research article scutellarin reduces endothelium

Research ArticleScutellarin Reduces Endothelium Dysfunction throughthe PKG-I Pathway

Xiaohua Du12 Chen Chen1 Min Zhang3 Donghua Cai1 Jiaqi Sun1 Jian Yang1 Na Hu1

Congji Ma3 Liyan Zhang2 Jun Zhang4 and Weimin Yang1

1School of Pharmaceutical Science and Yunnan Key Laboratory of Pharmacology for Natural Products Kunming Medical UniversityKunming 650500 China2Department of Respiratory Medicine First Affiliated Hospital of Kunming Medical University Kunming 650032 China3Pharmacy Department First Affiliated Hospital of Kunming Medical University Kunming 650032 China4Department of Clinical Pharmacy First Affiliated Hospital of Kunming Medical University Kunming 650032 China

Correspondence should be addressed to Jun Zhang zhangjunyang126com and Weimin Yang ywmbessieyeahnet

Received 4 May 2015 Revised 25 July 2015 Accepted 4 August 2015

Academic Editor Dan Hu

Copyright copy 2015 Xiaohua Du et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Purpose In this report we investigated the protective mechanism of scutellarin (SCU) in vitro and in vivo which could be involvedin endothelial cGMP-dependent protein kinase (PKG) vasodilator stimulated phosphoprotein (VASP) pathway and vascularendothelium dysfunction (EtD) Method Human brain microvascular endothelial cells (HBMECs) with hypoxia reoxygenation(HR) treatment and rats with cerebral ischemia reperfusion (CIR) treatment were applied Protein and mRNA expression of PKGVASP and p-VASP were evaluated by Western blot and RT-PCR methods Vascular EtD was assessed by using wire myographyto determine endothelium-dependent vasorelaxation in isolated rat basilar artery (BA) Result In cultured HBMECs SCU (01 1and 10120583M) increased cell viability mRNA protein level and phosphorylative activity of PKG and VASP against HR injury In HRmodel of BA SCU increased protein level of P-VASP In rat CIR model wire myography demonstrated that SCU (45 and 90mgkgiv) significantly reduced ischemic size by partially restoring the endothelium dependent vasodilation of BA PKG inhibitor Rp-8-Br-cGMPS (50 120583gkg iv) reversed this protection of SCU in CIR rats Conclusion SCU protects against cerebral vascular EtDthrough endothelial PKG pathway activation

1 Introduction

Vascular endothelium has complex physiological functionssuch as maintaining vascular tone inhibiting platelet aggre-gation reducing endothelial permeability reducing adhesionmolecules expression and inhibiting vascular smoothmusclecell (VSMC) proliferation [1] Endothelial cells in easily dam-aged anatomical functional interface are firstly affected by avariety of injuries including ischemia reperfusion (IR) thatstimulating effect leads to the damage of structural integrityand reduce of function and then endothelial dysfunction(EtD) occurs EtD may be an important basis for a numberof diseases [1 2] Vascular tension adjustment disorders andabnormal expression of adhesion molecules are the mainmanifests of EtD Study has shown that IR produces vascularEtD as defined by abrogated endothelium-dependent dilation

[2] In addition previous study indicated that hypoxia reoxy-genation (HR) caused selective inhibition of response toacetylcholine (ACh) in the cerebral arteries [3]

Ischemia reperfusion (IR) leads to tissue injury in variouspathophysiological conditions IR directly affects the vascularwall and luminal surface of blood vessel causing damagesincluding hemorrhage capillary plugging adhesion and infil-tration of granulocytes impaired vascular permeability andendothelial dysfunction (EtD) [4] Endothelial cells whilebeing particularly susceptible to IR injury play an activerole in IR-induced organ damage [5] EtD reduces perfusionto ischemia areas and thereby exacerbates tissue injury andsubsequent damage [6]

cGMP-dependent protein kinase (PKG) is a serinethre-onine protein kinase that is activated by cGMP Accumulat-ing evidences are demonstrating that PKG phosphorylates

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 430271 12 pageshttpdxdoiorg1011552015430271

2 Evidence-Based Complementary and Alternative Medicine

a number of biologically important targets which are neededto accomplish multiple cellular functions and its dysregula-tion has been incriminated in many diseases such as hyper-tension atherosclerosis chronic heart failure left ventricularhypertrophy ventricular remodelling IR injury diabetes andcancer [7] Among the three isoforms of PKG-I120572 PKG-I120573and PKG-II PKG-I120572 is the strongest vascular tonemodulatorregulating cell motility migration proliferation and vasculartone [8]

Moreover there was report that PKG is involved intestosterone-induced vasodilation of human umbilical artery[9] Upon activation PKG phosphorylates VASP which inturn activates downstream ion channels leading to vascularsmooth muscle relaxation and vasodilation Previous reportshave suggested that vasorelaxant effects of baicalin aremainlyattributed to voltage-dependent Ca2+ channel (VDCC) inhi-bition and BKCa channel activation through PKA and PKGpathways [10]

Vasodilator stimulated phosphoprotein (VASP) belongsto the EnaVASP protein family and is an important PKG-Isubstrate and actin regulatory protein Studies [11] suggestedthat phosphorylated VASP at serine 239 (p-VASP) has beenshown to be a usefulmonitor for PKG-I activity in intact cells

Scutellarin (SCU) 4101584056-trihydroxy flavonoid-7-glucu-ronide was reported to be the primary active ingredientof breviscapine which is a mixture of flavonoid glycosidesisolated from a Chinese traditional medicine plant Erigeronbreviscapus (Vant) Hand Mazz [12] The plant extracts andSCU have been used in China to treat a variety of disordersincluding cardiovascular cerebrovascular and inflammatorydiseases for many years [13] In animal studies SCU hasbeen reported to be neuroprotective in rat cerebral ischemiareperfusion (CIR) models [14] via augmentation of antioxi-dant defense capacity [13] In addition SCU prevented EtDin diabetic rats and inhibited translocation of protein kinaseC in diabetic thoracic aorta of the rat [15] Our earlierstudy [16 17] showed that relaxation effect of SCU on arterywas predominantly endothelium dependent and partiallyinvolved the catalase-sensitive nitric oxide synthase signalingpathway

Based on these observations we hypothesize that SCUreduces EtD through the PKG-I pathway To verify thishypothesis we test the protein level and mRNA expressionof PKG-I VASP and p-VASP in human brain microvascularendothelial cells (HBMECs) The effects of SCU on EtD ofbrain basilar artery (BA) and infarct size were checked in ratswith CIR injury

2 Materials and Methods

21 Chemicals and Drugs SCU was obtained from Kun-ming Longjin Pharmaceutical Co (Kunming China) Cellculture reagents DMEM modified RPMI-1640 mediumand fetal bovine serum were obtained from the Hyclone(Thermo Scientific USA) Other items include Wire Myo-graph System DMT (Danish Myo Technology CompanyDenmark) and Power Lab data recording and analyt-ical system (ADInstruments Ltd Australia) HBMECswere purchased from Yangsen Biology Limited Company

(Shanghai China) U46619 was purchased from CaymanChemical Company PKG inhibitor Rp-8-Br-cGMPS waspurchased from Santa Cruz Biotechnology (Dallas TXUSA) 3-(45-Dimethylthiazol-2-yl)-25-diphenyltetrazoliumbromide (MTT) triphenyl tetrazolium chloride (TTC) andACh were purchased from Sigma-Aldrich (St Louis MOUSA)

22 Animals Sprague-Dawley rats (180ndash220 g male andfemale in each half) were provided by the animal center ofKunming Medical University All animals were housed inmicroisolation under conditions of constant temperature andcontrolled illumination (light on 12-hour lightdark cycle)Food and water were available ad libitum All the animalsused in the experiment received humane care All surgicaland experimental procedures were in accordance with theinstitutional animal care guidelines The animal study wasapproved by the Animal Care and Use Committee of Kun-ming Medical University and conformed to the standards setby the Yunnan Experimental Animal Management Board

23 Methods

231 Endothelial Cell Culture and HR Treatment HBMECswere obtained from the Shanghai Yangsen BiochemicalTechnology Company (Shanghai China) and grown in1640 medium supplemented with 10 fetal bovine serumand 1 penicillinstreptomycin antibiotics Tightly confluentmonolayers of HBMECs from 4thminus15th passage were usedin all experiments In experiments checking the effects ofSCU under normal condition cells were treated with vehiclecontrol (NS) or SCU (01 10 and 100 120583M) at different con-centrations for 26 hours In experiments with HR treatmentscells were divided into 5 groups including control HRmodelHR + SCU 01 120583M HR + SCU 1 120583M and HR + SCU 10 120583Mgroup Control cells were cultured in parallel and kept innormal culture condition for the entire time period (26 h)Simulated HR injury was induced according to previouslydescribed procedures [18] with minor modifications BrieflyHBMECs were placed in a humidified hypoxic chamber(HF100 Heal Force Biotech Co Shanghai China) for 12 h ofhypoxia (5 CO

2+ 2 O

2+ 93 N

2) with medium free of

glucose and serum at 37∘C followed by 12 h of reoxygenation(5 CO

2+ 95 air) in complete medium containing glucose

and serum For HR + SCU groups cells were incubatedwith SCU at different concentrations for 2 h prior to hypoxiatreatment and during hypoxia (12 h) and reoxygenation (12 h)injury For control group cells were treated with vehiclecontrol (NS) for 26 h and for SCU groups cells were givendifferent concentrations of SCU for 26 h separately At the endof the experiment cell viability was examined using MTTassay as described below Furthermore cells of each groupwere also collected for RT-PCR assay and Western blottingassay as described in the following sections All experimentswere performed in triplicate

232 MTT Assay of Cell Viability For MTT assay cellswere plated in 96-well flat-bottomed plates at a densityof 3 times 104 cellsmL and 90120583Lwell Cells were cultured in

Evidence-Based Complementary and Alternative Medicine 3

normal culture condition or treatedwith simulatedHR injuryas described above After the treatments 20120583L of MTT(5mgmL) was added to each well and the plates were incu-bated for 4 h at 37∘CThen 100120583L of lysis buffer (20 sodiumdodecyl sulfate [SDS] in 50 NN-dimethylformamide con-taining 04 [v v] 1 N HCL and 05 [v v] 80 acetic acid)was added to eachwell and incubated overnight Cell viabilitywas determined by measuring the ability of metabolicallyactive cells to convert the yellow tetrazolium salt MTT(5mgmL PH = 74) into purple formazan crystals with amicroplate reader at 570 nm Results of three independentexperiments (each conducted in triplicate) were used forstatistical analysis

233 Western Blot Analysis The PKG-I VASP and p-VASPprotein levels in lyzed cell were examined by Westernanalysis Protein concentrations were determined by usingBCA Protein Assay Kit (Beyotime Biotechnology HaimenJiangsu China) Total protein (20120583g) was fractionated on10 SDS-PAGE and transferred to nitrocellulosemembranesMembranes were blocked with 10 defatted milk powdersolution at room temperature for 2 h and incubated overnightat 4∘C with the rabbit antibodies against VASP (concen-tration 1 500 Cell Signaling Technology Inc USA) p-VASP (Ser239) (concentration 1 2000 Santa Cruz Biotech-nology USA) and PKG-I (concentration 1 1000 Santa CruzBiotechnology USA) After three washes membranes wereincubated with horseradish peroxidase conjugated goat anti-rabbit IgG antibody (Santa Cruz Biotechnology USA) for15 h at room temperature Then four washes were repeatedand the protein was visualized with enhanced chemilumi-nescence kit (Sigma USA) The density values of bandswere quantified by densitometric analysis of scanned images(Scion Image 403) The relative protein ratio was calculatedby determining the integrated intensity of the bands of eachtreated group as a ratio of the control condition

234 RT-PCR Total RNAs were isolated from the cellsby using Trizol reagent (TaKaRa Japan) The sequences ofprimers (human PKG-I120572 and GAPDH) used in this studywere PKG-I120572 (forward AGCGGATCGAAGCAGGAG-GC and reverse TGACGGTCGCTGTCCGGGTA 728 bp)and GAPDH (forward AATCCCATCACCATCTTCC andreverse GAGTCCTTCCACGATACCAA 309 bp) respec-tively

Total RNA (1 120583g) was reverse-transcribed into cDNAusing a Quantscript RT Kit (Tiangen China) PCR wasperformed using a PCRMasterMix Kit (BioTeke China) in aGeneAmp PCR system 9600 (ABI Int) cDNA was amplifiedunder the thermocycling conditions as follows 3min initialdenaturation at 94∘C (1 cycle) 30 s denaturation at 94∘C (35cycles) 30 s annealing at 57∘C for human PKG andVASP and45 s extension at 72∘CThe last amplification was followed bya final 7min incubation at 72∘CPCRproductswere separatedby electrophoresis through 1 agarose gel stained withethidiumbromide and visualized byUV transillumination ina Tocan Gel Imaging System (Tocan Co Shanghai China)GAPDH was used as an internal control The mRNA levelwas calculated by determining the integrated intensity of

the bands of each treated group as a ratio of the control Eachsample was measured in triplicate and the mean thresholdcycle (Ct) value was calculated

235 Rat CIR Model and Evaluation of Cerebral InfarctVolume At room temperature (22 plusmn 2∘C) conditions therats with 10 chloral hydrate (0035mLkg) intraperitonealinjection of anesthesia were supinely fixed on the operatingtable giving the tail vein injection of drugs while startingsurgery The right external carotid artery was ligated andthen right middle cerebral artery was given reperfusion 24 hafter occlusion for 1 h [19] At the end of the reperfusionrats were decapitated The brains were rapidly removed andfrozen immediately at minus20∘C for 15min and then the brainswere cut into 2mm thick coronal sections which were stainedwith 1 TTC at 37∘C for 10min followed by fixation with 4paraformaldehyde for 1 hour Unstained areas were defined asischemic lesions whereas normal tissue was stained red Theinfarct areas were traced and quantified with IPP softwareInfarct areas of all sections were added to derive the totalinfarct area which was multiplied by the thickness of thebrain sections to obtain the infarct volume To compensatefor the effect of brain edema the corrected infarct volumewas calculated as previously described [20] Corrected infarctvolume equals total infarct volume multiply contralateralhemisphere volumeipsilateral hemisphere volume

To evaluate the effects of SCU on CIR injury SD ratswere divided into four groups (119899 = 10ndash12 in each group)sham CIRmodel and two SCU groups (45 or 90mgkg iv)In another experiment the influence of PKG inhibitor onthe effects of SCU was assessed The rats were divided intofour groups (119899 = 10ndash15 each) CIR model SCU (90mgkgiv) PKG inhibitor (50 120583gkg iv) and SCU (90mgkgiv) + PKG inhibitor (50120583gkg iv) treated group Druginfusion was initiated intravenously during surgery via thetail vein (2mLh) The sham and CIR model groups weregiven normal saline (NS)

236 Evaluation of EtD in Isolated BA from CIR Rats Theabove treated rats were sacrificed and the BA was gentlyexcised and rinsed off blood in 4∘C PSS buffer solution(140mM NaCl 47mM KCl 16mM CaCl

2 12mMMgSO

4

12mM MOPS 14mM Na2HPO4 002mM EDTA and

56mM D-glucose The PH of PSS was preadjusted to 74 at37∘C)The BA was cleaned of fat and surrounding tissue andthen was cut into approximately 1-2mm rings

An integrated wire myograph system (Model 620MDMT Asia Ltd Shanghai China) was applied with a MoticSMZ168-TL stereomicroscope to mount BA rings on 60 120583msteel wires in separated tissue baths of the wire myographsystem for tension recording The tissue baths were filledwith PSS solution (PH 74) at 37 plusmn 1∘C and aerated withO2 Washout was performed by draining and replacing the

bathing solution using a syringe Isometric tension signalswere recorded and data were collected by a PowerLab dataacquisition system (ADInstruments Asia Shanghai China)Each ring was stretched to an optimal tension of 1mN andpermitted to equilibrate for 90minutes before the experimentstarted The rings were contracted by U46619 (1120583molL)


and relaxed using cumulative addition of ACh (0001ndash1000 120583molL) to test the endothelial-dependent vasodilationby calculating EC

50and 119864max values

237 Preparation of HR Model of Isolated BA and Treatmentwith SCU Rats were sacrificed by intraperitoneal injection of10 (01mL100 g) urethane BA was removed and dissectedfree from brain tissues Isolated BA segments were placed inanaerobic sugar-free physical salt solution (PSS) with 3-(N-morpholino)propanesulfonic acid sodium polystyrene sul-fonate (MOPS) which leads to nitrogen hypoxia for 2 hoursthen the BA segments were changed into normal MOPS-PSS solution with restoring oxygen and glucose for 2 hoursDuring HR damage SCU (50120583M 100 120583M) was incubatedalong in SCU groups while HR model group was givenvehicle (NS) Control group was the normal BA At the end ofHR injury BA transferred into a precooled glass homogenizercontaining 200120583L precooled RIPA buffer (50mM Tris-HCl150mM NaCl 1mM EDTA 1mM PMSF 1mM Na

3VO4

1 mM NaF 1 NP-40 025 Na-deoxycholate 10 glyceroland 1mgmL of each of the phosphatase inhibitors aprotininleupeptin and pepstatin PH 74) Vessel tissues were thenhomogenized for 10min transferred to a 15mL Eppendorftube and centrifuged at 18000 rpm for 10min at 4∘C Thesupernatant was collected by decantation and stored at minus80∘Cuntil Western blot analysis

238 Calculations and Statistical Analysis Data were ex-pressed as means plusmn SEM Statistical analysis was performedusing statistical software Sigma Stat 35 The 119864max value rep-resents the maximal vasodilative response that drug causedEC50

is the concentration in which drug makes 50 of themaximum vasodilative effect Nonlinear regression analysisfor individual concentration-response curves was performedusing aHill algorithm in Sigma Plot 100 allowing for an indi-vidual geometric ldquoEC

50rdquo value to be calculated 119864max = [(the

maximal stress of precontraction minus the minimal stress)themaximal stress of precontraction] lowast 100 Comparisonswere made using one-way ANOVA analysis 119875 lt 005 wasconsidered statistically significant

3 Results

31 Effect of SCU on PKG-I and Cell Viability in NormalCultured HBMECs As seen from Figures 1(a) 1(b) and 1(d)SCU (01 1 and 10 120583M) treatment increased the protein levelsPKG-I p-VASP and VASP in normal HBMECs comparedwith control group In addition the ratio of p-VASP toVASP that indicated the activity of PKG was also increasedunder SCU treatment (Figure 1(c)) Particularly there wasapparent increase in protein levels of PKG-I P-VASP VASPandPKGactivity in normalHBMECswith SCU (1 and 10120583M)treatment

As shown in Figures 2(a) and 2(b) under normal con-dition SCU (01 1 and 10 120583M) incubation increased mRNAlevel of PKG-I120572 especially in SCU 1 120583M group

In our preliminary experiments checking the influence ofSCU on cell viability of normal HBMECs SCU with dose lt01 120583Mdid not cause significant changes in cell viability while

SCU with dose gt100 120583M exhibited cytotoxicity (data notshown)Therefore SCU at doses of 01 1 and 10120583Mwas usedin the present study As shown in Figure 2(c) results of MTTassay indicated that SCU 10 120583M incubation raised apparentlycell viability under normal culture condition while there arenot significant changes in SCU 01 and 1 120583M group

32 Effect of SCU on PKG-I and Cell Viability inHBMECswithHR Treatment The effects of SCU on protein expression ofPKG-I VASP and phosphorylation of VASP (p-VASP) underHR injury were shown in Figure 3 Compared with controlHR injury decreased p-VASP VASP and PKG especiallyp-VASP compared with model SCU preincubations couldsignificantly raise the protein expression of p-VASP VASPand PKG-I (Figures 3(a) 3(b) and 3(d)) HR injury alsoreduced PKG activity in model group (ratio of p-VASP andVASP) while SCU pretreatment could significantly augmentit especially in SCU 10 120583M (Figure 3(c))

As seen from Figures 4(a) and 4(b) the expressionof PKG-Ι120572 mRNA was significantly reduced in HR modelgroup while SCU (01 1 and 10 120583M) pretreatment increasedit sharply (Figure 4(b)) The results indicated SCU couldupregulatemRNA expression of PKG-I120572 and antagonized theHR-induced injury

As shown in Figure 4(c) HR injury greatly reduced cellviability of HBMECs and pretreatment of SCU (01 1 and10 120583M) could protect cells fromHR-induced injury especiallythe obvious effect of SCU (10 120583M) on viability (Figure 4(c))The data indicated that SCU has the protective effects onHBMECs against HR injury

33 The Effect of SCU and PKG Inhibitor in Rats with CIRTreatment As seen from Figures 5(a) and 5(b) comparedwithmodel group SCU (45 90mgkg) attenuated infarct sizein rats withCIR treatment especially in SCU45mgkg groupMoreover the values of vascular tensionwere reduced in SCU(90mgkg) group (Figure 5(c)) while the values of vasculartension in SCU (45mgkg) have no significant change

There was similar change in the values of BA cumulative-response curves of ACh EC

50 and 119864max in CIR rats CIR

injury caused BA EC50

and 119864max to be higher while inCIR rats handled with SCU (90mgkg) pretreatment thevalues of BA cumulative-response curves of ACh EC

50

and 119864max were low apparently These above results showedthat SCU (90mgkg) pretreatment has a protective effect onvascular EtD induced by CIR and endothelium-dependentvasodilation in response to AChwas significantly impaired inBA exposed to CIR treatment which showed that CIR modelwas successfully made

In order to check the mechanism of SCU the PKGinhibitor was used before the CIR rats were given SCUtreatment The results showed that PKG inhibitor reversedthe effects of SCU on improving infarct size and BA valuesof EC50and 119864max in CIR rats dealt with SCU As presented in

Figures 5(d) and 5(e) infarct sizes in rats treated with SCUand PKG inhibitor were bigger than those in rats handledwith SCU alone Meanwhile similar changes occurred in theEC50 119864max and the values of BA cumulative-response curves

of ACh in CIR rats handled with SCU and PKG inhibitor


Control

PKG-I

p-VASP

VASP

GAPDH

01 1 10

SCU (120583M)

(a)

Control 01 1 10

Prot

ein

leve

l (ra

tio o

f con

trol)

00

05

10

15

20

25

p-VASPVASP

lowast

lowast

lowastlowast

lowastlowast

SCU (120583M)

(b)

20

15

10

05

00Control 01 1 10

PKG

-I ac

tivity

(rat

io o

f p-V

ASP

VA

SP)

lowast lowast lowastlowast

SCU (120583M)

(c)

18

16

14

12

10

08

06

04

02

00Control 01 1 10

PKG

-I p

rote

in le

vel (

ratio

of c

ontro

l)lowastlowast

lowast

SCU (120583M)

(d)

Figure 1 The effect of SCU on the protein level of PKG-I VASP and p-VASP protein in normal HBMECs (a) Representative immunoblotand quantification of PKG-I p-VASP and VASP protein expression in normal HBMECs with SCU treatment (b) The ratio of p-VASP andVASP comparedwith control in normalHBMECswith SCU treatment Protein ratio of control was calculated by determining band integratedintensity as ratio of control (c) The ratio of p-VASP compared with VASP in normal HBMECs with SCU treatment (d) The ratio of PKG-Icompared with control in normal HBMECs with SCU treatment Protein ratio of control was calculated by determining band integratedintensity as ratio of control Normal HBMECs under normal culture condition cells were incubated with SCU (01 1 and 10120583M) for 24 hexcept control group One-way ANOVA on Rank followed by SNK test lowast119875 lt 005 lowastlowast119875 lt 001 compared to control Data are means plusmn SEM119899 = 3 independent experiments with independent culture

(Figure 5(c) and Table 1) The results indicated that PKGinhibitor reversed the effect of SCU on BA of rats with CIRtreatment through inhibiting improvement of endotheliumvasodilation and suggested that SCU might protect againstCIR via PKG signal pathway

34 The Effect of SCU on p-VASP in Isolated BA of Rats withHR Treatment The effects of SCU on protein expression ofphosphorylation of VASP (p-VASP) under HR injury wereshown in Figures 6(a) and 6(b) HR injury significantlydecreased p-VASP while SCU treatment could significantlyincrease the protein level of p-VASPThis shows that SCU canfight against HR injury in isolated BA blood vessel involvedin PKG pathway

4 Discussion

This study advances not only our understanding aboutthe regulation of VASP and PKG-I but also a mechanismunderlying the beneficial effects of SCU in treating cere-brovascular diseases In the present study we found firstly toour knowledge that SCU increased the protein and mRNAexpression of PKGandVASP inHBMECswithHR treatmentMoreover compared with model group SCU increased thecell viability and simultaneously raised the viability of PKG-IinHBMECswithHR treatmentThis suggested the protectiveeffects of SCU on the injury of HBMECs with HR involved inPKG-IVASP signaling pathway In our cell experiment wefound that the effect of SCU on normal HBMECs is sharply


PKG-I120572

GAPDH

Control 01 1 10

SCU (120583M)

(a)

Control 01 1 10

14

16

12

10

08

06

04

02

00

PKG

-I120572

mRN

A (r

atio

of c

ontro

l)

lowast

lowastlowast

SCU (120583M)

(b)

HBM

ECs v

iabi

lity

()

40

20

0

60

80

100

120

14024h lowast

Control 01 1 10

SCU (120583M)

(c)

Figure 2 The effect of SCU on mRNA expression of PKG-I and cell viability in normal HBMECs (a) Representative electrophotogramand quantification of PKG-I120572mRNA expression in normal HBMECs (b) The mRNA ratio of PKG-I120572 in normal HBMECs Ratio of controlwas calculated by determining band integrated intensity as ratio of control (c) The ratio of cell viability compared with control in normalHBMECs with SCU (01 1 and 10 120583M) treatment Cell viability was examined using MTT assay Normal HBMECs under normal culturecondition cells were incubated with SCU (01 1 and 10 120583M) for 24 h except control group One-way ANOVA on Rank followed by SNK testlowast119875 lt 005 lowastlowast119875 lt 001 compared to control Data are means plusmn SEM 119899 = 3 independent experiments with independent culture

weaker than that of HBMECs with HR treatment this resultshowed that SCU could fight against HR injury

In CIR rats SCU preincubations could significantlydecrease brain infarct size vascular tension and the valuesof BA cumulative-response curves of ACh EC

50 and 119864max

and increase protein level of p-VASP in HR BA Theseabove results showed that SCU (90mgkg) pretreatment hasa protective effect on vascular EtD induced by CIR andendothelium-dependent vasodilation in response toAChwassignificantly impaired in BA exposed toCIR treatment whichshowed that CIRmodel was successfullymadeMoreover ourresults showed that SCU decreased the cerebral infarct sizethis is consistentwith the previous studies by Lin et al [21 22]

In order to check the mechanism of SCU the PKGinhibitor was used before the CIR rats were given SCUtreatment The result shows that PKG inhibitor reversed theeffects of SCUon improving infarct size vascular tension andthe BA values of EC

50and 119864max in CIR rats dealt with SCU

This result indicated that PKG inhibitor reversed the effect

of SCU on BA of rats with CIR treatment through inhibitingimprovement of endothelium vasodilation and suggested thatSCU protects against CIR partially via PKG signal pathway

Endothelial cells play an important role in controllinglocal vascular tension In this study we proposed one ofthe mechanisms of SCU-attenuated EtD induced by IR viaPKG-IVASP signaling pathway Our results showed SCUattenuated the EC

50and 119864max values of ACh in isolated

BA induced by CIR but the PKG inhibitor reversed theeffects of SCU on improving EC

50and 119864max values of

BA in CIR rats This confirmed our hypothesis that SCUattenuated EtD induced by IR via PKG-IVASP signalingpathway and supported our grouprsquos previous studies that SCUvasorelaxation was predominantly endothelium dependentand involved nitric oxide synthase signaling pathway [17]Meanwhile we found that PKG inhibitor blocked the effect ofSCUon reducing the cerebral infarction sizeThismechanismcontributes at least in part to elucidating the beneficialeffects of SCU in CIR injury via PKG-I signal pathway


GAPDH

Control Model 01 1 10

p-VASP

VASP

PKG-I

SCU (120583M)

(a)

ModelControl 01 1 10

lowast

lowast

SCU (120583M)

Prot

ein

leve

l (ra

tio o

f con

trol )

00

02

04

06

08

10

12

14

16

p-VASPVASP

(b)

PKG

-I ac

tivity

(r

atio

of p

-VA

SPV

ASP

)

000204060810121416

lowast


SCU (120583M)

(c)

PKG

-I p

rote

in le

vel

(rat

io o

f con

trol )

00

05

10

15

20

25

lowast lowast


SCU (120583M)

(d)

Figure 3 The effect of SCU on the protein level of PKG-I VASP and p-VASP in HR HBMECs (a) Representative immunoblot andquantification of PKG-I p-VASP and VASP protein expression in HR HBMECs with SCU treatment (b) The ratio of p-VASP and VASPcompared with control in HRHBMECs with SCU treatment Protein ratio of control was calculated by determining band integrated intensityas ratio of control (c) The ratio of p-VASP compared with VASP in HR HBMECs with SCU treatment (d) The ratio of PKG-I comparedwith control in HR HBMECs with SCU treatment Protein ratio of control was calculated by determining band integrated intensity as ratioof control Control group cells were treated with vehicle control (NS) under normal culture condition for 26 h Model group cells wereincubated under normal culture condition for 2 h and then given HR treatment (hypoxia 12reoxygenation 12 hours) SCU groups cells wereincubated with SCU for 2 h prior to HR injury One-way ANOVA followed by SNK test 119875 lt 005 119875 lt 001 compared to model of HRgroup lowast119875 lt 005 lowastlowast119875 lt 001 compared to control group Data are means plusmn SEM 119899 = 3 independent experiments with independent culture

SCU may exert its protection effects in IR by prevent-ing generation of ROS directly scavenging ROS or indi-rectly through enhancement of cellular antioxidant enzymes[23 24] Moreover SCU could promote angiogenesis inendothelial cells [25] inhibit the apoptosis and the apopto-sis inducing factor pathway [26ndash28] and attenuate mucusproduction in vitro and in vivo involving the inhibition ofPKC-ERK signaling pathway [29] SCU benzyl ester hasa remarkable protective effect against myocardial ischemicinjury and the protective mechanism may associate withits antiapoptotic effect by inhibiting cytochrome C releaseand caspase-3 activation and attenuate inflammation [30]antitumor [31 32] antiviral [33] and neuroprotective effect[24] Long-term administration of SCU improved the cardiacfunction of MI rats by inhibiting interstitial fibrosis and

the mechanisms may involve the suppression of profibroticcytokine TGF1205731 expression and inhibition of p38 MAPKand ERK12 phosphorylation [22] SCU exerts protectiveeffects against IR injury through inhibiting PKC [15 34]However the current study demonstrates that the PKGVASPpathway plays an important role in pharmacological studiesof SCU in vivo combining in vitro IRmodelThis observationmay have further pharmacy implications because it maycontribute to the clarification of the mechanisms behindthe observed decreases in cardiocerebral vascular morbidityand mortality in patients receiving SCU or other flavonoidsStudies in another flavonoid baicalin suggest that vasodilatorproperties were attributed to endothelium-dependent relax-ation through the PKG pathway [10] There are evidencessuggesting that ischemia followed by reperfusion causes local


101 10Control Model

PKG-I120572

GAPDH

SCU (120583M)

(a)

00

02

04

06

08

10

12

14


lowast

PKG

-I120572

mRN

A (r

atio

of c

ontro

l)

SCU (120583M)

(b)

HBM

ECs v

iabi

lity

()

0

20

40

60

80

100

120

lowastlowast

lowast lowast lowast


SCU (120583M)

(c)

Figure 4The effect of scutellarin (SCU) onmRNA expression of PKG and cell viability inHRHBMECs (a) Representative electrophotogramand quantification of PKG-I120572 mRNA expression in HR HBMECs (b) The mRNA ratio of PKG-I120572 in HR HBMECs Ratio of control wascalculated by determining band integrated intensity as ratio of control (c) SCU increases cell viability in hypoxia reoxygenation- (HR-) treatedhuman brain microvascular endothelial cells (HBMECs) Cell viability was examined using MTT assay and expressed as ratio of controlControl group cells were treated with vehicle control (NS) under normal culture condition for 26 h Model group cells were incubated undernormal culture condition for 2 h and then given HR treatment (hypoxia 12reoxygenation 12 hours) One-way ANOVA followed by SNK test119875 lt 005 119875 lt 001 compared to model of HR group lowast119875 lt 005 lowastlowast119875 lt 001 compared to control group Data are means plusmn SEM 119899 = 3independent experiments with independent culture

dysfunction including EtD [35] Hypoxia and reoxygenationare two essential elements of ischemia and reperfusion injuryHRmay cause different forms of vascular injury such as hem-orrhage change in vascular permeability and EtD includingimpaired endothelium-dependent vasodilation Our studyshowed that IR caused EtD of BA with CIR and SCU hadprotective effect on the EtD of BA in vivo This observationwas also in accordance with our previous result that SCUcould relax isolated aortic rings of rat [17]

The roles of PKG-I isozymes have been documentedin many processes including gastrointestinal motility bloodflow neuronal plasticity erectile function lower urinary tractfunctions endothelial permeability and cardiac protection[7 36ndash38]Moreover therewas report that PKG is involved intestosterone-induced vasodilation of human umbilical artery

[9] Upon activation PKG phosphorylates VASP which inturn activates downstream ion channels leading to vascularsmooth muscle relaxation and vasodilation Previous reportshave suggested that cGMPPKGROScalmodulinCaMKIIsignaling pathwaymay regulate cardiomyocyte excitability byopening KATP channels and contribute to cardiac protectionagainst IR injury [39] In vascular endothelial cells PKG-I regulates cell motility migration and proliferation Thesefunctions are reported to be essential for vascular perme-ability and angiogenesis Experiments with PKG-I deficientvascular model systems have recently established that NOdonor-induced VASP phosphorylation is primarily mediatedby PKG-I [11] Our result showed that the actions of PKG-IVASP signaling may be a novel therapy target for IR injuryof BA


Sham

CIR model

(a)

SCU45 mgkg

SCU90 mgkg

(d)

CIR model

PKG inhibitor

SCU + PKG inhibitor

SCU90 mgkg

(b)

lowastlowast

SCU 45 mgkgCIR modelSCU 90 mgkg

(e)

lowast

ShamCIR modelSCU 90 mgkg

PKG inhibitor 50120583gkgSCU + PKG inhibitor

0

5

10

15

20

25

Cor

rect

ed in

farc

t siz

e (

)0

5

10

15

20

25

Cor

rect

ed in

farc

t siz

e (

)

CIR model 45 90Sham

SCU (mgkg )CIR

modelSCU + PKG

inhibitorSham SCU PKG

inhibitor

(c)

Rela

xatio

n of

BA

()

100

80

60

40

20

0

minus3 minus2 minus1 0 1 2

ACh (log 120583molL)

lowast

lowast

ShamCIR modelSCU 90mgkg

PKG inhibitorPKG inhibitor + SCU

CIR model

PKG inhibitor

SCU + PKG inhibitor

SCU90 mgkg

Figure 5The effect of SCU and PKG inhibitor in rats with CIR treatment (a) Representative images of triphenyl tetrazolium chloride (TTC)staining of ischemic brain slices from CIR (ischemia 60 minutesreperfusion 24 hours) rats with SCU treatment (b) Quantification of thecorrected cerebral infarct volume in CIR rats with SCU treatment 119899 = 10 rats in each group One-way ANOVA followed by SNK test versusmodel lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 (c) Cumulative concentration response curves of acetylcholine chloride (ACh) in isolated basilar artery(BA) segments from normal or CIR rats which were given SCU (90mgkg iv) or PKG inhibitor treatment Relaxation in response to AChis expressed as a percent of precontraction with U46199 (1120583molL) 119899 = 8 segments obtained from 3 rats with different treatments Two-wayANOVA test versus model lowast119875 lt 005 versus SCU

119875 lt 005 (d) Representative images of TTC staining of ischemic brain slices fromCIR (ischemia 60 minutesreperfusion 24 hours) rats with SCU (90mgkg iv) and PKG inhibitor (Rp-8-Br-cGMPS 50 120583gkg iv) aloneand combined (SCU 90mgkg + Rp-8-Br-cGMPS 50 120583gkg) treatments (e) Quantification of the corrected cerebral infarct size in CIR ratsadministered with SCU and PKG inhibitor 119899 = 10 rats in each group One-way ANOVA followed by SNK test versus model lowast119875 lt 005versus SCU 119875 lt 005 Data are means plusmn SEM


Table 1 Effect of SCU and PKG inhibitor administrations on ACh EC50and 119864max in BA rings

Group Dosage (iv) 119899 EC50(120583M) 119864max ( of U46619)

Sham NS 12 039 plusmn 014 7906 plusmn 657lowastlowast

Model NS 11 111 plusmn 047 3646 plusmn 490

SCU 45mgkg 8 NA 2247 plusmn 759

SCU 90mgkg 11 030 plusmn 017 6433 plusmn 440lowastlowast

PKG inhibitor 50120583gkg 8 NA 2524 plusmn 719

SCU + PKG inhibitor 90mgkg + 50 120583gkg 15 NA 151 plusmn 490lowast

Data are means plusmn SEM PKG inhibitor Rp-8-Br-cGMPS 119864max is the maximum vasodilative effect that ACh caused EC50 is the ACh concentration that makes50 of the maximum vasodilative effect 119899 BA segmentsrsquo number in each group blood vessel rings were obtained from 3-4 rats with different treatmentsBlood vessel rings in each group were pretreated by tail intravenous injection with SCU or NS NA not available as relaxative response is too weak to be testedNS normal saline one-way ANOVA on Rank Kruskal-Wallis test 119864max versus model lowast119875 lt 005 lowastlowast119875 lt 001 versus SCU (90mgkg) 119875 lt 005 119875 lt 001

Control 50 100HR model

120573-actin

SCU (120583M)

p-VASP

(a)

p-VA

SP p

rote

in le

vel (

the r

atio

of c

ontro

l)

12

10

08

06

04

02

00Control HR model 50 100

SCU (120583M)

lowast

lowastlowast

(b)

Figure 6The effect of SCUon p-VASP in isolated BA of rats withHR treatment (a) Representative immunoblot and quantification of p-VASPprotein expression in rat BA with CIR and SCU treatment (b)The ratio of p-VASP compared with control and CIR model in BA of rats withtreatment of SCU and CIR injury Protein ratios of control and model were calculated by the ratio of each bar compared to the control ratioand to the model ratio separately after the band integrated intensity as ratio of internal control (120573-actin) 119899 = 8 rats in each group One-wayANOVA followed by SNK test versus control lowast119875 lt 005 lowastlowast119875 lt 001 versus model 119875 lt 005 119875 lt 001 Data are means plusmn SEM

Among the downstream targets of PKG is vasodilator-stimulated phosphoprotein (VASP) a protein implicated inthe control of cytoskeletal dynamics and cell migration [40]Ser239 is the major site of action PKG Phosphorylation ofVASP at Ser239 by PKG inhibits growth of vascular smoothmuscle [41] in part by capping actin filaments resulting infilament retraction [42 43] The determination of P-VASPlevels could be a novel indicator of both PKG-I activity andendothelium integrity under physiological and pathophysio-logical conditions in human tissue [11] Our study suggestedthat VASP and P-VASP protein levels were depressed in IRmodel group but elevated after being given SCU treatmentwhich was accompanied with the PKG-I fluctuation and thechange of EtD of BA Our result showed that the actions ofPKG-IVASP signaling may be a novel therapy target for IRinjury of BA

In summary SCU produces a marked pharmacologicaction and regulates cellular function through multiple path-ways Our present study firstly provides important evidencethat SCU induced expression and activation of PKG-I andincreased VASP expression in normal cultured and HR

treatedHBMECs In CIR rats SCU treatment decreased cere-bral infarct size and augmented the endothelium-dependentrelaxation in isolated BA against EtD caused byCIRwhile theprotective effects of SCU could be reversed by PKG inhibitorThese suggest that SCU protects endothelial cells againstHR injury and improves endothelium-dependent relaxationwhich involves PKGVASP signaling pathway Our studiesprovide a new mechanism to explain cerebral protectiveeffects of SCU

Abbreviations

SCU ScutellarinHR Hypoxia reoxygenationCIR Cerebral ischemia reperfusionIR Ischemia reperfusionPKG cGMP-dependent protein kinaseEtD Endothelial dysfunctionBA Basilar arteryACh AcetylcholineHBMECs Human brain microvascular endothelial cells


p-VASP Phosphorylated VASPVASP Vasodilator stimulated phosphoprotein

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Weimin Yang designed experiments and revised the paperXiaohuaDuwrote the paperOther authors performed exper-iments Xiaohua Du and Chen Chen contributed equally tothis study

Acknowledgments

This work was partly supported by the National NaturalScience Foundation of China (nos 30960450 and 81173110)and Yunnan Provincial Science and Technology Depart-ment (nos 2011FA022 2014FA010 2012BC012 2014FB0372008CD054 and 2014BC012) The authors thankMr RenweiZhang and Xianer Fang for the gift of SCU compound Theyalso thankDr Yongxiao Cao and his groupmembers for theirtechnical assistance with wire myography and Yiping Wangand his group for their technical assistance with CIR animalmodel assessments

References

[1] A R Pries and W M Kuebler ldquoNormal endotheliumrdquo in TheVascular Endothelium I vol 176 of Handbook of ExperimentalPharmacology part 1 pp 1ndash40 Springer Berlin Germany 2006

[2] T W Hein C Zhang W Wang C-I Chang N Thengchaisriand L Kuo ldquoIschemia-reperfusion selectively impairs nitricoxide-mediated dilation in coronary arterioles counteractingrole of arginaserdquo The FASEB Journal vol 17 no 15 pp 2328ndash2330 2003

[3] H Xie P E Ray and B L Short ldquoNF-120581B activation plays a rolein superoxide-mediated cerebral endothelial dysfunction afterhypoxiareoxygenationrdquo Stroke vol 36 no 5 pp 1047ndash10522005

[4] H K Eltzschig and C D Collard ldquoVascular ischaemia andreperfusion injuryrdquo British Medical Bulletin vol 70 no 1 pp71ndash86 2004

[5] K Laude P Beauchamp CThuillez and V Richard ldquoEndothe-lial protective effects of preconditioningrdquo CardiovascularResearch vol 55 no 3 pp 466ndash473 2002

[6] R K Kharbanda M Peters B Walton et al ldquoIschemic precon-ditioning prevents endothelial injury and systemic neutrophilactivation during ischemia-reperfusion in humans in vivordquoCirculation vol 103 no 12 pp 1624ndash1630 2001

[7] S H Francis J L Busch and J D Corbin ldquocGMP-dependentprotein kinases and cGMP phosphodiesterases in nitric oxideand cGMP actionrdquo Pharmacological Reviews vol 62 no 3 pp525ndash563 2010

[8] H Sellak C-S Choi N B Dey and T M Lincoln ldquoTranscrip-tional and post-transcriptional regulation of cGMP-dependentprotein kinase (PKG-I) pathophysiological significancerdquo Car-diovascular Research vol 97 no 2 pp 200ndash207 2013

[9] E Cairrao A J Santos-Silva and I Verde ldquoPKG is involvedin testosterone-induced vasorelaxation of human umbilicalarteryrdquo European Journal of Pharmacology vol 640 no 1ndash3 pp94ndash101 2010

[10] Y-L Lin Z-K Dai R-J Lin et al ldquoBaicalin a flavonoidfrom Scutellaria baicalensisGeorgi activates large-conductanceCa2+-activated K+ channels via cyclic nucleotide-dependentprotein kinases in mesenteric arteryrdquo Phytomedicine vol 17 no10 pp 760ndash770 2010

[11] E Schulz N Tsilimingas R Rinze et al ldquoFunctional and bio-chemical analysis of endothelial (Dys)function and NOcGMPsignaling in human blood vessels with and without nitroglyc-erin pretreatmentrdquo Circulation vol 105 no 10 pp 1170ndash11752002

[12] M Gao W Huang and C-Z Liu ldquoSeparation of scutellarinfrom crude extracts of Erigeron breviscapus (vant) HandMazz by macroporous resinsrdquo Journal of Chromatography BAnalytical Technologies in the Biomedical and Life Sciences vol858 no 1-2 pp 22ndash26 2007

[13] H Guo L-M Hu S-X Wang et al ldquoNeuroprotective effectsof scutellarin against hypoxic-ischemic-induced cerebral injuryvia augmentation of antioxidant defense capacityrdquo ChineseJournal of Physiology vol 54 no 6 pp 399ndash405 2011

[14] X-M Hu M-M Zhou X-M Hu and F-D Zeng ldquoNeuropro-tective effects of scutellarin on rat neuronal damage inducedby cerebral ischemiareperfusionrdquo Acta Pharmacologica Sinicavol 26 no 12 pp 1454ndash1459 2005

[15] Y Su W Liu L Ma X Liu Z Liu and B Zhu ldquoScutellarininhibits translocation of protein kinase C in diabetic thoracicaorta of the ratrdquo Clinical and Experimental Pharmacology andPhysiology vol 39 no 2 pp 136ndash140 2012

[16] W Yang A Bofferding R M Lust and C J Wingard ldquoEffectof scutellarin on contractile behavior of isolated thoracic aortarings of the mouserdquoThe FASEB Journal vol 21 no 6 Article IDA1160 2007

[17] W Yang R M Lust A Bofferding and C J Wingard ldquoNitricoxide and catalase-sensitive relaxation by scutellarin in themouse thoracic aortardquo Journal of Cardiovascular Pharmacologyvol 53 no 1 pp 66ndash76 2009

[18] D S Burley P Ferdinandy and G F Baxter ldquoCyclic GMP andprotein kinase-G in myocardial ischaemia-reperfusion oppor-tunities and obstacles for survival signalingrdquo British Journal ofPharmacology vol 152 no 6 pp 855ndash869 2007

[19] Z Wang T Liu L Gan et al ldquoShikonin protects mousebrain against cerebral ischemiareperfusion injury through itsantioxidant activityrdquo European Journal of Pharmacology vol643 no 2-3 pp 211ndash217 2010

[20] X Zeng S Zhang L Zhang K Zhang and X Zheng ldquoA studyof the neuroprotective effect of the phenolic glucoside gastrodinduring cerebral ischemia in vivo and in vitrordquo Planta Medicavol 72 no 15 pp 1359ndash1365 2006

[21] L-L Lin A-J Liu J-G Liu X-H Yu L-P Qin and D-F SuldquoProtective effects of scutellarin and breviscapine on brain andheart ischemia in ratsrdquo Journal of Cardiovascular Pharmacologyvol 50 no 3 pp 327ndash332 2007

[22] Z Pan W Zhao X Zhang et al ldquoScutellarin alleviates intersti-tial fibrosis and cardiac dysfunction of infarct rats by inhibitingTGF1205731 expression and activation of p38-MAPK and ERK12rdquoBritish Journal of Pharmacology vol 162 no 3 pp 688ndash7002011


[23] H Hong and G-Q Liu ldquoScutellarin protects PC12 cells fromoxidative stress-induced apoptosisrdquo Journal of Asian NaturalProducts Research vol 8 no 6 pp 471ndash479 2006

[24] S Wang H Wang H Guo L Kang X Gao and L HuldquoNeuroprotection of Scutellarin is mediated by inhibition ofmicroglial inflammatory activationrdquo Neuroscience vol 185 pp150ndash160 2011

[25] Z-X Gao D-Y Huang H-X Li et al ldquoScutellarin promotes invitro angiogenesis in human umbilical vein endothelial cellsrdquoBiochemical and Biophysical Research Communications vol400 no 1 pp 151ndash156 2010

[26] H Dai J Gu L-Z Li L-M Yang H Liu and J-Y LildquoScutellarin benzyl ester partially secured the ischemic injuryby its anti-apoptosis mechanism in cardiomyocytes of neonatalratsrdquo Journal of Chinese Integrative Medicine vol 9 no 9 pp1014ndash1021 2011

[27] L-L Guo Z-Z Guan Y Huang Y-L Wang and J-S ShildquoThe neurotoxicity of 120573-amyloid peptide toward rat brainis associated with enhanced oxidative stress inflammationand apoptosis all of which can be attenuated by scutellarinrdquoExperimental and Toxicologic Pathology vol 65 no 5 pp 579ndash584 2013

[28] H-F Zhang X-M Hu L-X Wang S-Q Xu and F-D ZengldquoProtective effects of scutellarin against cerebral ischemia inrats evidence for inhibition of the apoptosis-inducing factorpathwayrdquo Planta Medica vol 75 no 2 pp 121ndash126 2009

[29] D-P Jiang Q Li J Yang J M Perelman V P Kolosovand X-D Zhou ldquoScutellarin attenuates human-neutrophil-elastase-inducedmucus production by inhibiting the PKC-ERKsignaling pathway in vitro and in vivordquoTheAmerican Journal ofChinese Medicine vol 39 no 6 pp 1193ndash1206 2011

[30] P Luo Z-H Tan Z-F ZhangH Zhang X-F Liu andZ-JMoldquoScutellarin isolated from Erigeron multiradiatus inhibits highglucose-mediated vascular inflammationrdquo Yakugaku Zasshivol 128 no 9 pp 1293ndash1299 2008

[31] H Xu and S Zhang ldquoScutellarin-induced apoptosis in HepG2hepatocellular carcinoma cells via a STAT3 pathwayrdquo Phytother-apy Research vol 27 no 10 pp 1524ndash1528 2013

[32] Y Feng S Zhang J Tu et al ldquoNovel function of scutellarinin inhibiting cell proliferation and inducing cell apoptosisof human Burkitt lymphoma Namalwa cellsrdquo Leukemia andLymphoma vol 53 no 12 pp 2456ndash2464 2012

[33] G-H Zhang QWang J-J Chen X-M Zhang S-C Tam andY-T Zheng ldquoThe anti-HIV-1 effect of scutellarinrdquo Biochemicaland Biophysical Research Communications vol 334 no 3 pp812ndash816 2005

[34] W Xu R-P Zha W-Y Wang and Y-P Wang ldquoEffects ofscutellarin on PKC120574 in PC12 cell injury induced by oxygen andglucose deprivationrdquoActa Pharmacologica Sinica vol 28 no 10pp 1573ndash1579 2007

[35] B H Neo S Kandhi M Ahmad and M S Wolin ldquoRedoxregulation of guanylate cyclase and protein kinase G in vascularresponses to hypoxiardquo Respiratory Physiology and Neurobiologyvol 174 no 3 pp 259ndash264 2010

[36] A Aicher CHeeschen S Feil et al ldquoCGMP-dependent proteinkinase I is crucial for angiogenesis and postnatal vasculogene-sisrdquo PLoS ONE vol 4 no 3 Article ID e4879 2009

[37] B Kemp-Harper and H H H W Schmidt ldquocGMP in thevasculaturerdquo in cGMP Generators Effectors and TherapeuticImplications vol 191 of Handbook of Experimental Pharmacol-ogy pp 447ndash467 Springer Berlin Germany 2009

[38] D Atochin E Buys H Swanson et al ldquoAbstract T P225 cGMP-dependent protein kinase I in smooth muscle cells protectsagainst stroke injury in micerdquo Stroke vol 45 supplement 1Article ID ATP225 2014

[39] Y Chai D-M Zhang and Y-F Lin ldquoActivation of cGMP-dependent protein kinase stimulates cardiac ATP-sensitivepotassium channels via a ROScalmodulinCaMKII signalingcascaderdquo PLoS ONE vol 6 no 3 Article ID e18191 2011

[40] J E Bear J J Loureiro I Libova R Fassler JWehland and F BGertler ldquoNegative regulation of fibroblastmotility by EnaVASPproteinsrdquo Cell vol 101 no 7 pp 717ndash728 2000

[41] L Chen G Daum K Chitaley et al ldquoVasodilator-stimulatedphosphoprotein regulates proliferation and growth inhibitionby nitric oxide in vascular smoothmuscle cellsrdquoArteriosclerosisThrombosis and Vascular Biology vol 24 no 8 pp 1403ndash14082004

[42] TM Gomez and E Robles ldquoThe great escape phosphorylationof EnaVASP by PKA promotes filopodial formationrdquo Neuronvol 42 no 1 pp 1ndash3 2004

[43] X Wang J L Pluznick D C Settles and S C SansomldquoAssociation of VASP with TRPC4 in PKG-mediated inhibitionof the store-operated calcium response in mesangial cellsrdquoAmerican Journal of Physiology - Renal Physiology vol 293 no6 pp F1768ndashF1776 2007

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


a number of biologically important targets which are neededto accomplish multiple cellular functions and its dysregula-tion has been incriminated in many diseases such as hyper-tension atherosclerosis chronic heart failure left ventricularhypertrophy ventricular remodelling IR injury diabetes andcancer [7] Among the three isoforms of PKG-I120572 PKG-I120573and PKG-II PKG-I120572 is the strongest vascular tonemodulatorregulating cell motility migration proliferation and vasculartone [8]

Moreover there was report that PKG is involved intestosterone-induced vasodilation of human umbilical artery[9] Upon activation PKG phosphorylates VASP which inturn activates downstream ion channels leading to vascularsmooth muscle relaxation and vasodilation Previous reportshave suggested that vasorelaxant effects of baicalin aremainlyattributed to voltage-dependent Ca2+ channel (VDCC) inhi-bition and BKCa channel activation through PKA and PKGpathways [10]

Vasodilator stimulated phosphoprotein (VASP) belongsto the EnaVASP protein family and is an important PKG-Isubstrate and actin regulatory protein Studies [11] suggestedthat phosphorylated VASP at serine 239 (p-VASP) has beenshown to be a usefulmonitor for PKG-I activity in intact cells

Scutellarin (SCU) 4101584056-trihydroxy flavonoid-7-glucu-ronide was reported to be the primary active ingredientof breviscapine which is a mixture of flavonoid glycosidesisolated from a Chinese traditional medicine plant Erigeronbreviscapus (Vant) Hand Mazz [12] The plant extracts andSCU have been used in China to treat a variety of disordersincluding cardiovascular cerebrovascular and inflammatorydiseases for many years [13] In animal studies SCU hasbeen reported to be neuroprotective in rat cerebral ischemiareperfusion (CIR) models [14] via augmentation of antioxi-dant defense capacity [13] In addition SCU prevented EtDin diabetic rats and inhibited translocation of protein kinaseC in diabetic thoracic aorta of the rat [15] Our earlierstudy [16 17] showed that relaxation effect of SCU on arterywas predominantly endothelium dependent and partiallyinvolved the catalase-sensitive nitric oxide synthase signalingpathway

Based on these observations we hypothesize that SCUreduces EtD through the PKG-I pathway To verify thishypothesis we test the protein level and mRNA expressionof PKG-I VASP and p-VASP in human brain microvascularendothelial cells (HBMECs) The effects of SCU on EtD ofbrain basilar artery (BA) and infarct size were checked in ratswith CIR injury

2 Materials and Methods

21 Chemicals and Drugs SCU was obtained from Kun-ming Longjin Pharmaceutical Co (Kunming China) Cellculture reagents DMEM modified RPMI-1640 mediumand fetal bovine serum were obtained from the Hyclone(Thermo Scientific USA) Other items include Wire Myo-graph System DMT (Danish Myo Technology CompanyDenmark) and Power Lab data recording and analyt-ical system (ADInstruments Ltd Australia) HBMECswere purchased from Yangsen Biology Limited Company

(Shanghai China) U46619 was purchased from CaymanChemical Company PKG inhibitor Rp-8-Br-cGMPS waspurchased from Santa Cruz Biotechnology (Dallas TXUSA) 3-(45-Dimethylthiazol-2-yl)-25-diphenyltetrazoliumbromide (MTT) triphenyl tetrazolium chloride (TTC) andACh were purchased from Sigma-Aldrich (St Louis MOUSA)

22 Animals Sprague-Dawley rats (180ndash220 g male andfemale in each half) were provided by the animal center ofKunming Medical University All animals were housed inmicroisolation under conditions of constant temperature andcontrolled illumination (light on 12-hour lightdark cycle)Food and water were available ad libitum All the animalsused in the experiment received humane care All surgicaland experimental procedures were in accordance with theinstitutional animal care guidelines The animal study wasapproved by the Animal Care and Use Committee of Kun-ming Medical University and conformed to the standards setby the Yunnan Experimental Animal Management Board

23 Methods

231 Endothelial Cell Culture and HR Treatment HBMECswere obtained from the Shanghai Yangsen BiochemicalTechnology Company (Shanghai China) and grown in1640 medium supplemented with 10 fetal bovine serumand 1 penicillinstreptomycin antibiotics Tightly confluentmonolayers of HBMECs from 4thminus15th passage were usedin all experiments In experiments checking the effects ofSCU under normal condition cells were treated with vehiclecontrol (NS) or SCU (01 10 and 100 120583M) at different con-centrations for 26 hours In experiments with HR treatmentscells were divided into 5 groups including control HRmodelHR + SCU 01 120583M HR + SCU 1 120583M and HR + SCU 10 120583Mgroup Control cells were cultured in parallel and kept innormal culture condition for the entire time period (26 h)Simulated HR injury was induced according to previouslydescribed procedures [18] with minor modifications BrieflyHBMECs were placed in a humidified hypoxic chamber(HF100 Heal Force Biotech Co Shanghai China) for 12 h ofhypoxia (5 CO

2+ 2 O

2+ 93 N

2) with medium free of

glucose and serum at 37∘C followed by 12 h of reoxygenation(5 CO

2+ 95 air) in complete medium containing glucose

and serum For HR + SCU groups cells were incubatedwith SCU at different concentrations for 2 h prior to hypoxiatreatment and during hypoxia (12 h) and reoxygenation (12 h)injury For control group cells were treated with vehiclecontrol (NS) for 26 h and for SCU groups cells were givendifferent concentrations of SCU for 26 h separately At the endof the experiment cell viability was examined using MTTassay as described below Furthermore cells of each groupwere also collected for RT-PCR assay and Western blottingassay as described in the following sections All experimentswere performed in triplicate

232 MTT Assay of Cell Viability For MTT assay cellswere plated in 96-well flat-bottomed plates at a densityof 3 times 104 cellsmL and 90120583Lwell Cells were cultured in










2 12mMMgSO

4









3VO4






3 Results













50






Control

PKG-I

p-VASP

VASP

GAPDH

01 1 10

SCU (120583M)

(a)

Control 01 1 10

Prot

ein

leve

l (ra

tio o

f con

trol)

00

05

10

15

20

25

p-VASPVASP

lowast

lowast

lowastlowast

lowastlowast

SCU (120583M)

(b)

20

15

10

05

00Control 01 1 10

PKG

-I ac

tivity

(rat

io o

f p-V

ASP

VA

SP)


SCU (120583M)

(c)

18

16

14

12

10

08

06

04

02

00Control 01 1 10

PKG

-I p

rote

in le

vel (

ratio

of c

ontro

l)lowastlowast

lowast

SCU (120583M)

(d)




4 Discussion



PKG-I120572

GAPDH

Control 01 1 10

SCU (120583M)

(a)

Control 01 1 10

14

16

12

10

08

06

04

02

00

PKG

-I120572

mRN

A (r

atio

of c

ontro

l)

lowast

lowastlowast

SCU (120583M)

(b)

HBM

ECs v

iabi

lity

()

40

20

0

60

80

100

120

14024h lowast

Control 01 1 10

SCU (120583M)

(c)




50 and 119864max












GAPDH


p-VASP

VASP

PKG-I

SCU (120583M)

(a)


lowast

lowast

SCU (120583M)

Prot

ein

leve

l (ra

tio o

f con

trol )

00

02

04

06

08

10

12

14

16

p-VASPVASP

(b)

PKG

-I ac

tivity

(r

atio

of p

-VA

SPV

ASP

)

000204060810121416

lowast


SCU (120583M)

(c)

PKG

-I p

rote

in le

vel

(rat

io o

f con

trol )

00

05

10

15

20

25

lowast lowast


SCU (120583M)

(d)





101 10Control Model

PKG-I120572

GAPDH

SCU (120583M)

(a)

00

02

04

06

08

10

12

14


lowast

PKG

-I120572

mRN

A (r

atio

of c

ontro

l)

SCU (120583M)

(b)

HBM

ECs v

iabi

lity

()

0

20

40

60

80

100

120

lowastlowast



SCU (120583M)

(c)






Sham

CIR model

(a)

SCU45 mgkg

SCU90 mgkg

(d)

CIR model

PKG inhibitor

SCU + PKG inhibitor

SCU90 mgkg

(b)

lowastlowast


(e)

lowast



0

5

10

15

20

25

Cor

rect

ed in

farc

t siz

e (

)0

5

10

15

20

25

Cor

rect

ed in

farc

t siz

e (

)

CIR model 45 90Sham

SCU (mgkg )CIR

modelSCU + PKG


inhibitor

(c)

Rela

xatio

n of

BA

()

100

80

60

40

20

0



lowast

lowast



CIR model

PKG inhibitor

SCU + PKG inhibitor

SCU90 mgkg














120573-actin

SCU (120583M)

p-VASP

(a)

p-VA

SP p

rote

in le

vel (

the r

atio

of c

ontro

l)

12

10

08

06

04

02


SCU (120583M)

lowast

lowastlowast

(b)





Abbreviations








Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























2 12mMMgSO

4









3VO4






3 Results













50






Control

PKG-I

p-VASP

VASP

GAPDH

01 1 10

SCU (120583M)

(a)

Control 01 1 10

Prot

ein

leve

l (ra

tio o

f con

trol)

00

05

10

15

20

25

p-VASPVASP

lowast

lowast

lowastlowast

lowastlowast

SCU (120583M)

(b)

20

15

10

05

00Control 01 1 10

PKG

-I ac

tivity

(rat

io o

f p-V

ASP

VA

SP)


SCU (120583M)

(c)

18

16

14

12

10

08

06

04

02

00Control 01 1 10

PKG

-I p

rote

in le

vel (

ratio

of c

ontro

l)lowastlowast

lowast

SCU (120583M)

(d)




4 Discussion



PKG-I120572

GAPDH

Control 01 1 10

SCU (120583M)

(a)

Control 01 1 10

14

16

12

10

08

06

04

02

00

PKG

-I120572

mRN

A (r

atio

of c

ontro

l)

lowast

lowastlowast

SCU (120583M)

(b)

HBM

ECs v

iabi

lity

()

40

20

0

60

80

100

120

14024h lowast

Control 01 1 10

SCU (120583M)

(c)




50 and 119864max












GAPDH


p-VASP

VASP

PKG-I

SCU (120583M)

(a)


lowast

lowast

SCU (120583M)

Prot

ein

leve

l (ra

tio o

f con

trol )

00

02

04

06

08

10

12

14

16

p-VASPVASP

(b)

PKG

-I ac

tivity

(r

atio

of p

-VA

SPV

ASP

)

000204060810121416

lowast


SCU (120583M)

(c)

PKG

-I p

rote

in le

vel

(rat

io o

f con

trol )

00

05

10

15

20

25

lowast lowast


SCU (120583M)

(d)





101 10Control Model

PKG-I120572

GAPDH

SCU (120583M)

(a)

00

02

04

06

08

10

12

14


lowast

PKG

-I120572

mRN

A (r

atio

of c

ontro

l)

SCU (120583M)

(b)

HBM

ECs v

iabi

lity

()

0

20

40

60

80

100

120

lowastlowast



SCU (120583M)

(c)






Sham

CIR model

(a)

SCU45 mgkg

SCU90 mgkg

(d)

CIR model

PKG inhibitor

SCU + PKG inhibitor

SCU90 mgkg

(b)

lowastlowast


(e)

lowast



0

5

10

15

20

25

Cor

rect

ed in

farc

t siz

e (

)0

5

10

15

20

25

Cor

rect

ed in

farc

t siz

e (

)

CIR model 45 90Sham

SCU (mgkg )CIR

modelSCU + PKG


inhibitor

(c)

Rela

xatio

n of

BA

()

100

80

60

40

20

0



lowast

lowast



CIR model

PKG inhibitor

SCU + PKG inhibitor

SCU90 mgkg














120573-actin

SCU (120583M)

p-VASP

(a)

p-VA

SP p

rote

in le

vel (

the r

atio

of c

ontro

l)

12

10

08

06

04

02


SCU (120583M)

lowast

lowastlowast

(b)





Abbreviations








Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















3VO4






3 Results













50






Control

PKG-I

p-VASP

VASP

GAPDH

01 1 10

SCU (120583M)

(a)

Control 01 1 10

Prot

ein

leve

l (ra

tio o

f con

trol)

00

05

10

15

20

25

p-VASPVASP

lowast

lowast

lowastlowast

lowastlowast

SCU (120583M)

(b)

20

15

10

05

00Control 01 1 10

PKG

-I ac

tivity

(rat

io o

f p-V

ASP

VA

SP)


SCU (120583M)

(c)

18

16

14

12

10

08

06

04

02

00Control 01 1 10

PKG

-I p

rote

in le

vel (

ratio

of c

ontro

l)lowastlowast

lowast

SCU (120583M)

(d)




4 Discussion



PKG-I120572

GAPDH

Control 01 1 10

SCU (120583M)

(a)

Control 01 1 10

14

16

12

10

08

06

04

02

00

PKG

-I120572

mRN

A (r

atio

of c

ontro

l)

lowast

lowastlowast

SCU (120583M)

(b)

HBM

ECs v

iabi

lity

()

40

20

0

60

80

100

120

14024h lowast

Control 01 1 10

SCU (120583M)

(c)




50 and 119864max












GAPDH


p-VASP

VASP

PKG-I

SCU (120583M)

(a)


lowast

lowast

SCU (120583M)

Prot

ein

leve

l (ra

tio o

f con

trol )

00

02

04

06

08

10

12

14

16

p-VASPVASP

(b)

PKG

-I ac

tivity

(r

atio

of p

-VA

SPV

ASP

)

000204060810121416

lowast


SCU (120583M)

(c)

PKG

-I p

rote

in le

vel

(rat

io o

f con

trol )

00

05

10

15

20

25

lowast lowast


SCU (120583M)

(d)





101 10Control Model

PKG-I120572

GAPDH

SCU (120583M)

(a)

00

02

04

06

08

10

12

14


lowast

PKG

-I120572

mRN

A (r

atio

of c

ontro

l)

SCU (120583M)

(b)

HBM

ECs v

iabi

lity

()

0

20

40

60

80

100

120

lowastlowast



SCU (120583M)

(c)






Sham

CIR model

(a)

SCU45 mgkg

SCU90 mgkg

(d)

CIR model

PKG inhibitor

SCU + PKG inhibitor

SCU90 mgkg

(b)

lowastlowast


(e)

lowast



0

5

10

15

20

25

Cor

rect

ed in

farc

t siz

e (

)0

5

10

15

20

25

Cor

rect

ed in

farc

t siz

e (

)

CIR model 45 90Sham

SCU (mgkg )CIR

modelSCU + PKG


inhibitor

(c)

Rela

xatio

n of

BA

()

100

80

60

40

20

0



lowast

lowast



CIR model

PKG inhibitor

SCU + PKG inhibitor

SCU90 mgkg














120573-actin

SCU (120583M)

p-VASP

(a)

p-VA

SP p

rote

in le

vel (

the r

atio

of c

ontro

l)

12

10

08

06

04

02


SCU (120583M)

lowast

lowastlowast

(b)





Abbreviations








Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control

PKG-I

p-VASP

VASP

GAPDH

01 1 10

SCU (120583M)

(a)

Control 01 1 10

Prot

ein

leve

l (ra

tio o

f con

trol)

00

05

10

15

20

25

p-VASPVASP

lowast

lowast

lowastlowast

lowastlowast

SCU (120583M)

(b)

20

15

10

05

00Control 01 1 10

PKG

-I ac

tivity

(rat

io o

f p-V

ASP

VA

SP)


SCU (120583M)

(c)

18

16

14

12

10

08

06

04

02

00Control 01 1 10

PKG

-I p

rote

in le

vel (

ratio

of c

ontro

l)lowastlowast

lowast

SCU (120583M)

(d)




4 Discussion



PKG-I120572

GAPDH

Control 01 1 10

SCU (120583M)

(a)

Control 01 1 10

14

16

12

10

08

06

04

02

00

PKG

-I120572

mRN

A (r

atio

of c

ontro

l)

lowast

lowastlowast

SCU (120583M)

(b)

HBM

ECs v

iabi

lity

()

40

20

0

60

80

100

120

14024h lowast

Control 01 1 10

SCU (120583M)

(c)




50 and 119864max












GAPDH


p-VASP

VASP

PKG-I

SCU (120583M)

(a)


lowast

lowast

SCU (120583M)

Prot

ein

leve

l (ra

tio o

f con

trol )

00

02

04

06

08

10

12

14

16

p-VASPVASP

(b)

PKG

-I ac

tivity

(r

atio

of p

-VA

SPV

ASP

)

000204060810121416

lowast


SCU (120583M)

(c)

PKG

-I p

rote

in le

vel

(rat

io o

f con

trol )

00

05

10

15

20

25

lowast lowast


SCU (120583M)

(d)





101 10Control Model

PKG-I120572

GAPDH

SCU (120583M)

(a)

00

02

04

06

08

10

12

14


lowast

PKG

-I120572

mRN

A (r

atio

of c

ontro

l)

SCU (120583M)

(b)

HBM

ECs v

iabi

lity

()

0

20

40

60

80

100

120

lowastlowast



SCU (120583M)

(c)






Sham

CIR model

(a)

SCU45 mgkg

SCU90 mgkg

(d)

CIR model

PKG inhibitor

SCU + PKG inhibitor

SCU90 mgkg

(b)

lowastlowast


(e)

lowast



0

5

10

15

20

25

Cor

rect

ed in

farc

t siz

e (

)0

5

10

15

20

25

Cor

rect

ed in

farc

t siz

e (

)

CIR model 45 90Sham

SCU (mgkg )CIR

modelSCU + PKG


inhibitor

(c)

Rela

xatio

n of

BA

()

100

80

60

40

20

0



lowast

lowast



CIR model

PKG inhibitor

SCU + PKG inhibitor

SCU90 mgkg














120573-actin

SCU (120583M)

p-VASP

(a)

p-VA

SP p

rote

in le

vel (

the r

atio

of c

ontro

l)

12

10

08

06

04

02


SCU (120583M)

lowast

lowastlowast

(b)





Abbreviations








Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















PKG-I120572

GAPDH

Control 01 1 10

SCU (120583M)

(a)

Control 01 1 10

14

16

12

10

08

06

04

02

00

PKG

-I120572

mRN

A (r

atio

of c

ontro

l)

lowast

lowastlowast

SCU (120583M)

(b)

HBM

ECs v

iabi

lity

()

40

20

0

60

80

100

120

14024h lowast

Control 01 1 10

SCU (120583M)

(c)




50 and 119864max












GAPDH


p-VASP

VASP

PKG-I

SCU (120583M)

(a)


lowast

lowast

SCU (120583M)

Prot

ein

leve

l (ra

tio o

f con

trol )

00

02

04

06

08

10

12

14

16

p-VASPVASP

(b)

PKG

-I ac

tivity

(r

atio

of p

-VA

SPV

ASP

)

000204060810121416

lowast


SCU (120583M)

(c)

PKG

-I p

rote

in le

vel

(rat

io o

f con

trol )

00

05

10

15

20

25

lowast lowast


SCU (120583M)

(d)





101 10Control Model

PKG-I120572

GAPDH

SCU (120583M)

(a)

00

02

04

06

08

10

12

14


lowast

PKG

-I120572

mRN

A (r

atio

of c

ontro

l)

SCU (120583M)

(b)

HBM

ECs v

iabi

lity

()

0

20

40

60

80

100

120

lowastlowast



SCU (120583M)

(c)






Sham

CIR model

(a)

SCU45 mgkg

SCU90 mgkg

(d)

CIR model

PKG inhibitor

SCU + PKG inhibitor

SCU90 mgkg

(b)

lowastlowast


(e)

lowast



0

5

10

15

20

25

Cor

rect

ed in

farc

t siz

e (

)0

5

10

15

20

25

Cor

rect

ed in

farc

t siz

e (

)

CIR model 45 90Sham

SCU (mgkg )CIR

modelSCU + PKG


inhibitor

(c)

Rela

xatio

n of

BA

()

100

80

60

40

20

0



lowast

lowast



CIR model

PKG inhibitor

SCU + PKG inhibitor

SCU90 mgkg














120573-actin

SCU (120583M)

p-VASP

(a)

p-VA

SP p

rote

in le

vel (

the r

atio

of c

ontro

l)

12

10

08

06

04

02


SCU (120583M)

lowast

lowastlowast

(b)





Abbreviations








Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















GAPDH


p-VASP

VASP

PKG-I

SCU (120583M)

(a)


lowast

lowast

SCU (120583M)

Prot

ein

leve

l (ra

tio o

f con

trol )

00

02

04

06

08

10

12

14

16

p-VASPVASP

(b)

PKG

-I ac

tivity

(r

atio

of p

-VA

SPV

ASP

)

000204060810121416

lowast


SCU (120583M)

(c)

PKG

-I p

rote

in le

vel

(rat

io o

f con

trol )

00

05

10

15

20

25

lowast lowast


SCU (120583M)

(d)





101 10Control Model

PKG-I120572

GAPDH

SCU (120583M)

(a)

00

02

04

06

08

10

12

14


lowast

PKG

-I120572

mRN

A (r

atio

of c

ontro

l)

SCU (120583M)

(b)

HBM

ECs v

iabi

lity

()

0

20

40

60

80

100

120

lowastlowast



SCU (120583M)

(c)






Sham

CIR model

(a)

SCU45 mgkg

SCU90 mgkg

(d)

CIR model

PKG inhibitor

SCU + PKG inhibitor

SCU90 mgkg

(b)

lowastlowast


(e)

lowast



0

5

10

15

20

25

Cor

rect

ed in

farc

t siz

e (

)0

5

10

15

20

25

Cor

rect

ed in

farc

t siz

e (

)

CIR model 45 90Sham

SCU (mgkg )CIR

modelSCU + PKG


inhibitor

(c)

Rela

xatio

n of

BA

()

100

80

60

40

20

0



lowast

lowast



CIR model

PKG inhibitor

SCU + PKG inhibitor

SCU90 mgkg














120573-actin

SCU (120583M)

p-VASP

(a)

p-VA

SP p

rote

in le

vel (

the r

atio

of c

ontro

l)

12

10

08

06

04

02


SCU (120583M)

lowast

lowastlowast

(b)





Abbreviations








Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















101 10Control Model

PKG-I120572

GAPDH

SCU (120583M)

(a)

00

02

04

06

08

10

12

14


lowast

PKG

-I120572

mRN

A (r

atio

of c

ontro

l)

SCU (120583M)

(b)

HBM

ECs v

iabi

lity

()

0

20

40

60

80

100

120

lowastlowast



SCU (120583M)

(c)






Sham

CIR model

(a)

SCU45 mgkg

SCU90 mgkg

(d)

CIR model

PKG inhibitor

SCU + PKG inhibitor

SCU90 mgkg

(b)

lowastlowast


(e)

lowast



0

5

10

15

20

25

Cor

rect

ed in

farc

t siz

e (

)0

5

10

15

20

25

Cor

rect

ed in

farc

t siz

e (

)

CIR model 45 90Sham

SCU (mgkg )CIR

modelSCU + PKG


inhibitor

(c)

Rela

xatio

n of

BA

()

100

80

60

40

20

0



lowast

lowast



CIR model

PKG inhibitor

SCU + PKG inhibitor

SCU90 mgkg














120573-actin

SCU (120583M)

p-VASP

(a)

p-VA

SP p

rote

in le

vel (

the r

atio

of c

ontro

l)

12

10

08

06

04

02


SCU (120583M)

lowast

lowastlowast

(b)





Abbreviations








Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Sham

CIR model

(a)

SCU45 mgkg

SCU90 mgkg

(d)

CIR model

PKG inhibitor

SCU + PKG inhibitor

SCU90 mgkg

(b)

lowastlowast


(e)

lowast



0

5

10

15

20

25

Cor

rect

ed in

farc

t siz

e (

)0

5

10

15

20

25

Cor

rect

ed in

farc

t siz

e (

)

CIR model 45 90Sham

SCU (mgkg )CIR

modelSCU + PKG


inhibitor

(c)

Rela

xatio

n of

BA

()

100

80

60

40

20

0



lowast

lowast



CIR model

PKG inhibitor

SCU + PKG inhibitor

SCU90 mgkg














120573-actin

SCU (120583M)

p-VASP

(a)

p-VA

SP p

rote

in le

vel (

the r

atio

of c

ontro

l)

12

10

08

06

04

02


SCU (120583M)

lowast

lowastlowast

(b)





Abbreviations








Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























120573-actin

SCU (120583M)

p-VASP

(a)

p-VA

SP p

rote

in le

vel (

the r

atio

of c

ontro

l)

12

10

08

06

04

02


SCU (120583M)

lowast

lowastlowast

(b)





Abbreviations








Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Acknowledgments


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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