research article interleukin-27 protects cardiomyocyte...

Research ArticleInterleukin-27 Protects Cardiomyocyte-Like H9c2 Cells againstMetabolic Syndrome Role of STAT3 Signaling

Wei-Lian Phan1 Yu-Tzu Huang2 and Ming-Chieh Ma2

1Department of Cardiology Cardinal Tien Hospital New Taipei City 23148 Taiwan2School of Medicine Fu Jen Catholic University New Taipei City 24205 Taiwan

Correspondence should be addressed to Ming-Chieh Ma med0041mailfjuedutw

Received 9 December 2014 Revised 6 March 2015 Accepted 13 March 2015

Academic Editor Daniela Parolaro

Copyright copy 2015 Wei-Lian Phan et alThis 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

The present results demonstrated that high glucose (G) salt (S) and cholesterol C (either alone or in combination) as mimickingextracellular changes in metabolic syndrome damage cardiomyocyte-like H9c2 cells and reduce their viability in a time-dependentmanner However the effects were greatest when cells were exposed to all three agents (GSC) The mRNA of glycoprotein (gp) 130and WSX-1 both components of the interleukin (IL)-27 receptor were present in H9c2 cells Although mRNA expression was notaffected by exogenous treatment with IL-27 the expression of gp130 mRNA (but not that ofWSX-1 mRNA) was attenuated by GSCTreatment of IL-27 to H9c2 cells increased activation of signal transducer and activator of transcription 3 (STAT3) and protectedcells fromGSC-induced cytochrome c release and cell damageTheprotective effects of IL-27were abrogated by the STAT3 inhibitorstattic The results of the present study clearly demonstrate that the STAT3 pathway triggered by anti-inflammatory IL-27 plays arole in protecting cardiomyocytes against GSC-mediated damage

1 Introduction

In humans metabolic syndrome (MS) is characterized by acombination of obesity hyperglycemia glucose intolerancedyslipidemia and hypertension [1] The risk of heart attack isthree times greater for those with MS than for those without[2] Previous studies show that MS increases mortality inpatients with acute myocardial infarction and both duringand after coronary artery bypass surgery [3ndash5]ThusMS ren-ders the myocardium intolerant to further injury includingischemia or mechanical damage This notion is supported bya recent study showing that MS increases apoptosis in ratcardiomyocytes after myocardial ischemiareperfusion (IR)injury via reactive oxygen species- (ROS-)mediated increasesin mitochondrial permeability [6] Thus it is necessary toexplore the mechanism underlying the effects of MS on themyocardium with a view on developing new treatments forheart diseases associated with metabolic disorders

Inflammation links MS and heart disease [7] Metabolicoverload triggers oxidative stress organelle dysfunctionand cell hypertrophy all of which generate a vicious self-amplifying cycle that leads to inflammation [1] For example

hypertrophy of adipose tissue (which is an active endocrineorgan) causes cell rupture this releases large amounts ofcytokines such as interleukin- (IL-) 6 tumor necrosis factor-120572 and adiponectin all of which cause inflammation [17 8] IL-6 has been identified as an inflammatory markerin MS however animal studies show that this cytokinehas protective effects in patients with unstable angina andameliorates IR injury [9ndash11] Interestingly these protectiveeffects are mostly related to the activation of signal trans-ducer and activator of transcription 3 (STAT3) IL-6 familycytokines activate glycoprotein (gp) 130 and phosphorylateSTAT3 which is then translocated to the cell nucleus where itactivates genes that control cell proliferation differentiationand survival [12ndash14] Increased STAT activity upregulates theexpression of several cardioprotective genes including B-celllymphoma-extra large which prevents myocardial apoptosisand inhibits vascular endothelial growth factor [15 16]

IL-27 belongs to the IL-6 family It is a heterodimericcytokine comprising Epstein-Barr virus-induced gene-3 andp28 (IL-27p28) [17] Its receptor is also a heterodimer com-prising gp130 and WSX-1 [18] IL-27 is primarily producedby dendritic cells (DCs) and regulates the proliferation and

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 689614 10 pageshttpdxdoiorg1011552015689614

2 BioMed Research International

activation of other immune cells [17 19] In the rat heart DCsreside close to endocardial blood vessels and align parallel tothe myocardium with their processes interdigitating betweenthe cardiomyocytes [20] Studies in the IR rat model showthat DCs accumulate within the zones bordering infarctedtissue 7 days after myocardial infarction [21] Moreover cir-culating levels of IL-27 are elevated in patients with coronaryartery disease [22] Taken together these results indicate thatIL-27 released by DCs located around peri-infarcted tissuesmay aid myocardial recovery from postischemic insult

Our previously results show that IL-27 attenuates IR-induced cardiac injury in isolated rat hearts and protectsH9c2 cells against severe hypoxia (2 oxygen) induced celldeath [23]These protective effects were blocked by the small-molecule STAT3 inhibitor stattic The aim of the presentstudy was therefore to examine whether IL-27 activates theSTAT3 pathway to protect H9c2 cells against phenotypicchanges associated with MS Because hyperglycemia hyper-tension and dyslipidemia are the most important symp-toms in MS and because of lack of definition of MS atcellular level we applied high glucose (G) high NaCl (S)andor cholesterol (C) treatment to mimic the phenotypeandor extracellular changes associated with MS We firstcompared the effects of G S and C either individuallyor in combination on cell viability and used RT-PCT toexamine their effects on IL-27 receptor expression in treatedH9c2 cells We then further examined the effect of IL-27 byexposing cells to the cytokine in the presence and absence ofstattic Quantitative changes in STAT3 activity under differenttreatment conditions were measured using enzyme-linkedimmunosorbent assay (ELISA) kits

2 Materials and Methods

21 Chemicals IL-27 and stattic (6-nitro-11-dioxide-benzo-[b]thiophene) were obtained fromRampDSystems (Minneapo-lis MN) andMerck (Darmstadt Germany) respectively andwere prepared in sterile water S G and water-soluble C 3-(45-cimethylthiazol-2-yl)-25-diphenyl tetrazolium bromide(MTT) and dimethyl sulfoxide (DMSO) were purchasedfrom Sigma-Aldrich (St Louis MO)

22 Culture of Cardiomyocyte-Like H9c2 Cells The cardio-myoblast cell line H9c2 was purchased from the Biore-source Collection and Research Center (Hsinchu Taiwan)The cell line was originally derived from the AmericanType Culture Collection line CRL-1446 All culture mediaand supplements were purchased from Thermo ScientificHyClone (South Logan UT) H9c2 cells were routinely cul-tured at 37∘C5CO

2inDulbeccorsquosmodified Eaglersquosmedium

containing 10 fetal bovine serum (FBS) 1 L-glutaminepenicillin (100UmLminus1) streptomycin (100mgmLminus1) andamphotericin B (025 120583gmLminus1) as described previously [24]The concentrations of S G andC in the culturemediumwere155mM 5mM and zero respectively Cells were subculturedwhen they reached 80 confluence (approximately every2-3 days) Two days before each experiment cells weresubcultured in 48 well plates (15 times 104well) and grown in

culture medium containing 5 FBS to avoid overstimula-tion of STAT3 Pilot results showed that cells adapted wellunder these conditions with no evident change in lactatedehydrogenase (LDH) release or MTT staining after 48 h ofincubation

On the day of the experiment cells were pretreated withsterile water (vehicle) IL-27 (150 ngmLminus1) or stattic (10 120583M)for 4 h Dosing was based on cell viability Next the cells wereexposed to G (25mM) S (250mM) and C (300120583gmLminus1)either alone or in different combinations (GS SC GC orGSC) for 4 8 24 or 48 h to mimic MS

23 Quantitative Real-Time PCR RNA was extracted usinga commercial kit (RareRNA Bio-East Technology TaipeiTaiwan) as previously described [25] and cDNA synthesizedat 42∘C for 45min (reaction mixture 2120583g RNA 5 120583g ofpoly(dT)15 oligonucleotide primer (Life Technologies) and200 units of reverse transcriptase (Moloney murine leukemiavirus Promega Madison WI)) Quantitative RT-PCR wasperformed in an ABI StepOne Plus system (Applied Biosys-tems Foster City CA) PCR was performed using 100 ng ofcDNA and 30 120583mol of primers (total reaction volume 20120583L)using the SYBR Green PCR master mix kit according tothe manufacturerrsquos instructions (Applied Biosystems) Thefollowing primers were used for PCR gp130 51015840-ATA CCTTAA ACA AGC TCC ACC TTC-31015840 (forward) and 51015840-AGTTTC ATT TCC AAT GAT GGT TCT-31015840 (reverse) WSX-1 51015840-GAA ACC CAA ATG AAG CCA AA-31015840 (forward)and 51015840-GCC TCC TGA CAT CTT CGG TA-31015840 (reverse)glyceraldehyde 3-phosphate dehydrogenase (GAPDH) 51015840-TTA GCA CCC CTG GCC AAG G-31015840 (forward) and 51015840-CTTACTCCTTGGAGGCCATG-31015840 (reverse)The cyclingconditions were as follows 95∘C for 20 s followed by 40cycles of 95∘C for 1 s and 60∘C for 20 s Melting curve analysiswas performed at the end of each PCR experiment Allreactions were run in duplicate The ΔCt (threshold cycle)was calculated by subtracting the rawCt values for the house-keeping gene (GAPDH) from the raw Ct values for the targetgene thereby providing information about relative changesin gene expression Changes in target gene expression werecalculated as 2minusΔCt and expressed as the fold change relativeto that in control cells

24 Cell CytotoxicityViability Assays To assess cytotoxicityLDH levels were measured in cell culture medium (20120583L) atdifferent time points using a commercial kit (Roche AppliedScience Indianapolis IN) as described previously [24] Theadvantage of LDH assay is that it allows cell viability tobe continuously monitored An MTT assay was used tomeasure cell viability Briefly cells were cultured for 48 h inthe presence or absence of the different agents The mediumwas then removed and the cells were washed three times withphosphate-buffered saline (PBS pH 74) MTT (5mgmLminus1 inPBS) was then added to cells for 4 h at 37∘C MTT formazancrystals were then dissolved in DMSO and the optical density(OD) measured in an ELISA plate reader (Amersham-Pharmacia Biotech Piscataway NJ) at 570 nm Cell viability

BioMed Research International 3

() was calculated using the following formula (viable cells) = (OD of treated sampleOD of control sample) times 100

25 Measurement of Phosphorylated STAT3 and Total STAT3Commercial ELISA kits (eBioscience San Diego CA) wereused to measure the levels of phosphorylated and totalSTAT3 according to the manufacturerrsquos instructions Brieflycells were lysed in lysis buffer supplied with the kit at roomtemperature for 20min The protein concentration in eachsamplewas thenmeasured using a colorimetric assay kit (Bio-Rad Hercules CA) to confirm that the amount of proteinwas sufficient for further tests The lysed sample and thepositive control (supplied with kit) were then transferredto the coating plate and mixed with an equivalent volumeof the antibody cocktail supplied with the kit The mixturewas then incubated in the dark for 2 h After washing thedetection reagent was added for 30min Finally stop solutionwas added to each well and the colorimetric reaction wasmeasured at 450 nm STAT3 activity was expressed as aratio of the amount of phosphorylated to total STAT3 afternormalization against the total protein concentration

26 Western Blot Analysis The expression of cytochrome c(an indicator of apoptosis) and actin was examined by immu-noblot analysis as described previously [24 25] Primaryantibodies against cytochrome c (cyto c) and actin wereobtained from Santa Cruz Biotechnology (1 2000 SantaCruz CA) Briefly equal amounts of cytosolic proteinwere separated in denaturing SDS polyacrylamide gels andelectrophoretically transferred to polyvinylidene difluoridemembranes (Amersham Little Chalfont UK) The mem-branes were then incubated with the appropriate primaryantibodies overnight at 4∘C After washing the membraneswere incubated for 1 h at room temperature with the corre-sponding horseradish peroxidase-conjugated secondary anti-bodies (1 200 Vector Laboratories Burlingame CA) Boundantibodies were visualized using an enhanced chemilumi-nescence kit (Amersham-Pharmacia Biotech) and Kodakfilm Band density was measured semiquantitatively usingan image analytical system (Diagnostic Instruments SterlingHeights MI) The amount of each protein was expressedrelative to the amount of actin

27 Statistical Analysis All data are expressed as the mean plusmnstandard error of the mean (SEM) Statistical analysis wasperformed using analysis of variance or analysis of variancefollowed by the Newman-Keuls method for multiple com-parisons between groups A 119875 value lt 005 was consideredsignificant

3 Results

31 High Glucose High Salt and Cholesterol Induce CellInjury Treatment of cells with G S or C alone led to atime-dependent increase in LDH release from H9c2 cells(Figure 1(a) open bars) In addition G or S alone led to areduction in cell viability after 48 h of treatment (Figure 1(b)open bars) Cell damage was more severe when cells were

exposed to a combination of all three agents (GSC) par-ticularly after 24 h and 48 h A significant reduction in cellviability was noted when cells were treated with S plus G or Cbut not when cells were treated with G plus C in the absenceof S However when cells were treated with GSC cell viabilitywas reduced to sim58 of that in control cells

Prior treatment with IL-27 markedly attenuated celldamage and death caused by G S or C alone (Figures1(a) and 1(b) gray bars) However significant changes wereonly observed after 24 h and 48 h of treatment with S Thesynergistic effects of GSC on cell viability were abrogated bypretreatment with IL-27

32 IL-27 Receptor Expression The mRNA for gp130 andWSX-1 was detected by RT-PCR indicating that H9c2 cellsexpress IL-27 receptors However the expression of gp130mRNA was markedly attenuated in cells treated with GSC(037plusmn011 versus 10plusmn013 in controls119875 lt 005 Figure 2(a))IL-27 had no effect on gp130 mRNA expression in eitherthe presence or absence of GSC (045 plusmn 012 in the IL-27 +GSC group) Neither GSC nor IL-27 affected the expressionof WSX-1 mRNA (Figure 2(b))

33 IL-27 Increases STAT3 Activity and Inhibits the Releaseof Cytochrome c Binding of IL-27 to gp130 triggers STAT3activation which then transduces downstream signals toelicit cellular responses [17] Therefore we next examinedwhether IL-27 affects STAT3 activity in H9c2 cells Prior totreatment with IL-27 cells showed similar levels of STAT3activity (Figure 3(a)) STAT3 activity was significantly higherin cells exposed to IL-27 alone for 8 24 and 48 h than invehicle-treated cells However IL-27 had no significant effecton LDH release (Figure 3(b)) Interestingly cells treated withGSC for 48 h showed lower STAT3 activity (024plusmn003 versus054 plusmn 004 in controls 119875 lt 005 Figure 3(c)) and increasedLDH release (339 plusmn 21 versus 28 plusmn 09ULminus1 in controls119875 lt 005 Figure 3(d)) Pretreatment with IL-27 preventedthe GSC-induced reduction in STAT3 activity (045 plusmn 004119875 lt 005 versus GSC group) and cell injury (179 plusmn 20ULminus1119875 lt 005 versus GSC group) GSC treatment significantlyincreased the expression of cytochrome c in the cytosolicfraction (118 plusmn 015 versus 062 plusmn 009 in controls 119875 lt005 Figure 3(e)) suggesting that GSC induces cytochromec release This GSC-induced increase in cytochrome c wasabrogated by IL-27 (076 plusmn 011 119875 lt 005 versus GSC group)

34 STAT3 Inhibition Abrogates IL-27-Mediated MyocardialProtection The above results clearly indicate that increasedSTAT3 activity plays a role in IL-27-mediated myocardialprotection Therefore we next examined the effect of aselective STAT3 inhibitor stattic on the effects of IL-27 Cellswere treated with stattic alone (dark gray bars) or stattic +IL-27 (light gray bars) 4 h prior to treatment with G S orC either alone or in different combinations Stattic alonehad no effect on LDH release by control cells (Figure 4(a))However there was no difference in LDH release or cellviability after cotreatment with G S and C alone or in


0

10

20

30

40

Control ControlG S(h)

C GS GC SC GSC G S C GS GC SC GSC

ab

ab

bc

def

a

cb

dfe

VehicleIL-27

4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448

lowast

lowast

lowast

lowast

LDH

(U Lminus1)

(a)

Con

trol

Con

trolG S C GS

GC SC

GSC G S C G

SG

C SCG

SC

lowast

Cel

l via

bilit

y (M

TT as

say

)

0

20

40

60

80

100

120

VehicleIL-27

abc

def

ab

deflowast

lowast

(b)

Figure 1 Cytotoxic effects of high glucose high sodium or high cholesterol on the viability of H9c2 cells (a) Cells were treated with glucose(25mM G) NaCl (250mM S) or cholesterol (300 120583gmLminus1 C) either alone or in different combinations for 4 8 24 and 48 h Cells weretreated with IL-27 4 h before treatment with G S or C Lactate dehydrogenase (LDH) release was measured as marker of cell injury (b) Cellswere cultured for 48 h in the presence of G S or C (either alone or in different combinations) and cell viability was monitored in an MTTassay 119873 = 6 of experiments performed at each time point lowast119875 lt 005 versus the control group a119875 lt 005 versus G b119875 lt 005 versus Sc119875 lt 005 versus C d119875 lt 005 versus GS e119875 lt 005 versus GC f119875 lt 005 versus the SC and

119875 lt 005 versus the vehicle-treated control atthe same time-point

different combinations and stattic or stattic + IL-27 (Figures4(a) and 4(b))

35 Stattic Reverses the Effects of IL-27 We next examinedwhether stattic abrogated the protective effects of IL-27 in

H9c2 cells Compared to Figure 3(c) stattic had no effecton reduction in STAT3 activity in cells treated with GSCbut led to a reduction in STAT3 activity in cells treatedwith IL-27 alone (Figure 5(a)) However it did reduce STAT3activity in cells treatedwith both IL-27 andGSC (Figure 5(a))


C GSC IL-27

gp13

0 m

RNA

fold

chan

ge

00

05

10

15

GSC

lowast

IL-27 +

(a)

C GSC IL-27

WSX

-1 m

RNA

fold

chan

ge

00

05

10

15

GSCIL-27 +

(b)

Figure 2 Expression of IL-27 receptor mRNAThe expression of gp130 (a) andWSX-1 (b) mRNA in control cells and cells treated with GSCandor IL-27 was examined by RT-PCR119873 = 6 of experiments performed in each group lowast119875 lt 005 versus the control (C) group 119875 lt 005versus the IL-27 group

Cotreatment with stattic or with stattic plus IL-27 increasedthe levels of GSC-mediated cell damage (Figure 5(b)) morethan those in Figure 3(d) However stattic did not affectthe GSC-induced increase in cytochrome c levels in thecytoplasm (Figure 5(c)) A similar increase in cytochromec expression was observed in GSC cells cotreated withstattic plus IL-27 These results clearly indicate that IL-27-mediated cardioprotection against GSC-induced cell damageand apoptosis is dependent upon STAT3 activation

4 Discussion

The results of the present study show that the IL-27STAT3pathway plays a role in protecting cardiomyocytes againstGSC-mediated damage First we showed that G S and C(either alone or in combination) damage H9c2 cells andreduce their viability however the effects were greatest whencells were exposed to all three agents (GSC) Second wefound that H9c2 cells expressed mRNA for gp130 and WSX-1 both components of the IL-27 receptor Although mRNAexpression was not affected by exogenous treatment with IL-27 the expression of gp130 mRNA (but not that of WSX-1 mRNA) was attenuated by GSC Third binding of IL-27to its receptor on H9c cells increased activation of STAT3and protected cells from GSC-induced cytochrome c releaseand cell damage Finally the protective effects of IL-27 wereabrogated by the STAT3 inhibitor stattic

Treatment with high glucose high sodium fatty acidsor cholesterol derivatives induce apoptosis in H9c2 cellsand adult rat cardiomyocytes [26ndash28] Here we showed thatGSC treatment caused cell damage and apoptosis in H9c2cardiomyoblasts High glucose high sodium andor fatty

acid levels can injure cardiomyocytes via severalmechanismsincluding oxidative stress activation of nuclear factor-120581Bincreased release of inflammatory cytokines (eg IL-1120573 IL-6 and TNF-120572) and apoptosis [26ndash28] Here we identifieda novel mechanism of cardiac cell injury induced by GSC-mediated suppression of STAT3 activity

Binding of growth factors or cytokines to gp130 acti-vates Janus kinase which in turn recruits and activatesSTAT3 which is then translocated to the nucleus where ittransactivates its target genes [29] We speculated that GSCimpairs the survival-promoting effects of growth factors orcytokines on cardiac cells thereby inducing cell death Thisnotion is supported by our observations of reduced STAT3activity and defects in gp130 mRNA expression in GSC-treated H9c2 cells Gp130 is utilized by several growth factorsand cytokines therefore reduced gp130 expressionmay affectcell fate We found that although IL-27 had no effect on theGSC-mediated reduction in gp130 mRNA expression it didinduce a significant increase in STAT3 activity This may bedue to the enhanced function of residual gp130 expressedby GSC-treated cells which triggers increased STAT3 acti-vation However because cardiomyocytes release cytokinesupon cardiac injury we cannot rule out the possibility thatimpaired release of such growth factors adversely affectsendogenous repair mechanisms [30] Interestingly neitherGSC nor IL-27 affected the expression of WSX-1 mRNATheassociation betweenWSX-1 and gp130 inhibits inflammation[31] Genetic defects in WSX-1 make animals more suscep-tible to lethal inflammation induced by parasitic infection[31]Therefore it may be that theWSX-1 expression observedin GSC-treated H9c2 protects the cells by allowing STAT3activation Further studies are needed to examine whether


(h)0 8 24 48

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(AU

)

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IL-27 Control

lowast

lowastlowast

(a)

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(U Lminus1)

IL-27 Control

(b)

STAT

3 ac

tivity

(AU

)

00

05

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lowast

lowast

+

GSCIL-27 +

(c)

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(U Lminus1)

lowast

+

C GSC IL-27GSCIL-27 +

(d)

Cyto

chro

me c

act

in (A

U)

00

05

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Cytochrome

Actin

C GSC IL-27

lowast

+

12kDa

42kDa

GSCIL-27 +C GSC IL-27

GSCIL-27 +

(e)

Figure 3 Effects of IL-27 on STAT3 activity and cytochrome c release from H9c2 cells (a) IL-27 increased the activity of STAT3 in a time-dependent manner compared with that in control (vehicle-treated) cells (b) Lactate dehydrogenase (LDH) release by control cells indicatesspontaneous turnover during cell growth Note that IL-27 had no effect on LDH release (c) Changes in STAT3 activity after 48 h of treatmentwith GSC IL-27 or GSC + IL-27 Note that IL-27 partially reversed the glucose sodium and cholesterol (GSC) induced reduction in STAT3activity (d) Effects of GSC and IL-27 on LDH release Note that the GSC-mediated increase in LDH release was attenuated by IL-27 (e)Apoptosis was evaluated by assessing the cytosolic levels of cytochrome c (Cyto c) Western blots showing the expression of cytochrome c inthree representative cultures Equal amounts of protein (20120583g per lane) were loadedThe bar graph shows changes in cytochrome c expression(as measured by densitometry) after normalization against actin119873 = 6 of experiments performed at each time-point or in each group AUarbitrary units lowast119875 lt 005 versus the zero time-point or the control (C) group 119875 lt 005 versus the IL-27 group +119875 lt 005 versus the GSCgroup


0

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lowast

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(U Lminus1)

IL-27Stattic +Stattic +

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(h)

4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448

(a)

Cel

l via

bilit

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TT as

say

)

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def

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clowast

lowast

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tic

Stat

tic+

IL-27G S C GS

GC SC

GSC G S C G

SG

C SCG

SC

Stattic + Stattic + IL-27 +

(b)

Figure 4 Effect of STAT3 inhibition on cell viability (a) Cells were treated with stattic (dark gray bars) or stattic plus IL-27 (light gray bars)4 h prior to treatment with glucose (25mM G) NaCl (250mM S) or cholesterol (300120583g mLminus1 C) either alone or in different combinationsfor 4 8 24 and 48 h Lactate dehydrogenase (LDH) was determined as a marker of cell injury (b) Cells were cultured for 48 h in the presenceof the indicated agents and viability was measured in anMTT assay119873 = 6 of experiments performed in each time-point of group lowast119875 lt 005versus the corresponding control groups (stattic alone or stattic + IL-27) a119875 lt 005 versus G b119875 lt 005 versus S c119875 lt 005 versus C d119875 lt 005versus GS e119875 lt 005 versus GC f119875 lt 005 versus SC

WSX-1 blockade exacerbates GSC-mediated cardiomyocyteinjury Moreover measurements of IL-27 receptor mRNAprobably do not reflect the protein expression on the surfaceof cells Further studies are required to examine the proteinlevels of gp130 and WSX-1 after MS andor IL-27 treatmentand this is an experimental limitation of the study

Though the present study did not explore the downstreameffects mediated by STAT3 previous studies show that STAT3

phosphorylation targets both nucleus andmitochondria [14]Key residues within the STAT3 protein that is tyr705 andser727must be phosphorylated formaximum transcriptionalactivity in the nucleus [14] Phosphorylation at these sitesis required for STAT3 to play its roles in cell survivalproliferation and differentiation and in preventing apoptosis[32] The commercial kit used in the present study wasable to detect STAT3 phosphorylation at both these crucial


Stattic

STAT

3 ac

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(AU

)

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GSCGSC IL-27Stattic + Stattic + Stattic +

IL-27 +

(a)

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(U Lminus1)

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IL-27 +

(b)

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Cytochrome

Actin

12kDa

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IL-27 +Stattic


IL-27 +

(c)

Figure 5 Effect of stattic on STAT3 activity and cytochrome c release (a) Changes in STAT3 activity after 48 h of treatment with statticstattic + GSC stattic + IL-27 or stattic + IL-27 + GSC Note that stattic enhanced the high glucose sodium and cholesterol (GSC) mediatedreduction in STAT3 activity and abrogated the STAT3-enhancing effects of IL-27 (b) Stattic increased LDH release in GSC-treated cells in thepresence or absence of IL-27 (c) Apoptosis was evaluated by measuring cytochrome c (Cyto c) release Western blots showing the expressionof cytochrome c in three representative cell cultures Equal amounts of protein (20120583g per lane) were loaded The bar graph shows changes inthe expression of cytochrome c in treated cells (after being normalized against actin) 119873 = 6 of experiments performed in each group AUarbitrary units lowast119875 lt 005 versus the stattic group 119875 lt 005 versus the stattic + IL-27 group

sites About 10 of cytosolic STAT3 resides within themitochondria where it binds to complex I or II in theelectron transport chain to driveATP synthesis [14 33]Theseinteractions however require that STAT3 be phosphorylatedonly on ser727 not tyr705 [14 33]Moreover a previous studyshowed that the mitochondrial distribution of STAT3 playsan important role in inhibiting mitochondrial permeabilitytransition pore (MPTP) opening [34] which allows therelease of cytochrome c and induces myocardial apoptosisand necrosis The results of the present study are consistentwith this as STAT3-mediated protection of H9c2 cells by IL-27 was associated with reduced cytochrome c release andwas abrogated by stattic As the MPTP opens in response tohigh concentrations of ROS and calcium we speculate thatIL-27-mediated STAT3 activation may improve antioxidant

defense and maintain calcium homeostasis Indeed trans-genic mouse hearts expressing constitutively active STAT3show upregulated expression of the free radical scavengermetallothionein [35] Further studies should explore the roleof STAT3 in protecting against calcium overload during heartfailure Calcium overload results from a lack of ATP infailed cardiomyocytes however STAT3 plays a unique rolein regulating ATP synthesis when targeted to mitochondria

STAT3 is an essential component of the Survivor Activat-ing Factor Enhancement (SAFE) pathway and as such it canreduce cardiomyocyte death at the time of reperfusion afterischemic insult [14] Thus several cardioprotective strate-gies and agents that activate STAT3 can successfully rescueinjured cardiomyocytes via the SAFEpathway such strategies


and agents include ischemic conditioning cardiotrophin-1 urocortin opioids insulin leptin resveratrol melatoninand erythropoietin [36ndash43] Though the present study didnot examine the effects of IR injury our previous datashow that IL-27-induced STAT3 activation is important forattenuating IR and hypoxic injury to rat hearts or H9c2cells respectively [23] Here we identified a novel role forIL-27 in cardioprotection via activation of STAT3 and theSAFE pathway Studies show that the diet-induced obesityand insulin resistance associated with MS reduce myocardialresistance to IR injury [44] Obesogenic hearts expresslow levels of molecules involved in the reperfusion injurysalvage kinase (RISK) pathway including Akt and glycogensynthase kinase 3120573 [44] Moreover previous findings in chickembryonic hearts reveal that protein kinases within the RISKpathway are activated downstream of STAT3 [45] Thereforefurther studies should examine whether the RISK pathwayparticipates in IL-27STAT3 signaling to protect against MS-mediated cardiac injury Furthermore a number of mech-anisms such as suppressors of cytokine signaling (SOCS)and the protein inhibitors of activated STATs (PIAS) alsoinhibit STAT activity as part of a negative feedback controlmechanism [12 14] It is not clear whether these negativeregulatory mechanisms are present in cardiomyocytes if sothen further studies should examine whether their functionsare affected by GSC or IL-27 Such studies may provide amore complete picture of the effects of IL-27 with respect tocardioprotection

Conflict of Interests

The authors declare no conflict of interests regarding thepublication of this paper

Authorsrsquo Contribution

Wei-Lian Phan and Yu-TzuHuang contributed equally to thiswork

Acknowledgments

This work was supported by grants from the Cardinal TienHospital (CTH101-1-2B01 CTH102-1-2B11) and the Ministryof Science and Technology (NSC101-2314-B-030-002-MY3)

References

[1] F Emanuela M Grazia D RMarco L Maria Paola F Giorgioand B Marco ldquoInflammation as a link between obesity andmetabolic syndromerdquo Journal of Nutrition and Metabolism vol2012 Article ID 476380 7 pages 2012

[2] B Isomaa P Almgren T Tuomi et al ldquoCardiovascular mor-bidity and mortality associated with the metabolic syndromerdquoDiabetes Care vol 24 no 4 pp 683ndash689 2001

[3] G Levantesi AMacchia RMarfisi et al ldquoMetabolic syndromeand risk of cardiovascular events after myocardial infarctionrdquoJournal of the American College of Cardiology vol 46 no 2 pp277ndash283 2005

[4] N Echahidi P Pibarot J-P Despres et al ldquoMetabolic syndromeincreases operative mortality in patients undergoing coronaryartery bypass grafting surgeryrdquo Journal of the American Collegeof Cardiology vol 50 no 9 pp 843ndash851 2007

[5] M Zeller P G Steg J Ravisy et al ldquoPrevalence and impact ofmetabolic syndrome on hospital outcomes in acute myocardialinfarctionrdquo Archives of Internal Medicine vol 165 no 10 pp1192ndash1198 2005

[6] A Xu J Liu P Liu M Jia H Wang and L Tao ldquoMitochon-drial translocation of Nur77 induced by ROS contributed tocardiomyocyte apoptosis in metabolic syndromerdquo Biochemicaland Biophysical Research Communications vol 446 no 4 pp1184ndash1189 2014

[7] R Monteiro and I Azevedo ldquoChronic inflammation in obesityand the metabolic syndromerdquo Mediators of Inflammation vol2010 Article ID 289645 10 pages 2010

[8] L F Van Gaal I L Mertens and C E De Block ldquoMechanismslinking obesity with cardiovascular diseaserdquo Nature vol 444no 7121 pp 875ndash880 2006

[9] R N Sanchez C K Chan S Garg et al ldquoInterleukin-6in retinal ischemia reperfusion injury in ratsrdquo InvestigativeOphthalmology and Visual Science vol 44 no 9 pp 4006ndash40112003

[10] K Matsushita S Iwanaga T Oda et al ldquoInterleukin-6solubleinterleukin-6 receptor complex reduces infarct size via inhibit-ing myocardial apoptosisrdquo Laboratory Investigation vol 85 no10 pp 1210ndash1223 2005

[11] Y Wang B Yin S Liu and S Xue ldquoCardioprotective effectby tumor necrosis factor-120572 and Interleukin-6 through late pre-conditioning in unstable angina patientsrdquo Archives of MedicalResearch vol 38 no 1 pp 80ndash85 2007

[12] W J Leonard and J J OrsquoShea ldquoJaks and STATs biologicalimplicationsrdquo Annual Review of Immunology vol 16 pp 293ndash322 1998

[13] M Z Kamran P Patil and R P Gude ldquoRole of STAT3 incancer metastasis and translational advancesrdquo BioMed ResearchInternational vol 2013 Article ID 421821 15 pages 2013

[14] R A Knight T M Scarabelli and A Stephanou ldquoSTATtranscription in the ischemic heartrdquo JAK-STAT vol 1 no 2 pp111ndash117 2012

[15] Y Fujio K Kunisada H Hirota K Yamauchi-Takihara andT Kishimoto ldquoSignals through gp130 upregulate bcl-x geneexpression via STAT1-binding cis-element in cardiacmyocytesrdquoJournal of Clinical Investigation vol 99 no 12 pp 2898ndash29051997

[16] T Osugi Y Oshima Y Fujio et al ldquoCardiac-specific activationof signal transducer and activator of transcription 3 promotesvascular formation in the heartrdquo Journal of Biological Chemistryvol 277 no 8 pp 6676ndash6681 2002

[17] S Pflanz J C Timans J Cheung et al ldquoIL-27 a heterodimericcytokine composed of EBI3 and p28 protein induces prolifera-tion of naiveCD4+ Tcellsrdquo Immunity vol 16 no 6 pp 779ndash7902002

[18] S Pflanz L Hibbert J Mattson et al ldquoWSX-1 and glycoprotein130 constitute a signal-transducing receptor for IL-27rdquo Journalof Immunology vol 172 no 4 pp 2225ndash2231 2004

[19] H Yoshida andMYoshiyuki ldquoRegulation of immune responsesby interleukin-27rdquo Immunological Reviews vol 226 no 1 pp234ndash247 2008

[20] D N J Hart and J W Fabre ldquoDemonstration and characteri-zation of Ia-positive dendritic cells in the interstitial connective


tissues of rat heart and other tissues but not brainrdquo Journal ofExperimental Medicine vol 154 no 2 pp 347ndash361 1981

[21] J Zhang Z-X Yu S Fujita M L Yamaguchi and V J FerransldquoInterstitial dendritic cells of the rat heart quantitative andultrastructural changes in experimental myocardial infarctionrdquoCirculation vol 87 no 3 pp 909ndash920 1993

[22] W Jin Y Zhao W Yan et al ldquoElevated circulating interleukin-27 in patients with coronary artery disease is associated withdendritic cells oxidized low-density lipoprotein and severity ofcoronary artery stenosisrdquoMediators of Inflammation vol 2012Article ID 506283 10 pages 2012

[23] M-C Ma B-W Wang T-P Yeh et al ldquoInterleukin-27 a novelcytokine induced by ischemia-reperfusion injury in rat heartsmediates cardioprotective effects via the gp130STAT3pathwayrdquoBasic Research in Cardiology In press

[24] J-S Lin Y-S ChenH-S Chiang andM-CMa ldquoHypoxic pre-conditioning protects rat hearts against ischaemia-reperfusioninjury role of erythropoietin on progenitor cell mobilizationrdquoJournal of Physiology vol 586 no 23 pp 5757ndash5769 2008

[25] M J Lu Y S Chen H S Huang and M C Ma ldquoHypoxic pre-conditioning protects rat hearts against ischemia-reperfusioninjury via the arachidonate12-lipoxygenasetransient receptorpotential vanilloid 1 pathwayrdquo Basic Research in Cardiology vol109 no 4 article 414 2014

[26] L Kui Z Weiwei L ling et al ldquoGhrelin inhibits apoptosisinduced by high glucose and sodium palmitate in adult ratcardiomyocytes through the PI3K-Akt signaling pathwayrdquo Reg-ulatory Peptides vol 155 no 1ndash3 pp 62ndash69 2009

[27] K Sathishkumar X Gao A C Raghavamenon N ParinandiW A Pryor and R M Uppu ldquoCholesterol secoaldehydeinduces apoptosis in H9c2 cardiomyoblasts through reactiveoxygen species involving mitochondrial and death receptorpathwaysrdquo Free Radical Biology and Medicine vol 47 no 5 pp548ndash558 2009

[28] W Xu W Wu J Chen et al ldquoExogenous hydrogen sulfideprotects H9c2 cardiac cells against high glucose-induced injuryby inhibiting the activities of the p38 MAPK and ERK12pathwaysrdquo International Journal of Molecular Medicine vol 32no 4 pp 917ndash925 2013

[29] J S Rawlings KM Rosler andD AHarrison ldquoThe JAKSTATsignaling pathwayrdquo Journal of Cell Science vol 117 no 8 pp1281ndash1283 2004

[30] T Aoyagi and T Matsui ldquoThe cardiomyocyte as a source ofcytokines in cardiac injuryrdquo Journal of Cell Science amp Therapyvol 1 supplement 5 2012

[31] A V Villarino E Huang and C A Hunter ldquoUnderstandingthe pro- and anti-inflammatory properties of IL-27rdquo Journal ofImmunology vol 173 no 2 pp 715ndash720 2004

[32] D E Levy and C K Lee ldquoWhat does Stat3 dordquoThe Journal ofClinical Investigation vol 109 no 9 pp 1143ndash1148 2002

[33] J Wegrzyn R Potla Y-J Chwae et al ldquoFunction of mitochon-drial Stat3 in cellular respirationrdquo Science vol 323 no 5915 pp793ndash797 2009

[34] K Boengler D Hilfiker-Kleiner G Heusch and R SchulzldquoInhibition of permeability transition pore opening by mito-chondrial STAT3 and its role in myocardial ischemiareperfu-sionrdquo Basic Research in Cardiology vol 105 no 6 pp 771ndash7852010

[35] YOshima Y Fujio T Nakanishi et al ldquoSTAT3mediates cardio-protection against ischemiareperfusion injury through metal-lothionein induction in the heartrdquoCardiovascular Research vol65 no 2 pp 428ndash435 2005

[36] G Heusch J Musiolik N Gedik and A Skyschally ldquoMito-chondrial STAT3 activation and cardioprotection by ischemicpostconditioning in pigs with regional myocardial ischemiareperfusionrdquoCirculationResearch vol 109 no 11 pp 1302ndash13082011

[37] L C Huffman S E Koch and K L Butler ldquoCoronary effluentfrom a preconditioned heart activates the JAK-STAT pathwayand induces cardioprotection in a donor heartrdquo The AmericanJournal of PhysiologymdashHeart and Circulatory Physiology vol294 no 1 pp H257ndashH262 2008

[38] J E Railson Z Liao B K Brar et al ldquoCardiotrophin-1 and uro-cortin cause protection by the same pathway and hypertrophyvia distinct pathways in cardiac myocytesrdquo Cytokine vol 17 no5 pp 243ndash253 2002

[39] E R Gross A K Hsu andG J Gross ldquoThe JAKSTAT pathwayis essential for opioid-induced cardioprotection JAK2 as amediator of STAT3Akt andGSK-3 betardquoTheAmerican Journalof PhysiologymdashHeart and Circulatory Physiology vol 291 no 2pp H827ndashH834 2006

[40] B N Fuglesteg N Suleman C Tiron et al ldquoSignal transducerand activator of transcription 3 is involved in the cardio-protective signalling pathway activated by insulin therapy atreperfusionrdquo Basic Research in Cardiology vol 103 no 5 pp444ndash453 2008

[41] C C T Smith R ADixon AMWynne et al ldquoLeptin-inducedcardioprotection involves JAKSTAT signaling that may belinked to the mitochondrial permeability transition porerdquoThe American Journal of PhysiologymdashHeart and CirculatoryPhysiology vol 299 no 4 pp H1265ndashH1270 2010

[42] K T Lamont S Somers L Lacerda L H Opie and SLecour ldquoIs red wine a SAFE sip away from cardioprotectionMechanisms involved in resveratrol- and melatonin-inducedcardioprotectionrdquo Journal of Pineal Research vol 50 no 4 pp374ndash380 2011

[43] FMarzoA LavorgnaGColuzzi et al ldquoErythropoietin in heartand vessels focus on transcription and signalling pathwaysrdquoJournal of Thrombosis and Thrombolysis vol 26 no 3 pp 183ndash187 2008

[44] I Wensley K Salaveria A C Bulmer D G Donner andE F Du Toit ldquoMyocardial structure function and ischaemictolerance in a rodent model of obesity with insulin resistancerdquoExperimental Physiology vol 98 no 11 pp 1552ndash1564 2013

[45] S Pedretti and E Raddatz ldquoSTAT3120572 interacts with nuclearGSK3beta and cytoplasmic RISK pathway and stabilizes rhythmin the anoxic-reoxygenated embryonic heartrdquo Basic Research inCardiology vol 106 no 3 pp 355ndash369 2011

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


activation of other immune cells [17 19] In the rat heart DCsreside close to endocardial blood vessels and align parallel tothe myocardium with their processes interdigitating betweenthe cardiomyocytes [20] Studies in the IR rat model showthat DCs accumulate within the zones bordering infarctedtissue 7 days after myocardial infarction [21] Moreover cir-culating levels of IL-27 are elevated in patients with coronaryartery disease [22] Taken together these results indicate thatIL-27 released by DCs located around peri-infarcted tissuesmay aid myocardial recovery from postischemic insult

Our previously results show that IL-27 attenuates IR-induced cardiac injury in isolated rat hearts and protectsH9c2 cells against severe hypoxia (2 oxygen) induced celldeath [23]These protective effects were blocked by the small-molecule STAT3 inhibitor stattic The aim of the presentstudy was therefore to examine whether IL-27 activates theSTAT3 pathway to protect H9c2 cells against phenotypicchanges associated with MS Because hyperglycemia hyper-tension and dyslipidemia are the most important symp-toms in MS and because of lack of definition of MS atcellular level we applied high glucose (G) high NaCl (S)andor cholesterol (C) treatment to mimic the phenotypeandor extracellular changes associated with MS We firstcompared the effects of G S and C either individuallyor in combination on cell viability and used RT-PCT toexamine their effects on IL-27 receptor expression in treatedH9c2 cells We then further examined the effect of IL-27 byexposing cells to the cytokine in the presence and absence ofstattic Quantitative changes in STAT3 activity under differenttreatment conditions were measured using enzyme-linkedimmunosorbent assay (ELISA) kits

2 Materials and Methods

21 Chemicals IL-27 and stattic (6-nitro-11-dioxide-benzo-[b]thiophene) were obtained fromRampDSystems (Minneapo-lis MN) andMerck (Darmstadt Germany) respectively andwere prepared in sterile water S G and water-soluble C 3-(45-cimethylthiazol-2-yl)-25-diphenyl tetrazolium bromide(MTT) and dimethyl sulfoxide (DMSO) were purchasedfrom Sigma-Aldrich (St Louis MO)

22 Culture of Cardiomyocyte-Like H9c2 Cells The cardio-myoblast cell line H9c2 was purchased from the Biore-source Collection and Research Center (Hsinchu Taiwan)The cell line was originally derived from the AmericanType Culture Collection line CRL-1446 All culture mediaand supplements were purchased from Thermo ScientificHyClone (South Logan UT) H9c2 cells were routinely cul-tured at 37∘C5CO

2inDulbeccorsquosmodified Eaglersquosmedium

containing 10 fetal bovine serum (FBS) 1 L-glutaminepenicillin (100UmLminus1) streptomycin (100mgmLminus1) andamphotericin B (025 120583gmLminus1) as described previously [24]The concentrations of S G andC in the culturemediumwere155mM 5mM and zero respectively Cells were subculturedwhen they reached 80 confluence (approximately every2-3 days) Two days before each experiment cells weresubcultured in 48 well plates (15 times 104well) and grown in

culture medium containing 5 FBS to avoid overstimula-tion of STAT3 Pilot results showed that cells adapted wellunder these conditions with no evident change in lactatedehydrogenase (LDH) release or MTT staining after 48 h ofincubation

On the day of the experiment cells were pretreated withsterile water (vehicle) IL-27 (150 ngmLminus1) or stattic (10 120583M)for 4 h Dosing was based on cell viability Next the cells wereexposed to G (25mM) S (250mM) and C (300120583gmLminus1)either alone or in different combinations (GS SC GC orGSC) for 4 8 24 or 48 h to mimic MS

23 Quantitative Real-Time PCR RNA was extracted usinga commercial kit (RareRNA Bio-East Technology TaipeiTaiwan) as previously described [25] and cDNA synthesizedat 42∘C for 45min (reaction mixture 2120583g RNA 5 120583g ofpoly(dT)15 oligonucleotide primer (Life Technologies) and200 units of reverse transcriptase (Moloney murine leukemiavirus Promega Madison WI)) Quantitative RT-PCR wasperformed in an ABI StepOne Plus system (Applied Biosys-tems Foster City CA) PCR was performed using 100 ng ofcDNA and 30 120583mol of primers (total reaction volume 20120583L)using the SYBR Green PCR master mix kit according tothe manufacturerrsquos instructions (Applied Biosystems) Thefollowing primers were used for PCR gp130 51015840-ATA CCTTAA ACA AGC TCC ACC TTC-31015840 (forward) and 51015840-AGTTTC ATT TCC AAT GAT GGT TCT-31015840 (reverse) WSX-1 51015840-GAA ACC CAA ATG AAG CCA AA-31015840 (forward)and 51015840-GCC TCC TGA CAT CTT CGG TA-31015840 (reverse)glyceraldehyde 3-phosphate dehydrogenase (GAPDH) 51015840-TTA GCA CCC CTG GCC AAG G-31015840 (forward) and 51015840-CTTACTCCTTGGAGGCCATG-31015840 (reverse)The cyclingconditions were as follows 95∘C for 20 s followed by 40cycles of 95∘C for 1 s and 60∘C for 20 s Melting curve analysiswas performed at the end of each PCR experiment Allreactions were run in duplicate The ΔCt (threshold cycle)was calculated by subtracting the rawCt values for the house-keeping gene (GAPDH) from the raw Ct values for the targetgene thereby providing information about relative changesin gene expression Changes in target gene expression werecalculated as 2minusΔCt and expressed as the fold change relativeto that in control cells

24 Cell CytotoxicityViability Assays To assess cytotoxicityLDH levels were measured in cell culture medium (20120583L) atdifferent time points using a commercial kit (Roche AppliedScience Indianapolis IN) as described previously [24] Theadvantage of LDH assay is that it allows cell viability tobe continuously monitored An MTT assay was used tomeasure cell viability Briefly cells were cultured for 48 h inthe presence or absence of the different agents The mediumwas then removed and the cells were washed three times withphosphate-buffered saline (PBS pH 74) MTT (5mgmLminus1 inPBS) was then added to cells for 4 h at 37∘C MTT formazancrystals were then dissolved in DMSO and the optical density(OD) measured in an ELISA plate reader (Amersham-Pharmacia Biotech Piscataway NJ) at 570 nm Cell viability






3 Results








0

10

20

30

40



ab

ab

bc

def

a

cb

dfe

VehicleIL-27

4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448

lowast

lowast

lowast

lowast

LDH

(U Lminus1)

(a)

Con

trol

Con

trolG S C GS

GC SC

GSC G S C G

SG

C SCG

SC

lowast

Cel

l via

bilit

y (M

TT as

say

)

0

20

40

60

80

100

120

VehicleIL-27

abc

def

ab

deflowast

lowast

(b)







C GSC IL-27

gp13

0 m

RNA

fold

chan

ge

00

05

10

15

GSC

lowast

IL-27 +

(a)

C GSC IL-27

WSX

-1 m

RNA

fold

chan

ge

00

05

10

15

GSCIL-27 +

(b)



4 Discussion






(h)0 8 24 48

STAT

3 ac

tivity

(AU

)

05

06

07

08

IL-27 Control

lowast

lowastlowast

(a)

(h)0 8 24 48

25

30

35

40

45

LDH

(U Lminus1)

IL-27 Control

(b)

STAT

3 ac

tivity

(AU

)

00

05

10

C GSC IL-27

lowast

lowast

+

GSCIL-27 +

(c)

0

10

20

30

40

LDH

(U Lminus1)

lowast

+


(d)

Cyto

chro

me c

act

in (A

U)

00

05

10

15

20

Cytochrome

Actin

C GSC IL-27

lowast

+

12kDa

42kDa


GSCIL-27 +

(e)



0

10

20

30

40

50


ab

ac

bc

defdef

c

ab

c

c

b

b

lowast

lowast

lowast lowast

lowast

lowast

LDH

(U Lminus1)


Stattic + IL-27 +

(h)

4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448

(a)

Cel

l via

bilit

y (M

TT as

say

)

0

20

40

60

80

100

120

abc

def

a

def

c

clowast

lowast

lowast

lowast

Stat

tic

Stat

tic+

IL-27G S C GS

GC SC

GSC G S C G

SG

C SCG

SC


(b)






Stattic

STAT

3 ac

tivity

(AU

)

00

05

10

lowast


IL-27 +

(a)

0

10

20

30

40

50lowast

LDH

(U Lminus1)

Stattic


IL-27 +

(b)

00

05

10

15

20

lowast

Cyto

chro

me c

act

in (A

U)

Cytochrome

Actin

12kDa

42kDa

Stattic


IL-27 +Stattic


IL-27 +

(c)











Acknowledgments


References




















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of






3 Results








0

10

20

30

40



ab

ab

bc

def

a

cb

dfe

VehicleIL-27

4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448

lowast

lowast

lowast

lowast

LDH

(U Lminus1)

(a)

Con

trol

Con

trolG S C GS

GC SC

GSC G S C G

SG

C SCG

SC

lowast

Cel

l via

bilit

y (M

TT as

say

)

0

20

40

60

80

100

120

VehicleIL-27

abc

def

ab

deflowast

lowast

(b)







C GSC IL-27

gp13

0 m

RNA

fold

chan

ge

00

05

10

15

GSC

lowast

IL-27 +

(a)

C GSC IL-27

WSX

-1 m

RNA

fold

chan

ge

00

05

10

15

GSCIL-27 +

(b)



4 Discussion






(h)0 8 24 48

STAT

3 ac

tivity

(AU

)

05

06

07

08

IL-27 Control

lowast

lowastlowast

(a)

(h)0 8 24 48

25

30

35

40

45

LDH

(U Lminus1)

IL-27 Control

(b)

STAT

3 ac

tivity

(AU

)

00

05

10

C GSC IL-27

lowast

lowast

+

GSCIL-27 +

(c)

0

10

20

30

40

LDH

(U Lminus1)

lowast

+


(d)

Cyto

chro

me c

act

in (A

U)

00

05

10

15

20

Cytochrome

Actin

C GSC IL-27

lowast

+

12kDa

42kDa


GSCIL-27 +

(e)



0

10

20

30

40

50


ab

ac

bc

defdef

c

ab

c

c

b

b

lowast

lowast

lowast lowast

lowast

lowast

LDH

(U Lminus1)


Stattic + IL-27 +

(h)

4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448

(a)

Cel

l via

bilit

y (M

TT as

say

)

0

20

40

60

80

100

120

abc

def

a

def

c

clowast

lowast

lowast

lowast

Stat

tic

Stat

tic+

IL-27G S C GS

GC SC

GSC G S C G

SG

C SCG

SC


(b)






Stattic

STAT

3 ac

tivity

(AU

)

00

05

10

lowast


IL-27 +

(a)

0

10

20

30

40

50lowast

LDH

(U Lminus1)

Stattic


IL-27 +

(b)

00

05

10

15

20

lowast

Cyto

chro

me c

act

in (A

U)

Cytochrome

Actin

12kDa

42kDa

Stattic


IL-27 +Stattic


IL-27 +

(c)











Acknowledgments


References




















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

10

20

30

40



ab

ab

bc

def

a

cb

dfe

VehicleIL-27

4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448

lowast

lowast

lowast

lowast

LDH

(U Lminus1)

(a)

Con

trol

Con

trolG S C GS

GC SC

GSC G S C G

SG

C SCG

SC

lowast

Cel

l via

bilit

y (M

TT as

say

)

0

20

40

60

80

100

120

VehicleIL-27

abc

def

ab

deflowast

lowast

(b)







C GSC IL-27

gp13

0 m

RNA

fold

chan

ge

00

05

10

15

GSC

lowast

IL-27 +

(a)

C GSC IL-27

WSX

-1 m

RNA

fold

chan

ge

00

05

10

15

GSCIL-27 +

(b)



4 Discussion






(h)0 8 24 48

STAT

3 ac

tivity

(AU

)

05

06

07

08

IL-27 Control

lowast

lowastlowast

(a)

(h)0 8 24 48

25

30

35

40

45

LDH

(U Lminus1)

IL-27 Control

(b)

STAT

3 ac

tivity

(AU

)

00

05

10

C GSC IL-27

lowast

lowast

+

GSCIL-27 +

(c)

0

10

20

30

40

LDH

(U Lminus1)

lowast

+


(d)

Cyto

chro

me c

act

in (A

U)

00

05

10

15

20

Cytochrome

Actin

C GSC IL-27

lowast

+

12kDa

42kDa


GSCIL-27 +

(e)



0

10

20

30

40

50


ab

ac

bc

defdef

c

ab

c

c

b

b

lowast

lowast

lowast lowast

lowast

lowast

LDH

(U Lminus1)


Stattic + IL-27 +

(h)

4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448

(a)

Cel

l via

bilit

y (M

TT as

say

)

0

20

40

60

80

100

120

abc

def

a

def

c

clowast

lowast

lowast

lowast

Stat

tic

Stat

tic+

IL-27G S C GS

GC SC

GSC G S C G

SG

C SCG

SC


(b)






Stattic

STAT

3 ac

tivity

(AU

)

00

05

10

lowast


IL-27 +

(a)

0

10

20

30

40

50lowast

LDH

(U Lminus1)

Stattic


IL-27 +

(b)

00

05

10

15

20

lowast

Cyto

chro

me c

act

in (A

U)

Cytochrome

Actin

12kDa

42kDa

Stattic


IL-27 +Stattic


IL-27 +

(c)











Acknowledgments


References




















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


C GSC IL-27

gp13

0 m

RNA

fold

chan

ge

00

05

10

15

GSC

lowast

IL-27 +

(a)

C GSC IL-27

WSX

-1 m

RNA

fold

chan

ge

00

05

10

15

GSCIL-27 +

(b)



4 Discussion






(h)0 8 24 48

STAT

3 ac

tivity

(AU

)

05

06

07

08

IL-27 Control

lowast

lowastlowast

(a)

(h)0 8 24 48

25

30

35

40

45

LDH

(U Lminus1)

IL-27 Control

(b)

STAT

3 ac

tivity

(AU

)

00

05

10

C GSC IL-27

lowast

lowast

+

GSCIL-27 +

(c)

0

10

20

30

40

LDH

(U Lminus1)

lowast

+


(d)

Cyto

chro

me c

act

in (A

U)

00

05

10

15

20

Cytochrome

Actin

C GSC IL-27

lowast

+

12kDa

42kDa


GSCIL-27 +

(e)



0

10

20

30

40

50


ab

ac

bc

defdef

c

ab

c

c

b

b

lowast

lowast

lowast lowast

lowast

lowast

LDH

(U Lminus1)


Stattic + IL-27 +

(h)

4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448

(a)

Cel

l via

bilit

y (M

TT as

say

)

0

20

40

60

80

100

120

abc

def

a

def

c

clowast

lowast

lowast

lowast

Stat

tic

Stat

tic+

IL-27G S C GS

GC SC

GSC G S C G

SG

C SCG

SC


(b)






Stattic

STAT

3 ac

tivity

(AU

)

00

05

10

lowast


IL-27 +

(a)

0

10

20

30

40

50lowast

LDH

(U Lminus1)

Stattic


IL-27 +

(b)

00

05

10

15

20

lowast

Cyto

chro

me c

act

in (A

U)

Cytochrome

Actin

12kDa

42kDa

Stattic


IL-27 +Stattic


IL-27 +

(c)











Acknowledgments


References




















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


(h)0 8 24 48

STAT

3 ac

tivity

(AU

)

05

06

07

08

IL-27 Control

lowast

lowastlowast

(a)

(h)0 8 24 48

25

30

35

40

45

LDH

(U Lminus1)

IL-27 Control

(b)

STAT

3 ac

tivity

(AU

)

00

05

10

C GSC IL-27

lowast

lowast

+

GSCIL-27 +

(c)

0

10

20

30

40

LDH

(U Lminus1)

lowast

+


(d)

Cyto

chro

me c

act

in (A

U)

00

05

10

15

20

Cytochrome

Actin

C GSC IL-27

lowast

+

12kDa

42kDa


GSCIL-27 +

(e)



0

10

20

30

40

50


ab

ac

bc

defdef

c

ab

c

c

b

b

lowast

lowast

lowast lowast

lowast

lowast

LDH

(U Lminus1)


Stattic + IL-27 +

(h)

4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448

(a)

Cel

l via

bilit

y (M

TT as

say

)

0

20

40

60

80

100

120

abc

def

a

def

c

clowast

lowast

lowast

lowast

Stat

tic

Stat

tic+

IL-27G S C GS

GC SC

GSC G S C G

SG

C SCG

SC


(b)






Stattic

STAT

3 ac

tivity

(AU

)

00

05

10

lowast


IL-27 +

(a)

0

10

20

30

40

50lowast

LDH

(U Lminus1)

Stattic


IL-27 +

(b)

00

05

10

15

20

lowast

Cyto

chro

me c

act

in (A

U)

Cytochrome

Actin

12kDa

42kDa

Stattic


IL-27 +Stattic


IL-27 +

(c)











Acknowledgments


References




















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

10

20

30

40

50


ab

ac

bc

defdef

c

ab

c

c

b

b

lowast

lowast

lowast lowast

lowast

lowast

LDH

(U Lminus1)


Stattic + IL-27 +

(h)

4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448 4 82448

(a)

Cel

l via

bilit

y (M

TT as

say

)

0

20

40

60

80

100

120

abc

def

a

def

c

clowast

lowast

lowast

lowast

Stat

tic

Stat

tic+

IL-27G S C GS

GC SC

GSC G S C G

SG

C SCG

SC


(b)






Stattic

STAT

3 ac

tivity

(AU

)

00

05

10

lowast


IL-27 +

(a)

0

10

20

30

40

50lowast

LDH

(U Lminus1)

Stattic


IL-27 +

(b)

00

05

10

15

20

lowast

Cyto

chro

me c

act

in (A

U)

Cytochrome

Actin

12kDa

42kDa

Stattic


IL-27 +Stattic


IL-27 +

(c)











Acknowledgments


References




















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Stattic

STAT

3 ac

tivity

(AU

)

00

05

10

lowast


IL-27 +

(a)

0

10

20

30

40

50lowast

LDH

(U Lminus1)

Stattic


IL-27 +

(b)

00

05

10

15

20

lowast

Cyto

chro

me c

act

in (A

U)

Cytochrome

Actin

12kDa

42kDa

Stattic


IL-27 +Stattic


IL-27 +

(c)











Acknowledgments


References




















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of







Acknowledgments


References




















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of
































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

research article interleukin-27 protects cardiomyocyte...

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