Activation of STAT3 is involved in neuroprotection by electroacupuncture pretreatment via cannabinoid CB1 receptors in rats

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    Activation of STAT3by electroacupunctuCB1 receptors in rats

    Heng Zhoua,1, Zhi Zhanga,1, HZijun Gaoa, Giovanni MarsicaaDepartment of Anesthesiology, Xijing Hospita

    e, 3307

    YLKTK). Neuronal

    usion. Our results

    of pSTAT3(Ser727)

    nt reduced infarct

    and decreased the

    Bax/Bcl-2 ratio following reperfusion. The benecial effects of EA were attenuated by PpYLKTK

    ncrease in pSTAT3

    ith siRNA blocked

    eas the two CB1R

    enhances STAT3

    t STAT3 activation

    b r a i n r e s e a r c h 1 5 2 9 ( 2 0 1 3 ) 1 5 4 1 6 4E-mail addresses: (Q. Wang), (L. Xiong).1Contributed equally to this work.0006-8993/$ - see front matter & 2013 Elsevier B.V. All rights reserved.

    Abbreviations: EA, electroacupuncture; STAT3, Signal transducers and activators of transcription 3; MCAO, middle cerebral artery

    occlusion; 2-AG, 2-arachidonylglycerol; AEA, N-arach-idonoylethanolamine-anandamide; ERK1/2, extracellular signal-regulated kinases1/2;

    PKC, protein kinase C epsilon; TTC, 2,3,5-triphenyltetrazolium chloride; TUNEL, deoxyuridine triphosphate nick-end labeling.nCorresponding authors. Fax: +86 29 8477 1262.& 2013 Elsevier B.V. All rights reserved.administered 30min before MACO, and PpYLKTK effectively reversed the i

    (Ser727) expression. Furthermore, CB1R antagonist or CB1R knockdown w

    the elevation of pSTAT3(Ser727) expression by EA pretreatment, wher

    agonists increased STAT3 activation. In conclusion, EA pretreatment

    activation via CB1R to protect against cerebral ischemia, suggesting tha

    may be a novel target for stroke intervention.Behavioralipsilateral ischemic penumbra. Infarct volumes and neurological scores wer

    after MACO in the presence or absence of the STAT3 inhibitor peptide (Pp

    apoptosis and the Bax/Bcl-2 ratio were also evaluated 24 h after reperf

    showed that EA pretreatment signicantly enhanced neuronal expression

    in the ischemic penumbra 6 h after reperfusion. Moreover, EA pretreatme

    volume, improved neurological outcome, inhibited neuronal apoptosisIschemic tolerance

    Strokeexpression of pSTAT3(Ser727), which is necessary for STAT3 activation, was examined in the

    e evaluated at 72 hKeywords:


    Middle cerebral artery occlusionplays an essential role in cell survival and proliferation. Therefore, we investigated whetherbINSERM U862, NeuroCentre Magendi

    a r t i c l e i n f o

    Article history:

    Accepted 3 July 2013

    Available online 20 July 2013is involved in neuroprotectionre pretreatment via cannabinoid

    aidong Weia, Feng Wanga, Fan Guoa,nob, Qiang Wanga,n, Lize Xionga,n

    l, Forth Military Medical University, Xi'an 710032, Shaanxi Province, China6 Bordeaux, France

    a b s t r a c t

    Pretreatment with electroacupuncture (EA) attenuates cerebral ischemic injury through the

    endocannabinoid system, although the molecular mechanisms mediate this neuroprotection

    are unknown. It is well-known that signal transducer and activator of transcription 3 (STAT3)

    STAT3 is involved in EA pretreatment-induced neuroprotection via cannabinoid CB1 receptors

    (CB1R) after transient focal cerebral ischemia in rats. Two hours after EA pretreatment, focal

    cerebral ischemia was induced by middle cerebral artery occlusion (MACO) for 120min. TheResearch Report

  • 1. Introduction

    Stroke is one of the leading causes of long-term disability andthe rst leading cause of death in China (Wu et al., 2012).Although recombinant tissue plasminogen activator (rtPA)has been shown to be an effective drug to treat stroke, it mustbe administered within 3.5 h of stroke onset, and only a fewpatients (less than 3%) can benet from this therapy (Li et al.,2012). The physical, emotional, and nancial toll strokeinicts on the patients and their families cannot be over-stated. Therefore, the development of new methods forstroke intervention is a major imperative.

    Electroacupuncture (EA), originating from Chinese tradi-tional acupuncture and modern electrotherapy, has beenproven to be effective not only in analgesia, but also for thereduction of ischemia/reperfusion injury of the heart andbrain (Xiong et al., 2003, Tsou et al., 2004, Wang et al., 2005,Zhou et al., 2012). Our previous study showed that 30 min ofEA pretreatment at the Baihui acupoint (GV 20) 2 h beforefocal cerebral ischemia was an effective method of inducingischemic tolerance (Xiong et al., 2003). Further studies foundthat EA pretreatment could modulate the endocannabinoidsystem by upregulating the production of the endocannabi-noids, including 2-arachidonylglycerol (2-AG) and N-arach-idonoylethanolamine-anandamide (AEA), which cause protec-tion through cannabinoid CB1 receptors (Wang et al., 2009).Notably, activation of extracellular signal-regulated kinases1/2(ERK1/2) and protein kinase C epsilon (PKC) was involved in EA

    pretreatment-induced ischemia tolerance via CB1R in the ratmodel of transient focal cerebral ischemia (Du et al., 2010, Wanget al., 2011). However, further research is still required to revealthe precise mechanism responsible for neuroprotection byEA pretreatment.

    Signal transducers and activators of transcription 3 (STAT3)plays a crucial role in cell survival and proliferation. Injury toneural cells could induce the activation of STAT3, and STAT3activation has been shown to cause neuroprotection (Dzienniset al., 2007, Dziennis and Alkayed, 2008). The phosphorylation ofSTAT3 on serine, induced by a PKC-Raf-mitogen-activatedprotein kinase (MAPK)/extracellular signal-regulated kinase(MEK)-p44/42 MAPK signaling pathway, proved to be necessaryfor a protective mechanism during heart ischemia (Xuan et al.,2001, Xuan et al., 2005); it has also been suggested as a criticalcontributor to neural cell protection (Kim et al., 2008).

    Based on these ndings, the present study was undertakento test the hypothesis that activation of STAT3 is involved in EApretreatment-induced neuroprotection via cannabinoid CB1receptors in a rat model of transient focal cerebral ischemia.

    2. Results

    2.1. EA pretreatment induced neuroprotection againstcerebral ischemia and reperfusion

    This experiment conrmed the benecial effect of EA pre-


    b r a i n r e s e a r c h 1 5 2 9 ( 2 0 1 3 ) 1 5 4 1 6 4 155Fig. 1 Infarct volumes and neurological scores at 72 h after5 - triphenyltetrazolium chloride staining of the cerebral infarafter reperfusion. (B) Quantication of infarct volume at 72 h

    infarct volume compared with the MCAO group (*Po0.05 vs. MCwith 120 min of MCAO. Pretreatment with EA signicantly impro(*Po0.05 vs. MCAO).erfusion in the rats (n8). (A) Representative 2, 3,in comparable sections of rat brain from three groups at 72 her reperfusion. The EA group had a signicantly reducedAO). (C) Neurological scores at 72 h after reperfusion in ratstreatment on brain ischemic injury that had been reportedved the neurological scores compared with the MCAO group

  • ingtlydco

    d c

    5 2previously. Infarct volume in the EA group was signicantlyreduced compared with the MCAO group 72 h after reperfu-sion (24.3871.586% vs. 34.6072.154%, t74.682, Po0.001,Bonferroni's Multiple Comparison Test), and pretreatmentwith EA improved the neurological scores (11.00, 10.012.75vs. 7.5, 6.259.0, P0.0026, Mann Whitney test, Fig. 1).

    2.2. EA pretreatment enhanced STAT3 activation aftercerebral ischemia and reperfusion

    Fig. 2 The expression of pSTAT3(Ser727) was evaluated usexpression of pSTAT3(Ser727) in the EA group was signicanand MCAO groups (*Po0.05 vs. MCAO, Po0.05 vs. Sham), anexpression of pSTAT3(Ser727) in the EA group was increased(Po0.05 vs. Sham), but no signicant signicance was foun

    b r a i n r e s e a r c h 1156EA pretreatment for 2 h prior to MCAO resulted in STAT3activation according to the results of Western blotting. Thesemi-quantitative analysis of the Western blots indicatedthat at 6 h after reperfusion, the content of pSTAT3(Ser727)in the peri-ischemic penumbra of the EA group (1.32070.1192) was signicantly higher compared with the Sham(0.422570.06329, t37.120, Po0.001, Bonferroni's MultipleComparison Test) and MCAO (0.625070.0750, t35.514,Po0.001, Bonferroni's Multiple Comparison Test) groups. Inaddition, compared with Sham group (0.245670.01066) thecontent of pSTAT3(Ser727) in the peri-ischemic penumbraof the MCAO and EA group was signicantly increased 24 hafter reperfusion (0.345770.02233, t33.886, Po0.05; 0.408870.01957, t36.337, Po0.001, Bonferroni's Multiple ComparisonTest), but no difference was found in the content of pSTAT3(Ser727) between EA and MCAO groups 24 h after reperfusion(Fig. 2).

    2.3. EA pretreatment up-regulated neuronal expressionof pSTAT3(Ser727) in the penumbra

    To evaluate the cellular localization of pSTAT3(Ser727),double-labeled immunouorescence was performed 6 h afterreperfusion in the Sham, MCAO and EA groups. Doubleimmunouorescence staining revealed that pSTAT3(Ser727)-positive cells co-localized with NeuN positive neurons (Fig. 3).2.4. STAT3 inhibitor peptide reversed the activationof STAT3 after EA pretreatment

    Increased phosphorylation of pSTAT3(Ser727) was blockedsignicantly by the injection of PpYLKTK (Pp) at a concentra-tion of 10 nmol/l 30 min before focal cerebral ischemia in theEA+Pp group (0.212570.03728) compared with the EA+PBSgroup (0.622570.04366, t34.921, P0.0003, Bonferroni's Mul-tiple Comparison Test, Fig. 4).western blotting (n4). (A) Western blots show that theincreased at 6 h after reperfusion compared with the Shamthe total STAT3 did not change. (B) Furthermore, thempared with the Sham group at 24 h after reperfusionompared with the MCAO group.

    9 ( 2 0 1 3 ) 1 5 4 1 6 42.5. STAT3 inhibitor peptide attenuated the neuroprotectioninduced by EA pretreatment

    EA pretreatment signicantly decreased infarct volume (21.0971.135%) at 72 h after reperfusion at 72 hours after reperfusionwhen compared with the MCAO (34.0170.9565, t79.470,Po0.0001, Bonferroni's Multiple Comparison Test) and EA+Pp (29.6971.234, t76.301, Po0.0001, Bonferroni's MultipleComparison Test) groups. EA pretreatment increased theneurological scores (11.00, 10-12.75) at 72 h after reper-fusion compared with the MCAO (7, 67.75, P0.0009, MannWhitney test) and EA+Pp (7.5, 6.258.75, P0.0013, MannWhitney test) groups and there were no statistical differencesin infarct volume and neurological scores between MCAOgroup and EA+Pp group (Fig. 5).

    Neuronal cell death was evaluated 24 h after reperfusion ineach group. Morphologically damaged neurons were positivefor TUNEL and caspase-3 staining. In the EA group, the numberof TUNEL (20.7572.869) and caspase-3 (3.50071.555) positivecells in the peri-ischemic penumbra were signicantlydecreased compared with the MCAO (57.0075.612, t36.852,Po0.001; 17.7572.626, t35.193, Po0.01, Bonferroni's MultipleComparison Test) and EA+Pp groups (45.5074.031, t34.678,Po0.01; 14.5072.398, t3 4.008, Po0.05, Bonferroni's MultipleComparison Test, Fig. 6).

  • 5 2b r a i n r e s e a r c h 1At 24 h after reperfusion, the ratio of Bax to Bcl-2 in theperi-ischemic penumbra of rats in the MCAO group washigher than that in the Sham group (1.43070.1989 vs.0.312570.0875, t35.691, Po0.001, Bonferroni's Multiple Com-parison Test). Compared with rats only subjected to MCAO,EA pretreatment markedly downregulated the ratio of Bax toBcl-2 (1.43070.1989 vs. 0.780070.1188, t33.310, Po0.05, Bon-ferroni's Multiple Comparison Test). Interestingly, PpYLKTKadministration before ischemia clearly suppressed the increase

    Fig. 4 Western blot analysis of pSTAT3(Ser727) at 6 h afterreperfusion with or without PpYLKTK treatment (n4).PpYLKTK (50 nM) remarkably reduced the expression ofpSTAT3(Ser727) compared with the EA+PBS group (#Po0.05vs. EA+PBS group).

    Fig. 3 Double immunouorescence staining for pSTAT3(Ser727penumbra 6 h after reperfusion (n4). pSTAT3(Ser727)-positive cwith neurons, and it seems that pSTAT3(Ser727) is expressed in) (in green) and neurons (in red) in the peri-ischemicells in the peri-ischemic penumbra were co-localized mainly9 ( 2 0 1 3 ) 1 5 4 1 6 4 157in Bcl-2 protein contents by EA pretreatment (1.67870.1257 vs.0.780070.1188, t3 4.570, Po0.01, Bonferroni's Multiple Com-parison Test, Fig. 7).

    2.6. Activation of STAT3 was mediated by thecannabinoid CB1 receptor

    The CB1R antagonist AM251 was administered intraperitone-ally 30 min before MCAO. At 6 h after reperfusion, theexpression of pSTAT3(Ser727) in the EA+AM251 group(0.620070.07118) was signicantly decreased compared withthe EA (1.39070.07382, t3 8.523, Po0.001, Bonferroni's Multi-ple Comparison Test) and EA+vehicle (1.46370.04679,t39.326, Po0.001, Bonferroni's Multiple Comparison Test)groups but no signicant difference was found comparedwith the MCAO group. For more evidence, we used CB1RsiRNA injected intracerebrally to interfere with CB1R. Theexpression of pSTAT3(Ser727) in the EA+CB1R siRNA(0.182670.03736) group was signicantly decreased comparedwith the EA (0.334470.03670, t33.602, Po0.05, Bonferroni'sMultiple Comparison Test) and EA+control siRNA groups(0.338770.02243, t33.706, Po0.05, Bonferroni's MultipleComparison Test).

    The two CB1R agonists WIN55,212-2 and ACEA were usedas to test whether they had an effect that was opposite toAM251 and CB1R siRNA. We found that the expression ofpSTAT3(Ser727) in the MCAO+WIN55,212-2 (1.17570.04787)group increased compared with the MCAO and MCAO

    both nucleus and cytoplasm.

  • EAC

    effel scs.

    5 2Fig. 5 Effect of pSTAT3(Ser727) inhibition with PpYLKTK oninfarct volume at 72 h after reperfusion in the rats with 2 h Mvolume, whereas PpYLKTK (50 nM) abolished the protectivePretreatment with EA signicantly increased the neurologicaabolished the protective effect of EA pretreatment (#Po0.05 vb r a i n r e s e a r c h 1158+vehicle groups (0.490070.06014, t39.511, Po0.001; 0.742570.04328, t36.005, Po0.001 Bonferroni's Multiple ComparisonTest). The expression of pSTAT3(Ser727) in the MCAO+ACEAgroup (0.498170.04738) was increased compared with theMCAO and MCAO+vehicle groups (0.220370.02722, t35.035,Po0.01; 0.281670.03975, t33.923, Po0.05 Bonferroni's Multi-ple Comparison Test, Fig. 8).

    3. Discussion

    Ischemic tolerance can be induced by sublethal attacks, andthere are several pretreatments that have been shown toinduce neuronal protection against ischemia-reperfusioninjury. Many of these treatments activate extacellular orintracellular signaling pathways by triggering endogenousprotective responses (Durukan and Tatlisumak, 2010). There-fore, further research is required to dene the precise mole-cular crosstalk and to optimize a novel way of dealing withcerebral ischemia.

    We reported that EA pretreatment could produce rapidischemic tolerance against lethal ischemia in a previous study(Xiong et al., 2003). Further research found that the adenosine A1receptor and the endocannabinoid system are involved inischemic tolerance induced by EA pretreatment (Wang et al.,2005, Wang et al., 2009). In addition, activation of ERK1/2 andPKC plays an important role in EA pretreatment-inducedneuronal p...


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