research article role of rutin on nitric oxide...

Research ArticleRole of Rutin on Nitric Oxide Synthesis inHuman Umbilical Vein Endothelial Cells

Azizah Ugusman1 Zaiton Zakaria1 Kien Hui Chua1

Nor Anita Megat Mohd Nordin1 and Zaleha Abdullah Mahdy2

1 Department of Physiology Faculty of Medicine Universiti Kebangsaan Malaysia Medical Centre Jalan Raja Muda Abdul Aziz50300 Kuala Lumpur Malaysia

2 Department of Obstetrics and Gynaecology Universiti Kebangsaan Malaysia Medical Centre Jalan Yaacob Latif 56000 CherasKuala Lumpur Malaysia

Correspondence should be addressed to Zaiton Zakaria zaitonukmgmailcom

Received 9 April 2014 Accepted 10 June 2014 Published 24 June 2014

Academic Editor Tullio Florio

Copyright copy 2014 Azizah Ugusman 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

Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) is a major antiatherogenic factor in the blood vesselOxidative stress plays an important role in the pathogenesis of various cardiovascular diseases including atherosclerosis Decreasedavailability of endothelial NO promotes the progression of endothelial dysfunction and atherosclerosis Rutin is a flavonoid withmultiple cardiovascular protective effects This study aimed to investigate the effects of rutin on eNOS and NO production incultured human umbilical vein endothelial cells (HUVEC) HUVEC were divided into four groups control oxidative stressinduction with 180120583MH

2O2 treatment with 300 120583Mrutin and concomitant induction with rutin andH

2O2for 24 hours HUVEC

treated with rutin produced higher amount of NO compared to control (119875 lt 001) In the oxidative stress-induced HUVECrutin successfully induced cellsrsquo NO production (119875 lt 001) Rutin promoted NO production in HUVEC by inducing eNOS geneexpression (119875 lt 005) eNOS protein synthesis (119875 lt 001) and eNOS activity (119875 lt 005) Treatment with rutin also led to increasedgene and protein expression of basic fibroblast growth factor (bFGF) in HUVEC Therefore upregulation of eNOS expression byrutin may be mediated by bFGF The results showed that rutin may improve endothelial function by augmenting NO productionin human endothelial cells

1 Introduction

Endothelial nitric oxide (NO) possesses various antiathero-sclerotic properties It is involved in the control of vasculartone and blood pressure by causing vasodilatation NO alsoinhibits various steps involved in atherogenesis such as oxi-dation of low density lipoprotein (LDL) platelet aggregationleucocytes adhesion and abnormal proliferation of vascularsmooth muscle cells [1] Loss of normal NO productionfrom the endothelium is a cardinal feature of endothelialdysfunction Based on the vasculoprotective effects of NOincreased endothelial NO synthesis has the potential to beused as a target in the prevention and treatment of cardio-vascular diseases [2]

Endothelial nitric oxide synthase (eNOS) is the majorenzyme responsible for NO production in the blood

vessels [3] NO synthesis increases when the level and activityof eNOS in the endothelial cells increase [4] NO synthesiscan also be modulated through regulation of eNOS geneexpression [5] Growth factors such as transforming growthfactor beta-1 (TGF-1205731) vascular endothelial growth factor(VEGF) and basic fibroblast growth factor (bFGF) werereported to upregulate eNOS gene expression [6]

Oxidative stress results from the imbalance between theprooxidative and the antioxidative defense mechanisms ofthe body The major source of endogenous reactive oxygenspecies (ROS) is generated from H

2O2[7] which has been

extensively used to induce oxidative stress in in vitro experi-ments [8 9] Oxidative stress plays an important role in thepathogenesis of atherosclerosis and cardiovascular diseasesby promoting endothelial dysfunction inflammation and

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 169370 9 pageshttpdxdoiorg1011552014169370

2 The Scientific World Journal

lipidlipoprotein peroxidation and lowering NO bioavailabil-ity [10] Loss of normalNOproduction from the endotheliumis a cardinal feature of endothelial dysfunction [11]

Flavonoids are a group of phenolic compounds which canbe found naturally in plants Epidemiological studies indicatethat increased intake of dietary flavonoids is associatedwith a decrease in the risk of cardiovascular diseases [12]The cardiovascular protective effects of flavonoids may bemediated by multiple mechanisms One possible pathway isby increasing eNOS expression and NO synthesis IncreasedNO produced by higher levels of eNOS might in turninhibit pathways leading to endothelial dysfunction andatherosclerosis [13]

Rutin (3310158404101584057-pentahydroxyflavone-3-rhamnogluco-side) is a flavonoid which can be found in buckwheat applegreen tea Betula pendula leaves and other sources [14 15] Ithas antioxidant [16] anti-inflammatory [17] and antiplatelet[18] activities Rutin supplementation causes lowering ofblood pressure in rats with metabolic syndrome [19] andrelaxation of ratsrsquo aortic rings [20]

Rutin is one of the active compounds found in Pipersarmentosum leaves [15] Piper sarmentosum is a creepingterrestrial herbaceous plant that belongs to the Piperaceaefamily It is commonly found in the tropical and subtropicalregions of the world such as the Asian and South East Asiaregions [21] Piper sarmentosum had been shown to promoteNO production in HUVEC [22] However the active com-pound responsible for the effect remains unclear Thereforethe present study was designed to look into the effects ofrutin on the eNOS system and NO synthesis in HUVECThe results of the present study may help in the preventionand treatment of endothelial dysfunction which is linkedto various cardiovascular diseases Furthermore beneficialresults from this study will also add to the scientific basis ofusing Piper sarmentosum as a supplement for cardiovascularhealth

2 Materials and Methods

21Materials Rutin(purity 95)hydrogenperoxide (H2O2)

and ethidium bromide were purchased from Sigma (StLouis USA) Collagenase type I was purchased from Gibco-Invitrogen Corp (Grand Island USA) Medium 200 andlow serum growth supplement (LSGS) were purchased fromCascade Biologics (Grand Island USA) TRI Reagent andpolyacryl carrier were purchased from Molecular ResearchCenter (Cincinnati USA) RNase and DNase free waterand SuperScript III First-Strand Synthesis SuperMix werepurchased from Invitrogen (Carlsbad USA) IQ SYBRGreenSupermix was purchased from Bio-Rad (Hercules USA)Quantikine human eNOS ELISA kit was purchased fromRampD Systems Inc (Minneapolis USA) Calbiochem nitricoxide synthase assay kit was purchased fromEMDChemicals(Darmstadt Germany) Bioxytech nitric oxide assay kit waspurchased from OxisResearch (Portland USA) Procartacytokine kit was purchased from Panomics (Fremont USA)

22 Cell Culture and Treatment Protocol Human umbilicalcords were obtained under sterile condition from labour

room in Hospital Kuala Lumpur Written consent was ob-tained from each subject and the present study was approvedby the Ethical Research Committee of Universiti KebangsaanMalaysia Medical Center (approval code FF-092-2010)HUVEC were obtained from umbilical cord veins by 01collagenase type I digestion Cells were grown inmedium 200supplemented with LSGS at 37∘C in a humidified atmosphereof 5 CO

2and 95 air HUVEC were confirmed by the

typical endothelial cell cobblestone morphology and thepositive expressions of von Willebrand factor and CD31 inimmunocytochemistry The culture medium was changedevery other day until the cells reached confluence HUVECfrom passage 3 at 80 confluency were used for experimentsThe cells were divided into four groups as follows controloxidative stress induction with 180 120583MH

2O2 treatment with

300 120583M rutin only and concomitant induction with 300 120583Mrutin and 180 120583M H

2O2 All treatments were given for 24

hoursThe dose of H2O2used was based on the IC

50of H2O2

adopted from a previous study [22] while 300 120583M rutin wasused as it significantly increased HUVEC viability by almost50 percent when induced with 180 120583MH

2O2[15]

23 Quantitative Reverse Transcription Polymerase ChainReaction (qPCR) for Analysis of eNOS TGF1205731 bFGF andVEGF mRNA Expression Following treatment for 24 hourstotal ribonucleic acid (RNA) from HUVEC was extractedusing TRI Reagent as previous research protocol [23] Pol-yacryl carrier was added to precipitate the total RNAExtracted RNA pellet was then washed with 75 ethanol anddried prior to dissolving it in RNase and DNase free waterExtracted total RNA was assessed for its purity and quantityusing Nanodrop ND-100 spectrophotometer (WilmingtonDE USA) and stored at minus80∘C before use ComplimentaryDNA (cDNA) was synthesized using SuperScript III First-Strand Synthesis SuperMix A total of 20120583L of volumereaction which consisted of 10 120583L of 2X RT reaction mix2 120583L of RT enzyme 5 120583L of total RNA and 3 120583L of DEPC-treated water was incubated at 25∘C for 10 minutes forprimer annealing then at 50∘C for 30 minutes for reversetranscription Following this the reaction was terminated at85∘C for 5 minutes chilled on ice for 1 minute and 1 120583Lof E coli RNase H was added to the mixture The cDNAwas further incubated at 37∘C for 20 minutes and storedat minus20∘C until use Subsequently qPCR was carried outto determine the mRNA expression level of eNOS TGF1205731bFGF and VEGF Glycerylaldehyde-3-phosphate dehydroge-nase (GAPDH) was used as the reference gene Primer 3software was used to design the primers from NIH GenBankdatabase The primer sequences for eNOS TGF1205731 bFGFand VEGF were listed in Table 1 The qPCR reaction wasperformed with 1 120583L of cDNA 5120583M of each forward andreverse primer and 125 120583L of IQ SYBR Green Supermixin BioRad iCycler (Bio-Rad USA) with reaction profile of40 cycles of 95∘C (10 seconds) and 61∘C (30 seconds) Thereaction kinetic of each primer set and protocol was verifiedwith melting profile and product size was further confirmedwith 2 agarose gel electrophoresis stained with ethidiumbromide The threshold cycle (CT) value was determined

The Scientific World Journal 3

Table 1 List of primers for qPCR analysis

mRNA target Genbank accession number Primer sequence PCR product size (bp)

GAPDH NM 002046 F tcc ctg agc tga acg gga agR gga gga gtg ggt gtc gct gt 217

eNOS NM 000603 F ttt gcc ctt atg gat gtg aagR cgc atc aaa gaa agc tca gtc 139

TGF1205731 NM 000358 F aac aca tca gag ctc cga gaaR gag gta tcg cca gga att gtt 141

VEGF NM 001033756 F ccc act gag gag tcc aac atR aaa tgc ttt ctc cgc tct ga 173

bFGF NM 002006 F ccg tta cct ggc tat gaa ggR act gcc cag ttc gtt tca gt 158

and the relative mRNA expression of eNOS TGF1205731 bFGFand VEGF was calculated as follows 2ΔΔCT with ΔΔCT =CT GAPDH minus CT gene of interest

24 Enzyme-Linked Immunosorbent Assay (ELISA) for eNOSProtein Analyses eNOS protein level of the culturedHUVECwas determined by using Quantikine human eNOS ELISAkit HUVEC were washed with phosphate-buffered saline(PBS) twice manually scraped from the culture flask andlysed with 400 120583L of lysis buffer The assay was performedusing 100120583L of the cell lysateThe cell lysate was pipetted intothe 96-well plate so that any eNOS present would be boundto the immobilized antibody in the plate After washing awayany unbound substances eNOS conjugate was added to thewellsThis was followed by addition of substrate solution andstop solutionThe optical density of eachwell was determinedat 450 nm using an ELISA microplate reader

25 Determination of eNOS Activity eNOS activity wasdetermined by using Calbiochem nitric oxide synthase assaykitThe principle of this assay was based on themeasurementof nitrite produced by eNOS in the sample in a timed reactionHUVEC were scraped from the culture flask homogenizedin PBS and centrifuged at 10000 g for 20 minutes Then thecell lysate in the supernatant solution was filtered througha 045120583m filter prior to ultracentrifugation at 100000 gfor 15 minutes A total of 40 120583L of the cell lysate wasdiluted with 20120583L of assay buffer Then the samples weremixed with NADPH nitrate reductase cofactor preparationsolution and lactate dehydrogenase (LDH) Total nitrite wasmeasured at 540 nm absorbance by reaction with Griessreagents (sulfanilamide and naphthalene-ethylenediaminedihydrochloride) Concentration of nitrite in the sample wascalculated using a standard curve The eNOS activity wasexpressed as nmol of nitritemin per mL of sample

26 Determination of Endothelial Nitric Oxide ProductionProduction of NO by HUVEC was measured as its stableoxidation product nitrite using Bioxytech nitric oxide assaykit Briefly 50120583L of the culture medium was diluted with35 120583L assay buffer and mixed with 10 120583L nitrate reductaseand 10 120583L NADH Following 20 minutes of incubation toconvert nitrate to nitrite total nitrite wasmeasured at 540 nm

absorbance by reaction with Griess reagents (sulfanilamideand naphthalene-ethylenediamine dihydrochloride)

27 Luminex Assay for TGF1205731 bFGF and VEGF ProteinAnalyses TGF1205731 bFGF and VEGF protein levels of thecultured HUVEC were obtained using Procarta cytokine kitin 96-well plate ELISA-based formats according to man-ufacturerrsquos instructions The sensitivity of the assay (limitof detection) was 1 pgmLcytokine [24] Following incuba-tion with antibody-conjugated beads detection antibodiesand streptavidin-phycoerythrin (SA-PE) complexes sampleswere analyzed with Luminex 100 instrument (Luminex Cor-poration) Fluorescence signals were collected and data wasexpressed in pgmL using internal standards as the mean ofthree individual experiments done in triplicate

28 Statistical Analysis Data was tested for normality usingKolmogorov-Smirnov test and all variables were normallydistributed Data was expressed as mean plusmn SEM Statisticalanalysis between two groups was performed using pairedStudentrsquos 119905-test using SPSS version 170 software Values of119875 lt 005 were considered statistically significant

3 Results

31 Effect of Rutin on eNOS mRNA Expression in HUVECeNOS mRNA expression in HUVEC treated with rutinincreased by 21-fold compared to the control group (119875 lt005) (Figure 1) In the oxidative stress-induced groupHUVEC treated with H

2O2showed a significant increase in

eNOSmRNAexpression by 16 times compared to the controlgroup (119875 lt 005) Concomitant treatment of HUVEC withboth rutin and H

2O2caused an increase in eNOS mRNA

expression by 18 times compared to the control group (119875 lt005)

32 Effect of Rutin on eNOS Protein Level in HUVEC eNOSprotein level in HUVEC treated with rutin (1864 plusmn 0088 times103 pgmL) increased significantly (119875 lt 001) compared to

the control (1441plusmn0113times103 pgmL) (Figure 2)TheH2O2-

induced group (1771 plusmn 0075 times 103 pgmL) also showeda significant increase in eNOS protein level compared tothe control (119875 lt 005) HUVEC induced with both rutin


0

05

1

15

2

25

3

Control Rutin

eNO

S m

RNA

expr

essio

n (fo

ld o

f con

trol)

H2O2 Rutin + H2O2

lowast

lowast

lowast

Figure 1 Bar chart showing eNOS mRNA expression in controlrutin H

2O2 and rutin + H

2O2groups Values are expressed as

means plusmn SEM of 119899 = 8 lowast119875 lt 005 versus control

05

07

09

11

13

15

17

19

21

23

Control Rutin H2O2 Rutin + H2O2

lowastlowastlowast

lowastlowast

eNO

S pr

otei

n le

vel (times10

3 pg

mL)

Figure 2 Bar chart showing eNOS protein level in control rutinH2O2 and rutin + H

2O2groups Values are expressed as means plusmn

SEM of 119899 = 8 lowast119875 lt 005 versus control lowastlowast119875 lt 001 versus controland 119875 lt 001 versus H2O2

and H2O2(2029 plusmn 0075 times 103 pgmL) showed a significant

increase in eNOS protein level compared to the control group(119875 lt 001) and H

2O2group (119875 lt 001)

33 Effect of Rutin on eNOS Activity in HUVEC eNOS activ-ity inHUVEC treatedwith rutin (4823plusmn0205times10minus2 nmolesmLmin) increased significantly (119875 lt 005) compared tothe control (4304plusmn0065times10minus2 nmolesmLmin) (Figure 3)The H

2O2-induced group (4573 plusmn 0118 times 10minus2 nmolesmL

min) also showed a significant increase in eNOS activitycompared to the control (119875 lt 005) HUVEC induced withboth rutin and H

2O2(4986 plusmn 0074 times 10minus2 nmolesmLmin)

showed a significant increase in eNOS activity compared tothe control group (119875 lt 001) and the H

2O2group (119875 lt 001)

34 Effect of Rutin on NO Production in HUVEC There wasa significant increase (119875 lt 001) in the level of NO producedby HUVEC treated with rutin (4095 plusmn 0203 120583M) compared

4

42

44

46

48

5

52


lowast

lowast

lowastlowast

eNO

S ac

tivity

(times10

minus2

nmol

esm

Lm

in)

Figure 3 Bar chart showing eNOS activity in control rutin H2O2

and rutin + H2O2groups Values are expressed as means plusmn SEM of

119899 = 8 lowast119875 lt 005 versus control lowastlowast119875 lt 001 versus control and119875 lt 001 versus H2O2

0

1

2

3

4

5

6

7


lowast

lowastlowast

lowastlowast

NO

leve

l (120583

M)

Figure 4 Bar chart showing NO level in control rutin H2O2 and

rutin +H2O2groups Values are expressed asmeansplusmn SEMof 119899 = 8

lowast

119875 lt 005 versus control lowastlowast119875 lt 001 versus control and 119875 lt 001

versus H2O2

to the control (1605plusmn 008 120583M) (Figure 4) HUVEC inducedwith H

2O2produced higher amount of NO (201plusmn0115 120583M)

compared to the control (119875 lt 001) The highest level ofNO was produced by HUVEC treated with both rutin andH2O2(565plusmn0683 120583M)whereby this increase was significant

compared to the control group (119875 lt 001) and the H2O2

group (119875 lt 001)

35 Effects of Rutin on TGF1205731 bFGF and VEGF mRNAExpression in HUVEC bFGF mRNA expression in HUVECtreated with rutin increased significantly (119875 lt 005) by 16times compared to the control (Figure 5) HUVEC treatedwith both rutin and H

2O2also showed higher level of bFGF

mRNA expression compared to the control (119875 lt 001) andthe H

2O2(119875 lt 001) groups There was no significant differ-

ence in the mRNA expression of TGF1205731 and VEGF

36 Effects of Rutin on TGF1205731 bFGF and VEGF ProteinLevel in HUVEC bFGF protein level in HUVEC treatedwith rutin (1169715 plusmn 34663 pgmL) increased significantly(119875 lt 001) compared to the control (946198 plusmn 44043 pgmL) (Figure 6) HUVEC treated with both rutin and H

2O2

also showed higher level of bFGF protein compared to thecontrol (119875 lt 005) and H

2O2(947696 plusmn 48933 pgmL)


0

05

1

15

2

Control Rutin

mRN

A ex

pres

sion

(fold

of c

ontro

l)

Treatment group

bFGFVEGF

H2O2 Rutin + H2O2

lowastlowast

TGF1205731

Figure 5 Bar chart showing TGF1205731 bFGF and VEGF mRNAexpression in control rutin H

2O2 and rutin + H

2O2groups Values

are expressed as means plusmn SEM of 119899 = 8 lowast119875 lt 005 versus control119875 lt 005 versus H2O2

0

200

400

600

800

1000

1200

1400

Control Rutin

Prot

ein

leve

l (pg

mL)

Treatment group

bFGFVEGF

lowastlowast lowast

H2O2 Rutin + H2O2

TGF1205731

Figure 6 Bar chart showing TGF1205731 bFGF and VEGF proteinlevel in control rutin H

2O2 and rutin + H

2O2groups Values are

expressed as means plusmn SEM of 119899 = 8 lowast119875 lt 005 versus controllowastlowast

119875 lt 001 versus control and 119875 lt 005 versus H2O2

(119875 lt 005) groups There was no significant difference inthe protein level of TGF1205731 and VEGF The increase in bFGFprotein level was in parallel with the increase in bFGFmRNAexpression (Figure 5)

4 Discussion

Results showed that rutin increased NO production byHUVEC Rutin also caused upregulation of eNOS mRNA

expression and increase in eNOS protein level and eNOSactivity The increase in eNOS mRNA expression causedmore eNOS protein to be synthesized The higher amountof eNOS protein led to a higher level of eNOS activity Thisresulted in an increase in the NO production by HUVECeNOS protein level was significantly increased in the com-bined rutin + H

2O2group compared to the H

2O2group (119875 lt

001) (Figure 2) However eNOS mRNA expression was notsignificantly increased when comparing between these twogroups (Figure 1) This could be due to the level of eNOSprotein in the rutin + H

2O2group which was high enough

to inhibit eNOS mRNA expression via negative feedbackmechanism [25]

Even though H2O2treatment alone increased NO pro-

duction the combined treatment of HUVEC with rutin andH2O2significantly increased NO production compared to

both control and H2O2groups The results suggested that

rutin may improve endothelial function by augmenting NOproduction in human endothelial cells Piper sarmentosumwas reported to enhance endothelial NO synthesis [22] Sincerutin is one of the major flavonoids found in Piper sarmen-tosum [15] it may play a role in modulating the stimulatoryeffect of Piper sarmentosum on NO production

An earlier study reported rutin to cause vasorelaxationin potassium- and phenylephrine-induced contractions inisolated rat thoracic aorta [20] The vasorelaxant effect ofrutin involved the release of NO from the endothelium aspretreatment with NO synthase inhibitor and NG-nitro-L-arginine methyl ester (L-NAME) attenuated the response[20] Rutin-treated rats with metabolic syndrome had lowerblood pressure and improved endothelial function Thehypotensive effect of rutin could be mediated by the increasein NO [19]

Oxidative stress can contribute to the development andprogression of atherosclerosis by promoting endothelial dys-function inflammation and lipid peroxidation and loweringNO bioavailability [10] In the present study oxidative stressinduction in HUVEC by addition of 180 120583MH

2O2increased

eNOS mRNA expression eNOS protein level eNOS activityand NO level (Figures 1 2 3 and 4) The responses to H

2O2

in this study were in accordance with earlier reports [10 26]NO level was higher in the H

2O2-treated group compared

to the control group This may be due to induction of NOproduction by H

2O2as part of the self-protective mechanism

of the cells The dose of H2O2used in this study was not

lethal to HUVEC therefore the cells were still able to increaseits endogenous NO production when being challenged byH2O2 However H

2O2also caused oxidative destruction of

the synthesized NO which explained why the increase inNO in the H

2O2-treated group was not as high as the other

groups like rutin and the combined rutin and H2O2groups

(Figure 4) H2O2-upregulated eNOS expression represents a

self-protectivemechanismof the endothelial cells tomaintainNObioactivity under conditions of enhanced oxidative stressH2O2also increases eNOS activity by inducing changes in the

phosphorylation status of the enzyme [27]Antioxidants are well known to enhance the biological

actions of NO by protecting NO against oxidative destruc-tion by ROS [27] Rutin was shown to exhibit antioxidant


Rutin

proliferation

(mitogenic effect)

concentration in the culture

ROS

Protection of NO

against oxidative

destruction

uarr HUVEC

uarr eNOS

uarr bFGF

uarr eNOS mRNA expression

uarr eNOS protein synthesis

uarr eNOS enzyme activity

uarr endothelial NO level

Figure 7 Schematic representation of mechanisms involved in rutin-mediated NO synthesis in HUVEC

properties [16] and cytoprotective effects against H2O2-

induced oxidative cell damage [15] Thus rutin may directlyprotect NO from oxidative destruction by H

2O2 Rutin also

enhanced NO production in HUVEC through increase ineNOS mRNA expression and protein synthesis as well as theenzyme activity (Figures 1 2 and 3) Thus all these mecha-nisms contributed to the increase in the NO level

In a previous study rutin significantly attenuatedH2O2-induced cytotoxicity and apoptosis in HUVEC in a

concentration-dependant manner [28] Reactive oxygenspecies (ROS) (superoxide H

2O2 and hydroxyl radicals) are

potent intracellular oxidants which were proposed as criticalregulators of apoptosis [29] Reduced glutathione (GSH) isa major antioxidant that protects cells from oxidative stressby scavenging peroxides in the mitochondria [30] H

2O2

may cause endothelial cell injury by inducing mitochondrialdysfunction which includes loss of mitochondrial membranepotential [31] Rutin protected HUVEC against H

2O2-

induced cytotoxicity by decreasing the intracellular ROSlevel increasing the intracellular GSH and restoring themitochondrial membrane potential along with the capacityof suppressing endothelial cell apoptosis [28]

Incubation of HUVEC with 50 100 and 200 120583M H2O2

for one hour was able to stimulate inducible nitric oxidesynthase (iNOS) mRNA and protein [32] Therefore theNO produced by the H

2O2-treated group may be also

contributed to iNOS apart from eNOS (Figure 4) Previousstudy showed that rutin suppressed iNOS gene transcriptionand NO production in lipopolysaccharide-stimulated RAW2647macrophages [33] Rutin also inhibited iNOS activity inthe kidneys of rats during ischemia-reperfusion injury [34]

Results of the present study also showed that rutinincreased bFGFmRNAand protein expression (Figures 5 and6) There were no significant changes in mRNA and protein

expression of TGF1205731 andVEGF Previous studies showed thatbFGF caused an increase in the eNOS expression in vitro andin vivo [35] Since rutin increased the expression of eNOS andbFGF it is suggested that upregulation of eNOS expressionby rutin may be mediated by bFGF However in the presentstudy the data was not enough to conclude the role of TGF1205731VEGF and bFGF in rutin-induced eNOS expression and NOproduction We advocate parallel experiments using specificinhibitor or siRNA in future

Incubation of bovine aortic endothelial cells with bFGFleads to increased eNOS mRNA expression eNOS proteinlevel and eNOS activity [36] Besides bFGF also stimulatedthe expression of eNOS mRNA and protein in ovine feto-placental artery endothelial cells [37] Intravenously admin-istered bFGF lowered blood pressure by causing systemicvasodilatation [38] bFGF-induced vasodilatation was atten-uated by coadministration of L-NAME showing that thevasodilatation was mediated by NO-dependent mechanism[39] Blood vessels of spontaneously hypertensive rats hadlow bFGF content [40] Restoration of bFGF to physiologicallevels either by systemic administration or by in vivo genetransfer significantly augmented the number of endothelialcells with positive immunostaining for eNOS correctedhypertension and improved vasorelaxation [40]

bFGF has a mitogenic effect whereby it may stimulateproliferation of various cells including endothelial cells [41]Rutin stimulated bFGF expression and bFGF had amitogeniceffect on endothelial cells This mitogenic effect may lead tothe increase in HUVEC culture proliferation Increase in thenumber of endothelial cells will cause higher concentrationof eNOS in the culture This may lead to increase in eNOSactivity and subsequently more NO production by HUVECThe mechanisms involved in rutin-promoting effects onendothelial NO production were summarized in Figure 7


bFGF stimulates eNOS expression via activation of themitogen-activated protein kinase (MAPK) p44 and p42pathways or also known as extracellular signal-regulatedkinases 12 (ERK or ERKs) Active ERK phosphorylatesseveral cytosolic and membrane-bound targets and upontranslocation from the cytoplasm into the nucleus activatesdifferent transcription factors thus also regulating gene tran-scription [42] The response to bFGF started when bFGFbinds to its receptor which contains tyrosine kinase domainThis may lead to phosphorylation and activation of MAPKp44 and p42 by MAPK kinase in the cytosol MAPK p44 andp42 will then be translocated from cytosol to nucleus whereit stimulates eNOS transcription [37 43ndash45] This activationwas inhibited by PD 98059 a specific MAPK kinase inhibitor[37] Since rutin increases bFGF which in turn increasesERK activity it may be postulated that rutin may also changeERK kinetic and its intracellular localization between thecytosol and the nucleus

However activation of eNOS in Chinese hamster ovary(CHO)-K1 cells is independent of theMAPK cascade [46] Inits inactive form eNOS is bound to caveolin 1 in caveolae atthe plasma membrane Dissociation of eNOS from caveolin1 and its translocation to the cytosol are important stepsin eNOS activation [47] In CHO-K1 cells bFGF activatessphingomyelinase to synthesize ceramide which in turnallows the dissociation of eNOS from caveolin 1 and itstranslocation to cytosol where it catalyzes the synthesis of NO[46]

The results also showed that there was no significantincrease in the VEGF mRNA and protein expression inresponse to H

2O2treatment (Figures 5 and 6) However pre-

vious study showed dose-dependent increase in the expres-sion of VEGF in HUVEC treated with 625ndash50120583M H

2O2

[48] Experimental results obtained with different HUVECisolates cannot easily be compared to each other because oftheir different donor origin [49] Watson et al [50] reportedfor example that the response to interleukin-8 stimulation isdifferent among several commercially available HUVEC andldquohome-isolatedrdquo primary culturedHUVEC Different growthmedia and growth conditions may also contribute to thevariations [50]

5 Conclusion

The results of the present study showed that rutin promotedNOproduction inHUVECby inducing eNOSmRNAexpres-sion protein synthesis and eNOS activity Rutinrsquos stimulatoryeffect on eNOS expression may be mediated by bFGF

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgments

This work was supported by Research Grants from Uni-versiti Kebangsaan Malaysia Medical Centre (FF-092-2010)and Ministry of Higher Education Malaysia (UKM-FF-03-FRGS0037-2010) The authors would like to thank Dr Thuan

D Bui from i-DNA Biotechnology Pte Ltd for his technicalassistance in running the Luminex assay Professor Dr SrijitDas for his assistance in editing the paper and the staff nursesin labour roomHospital Kuala Lumpur for their assistance inumbilical cord collections

References

[1] K M Naseem ldquoThe role of nitric oxide in cardiovascular dis-easesrdquoMolecular Aspects of Medicine vol 26 no 1-2 pp 33ndash652005

[2] L J Ignarro and C Napoli ldquoNovel features of nitric oxideendothelial nitric oxide synthase and atherosclerosisrdquo CurrentAtherosclerosis Reports vol 6 no 4 pp 281ndash287 2004

[3] U Forstermann and T Munzel ldquoEndothelial nitric oxide syn-thase in vascular disease From marvel to menacerdquo Circulationvol 113 no 13 pp 1708ndash1714 2006

[4] K Steinkamp-Fenske L Bollinger N Voller et al ldquoUrsolic acidfrom the Chinese herb Danshen (Salvia miltiorrhiza L) upreg-ulates eNOS and downregulates Nox4 expression in humanendothelial cellsrdquo Atherosclerosis vol 195 no 1 pp e104ndashe1112007

[5] H Li TWallerath TMunzel andU Forstermann ldquoRegulationof endothelial-typeNO synthase expression in pathophysiologyand in response to drugsrdquoNitric OxidemdashBiology and Chemistryvol 7 no 3 pp 149ndash164 2002

[6] S C Tai G B Robb and P A Marsden ldquoEndothelial nitricoxide synthase a new paradigm for gene regulation in theinjured blood vesselrdquo Arteriosclerosis Thrombosis and VascularBiology vol 24 no 3 pp 405ndash412 2004

[7] H Nohl A V Kozlov L Gille and K Staniek ldquoCell respirationand formation of reactive oxygen species facts and artefactsrdquoBiochemical Society Transactions vol 31 no 6 pp 1308ndash13112003

[8] XQ XiaoN T Lee P R Carlier Y Pang andY FHan ldquoBis(7)-tacrine a promising anti-Alzheimerrsquos agent reduces hydrogenperoxide-induced injury in rat pheochromocytoma cells com-parison with tacrinerdquo Neuroscience Letters vol 290 no 3 pp197ndash200 2000

[9] B Yang T N Oo and V Rizzo ldquoLipid rafts mediate H2O2

prosurvival effects in cultured endothelial cellsrdquo The FASEBJournal vol 20 no 9 pp 1501ndash1503 2006

[10] J Zhen H Lu X QWang N D Vaziri and X J Zhou ldquoUpreg-ulation of endothelial and inducible nitric oxide synthaseexpression by reactive oxygen speciesrdquo American Journal ofHypertension vol 21 no 1 pp 28ndash34 2008

[11] S Park W Jung S Moon et al ldquoChunghyuldan activates NOSmRNA expression and suppresses VCAM-1 mRNA expressionin human endothelial cellsrdquoCanadian Journal of Physiology andPharmacology vol 83 no 12 pp 1101ndash1108 2005

[12] I C W Arts and P C H Hollman ldquoPolyphenols and diseaserisk in epidemiologic studiesrdquoThe American Journal of ClinicalNutrition vol 81 no 1 supplement pp 317Sndash325S 2005

[13] M Appeldoorn D Venema T Peters et al ldquoSome phenoliccompounds increase the nitric oxide level in endothelial cellsin vitrordquo Journal of Agricultural and Food Chemistry vol 57 no17 pp 7693ndash7699 2009

[14] M Atanassova and V Bagdassarian ldquoRutin content in plantproductsrdquo Journal of the University of Chemical Technology andMetallurgy vol 44 no 2 pp 201ndash203 2009


[15] A Ugusman Z Zakaria C K Hui N A M M Nordin andZ A Mahdy ldquoFlavonoids of Piper sarmentosum and its cyto-protective effects against oxidative stressrdquo EXCLI Journal vol11 pp 705ndash714 2012

[16] R Guo P Wei and W Liu ldquoCombined antioxidant effects ofrutin and Vitamin C in Triton X-100 micellesrdquo Journal of Phar-maceutical and Biomedical Analysis vol 43 no 4 pp 1580ndash1586 2007

[17] T Kauss D Moynet J Rambert et al ldquoRutoside decreaseshuman macrophage-derived inflammatory mediators andimproves clinical signs in adjuvant-induced arthritisrdquo ArthritisResearch andTherapy vol 10 no 1 article R19 2008

[18] J Sheu G Hsiao P Chou M Shen and D Chou ldquoMechanismsinvolved in the antiplatelet activity of rutin a glycoside of theflavonol quercetin in human plateletsrdquo Journal of Agriculturaland Food Chemistry vol 52 no 14 pp 4414ndash4418 2004

[19] S K Panchal H Poudyal T V Arumugam and L BrownldquoRutin attenuates metabolic changes nonalcoholic steatohep-atitis and cardiovascular remodeling in high-carbohydratehigh-fat diet-fed ratsrdquo The Journal of Nutrition vol 141 no 6pp 1062ndash1069 2011

[20] M Ajay A H Gilani and M R Mustafa ldquoEffects of flavonoidson vascular smooth muscle of the isolated rat thoracic aortardquoLife Sciences vol 74 no 5 pp 603ndash612 2003

[21] T Rukachaisirikul P Siriwattanakit K Sukcharoenphol et alldquoChemical constituents and bioactivity of Piper sarmentosumrdquoJournal of Ethnopharmacology vol 93 no 2-3 pp 173ndash1762004

[22] A Ugusman Z Zakaria C K Hui and N A M M NordinldquoPiper sarmentosum increases nitric oxide production in oxida-tive stress A study on human umbilical vein endothelial cellsrdquoClinics vol 65 no 7 pp 709ndash714 2010

[23] A Ugusman Z Zakaria C K Hui and N A Megat MohdNordin ldquoPiper sarmentosum inhibits ICAM-1 and Nox4 geneexpression in oxidative stress-induced human umbilical veinendothelial cellsrdquo BMCComplementaryampAlternativeMedicinevol 11 no 1 article 31 2011

[24] A Hegde M Uttamchandani S MMoochhala andM BhatialdquoPlasma cytokine profiles in Preprotachykinin-A knockoutmice subjected to polymicrobial sepsisrdquo Molecular Medicinevol 16 no 1-2 pp 45ndash52 2010

[25] I Fleming and R Busse ldquoMolecular mechanisms involvedin the regulation of the endothelial nitric oxide synthaserdquoAmerican Journal of Physiology Regulatory Integrative andComparative Physiology vol 284 no 1 pp R1ndashR12 2003

[26] G R Drummond H Cai M E Davis S Ramasamy and D GHarrison ldquoTranscriptional and posttranscriptional regulationof endothelial nitric oxide synthase expression by hydrogenperoxiderdquoCirculation Research vol 86 no 3 pp 347ndash354 2000

[27] L J Ignarro R E Byrns D Sumi F de Nigris and C NapolildquoPomegranate juice protects nitric oxide against oxidativedestruction and enhances the biological actions of nitric oxiderdquoNitric Oxide vol 15 no 2 pp 93ndash102 2006

[28] G Gong Y Qin W Huang S Zhou X Yang and D LildquoRutin inhibits hydrogen peroxide-induced apoptosis throughregulating reactive oxygen species mediated mitochondrialdysfunction pathway in humanumbilical vein endothelial cellsrdquoEuropean Journal of Pharmacology vol 628 no 1ndash3 pp 27ndash352010

[29] N N Danial and S J Korsmeyer ldquoCell death critical controlpointsrdquo Cell vol 116 no 2 pp 205ndash219 2004

[30] P Kaur M Aschner and T Syversen ldquoGlutathione modulationinfluences methyl mercury induced neurotoxicity in primarycell cultures of neurons and astrocytesrdquo NeuroToxicology vol27 no 4 pp 492ndash500 2006

[31] H Cai ldquoHydrogen peroxide regulation of endothelial func-tion origins mechanisms and consequencesrdquo CardiovascularResearch vol 68 no 1 pp 26ndash36 2005

[32] M S Zadeh J-P Kolb D Geromin et al ldquoRegulation of ICAM-1CD54 expression on human endothelial cells by hydrogenperoxide involves inducible NO synthaserdquo Journal of LeukocyteBiology vol 67 no 3 pp 327ndash334 2000

[33] K Kazłowska T Hsu C-C Hou W-C Yang and G-J TsaildquoAnti-inflammatory properties of phenolic compounds andcrude extract from Porphyra dentatardquo Journal of Ethnopharma-cology vol 128 no 1 pp 123ndash130 2010

[34] A Korkmaz and D Kolankaya ldquoInhibiting inducible nitricoxide synthase with rutin reduces renal ischemiareperfusioninjuryrdquoCanadian Journal of Surgery vol 56 no 1 pp 6ndash14 2013

[35] H Li T Wallerath and U Forstermann ldquoPhysiological mech-anisms regulating the expression of endothelial-type NO syn-thaserdquo Nitric Oxide vol 7 no 2 pp 132ndash147 2002

[36] S K Kostyk S Kourembanas E LWheeler et al ldquoBasic fibrob-last growth factor increases nitric oxide synthase production inbovine endothelial cellsrdquoThe American Journal of PhysiologymdashHeart and Circulatory Physiology vol 269 no 5 pp H1583ndashH1589 1995

[37] J Zheng I M Bird A N Melsaether and R R MagnessldquoActivation of the mitogen-activated protein kinase cascadeis necessary but not sufficient for basic fibroblast growthfactor- and epidermal growth factor-stimulated expression ofendothelial nitric oxide synthase in ovine fetoplacental arteryendothelial cellsrdquo Endocrinology vol 140 no 3 pp 1399ndash14071999

[38] P Cuevas F Carceller S Ortega M Zazo I Nieto and GGimenez-Gallego ldquoHypotensive activity of fibroblast growthfactorrdquo Science vol 254 no 5035 pp 1208ndash1210 1991

[39] S Rosenblatt K Irikura C G Caday S P Finklestein and MA Moskowitz ldquoBasic fibroblast growth factor dilates rat pialarteriolesrdquo Journal of Cerebral Blood Flow amp Metabolism vol14 no 1 pp 70ndash74 1994

[40] P Cuevas M Garcıa-Calvo F Carceller et al ldquoCorrection ofhypertension by normalization of endothelial levels of fibroblastgrowth factor and nitric oxide synthase in spontaneously hyper-tensive ratsrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 93 no 21 pp 11996ndash120011996

[41] L Schweigerer G Neufeld J Friedman J A Abraham JC Fiddes and D Gospodarowicz ldquoCapillary endothelial cellsexpress basic fibroblast growth factor a mitogen that promotestheir own growthrdquo Nature vol 325 no 6101 pp 257ndash259 1987

[42] L Colucci-DrsquoAmato C Perrone-Capano and U di PorzioldquoChronic activation of ERK and neurodegenerative diseasesrdquoBioEssays vol 25 no 11 pp 1085ndash1095 2003

[43] C J Marshall ldquoSpecificity of receptor tyrosine kinase signalingtransient versus sustained extracellular signal-regulated kinaseactivationrdquo Cell vol 80 no 2 pp 179ndash185 1995

[44] D T Denhardt ldquoSignal-transducing protein phosphorylationcascades mediated by RasRho proteins in the mammalian cellthe potential for multiplex signallingrdquo Biochemical Journal vol318 part 3 pp 729ndash747 1996

[45] J Blenis ldquoSignal transduction via the MAP kinases proceed atyour own RSKrdquo Proceedings of the National Academy of Sciences


of the United States of America vol 90 no 13 pp 5889ndash58921993

[46] T Florio S Arena A Pattarozzi et al ldquoBasic fibroblast growthfactor activates endothelial nitric-oxide synthase in CHO-K1 cells via the activation of ceramide synthesisrdquo MolecularPharmacology vol 63 no 2 pp 297ndash310 2003

[47] I Fleming and R Busse ldquoSignal transduction of eNOS activa-tionrdquo Cardiovascular Research vol 43 no 3 pp 532ndash541 1999

[48] S K Nicholson G A Tucker and J M Brameld ldquoPhysio-logical concentrations of dietary polyphenols regulate vascularendothelial cell expression of genes important in cardiovascularhealthrdquo British Journal of Nutrition vol 103 no 10 pp 1398ndash1403 2010

[49] D Bouıs G A P Hospers C Meijer G Molema and N HMulder ldquoEndothelium in vitro a review of human vascularendothelial cell lines for blood vessel-related researchrdquo Angio-genesis vol 4 no 2 pp 91ndash102 2001

[50] C A Watson L Camera-Benson R Palmer-Crocker et alldquoVariability among human umbilical vein endothelial culturesrdquoScience vol 268 no 5209 pp 447ndash448 1995

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


lipidlipoprotein peroxidation and lowering NO bioavailabil-ity [10] Loss of normalNOproduction from the endotheliumis a cardinal feature of endothelial dysfunction [11]

Flavonoids are a group of phenolic compounds which canbe found naturally in plants Epidemiological studies indicatethat increased intake of dietary flavonoids is associatedwith a decrease in the risk of cardiovascular diseases [12]The cardiovascular protective effects of flavonoids may bemediated by multiple mechanisms One possible pathway isby increasing eNOS expression and NO synthesis IncreasedNO produced by higher levels of eNOS might in turninhibit pathways leading to endothelial dysfunction andatherosclerosis [13]

Rutin (3310158404101584057-pentahydroxyflavone-3-rhamnogluco-side) is a flavonoid which can be found in buckwheat applegreen tea Betula pendula leaves and other sources [14 15] Ithas antioxidant [16] anti-inflammatory [17] and antiplatelet[18] activities Rutin supplementation causes lowering ofblood pressure in rats with metabolic syndrome [19] andrelaxation of ratsrsquo aortic rings [20]

Rutin is one of the active compounds found in Pipersarmentosum leaves [15] Piper sarmentosum is a creepingterrestrial herbaceous plant that belongs to the Piperaceaefamily It is commonly found in the tropical and subtropicalregions of the world such as the Asian and South East Asiaregions [21] Piper sarmentosum had been shown to promoteNO production in HUVEC [22] However the active com-pound responsible for the effect remains unclear Thereforethe present study was designed to look into the effects ofrutin on the eNOS system and NO synthesis in HUVECThe results of the present study may help in the preventionand treatment of endothelial dysfunction which is linkedto various cardiovascular diseases Furthermore beneficialresults from this study will also add to the scientific basis ofusing Piper sarmentosum as a supplement for cardiovascularhealth

2 Materials and Methods

21Materials Rutin(purity 95)hydrogenperoxide (H2O2)

and ethidium bromide were purchased from Sigma (StLouis USA) Collagenase type I was purchased from Gibco-Invitrogen Corp (Grand Island USA) Medium 200 andlow serum growth supplement (LSGS) were purchased fromCascade Biologics (Grand Island USA) TRI Reagent andpolyacryl carrier were purchased from Molecular ResearchCenter (Cincinnati USA) RNase and DNase free waterand SuperScript III First-Strand Synthesis SuperMix werepurchased from Invitrogen (Carlsbad USA) IQ SYBRGreenSupermix was purchased from Bio-Rad (Hercules USA)Quantikine human eNOS ELISA kit was purchased fromRampD Systems Inc (Minneapolis USA) Calbiochem nitricoxide synthase assay kit was purchased fromEMDChemicals(Darmstadt Germany) Bioxytech nitric oxide assay kit waspurchased from OxisResearch (Portland USA) Procartacytokine kit was purchased from Panomics (Fremont USA)

22 Cell Culture and Treatment Protocol Human umbilicalcords were obtained under sterile condition from labour

room in Hospital Kuala Lumpur Written consent was ob-tained from each subject and the present study was approvedby the Ethical Research Committee of Universiti KebangsaanMalaysia Medical Center (approval code FF-092-2010)HUVEC were obtained from umbilical cord veins by 01collagenase type I digestion Cells were grown inmedium 200supplemented with LSGS at 37∘C in a humidified atmosphereof 5 CO

2and 95 air HUVEC were confirmed by the

typical endothelial cell cobblestone morphology and thepositive expressions of von Willebrand factor and CD31 inimmunocytochemistry The culture medium was changedevery other day until the cells reached confluence HUVECfrom passage 3 at 80 confluency were used for experimentsThe cells were divided into four groups as follows controloxidative stress induction with 180 120583MH

2O2 treatment with

300 120583M rutin only and concomitant induction with 300 120583Mrutin and 180 120583M H

2O2 All treatments were given for 24

hoursThe dose of H2O2used was based on the IC

50of H2O2

adopted from a previous study [22] while 300 120583M rutin wasused as it significantly increased HUVEC viability by almost50 percent when induced with 180 120583MH

2O2[15]

23 Quantitative Reverse Transcription Polymerase ChainReaction (qPCR) for Analysis of eNOS TGF1205731 bFGF andVEGF mRNA Expression Following treatment for 24 hourstotal ribonucleic acid (RNA) from HUVEC was extractedusing TRI Reagent as previous research protocol [23] Pol-yacryl carrier was added to precipitate the total RNAExtracted RNA pellet was then washed with 75 ethanol anddried prior to dissolving it in RNase and DNase free waterExtracted total RNA was assessed for its purity and quantityusing Nanodrop ND-100 spectrophotometer (WilmingtonDE USA) and stored at minus80∘C before use ComplimentaryDNA (cDNA) was synthesized using SuperScript III First-Strand Synthesis SuperMix A total of 20120583L of volumereaction which consisted of 10 120583L of 2X RT reaction mix2 120583L of RT enzyme 5 120583L of total RNA and 3 120583L of DEPC-treated water was incubated at 25∘C for 10 minutes forprimer annealing then at 50∘C for 30 minutes for reversetranscription Following this the reaction was terminated at85∘C for 5 minutes chilled on ice for 1 minute and 1 120583Lof E coli RNase H was added to the mixture The cDNAwas further incubated at 37∘C for 20 minutes and storedat minus20∘C until use Subsequently qPCR was carried outto determine the mRNA expression level of eNOS TGF1205731bFGF and VEGF Glycerylaldehyde-3-phosphate dehydroge-nase (GAPDH) was used as the reference gene Primer 3software was used to design the primers from NIH GenBankdatabase The primer sequences for eNOS TGF1205731 bFGFand VEGF were listed in Table 1 The qPCR reaction wasperformed with 1 120583L of cDNA 5120583M of each forward andreverse primer and 125 120583L of IQ SYBR Green Supermixin BioRad iCycler (Bio-Rad USA) with reaction profile of40 cycles of 95∘C (10 seconds) and 61∘C (30 seconds) Thereaction kinetic of each primer set and protocol was verifiedwith melting profile and product size was further confirmedwith 2 agarose gel electrophoresis stained with ethidiumbromide The threshold cycle (CT) value was determined
















3 Results










0

05

1

15

2

25

3

Control Rutin

eNO

S m

RNA

expr

essio

n (fo

ld o

f con

trol)

H2O2 Rutin + H2O2

lowast

lowast

lowast


2O2 and rutin + H



05

07

09

11

13

15

17

19

21

23


lowastlowastlowast

lowastlowast

eNO

S pr

otei

n le

vel (times10

3 pg

mL)






2O2group (119875 lt 001)






2O2group (119875 lt 001)


4

42

44

46

48

5

52


lowast

lowast

lowastlowast

eNO

S ac

tivity

(times10

minus2

nmol

esm

Lm

in)




0

1

2

3

4

5

6

7


lowast

lowastlowast

lowastlowast

NO

leve

l (120583

M)



lowast


versus H2O2





group (119875 lt 001)







2O2


2O2(947696 plusmn 48933 pgmL)


0

05

1

15

2

Control Rutin

mRN

A ex

pres

sion

(fold

of c

ontro

l)

Treatment group

bFGFVEGF

H2O2 Rutin + H2O2

lowastlowast

TGF1205731


2O2 and rutin + H

2O2groups Values


0

200

400

600

800

1000

1200

1400

Control Rutin

Prot

ein

leve

l (pg

mL)

Treatment group

bFGFVEGF

lowastlowast lowast

H2O2 Rutin + H2O2

TGF1205731


2O2 and rutin + H





4 Discussion




2O2group (119875 lt










2O2increased


2O2
















Rutin

proliferation

(mitogenic effect)


ROS

Protection of NO

against oxidative

destruction

uarr HUVEC

uarr eNOS

uarr bFGF








2O2 Rutin also






2O2


2O2-
















2O2


5 Conclusion




Acknowledgments



References



























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of
















3 Results










0

05

1

15

2

25

3

Control Rutin

eNO

S m

RNA

expr

essio

n (fo

ld o

f con

trol)

H2O2 Rutin + H2O2

lowast

lowast

lowast


2O2 and rutin + H



05

07

09

11

13

15

17

19

21

23


lowastlowastlowast

lowastlowast

eNO

S pr

otei

n le

vel (times10

3 pg

mL)






2O2group (119875 lt 001)






2O2group (119875 lt 001)


4

42

44

46

48

5

52


lowast

lowast

lowastlowast

eNO

S ac

tivity

(times10

minus2

nmol

esm

Lm

in)




0

1

2

3

4

5

6

7


lowast

lowastlowast

lowastlowast

NO

leve

l (120583

M)



lowast


versus H2O2





group (119875 lt 001)







2O2


2O2(947696 plusmn 48933 pgmL)


0

05

1

15

2

Control Rutin

mRN

A ex

pres

sion

(fold

of c

ontro

l)

Treatment group

bFGFVEGF

H2O2 Rutin + H2O2

lowastlowast

TGF1205731


2O2 and rutin + H

2O2groups Values


0

200

400

600

800

1000

1200

1400

Control Rutin

Prot

ein

leve

l (pg

mL)

Treatment group

bFGFVEGF

lowastlowast lowast

H2O2 Rutin + H2O2

TGF1205731


2O2 and rutin + H





4 Discussion




2O2group (119875 lt










2O2increased


2O2
















Rutin

proliferation

(mitogenic effect)


ROS

Protection of NO

against oxidative

destruction

uarr HUVEC

uarr eNOS

uarr bFGF








2O2 Rutin also






2O2


2O2-
















2O2


5 Conclusion




Acknowledgments



References



























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

05

1

15

2

25

3

Control Rutin

eNO

S m

RNA

expr

essio

n (fo

ld o

f con

trol)

H2O2 Rutin + H2O2

lowast

lowast

lowast


2O2 and rutin + H



05

07

09

11

13

15

17

19

21

23


lowastlowastlowast

lowastlowast

eNO

S pr

otei

n le

vel (times10

3 pg

mL)






2O2group (119875 lt 001)






2O2group (119875 lt 001)


4

42

44

46

48

5

52


lowast

lowast

lowastlowast

eNO

S ac

tivity

(times10

minus2

nmol

esm

Lm

in)




0

1

2

3

4

5

6

7


lowast

lowastlowast

lowastlowast

NO

leve

l (120583

M)



lowast


versus H2O2





group (119875 lt 001)







2O2


2O2(947696 plusmn 48933 pgmL)


0

05

1

15

2

Control Rutin

mRN

A ex

pres

sion

(fold

of c

ontro

l)

Treatment group

bFGFVEGF

H2O2 Rutin + H2O2

lowastlowast

TGF1205731


2O2 and rutin + H

2O2groups Values


0

200

400

600

800

1000

1200

1400

Control Rutin

Prot

ein

leve

l (pg

mL)

Treatment group

bFGFVEGF

lowastlowast lowast

H2O2 Rutin + H2O2

TGF1205731


2O2 and rutin + H





4 Discussion




2O2group (119875 lt










2O2increased


2O2
















Rutin

proliferation

(mitogenic effect)


ROS

Protection of NO

against oxidative

destruction

uarr HUVEC

uarr eNOS

uarr bFGF








2O2 Rutin also






2O2


2O2-
















2O2


5 Conclusion




Acknowledgments



References



























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

05

1

15

2

Control Rutin

mRN

A ex

pres

sion

(fold

of c

ontro

l)

Treatment group

bFGFVEGF

H2O2 Rutin + H2O2

lowastlowast

TGF1205731


2O2 and rutin + H

2O2groups Values


0

200

400

600

800

1000

1200

1400

Control Rutin

Prot

ein

leve

l (pg

mL)

Treatment group

bFGFVEGF

lowastlowast lowast

H2O2 Rutin + H2O2

TGF1205731


2O2 and rutin + H





4 Discussion




2O2group (119875 lt










2O2increased


2O2
















Rutin

proliferation

(mitogenic effect)


ROS

Protection of NO

against oxidative

destruction

uarr HUVEC

uarr eNOS

uarr bFGF








2O2 Rutin also






2O2


2O2-
















2O2


5 Conclusion




Acknowledgments



References



























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Rutin

proliferation

(mitogenic effect)


ROS

Protection of NO

against oxidative

destruction

uarr HUVEC

uarr eNOS

uarr bFGF








2O2 Rutin also






2O2


2O2-
















2O2


5 Conclusion




Acknowledgments



References



























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of







2O2


5 Conclusion




Acknowledgments



References



























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of












































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

research article role of rutin on nitric oxide...

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