Neuroprotective Effect of Resveratrol on Ischemia/ReperfusionInjury in Rats Through TRPC6/CREB Pathways

Yun Lin & Fang Chen & Jiancheng Zhang &

Tingting Wang & Xin Wei & Jing Wu & Yinglu Feng &

Zhongliang Dai & Qingping Wu

Received: 12 January 2013 /Accepted: 5 February 2013 /Published online: 23 February 2013# Springer Science+Business Media New York 2013

Abstract Previous studies have provided evidences that res-veratrol can protect the brain from ischemia/reperfusion injury;the mechanisms of its neuroprotective effects remain unknown.To investigate whether resveratrol has neuroprotective effectson ischemia and reperfusion injury and whether resveratrolexerts its neuroprotective effects through inhibition of calpainproteolysis of TRPC6, a transient middle cerebral artery occlu-sion (MCAO) model was employed in rats. Western blotanalysis was performed to detect the protein levels of aII-spectrin, transient receptor potential canonical (subtype) 6(TRPC6) and phosphorylated cAMP/Ca2+ response element-binding protein (p-CREB). The immunoreactivity of p-CREBand TRPC6 were measured by quantum dot-based immuno-fluorescence analysis. Our results showed that MCAO ratsshowed large cortical infarct volumes and neurological scores.By contrast, resveratrol, when applied for 7 days beforeMCAOonset, significantly reduced infarct volumes and enhanced neu-rological scores at 24 h after reperfusion, and these results wereaccompanied by elevated TRPC6 and p-CREB activity anddecreased calpain activity. When MEK or CaMKIV activitywas inhibited by the addition of PD98059 or KN62, theneuroprotective effects of resveratrol were attenuated, and weobserved a correlated decrease in CREB activity. Our resultsdemonstrated that resveratrol prevented the brain from

ischemia/reperfusion injury through the TRPC6–MEK–CREB and TRPC6–CaMKIV–CREB pathways.

Keywords Cerebral ischemia . CREB . Neuroprotection .

Rat . TRPC

Introduction

Stroke affects 15 million people every year worldwide (Vivienet al. 2011). The majority of ischemic stroke results from anacute thrombosis. Intravenous fibrinolysis is the main treat-ment option for acute stroke (Saver 2011; Zinkstok et al. 2011).Although thrombolytic therapy is successful in animal studies,the treatment has not been found to be effective in humans(Ginsberg 2008). Several mechanisms of neuronal injury instroke have been proposed (Choi 1996). However, the exactpathophysiological mechanisms remain unknown, and there isstill no effective treatment for lessening the resulting neuro-logical damage.

The transient receptor potential channel (TRPC) subfamilyof seven subunits can be divided into four subgroups based ontheir sequence similarities: TRPC1, TRPC2, TRPC4/5, andTRPC3/6/7 (Clapham 2003; Clapham et al. 2003; Montell etal. 2002a, b). TRPC6 has been shown to be a protective factorin promoting neuronal survival (Du et al. 2010). Intracellularcalcium ion (Ca2+) overload is considered to be the majorcause of neuronal death following cerebral ischemia. Theactivation of calpain by intracellular calcium overload leadsto proteolysis of TRPC6 (Du et al. 2010; Vosler et al. 2008). Inischemic stroke, TRPC6 channels in neurons are specificallydownregulated by calpain proteolysis. Blocking calpain-mediated degradation of TRPC6 channels promotes neuronalsurvival against focal cerebral ischemia (Du et al. 2010). The

Yun Lin, Fang Chen, and Jiancheng Zhang contributed equally to thiswork.

Y. Lin : F. Chen : J. Zhang : T. Wang :X. Wei : J. Wu :Y. Feng :Z. Dai :Q. Wu (*)Department of Anesthesiology, Union Hospital, Tongji MedicalCollege, Huazhong University of Science and Technology,1277 Jiefang Avenue,Wuhan 430022, Chinae-mail: wu_qingping1968@yahoo.com.cn

J Mol Neurosci (2013) 50:504–513DOI 10.1007/s12031-013-9977-8

downstream effector for TRPC6 activation is CREB (Jia et al.2007). Activated TRPC6 can activate CREB through theRas/MEK/ERK (Du et al. 2010) and CaM/CaMKIV pathways(Du et al. 2010; Tai et al. 2008; Zhou et al. 2008). Enhanced p-CREB activation has been shown to reduce infarct volumes inthe penumbra region of cerebral ischemia (Zhu et al. 2004).Therefore, inhibition of TRPC6 dissociation is an attractivenew therapeutic strategy for the treatment of ischemic stroke.

Resveratrol is a polyphenol found in grapes, berries, pea-nuts, and medicinal plants such as Polygonum cuspidatum.The most important dietary source of resveratrol is red wine(Baur and Sinclair 2006). Resveratrol has receivedwidespreadattention for its potential use as a therapeutic agent in theprevention and treatment of numerous diseases. Previousstudies suggest that resveratrol exhibits anti-inflammatory,anti-oxidant, and anti-carcinogenic properties (Csiszar 2011;Jackson et al. 2010; Subramanian et al. 2011; Wu and Hsieh2011). Several studies have illustrated that resveratrol isneuroprotective in instances of Parkinson’s disease (PD),Alzheimer’s disease (Hung et al. 2010), and ischemic stroke(Li et al. 2011; Mattson and Cheng 2006; Raval et al. 2006;Tsai et al. 2007). Although a number of studies have investi-gated resveratrol-mediated neuroprotection in ischemic injury,the mechanism by which resveratrol confers neuroprotectionon cerebral ischemia still remains incompletely understood.

The purpose of this study was to determine whetherresveratrol has neuroprotective effects on ischemic neuronsin a middle cerebral artery occlusion (MCAO) rat model.We also sought to verify whether resveratrol improves theneurological status of MCAO rats through the inhibition ofcalpain-mediated TRPC6 proteolysis and the subsequentactivation of CREB through the Ras/MEK/ERK andCaM/CaMKIV pathways.

Materials and Methods

Animals and Treatment

Healthy male Sprague-Dawley rats were purchased fromHunan weasleyg scene of experimental animals Co., Ltd.Experimental protocols were approved by the committee ofexperimental animals of Tongji Medical College andconformed to the internationally accepted ethical standards(Guide for the Care and Use of Laboratory Animals, NIHPublication 85-23, revised 1985). The rats were allowed freeaccess to food and water. Rats weighing 200 to 250 g wererandomly divided into six groups, and each group was againdivided into three subgroups (n = 12 per subgroup) accordingto the time of reperfusion after ischemia (2, 12, and 24 h afterreperfusion): (1) sham operation (group S; subgroup S2, S12,and S24), (2) MCAO (group I; subgroup I2, I12, and I24), (3)ischemia combined with resveratrol treatment (group R;

subgroup R2, R12, and R24), (4) ischemia combined withresveratrol plus PD98059 treatment (group P; subgroup P2,P12, and P24), (5) ischemia combined with resveratrol plusKN62 treatment (group K; subgroup K2, K12, and K24), and(6) ischemia combined with resveratrol plus PD98059 andKN62 treatment (group C; subgroup C2, C12, and C24).Another 36 rats were randomly divided into four groups (n =9 per group): (1) group I: MCAO group, (2) group M: MCAO+ PD98059 treatment, (3) group Ca: MCAO + KN62 treat-ment; and (4) group D: MCAO + PD98059 + KN62 treatment.

Resveratrol (200 mg/kg, i.p.; Sigma-Aldrich, St. Louis,MO, USA) or 1 % DMSO (Sigma-Aldrich, St. Louis, MO,USA) was continuously administered to rats for 6 days; onthe seventh day, the last administration was performed 1 hbefore surgery. Rats were given PD98059 (dissolved in 1 %DMSO; 0.75 mg/rat) by intraperitoneal injection 20 minbefore MCA occlusion. KN62 (dissolved in 1 % DMSO;5 μg/rat) was administered by intracerebroventricular (ICV)injection 20 min before MCA occlusion.

Focal Cerebral Ischemia

Transient focal cerebral ischemia was produced by theMCAO procedure as described previously (Longa et al.1989). Briefly, rats were anesthetized with chloral hydrate(400 mg/kg, i.p.). The right common carotid artery, externalcarotid artery, and internal carotid artery were isolated. A 4-0 monofilament nylon suture (Beijing Sunbio Biotech Co.Ltd., Beijing, China) with a rounded tip was introducedthrough the internal carotid artery until a slight resistancewas felt, thereby to block the origin of the middle cerebralartery. After 2 h of ischemia, reperfusion was accomplishedby withdrawing the filament. Sham-operated rats were ma-nipulated in the same way, but the MCA was not occluded.Body temperature was maintained between 36.5 and 37 °Cwith a heating pad during the entire experiment.

Measurement of the Infarct Volume

At 24 h after reperfusion, rats were decapitated, and the brainswere rapidly removed and frozen at −20 °C for 10 min. Theolfactory bulb, frontal pole, cerebellum, and part of the lowerbrain stemwere cut into five coronal sections, each 2-mm thick.The sections were stained with 2 % 2,3,5-triphenyltetrazoliumchloride (TTC; Sigma, St. Louis, MO, USA) for 30 min at 37 °C and then followed by overnight immersion in 4 % parafor-maldehyde. The infarct area of each TTC-stained section wasmeasured using a computerized image analysis system (ImageJ software). To compensate for the effect of brain edema, thecorrected infarct volume was calculated as follows: percent-age of corrected infarct volume = {[total lesion volume −(ipsilateral hemisphere volume − contralateral hemispherevolume)] / contralateral hemisphere volume} × 100.

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Neurological Deficit Evaluation

Neurological function of rats 24 h after reperfusion wasassessed using a standard scoring system (Menzies et al.1992). Five categories were scored as follows: 0 = no apparentdeficits, 1 = contralateral forelimb flexion, 2 = decreased gripof contralateral forelimb while the tail was pulled, 3 = spon-taneous movement in all directions and contralateral circling ifpulled by the tail, and 4 = spontaneous contralateral circling.Rats with scores of 0 or 4 were rejected.

Western Blot Analysis

At 2, 12, and 24 h after reperfusion, all the rats were euthanizedby decapitation, and the infarct side of the cortex washarvested. The tissues were immediately frozen in liquid ni-trogen and stored at −80 °C. Total protein extraction wasperformed using a commercially available kit (KGP250,Nanjing Keygen Biotech Co. Ltd., Nanjing, China) accordingto the manufacturer’s protocol. Total protein extracts wereprepared for protein determination and analyzed by Westernblotting for TRPC6 and aII-spectrin. Nuclear protein extractionwas performed according to a commercial protocol (FermentasInternational, Glen Burnie, MD, USA). Nuclear protein ex-tracts were stored at −80 °C prior to assaying protein expres-sion of p-CREB. Protein concentrationsweremeasured using aBCA protein assay. Equal amounts of protein from differentgroups were subjected to SDS-PAGE and then transferred ontopolyvinylidene difluoridemembranes. After blockingwith 5%nonfat dry milk in TBS-T buffer for 1 h, the membranes wereincubated with primary antibodies of anti-aII-spectrin(1:1,000; Enzo Life Sciences, Plymouth Meeting, PA, USA),anti-TRPC6 (1:1,000; Abcam, Cambridge, UK), anti-p-CREB(1:1,000; Cell Signaling Technology, Danvers, MA, USA),anti-lamin B1 (1:500; Bioworld, Minneapolis, MN, USA),and anti-GAPDH (1:1,000, Proteintech Group, Inc.) at 4 °Covernight, respectively. Three 10-min TBST elutions werefollowed by incubation with horseradish peroxidase-labeledsecond antibodies of anti-mouse IgG antibody or anti-rabbitIgG antibody (1:5,000; Proteintech Group, Inc., Wuhan,Hubei, China), respectively. Labeled proteins were detectedwith the ChemiDocXRS+ chemiluminescence imaging system(Bio-Rad, Hercules, CA, USA). Bands were quantified usinglab imaging software. The experiments were repeated intriplicate.

Quantum Dot-Based Immunofluorescence

At 2, 12, and 24 h after reperfusion, rats were perfused withcold 250 ml of 0.9 % saline followed by 100 ml of 4 %paraformaldehyde in PBS (pH7.4). The brains were thenrapidly removed, blocked, and embedded in paraffin.Paraffin-embedded brains were cut into 4-μm-thick sections

according to standard procedures. The paraffin sections (n =3) were incubated overnight with antibodies against TRPC6(1:100; Abcam, Cambridge, UK) and p-CREB (1:100; CellSignaling Technology, Danvers, MA, USA) at 4 °C afterblocking with BSA. The samples were then incubated with abiotinylated secondary antibody at 37 °C for 30 min. Afterblocking with BSA, the paraffin sections were incubated withstreptavidin-conjugated QDs605 (1:100; Wuhan JiayuanQuantum Dot Co, Ltd., Wuhan, Hubei, China). TRPC6- andp-CREB-positive cells were measured at ×200 magnificationsper visual field in the cortex, andwe analyzed three visual fieldsper section and three brain sections per rat. Fluorescent signalswere detected with a fluorescence microscope (BX51,Olympus, Tokyo, Japan), and signal intensities were collectedfor statistical analysis. Images were taken with a Dopplerimaging system (Nuance Fx, CRi, Hopkinton, MA, USA).

Statistical Analysis

All values are expressed as the mean ± SEM. ANOVAfollowed by a post hoc Newman–Keuls test was performedfor the statistical comparison of several groups. An unpairedt test was used for comparison of two groups. P < 0.05 wasconsidered to be statistically significant. Statistical analyseswere performed by SPSS version 12.0.

Results

Physiological Parameters

Cranial temperature, arterial oxygen tension (PaO2), arterialcarbon dioxide tension (PaCO2), and arterial pH from ratswere recorded before ischemia, 60 min after ischemia, and2, 12, and 24 h after reperfusion (n = 6 at each time point).No statistical significance was noted among different timepoints for any of the physiologic parameters including cra-nial temperature (37.4 ± 0.1 °C, 37.0 ± 0.2 °C, 37.4 ± 0.2 °C,37.6 ± 0.3 °C, and 37.3 ± 0.1 °C, respectively), PaO2 (97.1 ±5.4 mmHg, 96.3 ± 6.3 mmHg, 93.8 ± 6.1 mmHg, 95.2 ±5.5 mmHg, and 94.4 ± 6.9 mmHg, respectively), PaCO2

(37.6 ± 5.3 mmHg, 38.9 ± 4.4 mmHg, 36.7 ± 3.9 mmHg,35.9 ± 3.2 mmHg, and 39.2 ± 4.9 mmHg, respectively), orpH (7.38 ± 0.06, 7.35 ± 0.10, 7.34 ± 0.09, 7.37 ± 0.11, and7.35 ± 0.07, respectively).

Resveratrol-Ameliorated Neurological Dysfunctionsand Reduced Brain Infarction at 24 h After Reperfusion

TTC staining showed that pretreatment with resveratrolreduced the infarct volumes. The infarct volumes inresveratrol-treated rats were markedly smaller than thoseof rats in the MCAO group (P < 0.01). Compared with the

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resveratrol-treated group, the infarct areas of the PD98059-or KN62-treated groups were mainly invariant (P > 0.05).When both PD98059 and KN62 were administered, theinfarct volumes were significantly increased compared tothose of group R (P < 0.01; Fig. 1a).

The neurological deficit score was significantly increasedin the MCAO group compared with that of the sham-operated group at 2, 12, and 24 h after reperfusion (P <0.01; Fig. 1b). When pretreated with resveratrol, the scorewas significantly decreased at 24 h compared with that ofthe MCAO group (P < 0.01).

Pretreatment with Resveratrol Inhibited the Activationof Calpain and Reduced the Degradation of TRPC6in Ischemia/Reperfusion Injury

Western blot analysis showed that the protein levels of calpain-specific aII-spectrin breakdown products (SBDP) of 145 kDa(SBDP145) were significantly increased in theMCAO group at12 and 24 h after reperfusion compared with the sham-operatedgroup (P < 0.05; Fig. 2a, b). Resveratrol treatment significantlyreduced the protein levels of SBDP145 at 12 and 24 h afterreperfusion (P < 0.05 and P < 0.01, respectively).

The levels of TRPC6 in the cerebral cortex were decreasedconsiderably in the MCAO group as early as 2 h after

reperfusion (P < 0.01; Fig. 2c, d). This significant decreasewas also observed at 12 and 24 h (P < 0.01). Pretreatment withresveratrol did not reduce the degradation of TRPC6 in cerebralischemia at 2 h after reperfusion, but resveratrol pretreatmentinhibited the degradation of TRPC6 at 12 h (P < 0.01) and 24 h(P < 0.01). The delayed effect of resveratrol in inhibiting thedegradation of TRPC6 corresponded with a reduction incalpain activity, which suggested that resveratrol exerted itsneuroprotective effects through the inhibition of calpain-mediated TRPC6 degradation. Immunofluorescence resultsshowed a cytomembrane staining pattern of TRPC6 proteinin neurons of the cerebral cortex, and this finding was corrob-orated by the results of Western blot analysis (Fig. 2e, f).

PD98059 or KN62 had No Effect on Ischemic Strokein Rats at 24 h After Reperfusion

To study the effect of PD98059 or KN62 in the stroke rats,PD98059 or KN62 was administrated 20 min prior to theoperation. Interestingly, applying PD98059 or KN62 alone,no statistical significance in the protein levels of p-CREBwas noted (Fig. 3a). There was no significant differencebetween the four groups during the whole process of ische-mia and reperfusion through measuring the infarct volumesand neurological scores (Fig. 3b, c).

Fig. 1 Neuroprotective effects of resveratrol on ischemic stroke after24 h of reperfusion. a Representative TTC-stained brain sections (n = 6).b Neurological deficit score (n = 6). Group S: sham operation, group I:MCAO, group R: MCAO + resveratrol, group P: MCAO + resveratrol +

PD98059, group K:MCAO + resveratrol + KN62, and group C: MCAO +resveratrol + PD98059 + KN62. Values are mean ± SEM. **P < 0.01compared to group R

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Resveratrol Maintained the Levels of p-CREBvia Both the TRPC6-Ras/MEK/ERKand TRPC6-CaMKIV Pathways

Compared with the sham-operated group, the level of p-CREBprotein in theMCAO group was significantly decreased 12 and

24 h after reperfusion (P < 0.01), but not 2 h (P > 0.05).Pretreatment with resveratrol for 7 days significantly enhancedthe phosphorylation level of CREB at 12 and 24 h (P < 0.01;Fig. 4a, b).When PD98059 or KN62was administrated 20minprior to the operation, the p-CREB protein levels in theresveratrol-treated rats were significantly decreased at 12 and

Fig. 2 Immunoblot analyses of calpain-specific aII-spectrin break-down products of 145 kDa (SBDP145) (a) and TRPC6 (c) levels at2, 12, and 24 h after reperfusion. Graphs show the densitometricanalysis of the ratio of the densities of SBDP145/GAPDH band (n =3) (b) and TRPC6/GAPDH band (n = 3) (d). TRPC6 immunoreactivity

(×200) (e). Staining was present within the cell membrane. Quantifica-tion of the levels of TRPC6 in the ipsilateral cortex at the indicated timesor sham surgery (n = 3) (f). Group S, sham: sham operation, group I:MCAO, and group R: MCAO + resveratrol. Values are mean ± SEM.*P < 0.05 and **P < 0.01

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24 h (P < 0.01). Immunofluorescence analysis showed a nu-clear staining pattern of TRPC6 in neurons of the cerebralcortex, and we obtained similar results by Western blottinganalysis (Fig. 5a, b).

Discussion

In this study, we demonstrated that pretreatment with resvera-trol for 7 days significantly decreased neurological deficits andinfarct volumes accompanied by elevated TRPC6 and p-CREBactivity and decreased SBDP145 activity following cerebralischemia/reperfusion injury. These results were consistent withthe previous studies (Li et al. 2011; Mattson and Cheng 2006;Raval et al. 2006; Tsai et al. 2007). Our results also clearlyshowed that resveratrol exerted strong neuroprotective effects

against ischemic injury through the TRPC6–MEK–CREB andTRPC6–CaMKIV–CREB pathways.

The canonical transient receptor potential channels arenonselective cation channels that are expressed in a varietyof multicellular organisms with different functions (Montellet al. 2002a, b). Brain-derived neurotrophic factor (BDNF)is known to promote neuronal survival and differentiationand to guide axon extension both in vitro and in vivo. In ratcerebellar granule neuron cultures, TRPC is required forBDNF’s protective effect (Jia et al. 2007). Another researchshowed that TRPC6 was required for BDNF-inducedchemo-attraction of axonal growth cone attraction (Li et al.2005). In addition, TRPC6 promoted the survival of retinalganglion cells in retinal ischemic injury in a rat model ofretinal ischemia/reperfusion (Wang et al. 2010). After ische-mia, the protein level of TRPC6 in neurons was specifically

Fig. 3 Effect of PD98059 or KN62 in the stroke rats after 24 h ofreperfusion. a Western blot analysis of p-CREB (n = 3). Nuclear proteinextracts were prepared from the ipsilateral cortex. Quantification of p-CREB assessed by Western blot analysis was normalized to the expres-sion level of lamin B1. b Representative 2,3,5-triphenyltetrazolium

chloride staining of the cerebral infarct in the rat brain (n = 6). cQuantification of neurological scores (n = 6). Group S: sham operation,group I: MCAO, group M: MCAO + PD98059, group Ca: MCAO +KN62, and group D: MCAO + PD98059 + KN62. Values are mean ±SEM. *P < 0.05 compared to sham operation

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Fig. 4 Effects of resveratrol pretreatment on the protein levels of p-CREB at 2, 12, and 24 h after reperfusion. a Representative Westernblot showing the effect of resveratrol pretreatment on the protein levelsof p-CREB and the suppressive effects of PD98059 and KN62 at 2, 12,and 24 h after reperfusion. b Graphs show the densitometric anal-ysis of the ratio of the densities of p-CREB/lamin B1 band (n = 3).

Group S: sham operation, group I: MCAO, group R: MCAO +resveratrol, group P: MCAO + resveratrol + PD98059 (MEK inhibitor),group K: MCAO + resveratrol + KN62 (CaMKIV inhibitor), and groupC: MCAO + resveratrol + PD98059 + KN62. Values are mean ± SEM.*P < 0.05 and **P < 0.01.

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downregulated, and this downregulation was specifically me-diated via NMDA receptor-dependent calpain proteolysis.TRPC6 proteolysis contributed to ischemic neuronal cell death,and suppression of TRPC6 degradation promoted neuronalsurvival and prevented ischemic brain damage (Du et al.2010; Tai et al. 2008). Therefore, inhibition of the activity ofcalpain or reduction of TRPC6 degradation is likely an effectivemethod for protecting the brain from ischemia/reperfusion in-jury. In the present study, when rats suffered from ischemia, thecalpain activity increased 12 h after reperfusion and peaked at24 h. These results were mainly in agreement with the previousstudies (Neumar et al. 2001; Zhang et al. 2002). Our data alsoshowed that the protein levels and immune activity of TRPC6were lower in the ischemic group compared with the sham-operated group, beginning as early as 2 h after reperfusion andreaching its lowest level at 24 h. Resveratrol pretreatmentsignificantly reduced the activity of calpain at 12 and 24 h afterreperfusion. In addition, resveratrol significantly increased theprotein levels of TRPC6 at 12 and 24 h. Taken together, ourresults clearly demonstrated that resveratrol exerted its

neuroprotective effects by inhibiting calpain-mediated TRPC6degradation.

TRPC6-induced neuroprotection was executed by p-CREB. The nuclear transcription factor CREB has manyfunctions; the active form is p-CREB. Phosphorylation ofserine-133 in CREB allows it to contact its co-activator,CREB-binding protein/p300, and is necessary for its activa-tion. A previous study has provided evidence that p-CREBcan stimulate neurogenesis and prevent infarct expansion inthe penumbra region of cerebral ischemia (Zhu et al. 2004).Overexpression of TRPC6 channels markedly increasesCREB phosphorylation and enhances CREB-dependenttranscription (Jia et al. 2007). Blocking TRPC6 degradationmaintains phosphorylation of CREB and greatly preventsischemic brain damage (Du et al. 2010). A modest level ofCa2+ influx through TRPC6 channels leads to activation ofboth ERK and CaMK, which converge on CREB to pro-mote neuronal survival (Jia et al. 2007). We used PD98059and KN62 to investigate the pathway by which resveratrolprotected rats from cerebral ischemia. Our results

Fig. 5 Effects of resveratrol pretreatment on the immunoreactivity ofp-CREB at 2, 12, and 24 h after reperfusion. a p-CREB immunoreac-tivity (×200). Staining was present within the cell nucleus. b Quanti-fication of the levels of p-CREB in the ipsilateral cortex at the indicatedtimes or sham surgery (n = 3). Group S: sham operation, group I:

MCAO, group R: MCAO + resveratrol, group P: MCAO + resveratrol +PD98059 (MEK inhibitor), group K: MCAO + resveratrol + KN62(CaMKIV inhibitor), and group C: MCAO + resveratrol + PD98059 +KN62. Values are mean ± SEM. **P < 0.01

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demonstrated that when PD98059 or KN62 was applied20 min prior to the operation, p-CREB protein levels weresignificantly decreased at 24 h after reperfusion compared tothe levels measured in resveratrol-treated rats. However,protein levels of p-CREB in the KN62-treated group weremuch lower than those in the PD98059-treated group 12 hafter reperfusion, indicating that ICV injection of KN62induced its neuroprotective effects faster than intraperitonealinjection of PD98059. These results demonstrated that theactivation of CREB through the MEK and CaMKIV path-ways is a pivotal downstream effector for the protective effectof TRPC6 channels in neurons. Resveratrol blocked calpain-mediated degradation of TRPC6 channels and allowed elevat-ed Ca2+ levels to stimulate the Ras/MEK/ERK and CaMKIVpathways that converge on CREB activation, resulting in itsneuroprotection at 24 h after reperfusion.

In conclusion, our results clearly demonstrated thatpretreatment with resveratrol for 7 days significantly decreasedneurological deficits and the infarct volumes associated withelevated TRPC6 and p-CREB activity and decreasedSBDP145 activity after cerebral ischemia/reperfusion injury.This study provided in vivo evidence that resveratrol couldexert its neuroprotective effect against ischemic injurythrough the TRPC6–MEK–CREB and TRPC6–CaMKIV–CREB pathways. Our study provides theoretical support forresveratrol in the treatment of acute or subacute cerebralischemic stroke.

Conflict of interest The authors declare that they have no conflict ofinterest.

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