from the bregma). KAR antagonist NS102 dissolved in 1% 4

ACCEPTED MANUSCRIPT dimethlysulfoxide (DMSO) at a dose of 10 mM was injected (10 µl) 30 min before KA infusion. GSNO (0.1 mg/kg) dissolved in 0.9% NaCl was intracerebroventricularly injected (10 µl) in the rat 30 min before KA infusion. The rats were intraperitoneally injected with SNP (5 mg/kg, dissolved in physiological saline) three times at an interval of 1.5 h, with the first SNP performed

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30 min prior to KA infusion (Zhang, Yan et al. 2011, Liu, Yuan et al. 2013). DNCB (60 µg in 10 µl of 1% DMSO) was injected 40 min before KA infusion. 10 nmol of FasL AS-ODNs dissolved in 10 µl of TE buffer (10 mM Tris-HCl (pH 8.0), 1 Mm EDTA) were intracerebroventricularly injected in the rats at an interval of 24 hours for three consecutive days. The same dose of FasL

5’-CTCTCGGAGTTCTGCCAGCT-3’,

and

for

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S-ODNs or TE buffer was used as a control. The sequence for FasL AS-ODNs was FasL

S-ODNs

was

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5’-AGCTGGCAGAACTCCGAGAG-3’(Zhang, Yan et al. 2011, Liu, Yuan et al. 2013). 2.3 Seizure Model

Adult male Sprague-Dawley (SD) rats weighing 250±10 g were selected (Shanghai Experimental Animal Center, Chinese Academy of Science, China). All experiments were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of

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Laboratory Animals. Seizures were induced by unilateral intracerebroventricular injection of KA

dissolved in sterile saline at a dose of 0.6 µg/10 µl (Lerma, Paternain et al. 1993, Zhang, Yan et al. 2011). Animals were behaviorally monitored for seizure duration of at least 6 h after KA injection.

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The seizures were scored according to Racine (Racine 1972) as follows: class I, behavioral arrest and staring spells; class II, head bobbing and gnawing; class III, unilateral forelimb clonus; class

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IV, bilateral forelimb clonus; class V, severe seizures with loss of postural control, and class VI, seizure-induced death (Racine 1972). Only rats with class IV-V seizures were included in our analyses.

2.4 Sample Preparation

The rats subjected to KA treatment were decapitated three or six hours later, with the

hippocampal CA1 and CA3/DG region isolated, followed by immediate placement in liquid nitrogen for freezing and storage until the commencement of our experiment. When necessary, tissues were homogenized in an ice-cold homogenization buffer containing 50 mM MOPS (pH 7.4), 100 mM KCl, 320 mM sucrose, 50 mM NaF, 0.5 mM MgCl2, 0.2 mM dithiothreitol, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 20 mM sodium pyrophosphate, 20 mM β-phosphoglycerol, 5

ACCEPTED MANUSCRIPT 1 mM p-nitrophenyl phosphate, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, and 5 µg/ml of leupeptin, aprotinin and pepstatin A each. The homogenates were centrifuged at 1000 × g for 10 min at 4 °C. Supernatants were collected, and protein concentrations were determined by the BCA method. Samples were stored at -80℃ and were thawed only once until use.

S-nitrosylation

of

Trx1

was

detected

by

the

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2.5 S-nitrosylation assay Biotin-Switch

method

(Jaffrey,

Erdjument-Bromage et al. 2001, Forrester, Foster et al. 2009), in which the free thiols in S-nitrosylated proteins were blocked by methylation with methyl methanethiosulfonate (MMTS)

produce

free

thiols.

The

free

thiol

then

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and the cysteine residues that had been S-nitrosylated were reduced by a sodium ascorbate to covalently

linked

to

biotin-hexyl

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pyridyldithiopropionamide (HPDP), and thus S-nitrosylation was assayed by a biotin-based analysis. Briefly, the tissue homogenates were diluted with HEN buffer (250 mM HEPES, pH 7.7, 1 mM EDTA, 0.1 mM neocuproine, 1% Nonidet P-40, 150 mM NaCl, 1 mM PMSF, protease inhibitor mixture) and the resulting diluted solution were mixed with an equal volume of MMTS buffer (25 mM HEPES, pH 7.7, 5%SDS, 0.1 mM EDTA, 10 µM neocuproine, 20 mM MMTS)

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and incubated at 50℃ for 30 min with frequent vortexing. With the removal of unreacted MMTS by cold acetone precipitation, the precipitates were resuspended in HENS buffer containing 25 mM HEPES, pH 7.7, 0.1 mM EDTA, 10 µM neocuproine, and 1% sodium dodecyl sulfate (SDS).

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With the addition of 2 volumes of neutralization buffer (20 mM HEPES, pH 7.7, 1 mM EDTA, 100 mM NaCl, 0.5% Triton X-100), the samples were then modified with biotin in the following

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buffer (25 mM HEPES, pH 7.7, 0.1mM EDTA, 1% SDS, 10 µM neocuproine, 10 mM sodium ascorbate, and 0.2 mM biotin-HPDP). With the removal of free biotin-HPDP by cold acetone precipitation, biotinylated proteins were adsorbed onto streptavidin-agarose. The streptavidin adsorbates were then eluted by β-mercaptoethanol (100 mM), isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with an anti-Trx1 antibody. 2.6 Immunoprecipitation and Immunoblotting For immunoprecipitation, tissue homogenates (each containing 400 µg of proteins) were diluted fourfold with HEPES buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, and 1 mM each of EGTA, EDTA, PMSF and Na3VO4). Then the samples were 6

ACCEPTED MANUSCRIPT pre-incubated for 1h with 20 µl of protein A-sepharose CL-4B (Amersham Biosciences, Uppsala, Sweden) at 4°C and centrifuged to remove proteins adhered non-specifically from the A-sepharose. The supernatants were then incubated with specific antibodies (2-5 µg) for 4h or overnight at 4°C. With the addition of Protein A-sepharose mixture, the supernatant was incubated at 4°C for

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another 2h. Samples were centrifuged at 10,000 × g for 2 min at 4 °C, and the pellets were triple rinsed with immunoprecipitation buffer. Bound proteins were eluted by SDS-PAGE loading buffer and boiled at 100 °C for 5 min, then isolated by centrifugation. The supernatants were collected for immunoblot analysis.

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Immunoblotting (Western blot analysis) was performed with 15% SDS-PAGE. Then, proteins were electrotransferred onto nitrocellulose membrane (NC, pore size, 0.2 µm. Amersham

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Biosciences, Buckinghamshire, UK). At the end of blockage for 3 h in Tris-buffered saline with 0.1% Tween 20 (TBST) and 3% bovine serum albumin (BSA), membranes were incubated overnight at 4 °C with primary antibodies in TBST containing 3% BSA. Then membranes were rinsed and incubated with alkaline phosphatase-conjugated secondary antibodies in TBST for 2h for color development using nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate

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(NBT/BCIP) color substrate (Promega, Madison, WI, USA). The densities of the bands on the membrane were scanned and analyzed with an image analyzer (LabWorks Software; UVP, Upland, CA).

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2.7 Histological analysis and TUNEL staining

On the fifth day, rats subjected to KA treatment were perfusion-fixed with 4%

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paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4) under chloral hydrate anesthesia for histological analyses. Brains were removed quickly and further fixed with the same fixation solution overnight at 4°C. Post-fixed brains were embedded in paraffin, followed by preparation of coronal sections using a microtome (RM2155; Leica, Nussloch, Germany). Paraffin-embedded brain sections (5 µm) were deparaffinized with xylene, and rehydrated with ethanol at graded concentrations of 100–70% (v/v), followed by washing with distilled water. Then the sections were stained with 0.1% (w/v) cresyl violet to assess neuronal damage in the hippocampus by light microscopy. The number of surviving hippocampal CA1 or CA3/DG pyramidal cells per 1mm was counted as the neuronal density. TUNEL staining (an ApopTag Peroxidase in Situ Apoptosis Detection Kit) was performed 7

ACCEPTED MANUSCRIPT according to the manufacturer’s protocol with minor modifications. The paraffin-embedded coronal sections were deparaffinized, rehydrated, and then treated with protease K (20 µg/ml) for 15 min at room temperature. Following incubation in PBS (containing 0.1％ Triton X-100) and Methanol (containing 0.3% H2O2), the sections were then incubated with reaction buffer

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containing TdT enzyme at 37°C for 1h. The sections were rinsed in the stop/wash buffer and were further treated with anti-digoxygenin conjugate for 30 min at room temperature, followed by color development in peroxidase substrate. The nuclei were lightly counterstained with 0.5% methyl green.

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2.8 Data Analysis and Statistics

Values were expressed as mean ± standard deviation (SD) and were obtained from no fewer

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than three independent rats. Statistical analysis of the results was conducted with the Student’s t-test or one-way analysis of the variance (ANOVA) followed by the Duncan’s new multiple range method or Newman-Keuls test. P values <0.05 were considered significant. 3. Results 3.1. The KA-induced

Trx1 denitrosylation in the hippocampal CA1 and CA3/DG regions

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through the activation of GluR6-KAR

S-nitrosylation is required for the anti-apoptotic functions of Trx1 in cells (Haendeler, Hoffmann et al. 2002). Zhang et al. reported a concentration and time dependence of KA

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treatment by intracerebroventricular infusion. In this experiment, we also focused on the time course following KA injection at a dose of 0.6 µg/10 µl at 6 hours after KA injection (Zhang, Yan

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et al. 2011). Studies have indicated that KA injection induced the neuronal damage in hippocampal CA1 and CA3/DG regions mainly through GluR6-KAR. To verify whether KA exerts its effect via GluR6-KAR, we conducted our experiment with GluR6-KAR antagonist NS102. Our result revealed that KA injection can induce the denitrosylation of Trx1 and the GluR6-KAR antagonist NS102 could inhibit this denitrosylation, indicating that KA at the designated dose exerted its effects mainly through GluR6-KAR (Fig. 1). 3.2 Exogenous NO donors-inhibited Trx1 denitrosylation following KA treatment S-nitrosylation induced by endogenous NO and exogenous NO has been reported to regulate the activity of a number of metabolic enzymes, oxidoreductases, proteases, protein kinases and phosphatases both in vitro and in vivo, as well as respiratory proteins, receptor/ion channels and 8

ACCEPTED MANUSCRIPT transporters, cytoskeletal and structural components, transcription factors, and regulatory elements (including G proteins), etc. (Zhang, Li et al. 2012). We investigated whether exogenous NO could also influence the denitrosylation of Trx1 resulting in neuronal apoptosis. Thus, some exogenous NO donors such as SNP and GSNO were selected for the determination of their effects on the

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denitrosylation of Trx1 6h after KA infusion. The results of western blotting revealed that treatment with SNP and GSNO inhibited the denitrosylation of Trx1 as a negative control (Fig. 2). Our results supported the notion that KA-induced Trx1 denitrosylation can attenuated by exogenous NO.

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3.3 FasL antisense oligodeoxynucleotide-inhibited Trx1 denitrosylation

Previous studies have confirmed that KA injection can enhance the assembly of the

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GluR6-PSD95-MLK3 module and facilitate the c-Jun N-terminal Kinase (JNK) activation, which may up-regulate the expression of the FasL and ultimately contribute to Fas receptor-mediated apoptosis (Pei, Song et al. 2008, Yu, Xu et al. 2008, Zhang, Yan et al. 2011). We hypothesize that the denitrosylation of Trx1 is due to the upregulation of FasL expression. To test this possibility, we thereby administrated the FasL antisense oligodeoxynucleotides (FasL AS-ODNs) and FasL

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sense oligodeoxynucleotides (FasL S-ODNs) prior to KA infusion to investigate whether FasL AS-ODNs could down-regulate the expression of FasL. As shown in Fig. 3A, only FasL AS-ODNs could down-regulate the FasL expression, and hence the reduced proportion of

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Fas/FasL combination. Meanwhile, the injection of FasL AS-ODNs prior to KA treatment could inhibit the denitrosylation of Trx1, as shown in Fig. 3B. These results indicated that the Fas

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pathway played a role in GluR6-KA receptor-dependent Trx1 denitrosylation and that the inhibition of the Fas pathway could suppress the denitrosylation of Trx1. 3.4 The TrxR-inhibited Trx1 denitrosylation following KA treatment TrxR, a NADPH-dependent selenoflavoprotein, can reduce the disulfide in cysteine moieties

in proteins to the corresponding sulfhydrates. To ascertain the potential function of TrxR, we administered the TrxR inhibitors (DNCB) to observe the effect of TrxR on the denitrosylation of Trx1 6 hours after KA injection. As shown in Fig. 4, administration of DNCB significantly decreased the KA-induced denitrosylation levels of Trx1 in hippocampal CA1 and CA3/DG regions, which supported the hypothesis that TrxR could play an important role in the denitrosylation of Trx1. 9

ACCEPTED MANUSCRIPT 3.5 The Trx1 denitrosylation-induced caspase-3 activation It has been reported that the activation of Fas receptors could lead to the reinforced denitrosylation of procaspase-3, which facilitates the cleavage of procaspase-3 to activated caspase-3 (Mitchell, Morton et al. 2007, Wu, Liu et al. 2010). Moreover, KA infusion could

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induce the denitrosylation of procasepase-3 (Zhang, Yan et al. 2011). We also confirmed that KA injection could activate the Fas/FasL apoptotic pathway and increase the denitrosylation level of Trx1. To investigate whether Trx1 is involved in the denitrosylation of procaspase-3 and the activation of caspase-3, we injected the TrxR inhibitor DNCB 3 hours after KA treatment to detect

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procaspase-3 denitrosylation using the biotin-switch assay. As shown in Fig. 5A, procasepase3 denitrosylation was significantly inhibited in the DNCB group, which supported the hypothesis

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that Trx1 denitrosylation could lead to procasepase-3 denitrosylation. Subsequently, we tested the activation of caspase-3, with the treatment as above. The expression of procaspase-3 and its cleaved larger part (17/19kD) caspase-3 were determined by immunoblotting 6h after KA treatment. As shown in Fig. 5B, the activation of caspase-3 was significantly inhibited in the DNCB group, suggesting the denitrosylation of Trx1 could induce the activation of caspase-3.

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3.6 The protective effects of drugs against the KA-induced injury in hippocampal CA1 and CA3/DG pyramidal neurons through the inhibition of Trx1 denitrosylation To explore whether NS102, GSNO, SNP, DNCB and FasL AS-ODNs could play

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neuroprotection against the death of neurons induced by KA-injectiong Trx1 denitrosylation, cresyl violet staining was conducted to determine the survival of pyramidal neurons in the rat

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hippocampal CA1 and CA3/DG regions. From the Photomicrography of cresyl violet-stained brain sections we known that normal neurons in the pyramidal layer of the hippocampi in the normal saline group exhibited round nuclei, palely stained (Fig. 6Aa,b,c), whereas in the KA group (Fig. 6Ad,e,f) karyopyknosis was apparent, which was sign of cell death. The brain sections from rats pretreated with GSNO (Fig. 6Ag,h,i), SNP (Fig. 6Aj,k,l), NS102 (Fig. 6Ap,q,r), DNCB (Fig. 6As,t,u) and FasL AS-ODNs (Fig. 6Ay,z,A) prior KA injection demonstrated significant neuroprotection compared with those administered with KA alone. The pretreatment with FasL S-ODNs (Fig. 6AB,C,D), DMSO (Fig. 6Am,n,o), TE (Fig. 6Av,w,x) have no that neuroprotection. The population of viable cells within a length of 1 mm was calculated in the normal saline, KA alone, and GSNO, SNP, DMSO, NS102, DNCB, TE, FasL AS-ODNs and FasL S-ODNs prior KA 10

ACCEPTED MANUSCRIPT injection treatment groups, respectively. TUNEL staining was further employed to determine the apoptosis levels in hippocampal CA1 and CA3/DG pyramidal cells. As shown in Fig. 6Bd, e and f, a significant number of TUNEL-positive cells were observed on the fifth day after KA treatment, with some of them displaying the characteristic apoptotic morphological changes as

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karyopyknosis and nuclear hyperchromatism, and the same with DMSO group (Fig. 6Bm,n,o), TE group (Fig. 6Bv,w,x) and FasL S-ODNs (Fig. 6BB,C,D) group. Pretreatment with GSNO (Fig. 6Bg,h,i), SNP (Fig. 6Bj,k,l), NS102 (Fig. 6Bp,q,r), DNCB (Fig. 6Bs,t,u), and FasL AS-ODNs (Fig. 6By,z,A) significantly decreased the number of TUNEL-positive cells. The numbers of viable

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TUNEL-positive cells per 1 mm length were counted in the normal saline, KA alone, GSNO, SNP, DMSO, NS102, DNCB ,TE, FasL AS-ODNs and FasL S-ODNs treatment groups. The above

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results indicated that NS102, GSNO, SNP, DNCB and FasL AS-ODNs protected CA1 and CA3/DG pyramidal cells in the rat hippocampus from the KA-induced apoptosis. 4. Discussion

Our present experiment demonstrated for the first time that KA induces the denitrosylation of Trx1 through activates GluR6-containing KA receptor, which facilitated the up-expression of

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FasL, leading to procaspase-3 denitrosylation and the activation of caspase-3 resulting in neuronal apoptosis. Furthermore, our study also indicates that inhibiting the denitrosylation of Trx1 exerts neuroprotective effect after KA injection.

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S-nitrosylation and S-denitrosylation are crucial protein post-translation modifications which involve the regulation of protein function, such as phosphorylation and dephosphorylation, and

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play important roles in the regulation of the protein kinase activity under both physiological and pathological conditions(Moncada and Bolanos 2006, Benhar, Forrester et al. 2008). S-nitrosylation can positively or negatively regulate protein functions, and the same is true of S-denitrosylation (Lopez-Sanchez, Corrales et al. 2008). S-nitrosylation play a different role in different animal models and different physiological states. It has been shown that the pro-apoptosis kinase JNK3 and apoptosis signal-regulating kinase 1 (ASK1) were activated by S-nitrosylation during cerebral ischemia and reperfusion in the rat hippocampus, while Akt/PKB, the anti-apoptosis kinase, was inactivated by S-nitrosylation (Yasukawa, Tokunaga et al. 2005, Pei, Song et al. 2008, Liu, Yuan et al. 2013). It has been shown that the denitrosylation of procaspase-3 active caspase-3 leading to apoptosis (Sun, Hao et al. 2013). The activity of nNOS is regulated by 11

ACCEPTED MANUSCRIPT S-nitrosylation/denitrosylation (Qu, Miao et al. 2012). In our experiment, we confirmed that neuronal apoptosis was induced by the denitrosylation of Trx1, which was S-nitrosylated under basal conditions. A certain level of S-nitrosylation is needed to maintain the redox of Trx1, whereas excess denitrosylation will result in cell apoptosis after KA treatment. It has been

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indicated that nNOS is a critical source of NO and thereby mediates protein S-nitrosylation. nNOS was S-nitrosylated by exogenous NO donors and suppressed its enzymatic activity(Qu, Miao et al. 2012). We suppose the exogenous NO donors SNP and GSNO to decrease the denitrosylation of Trx1. The result agree with our hypothesis that exogenous NO donor can suppress the

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denitrosylation of Trx1.

Previous studies have demonstrated a potential signaling pathway in brain injury induced by

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KA injection. GluR6-containing KA receptor is activated that enhances the assembly of GluR6-PSD95-MLK3 signal module, and subsequently activates JNK downstream pathways (Li, Xu et al. 2010). Activated JNK phosphorylates c-Jun, a nuclear transcription factor, is, to increase AP-1 transcriptional activity, which modulates transcription of a number of apoptosis genes such as FasL(Lambert, Landau et al. 2003). So we speculated that GluR6 antagonist NS102 and FasL

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AS-ODNs which down-regulate the level of the FasL can decrease the Trx1 denitiosylation. Results from our current study suggest that the increased expression of FasL induced by GluR6 and JNK is involved in the denitrosylation of Trx1. Decreasing its expression by FasL AS-ODS

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attenuates the denitrosylation of Trx1. These results fit well with our hypothesis that KA receptor subunit GluR6-mediated Fas apoptotic pathway results in Trx1 denitiosylation and promoting cell

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It is known that apoptosis pathway includes intrinsic and extrinsic pathways. In our study, we

show that Trx1 is denitrosylated through Fas apoptotic pathway. Fas, a member of the tumor necrosis factor receptor family, is a classic apoptosis receptor due to its intracellular death domains(Lambert, Landau et al. 2003). Binding of FasL with Fas receptors induces trimerization, which recruits caspase-8 via the adapter protein Fas-associating protein with death domain (FADD) in the cytoplasm. The FasL-Fas-FADD-procaspase-8 forms death-inducing signaling complex and procaspase-8 autocatalyzes to activate downstream caspase-7 and caspase-3. Then Poly-ADP ribose polymerase is cleaved by caspase-3 resulting in cell apoptosis (Sun, Hao et al. 2013). Furthermore, the pro-apoptosis proteinase caspase-3 was activated by the denitrosylation of 12

ACCEPTED MANUSCRIPT procasepase-3 (S-nitrosylated at Cys163). In this study we report that the denitrosylation of Trx1 induce procasepase-3 denitrosylated and then cleave caspase-3. It have been reported that the Trx-TrxR system play an important role in protein S-nitrosylation. The denitrosylation of target proteins is achieved by the conversion of reduced

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Trx-(SH)2 to oxidized Trx-S2 with catalyst. Trx-S2 is reduced by NADPH, which is catalyzed by TrxR(Benhar, Forrester et al. 2008). To verify that Trx-TrxR system has a role in the denitrosylation of Trx1 by KA injection, we chose DNCB, the inhibitors of TrxR to investigate the role of Trx-TrxR system on Trx1 denitrosylation. Our results show that, DNCB can inhibit the

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denitrosylation of Trx1 induced by KA injection. From our result it has also been shown that DNCB can inhibit the denitrosylation of procaspase-3 and the activation of caspase-3 by

procaspase-3 denitrosylation.

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KA-injection. TrxR play an important role in the regulation of Trx1 denitrosylation and

Trx1 is a key redox modulator which is functionally conserved across a wide range of species, including plants, bacteria, and mammals. Trx1 acts on other proteins mainly via Trx-TrxR system, which is important to organisms in that it can regulate numerous signal transductions, and its

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dysfunctions have been implicated in several diseases, such as cancer, inflammation, and neurodegenerative and cardiovascular diseases(Lillig and Holmgren 2007). Meanwhile, Trx1 alone can directly interact with other proteins via the formation of disulphide bridges, such as

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transcription factors or the apoptosis signal-regulating kinase 1 (ASK1) (Yang, Wu et al. 2011). As is known, Trx1 has a conserved Cys-Gly-Pro-Cys (the former Cys being Cys32 and the latter

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Cys35) active center that undergoes reversible oxidation to the cystine disulfide (Haendeler, Hoffmann et al. 2002). Likewise, Trx1 has cysteine residues at sites 69 and 73, which are S-nitrosylated in normal physiological conditions. Trx1 S-nitrosylation at Cys69 is required to maintain redox and antiapoptotic functions of Trx1, and also affects the S-nitrosylation of other proteins. The S-nitrosylation of Trx1 at Cys73 is capable of specifically transferring a nitrosothiol to the catalytic nucleophile of caspase-3, and the resulting procaspase-3 interacts with Trx1 to inhibit cell apoptosis(Mitchell, Morton et al. 2007). Our results are consistent with the results that the denitrosylation of Trx1 induced by KA involved in the activation of caspase-3 resulting in cell apoptosis by Trx-TrxR pathway. In summary, we demonstrated that the intracerebroventricular infusion of KA resulted in the 13

ACCEPTED MANUSCRIPT denitrosylation of Trx1, which was facilitated by GluR6-KA receptor. Moreover, NS102, GSNO, SNP, DNCB and FasL AS-ODNs exerted their neuroprotective effects by decreasing the denitrosylation level of Trx1. Meanwhile, Trx1 inhibited the denitrosylation of procaspase-3. Since our present experiment was intended to investigate the effects of FasL on the KA-induced

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denitrosylation of Trx1, as to the mechanisms underpinning the denitrosylation of Trx1, further profound studies are needed so as to explore more ideal therapeutic approaches to epileptic seizures. Nevertheless, more studies are required to elucidate the mechanisms underlying GluR6-KA signaling. It will be of interest to determine the level of Trx1 denitrosylation, which

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might involve other regulators.

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Acknowledgements—This research was supported by the National Natural Science Foundation of China (81271267), “333 Program” of Jiangsu Province（BRA2015068）, Jiangsu Natural Science Foundation (BE2016645, BK20161153), Xuzhou Natural Science Foundation (KC15J0060, KC15SX009), Jiangsu Educational Science Foundation (16KJB310018), a keylab-open grant from the Jiangsu Province Key Laboratory of Anesthesiology of Xuzhou Medical University (KJS1504) and the Foundation of Xuzhou Medical University (2015KJ03) to Hongning Yang.

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Figure Legends

Fig. 1. Effect of the activation of GluR6-containing KAR on the denitrosylation of Trx1 in the rat hippocampal CA1 and CA3/DG regions. The effect of KAR inhibitor NS102 (10 mM) on the KA-induced denitrosylation of Trx1 at 6 h in hippocampal CA1 and CA3/DG regions. S-nitrosylation of Trx1 was examined by biotin-switch assay. Western blotting was performed with anti-Trx1 antibody to determine S-nitrosylation of Trx1. The western blotted bands were scanned and the intensities are expressed as the fold changes with respect to saline treatment. Values are represented as mean ± SD from three independent animals (n=3); *P<0.05 versus saline group. #P<0.05 versus the KA injection group. 16

ACCEPTED MANUSCRIPT Fig. 2. Effect of exogenous NO donors on the denitrosylation of Trx1 in the rat hippocampal CA1 and CA3/DG regions. The effects of GSNO (100 µg/kg) and SNP (5 mg/kg) on the denitrosylation of Trx1 were observed at 6 h after treatment with KA in hippocampal CA1 and CA3/DG regions. Samples underwent treatment with Biotin-Switch method prior to

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western-blotting. Bands were scanned, and the intensities were determined by optical density (OD) measurement. Data were expressed as mean ± SD from three independent animals and were represented as folds versus saline control. n=3. *P<0.05 versus saline group. #P<0.05 versus the KA injection group.

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Fig. 3. Effects of Fas/FasL pathway on the denitrosylation of Trx1. (A) Unilateral intracerebroventricular injections (10 nmol each) of FasL AS-ODNs and FasL S-ODNs were

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administered in the rats at an interval of 24 hours for three consecutive days prior to KA treatment. 6 hours after KA treatment, one of the samples underwent coimmunoprecipitation analysis of FasL with Fas followed by western blotting with a FasL antibody, with the remaining samples immunoblotted with Fas and Trx1 antibodies, respectively. The corresponding western blotted bands were scanned and the OD measurements are represented as the fold changes versus the

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saline control. Data are expressed as the mean ±SD from three independent animals (n=3), *P<0.05 versus the saline group; #P<0.05 versus the KA injection group. (B) Effects of FasL AS-ODNs on the denitrosylation of Trx1 were recorded at 6 h after KA treatment. Samples

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underwent treatment with Biotin-Switch method prior to western-blotting. Bands were scanned, and the intensities were determined by OD measurements. Data were expressed as mean ± SD

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from three independent animals and were represented as folds versus saline control. *P<0.05 versus saline group. #P<0.05 versus the KA injection group (n=3). Fig. 4. Effects of TrxR on the denitrosylation of Trx1. Effects of TrxR inhibitor DNCB on KA-induced Trx1 denitrosylation. Samples were treated with the biotin-switch assay: all the S-nitrosylated proteins were precipitated with streptavidin-agarose, followed by Western-blot analysis with anti-Trx1 antibody to detect S-nitrosylation of Trx1. Bands were scanned, and the intensities were determined by OD measurements. Values are represented as mean ± SD from three independent animals (n=3); *P<0.05 versus saline group. #P<0.05 versus the KA injection group. Fig. 5. Effects of Trx-TrxR system on the denitrosylation of procaspase-3 and the activation 17

ACCEPTED MANUSCRIPT of caspase3. (A) Effects of TrxR inhibitor DNCB (60 µg/10 µl) on the denitrosylation of procaspase-3 at 3 h after KA treatment. Samples were treated with the by the biotin-switch method, followed by Western-blot analysis. (B) Effect of TrxR inhibitor DNCB on the activation of caspase-3 induced by KA at 6 h. The expression level of procaspase-3 and its cleaved larger part

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(17/19 kD) caspase-3 after KA injection were examined at 6 hours by immunoblot analysis. The corresponding bands were scanned and the ODs were represented as folds versus saline control. Data were expressed as mean ± SD from three independent animals (n=3). *P<0.05 versus saline group. #P<0.05 versus the KA injection group.

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Fig. 6. Neuroprotection of the drugs that inhibit the denitrosylation of Trx1 against the KA-induced neuronal injuries to hippocampal CA1 and CA3/DG pyramidal neurons. (A)

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Cresyl violet staining was performed on sections from the hippocampus of rats subjected to treatment with saline (Fig. 6Aa,b,c), KA alone (Fig. 6Ad,e,f), or respective pretreatment with GSNO (Fig. 6Ag,h,i), SNP (Fig. 6Aj,k,l), DMSO (Fig. 6Am,n,o), NS102 (Fig. 6Ap,q,r), DNCB (Fig. 6As,t,u), TE (Fig. 6Av,w,x), FasL AS-ODNs (Fig. 6Ay,z,A) and FasL S-ODNs (Fig. 6AB,C,D) prior to KA treatment. Results were obtained from eight independent animals in each

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experimental group, with the results of a typical experiment demonstrated. The solid squares representing the CA1 and the dashed squares representing CA3/DG region in the left column are shown at higher magnification in the right columns. Scale bars: 200 µm (panel B, magnification

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×40 in the left column) and 10 µm (panel C and D, magnification ×400 in the middle and right columns). The numbers of viable cells were counted within a 1 mm region. *P<0.05 versus the

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saline group; #P<0.05 versus the KA injection group (n=8). (B) Representative hippocampal photomicrographs of TUNEL staining counterstained with methyl green. Rats were subjected to treatment with saline (Fig. 6Ba,b,c), KA alone (Fig. 6Bd,e,f), or respective pretreatment with GSNO (Fig. 6Bg,h,i), SNP (Fig. 6Bj,k,l), DMSO (Fig. 6Bm,n,o), NS102 (Fig. 6Bp,q,r), DNCB (Fig. 6Bs,t,u), TE (Fig. 6Bv,w,x), FasL AS-ODNs (Fig. 6By,z,A) and FasL S-ODNs (Fig. 6BB,C,D) prior to KA treatment. Results were obtained from eight independent animals in each experimental group, with the results of a typical experiment presented. Scale bars: 200 µm (panel B, Magnification: ×40 in the left column) and 10 µm (panel B and C, Magnification: ×400 in the middle and right columns). The numbers of viable TUNEL-positive cells were counted within 1 mm in length. *P<0.05 versus saline group. #P<0.05 versus the KA injection group, n=8. 18

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ACCEPTED MANUSCRIPT Kainate acid injection induced Thioredoxin 1 denitrosylated at cysteine 69. Thioredoxin 1 denitrosylated through the activation of GluR6-KAR. Thioredoxin 1 denitrosylation involved in Fas pathway.

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Thioredoxin 1 denitrosylation induced caspase-3 activation.