Post-occlusion administration of sodium butyrate attenuates cognitive impairment in a rat model of chronic cerebral hypoperfusion

Pharmacology, Biochemistry and Behavior 135 (2015) 53–59

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Post-occlusion administration of sodium butyrate attenuates cognitive impairment in a rat model of chronic cerebral hypoperfusion Hui Liu 1, Jun-Jian Zhang ⁎, Xiong Li 1, Ying Yang, Xiao-Feng Xie, Ke Hu Department of Neurology, Zhongnan Hospital, Wuhan University, Donghu Road 169#, Wuhan 430071, China Hubei Clinical Research Center for Dementia and Cognitive Impairment, Donghu Road 169#, Wuhan 430071, China

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Article history: Received 26 December 2014 Received in revised form 7 May 2015 Accepted 11 May 2015 Available online 23 May 2015 Keywords: Chronic cerebral hypoperfusion Cognitive impairment Histone acetylation NF-E2 related factor 2 Sodium butyrate

a b s t r a c t Chronic cerebral hypoperfusion (CCH) has been commonly associated with Alzheimer's disease and other types of dementia, but therapies that can improve cerebral blood flow displayed little effect on impaired cognition. Epigenetic intervention with histone deacetylase inhibitors, such as sodium butyrate (SB), on the other hand has been shown to improve cognition in several animal models of dementia. To investigate the effect of SB on cognitive impairment induced by CCH in rats, adult male SD rats were given intraperitoneal injections of SB at a daily dose of 840 mg/kg for 4 weeks, from the 29th day after permanent occlusion of bilateral common carotid arteries (2VO). Learning and memory were assessed by Morris water maze and novel object recognition. Following behavioral tests, western blotting of histone acetylation, of transcription factors, of neuronal/synaptic proteins, were performed using rat hippocampus and cortex. The data showed that SB treatment alleviated hippocampal dependent spatial learning disability in 2VO rats, and altered HDAC1/2 mRNA level, histone H4 acetylation and Nrf2 transcriptional activation in rat hippocampus. Accordingly, cognition-protective effect of SB appeared to be partially mediated by enhancing histone acetylation and hence by facilitating the transcription of Nrf2 downstream genes in the hippocampus. Thus, SB might be considered for putative treatment for CCH-related cognitive impairment. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Chronic cerebral hypoperfusion (CCH) is associated with cognitive dysfunctions in both Alzheimer's disease and other types of dementia (Iadecola, 2013; Osawa et al., 2004). Accordingly, therapies that increase cerebral blood flow have been proposed to improve the cognitive outcome. However, there is strong evidence in the literature that revealed no benefit in neuropsychological function either after carotid endarterectomy or after carotid artery stenting (De Rango et al., 2008). Thus, more effective interventions are urgently needed, particularly based on the underlying mechanisms of CCH induced cognitive dysfunction. Many pathophysiological changes might be involved in the development of CCH induced behavioral impairment, such as oxidative stress, deranged metabolism, protein synthesis abnormalities, neuronal damage, glia activation, and white matter lesions (Farkas et al., 2007; Liu et al., 2012).

⁎ Correspondence to: Jun-Jian Zhang, Department of Neurology, Zhongnan Hospital, Wuhan University, Donghu Road 169#, Wuhan 430071, China. Tel.: +86 27 67812885; fax: +86 27 68758670. E-mail address: [email protected] (J.-J. Zhang). 1 These two authors made equal contribution to this paper.

http://dx.doi.org/10.1016/j.pbb.2015.05.012 0091-3057/© 2015 Elsevier Inc. All rights reserved.

As epigenetics is proposed to be the site of nature and nurture integration, neuroepigenetics is becoming a focused research topic towards understanding learning and memory processes, as well as on therapies of cognitive disorders (Fischer et al., 2010). Acetylation of histones, which is thought to be a prevalent post-translational chromatin modification, has been shown to be involved in various stages of memory (Korzus et al., 2004; Vecsey et al., 2007). On the other hand, epigenetic intervention with histone deacetylase (HDAC) inhibitors such as sodium butyrate (SB), can improve cognition in several animal models of dementia (Fischer et al., 2007; Kilgore et al., 2010; Rane et al., 2012; Sharma and Singh, 2011). Moreover, SB exhibits the neuroprotective effect against cerebral ischemic damage (Kim et al., 2007). The neuroprotective and cognition-enhancing effects of histone deacetylase inhibitors could be attributed to the block of deacetylation of histones and other nuclear proteins and the alteration of the transcriptome in the brain (Graff and Tsai, 2013). As a result, it is hypothesized that SB treatment could be an attractive potentially therapeutic approach of CCH-related cognitive impairment. The present study is therefore designed to explore the effect of HDAC inhibition on cognitive disturbances in a rat model of chronic cerebral hypoperfusion (permanent ligation of bilateral common carotid arteries, 2VO), by administering SB after vascular occlusion. We also investigated whether the SB-induced cognitive protection is associated with enhanced histone acetylation and transcriptional activation.

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2. Materials and methods 2.1. Animals Male Sprague–Dawley (SD) rats (180–250 g) were obtained from the Experimental Animal Center of Hubei Province (Wuhan, China). The rats were group-housed with regular access to food and water and a regular 12-h light/dark cycle. All animals were treated in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the study protocol was approved by the Animal Ethics Committee of the Medical School of Wuhan University. Rats were randomly assigned to three groups: 2VO-SB group (n = 20), 2VO-saline group (n = 20), and sham group (n = 15). 2.2. Experimental protocol and surgery 2VO surgery was performed as described previously (Sun et al., 2010). Rats were anesthetized with 10% chloral hydrate (350 mg/kg, i.p.) and allowed to breathe spontaneously. Both common carotid arteries were exposed over a midline incision and dissections were made between the sternocleidomastoid and the sternohyoid muscles parallel to the trachea. The arteries were carefully freed from their adventitial sheath and vagus nerve, and were occluded by double ligation with 7–0 monofilament sutures. The sham group underwent the same surgery procedure without any artery ligation. The 2VO-SB group received four-week SB injection (840 mg/kg/d, i.p., SigmaAldrich, St. Louis, MO, USA) from the 29th day after the surgery, while both the sham group and the 2VO-saline group received a saline injection. The injection dose was used according to Fischer's report (Fischer et al., 2007). After the four-week SB treatment, rats were euthanized by decapitation under chloral hydrate anesthesia (10%, 350 mg/kg). The hippocampus and cortex were removed and stored in −80 °C for further measurements. 2.3. Behavioral tests Behavioral tests were conducted in the last nine days of SB administration (See Fig. 1). Hippocampal dependent learning and memory were measured by Morris water maze (MWM) test, while non-hippocampal dependent memory was measured by novel object recognition (NOR) test. Rats with vision impairment were excluded from behavioral tests. 2.3.1. MWM test The water maze comprised a circular pool (diameter: 120 cm; height: 60 cm; depth of water: 32 cm) with an unfeatured inner surface. The hidden platform (diameter: 9 cm) was submerged 2 cm below the water surface. Visual cues were posted over the walls of the testing room, by which rats could learn to locate the platform. Swimming paths were recorded by a computerized video tracking and analysis system (SMART-BS, Ethovision, Noldus Information Technology BV, Wageningen, the Netherlands). Rats were gently put into the water in one of four quadrants, facing the wall of the pool. The starting quadrant was varied randomly over the trials. Rats were allowed 60 s to find the escape platform. If a rat failed to find the platform within 60 s, it would

be placed on the platform. Each rat stayed on the platform for 20 s regardless of whether it found the platform. Rats underwent four trials every day with a constant interval of 1 h, lasting for five consecutive days. Escape latency, swim speed, and distance traveled before reaching the platform were measured and analyzed. On the sixth day, the animals were subjected to a 30-s trial without the platform (the probe trial). The time spent in the target quadrant where the platform was located during the training was recorded. 2.3.2. NOR test The experimental apparatus was a white open-field box (50 cm × 40 cm × 40 cm). The objects to be discriminated were white mark cups and colorful glass bottles. All rats were habituated in the box to decrease their novelty stress to the apparatus during the training trial. During habituation, the rats were allowed to freely explore the apparatus in the absence of objects for 10 min each for 2 days. After the second habituation, the rat was placed in the experimental apparatus and allowed to explore two identical objects for 10 min. One hour later, one of the familiar objects (cleaned) and a new object were placed in the same location. Then the rat was placed back in the box for 5 min. The time spent exploring each object and the total time spent exploring both objects were measured. The objects were thoroughly cleaned with 70% ethanol after each trial. A discrimination ratio was calculated as the difference in the time exploring novel and familiar objects, expressed as the ratio of the total time spent on exploring both objects. Rats showing a total exploration time less than 8 s were excluded. 2.4. Real-time polymerase chain reaction (PCR) Total RNA extraction was performed based on the manufacturer's protocol (Invitrogen, Carlsbad, CA, USA). The synthesis of cDNA and PCR amplification from the total RNA was performed by using the one-step real-time PT-PCR kit with SYBR Green (TOYOBO, Osaka, Japan). The PCR cycles were as follows: initial denaturation (50 °C, 2 min; 95 °C, 2 min), 40 cycles (95 °C, 15 s; annealing at 58 °C, 15 s; extension at 72 °C, 45 s) and final extension (72 °C, 10 min) followed by melting curve analysis. Specific amplification was confirmed by melting curve analysis and by analysis of amplified products by agarose gel electrophoresis. PCR primers used were listed in Table 1. The Ct values were normalized with the reference gene β-actin. The changes in each gene expression in the 2VO-saline and 2VO-SB groups were expressed as fold-change relative to sham rats, using the formula 2−△△Ct (Livak and Schmittgen, 2001). 2.5. Nuclear protein extraction Briefly, the tissue was homogenized in ice-cold hypotonic lysis buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mMMgCl2, 0.1 mM EDTA, 0.1 mM EGTA, and 1 mM dithiothreitol) containing proteinase inhibitors (0.5 mM phenyl–methylsulfonylfluoride, 2 μg/mL leupeptin, 2 μg/mL aprotinin, 0.5 mg/mL benzamidine, 5 mM NaF, 2 mM sodium pyrophosphate, and 1 mM sodium orthovanadate) and centrifuged (10,000 rpm) for 1 min at 4 °C. The nuclear pellets were washed with lysis buffer and

Fig. 1. Schematic representation of the experimental sodium butyrate intervention.

H. Liu et al. / Pharmacology, Biochemistry and Behavior 135 (2015) 53–59

3. Results

Table 1 PCR primer information. Genes

Primer-forward

Primer-reverse

HDAC1

5′-TGCTAATGTTGGGAGGAGGTG-3′

HDAC2

5′-GAGGGACGGTATAGATGACG AG-3′ 5′-GTTACACCGTCCGAAATGTTG-3′ 5′-CGAATCCAGCAAATCCTCAAC-3′ 5′-TCTGGATGATGCCAACGAGTC-3′

5′- CATATTGGAAGGGCTGAT GTG-3′ 5′-CACATTTAGCGTGACCTTTG AC-3′ 5′-GATGGAGCGTGAAATCTGGG-3′ 5′-GCAGTCACAAATCCCACAAAC-3′ 5′-CCTGGTCAGCAGTACCACAA ATA-3′ 5′-CACATTGCCAAACCACCACA-3′ 5′-ACAGCCGTGGCAGAACTATC-3′ 5′-GAAACTGAGTGTGAGGACCC ATC-3′ 5′-TAGGAGCCAGGGCAGTAATCT-3′

HDAC3 HDAC8 GCLc GCLm NQO1 HO-1

5′-CTGACATTGAAGCCCAGGAGT-3′ 5′-GTCCATTCCAGCCGACAAC-3′ 5′-GTGACAGAAGAGGCTAAGAC CGC-3′ β-Actin 5′-CGTTGACATCCGTAAAGACC TC-3′

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GCLc: γ-glutamylcysteine ligase-catalytic subunit; GCLm: γ-glutamylcysteine ligasemodulatory subunit; HDAC: histone deacetylase; HO-1: heme oxygenase-1; NQO1: NAD(P)H quinone oxidoreductase-1.

resuspended in an ice-cold, hypertonic, nuclear extract buffer (20 mM HEPES, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, and 1 mM dithiothreitol containing proteinase inhibitors), incubated on ice for 30 min with intermittent vortexing, and centrifuged (10,000 rpm) for 5 min at 4 °C. The protein concentration of the nuclear extracts was determined by the Bradford method. Total protein samples were extracted as described previously (Zhu et al., 2011). 2.6. Western blotting For protein electrophoresis and immunoblotting, samples (40 μg) were resolved by 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred onto 0.45 μm PVDF membranes at 200 mA for 1 h and stained with Ponceau red to confirm equal loading. Blots were blocked in 5% nonfat milk in TBST (20 mM Tris, 0.15 mM NaCl and 0.1% Tween-20) at room temperature for 1 h and then probed with primary antibodies in blocking buffer in 4 °C overnight. Primary antibodies used were: Anti-acetyl Histone H4 (Lys5/8/12/16, Millipore Corporation, Billerica, MA, USA), 1:2000 dilution; Anti-acetyl Histone H3 (Lys14, Millipore Corporation, Billerica, MA, USA), 1:2000 dilution; Anti-CREB (Millipore Chemicon International, Temecula, CA, USA), 1:1000 dilution; Anti-phospho-CREB (Millipore, Billerica, MA, USA), 1:1000 dilution; Anti-HIF-1α (BD transduction laboratories, San Diego, CA, USA), 1:1000 dilution; Anti-NF-κB (Abcam, Cambridge, MA, USA), 1:500 dilution; Anti-MAP-2 (Abcam, Cambridge, MA, USA), 1:1000 dilution; Anti-PSD95 (Gene Tex, Irvine, CA, USA), 1:1000 dilution; Anti-NeuN (Millipore, Billerica, MA, USA), 1:1000 dilution; Anti-BDNF (Millopore, Billerica, MA, USA), 1:1000 dilution; Anti-synaptophysin (Abcam, Cambridge, MA, USA), 1:500 dilution; Anti-Lamin B1 (Abcam, Cambridge, MA, USA), 1:1000 dilution; and Anti-β-actin (Santa Cruz, Dollas, TX, USA), 1:2000 dilution. The blot was visualized by chemiluminescence (SuperSignal West Pico, Pierce, Rockford, Illinois, USA) after incubation in an appropriate secondary antibody. Semiquantification of immunoreactive bands on x-ray film was achieved by analyses of optical density (HPIAS 2000, Tongji Qianping Company, Wuhan, China). Densities were normalized to β-actin or LaminB1. 2.7. Data analysis Data were presented as mean ± standard error of the mean (SEM). Analyses were performed using GraphPad Prism 5.01 (GraphPad Prism software Inc., San Diego, CA, US). Escape latencies in Morris water maze test were compared using two-way ANOVA, followed by a Bonferroni post hoc test. Other data was analyzed using one-way ANOVA, followed by a Bonferroni post hoc test. A p-value of less than 0.05 was considered to be statistically different.

3.1. Performances in learning and memory tests A two-way ANOVA was performed to examine the effect of SB and training time on the escape latency to find the hidden platform in the MWM test. All three groups showed a significant decrease in escape latency across the five-day training during spatial learning (F4, 170 = 66.76, P b 0.0001, Fig. 2A), indicating all rats were able to learn where the platform was located. The learning curve differed significantly between groups (F2, 170 = 31.08, P b 0.0001, Fig. 2A). The analysis followed by Bonferroni post hoc test showed a significantly higher escape latency in the 2VO-saline group compared to the sham group (P b 0.001, Day2–Day4; P b 0.01, Day5; Fig. 2A). The escape latency of the 2VO-SB group gradually decreased over the testing period compared with the 2VO-saline group (P b 0.01, Day 2; P b 0.05, Day 4; Fig. 2A). In the MWM probe trial, a one-way ANOVA showed a significant effect of the treatment (2VO and SB) on the performance of rats in the test (F2, 30 = 7.023, P = 0.0031, Fig. 2B). The 2VO-saline group showed a decreased dwell time in the target quadrant, compared with the sham group (P b 0.05, Fig. 2B). 2VO-SB rats spent significantly more time in the target quadrant than did 2VO-saline rats (P b 0.01, Fig. 2B). Distance swum during the probe trial did not differ between groups (F2, 16 = 0.6433, P = 0.5386, Fig. 2C). In the NOR test, a one-way ANOVA showed a significant effect of the treatment on the performances of rats in the test (F2, 19 = 5.904, P = 0.0101, Fig. 2D). The time percentage spent on exploring the novel object, represented by the discrimination ratio, was significantly less in 2VO-saline rats (P b 0.05, Fig. 2D), whereas SB administration was not able to reverse this trend (P N 0.05, Fig. 2D). 3.2. HDACs mRNA levels and histone acetylation Real-time PCR showed that the transcription of class I HDACs (HDAC1, 2, 3, 8) in both the hippocampus and the cortex was not significantly affected by 2VO (P N 0.05, Fig. 3A&B). However, the mRNA level of HDAC1 and 2 was up-regulated in the hippocampus of 2VO-SB rats (P b 0.05, Fig. 3A). Histone3 (H3, K14) and histone4 (H4, K5, 8, 12, 16) acetylation in both the hippocampus and the cortex was not significantly affected by 2VO (P N 0.05, Fig. 3C–E). SB treatment increased H4 acetylation only in the hippocampus (P b 0.01, Fig. 3C&D). 3.3. Transcription factors and neuronal/synaptic proteins Nuclear protein levels of four transcription factors [(phosphorylated) cAMP-response element binding protein, CREB/p-CREB; nuclear factor-κB, NF-κB; NF-E2 related factor 2, Nrf2; and hypoxia inducible factor-1α, HIF-1α] were assessed in the hippocampus, to reflect indirectly the activation of transcription by histone acetylation. As shown in Fig. 4A&B, only Nrf2 nuclear translocation in the hippocampus decreased in 2VO-saline rats (P b 0.05), and there was a trend of restoration after SB administration (P b 0.05). Immunoreactivity of neuronal/synaptic proteins (neuronal nuclei, NeuN; postsynaptic density protein 95, PSD-95; synaptophysin; and microtubule-associated protein 2, MAP-2) in hippocampal lysates was also not significantly changed in response to either 2VO or SB treatment (P N 0.05, Fig. 4C&D). 3.4. Transcription of Nrf2 down-stream genes To further explore the effect of SB on facilitating Nrf2-induced transcription, mRNA levels of Nrf2 down-steam genes (heme oxygenase 1, HO-1; NAD(P)H: quinone oxidoreductase 1, NQO-1; glutamatecysteine ligase catalytic/regulatory subunit, GCLc/GCLm) were measured.

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Fig. 2. The effect of the sodium butyrate treatment on learning and memory performance following 2VO surgery. (A) Spatial learning in the Morris water maze (MWM) test. Average escape latency (seconds) is shown for the 5 training sessions in the maze. (B) Spatial memory in the MWM test. The time in the target quadrant is shown. (C) Distance swum (cm) in the MWM during the probe trial as a measure for motor function. (D) Object recognition in the novel object recognition (NOR) test. ⁎⁎P b 0.01,⁎P b 0.05, 2VO-SB vs. 2VO-saline; △△△P b 0.001, △△ P b 0.01, 2VO-saline vs. sham. Group size: Sham n = 15, 2VO-saline n = 12, 2VO-SB n = 10. Values represent mean ± SEM.

Real-time PCR showed that four-week SB treatment up-regulated the hippocampal GCLc mRNA level (P b 0.05, Fig. 5).

4. Discussion In the present study, we investigated the effects of SB, an HDAC inhibitor, on cognitive impairment in a rat model of chronic cerebral hypoperfusion. There is ample evidence that histone acetylation plays an important role during learning and memory processes (Fischer et al., 2010). Our data confirmed that SB attenuated hippocampus-dependent learning and memory disability of 2VO rats when given at a postocclusion time point (from the 29th day after the 2VO surgery). To our knowledge, this is the first evidence for the cognition-protective role of SB in cognitive impairment induced by cerebral hemodynamic dysfunction. In line with our results, Sharma et al. (Sharma and Singh, 2011) found that 27-day administration (i.p.) of SB reversed the impairment of learning and memory in a rat model of streptozotocin diabetes induced vascular dementia. HDAC inhibition by suberanilohydroxamic acid and phenylbutyrate has also been revealed to reverse memory deficits in mouse models of Alzheimer's disease (Ricobaraza et al., 2009) and neurodegeneration (Fischer et al., 2007).

SB acts as a pan-HDAC inhibitor, which shows specificity to class I HDACs (HDAC1, 2, 3, 8) (Fischer et al., 2007; Kilgore et al., 2010). In particular, HDAC2 has been identified to be negatively related with cognition. HDAC2 knockout mice showed elevated learning and memory abilities, while the over-expression of HDAC2 led to an obvious dysfunction of memory formation (Guan et al., 2009). Thus, the mRNA levels of these HDACs were measured. Surprisingly, SB treatment in our experiments increased the transcription of class I HDACs in rat hippocampus, which was not significantly affected by experimental cerebral hypoperfusion. In fact, the mechanism of action of HDAC inhibitors is mainly based on the direct combination and interaction of the enzyme and the inhibitor with active site zinc (Marks et al., 2004). Thus the increase of mRNA levels may be attributed to a compensatory transcription due to SB-induced deficient enzyme activities. The potential compensatory mechanism, in turn, might lead to a tolerance to SB treatment, or even reverse the therapeutic effect when SB treatment is terminated, which should be taken into account for the implementation of SB. Moreover, the effect of SB on histone acetylation in 2VO model was assessed. Nuclear protein western blotting showed that the levels of acetylation of histone 3 at lysine 14 (H3K14) and histone 4 at lysine 5, 8, 12, 16 (H4K5, 8, 12, 16) in the brain were not affected by 2VO. In contrast, H4K5, 8, 12, 16 acetylation was up-regulated by SB in the

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Fig. 3. The effect of the sodium butyrate treatment on HDACs mRNA levels and histone acetylation following 2VO surgery. (A&B) Class I HDACs (HDAC1, 2, 3, 8) mRNA levels in the hippocampus and the cortex. (C) Representative images of western blotting of acetylated histones (Ac-H3, Ac-H4). (D&E) Acetylation of histone H3 and H4 in the hippocampus and the cortex. ⁎P b 0.05, ⁎⁎P b 0.01, 2VO-SB vs. 2VO-saline. Group size: Sham n = 3–6, 2VO-saline n = 3–6, 2VO-SB n = 3–6. Values represent mean ± SEM.

hippocampus of the 2VO-SB rats. Similarly, reduced H4K12 acetylation correlates with memory impairment in aged mice (Peleg et al., 2010). In addition, Kim et al. found a reduction in levels of acetylated histone H3 in the ischemic brain hemisphere of rats after permanent middle cerebral artery occlusion (pMCAO), whereas HDAC inhibitors, including SB, valproic acid, trichostatin A, maintained H3K14 acetylation (Kim et al., 2007). It appears that acetylated H4 levels commonly serve as an index of HDAC inhibition in animal models of dementia while H3 acetylation is more involved in cerebral ischemia. Interestingly, the mRNA levels of HDAC as well as histone acetylation, in the cortex, were not impacted by SB treatment. The NOR test also showed no improvement of the hippocampus-independent memory impairment in 2VO-SB rats. These data indicated that the protective effect of SB might be restricted to the hippocampus in this CCH rat model. Considering that the hippocampus is also the most favored brain region for the previous 2VO studies (Farkas et al., 2007), the following investigation focused only in the hippocampus. To identify the underlying mechanism of SB-induced cognitive enhancement, activation status of transcription factors which are involved in cerebral ischemia and cognitive dysfunction, was measured, HDAC inhibition results in chromatin altering to a more open conformation, which then favors transcriptional initiation of variable transcription factors, such as CREB. CREB pathway plays an important role in gene transcription required for synaptic plasticity. Inhibiting HDAC activity is reported to facilitate the HAT actions of CREB-binding protein (CBP), hence improving long-term potentiation (LTP) and memory (Vecsey et al., 2007). HDAC inhibition not only regulates gene transcription directly via enhancing histone acetylation but also selectively

modulates transcription factors via histone-independent hyper acetylation (Bolden et al., 2006). For instance, the p65 unit of NF-κB could be acetylated and thereby associates with CBP, which plays an important role in fear conditioning (Yeh et al., 2004). Moreover, Nrf2, a key transcription factor modulating anti-oxidative capacity in the brain, is also involved in the neuroprotective effects of HDAC inhibitors (Wang et al., 2012). Notably, HIF-1α, being critical in the endogenous neuroprotection during cerebral ischemia, is repressed by HDAC inhibitors, by either reducing functional HIF-1α levels or suppressing HIF-1α transactivation activity (Chen and Sang, 2011). Our data showed that, among the four transcription factors, only Nrf2 nuclear protein level in hippocampus was elevated by SB treatment. In our previous study investigating the change of hippocampal Nrf2 expression in 2VO rats, the DNA binding activity of Nrf2 was found to decreased 8 weeks after 2VO (Yang et al., 2014), which was in agreement with the downregulated nuclear Nrf2 level reported here. In the present investigation neuronal and synaptic proteins in hippocampus were also measured. In line with the slight increase of CREB/p-CREB transcriptional activation, the hippocampal levels of these proteins, generally related to cognitive performance, were not influenced by SB treatment. Together with the Nrf2 data, we inferred that SB-induced cognitive protection in 2VO model might be partly attributed to Nrf2 transcriptional activation. To find additional support for this view, we measured the mRNA levels of Nrf2 down-stream genes, which turned out to be correlated with Nrf2 transcriptional status. Nrf2 mediates the production of a battery of endogenous antioxidative enzymes, such as superoxide dismutases (SODs), catalase, glutathione peroxidases (GPx's), peroxiredoxins (Prx's), NQOs, and

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Fig. 4. The effect of the sodium butyrate treatment on the protein expression of four transcription factors and five neuronal/synaptic proteins following 2VO surgery. (A) Representative images of western blotting of CREB/p-CREB, NF-κB, Nrf2, HIF-1α. (B) The nuclear protein level of CREB/p-CREB, NF-κB, Nrf2, HIF-1α in the hippocampus. (C) Representative images of western blotting of NeuN, PSD-95, synaptophysin, MAP-2, and BDNF. (D) The total protein level of NeuN, PSD-95, synaptophysin, MAP-2, and BDNF in the hippocampus. ⁎P b 0.05, 2VO-SB vs. 2VO-saline; △P b 0.05, 2VO-saline vs. sham. Group size: Sham n = 4–6, 2VO-saline n = 4–6, 2VO-SB n = 4–6. Values represent mean ± SEM. CREB/p-CREB, (phosphorylated) cAMP-response element binding protein; HIF-1α, hypoxia inducible factor-1α; NF-κB, nuclear factor-κB; Nrf2, NF-E2 related factor-2. BDNF, brain-derived neurotrophic factor; MAP-2, microtubule-associated protein 2; NeuN, neuronal nuclei; PSD-95, postsynaptic density protein 95. 2VO: 2VO-saline group; SB: 2VO-SB group.

heme oxygenases (HOs) (Calkins et al., 2009). In cell cultures, Nrf2 activation is known to be neuroprotective against oxidative stressors and mitochondrial toxins (Calkins et al., 2005). The presence of induced Nrf2 has shown to contribute to neuroprotection in vivo, while Nrf2 deficiency led to increased damage (Satoh et al., 2008). On the other hand, oxidative stress has been regarded as a common pathophysiological change and an essential factor in the development of CCH related cognitive impairment (Liu and Zhang, 2012; Xu et al., 2010). Accordingly, the anti-oxidative effect of SB-induced Nrf2 transcriptional activation may contribute to the cognitive enhancement in 2VO-SB rats. In conclusion, SB treatment attenuated hippocampal dependent spatial learning disability in a rat model of CCH, which may be exerted partially through histone acetylation and hence the transcriptional activation of Nrf2 in the hippocampus. Therefore, SB should be considered for putative treatment for CCH-related cognitive impairment. Fig. 5. The effect of the sodium butyrate treatment on the mRNA level of Nrf2 downstream genes following 2VO surgery. ⁎P b 0.05, 2VO-SB vs. 2VO-saline. Group size: Sham n = 4–6, 2VO-saline n = 4–6, 2VO-SB n = 4–6. Values represent mean ± SEM. GCLc/ GCLm, glutamate-cysteine ligase catalytic/regulatory subunit; HO-1, heme oxygenase 1; NQO-1, NAD(P)H: quinone oxidoreductase 1.

Disclosure/conflict of interest None.

H. Liu et al. / Pharmacology, Biochemistry and Behavior 135 (2015) 53–59

Acknowledgments The authors thank Prof. Paul G. M. Luiten (Department of Molecular Neurobiology, University of Groningen, the Netherlands) for providing language help. This study was supported by the National Natural Science Foundation of China (Grant numbers: 81000471, 81171029) and the Fundamental Research Fund for the Central Universities of China (Grant number: 201130302020003). References Bolden, J.E., Peart, M.J., Johnstone, R.W., 2006. Anticancer activities of histone deacetylase inhibitors. Nat Rev Drug Discov 5, 769–784. Calkins, M.J., Jakel, R.J., Johnson, D.A., Chan, K., Kan, Y.W., Johnson, J.A., 2005. Protection from mitochondrial complex II inhibition in vitro and in vivo by Nrf2-mediated transcription. Proc Natl Acad Sci U S A 102, 244–249. Calkins, M.J., Johnson, D.A., Townsend, J.A., Vargas, M.R., Dowell, J.A., Williamson, T.P., et al., 2009. The Nrf2/ARE pathway as a potential therapeutic target in neurodegenerative disease. Antioxid Redox Signal 11, 497–508. Chen, S.Y., Sang, N.L., 2011. Histone deacetylase inhibitors: the epigenetic therapeutics that repress hypoxia-inducible factors. J Biomed Biotechnol 2011 Article ID 197946. http://dx.doi.org/10.1155/2011/197946. De Rango, P., Caso, V., Leys, D., Paciaroni, M., Lenti, M., Cao, P., 2008. The role of carotid artery stenting and carotid endarterectomy in cognitive performance: a systematic review. Stroke 39, 3116–3127. Farkas, E., Luiten, P.G.M., Bari, F., 2007. Permanent, bilateral common carotid artery occlusion in the rat: a model for chronic cerebral hypoperfusion-related neurodegenerative diseases. Brain Res Rev 54, 162–180. Fischer, A., Sananbenesi, F., Wang, X.Y., Dobbin, M., Tsai, L.H., 2007. Recovery of learning and memory is associated with chromatin remodelling. Nature 447 [178-U2]. Fischer, A., Sananbenesi, F., Mungenast, A., Tsai, L.H., 2010. Targeting the correct HDAC(s) to treat cognitive disorders. Trends Pharmacol Sci 31, 605–617. Graff, J., Tsai, L.H., 2013. The potential of HDAC inhibitors as cognitive enhancers. Annu Rev Pharmacol 53, 311–330. Guan, J.S., Haggarty, S.J., Giacometti, E., Dannenberg, J.H., Joseph, N., Gao, J., et al., 2009. HDAC2 negatively regulates memory formation and synaptic plasticity. Nature 459 55-U8. Iadecola, C., 2013. The pathobiology of vascular dementia. Neuron 80, 844–866. Kilgore, M., Miller, C.A., Fass, D.M., Hennig, K.M., Haggarty, S.J., Sweatt, J.D., et al., 2010. Inhibitors of class 1 histone deacetylases reverse contextual memory deficits in a mouse model of Alzheimer's disease. Neuropsychopharmacology 35, 870–880. Kim, H.J., Rowe, M., Ren, M., Hong, J.S., Chen, P.S., Chuang, D.M., 2007. Histone deacetylase inhibitors exhibit anti-inflammatory and neuroprotective effects in a rat permanent ischemic model of stroke: multiple mechanisms of action. J Pharmacol Exp Ther 321, 892–901. Korzus, E., Rosenfeld, M.G., Mayford, M., 2004. CBP histone acetyltransferase activity is a critical component of memory consolidation. Neuron 42, 961–972.

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