research article magnesium lithospermate b, an...

Research ArticleMagnesium Lithospermate B an Active Extract ofSalvia miltiorrhiza Mediates sGCcGMPPKG Translocation inExperimental Vasospasm

Chih-Zen Chang123 Shu-Chuan Wu2 and Aij-Lie Kwan12

1 Department of Surgery Faculty of Medicine School of Medicine Kaohsiung Medical University Kaohsiung Taiwan2Department of Neurosurgery Kaohsiung Medical University Hospital No 100 Tzyou 1st Road Kaohsiung 80752 Taiwan3Department of Surgery Kaohsiung Municipal Ta Tung Hospital Kaohsiung Taiwan

Correspondence should be addressed to Chih-Zen Chang changchihzen2002yahoocomtw

Received 24 January 2014 Accepted 25 February 2014 Published 2 April 2014

Academic Editor John H Zhang

Copyright copy 2014 Chih-Zen Chang et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Background Soluble guanylyl cyclases (sGCs) and Ras homolog gene family member A (rhoA)Ras homolog gene familykinase(rho-kinase) plays a role in vascular smoothmuscle relaxation in subarachnoid hemorrhage (SAH) It is of interest to examinethe effect of MLB on rhoAROCK and sGCcGMPPKG expressionMethods A rodent SAHmodel was employed Tissue sampleswere for sGC1205721 sGC1205731 PKG rhoA ROCK (Western blot) and cGMP (ELISA) measurement Results MLB morphologicallyimproved convolution of the internal elastic lamina distortion of endothelial wall and necrosis of the smooth muscle in the SAHrats Expressed cGMP sGC1205721 sGC1205731 and PKG in the SAH groups were reduced (119875 lt 001) and MLB precondition significantlyinduced cGMP sGC1205721 sGC1205731 and PKG L-NAME reversed the vasodilation effect of MLB reduced the bioexpression of PKG andcGMP (119875 lt 001) and tends to reduce sGC1205721 level and induce rhoA ROCK level in MLB precondition + SAH groups ConclusionThese results demonstrate that sGCcGMPPKG and NOET pathways play pivotal roles in SAH-induced vasospasm Throughactivating sGCcGMPPKG pathway and partially by inactivating rho-kinase in a NO-dependent mechanismMLB shows promiseto be an effective strategy for the treatment of this disease entity

1 Introduction

Subarachnoid hemorrhage (SAH) induced cerebral vasos-pasm becomes an important subcategory of stroke owingto its high mortality and morbidity which included delayedneurological deficit and cardiopulmonary abnormality [12] Till now the precise mechanism of this disease contourremains unclear Several molecular and cellular researchesimplicate two key hypotheses to cerebral vasospasm onecenter on the roles of nitric oxide [3] and endothelins [4] andanother converges on the role of ATP-AMP and rhoAROCKII [5] In the study of Carter et al [3] NO donors wereable to relax vascular smooth muscle in the episode ofvasospasm Through mediated on a large conductance Ca2+-dependent K+ channels cGMP-dependent protein kinase(PKG) phophorylates and rhoA NOmay attenuates vascular

smooth muscle sensitivity to calcium ions [6] In addi-tion increased evidence suggests that NO counteracts withendothelins as a vascular modulator in part by mediatingthe activation of RhoA-ROCK and MLC phosphatase [34] The study of eNOS knockout mice reticence to diabeticnephropathy also provides some insight into the modulationof soluble guanylyl cyclase (sGC) on NO signaling pathway[7]

The involvement of NOET-1 and rhoAROCK in SAHinduced vasomediated pathways is not completely under-stood It has been shown that NO (catalyzed by eNOsnNos and iNOs) an endothelium-derived relaxing factor(EDRF) augments with endothelin-1 and inhibits activationof rhoAROCK in rat thoracic aorta [8] ET-1 potentiates theconstriction of cerebrovascular smooth muscles induced byoxyhemoglobin through PKC and rhoArho kinase pathways

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 272101 9 pageshttpdxdoiorg1011552014272101

2 BioMed Research International

[4 7] This is compatible with the findings that a PKCinhibitor staurosporine can abolish ET-1 induced contrac-tion in rabbit basilar artery [9] Moreover lines of evidenceaccumulated to present have suggested that activated PKCplays a role in SAH induced vasospasm [10] For examplephorbol 12 13-dibutyrate a PKC activator can induce apotent and long-lasting contraction of the rabbit basilarartery [11] In a canine double-hemorrhage model the mem-brane translocation of PKC120575 from cytosol in the basilarartery was observed after the second injection of autologousblood on the 4th day when vasospasm was robust [10 12]Furthermore intrathecal injection of the PKC120575 inhibitorrottlerin inhibited hemorrhage induced vasospastic responseas well as the translocation of PKC120575 [10]

On the contrary the key event for triggering vasomotoreffect of NOET-1 in SAH on the rho-kinase pathway hasattracted less attention despite of documented involvementof rho-kinase in cerebral vasospasm following SAH Rhoa family of small G-proteins is composed of 3 membersrhoA rhoB and rhoC which behaves like a substantial role inintracellular signaling [8 13] Inactive rho is resided as aGDP-bound form in the cytosol If the vascular smooth musclecells were stimulated by vasoactive agents and subsequentGDP-GTP exchange rho may become activated and thentranslocated to the cell membrane where it interacts withits downstream effectors rho-kinase (ROCK) [13 14] TheROCK family includes two isoforms namely ROCK-I andROCK-II By phosphorylating myosin light chain phos-phatase (MLCP) and inhibiting the activity of phosphataseat the myosin-binding subunit rho-kinase is stimulated andpromotes smooth muscle contraction [15 16] In the studyof a canine two-hemorrhage model topical application of aROCK inhibitor Y-27632 dose-dependently decreased thespastic response Rho-kinase activity and phosphorylation ofMLCP in the basilar artery [17 18] Moreover intra-arterialadministration of fasudil another kind of ROCK inhibitor isalso proved to be effective for the treatment of vasospasm inSAH patients [19]

Besides ET-1 nitric oxide (NO) composed of endothe-lial NOs(eNOs) neuronal NOs(nNOs) and inducible NOs(iNOs) is believed to be an important regulator of thecerebral vascular tone [7 20] Upon synthesis NO bindingactivates soluble guanylyl cyclase (sGC) a heterodimericenzyme consisting of 120572 (120572

1 1205722 and 120572

3) and 120573 (120573

1 1205732 and

1205733) subunits [21] Activation of sGC leads to convert GTP

to cGMP which in turn activates cGMP-dependent proteinkinase (PKG) and cGMP gated ion channel among targetcells In blood vessels PKG can phosphorylate the inositol 14 5-triphosphate receptor and decrease Ca2+ concentrationand ultimately result in smooth muscle relaxation [9 2223] Under normal physiological conditions the productionsof ET-1 and NO yield a balanced cerebral vascular toneHowever enhanced generation of ET-1 along with impairedNO production (NO-dependent) or with excessively inducedNO (NO-independent) was noted in human and animalswith SAH [3] Nevertheless the effect of NOET-1 productionon the sGCcGMP signaling pathway has not been fullyinvestigated

In the present study we aimed to assess the neurologicaldeficits and plasma NOET-1 levels as well as the expressionsof PKC120575 rhoA ROCK-II and sGCcGMPPKG in the basilarartery of rats subject to SAH The effect of magnesiumlithospermate B (MLB) on the NOSsGCcGMPPKG andrhoAROCK signaling pathway in SAH was investigated

2 Materials and Methods

21Materials Anti-rabbit sGC1205721 and sGC1205731 and anti-mouse120573-actin antibodies were bought from Sigma-Aldrich (Shang-hai PRC) Anti-rabbit PKG anti-mouse PKC120575 anti-rabbitROCK-II anti-mouse rhoA and horseradish peroxidase-labeled goat anti-mouse IgG antibodies were obtained fromAbcam (Cambridge MA USA) BD Transduction Lab (BDBiosciences California USA) Upstate Biotech (NY USA)Santa Cruz Biotech (Santa Cruz Biotechnology Inc Cal-ifornia USA) and Chemicon International (CA USA)respectively CNMprotein extraction kits were fromBiochain(Hayward CA USA) ET-1 and cGMP ELISA kits wereproducts of Assay Designs (Enzo Life Sciences Inc NYUSA) and Cayman Chemical (Michigan USA) respectivelyMagnesium Lithospermate B was purified by Ms Wu SC(Kaohsiung Medical University Hospital Kaohsiung Tai-wan ROC) according to the modified protocol describedby Tanaka et al Dried Salvia miltiorrhiza were obtainedinoculation with methanol for 8 hr and then concentratedMLB was extracted by the aid of a Sephadex LH-20 multiplecolumn chromatography for four times (Pharmacia FineChemicals Piscataway NJ USA)

22 Animal Protocols All the experimental protocols wereapproved by the Kaohsiung Medical University Hospitalanimal research committee Forty-five male Sprague-Dawleyrats (BioLasco Taiwan Co Ltd authorized by Charles RiverLab) weighing 250ndash350 g were assigned into five groups (9animals each) Group 1 was designated as sham-operatedGroup 2 rats subjected to SAH only Group 3 SAH ratstreated with vehicle (01molL PBS) and Groups 4 and 5SAH animals received 10mgkgday MLB treatment at 24 hr(prevention protocol) and 1 hr (reversal protocol) after SAHrespectively To induce SAH rats were anesthetized witha mixture of ketamine (40mgkg) and xylazine (6mgkg)intraperitoneally (ip) and 03mL fresh blood was drawnfrom the central tail artery and injected into the craniocervi-cal junction using a stereotactic technique Further nine ani-mals were enrolled in the 01 120583L NG-nitro-L-arginine methylester (L-NAME) intrathecal injection plus 10mgkgdayMLBpreconditioning SAH rats

23 Hemodynamic Measurements Heart rate blood pres-sure and rectal temperature were monitored before and afterMLB treatment as well as at 48 hr after the induction ofSAH by a tail-cuff method (SC1000 Single Channel SystemHatteras Instruments North Carolina USA) and rectal ther-mometer (BIO-BRET-2-ISO FL USA)

24 Neurological Assessment Behavior assessment adapteda modified limb-placing tests (MLPT) [2] was performed

BioMed Research International 3

Table 1 Modified limb-placing test (MLPT)

Treatment GroupAmbulation Placingstepping reflex MDI

Normal 0 0 0SAH 128 plusmn 02 16 plusmn 016 288 plusmn 032

SAH + vehicle 122 plusmn 013 148 plusmn 022 27 plusmn 035

SAH + 1mgkg MLBPrevention 083 plusmn 016 078 plusmn 018

lowast161 plusmn 034

lowast

Treatment 082 plusmn 015 084 plusmn 013 166 plusmn 028lowast

L-NAME + prevention 132 plusmn 023 145 plusmn 016 277 plusmn 039

Results are expressed as the mean plusmn SEM 119899 = 9 lowast119875 lt 001 versus SAH condition by Mann-Whitney test

before and at 48 hr after the induction of SAH Motorfunction was assessed by two individuals who are blind to thetreatment status set which is composed of two limb-placingtasks of assessment of the sensorimotor assimilation of theforelimb and hindlimb as well as monitoring its responseto tactile and proprioceptive stimuli The summations ofneurological examination are scored as normal performance(0 point) incomplete performance (1 point) and no perfor-mance (2 points) which were shown in Table 1

25 Tissue Embedding Basilar artery was collected and themiddle third of each artery was left for further analysisThe arterial segment was rinsed in a 01molL PBS (pH74) repeatedly set in 1 osmium tetroxide in PBS at roomtemperature for 1 hr and then cleaned with PBS The vesselsegments were dehydrated and immersed in a 1 1 mixture ofpropylene oxide and epoxy resin overnight The specimenswere embedded in 100 epoxy resin and let to polymerizeat 60∘C for 48 hr the next day The BAs were cross-sectionedat a thickness of 05 120583m on an Ultracut E ultramicro-tome (Reichert-Jung Ultracut E Leica Wetzlar Germany)launched on glass slides and stained with 05 toluidineblue for morphometric analysis later The rest of tissue waspreserved in liquid N

2at minus70∘C until the measurements of

protein expression and cGMP levels

26 Basilar Artery Morphometric Studies Five selected BAcross sections of each animal were analyzed by two inves-tigators blinded to the set groups The cross-sectional areawas automatically measured using computer-assisted mor-phometry (Image-1Metamorph Imaging System UniversalImaging Corp) The average of five cross sections from agiven animal was collected as a single value for this animalGroup data are expressed as the means plusmn standard error ofthe means

27 Determination of Endothelial Nitric Oxide Synthase(eNOS) An isolated BA was quantified by adapting a com-mercial kit to check NOS (Bioxytech NOS Assay Kit OxisInternational Inc Portland OR USA) Briefly proteinextracted frommicrovessels was incubated with radiolabeledL-arginine in the presence or in the absence of 1mmolLNOS inhibitor NG-nitro-L-arginine methyl ester (L-NAMESigma-Aldrich Shanghai PRC) The reaction was finished

by the addition of 50mmolL HEPES buffer containing5mmolL EDTA Radiolabeled L-citrulline was counted afterremoval of excess L-arginine with an equilibrated resin andthen centrifuged

28 Western Blot to Evaluate RhoA ROCK-II sGC1205721 sGC1205731and PKC120575 Protein Expression Basilar arteries were homoge-nized for extraction of cytoplasm (buffer C) nuclear (bufferN) and membrane- (buffer M-) bound proteins as indicatedby the instructions of themanufacturer Expressions of PKC120575rhoA ROCK-II sGC1205721 sGC1205731 and PKG were determinedby Western blots using the Bio-Rad protein assay kit (Bio-Rad Lab Shanghai Co Ltd Pudong Shanghai PRC) fol-lowing the manufacturerrsquos instruction Sampling of equalamount (20120583glane) was seceded by a 10 polyacrylamidegel and conveyed to a polyvinylidene difluoride membrane(PerkinElmer Informatics Cambridge MA USA) A vaguebinding was blocked with a TBST buffer (50mM Tris-HClpH 76 150mMNaCl 01 Tween 20) mixed with 5 fatfree milk for 1 h at room temperature The membraneswere incubated overnight at 4∘C with one of the followingprimary antibodies rabbit anti-ROCK (1 1000) rabbit anti-sGC1205721 (1 1000) rabbit anti-sGC1205731 (1 1000) mouse anti-PKC (1 1000) and mouse anti-120573-actin (1 15000) The mem-branes were then washed six times per 5min with TBSTbuffer Appropriate dilution of secondary antibodies (1 1000)was incubated for 1 hr After washing with TBST for six timesthe protein bands were detected with the Signal Fire ECLreagent fromCell SignalingTechnology (CST) (Cell SignalingTechnology Inc MA USA)

29 ELISA for Tissue cGMP Levels Arterial blood was col-lected in heparin-containing tubes prior to the perfusionPlasma samples were frozen at minus70∘C until use The levelof cGMP in the homogenate of BA was measured using anELISA kit according to the instruction of the manufacturer

210 Statistical Analyses Group data are expressed as themeans plusmn SEM Comparison of the neurological deficit scoresbetween groups was performed by the Mann-Whitney testComparison of protein expression and biomarkers betweengroups was done using one-way ANOVA followed by Dun-nettrsquos test Differences were considered significant at a 119875 lt001


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0010203040506070809

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arte

ry(m

m3 )

(b)

Figure 1 Effect of magnesium lithospermate B (MLB) on endothelial NOS levels (a) and cross section of BAs (b) 10mgkg MLB wasadministered at 1 hr (prevention protocol P) and 24 hr (treatment protocol R) after animals were subjected to SAH BAs eNOS (rt-PCR)was measured Bottom panels represent micrographs of the cross section of BAs obtained from the healthy controls (A) the SAH only rats(B) the vehicle-treated SAH rats (C) SAH rats received 10mgkgday MLB treatment (D) SAH rats received 10mgkgday MLB preventivetreatment (E) and SAH rats received both 10mgkgday magnesium lithospermate B pretreatment and 1 120583g L-NAME (F) Standard bar =200 120583m lowast119875 lt 001 compared with the vehicle + SAH and MLB prevention group lowastlowast119875 lt 001 comparison between sham and the vehicle+ SAH groups lowastlowastlowast119875 lt 001 MLB prevention group compared with the L-NAME treatment group

119875 gt 005 compared MLB treatmentgroup with the sham group Data are mean plusmn SEM (119899 = 9group)

3 Results

31 General Observations By the end of the experimentthere were no statistically significant differences among thecontrol and experimental groups in the following physio-logical parameters evaluated which enrolled blood arterialgas mean arterial blood pressure heart rate and rectaltemperature (data not shown) No mortality and seriousmorbidity were observed within the experimental groups

Visual inspection during removal of the brain showed thatsubarachnoid blood clots had formed and covered the BAs inall animals subject to SAH

32 Tissue Morphometry The internal elastic lamina in thebasilar artery of SAH and SAH+vehicle groups showed sub-stantial corrugation when compared with that obtained fromthe controls (Figure 1(b)) Corrugation was less prominent


Membrane

Cytosol

(A) (B) (C) (D) (E)Ra

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MLB

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lowastlowastlowast

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SAH

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120573-Actin (43kD)

L-N

AM

E

ROCK

II ex

pres

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(rel

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roup

)

(b)

Figure 2 Inhibitory effect of MLB on RhoA translocation (a) and ROCK-II expression (b) in the BAs Expression of RhoA in the cytoplasmand membrane as well as ROCK-II was also determined by Western blot analysis (A) the healthy controls (B) the vehicle-treated SAH rats(C) SAH rats received 10mgkgday MLB treatment (D) SAH rats received 10mgkgday MLB preventive treatment and SAH rats receivedboth 10mgkgday MLB pretreatment and 1mgkg L-NAME as (E) (upper panel) The ratio of membrane bound to cytoplasm RhoA in theSAH group was set at 100 in (a) whereas the expression of ROCK-II (normalized using 120573-actin) was set the same in (b) Data are mean plusmnSEM (119899 = 9group) lowastlowastlowastlowastlowastlowast119875 lt 001 versus the vehicle +SAH MLB and L-NAME groups respectively

in the SAH + 10mgkgday MLB groups The cross-sectionalareas of BAs in the SAH and SAH + vehicle groups weresignificantly reduced when compared with the control group(048 plusmn 027 053 plusmn 018 and 093 plusmn 011 resp) Treatmentwith MLB significantly attenuated the decrease in both theprecondition and reversal groups in previous study

33 Neurological Deficit Using a modified limb-placing test(MLPT) shown previously both the scores of ambulation toassess the sensorimotor incorporation of the both forelimband hindlimb and placingstepping reflex as a reflex ofresponse to tactile and proprioceptive stimulation in the SAHgroups were significantly higher than the healthy controlsThe sum of scores from these two tests is referred to asmotor deficit index (MDI) The values of MDI in the SAHand SAH + vehicle groups were 288 plusmn 032 and 270 plusmn 035respectively comparedwith a score of 0 in the healthy controlTreatment with MLB significantly improved the MDI in theprevention and reversal groups (Table 1) Likewise paraplegiarate (defined as the percentage of rats with MDI ge 3 ineach group) was substantially decreased in both the MLBprecondition and treatment groups when compared with theSAH animals

34 Expressed eNOS Levels in BAs When compared with thehealthy controls CSF ET-1 levels in the SAH groups weresignificantly increased in the pilot study TreatmentwithMLBsignificantly decreased CSF ET-1 in both theMLB preventionand reversal groups to levels that were not aside from the

healthy controls The analysis of radiolabeled L-citrullineshowed higher eNOS protein in the control and both MLBprevention and treatment groups Groups precondition andtreatment with MLB tend to increase the expressed eNOSprotein in BAs when compared with that in SAH rats (119875 lt001 Figure 1(a)) Furthermore the effect of decreased ET-1and increased eNOS exerted by MLB was reversed by addingL-NAME (Figure 1(b))

35 RhoA Translocation and ROCK-II Expression in theBAs Activated rhoA translocated from the cytoplasm tomembrane was evaluated In the BAs the level of membrane-bound rhoA was significantly increased in rats subject toSAH when compared with the normal control (Figure 2(a))MLB significantly reduced membrane-bound rhoA whiletreatment with vehicle showed no significant differencewhen compared with the SAH only group

The pattern of ROCK-II expression in the BAs resembledthat observedwith levels ofmembrane-boundPKC120575 or rhoAROCK-II expression was significantly increased in the SAHand SAH + vehicle groups when compared with the controlsand treatment with MLB reduced the expression of ROCK-II (Figure 2(b))The administration of L-NAME significantlyincreased the bio-expression of rhoA and ROCK-II in theMLB + SAH groups when compared with the MLB groups(Figure 2)

36 Expression of sGC and Associated Downstream cGMPPKG Pathway In contrast to the expression of ROCK-II in


002040608

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GC1205721

(rel

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Figure 3 Up-regulation of sGC1205721 (a) and sGC1205731 (b) by MLB in the BAs Expression of sGC1205721 and sGC1205731 was determined by Western blotanalysis and standardized by using 120573-actin The expression of sGC1205721 and sGC1205731 in the SAH group were set at 100 All values are mean plusmnSEM (119899 = 9group) lowast119875 lt 001 compared with the vehicle + SAH groups lowastlowast005 gt 119875 gt 001 comparison with the vehicle + SAH groupand 119875 gt 005 comparison among the vehicle plus SAH MLB prevention and treatment groups All groups are equal to those publicized in

Figure 2

the BAs of SAH rats the level of sGC1205721 and sGC1205731 wassignificantly increased in the MLB pretreatment SAH groupcompared with the SAH groups while treatment with MLBfailed to reduce sGC1205721 and sGC1205731 to a significant level whencompared with that in the SAH rats L-NAME decreased theexpressions of these two enzymes to levels similar to those ofthe SAH groups which was induced by MLB pretreatment(Figures 3(a) and 3(b))

Comparative to the reduced sGC1205721 and sGC1205731 expres-sions the levels of cGMP were significantly decreased in allSAH animals Administration of MLB in both the preventionand reversal protocols significantly attenuated the reductionof cGMP production in the BAs of the SAH rats (Figure 5)This increased production of cGMP upon MLB treatmentalso resulted in an increased expression of PKG in the BAsof the SAH rats which if untreated showed a significantreduction in PKG expression when compared with the SAHcontrols (Figure 4) Besides the administration of L-NAMEshowed to be effective on the reduction of cGMP level similarto that of SAH groups

37 Translocated PKC120575 in Cytoplasm to Membrane Contraryto that found with rhoA activated PKC120575 was translocatedfrom cytoplasm to membrane The ratio of membrane tocytoplasm PKC120575 expression in the BA of the SAH rats wasset as a standard reference Only half of PKC120575wasmembranebound in normal animals pretreatment with MLB at 1hrafter SAH induced the levels of activted PKC120575 to the normalcontrol The treatment of L-NAME reduced MLBrsquos effect onthe translocation of activated PKC120575 (Figure 4)

4 Discussion

Cerebral vasospasm following spontaneous SAH remains theleading factor contributing to the mortality and morbidityin patients after aneurysm rupture Even numerous basicand clinical trials conducted the neurological outcomes forSAH patients are still disappointing [5] Lines of evidenceindicate that both inflammatory and noninflammatory fac-tors are enrolled in the development and maintenance ofcerebral vasospasm Various components of the inflamma-tion have been incriminated in the pathogenesis of cerebralvasospasm including adhesion molecules [15] cytokines [8]immunoglobulin [8] complement [24 25] and endothelins[26] at both the cellular and molecular basis Among themET-1 derived fromcerebral arteries endothelial cells has beenimplicated as a potent vasoconstrictor in mediating SAH-induced vasospasm Continuous effort pointed to dissectmodules besides SAH induced inflammatory response NObecomes a critical role to mediate vascular dilatation In thestudy of Deng et al [27] small nanomolar concentration ofNO catalyzed by NO synthase is produced by endothelialcells which then disperses into vascular smooth muscle(VSM) and acts on the NO-sensitive sGC12057211205731 and 12057221205731 Theactivation of sGC and another enzyme phosphodiesterases(PDEs) existed in platelets and astrocytes is involved in theproduction and maintenance of cGMP Through activatingcGMP-dependent protein kinase (PKG) cGMP is able tomediate the vascular muscle relaxation The present studyshowed that MLB significantly improved the motor functionindex and lowered the rate of paraplegia in the SAH ratsIn addition treatment with MLB significantly increased the


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Figure 4 Suppressed PKC120575 translocation by MLB in the BAsThe bio-expression of PKC120575 from the cytoplasm to membrane wasdetermined byWestern blot analysis The ratio of membrane boundto cytoplasm PKC120575 in the SAH group was set at 100 Data areshown as mean plusmn SEM (119899 = 9group) lowastlowastlowast119875 lt 001 the vehicle+SAH versus Sham and MLB prevention groups respectively and119875 gt 005 compared with SAH rats pre-treatment with MLB and

L-NAME All groups are identical to those shown in the legend ofFigure 2

levels of eNOs sGC1205721 and cGMP as well as PKG The addi-tion of NOS inhibitor L-NAME decreased the bioexpressionof activation of sGC1205721 sGC1205731 cGMP and PKG Theseresults suggest that the beneficial effects of MLB in cerebralvasospasm after SAH may through additive influence on theNOsGCcGMPPKG pathway

As described herein another concept stated by Brandeset al [7] indicated through binding to adenosine A2Areceptors activated NOS was able to increase NO andconcomitantly disable the PKC120575 and rhoArho kinase-IIpathways in animals subject to SAH PKC120575 and rhoA as wellas the expression of rho-kinase were thought to be the mainfactors contributing to the vascular spastic response alongwith enhanced components in the sGCcGMPPKG pathwayTreatment withMLB induced the activatedNOS and normal-ized the expression of the two vascular regulatory pathways[3] These results consisted with the reports showing thatoxyhemoglobin is a major causative component of bloodclot for cerebral vasospasm following SAH [26] and that ET-1 potentiates the oxyhemoglobin-induced cerebrovascularsmoothmuscle contraction via the rhoArho kinase and PKCpathways [28 29] In our previous study MLB can reduceCSF ET-1 level through a NO dependent mechanism [3] Inthis study the level of sGC1205721 and sGC1205731 was reduced in theinduction of SAH The administration of L-NAME reversesthe enhanced effect of MLB on sGC1205721 expression

In contrast to the activation of the two aforementionedvasoregulatory pathways SAH resulted in decreasedexpression of the NO-mediated vasodilatory pathway

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Figure 5 Induced cGMP level by MLB in the BAs The levels ofcGMP (ELISA) was determined PKG expression in the SAH groupwas set at 100 Data are mean plusmn SEM (119899 = 9group) lowastlowastlowast119875 lt 001versus the vehicle plus SAH group to the normal controls and MLBgroups respectively

119875 gt 005 comparison among the vehicle plusSAH L-NAME and MLB prevention group

sGCcGMPPKG (Figures 2ndash4) MLB is an active componentextracted from the root of Salvia miltiorrhiza (SM)Administration of extract of SM has been shown to attenuatevasoactive mediator and impede the production of ET-1in patients with congenital heart failure and pulmonaryhypertension during cardiac surgery [1 30] MLB as themain component plays a dual role of antioxidant and potentfibronectin antagonist to holt platelet aggregation which isable to lessen ischemia associated myocardial damage Inthe present study MLB is proved to be able to improve theendothelial function of vascular wall by suppressing the levelof CSF ET-1 and stimulating the sGCcGMPPKG pathwayvia increased production of NO through the inducibleNO synthase as seen in the rodent streptozotocin-inducedrenal injury [2] In the study of human umbilical vascularendothelial cells (HUVECs) cryptotanshinone a nonpolaractive compound similar to MLB attenuates the level ofET-1 in the medium as well as the level of TNF-120572 [30]This compound is able to inhibit bioactivation of TNF-120572-induced NF-120581B and is protective to homocysteine-inducedendothelium dysfunction [1] and in the other studies ahigh dosage cryptotanshinone is able to impede angiotensinconverting enzyme (ACE) reduce blood pressure expandarteries enhance microcirculation and albeit atheroscleroticplaque formation [2] What is more MLB was reported toprotect cerebrum against ischemia reperfusion injury andattenuate the infarct area in a focal cerebral occlusion animalmodel [1] Since MLB is composed of a Mg2+ cation and acaffeic acid tetramer the Mg2+ salt bridged against MLB isbelieved to play an adjunct role in blocking excitotoxicity-induced calcium ion influx into smooth muscle cells whichare overstimulated by SAH Mg2+ cation is capable ofmodulating the L-type calcium channel and augments the


vasodilation effect of NO related pathways in the cardiacsmooth muscle [22 31]

In summary this study shows that MLB could inducethe levels of eNOs improve the motor function indexactivate PKC120575 sGCcGMPPKG pathway and tend to reducerhoArho kinase-II pathway in SAH rats It is likely thatthis compound exerts these beneficial effects mainly via itsorganic caffeic acid tetramer component which exerts thedual NO related sGCcGMPPKG and a G-protein ligandmediated PKC120575 pathways Reduction of rhoAROCK II acti-vation through a NO dependent manner also lends evidenceto its antivasospastic effect However the affinity of MLB tothe adenosine A2A receptor needed to be clarified later

Abbreviations

BA Basilar arterycGMP Cyclic -3101584051015840-guanosine monophosphateCSF Cerebrospinal fluidETs EndothelinsGTP Guanosine triphosphateHRP Horseradish peroxidaseIEL Internal elastic laminaip IntraperitoneallyLCA Leukocyte common antigenMLB Magnesium Lithospermate BNO Nitric oxidePDEs PhosphodiesterasesPKG cGMP-dependent protein kinasePBS Phosphate-buffered salineRhoA Ras homolog gene family member AROCK RhoARho-kinaseSAH Subarachnoid hemorrhagesGC-1205721 -1205731 Soluble guanylyl cyclase -1205721 -1205731VSM Vascular smooth muscle

Conflict of Interests

The authors of this paper disclose no financial conflict ofinterests

Acknowledgment

This work was supported by The National Science CouncilROC under Grant NSC99-2314-B-037-060-MY2

References

[1] G Grasso ldquoAn overview of new pharmacological treatments forcerebrovascular dysfunction after experimental subarachnoidhemorrhagerdquo Brain Research Reviews vol 44 no 1 pp 49ndash632004

[2] S G Keyrouz and M N Diringer ldquoClinical review preventionand therapy of vasospasm in subarachnoid hemorrhagerdquo Criti-cal Care vol 11 no 4 article 220 2007

[3] R W Carter M Begaye and N L Kanagy ldquoAcute and chronicNOS inhibition enhances 1205722-adrenoreceptor-stimulated RhoAand Rho kinase in rat aortardquo The American Journal of

PhysiologymdashHeart and Circulatory Physiology vol 283 no 4pp H1361ndashH1369 2002

[4] D M Arrick and W G Mayhan ldquoInhibition of endothelin-1receptors improves impaired nitric oxide synthase-dependentdilation of cerebral arterioles in type-1 diabetic ratsrdquo Microcir-culation vol 17 no 6 pp 439ndash446 2010

[5] M C Gong I Gorenne P Read et al ldquoRegulation by GDIof RhoARho-kinase-induced Ca2+ sensitization of smoothmuscle myosin IIrdquo The American Journal of PhysiologymdashCellPhysiology vol 281 no 1 pp C257ndashC269 2001

[6] L J Janssen T Tazzeo J Zuo E Pertens and S Keshavjee ldquoKC1evokes contraction of airway smooth muscle via activation ofRhoA and Rho-kinaserdquo The American Journal of PhysiologymdashLung Cellular and Molecular Physiology vol 287 no 4 ppL852ndashL858 2004

[7] R P Brandes D Y Kim F H Schmitz-Winnenthal et alldquoIncreased nitrovasodilator sensitivity in endothelial nitricoxide synthase knockoutmice Role of soluble guanylyl cyclaserdquoHypertension vol 35 no 1 pp 231ndash236 2000

[8] S Duchemin M Boily N Sadekova and H Girouard ldquoThecomplex contribution of NOS interneurons in the physiologyof cerebrovascular regulationrdquo Front Neural Circuits vol 6 no51 pp 1ndash19 2012

[9] M A Schwartz and A R Horwitz ldquoIntegrating adhesionprotrusion and contraction during cell migrationrdquo Cell vol125 no 7 pp 1223ndash1225 2006

[10] K Obara S Nishizawa M Koide et al ldquoInteractive roleof protein kinase C-120575 with Rho-kinase in the developmentof cerebral vasospasm in a canine two-hemorrhage modelrdquoJournal of Vascular Research vol 42 no 1 pp 67ndash76 2005

[11] Y Watanabe F M Faraci and D D Heistad ldquoActivation ofrho-associated kinase during augmented contraction of thebasilar artery to serotonin after subarachnoid hemorrhagerdquoThe American Journal of PhysiologymdashHeart and CirculatoryPhysiology vol 288 no 6 pp 2653ndash2658 2005

[12] M Sato E Tani H Fujikawa and K Kaibuchi ldquoInvolvement ofRho-kinase-mediated phosphorylation of myosin light chain inenhancement of cerebral vasospasmrdquo Circulation Research vol87 no 3 pp 195ndash200 2000

[13] Y Funakoshi T Ichiki H Shimokawa et al ldquoRho-kinasemediates angiotensin II-induced monocyte chemoattractantprotein-1 expression in rat vascular smooth muscle cellsrdquoHypertension vol 38 no 1 pp 100ndash104 2001

[14] M Higashi H Shimokawa T Hattori et al ldquoLong-terminhibition of Rho-Kinase suppresses angiotensin II-inducedcardiovascular hypertrophy inRats in vivo effect on endothelialNAD(P)H oxidase systemrdquo Circulation Research vol 93 no 8pp 767ndash775 2003

[15] G Loirand P Guerin and P Pacaud ldquoRho kinases in cardiovas-cular physiology and pathophysiologyrdquo Circulation Researchvol 98 no 3 pp 322ndash334 2006

[16] Y Rikitake H H Kim Z Huang et al ldquoInhibition of Rhokinase (ROCK) leads to increased cerebral blood flow andstroke protectionrdquo Stroke vol 36 no 10 pp 2251ndash2257 2005

[17] K Ishiko T Sakoda T Akagami et al ldquoHyperglycemia inducedcell growth and gene expression via the serum response elementthrough rhoa and rho-kinase in vascular smooth muscle cellsrdquoPreparative Biochemistry and Biotechnology vol 40 no 2 pp139ndash151 2010

[18] M F Olson ldquoApplications for ROCKkinase inhibitionrdquoCurrentOpinion in Cell Biology vol 20 no 2 pp 242ndash248 2008


[19] T Ishizaki M Uehata I Tamechika et al ldquoPharmacologicalproperties of Y-27632 a specific inhibitor of rho- associatedkinasesrdquo Molecular Pharmacology vol 57 no 5 pp 976ndash9832000

[20] A K Vellimana E Milner T D Azad et al ldquoEndothelial nitricoxide synthase mediates endogenous protection against sub-arachnoid hemorrhage-induced cerebral vasospasmrdquo Strokevol 42 no 3 pp 776ndash782 2011

[21] C Pagiatakis J W Gordon S Ehyai and J C McDermottldquoA novel RhoAROCK-CPI-17-MEF2C signaling pathway reg-ulates vascular smooth muscle cell gene expressionrdquo Journal ofBiological Chemistry vol 287 no 11 pp 8361ndash8370 2012

[22] L D Longo Y Zhao W Long et al ldquoDual role of PKCin modulating pharmacomechanical coupling in fetal andadult cerebral arteriesrdquo The American Journal of PhysiologymdashRegulatory Integrative and Comparative Physiology vol 279 no4 pp R1419ndashR1429 2000

[23] A P Somlyo and A V Somlyo ldquoSignal transduction throughthe RhoARho-kinase pathway in smooth musclerdquo Journal ofMuscle Research and Cell Motility vol 25 no 8 pp 613ndash6152004

[24] K Mori T Yamamoto M Miyazaki et al ldquoOptimal cere-brospinal fluid magnesium ion concentration for vasodilatoryeffect and duration after intracisternal injection of magnesiumsulfate solution in a canine subarachnoid hemorrhage modelrdquoJournal of Neurosurgery vol 114 no 4 pp 1168ndash1175 2011

[25] C Muroi J K Burkhardt M Hugelshofer M Seule KMishima and E Keller ldquoMagnesium and the inflammatoryresponse potential pathophysiological implications in theman-agement of patients with aneurysmal subarachnoid hemor-rhagerdquoMagnesium Research vol 25 no 2 pp 64ndash71 2012

[26] J T Tzen T R Jinn Y C Chen et al ldquoMagnesium lithosper-mate B possesses inhibitory activity on Na+ K+-ATPase andneuroprotective effects against ischemic strokerdquo Acta Pharma-cologica Sinica vol 28 no 5 pp 609ndash615 2007

[27] J T Deng C Sutherland D L Brautigan M Eto and M PWalsh ldquoPhosphorylation of the myosin phosphatase inhibitorsCPI-17 and PHI-1 by integrin-linked kinaserdquo BiochemicalJournal vol 367 no 2 pp 517ndash524 2002

[28] J Y Han J Y Fan Y Horie et al ldquoAmeliorating effects of com-pounds derived from Salvia miltiorrhiza root extract on micro-circulatory disturbance and target organ injury by ischemia andreperfusionrdquo Pharmacology andTherapeutics vol 117 no 2 pp280ndash295 2008

[29] W S Park Y K Son J Han et al ldquoStaurosporine inhibitsvoltage-dependent K+ current through a PKC-independentmechanism in isolated coronary arterial smooth muscle cellsrdquoJournal of Cardiovascular Pharmacology vol 45 no 3 pp 260ndash269 2005

[30] Y J Lee D G Kang J S Kim and H S Lee ldquoEffect of Buddlejaofficinalis on high-glucose-induced vascular inflammation inhuman umbilical vein endothelial cellsrdquo Experimental Biologyand Medicine vol 233 no 6 pp 694ndash700 2008

[31] S Iwabuchi T Yokouchi M Hayashi H Uehara M Uedaand H Samejima ldquoIntra-arterial administration of fasudilhydrochloride for vasospasm following subarachnoid hemor-rhagemdashanalysis of time-density curve with digital subtractionangiographyrdquo Neurologia Medico-Chirurgica vol 46 no 11 pp535ndash539 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


[4 7] This is compatible with the findings that a PKCinhibitor staurosporine can abolish ET-1 induced contrac-tion in rabbit basilar artery [9] Moreover lines of evidenceaccumulated to present have suggested that activated PKCplays a role in SAH induced vasospasm [10] For examplephorbol 12 13-dibutyrate a PKC activator can induce apotent and long-lasting contraction of the rabbit basilarartery [11] In a canine double-hemorrhage model the mem-brane translocation of PKC120575 from cytosol in the basilarartery was observed after the second injection of autologousblood on the 4th day when vasospasm was robust [10 12]Furthermore intrathecal injection of the PKC120575 inhibitorrottlerin inhibited hemorrhage induced vasospastic responseas well as the translocation of PKC120575 [10]

On the contrary the key event for triggering vasomotoreffect of NOET-1 in SAH on the rho-kinase pathway hasattracted less attention despite of documented involvementof rho-kinase in cerebral vasospasm following SAH Rhoa family of small G-proteins is composed of 3 membersrhoA rhoB and rhoC which behaves like a substantial role inintracellular signaling [8 13] Inactive rho is resided as aGDP-bound form in the cytosol If the vascular smooth musclecells were stimulated by vasoactive agents and subsequentGDP-GTP exchange rho may become activated and thentranslocated to the cell membrane where it interacts withits downstream effectors rho-kinase (ROCK) [13 14] TheROCK family includes two isoforms namely ROCK-I andROCK-II By phosphorylating myosin light chain phos-phatase (MLCP) and inhibiting the activity of phosphataseat the myosin-binding subunit rho-kinase is stimulated andpromotes smooth muscle contraction [15 16] In the studyof a canine two-hemorrhage model topical application of aROCK inhibitor Y-27632 dose-dependently decreased thespastic response Rho-kinase activity and phosphorylation ofMLCP in the basilar artery [17 18] Moreover intra-arterialadministration of fasudil another kind of ROCK inhibitor isalso proved to be effective for the treatment of vasospasm inSAH patients [19]

Besides ET-1 nitric oxide (NO) composed of endothe-lial NOs(eNOs) neuronal NOs(nNOs) and inducible NOs(iNOs) is believed to be an important regulator of thecerebral vascular tone [7 20] Upon synthesis NO bindingactivates soluble guanylyl cyclase (sGC) a heterodimericenzyme consisting of 120572 (120572

1 1205722 and 120572

3) and 120573 (120573

1 1205732 and

1205733) subunits [21] Activation of sGC leads to convert GTP

to cGMP which in turn activates cGMP-dependent proteinkinase (PKG) and cGMP gated ion channel among targetcells In blood vessels PKG can phosphorylate the inositol 14 5-triphosphate receptor and decrease Ca2+ concentrationand ultimately result in smooth muscle relaxation [9 2223] Under normal physiological conditions the productionsof ET-1 and NO yield a balanced cerebral vascular toneHowever enhanced generation of ET-1 along with impairedNO production (NO-dependent) or with excessively inducedNO (NO-independent) was noted in human and animalswith SAH [3] Nevertheless the effect of NOET-1 productionon the sGCcGMP signaling pathway has not been fullyinvestigated

In the present study we aimed to assess the neurologicaldeficits and plasma NOET-1 levels as well as the expressionsof PKC120575 rhoA ROCK-II and sGCcGMPPKG in the basilarartery of rats subject to SAH The effect of magnesiumlithospermate B (MLB) on the NOSsGCcGMPPKG andrhoAROCK signaling pathway in SAH was investigated

2 Materials and Methods

21Materials Anti-rabbit sGC1205721 and sGC1205731 and anti-mouse120573-actin antibodies were bought from Sigma-Aldrich (Shang-hai PRC) Anti-rabbit PKG anti-mouse PKC120575 anti-rabbitROCK-II anti-mouse rhoA and horseradish peroxidase-labeled goat anti-mouse IgG antibodies were obtained fromAbcam (Cambridge MA USA) BD Transduction Lab (BDBiosciences California USA) Upstate Biotech (NY USA)Santa Cruz Biotech (Santa Cruz Biotechnology Inc Cal-ifornia USA) and Chemicon International (CA USA)respectively CNMprotein extraction kits were fromBiochain(Hayward CA USA) ET-1 and cGMP ELISA kits wereproducts of Assay Designs (Enzo Life Sciences Inc NYUSA) and Cayman Chemical (Michigan USA) respectivelyMagnesium Lithospermate B was purified by Ms Wu SC(Kaohsiung Medical University Hospital Kaohsiung Tai-wan ROC) according to the modified protocol describedby Tanaka et al Dried Salvia miltiorrhiza were obtainedinoculation with methanol for 8 hr and then concentratedMLB was extracted by the aid of a Sephadex LH-20 multiplecolumn chromatography for four times (Pharmacia FineChemicals Piscataway NJ USA)

22 Animal Protocols All the experimental protocols wereapproved by the Kaohsiung Medical University Hospitalanimal research committee Forty-five male Sprague-Dawleyrats (BioLasco Taiwan Co Ltd authorized by Charles RiverLab) weighing 250ndash350 g were assigned into five groups (9animals each) Group 1 was designated as sham-operatedGroup 2 rats subjected to SAH only Group 3 SAH ratstreated with vehicle (01molL PBS) and Groups 4 and 5SAH animals received 10mgkgday MLB treatment at 24 hr(prevention protocol) and 1 hr (reversal protocol) after SAHrespectively To induce SAH rats were anesthetized witha mixture of ketamine (40mgkg) and xylazine (6mgkg)intraperitoneally (ip) and 03mL fresh blood was drawnfrom the central tail artery and injected into the craniocervi-cal junction using a stereotactic technique Further nine ani-mals were enrolled in the 01 120583L NG-nitro-L-arginine methylester (L-NAME) intrathecal injection plus 10mgkgdayMLBpreconditioning SAH rats

23 Hemodynamic Measurements Heart rate blood pres-sure and rectal temperature were monitored before and afterMLB treatment as well as at 48 hr after the induction ofSAH by a tail-cuff method (SC1000 Single Channel SystemHatteras Instruments North Carolina USA) and rectal ther-mometer (BIO-BRET-2-ISO FL USA)

24 Neurological Assessment Behavior assessment adapteda modified limb-placing tests (MLPT) [2] was performed








lowast















eNO

S re

lativ

e to

sham

(fol

ds)

0

02

04

06

08

1

12

Sham SAH

SAH

V T P PMLB

lowastlowast

lowast

L-N

AM

E

lowastlowastlowast

0

500

1000

1500

2000

2500

3000

Sham SAH V T P PMLB

CSF

ET le

vel (

pgd

L)

SAH

L-N

AM

E

(a)

0010203040506070809

1

Sham SAH V T P PMLB

SAH

lowastlowastlowast

lowastlowastlowast

(B)(A) (C) (D) (E) (F)

L-N

AM

E

Lum

en ar

ea o

f bas

ilar

arte

ry(m

m3 )

(b)



3 Results





Membrane

Cytosol

(A) (B) (C) (D) (E)Ra

tio o

f mem

bran

ecy

topl

asm

Rho

A (f

olds

)

0

02

04

06

08

1

12

Sham SAH V T P P

MLB

SAH

lowastlowastlowast

lowastlowastlowast

L-N

AM

E

Rho A (25kD)

(a)

(A) (B) (C) (D) (E)

0

02

04

06

08

1

12

14

Sham SAH V T P PMLB

SAH

lowast

lowastlowast

ROCK II (150kD)

120573-Actin (43kD)

L-N

AM

E

ROCK

II ex

pres

sion

(rel

ativ

e fol

d to

SA

H g

roup

)

(b)










002040608

112141618

2

Sham SAH V T P PMLB

SAH

(A) (B) (C) (D) (E)

lowast

lowastlowast

L-N

AM

E

Ratio

of s

GC1205721

(rel

ativ

e to

SAH

gro

up)

sGC1205721 (80kD)

120573-Actin (43kD)

(a)

(A) (B) (C) (D) (E)

0

05

1

15

2

25

Sham SAH V T P P

MLBSAH

lowastlowast

lowast

L-N

AM

E

sGC1205731 (80kD)

120573-Actin (43kD)

Ratio

of s

GC1205731

(rel

ativ

e to

SAH

gro

up)

(b)


Figure 2




4 Discussion



002040608

112141618

2

Sham SAH V T P P

MLBSAH

PKG120575 (75kD)

120573-Actin (43kD)

lowastlowastlowast

L-N

AM

E

(A) (B) (C) (D) (E)

PKG

expr

essio

n(fo

ld re

lativ

e to

SAH

gro

up)






cGM

P le

vel (

pmol

mg

prot

ein)

0

001

002

003

004

005

006

007

008

Sham SAH V T P PMLB

SAH

lowastlowastlowast

L-N

AM

E







Abbreviations




Acknowledgment


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























lowast















eNO

S re

lativ

e to

sham

(fol

ds)

0

02

04

06

08

1

12

Sham SAH

SAH

V T P PMLB

lowastlowast

lowast

L-N

AM

E

lowastlowastlowast

0

500

1000

1500

2000

2500

3000

Sham SAH V T P PMLB

CSF

ET le

vel (

pgd

L)

SAH

L-N

AM

E

(a)

0010203040506070809

1

Sham SAH V T P PMLB

SAH

lowastlowastlowast

lowastlowastlowast

(B)(A) (C) (D) (E) (F)

L-N

AM

E

Lum

en ar

ea o

f bas

ilar

arte

ry(m

m3 )

(b)



3 Results





Membrane

Cytosol

(A) (B) (C) (D) (E)Ra

tio o

f mem

bran

ecy

topl

asm

Rho

A (f

olds

)

0

02

04

06

08

1

12

Sham SAH V T P P

MLB

SAH

lowastlowastlowast

lowastlowastlowast

L-N

AM

E

Rho A (25kD)

(a)

(A) (B) (C) (D) (E)

0

02

04

06

08

1

12

14

Sham SAH V T P PMLB

SAH

lowast

lowastlowast

ROCK II (150kD)

120573-Actin (43kD)

L-N

AM

E

ROCK

II ex

pres

sion

(rel

ativ

e fol

d to

SA

H g

roup

)

(b)










002040608

112141618

2

Sham SAH V T P PMLB

SAH

(A) (B) (C) (D) (E)

lowast

lowastlowast

L-N

AM

E

Ratio

of s

GC1205721

(rel

ativ

e to

SAH

gro

up)

sGC1205721 (80kD)

120573-Actin (43kD)

(a)

(A) (B) (C) (D) (E)

0

05

1

15

2

25

Sham SAH V T P P

MLBSAH

lowastlowast

lowast

L-N

AM

E

sGC1205731 (80kD)

120573-Actin (43kD)

Ratio

of s

GC1205731

(rel

ativ

e to

SAH

gro

up)

(b)


Figure 2




4 Discussion



002040608

112141618

2

Sham SAH V T P P

MLBSAH

PKG120575 (75kD)

120573-Actin (43kD)

lowastlowastlowast

L-N

AM

E

(A) (B) (C) (D) (E)

PKG

expr

essio

n(fo

ld re

lativ

e to

SAH

gro

up)






cGM

P le

vel (

pmol

mg

prot

ein)

0

001

002

003

004

005

006

007

008

Sham SAH V T P PMLB

SAH

lowastlowastlowast

L-N

AM

E







Abbreviations




Acknowledgment


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















eNO

S re

lativ

e to

sham

(fol

ds)

0

02

04

06

08

1

12

Sham SAH

SAH

V T P PMLB

lowastlowast

lowast

L-N

AM

E

lowastlowastlowast

0

500

1000

1500

2000

2500

3000

Sham SAH V T P PMLB

CSF

ET le

vel (

pgd

L)

SAH

L-N

AM

E

(a)

0010203040506070809

1

Sham SAH V T P PMLB

SAH

lowastlowastlowast

lowastlowastlowast

(B)(A) (C) (D) (E) (F)

L-N

AM

E

Lum

en ar

ea o

f bas

ilar

arte

ry(m

m3 )

(b)



3 Results





Membrane

Cytosol

(A) (B) (C) (D) (E)Ra

tio o

f mem

bran

ecy

topl

asm

Rho

A (f

olds

)

0

02

04

06

08

1

12

Sham SAH V T P P

MLB

SAH

lowastlowastlowast

lowastlowastlowast

L-N

AM

E

Rho A (25kD)

(a)

(A) (B) (C) (D) (E)

0

02

04

06

08

1

12

14

Sham SAH V T P PMLB

SAH

lowast

lowastlowast

ROCK II (150kD)

120573-Actin (43kD)

L-N

AM

E

ROCK

II ex

pres

sion

(rel

ativ

e fol

d to

SA

H g

roup

)

(b)










002040608

112141618

2

Sham SAH V T P PMLB

SAH

(A) (B) (C) (D) (E)

lowast

lowastlowast

L-N

AM

E

Ratio

of s

GC1205721

(rel

ativ

e to

SAH

gro

up)

sGC1205721 (80kD)

120573-Actin (43kD)

(a)

(A) (B) (C) (D) (E)

0

05

1

15

2

25

Sham SAH V T P P

MLBSAH

lowastlowast

lowast

L-N

AM

E

sGC1205731 (80kD)

120573-Actin (43kD)

Ratio

of s

GC1205731

(rel

ativ

e to

SAH

gro

up)

(b)


Figure 2




4 Discussion



002040608

112141618

2

Sham SAH V T P P

MLBSAH

PKG120575 (75kD)

120573-Actin (43kD)

lowastlowastlowast

L-N

AM

E

(A) (B) (C) (D) (E)

PKG

expr

essio

n(fo

ld re

lativ

e to

SAH

gro

up)






cGM

P le

vel (

pmol

mg

prot

ein)

0

001

002

003

004

005

006

007

008

Sham SAH V T P PMLB

SAH

lowastlowastlowast

L-N

AM

E







Abbreviations




Acknowledgment


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Membrane

Cytosol

(A) (B) (C) (D) (E)Ra

tio o

f mem

bran

ecy

topl

asm

Rho

A (f

olds

)

0

02

04

06

08

1

12

Sham SAH V T P P

MLB

SAH

lowastlowastlowast

lowastlowastlowast

L-N

AM

E

Rho A (25kD)

(a)

(A) (B) (C) (D) (E)

0

02

04

06

08

1

12

14

Sham SAH V T P PMLB

SAH

lowast

lowastlowast

ROCK II (150kD)

120573-Actin (43kD)

L-N

AM

E

ROCK

II ex

pres

sion

(rel

ativ

e fol

d to

SA

H g

roup

)

(b)










002040608

112141618

2

Sham SAH V T P PMLB

SAH

(A) (B) (C) (D) (E)

lowast

lowastlowast

L-N

AM

E

Ratio

of s

GC1205721

(rel

ativ

e to

SAH

gro

up)

sGC1205721 (80kD)

120573-Actin (43kD)

(a)

(A) (B) (C) (D) (E)

0

05

1

15

2

25

Sham SAH V T P P

MLBSAH

lowastlowast

lowast

L-N

AM

E

sGC1205731 (80kD)

120573-Actin (43kD)

Ratio

of s

GC1205731

(rel

ativ

e to

SAH

gro

up)

(b)


Figure 2




4 Discussion



002040608

112141618

2

Sham SAH V T P P

MLBSAH

PKG120575 (75kD)

120573-Actin (43kD)

lowastlowastlowast

L-N

AM

E

(A) (B) (C) (D) (E)

PKG

expr

essio

n(fo

ld re

lativ

e to

SAH

gro

up)






cGM

P le

vel (

pmol

mg

prot

ein)

0

001

002

003

004

005

006

007

008

Sham SAH V T P PMLB

SAH

lowastlowastlowast

L-N

AM

E







Abbreviations




Acknowledgment


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















002040608

112141618

2

Sham SAH V T P PMLB

SAH

(A) (B) (C) (D) (E)

lowast

lowastlowast

L-N

AM

E

Ratio

of s

GC1205721

(rel

ativ

e to

SAH

gro

up)

sGC1205721 (80kD)

120573-Actin (43kD)

(a)

(A) (B) (C) (D) (E)

0

05

1

15

2

25

Sham SAH V T P P

MLBSAH

lowastlowast

lowast

L-N

AM

E

sGC1205731 (80kD)

120573-Actin (43kD)

Ratio

of s

GC1205731

(rel

ativ

e to

SAH

gro

up)

(b)


Figure 2




4 Discussion



002040608

112141618

2

Sham SAH V T P P

MLBSAH

PKG120575 (75kD)

120573-Actin (43kD)

lowastlowastlowast

L-N

AM

E

(A) (B) (C) (D) (E)

PKG

expr

essio

n(fo

ld re

lativ

e to

SAH

gro

up)






cGM

P le

vel (

pmol

mg

prot

ein)

0

001

002

003

004

005

006

007

008

Sham SAH V T P PMLB

SAH

lowastlowastlowast

L-N

AM

E







Abbreviations




Acknowledgment


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















002040608

112141618

2

Sham SAH V T P P

MLBSAH

PKG120575 (75kD)

120573-Actin (43kD)

lowastlowastlowast

L-N

AM

E

(A) (B) (C) (D) (E)

PKG

expr

essio

n(fo

ld re

lativ

e to

SAH

gro

up)






cGM

P le

vel (

pmol

mg

prot

ein)

0

001

002

003

004

005

006

007

008

Sham SAH V T P PMLB

SAH

lowastlowastlowast

L-N

AM

E







Abbreviations




Acknowledgment


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Abbreviations




Acknowledgment


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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