research article natural antioxidant-isoliquiritigenin...

Hindawi Publishing CorporationMediators of InflammationVolume 2013 Article ID 390890 10 pageshttpdxdoiorg1011552013390890

Research ArticleNatural Antioxidant-Isoliquiritigenin AmelioratesContractile Dysfunction of Hypoxic Cardiomyocytes viaAMPK Signaling Pathway

Xiaoyu Zhang12 Ping Zhu3 Xiuying Zhang4 Yina Ma2 Wenguang Li1 Ji-Mei Chen3

Hui-Ming Guo3 Richard Bucala5 Jian Zhuang3 and Ji Li2

1 Institute of Physiology School of Basic Medicine Sciences Lanzhou University Lanzhou 730000 China2Department of Pharmacology and Toxicology School of Medicine and Biomedical SciencesUniversity at Buffalo-SUNY University of New York Buffalo NY 14214 USA

3Department of Cardiovascular Surgery Guangdong Cardiovascular Institute Guangdong General HospitalGuangdong Academy of Medical Sciences Guangzhou 510080 China

4Department of Emergency Gansu Provincial Hospital Lanzhou 730000 China5 Department of Internal Medicine Yale University School of Medicine New Haven CT 06520 USA

Correspondence should be addressed to Jian Zhuang zhuangjianzggdyahoocn and Ji Li jli23buffaloedu

Received 16 July 2013 Accepted 14 August 2013

Academic Editor Fatih Arslan

Copyright copy 2013 Xiaoyu Zhang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Isoliquiritigenin (ISL) a simple chalcone-type flavonoid is derived from licorice compounds and is mainly present in foodsbeverages and tobacco Reactive oxygen species (ROS) is a critical factor involved in modulating cardiac stress response signalingduring ischemia and reperfusion We hypothesize that ISL as a natural antioxidant may protect heart against ischemic injury viamodulating cellular redox status and regulating cardioprotective signaling pathwaysThe fluorescent probe H

2DCFDAwas used to

measure the level of intracellular ROSThe glucose uptake was determined by 2-deoxy-D-glucose-3H accumulationThe IonOptixSystem measured the contractile function of isolated cardiomyocytes The results demonstrated that ISL treatment markedlyameliorated cardiomyocytes contractile dysfunction caused by hypoxia ISL significantly stimulated cardioprotective signalingAMP-activated protein kinase (AMPK) and extracellular signal-regulated kinase (ERK) signaling pathways The ROS fluorescentprobe H

2DCFDA determination indicated that ISL significantly reduced cardiac ROS level during hypoxiareoxygenation

Moreover ISL reduced the mitochondrial potential (Δ120595) of isolated mouse cardiomyocytes Taken together ISL as a naturalantioxidant demonstrated the cardioprotection against ischemic injury that may attribute to the activation of AMPK and ERKsignaling pathways and balance of cellular redox status

1 Introduction

Myocardial infarction is one of the major causes of death inthe world Although restoration of blood flow is the only wayto save the myocardium from necrosis reperfusion-inducedinjury is at the background of a highmortality rate [1] Exten-sive studies showed that myocardial ischemia-reperfusion(IR) injury is associatedwith increased generation of reactiveoxygen species (ROS) The ROS may result in depressedcontractile function arrhythmias depletion of endogenousantioxidant network and membrane permeability changes[2] There is evidence that AMP-activated protein kinase

(AMPK) signaling pathway is involved in cardiac redoxregulation [3ndash6] Our laboratory and others have providedclear evidence that AMPK plays a critical role in protectionagainst ischemiareperfusion injury in the heart [7ndash14]

Isoliquiritigenin (ISL) a simple chalcone-type flavonoidis derived from licorice compounds and present in foodsbeverages and tobacco [15] It has been reported to possessa wide range of biological and pharmacological activitiesincluding antioxidative activity [16] antiplatelet aggregationeffects [17 18] antitumor activities [19] and estrogenicproperties [20] It has been reported that pretreatment withISL markedly decreased the severity of reperfusion-induced

2 Mediators of Inflammation

arrhythmias and myocardial infarct size and reduced theactivities of lactate dehydrogenase (LDH) and creatininephosphokinase (CPK) [18] Increased JAK2STAT3 phos-phorylation in the heart by ISL appears to the mecha-nism by which ISL protects the heart against ischemia andreperfusion injury [18] To further characterize the cardio-protective effects of ISL on cardiomyocytes under hypoxicstress we isolated mouse cardiomyocytes to investigate theeffects of ISL on cardiomyocytes contractile functions duringhypoxiareoxygenation and the signaling mechanism thatmediated ISL action on cardiomyocytes

2 Materials and Methods

21 Drugs and Chemicals Isoliquiritigenin (ISL) was pur-chased from Sigma (St Louis MO) (Scheme 1) ISL was dis-solved in dimethyl sulfoxide (DMSO) to produce a stock solu-tion of 10mmolL and theDMSOfinal concentrationwas lessthan 001 (v v) Other chemicals were of analytical purity

22 Animals andCell Line 8ndash12-week-oldmale FVBNJmicewere purchased from the Jackson Laboratory (Bar HarborME USA) All animals were kept in the institutional animalfacility at the State University of New York (SUNY) at Buffaloand were fed ad libitum All animal procedures used in thisstudy were approved by the Institutional Animal Care andUse Committees at the State University of New York (SUNY)at Buffalo

23 Isolation of Mouse Cardiomyocytes Cardiomyocyteswere enzymatically isolated as previously described [21]Briefly adult mouse hearts were removed and perfused withoxygenated (5 CO

295 O

2) Krebs-Henseleit bicarbonate

(KHB) buffer containing (in mM) 118 NaCl 47 KCl 125CaCl2 12 MgSO

4 12 KH

2PO4 25 NaHCO

3 10 HEPES

and 111 Glucose All the chemicals were purchased fromSigma (St Louis MO USA) Hearts were then perfusedwith a Ca2+-free KHB containing Liberase Blendzyme 4(Hoffmann-La Roche Inc Indianapolis IN USA) for 15ndash20min After perfusion left ventricles were removed andminced to disperse cardiomyocytes in Ca2+-free KHB bufferExtracellular Ca2+ was added incrementally back to 125mMOnly rod-shaped myocytes with clear edges were selectedfor pharmacological test and cell contractility studies Thecardiomyocyteswere treatedwith ISL (50 100120583M) forAMPKsignaling ROS levelmitochondrialmembrane potential andglucose uptake measurements

24 Hypoxia Treatment Isolated mouse cardiomyocyteswere subjected to two groups (normal and hypoxic groups)Hypoxic groups were kept at 37∘C in a humidified sealedchamber under a humidified atmosphere of 5 CO

2and

95 N2for 20min Normal groups were placed into a water-

jacketed incubator at 37∘C during the same period

25 Cardiomyocytes ShorteningRelengthening MeasurementThe mechanical properties of ventricular myocytes were

OHHO

O

OH

Isoliquiritigenin

Scheme 1

assessed using a SoftEdge MyoCam system (IonOptix Cor-poration Milton MA USA) [22] In brief left ventricularmyocytes were placed in a chamber mounted on the stageof an inverted microscope (Olympus IX-70 Center ValleyPA USA) and incubated at 25∘C with a buffer containing(in mM) 131 NaCl 4 KCl 1 CaCl

2 1 MgCl

2 10 Glucose

and 10 HEPES at pH 74 The cells were stimulated withsuprathreshold voltage at a frequency of 05Hz 3msecduration using a pair of platinum wires placed on oppositesides of the chamber and connected to an electrical stimu-lator (FHC Inc Brunswick NE USA) The myocytes beingstudied were displayed on a computer monitor using anIonOptix MyoCam camera An IonOptix SoftEdge softwarewas used to capture changes in cell length during shorteningand relengthening Cell shortening and relengthening wereassessed using the following indices peak shortening (PS)the amplitude myocytes shortened on electrical stimulationindicative of peak ventricular contractility time-to-PS (TPS)the duration of myocyte shortening an indicative of systolicduration time-to-90 relengthening (TR90) the duration toreach 90 relengthening an indicative of diastolic duration(90 rather 100 relengthening was used to avoid noisysignal at baseline concentration) and maximal velocitiesof shorteningrelengthening maximal slope (derivative) ofshortening and relengthening phases indicative of maximalvelocities of ventricular pressure increasedecrease In thecase of altering stimulus frequency the steady-state contrac-tion of myocytes was achieved (usually after the first 5-6beats) before PS amplitude was recorded

26 Intracellular Ca2+ Transient Measurements Isolated car-diomyocytes were loaded with fura-2AM (05 120583M) for15min and fluorescence measurements were recorded with adual-excitation fluorescence photomultiplier system (IonOp-tix) Myocytes were placed on an Olympus IX-70 invertedmicroscope and imaged through a Fluor (St Louis MOUSA) times40 oil objective Cells were exposed to light emittedby a 75W lamp and passed through either a 360 or a 380 nmfilter while being stimulated to contract at 05Hz Fluores-cence emissions were detected between 480 and 520 nm bya photomultiplier tube after first illuminating the cells at360 nm for 05 sec and then at 380 nm for the duration ofthe recording protocol (333Hz sampling rate) The 360 nmexcitation scan was repeated at the end of the protocol andqualitative changes in intracellular Ca2+ concentration were

Mediators of Inflammation 3

inferred from the ratio of fura-2 fluorescence intensity attwo wavelengths (360380) Fluorescence decay time wasmeasured as an indication of the intracellular Ca2+ clearingrate Both single and biexponential curve fit equations wereapplied to calculate the intracellular Ca2+ decay constant [21]

27 Determination of Reactive Oxygen Species (ROS) ROSwere detected as previously described [23 24] Briefly 5-(6)-chloromethyl-2101584071015840-dichlorodihydrofluorescein diacetate(CM-H

2DCFDA Molecular Probes Eugene OR USA) is a

cell-permeant indicator for ROS that is nonfluorescent untilremoval of the acetate groups by intracellular esterases andoxidation occurs within the cells It is intracellularly deesteri-fied and turns into highly fluorescent DCF upon oxidationIsolated cardiomyocytes were suspended in HEPES-salinebuffer and preincubation with 10 120583M H

2DCFDA at 37∘C

for 30min in the darkness After cells were washed twicefluorescence intensity was read at excitation wavelength of485 nm and emission wavelength of 530 nm in a fluorescenceplate reader (Microplate fluorometer Spectra GEMINIXSMolecular Device USA) The wells containing ISL but notH2DCFDA were used as blanks The production of ROS is

expressed as fluorescence intensity in relative to untreatedcontrol cells

28 Assessment of Mitochondrial Membrane Potential (Δ120595)The Δ120595 was measured using 5 51015840661015840-Tetrachloro-1 11015840331015840-tetraethylbenzimidazolocarbocyanine iodide (JC-1 SigmaSt Louis MO USA) Briefly JC-1 is a positively chargedfluorescent compound which is taken up by mitochon-dria proportionally to the inner mitochondrial membranepotential [25] When a critical concentration is exceededJC-1 monomer forms J-aggregates and becomes fluorescentred altering the fluorescence properties of the compoundThus the ratio of red (J-aggregate) green (monomeric JC-1)emission is directly proportional to the mitochondrial mem-brane potential Isolated cardiomyocytes were suspended inHEPES-saline buffer and preincubation with 10 120583M JC-1 for10min at 37∘C After cells were washed twice fluorescence ofeach sample was read at excitation wavelength of 490 nm andemission wavelength of 530 nm and 590 nm in a fluorescenceplate reader (Microplate fluorometer Spectra GEMINIXSMolecular Device USA) Results in fluorescence intensitywere expressed as 590 to 530 nm emission ratio

29 Immunoblotting Analysis and Antibodies Immunoblot-ting was performed as previously described [14 24 26] Iso-lated cardiomyocytes proteins were resolved by SDS-PAGEand transferred onto polyvinylidene difluoride membranes(Bio-Rad Hercules CA USA) For reprobing membraneswere stripped with 50mM Tris-HCl 2 SDS and 01M 120573-mercaptoethanol (pH 68)Membranes were blockedwith 5nonfat milk in TBS (pH 74) containing 01 Tween-20 for 1 hand subsequently incubated with primary antibodies (1 1000dilution) at 4∘C for overnight Immunoreactive bandswere detected using anti-rabbit horseradish peroxidase-conjugated secondary antibodies and visualized using chemi-luminescent substrate (ECL) Rabbit polyclonal antibodies

against phospho-AMPK (Thr172) total AMPK120572 phospho-ACC phospho-Akt phospho-ERK (Thr202Tyr204) phospho-p38 MAPK (Thr180Tyr182) phospho-STAT3 and GAPDHwere purchased from Cell Signaling (Danvers MA USA)The densities of immunoblot bands were analyzed using ascanning densitometer (model GS-800 Bio-Rad) coupledwith Bio-Rad personal computer analysis software [27ndash29]

210 Glucose Uptake 2-Deoxy-d-[1-3H] glucose accumula-tion in H9c2 cells was performed as previously described[30 31] H9c2 cells grown in 6-well plates were washed twicewith serum-freeDMEMand incubatedwith 2mL of the samemedium at 37∘C for 2 h The cells were washed 3 times withKrebs-Ringer-HEPES (KRH) buffer and incubated with 2mLKRH buffer at 37∘C for 30min Insulin (10 nM Sigma StLouis MO) andor ISL (50 100120583M) were then added toH9c2 Glucose uptake was initiated by the addition of 01mLKRH buffer and 2-deoxy-d-[1-3H] glucose (021 CimL GEHealthcare Piscataway NJ USA) and 5mM glucose as finalconcentrations Glucose uptake was terminated by washingthe cells three times with cold PBS The cells were lysedovernight with 1mL 05M NaOH and 01 SDS (wv) Theradioactivity retained by the cell lysates was determined by ascintillation counter (Beckmann LC 6000IC) and normalizedto protein amountmeasuredwith aMicro BCAProteinAssayKit (Pierce Chemical Rockford IL USA)

211 Statistical Analysis For each experimental series dataare presented as mean plusmn SE Statistical comparisons weremade using analysis of variance (ANOVA) Differences with119875 lt 005 were considered statistically significant

3 Results

31 ISL Ameliorated Cardiomyocyte Contractile DysfunctionInduced by Hypoxia To determine whether ISL protectscardiomyocytes against hypoxic injury we investigated thecardiomyocyte contractility when they were exposed tohypoxia atmosphereThemechanical properties of cardiomy-ocyte contractility were obtained under extracellular Ca2+of 10mM and a stimulus frequency of 05Hz As shownin Figure 1 ISL (100 120583M) treatment did not affect restingcardiomyocyte contractile function under the normal orhypoxic condition However during hypoxic conditionsthe cardiomyocytes displayed severe impaired peak short-ening (PS) (Figure 1(b)) and reduced maximal velocity ofshorteningrelengthening (+119889119871119889119905 minus119889119871119889119905) (Figures 1(c)and 1(d)) while ISL treatment significantly ameliorates thecontractile dysfunction of cardiomyocytes as reflected byboth peak shortening and maximal velocity of shorten-ingrelengthening (Figures 1(c) and 1(d)) Moreover hypoxiacaused the prolonged time-to-peak shortening (TPS) andtime-to-90 relengthening (TR90) of cardiomyocytes (Fig-ures 1(e) and 1(f)) however ISL (100 120583M)markedly inhibitedthe hypoxia-induced prolonged TPS and TR90 of cardiomy-ocytes (Figures 1(e) and 1(f)) These results suggest that ISLprotects cardiomyocytes from hypoxia-induced contractiledysfunction


100

50

Normoxia Hypoxia

Resti

ng ce

ll le

ngth(120583

m)

0

(a)

Normoxia Hypoxia

Peak

shor

teni

ng (

cell

leng

th)

0

2

4

6

lowast

dagger

(b)

200

100

0

Normoxia Hypoxia

lowast

dagger

+dL

dt(120583

ms)

(c)

Normoxia Hypoxia

lowast

dagger

minus50

minus150

minus250

minusdL

dt(120583

ms)

(d)

02

01

00

Normoxia Hypoxia

lowast

dagger

TPS

(s)

VehicleISL 100120583M

(e)

03

02

01

00

Normoxia Hypoxia

lowast

dagger

TR90

(s)


(f)

Figure 1 Contractile properties of cardiomyocytes from vehicle and ISL treatment after being exposed to hypoxia (a) Resting cell length(b) peak shortening (PS normalized to cell length) (c) maximal velocity of shortening (+119889119871119889119905) (d) relengthening (minus119889119871119889119905) (e) time-to-peak shortening (TPS) (f) time-to-90 relengthening (TR90) Values are means plusmn SE 119899 = 50ndash60 cells per group lowast119875 lt 005 versus normoxiavehicle dagger119875 lt 005 versus hypoxia vehicle

32 The Intracellular Ca2+ Properties of Cardiomyocytes Toexplore the potential mechanisms involved in the protectionof ISL against hypoxic cardiomyocyte contractile defectintracellular Ca2+ homeostasis was evaluated using the flu-orescence dye fura-2AM [32] The results revealed thathypoxia caused an elevation of the resting intracellular Ca2+levels in isolated cardiomyocytes (Figure 2(a)) and reducedintracellular Ca2+ clearance with prolonging the fluorescencedecay time (both single and biexponential decays Figures2(c) and 2(d)) as compared with cardiomyocytes undernormoxia conditions ISL (100120583M) did not elicit any overt

effect on resting intracellular Ca2+ and fluorescence decaytime in nonhypoxic conditions (Figures 2(a)ndash2(d)) butmarkedly recovered the elevated resting intracellular Ca2+levels and reduced intracellular Ca2+ clearance in isolatedcardiomyocytes under hypoxic conditions (Figure 2) How-ever hypoxia did not change electrically stimulated rise inintracellular Ca2+ levels (Figure 2(b))

33 ISL Stimulated Cardioprotective Signaling Pathways Ourgroup and others provided evidence that AMP-activated pro-tein kinase (AMPK) is a critical signaling in cardioprotection


000

025

050

075

100

Intr

acel

lula

rCa2+

(360

380

ratio

)

lowast

dagger

Normoxia Hypoxia

(a)

000

025

050

Normoxia Hypoxia

Rise

in in

trac

ellu

larC

a2+

(Δ360380

)

(b)

00

01

02

03

Intr

acel

lula

rCa2+

deca

y ra

te (s

)

Normoxia Hypoxia

lowast

dagger


(c)

00

01

02

Normoxia Hypoxia

lowast

dagger

Intr

acel

lula

rCa2+

deca

y ra

te (s

)


(d)

Figure 2 Intracellular Ca2+ properties of cardiomyocytes (a)The intracellular Ca2+ levels (b) the rise in intracellular Ca2+ levels in responseto electrical stimulus (c) the first exponential decay constant of intracellular Ca2+ (d) the biexponential decay constant of intracellular Ca2+

in response to hypoxia (20min) Values are means plusmn SE 119899 = 60ndash90 cells per group lowast119875 lt 005 versus normoxia vehicle dagger119875 lt 005 versushypoxia vehicle

against ischemic injury [7 11ndash13] To define the mechanisminvolved in the cardioprotective effect of ISL AMPK signal-ing pathways were detected in isolated cardiomyocytes inresponse to ISL treatmentThe results showed that ISL signif-icantly triggeredAMPKThr172 phosphorylation as comparedwith vehicle group (Figure 3(a)) In parallel with AMPKactivation the downstream targets of AMPK the phospho-rylation of acetyl CoA carboxylase (ACC) was induced byISL treatment (Figure 3(b)) Intriguingly ISL treatment alsoinduced extracellular signal-regulated kinase (ERK) signalingpathway in the cardiomyocytes (Figure 3(c)) These datasuggest that ISL treatment can induce phosphorylation of 120572catalytic subunit at Thr172 of AMPK and trigger a survivalsignaling ERK activation

34 ISL Decreased the Intracellular ROS Level in IsolatedCardiomyocytes Upon reperfusion of the myocardium afterischemiahypoxia there is a rapid increase in intracellularcalcium that will induce the opening of the mitochondrialpermeability transition pore (mPTP) [33] Uncoupling of theelectron transport chain within the mitochondria leads to

the release of destructive reactive oxygen species (ROS) [34]this increase in ROS is a significant contributor to the celldeath seen at the onset of reperfusion [33] The fluorescentprobeH

2DCFDAwas used tomeasure the effect of ISL on the

level of intracellular ROS in isolated cardiomyocytes underhypoxiareoxygenation conditions As shown in Figure 4(a)ROS level of cardiomyocytes under hypoxiareoxygenationwas much higher than that of vehicle normoxia group(119875 lt 001 versus vehicle normoxia) ISL treatment sig-nificantly decreased the intracellular ROS levels of isolatedcardiomyocytes during hypoxiareoxygenation (119875 lt 005versus vehicle hypoxia) It is suggested that ISL demonstratedcardioprotection against the hypoxia-induced contractiledysfunction through modulating the cellular redox status ofcardiomyocytes

35 ISL Reduced the Mitochondrial Membrane Potential ofCardiomyocytes There is evidence that AMPK signalingpathway is involved in regulation of mitochondrial mem-brane potential (Δ120595) [35] To understand the mechanismsby which ISL activates cardiac AMPK signaling pathway


4

2

0

p-A

MPK

rel

ativ

e uni

ts

lowast

Vehicle ISL (100 120583M)


p-AMPK (Thr172)

AMPK120572

(a)

4

2

0p-

ACC

relat

ive u

nits

lowast


GAPDH


p-ACC (Ser79)

(b)

4

2

0

6

p-ER

K re

lativ

e uni

ts

lowast


GAPDH


p-ERK (Thr202Tyr204)

(c)

Figure 3 ISL treatment stimulated cardiacAMP-activated protein kinase (AMPK) andERK signaling pathways Representative immunoblotsof isolated mouse cardiomyocytes showed phosphorylation of (a) AMPK atThr172 (p-AMPK) (b) ACC (Ser79) and (c) ERK PhosphorylatedAMPK was quantified relative to total AMPK120572 Phosphorylated ACC and ERK were quantified relative to GAPDH Values are expressed asmeans plusmn SE (119899 = 3ndash6) lowast119875 lt 005 versus vehicle

the mitochondrial membrane potential (Δ120595) of cardiomy-ocytes was assessed using JC-1 a lipophilic fluorophore thatforms J-aggregates in proportion to its intramitochondrialconcentration Isolated cardiomyocytes were preincubatedfor 20min with 10120583M JC-1 rinsed thoroughly and treatedwith ISL accordingly Figure 4(b) represented the ratio ofredgreen fluorescence corresponding to JC-1 in J-aggregateversus monomeric form The results demonstrated that ISLtreatment significantly reduced JC-1 dye accumulation anddecreased J-aggregate formation in cardiomyocyte mito-chondria in an independent manner which indicated thatISL caused mitochondrial membrane depolarizationThe Δ120595reductionmay contribute to the activation of AMPK inducedby ISL

36 ISL Stimulated Glucose Uptake in the CardiomyocytesTo explore whether ISL-activated cardiac AMPK signalingmodulates glucose metabolism in the cardiomyocytes theeffect of ISL on glucose uptake in cardiomyocytes was inves-tigated using the 2-deoxy-D-1-3H-glucose uptake assay [31]As shown in Figure 4(c) ISL significantly stimulated glucoseuptake in the isolated cardiomyocytes (119875 lt 001 versusvehicle) Interestingly ISL treatment can enhance insulin-induced glucose uptake of cardiomyocytes (Figure 4(c))

4 Discussion

ROS have been implicated in the pathogenesis of stress-induced injury including myocardial ischemiareperfusion


H2-D

CFD

A fl

uore

scen

ce0

4

8

12

Normoxia Hypoxia

lowast

dagger


(a)

0

2

4

6

8

lowast

JC-1

ratio

(590

530

nm)

Vehicle ISL 100120583M


(b)

0

5

10

15

Glu

cose

upt

ake (

cpm

mg

prot

ein)

lowast

lowastlowast

dagger

Vehicle ISL 100120583M+

minus

+

+

minus

+ Insulin 10nM


(c)

Figure 4 (a) ISL reduced the intracellular ROS levels in isolated mouse cardiomyocytes during hypoxiareoxygenation Intracellular ROSlevels were measured by the fluorescent probe H

2DCFDA after treatment with ISL (100120583M) or DMSO (vehicle) ROS production was

expressed as fluorescence intensity relative to untreated control cells Data are presented as means plusmn SE (119899 = 4ndash6) lowast119875 lt 005 versus normoxiavehicle dagger119875 lt 005 versus hypoxia vehicle (b) ISL reducedmitochondrialmembrane potential (Δ120595) in isolated cardiomyocytesMitochondrialmembrane potential (Δ120595) was measured by JC-1 fluorescence assay The result was presented as the ratio of redgreen fluorescence measuredat 590 nm and 530 nm respectively Values are means plusmn SE (119899 = 6ndash10) lowast119875 lt 001 versus vehicle (c) ISL treatment augmented glucose uptakeof cardiomyocytesThe cardiomyocytes were preincubated for 30min with or without ISL (100120583M) andor insulin (10 nM) before addition of2-deoxy-[1-3H]glucose for additional 30min to measure glucose uptake Values are means plusmn SE for 5 experiments lowast119875 lt 005 versus vehicledagger

119875 lt 005 versus insulin alone

injury ROS generation intracellularly contributes to contrac-tile dysfunction and cell death during simulated ischemiareperfusion in a perfused cardiomyocyte model [2 36]Extensive studies showed that some herbal extracts oractive components of herbs exhibited antioxidant effects [18]Although these studies have implicated antioxidative and car-dioprotective effects of ISL whether these actions of ISL cancorrelate with its cardiomyocyte contractile function is notwell understood In the present study ISL as a natural antiox-idant did not affect cardiomyocyte contractile function underthe normal condition However cardiomyocytes displayedsevere impaired contractile functions while ISL markedlyameliorated the hypoxia-caused contractile dysfunction ofcardiomyocytes Additionally ISL did not elicit any overteffect on resting intracellular Ca2+ and fluorescence decaytime in nonhypoxic conditions but it markedly elevated theelectrically stimulated intracellular Ca2+ levels and recoveredthe elevated resting intracellular Ca2+ levels and reduced

intracellular Ca2+ clearance in hypoxia-isolated cardiomy-ocytesThe explanation ofmechanical defects observed in ourstudy may be the impaired intracellular Ca2+ handling Thereduction of intracellular Ca2+ clearance is likely responsiblefor prolonged relaxation duration (TR90) and reduced PSin hypoxic cardiomyocytes Meanwhile the ROS level ofhypoxic cardiomyocytes is much higher than that of nor-mal cardiomyocytes which may contribute to the contrac-tile dysfunction of cardiomyocytes When cardiomyocyteswere exposed to hypoxia atmosphere ISL treatment signifi-cantly decreased the intracellular ROS level and amelioratedthe contractile dysfunction of cardiomyocytes GenerallyHypoxia-induced ROS production may cause the membranelipid peroxidation and protein denaturation that disturbedCa2+ transportation and mitochondrial membrane potentialTherefore the antioxidative activity of ISL could reduce theintracellular ROS levels and ameliorate the Ca2+ transporta-tion and mitochondrial membrane potential all of which


contribute to the improvement of contractile function ofcardiomyocytes under hypoxic stress conditions

Currently AMP-activated protein kinase (AMPK) path-way was revealed to be one of the signaling pathways thatprotect against cardiac ischemia [7 37ndash39] AMPK is a stress-sensitive kinase that can be activated by ATP depletion suchas hypoxia [5] ischemia [13] and exercise [40] ActivatedAMPK can phosphorylate Acetyl-CoA carboxylase (ACC) toinhibit its activity involved in fatty acid synthesis [41] Otherdownstream effects of AMPK pathways include glucoseuptake [42 43] glycolysis [44] and fatty acid oxidation [45]which favor the ATP production that supply enough energyfor cell living under the stress conditions AMPK promotesglucose transport maintains ATP stores and prevents injuryand apoptosis during ischemia [39] Our results showed thatISL stimulated AMPK Thr172 phosphorylation and activa-tion in the isolated cardiomyocytes ISL also significantlystimulated the AMPK downstream effector glucose uptakein the cardiomyocytes These data strongly suggest that ISLmay directly trigger cardiac AMPK signaling pathway thatmodulates glucose homeostasis to protect hypoxia-inducedcardiomyocytes injury

AMPK has several direct molecular targets on the heartbut also may interact with other stress-signaling pathways

On the other hand ERK activation is antiapoptotic inmost tissues [46] Our results demonstrated that ISL as anatural antioxidant triggers ERK signaling in the isolatedcardiomyocytes even though the molecular mechanism bywhich ISL activates the cardiac ERK pathway needs to becharacterized in future studies

In conclusion ISL demonstrated cardioprotection againstcontractile dysfunction caused by hypoxiareoxygenationThe mechanisms of cardioprotection of ISL are associatedwith the activation of cardioprotective signaling pathwaysand modulation of intracellular redox status in the car-diomyocytes Therefore ISL is a potential small molecule fortreatment of ischemic heart diseases in the future In terms ofour previous studies [10 24 27 38 47] regarding the clinicalsetting it may be beneficial to phosphorylate AMPK duringischemic injury in patients suffering from acute myocardialinfarction For this reason ISL could be administrated justprior to percutaneous coronary intervention (PCI) to reducethe ischemiareperfusion injury

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contributions

Xiaoyu Zhang Ping Zhu and Xiuying Zhang are equallycontributed to this work

Acknowledgments

This study was supported by China Scholarship Counciland Gansu Province Natural Science Foundation of China(no 0710RJZA037 no 1208RJZA231) to Xiaoyu Zhang

American Heart Association SDG 0835169N and 12GRNT11620029 to Ji Li American Diabetes Association BasicSciences Grant 1-11-BS-92 to Ji Li the Major Interna-tional (Regional) Joint Research Project 2008DFA31140 and2010DFA32660 to Ping Zhu and Guangdong Natural ScienceFund 10251008002000002 and S2011010005836 to Ping Zhuand Chinese Science and Technology Support Program2011BAI11B22 to Jian Zhuang

References

[1] M Paroczai E Roth G Matos G Temes J Lantos andE Karpati ldquoEffects of bisaramil on coronary-occlusion-reperfusion injury and free-radical-induced reactionsrdquo Phar-macological Research vol 33 no 6 pp 327ndash336 1996

[2] W-T Lin S-C Yang K-T Chen C-C Huang and N-Y LeeldquoProtective effects of L-arginine on pulmonary oxidative stressand antioxidant defenses during exhaustive exercise in ratsrdquoActa Pharmacologica Sinica vol 26 no 8 pp 992ndash999 2005

[3] T Toyoda T Hayashi LMiyamoto et al ldquoPossible involvementof the1205721 isoformof 51015840AMP-activated protein kinase in oxidativestress-stimulated glucose transport in skeletal musclerdquo Ameri-can Journal of Physiology Endocrinology and Metabolism vol287 no 1 pp E166ndashE173 2004

[4] M-H Zou S S Kirkpatrick B J Davis et al ldquoActivation ofthe AMP-activated protein kinase by the anti-diabetic drugmetformin in vivo role of mitochondrial reactive nitrogenspeciesrdquo Journal of Biological Chemistry vol 279 no 42 pp43940ndash43951 2004

[5] M-H Zou X-Y Hou C-M Shi et al ldquoActivation of 51015840-AMP-activated kinase is mediated through c-Src and phos-phoinositide 3-kinase activity during hypoxia-reoxygenation ofbovine aortic endothelial cells role of peroxynitriterdquo Journal ofBiological Chemistry vol 278 no 36 pp 34003ndash34010 2003

[6] P Song and M-H Zou ldquoRegulation of NAD(P)H oxidases byAMPK in cardiovascular systemsrdquo Free Radical Biology andMedicine vol 52 no 9 pp 1607ndash1619 2012

[7] L H Young J Li S J Baron and R R Russell ldquoAMP-activatedprotein kinase a key stress signaling pathway in the heartrdquoTrends in Cardiovascular Medicine vol 15 no 3 pp 110ndash1182005

[8] J Wang H Ma X Zhang et al ldquoA novel AMPK activatorfrom Chinese herb medicine and ischemia phosphorylate thecardiac transcription factor FOXO3rdquo International Journal ofPhysiology Pathophysiology and Pharmacology vol 1 no 2 pp116ndash126 2009

[9] J Wang and J Li ldquoActivated protein C a potential cardio-protective factor against ischemic injury during ischemiareperfusionrdquo American Journal of Translational Research vol 1no 4 pp 381ndash392 2009

[10] H Ma J Wang D P Thomas et al ldquoImpaired macrophagemigration inhibitory factor-amp-activated protein kinase acti-vation and ischemic recovery in the senescent heartrdquo Circula-tion vol 122 no 3 pp 282ndash292 2010

[11] A Morrison X Yan C Tong and J Li ldquoAcute rosiglita-zone treatment is cardioprotective against ischemia-reperfusioninjury by modulating AMPK Akt and JNK signaling innondiabetic micerdquo American Journal of Physiology Heart andCirculatory Physiology vol 301 no 3 pp H895ndashH902 2011

[12] R Shibata K Sato D R Pimentel et al ldquoAdiponectin pro-tects against myocardial ischemia-reperfusion injury through


AMPK- and COX-2-dependentmechanismsrdquoNatureMedicinevol 11 no 10 pp 1096ndash1103 2005

[13] R R Russell III J Li D L Coven et al ldquoAMP-activated proteinkinase mediates ischemic glucose uptake and prevents postis-chemic cardiac dysfunction apoptosis and injuryrdquo Journal ofClinical Investigation vol 114 no 4 pp 495ndash503 2004

[14] E J Miller J Li L Leng et al ldquoMacrophage migrationinhibitory factor stimulates AMP-activated protein kinase inthe ischaemic heartrdquo Nature vol 451 no 7178 pp 578ndash5822008

[15] X Zhang E D Yeung J Wang et al ldquoIsoliquiritigenin anatural anti-oxidant selectively inhibits the proliferation ofprostate cancer cellsrdquo Clinical and Experimental Pharmacologyand Physiology vol 37 no 8 pp 841ndash847 2010

[16] D Li Z Wang H Chen et al ldquoIsoliquiritigenin inducesmonocytic differentiation of HL-60 cellsrdquo Free Radical Biologyand Medicine vol 46 no 6 pp 731ndash736 2009

[17] M Tawata K Aida T Noguchi et al ldquoAnti-platelet actionof isoliquiritigenin an aldose reductase inhibitor in licoricerdquoEuropean Journal of Pharmacology vol 212 no 1 pp 87ndash921992

[18] W An J Yang and Y Ao ldquoMetallothionein mediates car-dioprotection of isoliquiritigenin against ischemia-reperfusionthrough JAK2STAT3 activationrdquo Acta Pharmacologica Sinicavol 27 no 11 pp 1431ndash1437 2006

[19] S A Chowdhury K Kishino R Satoh et al ldquoTumor-specificityand apoptosis-inducing activity of stilbenes and flavonoidsrdquoAnticancer Research vol 25 no 3 B pp 2055ndash2063 2005

[20] S Tamir M Eizenberg D Somjen S Izrael and J VayaldquoEstrogen-like activity of glabrene and other constituents iso-lated from licorice rootrdquo Journal of Steroid Biochemistry andMolecular Biology vol 78 no 3 pp 291ndash298 2001

[21] J Ren J R Privratsky X Yang F Dong and E C Carl-son ldquoMetallothionein alleviates glutathione depletion-inducedoxidative cardiomyopathy in murine heartsrdquo Critical CareMedicine vol 36 no 7 pp 2106ndash2116 2008

[22] Q Li A F Ceylan-Isik J Li and J Ren ldquoDeficiency of insulin-like growth factor 1 reduces sensitivity to aging-associatedcardiomyocyte dysfunctionrdquo Rejuvenation Research vol 11 no4 pp 725ndash733 2008

[23] V G Martinez K J Williams I J Stratford M Clynes andR OrsquoConnor ldquoOverexpression of cytochrome P450 NADPHreductase sensitises MDA 231 breast carcinoma cells to 5-fluorouracil possible mechanisms involvedrdquo Toxicology inVitro vol 22 no 3 pp 582ndash588 2008

[24] C Tong A Morrison S Mattison et al ldquoImpaired SIRT1nucleocytoplasmic shuttling in the senescent heart duringischemic stressrdquoThe FASEB Journal 2013

[25] S T SmileyMReers CMottola-Hartshorn et al ldquoIntracellularheterogeneity in mitochondrial membrane potentials revealedby a J-aggregate-forming lipophilic cation JC-1rdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 88 no 9 pp 3671ndash3675 1991

[26] J Wang Y Wang J Gao et al ldquoAntithrombin is protectiveagainst myocardial ischemia and reperfusion injuryrdquo Journal ofThrombosis and Haemostasis vol 11 pp 1020ndash1028 2013

[27] J Wang L Yang A R Rezaie and J Li ldquoActivated proteinC protects against myocardial ischemicreperfusion injurythrough AMP-activated protein kinase signalingrdquo Journal ofThrombosis and Haemostasis vol 9 no 7 pp 1308ndash1317 2011

[28] A Morrison C Tong J H Lee M karin and J Li ldquoSestrin2mediates the LKB1-AMPK signaling cascade in the ischemicheartrdquo Circulation vol 124 abstract A93 2011

[29] C Tong A Morrison X Yan et al ldquoMacrophage migrationinhibitory factor deficiency augments cardiac dysfunction inType 1 diabetic murine cardiomyocytesrdquo Journal of Diabetesvol 2 no 4 pp 267ndash274 2010

[30] J Li E J Miller J Ninomiya-Tsuji R R Russell III and L HYoung ldquoAMP-activated protein kinase activates p38 mitogen-activated protein kinase by increasing recruitment of p38MAPK to TAB1 in the ischemic heartrdquoCirculation Research vol97 no 9 pp 872ndash879 2005

[31] P Zhao J Wang H Ma et al ldquoA newly synthetic chromiumcomplex-Chromium (d-phenylalanine)3 activates AMP-activated protein kinase and stimulates glucose transportrdquoBiochemical Pharmacology vol 77 no 6 pp 1002ndash1010 2009

[32] P Zhao J Wang L He et al ldquoDeficiency in TLR4 signaltransduction ameliorates cardiac injury and cardiomyocytecontractile dysfunction during ischemiardquo Journal of Cellularand Molecular Medicine vol 13 no 8 A pp 1513ndash1525 2009

[33] D M Yellon and D J Hausenloy ldquoMyocardial reperfusioninjuryrdquo The New England Journal of Medicine vol 357 no 11pp 1074ndash1135 2007

[34] P Ferdinandy R Schulz and G F Baxter ldquoInteraction of car-diovascular risk factors with myocardial ischemiareperfusioninjury preconditioning and postconditioningrdquo Pharmacologi-cal Reviews vol 59 no 4 pp 418ndash458 2007

[35] D Konrad A Rudich P J Bilan et al ldquoTroglitazone causesacute mitochondrial membrane depolarisation and an AMPK-mediated increase in glucose phosphorylation in muscle cellsrdquoDiabetologia vol 48 no 5 pp 954ndash966 2005

[36] T L Vanden Hoek C Li Z Shao P T Schumacker and L BBecker ldquoSignificant levels of oxidants are generated by isolatedcardiomyocytes during ischemia prior to reperfusionrdquo Journalof Molecular and Cellular Cardiology vol 29 no 9 pp 2571ndash2583 1997

[37] A Morrison and J Li ldquoPPAR-120574 and AMPKmdashdvantageous tar-gets for myocardial ischemiareperfusion therapyrdquo BiochemicalPharmacology vol 82 no 3 pp 195ndash200 2011

[38] R Costa A Morrison J Wang C Manithody J Li and AR Rezaie ldquoActivated protein C modulates cardiac metabolismand augments autophagy in the ischemic heartrdquo Journal ofThrombosis and Haemostasis vol 10 pp 1736ndash1744 2012

[39] A Moussa and J Li ldquoAMPK in myocardial infarction anddiabetes the yinyang effectrdquo Acta Pharmaceutica Sinica B vol2 pp 368ndash378 2012

[40] D L Coven X Hu L Cong et al ldquoPhysiological role of AMP-activated protein kinase in the heart graded activation duringexerciserdquo American Journal of Physiology Endocrinology andMetabolism vol 285 no 3 pp E629ndashE636 2003

[41] N Kudo J G Gillespie L Kung et al ldquoCharacterization of5rsquoAMP-activated protein kinase activity in the heart and itsrole in inhibiting acetyl-CoA carboxylase during reperfusionfollowing ischemiardquo Biochimica et Biophysica Acta vol 1301 no1-2 pp 67ndash75 1996

[42] J Li X Hu P Selvakumar et al ldquoRole of the nitric oxidepathway in AMPK-mediated glucose uptake and GLUT4translocation in heart musclerdquo American Journal of PhysiologyEndocrinology and Metabolism vol 287 no 5 pp E834ndashE8412004

[43] R R Russell III R Bergeron G I Shulman and L H YoungldquoTranslocation of myocardial GLUT-4 and increased glucose


uptake through activation of AMPK by AICARrdquo AmericanJournal of Physiology Heart and Circulatory Physiology vol 277no 2 pp H643ndashH649 1999

[44] S B Joslashrgensen J N Nielsen J B Birk et al ldquoThe 1205722-51015840 AMP-activated protein kinase is a site 2 glycogen synthase kinase inskeletal muscle and is responsive to glucose loadingrdquo Diabetesvol 53 no 12 pp 3074ndash3081 2004

[45] D G Hardie and D Carling ldquoThe AMP-activated proteinkinase Fuel gauge of the mammalian cellrdquo European Journalof Biochemistry vol 246 no 2 pp 259ndash273 1997

[46] B B Whitlock S Gardai V Fadok D Bratton and P MHenson ldquoDifferential roles for 120572(M)1205732 integrin clustering oractivation in the control of apoptosis via regulation of Akt andERK survival mechanismsrdquo Journal of Cell Biology vol 151 no6 pp 1305ndash1320 2000

[47] J Wang C Tong X Yan et al ldquoLimiting cardiac ischemicinjury by pharmacologic augmentation of MIF-AMPK signaltransductionrdquo Circulation vol 128 pp 225ndash236 2013

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


arrhythmias and myocardial infarct size and reduced theactivities of lactate dehydrogenase (LDH) and creatininephosphokinase (CPK) [18] Increased JAK2STAT3 phos-phorylation in the heart by ISL appears to the mecha-nism by which ISL protects the heart against ischemia andreperfusion injury [18] To further characterize the cardio-protective effects of ISL on cardiomyocytes under hypoxicstress we isolated mouse cardiomyocytes to investigate theeffects of ISL on cardiomyocytes contractile functions duringhypoxiareoxygenation and the signaling mechanism thatmediated ISL action on cardiomyocytes

2 Materials and Methods

21 Drugs and Chemicals Isoliquiritigenin (ISL) was pur-chased from Sigma (St Louis MO) (Scheme 1) ISL was dis-solved in dimethyl sulfoxide (DMSO) to produce a stock solu-tion of 10mmolL and theDMSOfinal concentrationwas lessthan 001 (v v) Other chemicals were of analytical purity

22 Animals andCell Line 8ndash12-week-oldmale FVBNJmicewere purchased from the Jackson Laboratory (Bar HarborME USA) All animals were kept in the institutional animalfacility at the State University of New York (SUNY) at Buffaloand were fed ad libitum All animal procedures used in thisstudy were approved by the Institutional Animal Care andUse Committees at the State University of New York (SUNY)at Buffalo

23 Isolation of Mouse Cardiomyocytes Cardiomyocyteswere enzymatically isolated as previously described [21]Briefly adult mouse hearts were removed and perfused withoxygenated (5 CO

295 O

2) Krebs-Henseleit bicarbonate

(KHB) buffer containing (in mM) 118 NaCl 47 KCl 125CaCl2 12 MgSO

4 12 KH

2PO4 25 NaHCO

3 10 HEPES

and 111 Glucose All the chemicals were purchased fromSigma (St Louis MO USA) Hearts were then perfusedwith a Ca2+-free KHB containing Liberase Blendzyme 4(Hoffmann-La Roche Inc Indianapolis IN USA) for 15ndash20min After perfusion left ventricles were removed andminced to disperse cardiomyocytes in Ca2+-free KHB bufferExtracellular Ca2+ was added incrementally back to 125mMOnly rod-shaped myocytes with clear edges were selectedfor pharmacological test and cell contractility studies Thecardiomyocyteswere treatedwith ISL (50 100120583M) forAMPKsignaling ROS levelmitochondrialmembrane potential andglucose uptake measurements

24 Hypoxia Treatment Isolated mouse cardiomyocyteswere subjected to two groups (normal and hypoxic groups)Hypoxic groups were kept at 37∘C in a humidified sealedchamber under a humidified atmosphere of 5 CO

2and

95 N2for 20min Normal groups were placed into a water-

jacketed incubator at 37∘C during the same period

25 Cardiomyocytes ShorteningRelengthening MeasurementThe mechanical properties of ventricular myocytes were

OHHO

O

OH

Isoliquiritigenin

Scheme 1

assessed using a SoftEdge MyoCam system (IonOptix Cor-poration Milton MA USA) [22] In brief left ventricularmyocytes were placed in a chamber mounted on the stageof an inverted microscope (Olympus IX-70 Center ValleyPA USA) and incubated at 25∘C with a buffer containing(in mM) 131 NaCl 4 KCl 1 CaCl

2 1 MgCl

2 10 Glucose

and 10 HEPES at pH 74 The cells were stimulated withsuprathreshold voltage at a frequency of 05Hz 3msecduration using a pair of platinum wires placed on oppositesides of the chamber and connected to an electrical stimu-lator (FHC Inc Brunswick NE USA) The myocytes beingstudied were displayed on a computer monitor using anIonOptix MyoCam camera An IonOptix SoftEdge softwarewas used to capture changes in cell length during shorteningand relengthening Cell shortening and relengthening wereassessed using the following indices peak shortening (PS)the amplitude myocytes shortened on electrical stimulationindicative of peak ventricular contractility time-to-PS (TPS)the duration of myocyte shortening an indicative of systolicduration time-to-90 relengthening (TR90) the duration toreach 90 relengthening an indicative of diastolic duration(90 rather 100 relengthening was used to avoid noisysignal at baseline concentration) and maximal velocitiesof shorteningrelengthening maximal slope (derivative) ofshortening and relengthening phases indicative of maximalvelocities of ventricular pressure increasedecrease In thecase of altering stimulus frequency the steady-state contrac-tion of myocytes was achieved (usually after the first 5-6beats) before PS amplitude was recorded

26 Intracellular Ca2+ Transient Measurements Isolated car-diomyocytes were loaded with fura-2AM (05 120583M) for15min and fluorescence measurements were recorded with adual-excitation fluorescence photomultiplier system (IonOp-tix) Myocytes were placed on an Olympus IX-70 invertedmicroscope and imaged through a Fluor (St Louis MOUSA) times40 oil objective Cells were exposed to light emittedby a 75W lamp and passed through either a 360 or a 380 nmfilter while being stimulated to contract at 05Hz Fluores-cence emissions were detected between 480 and 520 nm bya photomultiplier tube after first illuminating the cells at360 nm for 05 sec and then at 380 nm for the duration ofthe recording protocol (333Hz sampling rate) The 360 nmexcitation scan was repeated at the end of the protocol andqualitative changes in intracellular Ca2+ concentration were






2DCFDA at 37∘C








3 Results



100

50

Normoxia Hypoxia

Resti

ng ce

ll le

ngth(120583

m)

0

(a)

Normoxia Hypoxia

Peak

shor

teni

ng (

cell

leng

th)

0

2

4

6

lowast

dagger

(b)

200

100

0

Normoxia Hypoxia

lowast

dagger

+dL

dt(120583

ms)

(c)

Normoxia Hypoxia

lowast

dagger

minus50

minus150

minus250

minusdL

dt(120583

ms)

(d)

02

01

00

Normoxia Hypoxia

lowast

dagger

TPS

(s)


(e)

03

02

01

00

Normoxia Hypoxia

lowast

dagger

TR90

(s)


(f)






000

025

050

075

100

Intr

acel

lula

rCa2+

(360

380

ratio

)

lowast

dagger

Normoxia Hypoxia

(a)

000

025

050

Normoxia Hypoxia

Rise

in in

trac

ellu

larC

a2+

(Δ360380

)

(b)

00

01

02

03

Intr

acel

lula

rCa2+

deca

y ra

te (s

)

Normoxia Hypoxia

lowast

dagger


(c)

00

01

02

Normoxia Hypoxia

lowast

dagger

Intr

acel

lula

rCa2+

deca

y ra

te (s

)


(d)










4

2

0

p-A

MPK

rel

ativ

e uni

ts

lowast



p-AMPK (Thr172)

AMPK120572

(a)

4

2

0p-

ACC

relat

ive u

nits

lowast


GAPDH


p-ACC (Ser79)

(b)

4

2

0

6

p-ER

K re

lativ

e uni

ts

lowast


GAPDH



(c)




4 Discussion



H2-D

CFD

A fl

uore

scen

ce0

4

8

12

Normoxia Hypoxia

lowast

dagger


(a)

0

2

4

6

8

lowast

JC-1

ratio

(590

530

nm)



(b)

0

5

10

15

Glu

cose

upt

ake (

cpm

mg

prot

ein)

lowast

lowastlowast

dagger


minus

+

+

minus

+ Insulin 10nM


(c)

















Acknowledgments



References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















2DCFDA at 37∘C








3 Results



100

50

Normoxia Hypoxia

Resti

ng ce

ll le

ngth(120583

m)

0

(a)

Normoxia Hypoxia

Peak

shor

teni

ng (

cell

leng

th)

0

2

4

6

lowast

dagger

(b)

200

100

0

Normoxia Hypoxia

lowast

dagger

+dL

dt(120583

ms)

(c)

Normoxia Hypoxia

lowast

dagger

minus50

minus150

minus250

minusdL

dt(120583

ms)

(d)

02

01

00

Normoxia Hypoxia

lowast

dagger

TPS

(s)


(e)

03

02

01

00

Normoxia Hypoxia

lowast

dagger

TR90

(s)


(f)






000

025

050

075

100

Intr

acel

lula

rCa2+

(360

380

ratio

)

lowast

dagger

Normoxia Hypoxia

(a)

000

025

050

Normoxia Hypoxia

Rise

in in

trac

ellu

larC

a2+

(Δ360380

)

(b)

00

01

02

03

Intr

acel

lula

rCa2+

deca

y ra

te (s

)

Normoxia Hypoxia

lowast

dagger


(c)

00

01

02

Normoxia Hypoxia

lowast

dagger

Intr

acel

lula

rCa2+

deca

y ra

te (s

)


(d)










4

2

0

p-A

MPK

rel

ativ

e uni

ts

lowast



p-AMPK (Thr172)

AMPK120572

(a)

4

2

0p-

ACC

relat

ive u

nits

lowast


GAPDH


p-ACC (Ser79)

(b)

4

2

0

6

p-ER

K re

lativ

e uni

ts

lowast


GAPDH



(c)




4 Discussion



H2-D

CFD

A fl

uore

scen

ce0

4

8

12

Normoxia Hypoxia

lowast

dagger


(a)

0

2

4

6

8

lowast

JC-1

ratio

(590

530

nm)



(b)

0

5

10

15

Glu

cose

upt

ake (

cpm

mg

prot

ein)

lowast

lowastlowast

dagger


minus

+

+

minus

+ Insulin 10nM


(c)

















Acknowledgments



References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















100

50

Normoxia Hypoxia

Resti

ng ce

ll le

ngth(120583

m)

0

(a)

Normoxia Hypoxia

Peak

shor

teni

ng (

cell

leng

th)

0

2

4

6

lowast

dagger

(b)

200

100

0

Normoxia Hypoxia

lowast

dagger

+dL

dt(120583

ms)

(c)

Normoxia Hypoxia

lowast

dagger

minus50

minus150

minus250

minusdL

dt(120583

ms)

(d)

02

01

00

Normoxia Hypoxia

lowast

dagger

TPS

(s)


(e)

03

02

01

00

Normoxia Hypoxia

lowast

dagger

TR90

(s)


(f)






000

025

050

075

100

Intr

acel

lula

rCa2+

(360

380

ratio

)

lowast

dagger

Normoxia Hypoxia

(a)

000

025

050

Normoxia Hypoxia

Rise

in in

trac

ellu

larC

a2+

(Δ360380

)

(b)

00

01

02

03

Intr

acel

lula

rCa2+

deca

y ra

te (s

)

Normoxia Hypoxia

lowast

dagger


(c)

00

01

02

Normoxia Hypoxia

lowast

dagger

Intr

acel

lula

rCa2+

deca

y ra

te (s

)


(d)










4

2

0

p-A

MPK

rel

ativ

e uni

ts

lowast



p-AMPK (Thr172)

AMPK120572

(a)

4

2

0p-

ACC

relat

ive u

nits

lowast


GAPDH


p-ACC (Ser79)

(b)

4

2

0

6

p-ER

K re

lativ

e uni

ts

lowast


GAPDH



(c)




4 Discussion



H2-D

CFD

A fl

uore

scen

ce0

4

8

12

Normoxia Hypoxia

lowast

dagger


(a)

0

2

4

6

8

lowast

JC-1

ratio

(590

530

nm)



(b)

0

5

10

15

Glu

cose

upt

ake (

cpm

mg

prot

ein)

lowast

lowastlowast

dagger


minus

+

+

minus

+ Insulin 10nM


(c)

















Acknowledgments



References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















000

025

050

075

100

Intr

acel

lula

rCa2+

(360

380

ratio

)

lowast

dagger

Normoxia Hypoxia

(a)

000

025

050

Normoxia Hypoxia

Rise

in in

trac

ellu

larC

a2+

(Δ360380

)

(b)

00

01

02

03

Intr

acel

lula

rCa2+

deca

y ra

te (s

)

Normoxia Hypoxia

lowast

dagger


(c)

00

01

02

Normoxia Hypoxia

lowast

dagger

Intr

acel

lula

rCa2+

deca

y ra

te (s

)


(d)










4

2

0

p-A

MPK

rel

ativ

e uni

ts

lowast



p-AMPK (Thr172)

AMPK120572

(a)

4

2

0p-

ACC

relat

ive u

nits

lowast


GAPDH


p-ACC (Ser79)

(b)

4

2

0

6

p-ER

K re

lativ

e uni

ts

lowast


GAPDH



(c)




4 Discussion



H2-D

CFD

A fl

uore

scen

ce0

4

8

12

Normoxia Hypoxia

lowast

dagger


(a)

0

2

4

6

8

lowast

JC-1

ratio

(590

530

nm)



(b)

0

5

10

15

Glu

cose

upt

ake (

cpm

mg

prot

ein)

lowast

lowastlowast

dagger


minus

+

+

minus

+ Insulin 10nM


(c)

















Acknowledgments



References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















4

2

0

p-A

MPK

rel

ativ

e uni

ts

lowast



p-AMPK (Thr172)

AMPK120572

(a)

4

2

0p-

ACC

relat

ive u

nits

lowast


GAPDH


p-ACC (Ser79)

(b)

4

2

0

6

p-ER

K re

lativ

e uni

ts

lowast


GAPDH



(c)




4 Discussion



H2-D

CFD

A fl

uore

scen

ce0

4

8

12

Normoxia Hypoxia

lowast

dagger


(a)

0

2

4

6

8

lowast

JC-1

ratio

(590

530

nm)



(b)

0

5

10

15

Glu

cose

upt

ake (

cpm

mg

prot

ein)

lowast

lowastlowast

dagger


minus

+

+

minus

+ Insulin 10nM


(c)

















Acknowledgments



References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















H2-D

CFD

A fl

uore

scen

ce0

4

8

12

Normoxia Hypoxia

lowast

dagger


(a)

0

2

4

6

8

lowast

JC-1

ratio

(590

530

nm)



(b)

0

5

10

15

Glu

cose

upt

ake (

cpm

mg

prot

ein)

lowast

lowastlowast

dagger


minus

+

+

minus

+ Insulin 10nM


(c)

















Acknowledgments



References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























Acknowledgments



References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article natural antioxidant-isoliquiritigenin...

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