Research Article Treatment of Stress Urinary Incontinence by ...Research Article Treatment of Stress...

Research ArticleTreatment of Stress Urinary Incontinence byCinnamaldehyde the Major Constituent of the ChineseMedicinal Herb Ramulus Cinnamomi

Yung-Hsiang Chen12 Yu-Ning Lin1 Wen-Chi Chen12

Wen-Tsong Hsieh1 and Huey-Yi Chen12

1 Department of Pharmacology Graduate Institute of Integrated Medicine College of Chinese MedicineChina Medical University Taichung 40402 Taiwan

2Departments of Medical Research Urology and Obstetrics and Gynecology China Medical University HospitalTaichung 40402 Taiwan

Correspondence should be addressed to Huey-Yi Chen d888208ms45hinetnet

Received 16 October 2013 Accepted 3 February 2014 Published 10 March 2014

Academic Editor Evan Paul Cherniack

Copyright copy 2014 Yung-Hsiang Chen 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

Stress urinary incontinence (SUI) is a common disorder in middle-aged women and the elderly population Although surgicaltreatment of SUI has progressed pharmacological therapies remain unelucidated We screened potential herbal medicines againstSUI with an ex vivo organ bath assay Ramulus Cinnamomi and its major constituent cinnamaldehyde cause a high contractile forceof the urethra and a low contractile force of blood vessels Cinnamaldehyde dose-dependently reduced lipopolysaccharide-inducednitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in RAW 2647 cells In the vaginal distension-(VD-) induced SUI model in mice cinnamaldehyde significantly reversed the VD-induced SUI physical signs and reducedblood pressure Cinnamaldehyde may offer therapeutic potential against SUI without the possible side effect of hypertensionThe modulation of several SUI-related proteins including myosin iNOS survival motor neuron (SMN) protein and superoxidedismutase 3 (SOD3) may play some crucial roles in the therapeutic approach against SUI This information may offer clues to thepathogenesis of SUI and open additional avenues for potential therapy strategies

1 Introduction

Stress urinary incontinence (SUI) is a common disorderin recent decades given the steep increases in the elderlypopulation [1 2] It is also a common medical conditionaffecting middle-aged women worldwide Approximately40of youngerwomen (20ndash45 years old) suffer fromSUI thatsignificantly affects their quality of life [3] Although progresshas been made in the surgical treatment of SUI pharma-cological therapy for SUI is poorly understood Thereforedrug discovery and development for SUI is urgent An incon-clusive therapy for SUI which causes urethral contractionsincludes 120572-adrenergic agonists such as ephedrine phenyle-phrine (PE)midodrine norfenefrine phenylpropanolamine

and imipramine Treatment of women with SUI using 120572-adrenergic stimulating drugs was withdrawn because ofunwanted side effects including insomnia restlessness ele-vated blood pressure arrhythmia chest pain and headache[4]

Since conventional therapy is not completely efficaciousand older individuals may be unwilling to undergo surgicaltreatment alternative treatments may be potentially usedas an adjunctive therapy for SUI episodes [5] Reportedtraditional Chinese herbal medicines for SUI include severalherbs and polyherbal formulae but evidence regarding thebenefits is often lacking Recently integrative medicine hasbeen commonly used for health conditions in middle-agedwomen and several herbal therapies have been tested to

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 280204 10 pageshttpdxdoiorg1011552014280204

2 Evidence-Based Complementary and Alternative Medicine

determine if they ameliorate the physical signs of SUI or notIn an uncontrolled trial subjects were given a Chinese herbalformula 78 of them experienced decreased frequency ofincontinent episodes [6] suggesting the potential applicationof Chinese herbal medicine for SUI treatment [7ndash9]

Comprehending the urethral function and pharmacologymay lead to the development of promising new agents [10]which may be useful in the management of SUI in women[4] Because of concerns about 120572-adrenergic agonists bothpractitioners and patients are seeking alternatives to clinical120572-adrenergic stimulating drugs for managing SUI physicalsign In the present study several candidate herbal medicineswere screened and botanical agents for SUI were proposedfor development This study established an ex vivo organbath in vitro cell culture and in vivo animal SUI modelsto evaluate potential botanical agents against SUI and toexplore the possible mechanisms Among these botanicalherbs Ramulus Cinnamomi (the dry twig of Cinnamomumcassia Presl a plant in the family Lauraceae) and its majorconstituent cinnamaldehyde caused a high contractile forceof the urethra and a low contractile force of blood vesselsPlant-derived essential oils have long been used as flavoringagents in agricultural food and beverages and as naturalagents for food preservation Cinnamomum cassia bark oilknown as cinnamon oil is commonly used in the foodindustry because of its special aroma Cinnamaldehyde iscommonly used in agricultural food and beverages and withgreat commercial and possible medical value [11] Theseanalyses will give an insight of pharmacologic treatment ofSUI This information may offer clues to the pathogenesisof SUI and open additional avenues for potential therapystrategies

2 Materials and Methods

21 Organ Bath Experiment for Potential SUI Drug ScreeningOrgan bath experiment was used for functional evaluationof contractile urethra and blood vessel ex vivo for thecontractility study [12 13] female pig urethra and bloodvessel were prepared from the basal part of the bladderbody and renal artery respectively [13] Urethra and renalartery were mounted between platinum-plate electrodes andsecured by small clips in a double-jacketed organ bathcontaining 20mL Krebsrsquo solution aerated with 95 O

2and

5 CO2to obtain a pH of 74 at 37∘C The composition of

the Krebsrsquo solution was (mM) 133 NaCl 47 KCl 25 CaCl2

163 NaHCO3 135 NaH

2PO4 06 MgSO

4 and 78 dextrose

Isometric contraction was recorded with a computerizeddata acquisition program (Biobench National InstrumentsCorporation TX USA) at a rate of 50Hz and stored on ahard drive for later analyses At the end of the experimentthe length and weight of each muscle strip between thesuspension clips weremeasuredThe 120572-adrenergic agonist PEwas used as a positive control

22 Cell Culture Raw 2647 cells were purchased as cryop-reserved cultures from Bioresource Collection and ResearchCenter (Hsinchu Taiwan) 1 times 106 cells were seeded into 100

mm Petri dishes and incubated at 37∘C in 90 Dulbeccorsquosmodified Eaglersquos medium with 4mM L-glutamine adjustedto contain 15 gL sodium bicarbonate and 45 gL glucose+ 10 fetal bovine serum Primary human bladder smoothmuscle cells (HBdSMC) (ScienCell CA USA) were grownin the smooth muscle cell medium (SMCM Catalog number1101) By which time the cells had reached 90 confluencethe cells were plated at 96-well or 10 cm plates for MTT assayor Western blot [14 15] respectively

23MTTAssay for Cell Viability Cells were grown in 96-wellplates at a concentration of 3times 104 cellswell for 24 h and thenserum-freemediumwith different drugs was addedThe cellswere collected after 24 h later Mitochondrial dehydrogenaseactivity was measured as an index of cell viability using the 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazoliumbromide(MTT) assays [16] In brief MTT (05mgmL) was appliedto the cells for 4 h to allow the conversion of MTT into for-mazan crystals the cells were lysed with dimethyl sulfoxide(DMSO) and the absorbance was read at 570 and 650 nmwith a DIAS Microplate Reader (Molecular Devices CAUSA) The reduction in optical density caused by treatmentwas used as a measurement of cell viability normalized to thecells incubated in control medium which were considered100 viable

24 Nitric Oxide Assay Cells were grown in 96-well platesat a concentration of 3 times 104 cellswell for 24 h and thenserum-free medium with different drugs was added Culturesupernatants were collected after 24 h later Nitrite levelswere measured by addition of 01mL of the Griess reagent(15 sulfanilamide and 01 naphthylethylenediamine dihy-drochloride in 25 H

3PO4) to 01mL of culture supernatant

in 96-well plates leaving the plates in the dark for 10min andmeasuring the color intensity with an automated microtiterplate reader at 550 nm [17]

25 Experimental Animals and Study Design Twenty virginfemale C57BL6 strain mice aged approximately 6ndash8 weekswere randomized into 4 groups a noninstrumented controlgroup the other mice undergoing vaginal distension (VD)for 1 h with 8mm dilators (compatible with the diameterof mouse newborn head) were randomized to receive PE(25 120583gkg ip) [18] cinnamaldehyde (150mgkg ip) [19] orthe vehicle for 3 days Four days after VD mice underwentsuprapubic bladder tubing (SPT) placement Six days afterVD leak point pressure (LPP) and maximal urethral closurepressure (MUCP) measurements were assessed in thesemice under urethane (1 gkg ip) anesthesia [20 21] Bloodpressure (BP) and heart rate (HR) were measured using aBPMonitor for Rats and Mice Model MK-2000 (MuromachiKikai Tokyo Japan) according to the instructions of themanufacturer [22] Aftermeasuring LPPMUCP BP andHRthe animals were sacrificed and the urethras were removedfor Western blot analysis All experimental protocols wereapproved by the Institutional Animal Care and Use Commit-tee of China Medical University

Evidence-Based Complementary and Alternative Medicine 3

26 Vaginal Distension Mice in the VD groups were anes-thetized with 15 isoflurane To avoid rupturing the vaginavaginal accommodation of Hegarrsquos dilators was achievedby sequentially inserting and removing different increasingsizes of Hegarrsquos dilators that were lubricated with Surgilube(Fougera Melville NY USA) Finally for the VD group an8mmdilator was lubricated and inserted into the vagina [23]After 1 h the 8mm dilator was removed and the animalwas allowed to awaken from the anesthesia spontaneouslyThe noninstrumented control group did not undergo vaginaldilation

27 Suprapubic Tube Implantation The surgical procedurewas carried out under 15 isoflurane anesthesia [23] Twodays after VD an SPT (PE-10 tubing Clay Adams Parsip-pany NJ USA) was implanted in the bladder Key pointsof the operation were as follows (1) a midline longitudinalabdominal incision was made 05 cm above the urethralmeatus (2) a small incision was made in the bladder walland PE-10 tubing with a flared tip was implanted in thebladder dome and (3) a purse-string suture with 8-0 silkwas tightened around the catheter which was tunneledsubcutaneously to the neck where it exited the skin

28 LPP and MUCP Measurement Two days after implant-ing the bladder catheter the LPP was assessed in thesemice under urethane anesthesia The bladder catheter wasconnected to both a syringe pump and a pressure transducerPressure and force transducer signals were amplified anddigitized for computer data collection at 10 samplessecond(PowerLabs AD Instruments Bella Vista Australia) Themice were placed supine at the level of zero pressure whilebladders were filled with room temperature saline at 1mLhthrough the bladder catheter If a mouse voided the bladderwas emptied manually using Credersquos maneuver The averagebladder capacity of each mouse was determined after 3ndash5 voiding cycles Subsequently the LPP and MUCP weremeasured in the following manner [23 24] When half-bladder capacity was reached gentle pressure with one fingerwas applied to the mousersquos abdomen Pressure was gentlyincreased until urine leaked at which time the externallyapplied pressure was quickly removed The peak bladderpressure was taken as the LPP At least three data wereobtained for each animal and the mean LPP was calculated

Urethral pressure profile (UPP) was assessed in thesemice under urethane (1 gkg ip) anesthesia The bladdercatheter (PE-10 tubing Clay Adams Parsippany NJ USA)was connected to a syringe pump with room temperaturesaline at 1mLh The urethral catheter was connected toa pressure transducer A withdrawal speed of 10120583m perminute was used Pressure and force transducer signals wereamplified and digitized for computer data collection at 10samplessecond (PowerLabs AD Instruments Bella VistaAustralia) Three successive profiles were obtained in thesupine position The urethral closure pressure (Pclose) isthe difference between the urethral pressure (Pure) and thebladder pressure (Pves) Pclose = Pureminus Pves [25]Maximum

urethral pressure (MUP) and MUCP were determined fromthe UPP measurements taken

29 Western Blot Analysis Cell extract or urethral tissuewas prepared by homogenization of cells in a lysis buffercontaining 1 IGEPAL CA-630 05 sodium deoxycholate01 sodium dodecyl sulfate aprotinin (10mgmL) leu-peptin (10mgmL) and phosphate-buffered saline (PBS)Cell lysates containing 100 120583g of protein were subjected tosodium dodecyl sulfate polyacrylamide gel electrophoresisand then transferred to a polyvinylidene fluoride membrane(Millipore Corp Bedford MA USA) The membrane wasstained with Ponceau S to verify the integrity of the trans-ferred proteins and to monitor the unbiased transfer of allprotein samples Detection of myosin inducible nitric oxidesynthase (iNOS) survival motor neuron (SMN) protein120572-adrenergic receptor 1a (AdR1a) extracellular superoxidedismutase (SOD3) and glyceraldehyde 3-phosphate dehy-drogenase (GAPDH) on the membranes was performed withan electrochemiluminescence kit (Amersham Life SciencesInc ArlingtonHeights IL USA) with the use of the antibodyderived from rabbit (anti-myosin antibody 1 200 dilutionMillipore MA USA anti-iNOS antibody 1 200 dilutionAbcam Cambridge UK anti-AdR1a antibody 1 200 dilu-tion Abcam Cambridge UK and anti-GAPDH antibody1 3000 dilution Abcam Cambridge UK) The intensity ofeach band was quantified using a densitometer (MolecularDynamics Sunnyvale CA USA) [26 27]

210 Statistical Analysis The data are presented as mean plusmnstandard deviation (SD) for each group Statistical differencesamong groups were determined by one-way analysis ofvariance (ANOVA) followed by Fisherrsquos LSD as a post hoc testAll statistical tests were two-sided A 119875 value less than 005was considered statistically significant All calculations wereperformed using the Statistical Package for Social Sciences(SPSS forWindows release 80 SPSS Inc Chicago IL USA)

3 Results

31 Organ Bath Study Some herbal medicines may have asympathomimetic effect [28] The organ bath experiment isthe gold standard for functional evaluation of contractiletissues ex vivo such as the urinary tract (including ureters theurinary bladder and the urethra) and blood vessels [29 30]Figures 1(a) and 1(b) show that the 120572-adrenergic agonistPE has high contractile force of both urethra and arteryin the organ bath experiment Ramulus Cinnamomi wasfound to have the highest efficacy for stimulation of urethralcontraction Therefore we tested the effect of the majorconstituent of the Ramulus Cinnamomi cinnamaldehydeand found that cinnamaldehyde has a stimulating effect onurethral contraction but less effect on renal artery contraction(Figures 1(a)ndash1(c))

32 Effect of Cinnamaldehyde on NO Production and ProteinExpressions in the Cell Culture Model The primary etiologi-cal factor of SUI is vaginal delivery [31] which may provide


PE (M) Cinnamaldehyde (M)10minus5 10minus6 10minus410minus5

4min

1g

(a)


4min

1g

(b)

Cinnamaldehyde (M)

Con

trac

tion

()

0

20

40

60

80

100

UrethraArtery

10minus510minus8 10minus410minus610minus70

(c)

Figure 1 Urethra and artery contraction in organ bath experiment Representative original traces of the contractile responses to PE andcinnamaldehyde in female pig urethra (a) and artery (b) Contractions induced by 10minus5M of PE in the control media were taken as 100 (c)Graphic representation of concentration-response curves

ischemic [32] and inflammatory damage to the urogenitaltract [33] Ischemia and inflammation cause lower urinarytract dysfunction and activate iNOS expression therebyincreasing NO synthesis and resulting in urethral relaxation[4 21] Thus we tested the effect of cinnamaldehyde onthe NO production in vitro and iNOS expression in themouse leukemic monocyte macrophage cell line Raw 2647The effect of cinnamaldehyde on RAW 2647 cell viabil-ity was first determined by an MTT assay Cells culturedwith cinnamaldehyde at the concentrations 0 625 125and 25 120583M for 24 h did not change cell viability howevercinnamaldehyde significantly reduced cell viability at highconcentrations (50 and 100 120583M) (Figure 2(a)) Therefore theeffect of cinnamaldehyde (le25 120583M) on LPS-induced NOproduction in RAW 2647 macrophages was investigatedNitrite accumulated in the culture medium was estimatedby the Griess reaction as an index for NO release from the

cells After treatment with LPS (500 ngmL) for 24 h thenitrite concentration increased in the medium When RAW2647 macrophages were treated with different concentra-tions of cinnamaldehyde together with LPS for 24 h the cin-namaldehyde dose-dependently reduced nitrite production(Figure 2(b)) In parallel cinnamaldehyde dose-dependentlyinhibited LPS-induced iNOS expression (Figure 2(c))

Our preliminary data of proteomic analysis related to SUIfollowingVD found 68 differentially expressed proteins of theurethra (unpublished data) Majority of the VD-modulatedproteins were involved in muscle contraction metabolitesand energy oxidative stress regulation of apoptosis andglycolysis Since human urethral smooth muscle cells arenot commercially available primary human bladder smoothmuscle cells (HBdSMC) were used as a urethral cell cul-ture model to explore the effects of cinnamaldehyde onthe expression of several candidate proteins that related to


Cel

l via

bilit

y (

of c

ontro

l)

0

20

40

60

80

100

120

0 3125 625 125 25 50 100Cinnamaldehyde (120583M)

lowast

lowast

(a)

Control LPS 3125 625 125 25

NO

pro

duct

ion

( o

f con

trol)

0

200

400

600

800

+Cinnamaldehyde (120583M)

lowast

(b)

Control LPS 625 125 25

iNO

S ex

pres

sion

(fold

of c

ontro

l)

0

2

4

6

8

iNOS

GAPDH

LPSCinnamaldehyde 625 125 25


lowast

(120583M)++++minus

minus minus

minus130kD

minus37kD

(c)

Myosin iNOS SMN AdR1a SOD3

Prot

ein

expr

essio

n(fo

ld o

f con

trol)

0

1

2

3

4

5

Control 625 125 25

iNOS

GAPDH

Cinnamaldehyde 625 125 25

SMN

SOD3

Myosin

AdR1a

Cinnamaldehyde (120583M)

lowast

lowastlowast

lowast

lowast

lowast

(120583M)minus

minus220 kD

minus130kD

minus66kD

minus52kD

minus35kD

minus37kD

(d)

Figure 2 (a) Raw 2647 cell viability after culture with cinnamaldehyde for 24 h as determined by MTT assay (b) Effects of cinnamaldehydeon LPS-induced NO production of RAW 2647 macrophages Cells were incubated for 24 h with 500 ngmL of LPS in the absence or presenceof cinnamaldehyde Nitrite concentration in the medium was determined using Griess reagent (c) Inhibition of iNOS protein expressionby cinnamaldehyde in LPS-stimulated RAW 2647 cells (d) Modulation of myosin iNOS SMN AdR1a and SOD3 protein expressions incinnamaldehyde-treated HBdSMCs GADPH was used as a loading control The calculated data were presented as mean plusmn SD for at leastthree different experiments lowast119875 lt 005 compared to control group 119875 lt 005 compared to LPS group


0

10

20

30

40

50

Control VD PE Cinnamaldehyde

lowast

+VD

LPP

(cm

H2O

)

(a)

0

10

20

30

40

50


lowast

+VD

MU

CP (c

m H

2O

)

(b)

0

20

40

60

80

100

120

140

160


+VD

BP (m

m H

g)

(c)

HR

(bea

tsm

in)

0

200

400

600

800


+VD

(d)

Figure 3 (a) LPP (b) MUCP (c) BP and (d) HR values on the sixth day after VD in the different groups Each bar represents the mean plusmn SDof five individual mice lowast119875 lt 005 compared to control group 119875 lt 005 compared to VD group

SUI Figure 2(d) shows that low-dose (625 andor 125 120583M)cinnamaldehyde significantly induced myosin SMN andSOD3 expressions

33 Treatment of SUI by Cinnamaldehyde in the Mice VDModel VD has been used for creation of SUI in rats asevidenced by LPP andMUCP on urodynamic testing [20 21]Figures 3(a) and 3(b) show that LPP andMUCP respectivelywere significantly decreased in the VD group as compared tothe uninstrumented control group treatment with both PE

and cinnamaldehyde significantly reversed the SUI physicalsigns However as anticipated BP was slightly increased inthe PE group although this did not reach statistical sig-nificance cinnamaldehyde treatment significantly decreasedthe BP as compared with the results of both control andVD groups (Figure 3(c)) No significant change was foundin HR amount in all experimental groups (Figure 3(d)) Theresults provide evidence that cinnamaldehyde induces highcontractile force of the urethra and low contractile force ofblood vessels in the animal models Cinnamaldehyde may



Prot

ein

expr

essio

n(fo

ld o

f con

trol)

0

1

2

3

4

5

Control VD

iNOS

GAPDH


SMN

SOD3

Myosin

AdR1a

VD+

minus220 kD

minus130kD

minus66kD

minus52kD

minus35kD

minus37kD

lowast

+PE+Cinnamaldehyde

Figure 4 Myosin iNOS SMN AdR1a and SOD3 expressions onthe sixth day afterVD in the different groups Each bar represents themean plusmn SD of five individual mice lowast119875 lt 005 compared to controlgroup 119875 lt 005 compared to VD group

offer therapeutic potential against SUI without the possibleside effect of hypertension

34 Protein Expressions in the SUI Mice Model We usedWestern blot analysis to confirm several candidate proteinsfrom preliminary data of proteomic analysis related to SUIfollowing VD and tried to explore the possible in vivomechanisms of cinnamaldehyde on SUI Figure 4 shows thatmyosin expression in the urethra was significantly decreasedin the VD group as compared with that in the control groupPE and cinnamaldehyde treatments significantly reversedmyosin downregulation No statistical change was foundin iNOS and AdR1a expressions PE treatment significantlyincreased the expressions of SMN and SOD3

4 Discussion

In the present study ex vivo organ bath assay revealedthat Ramulus Cinnamomi extract and its major constituentcinnamaldehyde cause high contractile force of urethra andlow contractile force of blood vessels In the VD-inducedSUI model in mice cinnamaldehyde treatment significantlyreversed the SUI physical signs without the side effect ofhypertensionThemodulation of several SUI-related proteins

including myosin iNOS SMN AdR1a and SOD3 may playsome crucial roles in the SUI progression and medicaltreatment

Integrative medicine is commonly used for health con-ditions in middle-aged women and the traditional Chineseherbal medicines have been used to ameliorate the physicalsigns of SUI but evidence regarding the benefits is stilllacking Ramulus Cinnamomi is the twig of an evergreen treebelonging to the Lauraceae family It is commonly used asChinese medicine for gastritis dyspepsia blood circulationdisturbances inflammation and also incontinence [11] Itsextracts contain several active components such as essentialoils including cinnamaldehyde [34] This study providesthe first evidence that cinnamaldehyde increased urethracontraction but had less effect on renal artery contrac-tion suggesting its potential on SUI treatment A possiblereason is that urethral and arterial smooth muscle cellshave very different pharmacological profiles [35] such asthe different distribution of adrenoceptors in these tissuesSince one of the more obvious side effects of 120572-agonists isthe stimulation of vasomotor responses and BP elevationsuggesting the complementary value of cinnamaldehyde toconventional PE treatment on SUI Moreover cinnamalde-hyde has pleiotropic pharmacological properties such asantioxidant anti-inflammatory and antiskin aging activities[36] suggesting the possible advantages of cinnamaldehydeover PE in SUI treatment

The primary etiological factor of SUI is vaginal delivery[31] whichmay provide ischemic [32] and inflammatory [37]damage to the urogenital tract [33] Ischemia and inflamma-tion cause lower urinary tract dysfunction [37] activate iNOSexpression and increase NO synthesis resulting in urethralrelaxation [4 21] In vitro models such as macrophage orother cell lines are useful materials with a steady high-level production of NO These results are consistent withprevious studies [11 38 39] that cinnamaldehyde inhibitsLPS-induced iNOS expression and NO production [40] Italso suggests the possible inhibitory effect of cinnamaldehydeon macrophage-mediated inflammatory responses Interest-ingly low-dose cinnamaldehyde appears to slightly enhanceiNOS in the HBdSMCs although this did not reach sta-tistical significance NOS activation as a result of trauma(calcium influx) leads to NO production which activates itsdownstream receptors This event may lead to one or moresystemic effects including dilatation in the cardiovascularrespiratory and urinary systems [41] However NO has dualrole as a protective and toxic molecule This paradoxicaldichotomy is leaving investigators with a double challengeto determine the net impact of NO [42] Cinnamaldehydehas been shown to induce endothelium-dependent and -independent vasorelaxant action [43 44] The other molec-ular mechanisms by which cinnamaldehyde inhibits urethralcontraction need to be further elucidated A comprehensiveand dynamic view of the cascade of molecular and cellularevents underlying urinary biology will allow investigators toexploit the potential properties of NO

Myosin molecules consist of two heavy chains Myosinheavy chains play a crucial role inmodulating smoothmusclecontraction [45] In an animalmodel increasing load induces


significant smooth muscle hypertrophy which is associatedwith a downregulation of myosin heavy chain expressionThis contributes to the decreased smoothmuscle contractility[46] In this study the protein expression of myosin in bothHBdSMCs and mice urethral tissues was examined Theresults indicated that low-dose cinnamaldehyde enhancedsmooth muscle myosin protein levels In the VD micemyosin expression in the urethra was significantly decreasedand cinnamaldehyde treatment could markedly reverse themyosin downregulation It is possible that VD in the urethrainduces dysfunction of the smooth muscle cells whichalters contractility leading to altered urethral performanceand decreased compliance These data suggest the potentialpharmacologic effect of cinnamaldehyde on smooth musclecontractile properties with their myosin expression

SMN protein is necessary for the assembly of Sm proteinsonto the small nuclear RNAs to form small nuclear ribonucle-oproteins (snRNPs) [47] The mutations of SMN gene resultin spinal muscular atrophy a common neurodegenerativedisease However the viability and sensitivity to stressesof other cell types also need to be determined Liu etal established HeLa stable cell line with inducible SMNknockdown to study its viability and sensitivity to oxidativestress Their results suggested that the there was only a slightdecrease in the proliferative rate of SMN knockdown cellsIn contrast after H

2O2reached certain concentrations the

survival rate of SMNknockdown cells decreased significantlyTheir data indicate that SMN knockdown alone is not criticalto cell viability However when SMN knockdown cells areunder stress such as oxidative stress [48] their survival ratemay significantly decrease [47] In parallel SOD3 (extra-cellular superoxide dismutase EC-SOD) was shown to bethe predominant form in extracellular fluids Many studieshave focused on the antioxidative effect of SOD3 in the lungand vascular wall where it is highly expressed Thus theroles of SOD3 as an antioxidative enzyme in ROS-mediatedischemia and inflammation have been extensively studiedHowever Kwon et al revealed that the role of SOD3 ininflammation is not simple because of radical scavenging itaffects immune responses and signal initiation [49] as VDmay provide ischemic and oxidative damage to the urogenitaltract [33] This data showed that cinnamaldehyde and PEinduced SMN and SOD3 inHBdSMCs and urethral tissues ofmice respectively suggesting that SMN and SOD3 may playimportant roles in pharmacological prevention of SUI

Traditional 120572-adrenergic stimulating drugs cause ure-thral contraction and have unwanted side effects includingvasomotor stimulation restlessness and insomnia [4 12]Sympathetically mediated urethral tone is essential for themaintenance of continence and involves the activation ofpostjunctional 120572

1-adrenoceptorsThe contraction of urethral

circular smooth muscle is mediated via 1205721-adrenoceptors

with the pharmacological characteristics [50] Yono et alshowed that doxazosin (120572-blocker) treatment caused anupregulation in the mRNA levels of 120572

1-adrenoceptors in the

rat urethra indicating that chronic doxazosin treatment maycause an alteration in the properties of 120572

1-adrenoceptors

[51] These results showed that cinnamaldehyde did notsignificantly modify the AdR1a expression suggesting that

alteration in the properties of 1205721-adrenoceptors is probably

not involved in cinnamaldehyde pharmacology

5 Conclusion

The major constituent of the Chinese medicinal herb Ramu-lus Cinnamomi cinnamaldehyde significantly reversed theVD-induced SUI physical signs without the side effect ofhypertensionThemodulation of several SUI-related proteinsincluding myosin iNOS SMN and SOD3 may play somecrucial roles in the therapeutic approach against SUI Theexact mechanism underlying its effects remains unclear andfurther experimental and clinical studies are required to elu-cidate the mechanism(s) responsible for its pharmacologicalactivities

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgments

This work was supported by Taiwan National Science Coun-cil (NSC101-2314-B-039-018 and NSC101-2314-B-039-025)China Medical University Hospital (DMR-102-059) and inpart by the Taiwan Department of Health Clinical Trial andResearch Center of Excellence (DOH102-TD-B-111-004)

References

[1] N B Erichsen and A Bussing ldquoSpiritual needs of elderly livingin residentialnursing homesrdquo Evidence-Based Complementaryand Alternative Medicine vol 2013 Article ID 913247 10 pages2013

[2] M S Ju S Lee I Bae et al ldquoEffects of aroma massage on homeblood pressure ambulatory blood pressure and sleep qualityin middle-aged women with hypertensionrdquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID403251 7 pages 2013

[3] C H van der Vaart J R J de Leeuw J P W R Roovers and AP M Heintz ldquoThe effect of urinary incontinence and overactivebladder symptoms on quality of life in young womenrdquo BJUInternational vol 90 no 6 pp 544ndash549 2002

[4] A E Canda M G Cinar B Turna and M O Sahin ldquoPharma-cologic targets on the female urethrardquo Urologia Internationalisvol 80 no 4 pp 341ndash354 2008

[5] E P Cherniack ldquoBiofeedback and other therapies for thetreatment of urinary incontinence in the elderlyrdquo AlternativeMedicine Review vol 11 no 3 pp 224ndash231 2006

[6] Y Murakami ldquoClinical effect of Hotyuekkito (Buzhongyiqi-tang) on symptoms due to renal ptosis and stress incontinencerdquoActa Urologica Japonica vol 34 no 10 pp 1841ndash1843 1988

[7] Y H Lee S H Hyun and S Y Choung ldquoEffect of herbal extractmixture on menopausal urinary incontinence in ovariec-tomized ratsrdquo BioFactors vol 26 no 3 pp 171ndash178 2006

[8] D M Wu and Y F Song ldquoEffect of ginsenoside-Rg1 on theproliferation of paraurethral fascia fibroblasts derived fromwomen suffering from stress urinary incontinencerdquo ZhonghuaFu Chan Ke Za Zhi vol 43 no 11 pp 835ndash838 2008


[9] Z Y Yang and X Qin ldquoMoxibustion combined with Chinesemedicine for 20 cases of urinary frequence and urinary inconti-nence in middle aged and elderly womenrdquo Zhongguo Zhen Jiuvol 32 no 11 pp 1029ndash1030 2012

[10] W-C Chen S-Y Wu H-P Liu et al ldquoIdentification ofmelaminecyanuric acid-containing nephrolithiasis by infraredspectroscopyrdquo Journal of Clinical Laboratory Analysis vol 24no 2 pp 92ndash99 2010

[11] J-C Liao J-S Deng C-S Chiu et al ldquoAnti-inflammatoryactivities of Cinnamomum cassia constituents in vitro and invivordquo Evidence-based Complementary and AlternativeMedicinevol 2012 Article ID 429320 2012

[12] A Alhasso C M Glazener R Pickard and J Nrsquodow ldquoAdren-ergic drugs for urinary incontinence in adultsrdquo CochraneDatabase of Systematic Reviews no 3 Article ID CD0018422005

[13] T Ramalingam N T Durlu-Kandilci and A F Brading ldquoAcomparison of the contractile properties of smooth musclefrom pig urethra and internal anal sphincterrdquoNeurourology andUrodynamics vol 29 no 7 pp 1326ndash1331 2010

[14] P-L Liu J-R Tsai C-C Chiu et al ldquoDecreased expressionof thrombomodulin is correlated with tumor cell invasivenessand poor prognosis in nonsmall cell lung cancerrdquo MolecularCarcinogenesis vol 49 no 10 pp 874ndash881 2010

[15] F-Y Lin Y-W Lin C-Y Huang et al ldquoGroEL1 a heatshock protein 60 of chlamydia pneumoniae induces lectin-like oxidized low-density lipoprotein receptor 1 expression inendothelial cells and enhances atherogenesis in hypercholes-terolemic rabbitsrdquo Journal of Immunology vol 186 no 7 pp4405ndash4414 2011

[16] A A Welder ldquoA primary culture system of adult rat heart cellsfor the evaluation of cocaine toxicityrdquo Toxicology vol 72 no 2pp 175ndash187 1992

[17] L A J Mur J Mandon S M Cristescu F J M Harrenand E Prats ldquoMethods of nitric oxide detection in plants acommentaryrdquo Plant Science vol 181 no 5 pp 509ndash519 2011

[18] X Sun K M Kaltenbronn T H Steinberg and K J BlumerldquoRGS2 is amediator of nitric oxide action on blood pressure andvasoconstrictor signalingrdquoMolecular Pharmacology vol 67 no3 pp 631ndash639 2005

[19] S Y Wang C W Yang J W Liao W W Zhen F H Chuand S T Chang ldquoEssential oil from leaves of Cinnamomumosmophloeum acts as a xanthine oxidase inhibitor and reducesthe serum uric acid levels in oxonate-induced micerdquo Phy-tomedicine vol 15 no 11 pp 940ndash945 2008

[20] T W Cannon E M Wojcik C L Ferguson S Saraga CThomas and M S Damaser ldquoEffects of vaginal distension onurethral anatomy and functionrdquo BJU International vol 90 no4 pp 403ndash407 2002

[21] H-Y Chen Y-N Lin Y-H Chen and W-C Chen ldquoStressurinary incontinence following vaginal trauma involves remod-eling of urethral connective tissue in female micerdquo EuropeanJournal of Obstetrics Gynecology and Reproductive Biology vol163 no 2 pp 224ndash229 2012

[22] S Hikoso O Yamaguchi Y Nakano et al ldquoThe I120581B Kinase120573Nuclear factor 120581b signaling pathway protects the heart fromhemodynamic stress mediated by the regulation of manganesesuperoxide dismutase expressionrdquo Circulation Research vol105 no 1 pp 70ndash79 2009

[23] Y-H Lin G Liu and F Daneshgari ldquoA mouse model ofsimulated birth trauma induced stress urinary incontinencerdquoNeurourology andUrodynamics vol 27 no 4 pp 353ndash358 2008

[24] T W Cannon and M S Damaser ldquoEffects of anesthesia oncystometry and leak point pressure of the female ratrdquo LifeSciences vol 69 no 10 pp 1193ndash1202 2001

[25] P Hilton and S L Stanton ldquoUrethral pressure measurementby microtransducer the results in symptom-free women andin those with genuine stress incontinencerdquo British Journal ofObstetrics and Gynaecology vol 90 no 10 pp 919ndash933 1983

[26] T-L Yang F-Y Lin Y-H Chen et al ldquoSalvianolic acidB inhibits low-density lipoprotein oxidation and neointimalhyperplasia in endothelium-denuded hypercholesterolaemicrabbitsrdquo Journal of the Science of Food and Agriculture vol 91no 1 pp 134ndash141 2011

[27] W-H Yin Y-H Chen J Wei et al ldquoAssociations betweenendothelin-1 and adiponectin in chronic heart failurerdquo Cardi-ology vol 118 no 4 pp 207ndash216 2011

[28] J V Pinkerton DW Stovall and R S Kightlinger ldquoAdvances inthe treatment of menopausal symptomsrdquo Womenrsquos Health vol5 no 4 pp 361ndash384 2009

[29] A Malmgren B Uvelius K-E Andersson and P-O Anders-son ldquoUrinary bladder function in rats with hereditary diabetesinsipidus a cystometrical and in vitro evaluationrdquo Journal ofUrology vol 148 no 3 pp 930ndash934 1992

[30] C H Fry ldquoExperimental models to study the physiologypathophysiology and pharmacology of the lower urinary tractrdquoJournal of Pharmacological and Toxicological Methods vol 49no 3 pp 201ndash210 2004

[31] D H Thom S K van den Eeden and J S Brown ldquoEvaluationof parturition and other reproductive variables as risk factorsfor urinary incontinence in later liferdquoObstetrics andGynecologyvol 90 no 6 pp 983ndash989 1997

[32] M S Damaser C Whitbeck P Chichester and R M LevinldquoEffect of vaginal distension on blood flow and hypoxia of uro-genital organs of the female ratrdquo Journal of Applied Physiologyvol 98 no 5 pp 1884ndash1890 2005

[33] G Lin A W Shindel L Banie et al ldquoMolecular mechanismsrelated to parturition-induced stress urinary incontinencerdquoEuropean Urology vol 55 no 5 pp 1213ndash1223 2009

[34] Z-D He C-F Qiao Q-B Han et al ldquoAuthentication andquantitative analysis on the chemical profile of Cassia Bark(Cortex Cinnamomi) by high-pressure liquid chromatographyrdquoJournal of Agricultural and Food Chemistry vol 53 no 7 pp2424ndash2428 2005

[35] M C Michel and M M Barendrecht ldquoPhysiological andpathological regulation of the autonomic control of urinarybladder contractilityrdquo Pharmacology and Therapeutics vol 117no 3 pp 297ndash312 2008

[36] J Cocchiara C S Letizia J Lalko A Lapczynski and A MApi ldquoFragrance material review on cinnamaldehyderdquo Food andChemical Toxicology vol 43 no 6 pp 867ndash923 2005

[37] C H Fry F Daneshgari K Thor et al ldquoAnimal models andtheir use in understanding lower urinary tract dysfunctionrdquoNeurourology and Urodynamics vol 29 no 4 pp 603ndash6082010

[38] H-S Lee B-S Kim and M-K Kim ldquoSuppression effect ofCinnamomum cassia bark-derived component on nitric oxidesynthaserdquo Journal of Agricultural and Food Chemistry vol 50no 26 pp 7700ndash7703 2002

[39] S H Lee S Y Lee D J Son et al ldquoInhibitory effect of 21015840-hydroxycinnamaldehyde on nitric oxide production throughinhibition of NF-120581B activation in RAW2647 cellsrdquo BiochemicalPharmacology vol 69 no 5 pp 791ndash799 2005


[40] B H Kim Y G Lee J Lee J Y Lee and J Y Cho ldquoRegulatoryeffect of cinnamaldehyde on monocytemacrophage-mediatedinflammatory responsesrdquoMediators of Inflammation vol 2010Article ID 529359 9 pages 2010

[41] TThippeswamy J S McKay J P Quinn and R Morris ldquoNitricoxide a biological double-faced janusmdashis this good or badrdquoHistology and Histopathology vol 21 no 4-6 pp 445ndash4582006

[42] S Mocellin V Bronte and D Nitti ldquoNitric oxide a doubleedged sword in cancer biology searching for therapeuticopportunitiesrdquo Medicinal Research Reviews vol 27 no 3 pp317ndash352 2007

[43] A Yanaga H Goto T Nakagawa H Hikiami N Shiba-hara and Y Shimada ldquoCinnamaldehyde induces endothelium-dependent and -independent vasorelaxant action on isolated rataortardquo Biological and Pharmaceutical Bulletin vol 29 no 12 pp2415ndash2418 2006

[44] Y-L XueH-X Shi FMurad andK Bian ldquoVasodilatory effectsof cinnamaldehyde and itsmechanismof action in the rat aortardquoVascular Health and Risk Management vol 7 pp 273ndash280 2011

[45] G Lin TM Fandel AW Shindel et al ldquoModulation of smoothmuscle tonus in the lower urinary tract interplay of myosinlight-chain kinase (MLCK) and MLC phosphatase (MLCP)rdquoBJU International vol 108 no 2 pp E66ndashE70 2011

[46] VK Lin and JDMcConnell ldquoEffects of obstruction on bladdercontractile proteinsrdquo Progress in clinical and biological researchvol 386 pp 263ndash269 1994

[47] T-Y Liu C-Y Yuo C-H Kao et al ldquoHydrogen peroxidedecreases the survival rate of HeLa cells with stable knockdownof survivalmotor neuron proteinrdquoKaohsiung Journal ofMedicalSciences vol 27 no 3 pp 102ndash107 2011

[48] L Wan E Ottinger S Cho and G Dreyfuss ldquoInactivation ofthe SMN Complex by Oxidative Stressrdquo Molecular Cell vol 31no 2 pp 244ndash254 2008

[49] M J Kwon B Kim Y S Lee and T Y Kim ldquoRole of superoxidedismutase 3 in skin inflammationrdquo Journal of DermatologicalScience vol 67 no 2 pp 81ndash87 2012

[50] K Bagot and R Chess-Williams ldquoAlpha1AL-adrenoceptorsmediate contraction of the circular smooth muscle of the pigurethrardquo Autonomic and Autacoid Pharmacology vol 26 no 4pp 345ndash353 2006

[51] M Yono H E Foster Jr D Shin W Takahashi M Pouresmailand J Latifpour ldquoDoxazosin-induced up-regulation of 1205721A-adrenoceptor mRNA in the rat lower urinary tractrdquo CanadianJournal of Physiology and Pharmacology vol 82 no 10 pp 872ndash878 2004

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


determine if they ameliorate the physical signs of SUI or notIn an uncontrolled trial subjects were given a Chinese herbalformula 78 of them experienced decreased frequency ofincontinent episodes [6] suggesting the potential applicationof Chinese herbal medicine for SUI treatment [7ndash9]

Comprehending the urethral function and pharmacologymay lead to the development of promising new agents [10]which may be useful in the management of SUI in women[4] Because of concerns about 120572-adrenergic agonists bothpractitioners and patients are seeking alternatives to clinical120572-adrenergic stimulating drugs for managing SUI physicalsign In the present study several candidate herbal medicineswere screened and botanical agents for SUI were proposedfor development This study established an ex vivo organbath in vitro cell culture and in vivo animal SUI modelsto evaluate potential botanical agents against SUI and toexplore the possible mechanisms Among these botanicalherbs Ramulus Cinnamomi (the dry twig of Cinnamomumcassia Presl a plant in the family Lauraceae) and its majorconstituent cinnamaldehyde caused a high contractile forceof the urethra and a low contractile force of blood vesselsPlant-derived essential oils have long been used as flavoringagents in agricultural food and beverages and as naturalagents for food preservation Cinnamomum cassia bark oilknown as cinnamon oil is commonly used in the foodindustry because of its special aroma Cinnamaldehyde iscommonly used in agricultural food and beverages and withgreat commercial and possible medical value [11] Theseanalyses will give an insight of pharmacologic treatment ofSUI This information may offer clues to the pathogenesisof SUI and open additional avenues for potential therapystrategies

2 Materials and Methods

21 Organ Bath Experiment for Potential SUI Drug ScreeningOrgan bath experiment was used for functional evaluationof contractile urethra and blood vessel ex vivo for thecontractility study [12 13] female pig urethra and bloodvessel were prepared from the basal part of the bladderbody and renal artery respectively [13] Urethra and renalartery were mounted between platinum-plate electrodes andsecured by small clips in a double-jacketed organ bathcontaining 20mL Krebsrsquo solution aerated with 95 O

2and

5 CO2to obtain a pH of 74 at 37∘C The composition of

the Krebsrsquo solution was (mM) 133 NaCl 47 KCl 25 CaCl2

163 NaHCO3 135 NaH

2PO4 06 MgSO

4 and 78 dextrose

Isometric contraction was recorded with a computerizeddata acquisition program (Biobench National InstrumentsCorporation TX USA) at a rate of 50Hz and stored on ahard drive for later analyses At the end of the experimentthe length and weight of each muscle strip between thesuspension clips weremeasuredThe 120572-adrenergic agonist PEwas used as a positive control

22 Cell Culture Raw 2647 cells were purchased as cryop-reserved cultures from Bioresource Collection and ResearchCenter (Hsinchu Taiwan) 1 times 106 cells were seeded into 100

mm Petri dishes and incubated at 37∘C in 90 Dulbeccorsquosmodified Eaglersquos medium with 4mM L-glutamine adjustedto contain 15 gL sodium bicarbonate and 45 gL glucose+ 10 fetal bovine serum Primary human bladder smoothmuscle cells (HBdSMC) (ScienCell CA USA) were grownin the smooth muscle cell medium (SMCM Catalog number1101) By which time the cells had reached 90 confluencethe cells were plated at 96-well or 10 cm plates for MTT assayor Western blot [14 15] respectively

23MTTAssay for Cell Viability Cells were grown in 96-wellplates at a concentration of 3times 104 cellswell for 24 h and thenserum-freemediumwith different drugs was addedThe cellswere collected after 24 h later Mitochondrial dehydrogenaseactivity was measured as an index of cell viability using the 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazoliumbromide(MTT) assays [16] In brief MTT (05mgmL) was appliedto the cells for 4 h to allow the conversion of MTT into for-mazan crystals the cells were lysed with dimethyl sulfoxide(DMSO) and the absorbance was read at 570 and 650 nmwith a DIAS Microplate Reader (Molecular Devices CAUSA) The reduction in optical density caused by treatmentwas used as a measurement of cell viability normalized to thecells incubated in control medium which were considered100 viable

24 Nitric Oxide Assay Cells were grown in 96-well platesat a concentration of 3 times 104 cellswell for 24 h and thenserum-free medium with different drugs was added Culturesupernatants were collected after 24 h later Nitrite levelswere measured by addition of 01mL of the Griess reagent(15 sulfanilamide and 01 naphthylethylenediamine dihy-drochloride in 25 H

3PO4) to 01mL of culture supernatant

in 96-well plates leaving the plates in the dark for 10min andmeasuring the color intensity with an automated microtiterplate reader at 550 nm [17]

25 Experimental Animals and Study Design Twenty virginfemale C57BL6 strain mice aged approximately 6ndash8 weekswere randomized into 4 groups a noninstrumented controlgroup the other mice undergoing vaginal distension (VD)for 1 h with 8mm dilators (compatible with the diameterof mouse newborn head) were randomized to receive PE(25 120583gkg ip) [18] cinnamaldehyde (150mgkg ip) [19] orthe vehicle for 3 days Four days after VD mice underwentsuprapubic bladder tubing (SPT) placement Six days afterVD leak point pressure (LPP) and maximal urethral closurepressure (MUCP) measurements were assessed in thesemice under urethane (1 gkg ip) anesthesia [20 21] Bloodpressure (BP) and heart rate (HR) were measured using aBPMonitor for Rats and Mice Model MK-2000 (MuromachiKikai Tokyo Japan) according to the instructions of themanufacturer [22] Aftermeasuring LPPMUCP BP andHRthe animals were sacrificed and the urethras were removedfor Western blot analysis All experimental protocols wereapproved by the Institutional Animal Care and Use Commit-tee of China Medical University









3 Results





4min

1g

(a)


4min

1g

(b)

Cinnamaldehyde (M)

Con

trac

tion

()

0

20

40

60

80

100

UrethraArtery


(c)






Cel

l via

bilit

y (

of c

ontro

l)

0

20

40

60

80

100

120


lowast

lowast

(a)

Control LPS 3125 625 125 25

NO

pro

duct

ion

( o

f con

trol)

0

200

400

600

800


lowast

(b)


iNO

S ex

pres

sion

(fold

of c

ontro

l)

0

2

4

6

8

iNOS

GAPDH



lowast

(120583M)++++minus

minus minus

minus130kD

minus37kD

(c)


Prot

ein

expr

essio

n(fo

ld o

f con

trol)

0

1

2

3

4

5

Control 625 125 25

iNOS

GAPDH


SMN

SOD3

Myosin

AdR1a


lowast

lowastlowast

lowast

lowast

lowast

(120583M)minus

minus220 kD

minus130kD

minus66kD

minus52kD

minus35kD

minus37kD

(d)



0

10

20

30

40

50


lowast

+VD

LPP

(cm

H2O

)

(a)

0

10

20

30

40

50


lowast

+VD

MU

CP (c

m H

2O

)

(b)

0

20

40

60

80

100

120

140

160


+VD

BP (m

m H

g)

(c)

HR

(bea

tsm

in)

0

200

400

600

800


+VD

(d)







Prot

ein

expr

essio

n(fo

ld o

f con

trol)

0

1

2

3

4

5

Control VD

iNOS

GAPDH


SMN

SOD3

Myosin

AdR1a

VD+

minus220 kD

minus130kD

minus66kD

minus52kD

minus35kD

minus37kD

lowast

+PE+Cinnamaldehyde




4 Discussion

















1-adrenoceptors




5 Conclusion




Acknowledgments


References



























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























3 Results





4min

1g

(a)


4min

1g

(b)

Cinnamaldehyde (M)

Con

trac

tion

()

0

20

40

60

80

100

UrethraArtery


(c)






Cel

l via

bilit

y (

of c

ontro

l)

0

20

40

60

80

100

120


lowast

lowast

(a)

Control LPS 3125 625 125 25

NO

pro

duct

ion

( o

f con

trol)

0

200

400

600

800


lowast

(b)


iNO

S ex

pres

sion

(fold

of c

ontro

l)

0

2

4

6

8

iNOS

GAPDH



lowast

(120583M)++++minus

minus minus

minus130kD

minus37kD

(c)


Prot

ein

expr

essio

n(fo

ld o

f con

trol)

0

1

2

3

4

5

Control 625 125 25

iNOS

GAPDH


SMN

SOD3

Myosin

AdR1a


lowast

lowastlowast

lowast

lowast

lowast

(120583M)minus

minus220 kD

minus130kD

minus66kD

minus52kD

minus35kD

minus37kD

(d)



0

10

20

30

40

50


lowast

+VD

LPP

(cm

H2O

)

(a)

0

10

20

30

40

50


lowast

+VD

MU

CP (c

m H

2O

)

(b)

0

20

40

60

80

100

120

140

160


+VD

BP (m

m H

g)

(c)

HR

(bea

tsm

in)

0

200

400

600

800


+VD

(d)







Prot

ein

expr

essio

n(fo

ld o

f con

trol)

0

1

2

3

4

5

Control VD

iNOS

GAPDH


SMN

SOD3

Myosin

AdR1a

VD+

minus220 kD

minus130kD

minus66kD

minus52kD

minus35kD

minus37kD

lowast

+PE+Cinnamaldehyde




4 Discussion

















1-adrenoceptors




5 Conclusion




Acknowledgments


References



























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















4min

1g

(a)


4min

1g

(b)

Cinnamaldehyde (M)

Con

trac

tion

()

0

20

40

60

80

100

UrethraArtery


(c)






Cel

l via

bilit

y (

of c

ontro

l)

0

20

40

60

80

100

120


lowast

lowast

(a)

Control LPS 3125 625 125 25

NO

pro

duct

ion

( o

f con

trol)

0

200

400

600

800


lowast

(b)


iNO

S ex

pres

sion

(fold

of c

ontro

l)

0

2

4

6

8

iNOS

GAPDH



lowast

(120583M)++++minus

minus minus

minus130kD

minus37kD

(c)


Prot

ein

expr

essio

n(fo

ld o

f con

trol)

0

1

2

3

4

5

Control 625 125 25

iNOS

GAPDH


SMN

SOD3

Myosin

AdR1a


lowast

lowastlowast

lowast

lowast

lowast

(120583M)minus

minus220 kD

minus130kD

minus66kD

minus52kD

minus35kD

minus37kD

(d)



0

10

20

30

40

50


lowast

+VD

LPP

(cm

H2O

)

(a)

0

10

20

30

40

50


lowast

+VD

MU

CP (c

m H

2O

)

(b)

0

20

40

60

80

100

120

140

160


+VD

BP (m

m H

g)

(c)

HR

(bea

tsm

in)

0

200

400

600

800


+VD

(d)







Prot

ein

expr

essio

n(fo

ld o

f con

trol)

0

1

2

3

4

5

Control VD

iNOS

GAPDH


SMN

SOD3

Myosin

AdR1a

VD+

minus220 kD

minus130kD

minus66kD

minus52kD

minus35kD

minus37kD

lowast

+PE+Cinnamaldehyde




4 Discussion

















1-adrenoceptors




5 Conclusion




Acknowledgments


References



























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Cel

l via

bilit

y (

of c

ontro

l)

0

20

40

60

80

100

120


lowast

lowast

(a)

Control LPS 3125 625 125 25

NO

pro

duct

ion

( o

f con

trol)

0

200

400

600

800


lowast

(b)


iNO

S ex

pres

sion

(fold

of c

ontro

l)

0

2

4

6

8

iNOS

GAPDH



lowast

(120583M)++++minus

minus minus

minus130kD

minus37kD

(c)


Prot

ein

expr

essio

n(fo

ld o

f con

trol)

0

1

2

3

4

5

Control 625 125 25

iNOS

GAPDH


SMN

SOD3

Myosin

AdR1a


lowast

lowastlowast

lowast

lowast

lowast

(120583M)minus

minus220 kD

minus130kD

minus66kD

minus52kD

minus35kD

minus37kD

(d)



0

10

20

30

40

50


lowast

+VD

LPP

(cm

H2O

)

(a)

0

10

20

30

40

50


lowast

+VD

MU

CP (c

m H

2O

)

(b)

0

20

40

60

80

100

120

140

160


+VD

BP (m

m H

g)

(c)

HR

(bea

tsm

in)

0

200

400

600

800


+VD

(d)







Prot

ein

expr

essio

n(fo

ld o

f con

trol)

0

1

2

3

4

5

Control VD

iNOS

GAPDH


SMN

SOD3

Myosin

AdR1a

VD+

minus220 kD

minus130kD

minus66kD

minus52kD

minus35kD

minus37kD

lowast

+PE+Cinnamaldehyde




4 Discussion

















1-adrenoceptors




5 Conclusion




Acknowledgments


References



























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

10

20

30

40

50


lowast

+VD

LPP

(cm

H2O

)

(a)

0

10

20

30

40

50


lowast

+VD

MU

CP (c

m H

2O

)

(b)

0

20

40

60

80

100

120

140

160


+VD

BP (m

m H

g)

(c)

HR

(bea

tsm

in)

0

200

400

600

800


+VD

(d)







Prot

ein

expr

essio

n(fo

ld o

f con

trol)

0

1

2

3

4

5

Control VD

iNOS

GAPDH


SMN

SOD3

Myosin

AdR1a

VD+

minus220 kD

minus130kD

minus66kD

minus52kD

minus35kD

minus37kD

lowast

+PE+Cinnamaldehyde




4 Discussion

















1-adrenoceptors




5 Conclusion




Acknowledgments


References



























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Prot

ein

expr

essio

n(fo

ld o

f con

trol)

0

1

2

3

4

5

Control VD

iNOS

GAPDH


SMN

SOD3

Myosin

AdR1a

VD+

minus220 kD

minus130kD

minus66kD

minus52kD

minus35kD

minus37kD

lowast

+PE+Cinnamaldehyde




4 Discussion

















1-adrenoceptors




5 Conclusion




Acknowledgments


References



























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























1-adrenoceptors




5 Conclusion




Acknowledgments


References



























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Treatment of Stress Urinary Incontinence by ...Research Article Treatment of Stress...

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Transcript of Research Article Treatment of Stress Urinary Incontinence by ...Research Article Treatment of Stress...