Research Article Cigarette Smoke Extract-Induced Oxidative ...downloads.hindawi.com/journals/omcl/2016/4676289.pdfResearch Article Cigarette Smoke Extract-Induced Oxidative Stress

Research ArticleCigarette Smoke Extract-Induced Oxidative Stress andFibrosis-Related Genes Expression in Orbital Fibroblasts fromPatients with Gravesrsquo Ophthalmopathy

Hui-Chuan Kau12 Shi-Bei Wu3 Chieh-Chih Tsai2

Catherine Jui-Ling Liu2 and Yau-Huei Wei345

1Department of Ophthalmology Koo Foundation Sun Yat-Sen Cancer Center Taipei 112 Taiwan2Department of Ophthalmology Taipei Veterans General Hospital and National Yang-Ming University Taipei 112 Taiwan3Department of Biochemistry and Molecular Biology National Yang-Ming University Taipei 112 Taiwan4Department of Medicine Mackay Medical College New Taipei City 252 Taiwan5Institute of Biomedical Sciences Mackay Medical College New Taipei City 252 Taiwan

Correspondence should be addressed to Chieh-Chih Tsai cctsai1234yahoocom

Received 20 February 2016 Revised 9 May 2016 Accepted 10 May 2016

Academic Editor Guido Haenen

Copyright copy 2016 Hui-Chuan Kau et alThis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Cigarette smoking is the most important risk factor for the development or deterioration of Gravesrsquo ophthalmopathy Smoke-induced increased generation of reactive oxygen species may be involved However it remains to be clarified how orbital fibroblastsare affected by cigarette smoking Our study demonstrated that Gravesrsquo orbital fibroblasts have exaggerated response to cigarettesmoke extract challenge along with increased oxidative stress fibrosis-related genes expression especially connective tissue growthfactor and intracellular levels of transforming growth factor-1205731 and interleukin-1120573Thefindings obtained in this study provide someclues for the impact of cigarette smoking on Gravesrsquo ophthalmopathy and offer a theoretical basis for the potential and rational useof antioxidants in treating Gravesrsquo ophthalmopathy

1 Introduction

Gravesrsquo ophthalmopathy (GO) also called Gravesrsquo orbitopa-thy thyroid-associated orbitopathy or thyroid eye disease isa cosmetically disfiguring and potentially vision-threateningdisease Although the pathophysiology of GO is still notfully clarified it is known as a complex interplay processbetween multiple endogenous and environmental factors [1ndash4] Cigarette smoking is the most important environmentaland risk factor for the development or deterioration of GOand the risk increases in parallel with the current numberof cigarettes smoked per day [5ndash7] Furthermore cigarettesmoking is associatedwith poor response to treatment forGO[8ndash10] and quitting smoking currently is the only methodof GO prevention [11 12] In the study by Planck et alsome adipocyte-related immediate early genes interleukin-(IL-) 1120573 and IL-6 were overexpressed in smokers withsevere active GO compared to nonsmokers indicating that

smoking activates pathways associated with adipogenesisand inflammation [13] However the exact mechanismsunderlying the deleterious effect of smoking in GO remainto be identified It has been proposed that smoke may inducethe generation of reactive oxygen species (ROS) in the orbitalsocket either through direct contact with the surroundingsinuses or indirectly through the blood circulation [14]Cigarette smoke extract (CSE) has been reported to stimulateadipocyte differentiation in cultured orbital fibroblasts bysynergizing with either IL-1 or ROS [12 14] In our previousstudy we demonstrated that ROS could induce the proteinexpression of connective tissue growth factor (CTGF) animportant fibrogenic factor in culturedGOorbital fibroblasts[15] The aim of the present study is to investigate thechange of oxidative stress fibrotic-related genes expressionand intracellular cytokines in the primary cultures of orbitalfibroblasts in response to CSE We also assessed whetheror not CSE-induced oxidative stress fibrotic-related genes

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 4676289 10 pageshttpdxdoiorg10115520164676289

2 Oxidative Medicine and Cellular Longevity

expression and intracellular cytokines in the GO orbitalfibroblasts could be reduced by pretreatment of the cells withantioxidants

2 Materials and Methods

21 Patients and Tissues Acquisition The surgical specimensof 5 patients with GO (GO1ndashGO5) during orbital decom-pression surgery (one man and four women mean age 37years) and the specimens of 5 age- and sex-matched patients(N1ndashN5) (one man and four women mean age 36 years)who received oculoplastic surgery for noninflammatory con-ditions were used in this study All specimens were collectedin accordance with the Declaration of Helsinki and withinformed consent of the patients All GO patients achievedstable euthyroidism with antithyroid medications for at least6 months before surgery and are maintained in the inactivestage of GO In addition all study subjects had not receivedspecific treatment (systemic steroids or radiotherapy) forGO Exclusion criteria include ocular diseases other thanGO alcohol drinking regular ingestion of antioxidants andpregnancy In addition the patients suffering from chronicor acute diseases such as diabetes mellitus hyperlipidemiadiseases of the lung liver or kidney cancer other endocrinedysfunctions and immunological or inflammatory disorderswere also excluded

22 Primary Cultures of Orbital Fibroblasts The primarycultures of orbital fibroblasts were established according toour previous study [16] Briefly the orbital tissues wereminced aseptically in phosphate-buffered saline (PBS pH 73)and then incubated with a sterile solution containing 05collagenase and dispase (Sigma-Aldrich Chemical Co StLouis MO USA) for 24 hours in an incubator filled withan atmosphere of 5 CO

2and kept at 37∘C The mixture

of digested orbital tissues was pelleted by centrifugation at1000timesg and then resuspended inDulbeccorsquosModified EaglersquosMedium (DMEM purchased from Gibco Life TechnologiesGaithersburg MD USA) containing 10 fetal bovine serum(FBS) and a cocktail of antibiotics (Biological Industries Kib-butz Beit Haemek Israel) which was composed of 100UmLpenicillin G and 100 120583gmL streptomycin sulfate (BiologicalIndustries Kibbutz Beit Haemek Israel)The cultured orbitalfibroblasts were used between the 3rd and 5th passages andthe cell cultures at the same passage number were used forthe same set of experiments

23 Preparation of Cigarette Smoke Extract (CSE) CSE wasprepared according to prior studies with minor modification[12 14] The commercial cigarettes (The Longlife tobaccopackage purchased from Taipei Taiwan) were used in thisstudy and each cigarette contained 10mg tar and 08mgnicotine Ten pieces of cigarettes were smoked continuouslyby a pump-smoke machine and this smoke was used togenerate 200mL of prewarmed CSE-PBS solution Eachcigarette was smoked for 3min and the pH of CSE-PBSsolution was adjusted to 74 and then passed through a 022-120583m pore size filter (Millipore Corporation Billerica MAUSA) to remove large particulates and bacteriaThe CSE-PBS

solution is defined as 100 CSE and this CSE will bediluted with DMEM in the following experiments CSEpreparation is standardized by measuring the absorbance ata wavelength of 320 nm (optical density = 20ndash22) and thepattern of absorbance observed at a wavelength of 320 nmshows insignificant variation between different preparationsof CSE CSE concentrations in the current study are rangedfrom 0 to 15

24 Treatment of Orbital Fibroblasts with CSE and Antiox-idants After washing with PBS buffer (pH 74) twice theorbital fibroblasts were treated with various concentrationsof CSE ranging from 1 to 15 for 24 hours To investigatewhether the effect of CSE could be blocked by antioxi-dants we pretreated the orbital fibroblasts with 1mM N-acetylcysteine (NAC) or 2mM vitamin C (VitC) for 1 hourfollowed by the induction of 5 CSE treatment for another24 hours respectively

25 Determination of Cell Viability Cell viability was mea-sured by the Trypan blue exclusion assay and was countedby using a hemocytometer The number of viable cells wasdetermined on the basis of their exclusion of 04 Trypanblue (Sigma-Aldrich St Louis MO USA) The relative cellviability was normalized by the value of cells without CSEtreatment andwas expressed asmeanplusmn SD of the results fromthree independent experiments

26 Measurement of ROS Content The probes from 2101584071015840-dichlorofluorescin diacetate (DCFH-DA Molecular ProbesEugene OR USA) will be used to evaluate the intra-cellular H

2O2contents [17] After incubation of orbital

fibroblasts with 20120583M DCFH-DA at 37∘C for 20min cellswere trypsinized and then resuspended in 05mL of PBSbuffer (pH 74) and analyzed with a flow cytometer (ModelEPICS XL-MCL Beckman-Coulter Miami FL USA) Theexcitation wavelength is set at 488 nm and the intensity ofemitted fluorescence of a total of 10000 cells at 525 nmis recorded on channel FL1 for the DCFH-DA probeData were analyzed by the EXPO32 software (Beckman-Coulter Miami FL USA) The intracellular H

2O2con-

tents in the treated cells were presented as relative valuescompared with that of the cells without H

2O2or CSE

treatment

27 Determination of Lipid Peroxidation The lipid peroxida-tion product malondialdehyde (MDA) in cultured orbitalfibroblasts was measured by the spectrophotometric assay kit(MDA-586 OxisResearch Inc Portland OR USA) accord-ing to themanufacturerrsquos instructions [18]TheMDA is quan-tified in the reaction with a chromogenic regent N-methyl-2-phenylindole to form an intensely colored carbocyaninedye with a maximum absorbance at 586 nm The method isspecific for MDA instead of other lipid peroxidation prod-ucts such as 4-hydroxyalkenal because they cannot producesignificant absorbance at 586 nm under the experimentalconditions AnMDA standard curve was established by usingtheMDA samples at the concentration range of 0ndash50 120583M andthe MDA levels in orbital fibroblasts were normalized to cell

Oxidative Medicine and Cellular Longevity 3

numbers (106 cells) The results were expressed as mean plusmn SDof the results from three independent experiments

28 Western Blot Analysis An aliquot of 50120583g proteinswas separated on 10 SDS-PAGE and blotted onto a pieceof the PVDF membrane (Amersham-Pharmacia BiotechInc Buckinghamshire UK) After blocking by 5 skimmilk in the TBST buffer (50mM Tris-HCl 150mM NaCland 01 Tween 20 pH 74) at room temperature for 1hour the membrane was incubated for another 1 hour withthe primary antibody at room temperature After washingthree times with the TBST buffer the membrane was incu-bated with a horseradish peroxidase- (HRP-) conjugatedsecondary antibody for another 1 hour at room temperatureAn enhanced chemiluminescence detection kit (Amersham-Pharmacia Biotech Inc Buckinghamshire UK) was usedto detect the protein signals with a Fuji X-ray film (FujiFilm Corp Tokyo Japan) and the intensities of signals werequantified by ImageScanner III with LabScan 60 software(GE Healthcare BioSciences Corp Piscataway NJ USA)The antibodies of HO-1 (SC-10789) and CTGF (SC-14939)were purchased from Santa Cruse Biotechnology Inc (CAUSA) and 120573-actin (A1978) was purchased from Sigma-Aldrich Chemical Co (MO USA) respectively All data wereexpressed as mean plusmn SD of the results obtained from threeindependent experiments

29 Real-Time Reverse Transcriptase-Polymerase Chain Reac-tion (RT-PCR) Theexpression levels of fibrosis-related geneswere determined by SYBR green-based real-time quantitativePCR Briefly the total cellular RNA from orbital fibroblastslysates was extracted with a chloroform solution after addingthe TRIZol reagent (MO USA) The extracted RNA wasprecipitated with isopropanol solution dried on ice anddissolved in DEPC-H

2O An aliquot of 5 120583g RNA was

reverse-transcribed to cDNA with the Ready-to-Go RT-PCR kit (Amersham Biosciences Uppsala Sweden) at 42∘Covernight Quantitative RT-PCR was performed using theSYBR Green Master kit (Sigma-Aldrich) according to themanufacturerrsquos instructions [18] The primer pairs were 51015840-CTCAACACGGGAAACCTCAC-31015840 and 51015840-CGCTCCACC-AACTAAGAACG-31015840 for 18S rRNA 51015840-CTGCAGGCTAGA-GAAGCAGAG-31015840 and 51015840-GATGCACTTTTTGCCCTTCT-31015840 for CTGF 51015840-CTGGCCGAAAATACATTGTAA-31015840 and51015840-CCACAGTCGGGTCAGGAG-31015840 for fibronectin and 51015840-GGACATCCACTTCCACAGC-31015840 and 51015840-GGTCATCGT-CGCCTTCTC-31015840 for apolipoprotein J respectively ThemRNA expression level of each gene in the orbital fibroblastswas normalized with the mRNA level of the 18S rRNA generespectively

210 Measurement of the Intracellular Cytokine Content Thehuman transforming growth factor-beta 1 (TGF-1205731 catalogDB100B) and interleukin-1120573 (IL-1120573 catalog DLB50) levelsin cell culture supernatant were quantified with enzyme-linked immunosorbent assay kits purchased from RampDSystems Inc (Minneapolis MN) Briefly about 105 orbitalfibroblasts were seeded in a 35-cm culture dish and incubatedfor 48 hours at 37∘C in a cell incubator with an atmosphere

of 5 CO2followed by treatment of 5 CSE for another

24 hours or the cells were pretreated with 1mM NAC or2mM VitC for 1 hour followed by the induction of CSEtreatment for another 24 hours respectively According to themanufacturerrsquos recommendation and our previous study [19]cell culture supernatant was centrifuged at 12000timesg at 4∘Cand the aliquots were immediately assayedThe standards forTGF-1205731 and IL-1120573 were used in a range of 0ndash200 pgmL andthe results were normalized by the cell number and expressedas pg104 cells

211 Statistical Analysis The Microsoft Excel 2010 statisticalpackage and SigmaPlot software version 123 (Systat SoftwareInc San Jose CA USA) were used to analyze the resultsand data were presented as means plusmn standard deviation (SD)of the results obtained from three independent experimentsThe significance level of the difference between the controland the experimental groups was determined by Studentrsquos 119905-test A difference was considered statistically significant whenthe lowast119901 value lt 005 and lowastlowast119901 value lt 001 respectively

3 Results

31 CSE-Induced Cytotoxicity and Oxidative Stress in theOrbital Fibroblasts In order to investigate the cytotoxic effectof smoke extracts in the orbital fibroblasts we treated theorbital fibroblasts with various concentrations of CSE for 24hoursThe effect of CSE ranging from0 to 15 on the viabilityof the orbital fibroblasts from patients with GO (GO1ndashGO5)and normal subjects (N1ndashN5) as determined with the Trypanblue exclusion assay is illustrated in Figure 1 The data showthat both normal and GO fibroblasts were reduced in a dose-dependent manner respectively (Figure 1(a)) The differencein cell viability between normal and GO orbital fibroblastswas statistically significant upon treatment with 5CSE (Fig-ure 1(b) 85 versus 62 119901 = 00374) On the other hand wealso observed the CSE-induced oxidative stress and oxidativeresponse in the GO orbital fibroblasts After treatment of GOorbital fibroblasts with various concentrations of CSE (0ndash15) for 24 hours the intracellular ROS measured by DCFstaining with a flow cytometer and the heme oxygenase-1 (HO-1) protein expression with Western blot were bothincreased in a dose-dependent manner (Figure 2)

32 Susceptible to 5 CSE-Induced Oxidative Stress andOxidative Damage in the GO Orbital Fibroblasts as Comparedto Those of Normal Controls and the Protective Role ofAntioxidants Due to the fact that the exposure of 5 CSEcould significantly reduce the cell viability in the GO orbitalfibroblasts as compared to those in the normal controlswe decided to treat normal and GO orbital fibroblasts with5 CSE in the following experiments After treatment oforbital fibroblasts with 5 CSE for 24 hours the intracellularROS measured by DCF staining was significantly increasedin both normal and GO orbital fibroblasts respectively(Figure 3(a) 119901 = 00347 and 119901 = 00021) In additionthe lipid peroxidation marker malondialdehyde (MDA) wasalso significantly increased in both normal and GO orbitalfibroblasts respectively after the addition of 5 CSE for


Cel

l via

bilit

y (

)

30

40

50

60

70

80

90

100

110

CSE ()0 1 2 5 10 15

lowast

lowastlowast

lowastlowast

Normal subjects (N1ndashN5)GO patients (GO1ndashGO5)

(a)C

ell v

iabi

lity

()

0

20

40

60

80

100

120

lowastp = 00374

lowastp = 0041 lowastp = 00158

Control5 CSE

GO patients(n = 5)

Normal subjects(n = 5)

(b)

Figure 1 Susceptible to cigarette smoke extract- (CSE-) caused cytotoxicity in the GO orbital fibroblasts as compared to those of normalcontrols (a) After treatment of orbital fibroblasts from GO patients (GO1ndashGO5) and age-matched normal subjects (N1ndashN5) with variousconcentrations of CSE ranging from 0 to 15 for 24 hours the cell viability was determined by the Trypan blue exclusion assay (b)Themeanvalues of cell viability by 5 CSE treatment were shown in the histogram and data were presented as means plusmn SD of the results from threeindependent experiments (lowast119901 lt 005 lowastlowast119901 lt 001 versus the indicated group)

24 hours (Figure 3(b) 119901 = 00186 and 119901 = 00032)Moreover we noted that the induction ratio of intracellularROS and MDA levels after treatment with 5 CSE was morepronounced in the GO orbital fibroblasts than those in thenormal controls respectively (Figures 3(a) and 3(b) 119901 =00273 and 119901 = 00075) On the other hand we also observedthe protective effects of NAC and VitC on CSE-inducedoxidative stress and oxidative damage in the GO orbitalfibroblasts respectively Preincubation with 1mM NAC or2mM VitC for 1 hour respectively significantly decreased5 CSE-induced elevations of intracellular ROS measuredby DCF staining in the GO orbital fibroblasts (Figure 3(c)119901 = 00451 and 119901 = 00071) A significant reduction in 5CSE-induced elevations of MDA contents was also obtainedafter the GO orbital fibroblasts were preincubated with 1mMNACor 2mMVitC respectively (Figure 3(d)119901 = 00382 and119901 = 00064)

33 Susceptible to 5CSE-InducedChanges of Fibrosis-RelatedGenes Expression in theGOOrbital Fibroblasts as Compared toThose of Normal Controls Previously we have shown that theelevated intracellular oxidative stress was associated with theincrease of fibrosis-related genes expression in the GO orbitalfibroblasts [15] In this study we further investigated whether5 CSE-induced ROS could lead to inducing the fibrosis-related genes expression in the orbital fibroblasts By a SYBRgreen-based RT-PCR we observed the significant elevationin the levels of fibrosis-related genes expression includingapolipoprotein J fibronectin and CTGF in both normal andGO orbital fibroblasts after treatment of 5 CSE for 24 hours(Table 1 119901 = 00431 versus 119901 = 00085 119901 = 00318 versus

119901 = 00033 and 119901 = 00441 versus 119901 = 0064 resp) Inaddition the induction ratio of apolipoprotein J fibronectinand CTGF by 5 CSE was more pronounced in the GOorbital fibroblasts than those in the normal controls (Table 1119901 = 00086 119901 = 00031 and 119901 = 00054 resp)

34 Inhibition of 5 CSE-Induced Fibrosis-Related GenesExpression by Antioxidants in GO Orbital Fibroblasts Toinvestigate whether 5 CSE-induced fibrosis-related genesexpression in GO orbital fibroblasts could be blocked byantioxidants we pretreated the orbital fibroblasts with 1mMNAC or 2mM vitamin C respectively for 1 hour followed bythe 5CSE treatmentThe results showed that preincubationof cells with 1mM NAC or 2mM VitC could significantlyinhibit the 5 CSE-induced fibrosis-related genes expressionincluding apolipoprotein J fibronectin and CTGF in theGO orbital fibroblasts by a SYBR green-based RT-PCR(Table 2) The inhibition ratio by 1mM NAC treatment forapolipoprotein J fibronectin andCTGF is 14 17 and 13respectively (119901 = 00437 119901 = 00251 and 119901 = 00470 resp)The inhibition ratio in the GO orbital fibroblasts by 2mMvitamin C treatment for apolipoprotein J fibronectin andCTGF is 24 28 and 27 respectively (119901 = 00294 119901 =00085 and119901 = 00224 resp) Accordingly we also examinedCSE-induced expression levels of CTGF protein by Westernblot The result showed that the protein expression of CTGFwas significantly increased in the GO orbital fibroblasts afterthe addition of 5 CSE for 24 hours (Figure 4 119901 = 00041)Besides the pretreatment of GO fibroblasts with 2mM VitCcould also inhibit 43 of the elevations in the CTGF proteinexpression (Figure 4 119901 = 00371)


0 1 2 5 10 15

Relat

ive D

CF in

tens

ity (

)

0

50

100

150

200

250

CSE ()

lowastlowast

lowast

lowastlowast

lowast

(a)

HO-1

CSE ()

Rela

tive i

nten

sity

(fold

s)0

2

4

6

8

10

12

0 2 5 10 15CSE ()

lowastlowast

lowastlowast

lowastlowast

lowastlowast

120573-actin

0 2 5 10 15

(b)

Figure 2 CSE-induced oxidative stress and oxidative response in a dose-dependent manner in the GO orbital fibroblasts (a) After treatmentof GO orbital fibroblasts with various concentrations of CSE ranging from 0 to 15 for 24 hours the intracellular ROS was measured byDCF staining with a flow cytometer and (b) the HO-1 protein expression was determined byWestern blot The representative histogram wasconstructed on the basis of the results from three independent experiments and data were presented as means plusmn SD (lowast119901 lt 005 lowastlowast119901 lt 001versus the control group without CSE treatment)

Table 1 The induction ratio of fibrotic-related genes expression in the orbital fibroblasts from normal subjects and GO patients before andafter 5 CSE treatment

Fibrosis-related genes Basal levels(mean plusmn SD)a

5 CSE-treated(mean plusmn SD)a

Induction ratio ()(mean plusmn SD) p value

Apolipoprotein JNormal (119899 = 5) 10836 plusmn 563 13549 plusmn 974 12583 plusmn 1174 00431GO (119899 = 5) 17354 plusmn 667 30167 plusmn 1273 16896 plusmn 1547 00085

119901 = 00381 119901 = 00086

FibronectinNormal (119899 = 5) 10538 plusmn 583 14737 plusmn 947 13984 plusmn 1437 00318GO (119899 = 5) 26345 plusmn 1037 48421 plusmn 1587 18047 plusmn 1671 00033

119901 = 00046 119901 = 00031

CTGFNormal (119899 = 5) 11738 plusmn 422 15857 plusmn 842 13509 plusmn 1288 00441GO (119899 = 5) 22307 plusmn 1417 37922 plusmn 1485 16974 plusmn 1153 00064

119901 = 00074 119901 = 00054aThe expression levels from 5 normal subjects and 5 GO patients were normalized to each individual 18S rRNA gene expression followed by adjusting to N1whose expression was defined as 100

35 Susceptible to 5 CSE-Induced Changes of IntracellularCytokines in the GO Orbital Fibroblasts as Compared to Thoseof Normal Controls and the Protective Role of AntioxidantsThe changes in the intracellular cytokines after stimulationof orbital fibroblasts with 5 CSE are shown in Table 3 Basallevels of TGF-1205731 and IL-1120573 were significantly higher in the

GO orbital fibroblasts as compared to those of the normalcontrols (119901 = 0004 and 119901 = 0008 resp) In addition CSEinduced significant increase in TGF-1205731 and IL-1120573 levels inGO orbital fibroblasts as compared to the respective controls(119901 = 0006 and 119901 = 0005 resp) Moreover the inductionratio of TGF-1205731 and IL-1120573 after stimulation with 5 CSE


Relat

ive D

CF in

tens

ity (

)

0

50

100

150

200

Control5 CSE

lowastp = 00273

lowastp = 00347

lowastlowastp = 00021

GO patients(n = 5)


(a)

0

100

200

300

400

500

lowastp = 00186



MD

A (n

mol

per

106

cells

)

Control5 CSE

GO patients(n = 5)


(b)

Relat

ive D

CF in

tens

ity (

)

0

50

100

150

200

250

5 C

SE

5 C

SE2

mM

VitC

+

2m

M V

itC

5 C

SE1

mM

NAC

+

1m

M N

AC

lowastlowastp = 00047lowastp = 00451


GO

pat

ient

s(n

=5)

(c)

0

100

200

300

400

500

5 C

SE

5 C

SE2

mM

VitC

+

2m

M V

itC

5 C

SE1

mM

NAC

+

1m

M N

AC

MD

A (n

mol

per

106

cells

)

lowastlowastp = 00035 lowastp = 00382


GO

pat

ient

s(n

=5)

(d)

Figure 3 Susceptible to CSE-induced oxidative stress and oxidative damage in the GO orbital fibroblasts as compared to those of normalcontrols and the role of antioxidants (a) After treatment of orbital fibroblasts from GO patients (GO1ndashGO5) and normal subjects (N1ndashN5)with 5 CSE for 24 hours the intracellular ROS and (b) MDA levels were determined as described in Materials and Methods (c) Afterpretreatment of GO orbital fibroblasts with 1mMNAC or 2mM vitamin C (VitC) for 1 hour followed by the addition of 5 CSE for another24 hours the intracellular ROS and (d) MDA levels were determined The results were from three independent experiments and data werepresented as means plusmn SD (lowast119901 lt 005 lowastlowast119901 lt 001 versus the indicated group)

was more pronounced in GO orbital fibroblasts than thosein the normal controls (119901 = 0008 and 119901 = 0003 resp)Table 4 showed a significant reduction in 5 CSE-inducedelevations of intracellular TGF-1205731 and IL-1120573 after the GOorbital fibroblasts were pretreatedwith 1mMNAC (119901 = 0037and119901 = 0028 resp) or 2mMVitC (119901 = 0008 and119901 = 0003resp)

4 Discussion

Evidence is mounting that oxidative stress plays an importantrole in the development of GO [17 20ndash22] We demonstratedin this study that CSE elicited more pronounced response ofoxidative stress in GO orbital fibroblasts More importantlythis is the first study to reveal that CSE could induce


Table 2The inhibition ratio of 5 CSE-induced fibrotic-related genes expression by antioxidants in the orbital fibroblasts fromGO patients

Fibrosis-relatedgenes


1mM NAC +5 CSE-treated(mean plusmn SD)a

Inhibition ratio ()(mean plusmn SD)

2mM VitC+ 5 CSE-treated(mean plusmn SD)a


Apolipoprotein JGO (119899 = 5) 28517 plusmn 2247 24518 plusmn 2033 1402 plusmn 327 21732 plusmn 1853 2379 plusmn 847

119901 = 00437 119901 = 00294

FibronectinGO (119899 = 5) 49635 plusmn 2864 41211 plusmn 2672 1696 plusmn 448 35538 plusmn 2329 2840 plusmn 505

119901 = 00251 119901 = 00085

CTGFGO (119899 = 5) 35032 plusmn 2573 30532 plusmn 2337 1282 plusmn 567 25429 plusmn 2177 2741 plusmn 967


Table 3The induction ratio of intracellular cytokine in the orbital fibroblasts from normal subjects and GO patients before and after 5 CSEtreatment

Cytokinesspecies

Basal levels(mean plusmn SD)

5 CSE-treated(mean plusmn SD)


TGF-1205731 (pg per 104 cells)Normal (n = 5) 8746 plusmn 1388 11705 plusmn 1811 13383 plusmn 1061 0041GO (n = 5) 14867 plusmn 1877 28237 plusmn 1629 18967 plusmn 1255 0006

119901 = 0004 119901 = 0008

IL-1120573 (pg per 104 cells)Normal (119899 = 5) 5705 plusmn 973 6819 plusmn 833 11907 plusmn 722 0033GO (119899 = 5) 7583 plusmn 752 12238 plusmn 1137 16158 plusmn 973 0005

119901 = 0008 119901 = 0003

fibrosis-related genes expression especially CTGF in the GOorbital fibroblasts as compared with those of normal controlsIn addition pretreatment with antioxidants such as NACand vitamin C could confer significant protection against theinfluence of CSE on oxidative damage fibrosis-related genesexpression and induction of TGF-1205731 and IL-1120573

Orbital fibroblasts one of the major target cells in GOare associated with many GO-related pathologic conditionsincluding oxidative stress [14 15 23] Oxidative stress alsohas been suggested to play a role on the deleterious impactof smoking in GO [14] The present study provided evidencethat the GO orbital fibroblasts were more susceptible toCSE-induced cytotoxicity and oxidative stress than thoseof normal controls Accumulation of CSE-induced ROSmay cause more oxidative damage including oxidative DNAdamage and lipid peroxidation which could explain in partour previous observation that smokers had significant higherurinary 8-hydroxy-21015840-deoxyguanosine (8-OHdG) than didnever smokers in GO patients [24] In addition increasedgeneration of ROS especially the superoxide anions andhydrogen peroxide can stimulate proliferation of GO orbitalfibroblasts [19] and induce the production of proinflam-matory cytokines [25] which all are key pathological fea-tures in GO Moreover cigarette smoke-mediated oxidativestress could recruit inflammatory and immune cells such

as lymphocytes macrophages and neutrophils and activatesome proinflammatory mediators [26] which may exacer-bate the inflammation and tissue remodeling processes ofGO

The disease course of GO is characterized not only byearly inflammatory process but also by tissue remodelingandor fibrosis Although fibrosis represents a quiescent stagein GO it may cause much of the substantial morbidity whichis often unresponsive to conventional medical treatment andrequires surgical intervention Oxidative stress is known asa factor that can induce various pathological fibrosis [2728] In current study we also noted that cigarette smoke-mediated oxidative stress could induce fibrotic-related genesexpression including apolipoprotein J fibronectin andCTGFin the GO orbital fibroblasts and these effects could beinhibited by pretreatment with antioxidants ApolipoproteinJ fibronectin and CTGF are commonly known as impor-tant fibrogenic factors Although it remains to be clarifiedwhether apolipoprotein J plays as a fibrosis biomarker oradaptive response in the development of fibrotic process ofGO CTGF has been shown to be substantially involved inthe pathogenesis of various fibrotic disorders such as liverheart kidney and ocular fibrosis [29ndash32] CTGF can exhibitdiverse cellular functions including extracellular matrixproduction cell migration proliferation and differentiation


Table 4 The inhibition ratio of 5 CSE-induced intracellular cytokine by antioxidants in the GO orbital fibroblasts

Cytokinesspecies


1mM NAC+ 5 CSE-treated(mean plusmn SD)


2mM VitC+ 5 CSE-treated(mean plusmn SD)

Inhibition ratio()

(mean plusmn SD)TGF-1205731 (pg per 104 cells)GO (119899 = 5) 26591 plusmn 1367 17708 plusmn 1405 3341 plusmn 1239 15339 plusmn 922 4232 plusmn 1125

119901 = 0037 119901 = 0008

IL-1120573 (pg per 104 cells)GO (119899 = 5) 13977 plusmn 820 9283 plusmn 937 3309 plusmn 733 8127 plusmn 726 4185 plusmn 528

119901 = 0028 119901 = 0003

CTGF

GO1 GO2

GO4 GO5

CTGF

120573-actin

120573-actin

GO3

Ctr VitC VitC + CSECSE

Ctr VitC VitC + CSECSE Ctr VitC VitC + CSECSE


(a)

CTG

F ex

pres

sion

(rel

ated

to 120573

-act

in ex

pres

sion)

0

1

2

3

4

5

GO 5 CSE2mM VitC5 CSE

2mM VitC +


(b)

Figure 4 Inhibition of CSE-induced fibrotic markers by pretreatment of the GO orbital fibroblasts with antioxidants (a) After pretreatmentof orbital fibroblasts from GO patients (GO1ndashGO5) with 2mM VitC for 1 hour followed by the addition of 5 CSE for another 24 hours theCTGF protein expression was determined by Western blot respectively (b) The levels of the CTGF protein expression were normalized toeach of corresponding 120573-actin expression levels and were adjusted to GO1 without CSE andor vitamin C (VitC) treatment whose CTGFexpression was defined as 100The representative histogramwas constructed on the basis of the results from three independent experimentsand data were presented as means plusmn SD (lowast119901 lt 005 lowastlowast119901 lt 001 versus the group without CSE treatment) Ctr without CSE treatment


Importantly CTGF is critical for TGF-120573-mediated fibroblast-myofibroblast transdifferentiation and subsequent depositionof extracellular matrix [33] which may contribute to thetissue remodeling and fibrosis process in GO It has alsobeen reported that periodontal fibrosis can be promotedby nicotine from smoking via effects on CTGF [34] Takentogether previous reports and our findings in this study mayexplain in part why smoking is associatedwith severeGO andpoor response to immunosuppressive therapy in GO

We previously revealed that low concentrations of hydro-gen peroxide can induce the production of proinflammatorycytokines such as TGF-1205731 and IL-1120573 in GO orbital fibroblasts[19] The observations in this study further show that 5CSE induced higher intracellular levels of TGF-1205731 and IL-1120573in GO orbital fibroblasts than those in the normal controlsMoreover 5 CSE-induced elevation of TGF-1205731 and IL-1120573in GO orbital fibroblasts was abolished by the antioxidanttreatment TGF-1205731 a potent fibrogenic factor has beenreported to modulate the proliferation of fibroblasts andtissue fibrosis [35 36] IL-1120573 an important proinflammatorycytokine in GO has been shown to stimulate hyaluronansynthesis in orbital fibroblasts [37] Fibroblast proliferationtissue fibrosis and hyaluronan accumulation are all impor-tant pathological features in the clinical expression of GOCollectively these findings suggest that oxidative stress playsan important role on the deteriorative effect of cigarettesmoking on GO

In conclusion this study demonstrated that GO fibrob-lasts have exaggerated response to cigarette smoke extractchallenge along with increased oxidative stress fibrosis-related genes expression and intracellular levels of TGF-1205731 and IL-1120573 The findings obtained in this study providesome clues for the impact of cigarette smoking on GO andoffer a theoretical basis for the potential and rational use ofantioxidants in treating GO

Competing Interests

None of the authors has any commercial interest in thematerial mentioned herein

Acknowledgments

This study was supported by Grants MOST104-2314-B-075-056-MY2 andMOST104-2320-B-715-006-MY2 from theMinistry of Science and Technology Executive Yuan Taiwanand a Grant V105-C-022 from Taipei Veterans GeneralHospital Taiwan The authors would like to express theirappreciation of the technical support and service of the corefacilities at National Yang-Ming University and at MackayMedical College

References

[1] R S Bahn ldquoCurrent Insights into the pathogenesis of GravesrsquoophthalmopathyrdquoHormone and Metabolic Research vol 47 no10 pp 773ndash778 2015

[2] J J Khong A A McNab P R Ebeling J E Craig and DSelva ldquoPathogenesis of thyroid eye disease review and update

on molecular mechanismsrdquo British Journal of Ophthalmologyvol 100 no 1 pp 142ndash150 2015

[3] B S Prabhakar R S Bahn and T J Smith ldquoCurrent perspectiveon the pathogenesis of gravesrsquo disease and ophthalmopathyrdquoEndocrine Reviews vol 24 no 6 pp 802ndash835 2003

[4] R S Douglas and S Gupta ldquoThe pathophysiology of thyroideye disease implications for immunotherapyrdquo Current Opinionin Ophthalmology vol 22 no 5 pp 385ndash390 2011

[5] M N Stan and R S Bahn ldquoRisk factors for development ordeterioration of Gravesrsquo ophthalmopathyrdquo Thyroid vol 20 no7 pp 777ndash783 2010

[6] L Bartalena ldquoPrevention of Gravesrsquo ophthalmopathyrdquo BestPractice and Research Clinical Endocrinology and Metabolismvol 26 no 3 pp 371ndash379 2012

[7] J Pfeilschifter and R Ziegler ldquoSmoking and endocrine ophthal-mopathy impact of smoking severity and current vs lifetimecigarette consumptionrdquo Clinical Endocrinology vol 45 no 4pp 477ndash481 1996

[8] L Xing L YeW Zhu et al ldquoSmoking was associated with poorresponse to intravenous steroids therapy in Gravesrsquo ophthal-mopathyrdquo British Journal of Ophthalmology vol 99 no 12 pp1686ndash1691 2015

[9] A Eckstein B Quadbeck G Mueller et al ldquoImpact of smokingon the response to treatment of thyroid associated ophthal-mopathyrdquo British Journal of Ophthalmology vol 87 no 6 pp773ndash776 2003

[10] R S Prabhu L Liebman T Wojno B Hayek W A Halland I Crocker ldquoClinical outcomes of radiotherapy as initiallocal therapy for Gravesrsquo ophthalmopathy and predictors of theneed for post-radiotherapy decompressive surgeryrdquo RadiationOncology vol 7 article 95 2012

[11] L Bartalena W M Wiersinga and A Pinchera ldquoGravesrsquoophthalmopathy state of the art and perspectivesrdquo Journal ofEndocrinological Investigation vol 27 no 3 pp 295ndash301 2004

[12] T J Cawood P Moriarty C OrsquoFarrelly and D OrsquoShea ldquoSmok-ing and thyroid-associated ophthalmopathy a novel explana-tion of the biological linkrdquo Journal of Clinical Endocrinology andMetabolism vol 92 no 1 pp 59ndash64 2007

[13] T Planck B Shahida H Parikh et al ldquoSmoking inducesoverexpression of immediate early genes in active GravesrsquoophthalmopathyrdquoThyroid vol 24 no 10 pp 1524ndash1532 2014

[14] J S YoonH J LeeMK Chae S Y Lee andE J Lee ldquoCigarettesmoke extract-induced adipogenesis in Gravesrsquo orbital fibrob-lasts is inhibited by quercetin via reduction in oxidative stressrdquoJournal of Endocrinology vol 216 no 2 pp 145ndash156 2013

[15] C Tsai S Wu P Chang Y Wei and P Soares ldquoAlteration ofConnective Tissue Growth Factor (CTGF) expression in orbitalfibroblasts from patients with gravesrsquo ophthalmopathyrdquo PLoSONE vol 10 no 11 article e0143514 2015

[16] C-C Tsai S-B Wu C-Y Cheng et al ldquoIncreased responseto oxidative stress challenge in Gravesrsquo ophthalmopathy orbitalfibroblastsrdquoMolecular Vision vol 17 pp 2782ndash2788 2011

[17] C-C Tsai S-B Wu C-Y Cheng et al ldquoIncreased oxidativeDNA damage lipid peroxidation and reactive oxygen speciesin cultured orbital fibroblasts from patients with Gravesrsquo oph-thalmopathy evidence that oxidative stress has a role in thisdisorderrdquo Eye vol 24 no 9 pp 1520ndash1525 2010

[18] H-T Lee C-S Lin C-S Lee C-Y Tsai and Y-H WeildquoIncreased 8-hydroxy-21015840-deoxyguanosine in plasma anddecreased mRNA expression of human 8-oxoguanine DNAglycosylase 1 anti-oxidant enzymes mitochondrial biogenesis-related proteins and glycolytic enzymes in leucocytes in patients


with systemic lupus erythematosusrdquo Clinical and ExperimentalImmunology vol 176 no 1 pp 66ndash77 2014

[19] C-C Tsai S-B Wu S-C Kao H-C Kau F-L Lee and Y-HWei ldquoThe protective effect of antioxidants on orbital fibroblastsfrom patients with Gravesrsquo ophthalmopathy in response tooxidative stressrdquoMolecular Vision vol 19 pp 927ndash934 2013

[20] M Zarkovic ldquoThe role of oxidative stress on the pathogenesis ofGravesrsquo diseaserdquo Journal of Thyroid Research vol 2012 ArticleID 302537 5 pages 2012

[21] C Marcocci and L Bartalena ldquoRole of oxidative stress andselenium in Gravesrsquo hyperthyroidism and orbitopathyrdquo Journalof Endocrinological Investigation vol 36 no 10 supplement pp15ndash20 2013

[22] L Bartalena M L Tanda E Piantanida and A Lai ldquoOxidativestress and Gravesrsquo ophthalmopathy in vitro studies and ther-apeutic implicationsrdquo BioFactors vol 19 no 3-4 pp 155ndash1632003

[23] S Hwang JW Byun J S Yoon and E J Lee ldquoInhibitory effectsof 120572-lipoic acid on oxidative stress-induced adipogenesis inorbital fibroblasts from patients with graves ophthalmopathyrdquoMedicine vol 95 no 2 article e2497 2016

[24] C-C Tsai C-Y Cheng C-Y Liu et al ldquoOxidative stress inpatients with Gravesrsquo ophthalmopathy relationship betweenoxidative DNA damage and clinical evolutionrdquo Eye vol 23 no8 pp 1725ndash1730 2009

[25] H B Burch S Lahiri R S Bahn and S Barnes ldquoSuperoxideradical production stimulates retroocular fibroblast prolifera-tion in gravesrsquo ophthalmopathyrdquoExperimental Eye Research vol65 no 2 pp 311ndash316 1997

[26] S Rajendrasozhan S-R Yang I Edirisinghe H Yao DAdenuga and I Rahman ldquoDeacetylases and NF-120581B in redoxregulation of cigarette smoke-induced lung inflammation epi-genetics in pathogenesis of COPDrdquo Antioxidants and RedoxSignaling vol 10 no 4 pp 799ndash811 2008

[27] M Aragno R Mastrocola G Alloatti et al ldquoOxidative stresstriggers cardiac fibrosis in the heart of diabetic ratsrdquoEndocrinol-ogy vol 149 no 1 pp 380ndash388 2008

[28] V Sanchez-Valle N C Chavez-Tapia M Uribe and NMendez-Sanchez ldquoRole of oxidative stress and molecularchanges in liver fibrosis a reviewrdquoCurrentMedicinal Chemistryvol 19 no 28 pp 4850ndash4860 2012

[29] M Abou-Shady H Friess A Zimmermann et al ldquoConnectivetissue growth factor in human liver cirrhosisrdquo Liver vol 20 no4 pp 296ndash304 2000

[30] H Yokoi A SugawaraMMukoyama et al ldquoRole of connectivetissue growth factor in profibrotic action of transforminggrowth factor-120573 a potential target for preventing renal fibrosisrdquoAmerican Journal of Kidney Diseases vol 38 no 4 pp S134ndashS138 2001

[31] A Daniels M van Bilsen R Goldschmeding G J van DerVusse and F A van Nieuwenhoven ldquoConnective tissue growthfactor and cardiac fibrosisrdquoActa Physiologica vol 195 no 3 pp321ndash338 2009

[32] S He Y Chen R Khankan et al ldquoConnective tissue growthfactor as a mediator of intraocular fibrosisrdquo Investigative Oph-thalmology and Visual Science vol 49 no 9 pp 4078ndash40882008

[33] P A Folger D Zekaria G Grotendorst and S K MasurldquoTransforming growth factor-120573mdashstimulated connective tissuegrowth factor expression during corneal myofibroblast differ-entiationrdquo Investigative Ophthalmology and Visual Science vol42 no 11 pp 2534ndash2541 2001

[34] H Takeuchi S Kubota E Murakashi et al ldquoNicotine-inducedCCN2 from smoking to periodontal fibrosisrdquo Journal of DentalResearch vol 89 no 1 pp 34ndash39 2010

[35] N Khalil Y D Xu R OrsquoConnor and V Duronio ldquoProliferationof pulmonary interstitial fibroblasts is mediated by transform-ing growth factor-1205731-induced release of extracellular fibroblastgrowth factor-2 and phosphorylation of p38 MAPK and JNKrdquoThe Journal of Biological Chemistry vol 280 no 52 pp 43000ndash43009 2005

[36] A E Heufelder and R S Bahn ldquoModulation of Gravesrsquo orbitalfibroblast proliferation by cytokines and glucocorticoid recep-tor agonistsrdquo Investigative Ophthalmology and Visual Sciencevol 35 no 1 pp 120ndash127 1994

[37] Y K Wong K T Tang J C Wu J J Hwang and H SWang ldquoStimulation of hyaluronan synthesis by interleukin-1beta involves activation of protein kinase C betaII in fibroblastsfrom patients with Gravesrsquo ophthalmopathyrdquo Journal of CellularBiochemistry vol 82 no 1 pp 58ndash67 2001

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


expression and intracellular cytokines in the GO orbitalfibroblasts could be reduced by pretreatment of the cells withantioxidants

2 Materials and Methods

21 Patients and Tissues Acquisition The surgical specimensof 5 patients with GO (GO1ndashGO5) during orbital decom-pression surgery (one man and four women mean age 37years) and the specimens of 5 age- and sex-matched patients(N1ndashN5) (one man and four women mean age 36 years)who received oculoplastic surgery for noninflammatory con-ditions were used in this study All specimens were collectedin accordance with the Declaration of Helsinki and withinformed consent of the patients All GO patients achievedstable euthyroidism with antithyroid medications for at least6 months before surgery and are maintained in the inactivestage of GO In addition all study subjects had not receivedspecific treatment (systemic steroids or radiotherapy) forGO Exclusion criteria include ocular diseases other thanGO alcohol drinking regular ingestion of antioxidants andpregnancy In addition the patients suffering from chronicor acute diseases such as diabetes mellitus hyperlipidemiadiseases of the lung liver or kidney cancer other endocrinedysfunctions and immunological or inflammatory disorderswere also excluded

22 Primary Cultures of Orbital Fibroblasts The primarycultures of orbital fibroblasts were established according toour previous study [16] Briefly the orbital tissues wereminced aseptically in phosphate-buffered saline (PBS pH 73)and then incubated with a sterile solution containing 05collagenase and dispase (Sigma-Aldrich Chemical Co StLouis MO USA) for 24 hours in an incubator filled withan atmosphere of 5 CO

2and kept at 37∘C The mixture

of digested orbital tissues was pelleted by centrifugation at1000timesg and then resuspended inDulbeccorsquosModified EaglersquosMedium (DMEM purchased from Gibco Life TechnologiesGaithersburg MD USA) containing 10 fetal bovine serum(FBS) and a cocktail of antibiotics (Biological Industries Kib-butz Beit Haemek Israel) which was composed of 100UmLpenicillin G and 100 120583gmL streptomycin sulfate (BiologicalIndustries Kibbutz Beit Haemek Israel)The cultured orbitalfibroblasts were used between the 3rd and 5th passages andthe cell cultures at the same passage number were used forthe same set of experiments

23 Preparation of Cigarette Smoke Extract (CSE) CSE wasprepared according to prior studies with minor modification[12 14] The commercial cigarettes (The Longlife tobaccopackage purchased from Taipei Taiwan) were used in thisstudy and each cigarette contained 10mg tar and 08mgnicotine Ten pieces of cigarettes were smoked continuouslyby a pump-smoke machine and this smoke was used togenerate 200mL of prewarmed CSE-PBS solution Eachcigarette was smoked for 3min and the pH of CSE-PBSsolution was adjusted to 74 and then passed through a 022-120583m pore size filter (Millipore Corporation Billerica MAUSA) to remove large particulates and bacteriaThe CSE-PBS

solution is defined as 100 CSE and this CSE will bediluted with DMEM in the following experiments CSEpreparation is standardized by measuring the absorbance ata wavelength of 320 nm (optical density = 20ndash22) and thepattern of absorbance observed at a wavelength of 320 nmshows insignificant variation between different preparationsof CSE CSE concentrations in the current study are rangedfrom 0 to 15

24 Treatment of Orbital Fibroblasts with CSE and Antiox-idants After washing with PBS buffer (pH 74) twice theorbital fibroblasts were treated with various concentrationsof CSE ranging from 1 to 15 for 24 hours To investigatewhether the effect of CSE could be blocked by antioxi-dants we pretreated the orbital fibroblasts with 1mM N-acetylcysteine (NAC) or 2mM vitamin C (VitC) for 1 hourfollowed by the induction of 5 CSE treatment for another24 hours respectively

25 Determination of Cell Viability Cell viability was mea-sured by the Trypan blue exclusion assay and was countedby using a hemocytometer The number of viable cells wasdetermined on the basis of their exclusion of 04 Trypanblue (Sigma-Aldrich St Louis MO USA) The relative cellviability was normalized by the value of cells without CSEtreatment andwas expressed asmeanplusmn SD of the results fromthree independent experiments

26 Measurement of ROS Content The probes from 2101584071015840-dichlorofluorescin diacetate (DCFH-DA Molecular ProbesEugene OR USA) will be used to evaluate the intra-cellular H

2O2contents [17] After incubation of orbital

fibroblasts with 20120583M DCFH-DA at 37∘C for 20min cellswere trypsinized and then resuspended in 05mL of PBSbuffer (pH 74) and analyzed with a flow cytometer (ModelEPICS XL-MCL Beckman-Coulter Miami FL USA) Theexcitation wavelength is set at 488 nm and the intensity ofemitted fluorescence of a total of 10000 cells at 525 nmis recorded on channel FL1 for the DCFH-DA probeData were analyzed by the EXPO32 software (Beckman-Coulter Miami FL USA) The intracellular H

2O2con-

tents in the treated cells were presented as relative valuescompared with that of the cells without H

2O2or CSE

treatment

27 Determination of Lipid Peroxidation The lipid peroxida-tion product malondialdehyde (MDA) in cultured orbitalfibroblasts was measured by the spectrophotometric assay kit(MDA-586 OxisResearch Inc Portland OR USA) accord-ing to themanufacturerrsquos instructions [18]TheMDA is quan-tified in the reaction with a chromogenic regent N-methyl-2-phenylindole to form an intensely colored carbocyaninedye with a maximum absorbance at 586 nm The method isspecific for MDA instead of other lipid peroxidation prod-ucts such as 4-hydroxyalkenal because they cannot producesignificant absorbance at 586 nm under the experimentalconditions AnMDA standard curve was established by usingtheMDA samples at the concentration range of 0ndash50 120583M andthe MDA levels in orbital fibroblasts were normalized to cell











3 Results




Cel

l via

bilit

y (

)

30

40

50

60

70

80

90

100

110

CSE ()0 1 2 5 10 15

lowast

lowastlowast

lowastlowast


(a)C

ell v

iabi

lity

()

0

20

40

60

80

100

120

lowastp = 00374


Control5 CSE

GO patients(n = 5)


(b)







0 1 2 5 10 15

Relat

ive D

CF in

tens

ity (

)

0

50

100

150

200

250

CSE ()

lowastlowast

lowast

lowastlowast

lowast

(a)

HO-1

CSE ()

Rela

tive i

nten

sity

(fold

s)0

2

4

6

8

10

12

0 2 5 10 15CSE ()

lowastlowast

lowastlowast

lowastlowast

lowastlowast

120573-actin

0 2 5 10 15

(b)







119901 = 00381 119901 = 00086


119901 = 00046 119901 = 00031






Relat

ive D

CF in

tens

ity (

)

0

50

100

150

200

Control5 CSE

lowastp = 00273

lowastp = 00347


GO patients(n = 5)


(a)

0

100

200

300

400

500

lowastp = 00186



MD

A (n

mol

per

106

cells

)

Control5 CSE

GO patients(n = 5)


(b)

Relat

ive D

CF in

tens

ity (

)

0

50

100

150

200

250

5 C

SE

5 C

SE2

mM

VitC

+

2m

M V

itC

5 C

SE1

mM

NAC

+

1m

M N

AC



GO

pat

ient

s(n

=5)

(c)

0

100

200

300

400

500

5 C

SE

5 C

SE2

mM

VitC

+

2m

M V

itC

5 C

SE1

mM

NAC

+

1m

M N

AC

MD

A (n

mol

per

106

cells

)



GO

pat

ient

s(n

=5)

(d)



4 Discussion











119901 = 00437 119901 = 00294


119901 = 00251 119901 = 00085




Cytokinesspecies





119901 = 0004 119901 = 0008


119901 = 0008 119901 = 0003







Cytokinesspecies





Inhibition ratio()


119901 = 0037 119901 = 0008


119901 = 0028 119901 = 0003

CTGF

GO1 GO2

GO4 GO5

CTGF

120573-actin

120573-actin

GO3




(a)

CTG

F ex

pres

sion

(rel

ated

to 120573

-act

in ex

pres

sion)

0

1

2

3

4

5


2mM VitC +


(b)






Competing Interests


Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























3 Results




Cel

l via

bilit

y (

)

30

40

50

60

70

80

90

100

110

CSE ()0 1 2 5 10 15

lowast

lowastlowast

lowastlowast


(a)C

ell v

iabi

lity

()

0

20

40

60

80

100

120

lowastp = 00374


Control5 CSE

GO patients(n = 5)


(b)







0 1 2 5 10 15

Relat

ive D

CF in

tens

ity (

)

0

50

100

150

200

250

CSE ()

lowastlowast

lowast

lowastlowast

lowast

(a)

HO-1

CSE ()

Rela

tive i

nten

sity

(fold

s)0

2

4

6

8

10

12

0 2 5 10 15CSE ()

lowastlowast

lowastlowast

lowastlowast

lowastlowast

120573-actin

0 2 5 10 15

(b)







119901 = 00381 119901 = 00086


119901 = 00046 119901 = 00031






Relat

ive D

CF in

tens

ity (

)

0

50

100

150

200

Control5 CSE

lowastp = 00273

lowastp = 00347


GO patients(n = 5)


(a)

0

100

200

300

400

500

lowastp = 00186



MD

A (n

mol

per

106

cells

)

Control5 CSE

GO patients(n = 5)


(b)

Relat

ive D

CF in

tens

ity (

)

0

50

100

150

200

250

5 C

SE

5 C

SE2

mM

VitC

+

2m

M V

itC

5 C

SE1

mM

NAC

+

1m

M N

AC



GO

pat

ient

s(n

=5)

(c)

0

100

200

300

400

500

5 C

SE

5 C

SE2

mM

VitC

+

2m

M V

itC

5 C

SE1

mM

NAC

+

1m

M N

AC

MD

A (n

mol

per

106

cells

)



GO

pat

ient

s(n

=5)

(d)



4 Discussion











119901 = 00437 119901 = 00294


119901 = 00251 119901 = 00085




Cytokinesspecies





119901 = 0004 119901 = 0008


119901 = 0008 119901 = 0003







Cytokinesspecies





Inhibition ratio()


119901 = 0037 119901 = 0008


119901 = 0028 119901 = 0003

CTGF

GO1 GO2

GO4 GO5

CTGF

120573-actin

120573-actin

GO3




(a)

CTG

F ex

pres

sion

(rel

ated

to 120573

-act

in ex

pres

sion)

0

1

2

3

4

5


2mM VitC +


(b)






Competing Interests


Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Cel

l via

bilit

y (

)

30

40

50

60

70

80

90

100

110

CSE ()0 1 2 5 10 15

lowast

lowastlowast

lowastlowast


(a)C

ell v

iabi

lity

()

0

20

40

60

80

100

120

lowastp = 00374


Control5 CSE

GO patients(n = 5)


(b)







0 1 2 5 10 15

Relat

ive D

CF in

tens

ity (

)

0

50

100

150

200

250

CSE ()

lowastlowast

lowast

lowastlowast

lowast

(a)

HO-1

CSE ()

Rela

tive i

nten

sity

(fold

s)0

2

4

6

8

10

12

0 2 5 10 15CSE ()

lowastlowast

lowastlowast

lowastlowast

lowastlowast

120573-actin

0 2 5 10 15

(b)







119901 = 00381 119901 = 00086


119901 = 00046 119901 = 00031






Relat

ive D

CF in

tens

ity (

)

0

50

100

150

200

Control5 CSE

lowastp = 00273

lowastp = 00347


GO patients(n = 5)


(a)

0

100

200

300

400

500

lowastp = 00186



MD

A (n

mol

per

106

cells

)

Control5 CSE

GO patients(n = 5)


(b)

Relat

ive D

CF in

tens

ity (

)

0

50

100

150

200

250

5 C

SE

5 C

SE2

mM

VitC

+

2m

M V

itC

5 C

SE1

mM

NAC

+

1m

M N

AC



GO

pat

ient

s(n

=5)

(c)

0

100

200

300

400

500

5 C

SE

5 C

SE2

mM

VitC

+

2m

M V

itC

5 C

SE1

mM

NAC

+

1m

M N

AC

MD

A (n

mol

per

106

cells

)



GO

pat

ient

s(n

=5)

(d)



4 Discussion











119901 = 00437 119901 = 00294


119901 = 00251 119901 = 00085




Cytokinesspecies





119901 = 0004 119901 = 0008


119901 = 0008 119901 = 0003







Cytokinesspecies





Inhibition ratio()


119901 = 0037 119901 = 0008


119901 = 0028 119901 = 0003

CTGF

GO1 GO2

GO4 GO5

CTGF

120573-actin

120573-actin

GO3




(a)

CTG

F ex

pres

sion

(rel

ated

to 120573

-act

in ex

pres

sion)

0

1

2

3

4

5


2mM VitC +


(b)






Competing Interests


Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 1 2 5 10 15

Relat

ive D

CF in

tens

ity (

)

0

50

100

150

200

250

CSE ()

lowastlowast

lowast

lowastlowast

lowast

(a)

HO-1

CSE ()

Rela

tive i

nten

sity

(fold

s)0

2

4

6

8

10

12

0 2 5 10 15CSE ()

lowastlowast

lowastlowast

lowastlowast

lowastlowast

120573-actin

0 2 5 10 15

(b)







119901 = 00381 119901 = 00086


119901 = 00046 119901 = 00031






Relat

ive D

CF in

tens

ity (

)

0

50

100

150

200

Control5 CSE

lowastp = 00273

lowastp = 00347


GO patients(n = 5)


(a)

0

100

200

300

400

500

lowastp = 00186



MD

A (n

mol

per

106

cells

)

Control5 CSE

GO patients(n = 5)


(b)

Relat

ive D

CF in

tens

ity (

)

0

50

100

150

200

250

5 C

SE

5 C

SE2

mM

VitC

+

2m

M V

itC

5 C

SE1

mM

NAC

+

1m

M N

AC



GO

pat

ient

s(n

=5)

(c)

0

100

200

300

400

500

5 C

SE

5 C

SE2

mM

VitC

+

2m

M V

itC

5 C

SE1

mM

NAC

+

1m

M N

AC

MD

A (n

mol

per

106

cells

)



GO

pat

ient

s(n

=5)

(d)



4 Discussion











119901 = 00437 119901 = 00294


119901 = 00251 119901 = 00085




Cytokinesspecies





119901 = 0004 119901 = 0008


119901 = 0008 119901 = 0003







Cytokinesspecies





Inhibition ratio()


119901 = 0037 119901 = 0008


119901 = 0028 119901 = 0003

CTGF

GO1 GO2

GO4 GO5

CTGF

120573-actin

120573-actin

GO3




(a)

CTG

F ex

pres

sion

(rel

ated

to 120573

-act

in ex

pres

sion)

0

1

2

3

4

5


2mM VitC +


(b)






Competing Interests


Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Relat

ive D

CF in

tens

ity (

)

0

50

100

150

200

Control5 CSE

lowastp = 00273

lowastp = 00347


GO patients(n = 5)


(a)

0

100

200

300

400

500

lowastp = 00186



MD

A (n

mol

per

106

cells

)

Control5 CSE

GO patients(n = 5)


(b)

Relat

ive D

CF in

tens

ity (

)

0

50

100

150

200

250

5 C

SE

5 C

SE2

mM

VitC

+

2m

M V

itC

5 C

SE1

mM

NAC

+

1m

M N

AC



GO

pat

ient

s(n

=5)

(c)

0

100

200

300

400

500

5 C

SE

5 C

SE2

mM

VitC

+

2m

M V

itC

5 C

SE1

mM

NAC

+

1m

M N

AC

MD

A (n

mol

per

106

cells

)



GO

pat

ient

s(n

=5)

(d)



4 Discussion











119901 = 00437 119901 = 00294


119901 = 00251 119901 = 00085




Cytokinesspecies





119901 = 0004 119901 = 0008


119901 = 0008 119901 = 0003







Cytokinesspecies





Inhibition ratio()


119901 = 0037 119901 = 0008


119901 = 0028 119901 = 0003

CTGF

GO1 GO2

GO4 GO5

CTGF

120573-actin

120573-actin

GO3




(a)

CTG

F ex

pres

sion

(rel

ated

to 120573

-act

in ex

pres

sion)

0

1

2

3

4

5


2mM VitC +


(b)






Competing Interests


Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























119901 = 00437 119901 = 00294


119901 = 00251 119901 = 00085




Cytokinesspecies





119901 = 0004 119901 = 0008


119901 = 0008 119901 = 0003







Cytokinesspecies





Inhibition ratio()


119901 = 0037 119901 = 0008


119901 = 0028 119901 = 0003

CTGF

GO1 GO2

GO4 GO5

CTGF

120573-actin

120573-actin

GO3




(a)

CTG

F ex

pres

sion

(rel

ated

to 120573

-act

in ex

pres

sion)

0

1

2

3

4

5


2mM VitC +


(b)






Competing Interests


Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Cytokinesspecies





Inhibition ratio()


119901 = 0037 119901 = 0008


119901 = 0028 119901 = 0003

CTGF

GO1 GO2

GO4 GO5

CTGF

120573-actin

120573-actin

GO3




(a)

CTG

F ex

pres

sion

(rel

ated

to 120573

-act

in ex

pres

sion)

0

1

2

3

4

5


2mM VitC +


(b)






Competing Interests


Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Competing Interests


Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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