Research Article Anti-Inflammatory Effect of Recreational ...downloads.hindawi.com/journals/omcl/2014/925981.pdf · Research Article Anti-Inflammatory Effect of Recreational Exercise

Research ArticleAnti-Inflammatory Effect of Recreational Exercise inTNBS-Induced Colitis in Rats Role of NOSHOMPO System

Zita Szalai1 Andraacutes Szaacutesz23 Istvaacuten Nagy3 Laacuteszloacute G Puskaacutes45

Krisztina Kupai1 Adeacutel Kiraacutely1 Anikoacute Magyarineacute Berkoacute1 Anikoacute Poacutesa1

Gerda Strifler3 Zoltaacuten Baraacuteth8 Lajos I Nagy4 Renaacuteta Szaboacute1 Imre Paacutevoacute1

Zsolt Murlasits6 Mariann Gyoumlngyoumlsi7 and Csaba Varga1

1 Department of Physiology Anatomy and Neuroscience University of Szeged Kozep Fasor 52 Szeged 6726 Hungary2 Institute of Physical Education and Sport Sciences University of Szeged Szeged 6725 Hungary3 Institute of Biochemistry Biological Research Centre of the Hungarian Academy of Sciences Szeged 6726 Hungary4AVIDIN Ltd Szeged 6726 Hungary5 Institute of Genetics Biological Research Center of the Hungarian Academy of Sciences Szeged 6726 Hungary6 Semmelweis University Faculty of Physical Education and Sport Sciences Budapest 1123 Hungary7Department of Cardiology Medical University of Vienna 1090 Wien Austria8University of Szeged Faculty of Dentistry and Department of Orthodontics and Pediatric Dentistry Szeged 6720 Hungary

Correspondence should be addressed to Csaba Varga vacsbiou-szegedhu

Received 26 June 2013 Revised 10 December 2013 Accepted 12 December 2013 Published 6 February 2014

Academic Editor Pedro Tauler Riera

Copyright copy 2014 Zita Szalai et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

There are opposite views in the available literature Whether physical exercise has a protective effect or not on the onset ofinflammatory bowel disease (IBD) Therefore we investigated the effects of recreational physical exercise before the induction ofcolitis After 6 weeks of voluntary physical activity (running wheel) male Wistar rats were treated with TNBS (10mg) 72 hrs aftertrinitrobenzene sulphonic acid (TNBS) challenge wemeasured colonic gene (TNF-120572 IL-1120573 CXCL1 and IL-10) and protein (TNF-120572)expressions of various inflammatory mediators and enzyme activities of heme oxygenase (HO) nitric oxide synthase (NOS) andmyeloperoxidase (MPO) enzymes Wheel running significantly increased the activities of HO constitutive NOS (cNOS) isoformFurthermore 6 weeks of running significantly decreased TNBS-induced inflammatorymarkers including extent of lesions severityof mucosal damage and gene expression of IL-1120573 CXCL1 and MPO activity while IL-10 gene expression and cNOS activity wereincreased iNOS activity decreased and the activity of HO enzyme increased but not significantly compared to the sedentaryTNBS-treated group In conclusion recreational physical exercise can play an anti-inflammatory role by downregulating the geneexpression of proinflammatory mediators inducing anti-inflammatory mediators and modulating the activities of HO and NOSenzymes in a rat model of colitis

1 Introduction

Inflammatory bowel disease (IBD) is a chronic disease ofthe gastrointestinal tract which primarily comprises Crohnrsquosdisease (CD) and ulcerative colitis Physical exercise canbe either harmful or beneficial for the gastrointestinal (GI)tract depending on the training variables such as exerciseintensity For instance it is believed that the reduced bloodflow to the gut during exercise can disturb the GI system [1]

Although there are similar studies suggesting the preventiveeffect of active lifestyle on the onset of IBD the availableliterature is weak and contradictory [2 3]

In the healthy gut interleukin-10 (IL-10) is the keycytokine responsible for the anti-inflammatory environ-ment primarily through the suppression of proinflammatorycytokines [4] Once the anti- and proinflammatory balanceof the intestinal immune system is disturbed the activatedand stimulated macrophages and T lymphocytes release

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2014 Article ID 925981 11 pageshttpdxdoiorg1011552014925981

2 Oxidative Medicine and Cellular Longevity

proinflammatory mediators such as tumour necrosis factor-120572 (TNF-120572) interleukin-1120573 (IL-1120573) and chemokine ligand1 (CXCL1) The importance of anti- and proinflammatorybalance is reflected in the efficacy of anti-TNF-120572 therapy inthe treatment of IBD [5] TNF-120572 inhibitors have proven suc-cessful in inducing and maintaining remission of moderate-to-severe IBD In addition CXCL1 is a chemoattractant forneutrophils and plays a role in inflammation increased ilealand colonic CXCL1 gene expression was also reported in IBDpatients [6]

IBD is associated with enhanced production of reactivemetabolites of oxygen and nitrogen (RONS) Moreoveroxidative stress plays an important role in the pathogen-esis of this disease [7] Heme oxygenase-1 (HO-1) theinducible isoform of heme oxygenase enzymes is a stress-responsive enzyme which can be induced by oxidative stressinflammatory cytokines and many other molecules HO-1 is thought to play an important role in the protectionof tissues from oxidative injuries and inflammation [8ndash10]These beneficial effects of HO-1 are mediated in part by theantioxidant and anti-inflammatory end products of hemebreakdown namely CO and bilirubin which can inhibitthe expression of proinflammatory cytokines such as TNF-120572 and IL-1120573 Several molecules such as interleukin-10 (IL-10) function through an HO-1 dependent mechanism [11 12]Pharmacological studies suggest a beneficial role for HO-1in modulating colitis Upregulation of HO-1 by heme andcadmium chloride can ameliorate while inhibition of theenzyme by tin protoporphyrin can aggravate experimentalinflammation in the colon [13] 5-Aminosalicylic-acid aclinically approved anticolitic agent can exert beneficialeffects in vivo partially through the induction or activationof HO-1 [14]

Another enzyme involved in intestinal inflammation andoxidative stress is nitric oxide synthase [15] The constitutiveproduction of NO derived from the constitutive isoforms ofthe enzyme namely neuronal NOS (nNOS) and endothelialNOS (eNOS) plays a role in various physiological processesin the GI mucosa such as the regulation of microvascularand epithelial permeability and the maintenance of adequateperfusion [16] On the other hand the effects of iNOS-produced [15]NO can be beneficial or detrimental dependingon the amount duration and anatomical site of synthesis[16 17] In experimental colitis the downregulation of nNOSand the overproduction of NO by iNOS are observed [18]For example proinflammatory cytokines such as TNF-120572 caninduceNOproduction and iNOS activity in colonic epithelialcells [17]

Earlier studies have focusedmainly on the role of exercisein the treatment of extraintestinal manifestations of IBD [1920] However the effects of physical exercise on the inflamedgut are still inconsistent Therefore the aims of the presentstudy were to investigate the effects of recreational physicalexercise (1) on the colonic damage in a TNBS rat model(damage score lesion) (2) on the gene expression (TNF-120572IL-1120573 CXCL1 and IL-10) and protein concentration (TNF-120572)of inflammatory mediators in experimental acute colitis and(3) on the activity of oxidativeantioxidative enzymes (MPOHO andNOS) in the inflamed colon of ratsWe hypothesized

that voluntary exercise prior to TNBS challenge will reducecolonic damage and oxidative and inflammatory mediatorgene and protein expressions

2 Materials and Methods

21 Animals Male Wistar rats (180ndash220 g) were housed ingroups Food was withdrawn overnight before induction ofcolitis otherwise the animals had access to food anddrinkingwater ad libitum throughout the experiments The animalcare and research protocols were in accordance with theguidelines of the University of Szeged

22 Experimental Design The animals were randomlydivided into four groups 1 sedentary control (nonrunningnon-colitis-induced 119899 = 13) 2 running control (runningnon-colitis-induced 119899 = 18) 3 sedentary TNBS (119899 = 14)and 4 running TNBS groups (119899 = 18) (Figure 1) Rats of therunning groups were placed in cages with a running wheel(Acellabor Ltd Budapest Hungary) Before the beginningof experimental period rats were allowed to accommodateto the running wheel for a week The activity of the rats onthe running wheel was monitored by a bicycle computerattached to each wheel After 6 weeks of self-administeredphysical exercise colitis was induced by 246-trinitrobenzenesulphonic acid (TNBS once 10mg in 025mL of 50ethanol wv) The intracolonic administration of TNBSwas performed with an 8 cm long plastic catheter undertransient ether anaesthesia (the method originally describedby Morris et al [21]) The control groups did not receiveany treatment Physical activity was continued followingTNBS treatment Rats were weighed twice a week untilthe induction of colitis and then daily following the TNBSchallenge 72 hours after the induction of colitis the animalswere sacrificed and the distal 8 cm portion of the colonwas dissected longitudinally opened gently rinsed withice-cold physiological saline and photographed (PanasonicLumix DMC-TZ6 digital camera) for the determination ofmacroscopic colonic inflammatory damage The colon wasweighed and divided into longitudinal segments to be usedfor the following molecular and biochemical analyses

23 Damage Score and Lesion Measurement The extentof macroscopically apparent inflammation ulceration andtissue disruption was determined in a randomized mannerfrom the colour images using a proprietary computerizedplanimetry software developed in our laboratory (Stat 2 1 1)The area of macroscopically visible mucosal involvement wascalculated and expressed as the percentage of the total colonicsegment area under study

The degree of colonic inflammation was scored on a 0ndash11scale in a randomized blinded fashion The criteria has beenadapted from what has been used previously 0 = no damage1 = focal hyperemia and no ulcers 2 = ulceration withouthyperemia or bowel wall thickening 3 = ulceration withinflammation at 1 site 4 = more than 2 sites of ulceration andinflammation 5 = more than 2 major sites of ulceration andinflammation or 1 site of ulcerationinflammation extending

Oxidative Medicine and Cellular Longevity 3

Sedentary control

Sedentary TNBS

TNBS challenge

Accommodation

Running control

Running TNBS

Weekly measuring of

the body weight

Daily measuring of the body weight

Sacrifce

Measuring of

Te activity ofTe relative gene expression ofScorelesion

Te concentration of

Timeline

Sedentary Running

Week 0

Week 1ndash6 TrainingSitting

Week 7

Experimental design

Groups

TNF-120572TNF-120572IL-1120573

CXCL1IL-10

challenge72h afer TNBS

∙ MPO

∙ iNOS∙ cNOS∙ HO

Figure 1 Diagram showing the experimental design

gt1 cm along the length of the colon and 6ndash11 = the score isincreased by 1 for each additional centimetre of involvement[14]

24 Quantification of TNF-120572 Protein with the AlphaLISAAssay The rat large intestine was immediately frozen inliquid nitrogen after dissection During protein extractionthe tissues were stored on ice We used 1mL RIPA buffer(150mMNaCl (Molar Chemicals Budapest Hungary)1 Nonidet P-40 (Sigma-Aldrich) 05 Na-deoxycholate(Sigma-Aldrich) 01 sodium dodecyl sulfate (Sigma-Aldrich) and 50mM Tris-HCl (pH 80) (Molar Chemicals))for protein extraction from 40mg tissue supplementedwith 20120583L NEM (N-ethyl-maleinimide) and 20 120583L phenyl-methane-sulfofluoride (PMSF) protease inhibitors Wehomogenized the tissue samples with Ultra-Turrax T25(IKA-Labortechnik Staufen Germany) tissue homogenizeron ice for 1min The homogenates were centrifuged for10min at 10000timesg at 4∘C and the concentration of thesupernatant was measured with a NanoDrop ND-1000spectrophotometer (NanoDrop Technologies WilmingtonUSA) All samples were then diluted to a uniform 05mgmLconcentration Until further analyses samples were stored atminus20∘C (stock samples were stored at minus80∘C)

For the quantification of TNF-120572 we used the AlphaL-ISA bead based technique which was developed and issupported by Perkin Elmer (Perkin Elmer MassachusettsUSA) Alpha Technology is a highly sensitive assay idealfor the measurement of protein interaction We used 5 120583Lvolume from05mgmLprotein solutionThefinal amount ofAlphaLISA Anti-Analyte Acceptor beads was set at 10120583gmLconcentration while the final amount of Streptavidin-Donorbeads was set at 40 120583gmL The final working concentrationof Biotinylated Antibody Anti-Analyte was 1 nM All incu-bations were under subdued laboratory lighting (lt100 lux)

The samples were measured in 50 120583L final volume filledin 384-well polystyrene plates (Tomtec Plastik BudapestHungary) with an EnVision-Alpha Reader (PerkinElmer)

25 Myeloperoxidase Activity To measure myeloperoxidaseactivity we employed a modification of the method describedby Bradley et al [22] The 8 cm longitudinal strips ofthe colon were weighed homogenized (Ultra-Turrax T25IKA-Labortechnik 13500 revmin twice for 30 s 250mgcolonmL buffer) in ice-cold phosphate buffer (50mMpH 60) containing 05 hexadecyltrimethylammonium-bromide freeze-thawed three times and then centrifuged at10000timesg for 15min at 4∘C A 12 120583L aliquot of the supernatantwas then mixed with 280120583L phosphate buffer (50mM pH60) containing 0167mgmL O-adenosine dihydrochloride(Sigma-Aldrich) and the reaction was started with 10120583L003 hydrogen peroxide and assayed spectrophotometri-cally at 490 nm after 90 sec shaking (Benchmark MicroplateReader Bio-Rad Labs Hercules CA) MPO activity wasexpressed as mUmg protein

26 Heme Oxygenase Activity Heme oxygenase activitywas assessed by measuring bilirubin formation with slightmodifications form what has been described by Tenhunenet al [23] The segment of the colon was homogenized(Ultra-Turrax T25 13500s twice for 30 sec) in ice-cold10mM N-[2-hydroxyethyl] piperazine-N1015840-[2-ethanesulfonicacid] (HEPES Sigma-Aldrich) 32mM sucrose (Sigma-Aldrich) 1mM dithiothreitol (DTT Sigma-Aldrich) 01mMEDTA 10 120583gmL soybean trypsin inhibitor (Sigma-Aldrich)10 120583gmL leupeptin (Sigma-Aldrich) and 2 120583gmL aprotinin(Sigma-Aldrich) at pH 74 The supernatant was collectedby centrifugation for 30min at 20000timesg at 4∘C Incubationwas carried out in the dark at 37∘C for 60min with thereaction mixture containing the following ingredients in a


final volume of 15mL 2mM glucose 6-phosphate (Sigma-Aldrich) 014UmL glucose 6-phosphate dehydrogenase(Sigma-Aldrich) 15120583M heme 150 120583M 120573-nicotinamide ade-nine dinucleotide phosphate (120573-NADPH Sigma-Aldrich)120120583gmL rat liver cytosol as a source of biliverdin reductase2mM MgCl

2 100mM potassium phosphate buffer and

150 120583L of supernatant The reaction was stopped by placingthe samples on iceThe bilirubin formed was calculated fromthe difference between optical densities obtained at 460 and530 nm One unit of heme oxygenase activity was defined asthe amount of bilirubin (nmol) producedhourmg protein

27 Nitric Oxide Synthase Activity Nitric oxide synthaseactivity was determined by quantifying the conversion of[14C]-radiolabelled L-arginine to citrulline by a previouslydescribed method with some minor modifications [24]A segment of colon was homogenized as described forHO activity Homogenates were centrifuged for 30min at20000timesg at 4∘C Samples (40 120583L) were incubated for 10minat 37∘C in 100 120583L of assay buffer (50mM KH

2PO4 10mM

MgCl2 50mML-valine 02mMCaCl

2 10mMDTT 10mM

L-citrulline 155 nM L-arginine 30 120583M flavin adenine din-ucleotide 30 120583M flavin mononucleotide 30120583M tetrahydro-L-biopterin dihydrochloride 450120583M 120573-NADPH and 12 pMof [14C]-L-arginine monohydrochloride (all from Sigma-Aldrich)) The reaction was terminated by the addition of05mL of 1 1 (vv) suspension of ice-cold DOWEX (Na+-form) in distilled water The mixture was resuspendedwith the addition of 850 120583L of ice-cold distilled water Thesupernatant (970 120583L) was removed and radioactivity wasdetermined by scintillation counting Calcium-dependencyof the NOS activity was determined by the addition of 10 120583Lof ethylene glycol-bis (120573-aminoethyl ether) tetraacetic acid(EGTA 1mM Sigma-Aldrich) NOS activity was confirmedby inhibition with 10 120583L of N120596-nitro-L-arginine-methylester(LNNA 37mM Sigma-Aldrich) InducibleNOSwas definedas the citrulline formation that was inhibited by LNNA butnot by EGTA The constitutive NOS activity was calculatedfrom the difference between citrulline formation that wasinhibited by EGTA and the total activity As the nature ofthe constitutive isoform (eNOS or nNOS) was not deter-mined this activity is referred to as cNOS NOS activity wasexpressed as pmolminmg protein

28 Protein Determination for HO NOS and MPO ActivityUsing a commercial protein assay kit (Bio-Rad Labs) aliquots(20120583L) of the diluted samples (15times or 25times with distilledwater) were mixed with 980120583L of distilled water with 200120583LBradford reagent added to each sample After mixing andfollowing 10min incubation the samples were assayed spec-trophotometrically at 595 nm Protein level was expressed asmg proteinmL

29 RNA Extraction Reverse Transcription and QuantitativeReverse Transcriptase Polymerase Chain Reaction (QPCR)The samples were homogenized in 1mL of TRIzol reagent(Life Technologies Carlsbad CA) with ultra-turrax T-18basic homogenizer (2times30 sec at 5000 rpm) one-third volume

of chloroform (Sigma-Aldrich) was added to the suspen-sion with vigorous vortexing Samples were centrifuged at13000 rpm for 10 minutes and total RNA was extractedfrom the upper phase by using RNeasy Plus Mini Kits(Qiagen Germany) according to themanufacturerrsquos protocolIn parallel peripheral bloodmononuclear cells (PBMC) wereisolated from the blood of the same animals by Ficoll PaquePlus (GE Healthcare UK) density gradient centrifugationTotal RNA from PBMCwas also extracted using RNeasy PlusMini Kits (Qiagen)The quality and quantity of isolated RNAwere determined using NanoDrop and Bioanalyzer (AgilentSanta Clara USA) measurements

cDNA was synthesized from 100 ng of total RNA byusing High Capacity RNA to cDNA Kit or SuperScriptVilo cDNA Synthesis Kit (both from Life Technologies)according to the manufacturerrsquos instructions cDNA levelswere determined by QPCR using StepOne Plus Real-TimePCR System (Life Technologies) Reactions were performedwith Power SybrGreen Master Mix (Life Technologies) withthe following primer sets

TNF-120572 sense GCTCCCTCTCATCAGTTCCATNF-120572 antisense GGCTTGTCACTCGAGTTT-TGACXCL1 sense CATTAATATTTAACGATGTGGAT-GCGTTTCACXCL1 antisense GCCTACCATCTTTAAACT-GCACAAT [25]IL-1120573 sense CAGGAAGGCAGTGTCACTCAIL-1120573 antisense AGACAGCACGAGGCATTTTTIL-10 sense CCTGCTCCTACTGGCTGGAGIL-10 antisense TTGTTCAGCTGGTCCTTCTT

To avoid false-positive results due to amplification of con-taminating genomic DNA in the cDNA preparation we usedprimers spanning exon-exon junctions All measurementswere performed in duplicates with at least four biologicalreplicates The ratio of each mRNA relative to the 18S rRNA(assay ID Hs99999901 Life Technologies) was calculatedusing the ΔΔ119862

119879method

210 Data Representation and Statistical Analysis Resultsof the body weight change damage score lesion and theactivity of MPO HO cNOS and iNOS are shown as meanplusmn SEM statistical comparisons were performed by two-tailed Studentrsquos t-test Statistical analysis of the TNF-120572 proteinsecretionwas performed using one-wayANOVAQPCR dataare presented as data points and median the significance ofthe differences between samples was determined by applyingthe Newman-Keuls test using GraphPad Prism forWindowsIn all statistical comparisons a probability (119875) value of lessthan 005 was considered significant

3 Results

31 Alteration of Body Weight after Running and TNBSTreatment Running distance averaged 2600 metersdayrat


1 11 21 31 41

100

125

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Sedentary controlRunning control

Days

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wei

ght c

hang

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(a)

0 1 2 380

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Days

Body

wei

ght c

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e (

)

(b)

Figure 2 Body weight changes during the 6 weeks of running (a) and after the TNBS treatment (b) Results are shown as mean plusmn SEM(lowast119875 lt 005 compared to the sedentary nontreated control group 119899 = 34ndash43)

We did not find any differences between the body weightsof sedentary and running groups during the 6 weeks ofexercise before TNBS administration (Figure 2(a)) On theother hand there was a progressive decrease in body weightafter the induction of colonic inflammation in the TNBStreated groups (Figure 2(b))

32 Effects of 6-Week Running and TNBS Challenge onDamage Score Mucosal Lesions and MPO Activity Theseverity of mucosal damage was scored on a 0ndash11 scale by arandomized blinded fashion 6 weeks of running before theTNBS challenge significantly (119875 lt 0001) decreased the levelof this inflammatory damage score marker from 76 plusmn 03 to66 plusmn 03 (Figure 3(a))

Challenge with TNBS caused hemorrhagic and necroticdamage of the colonic mucosa as determined macroscopi-cally 72 hours after TNBS administration In the sedentarynonrunning group the level of damage reached 546 plusmn 263of the total colonic area We detected a significant decrease(429 plusmn 321 119875 lt 001) in macroscopic injury in the colonsof the trained TNBS-treated group (Figures 3(b) and 3(c))

Following treatment with TNBS there was a thirtyfoldincrease in colonic MPO activity suggesting massive neu-trophil infiltration [22] Wheel running significantly (119875 lt001) attenuated the elevation of MPO activity from 8806 plusmn793 to 5684 plusmn 599mUmg protein (Figure 4)

33 The Effect of Physical Exercise on the Gene ExpressionProfiles of Selected Pro- and Anti-Inflammatory Mediators inTNBS-Induced Experimental Acute Colitis In PBMCs thephysical exercise alone significantly (119875 lt 005) downreg-ulated the gene expression of IL-1120573 CXCL1 and IL-10 ascompared to the inactive group (Figure 5(a)) TNBS treat-ment alone only downregulated the expression of CXCL1and had no effect on the expression of TNF-120572 IL-1120573 and IL-10 transcripts Interestingly exercise before TNBS treatmentdownregulated the expression of CXCL1 and IL-10 Notably

the downregulation of IL-10 was significant as compared tonot only absolute controls but also to TNBS treatment alone(Figure 5(a))

In contrast to the downregulated expression patterndetected in PBMCs physical exercise alone did not alterthe gene expression of the monitored inflammatory medi-ators in the colon when compared to sedentary controls(Figure 5(b)) Yet TNBS treatment alone induced markedupregulation (119875 lt 005) in the expression of pro- but notanti-inflammatorymediators (IL-1120573 CXCL1 and IL-10 resp)in the colon Furthermore exercise significantly attenuatedTNBS-induced upregulation of proinflammatory mediators(IL-1120573 CXCL1) and significantly increased expression of anti-inflammatory IL-10 (Figure 5(b))

34 Effect of Running andor TNBS Challenge on ColonicTNF-120572 Protein Content 6-week running had no significanteffect on the colonic content of TNF-120572 In the inflamedcolons we found a doubling of TNF-120572 concentration in thesedentary group (5784plusmn11847 pgmL) and although 6-weekrunning before TNBS treatment slightly decreased TNF-120572levels (49616plusmn8777 pgmL) the decrease was not significantcompared to the sedentary TNBS group (Figure 6(a))

35 Changes in Colonic HO Activity by Physical Exerciseandor TNBS Treatment Voluntary physical exercise sig-nificantly increased HO activity from 13 plusmn 02 to 28 plusmn03 nmol bilirubinhmg protein (119875 lt 0001) Treatment withTNBS alone led to even higher HO activity (69 plusmn 04 nmolbilirubinhmg protein 119875 lt 0001) after 6 weeks of exerciseslightly increased the HO activity but this difference wasnot significant between the running TNBS group and thenonrunning TNBS group (Figure 6(b))

36 Effects of TNBS Treatment andor Physical Exercise onColonic cNOS Activity The 6-week running alone causeda significant (119875 lt 005) increase in cNOS activity


0

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lowast lowast lowast

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age s

core

(sca

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(a)

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lowastlowast

(b)

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Running control

Sedentary TNBS

Running TNBS

(c)

Figure 3 Effects of 6 weeks of running on macroscopic colonic inflammatory damage score (a) and lesion (b) in TNBS-induced colitis after72 hrs Results are shown asmean plusmn SEM (lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 compared to the sedentary TNBS group 119899 = 13ndash19) Representativeimages of freewheel running and induction of colonic inflammation sedentary control and running control sedentary TNBS running TNBS(c)

In the nonrunning TNBS group we have measured a sig-nificantly decreased (119875 lt 0001) cNOS activity (from3211 plusmn 3516 to 1089 plusmn 256 pmolminmg protein) com-pared to the nontreated control group Voluntary exercisebefore TNBS administration significantly (119875 lt 0001)augmented cNOS activity from 1089 plusmn 256 to 3339 plusmn323 pmolminmg protein compared to the nonrunningTNBS group (Figure 7(a))

37 Effects of TNBS Treatment andor Physical Exerciseon Colonic iNOS Activity Inflammation caused by TNBSchallenge significantly elevated (119875 lt 001) iNOS activity(from 214 plusmn 549 to 2175 plusmn 2643 pmolminmg protein)independently of exercise Importantly the 6-week runningbefore TNBS treatment significantly (119875 lt 005) reduced

this increase of iNOS activity from 2175 plusmn 2643 to 1289 plusmn1582 pmolminmg protein (Figure 7(b))

4 Discussion

The results of the present study demonstrate that 6-weekphysical exercise before TNBS challenge can ameliorate theseverity and extent of colonic damage caused by TNBStreatment in rats by downregulating the expression of proin-flammatory mediators inducing the expression of anti-inflammatory mediators and affecting the activity of cNOSand iNOS enzymes

In this study we used the 246-trinitrobenzene-sulfonic-acid- (TNBS-) induced rat acute colitis model in which theinflammatory response is due to the generation of transmural


0

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activ

ity (m

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cont

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TNBS

lowastlowast

Figure 4 MPO activity in rats treated with TNBS after 6 weeks ofresting or 6 weeks of running Results are shown as mean plusmn SEM(lowastlowast119875 lt 001 compared to the sedentary TNBS group 119899 = 7ndash16)

oxidative stress and release of proinflammatory mediators[26 27] We used a voluntary wheel running model to mimicleisure-type physical exercise which induces lower degree ofstress compared to swimming or treadmill exercise protocolsdue to the reduced handling of animals and stress-inducingexercise motivators [28]

The body weights after 6 weeks of running did notshow any differences compared to the nonrunning groupshowever body composition was not examined

Intracolonic administration of TNBS causes an acutesevere transmural inflammation whichmimics several clini-cal and morphological features of Crohnrsquos disease [26] In thesedentary colitis group on average 50 of the 8 cm colonicportion was inflamed with ulceration and we have also founda 30-fold elevation in MPO activity

In order to determine if gene expression changes ofPBMCs reflect that of the inflamed colon we have monitoredthe gene expression changes of well-known inflammatorymarkers Interestingly we found that 6 weeks of runningalone downregulated the expression of both pro- and anti-inflammatory mediators (IL-1120573 CXCL1 and IL-10 resp)in PMBCs but not in the colon In addition in the colonphysical exercise only attenuated the expression level of anti-inflammatory IL-10 compared to sedentary TNBS-treatedanimals

In contrast to PBMCs TNBS induced marked upreg-ulation of proinflammatory cytokines IL-1120573 and CXCL1 inthe colon which is in good agreement with previous studiesshowing elevated expression of these molecules in IBD [6]Importantly recreational physical exercise downregulatedTNBS-induced expression of both cytokines In parallelwe detected markedly elevated IL-10 expression in runningTNBS animals These observations together with thoseshowing decreased level of damage score in physically activeanimals suggest that physical activity indeed ameliorates

the extent of the inflammation caused by TNBS However itremains to be determined if physical activity shows a similarprotective effect in patients with chronic IBD

Although there was no change in TNF-120572 gene expressionwe found a significantly increased TNF-120572 protein content inthe inflamed colon with TNBS in accordance with earlierfindings [26 29] We hypothesize that polymorphism inTNF-120572 promoter regions could be responsible for discrepan-cies between gene and protein expression level [30] Howeverno significant decrease in TNF-120572 protein content was seenafter exercise in the TNBS-treated group compared to thenonrunning TNBS-treated group It is feasible that exerciseimpacts inflammation through pathways other than TNF-120572because several other markers were attenuated in our studyindicating the effectiveness of this intervention against IBD

The elevated HO activity suggests the involvement ofoxidative stress in colonic damage Earlier findings demon-strated the ability of chemically induced HO to reduce theextent and intensity of experimental colonic inflammation[14] Several studies demonstrated that long-lasting exercisecan induce antioxidant mechanisms in a variety of tissuesprobably as a defensive response to oxidative stress [31]Brooks et al [32] demonstrated that 8 weeks of treadmillrunning reduced the release of ROS andNO from contractingmuscles and increased skeletal muscle antioxidant contentsuch as glutathione and protein thiols in mice George et al[33] found an elevated activity and expression of superoxidedismutase and HO-1 in the plasma and renal proximaltubules of rats after treadmill training for 12 weeks Ourobservations that a 6-week voluntary exercise can inducecolonic HO activity also agree with previous data supportingthe antioxidant effects of moderate training Along theselines Kasimay et al demonstrated the protective role of 6weeks of wheel running against oxidative colonic injury in amodel of acetic acidinduced intestinal inflammation [34]

We found opposite changes of cNOS and iNOS activity inthe inflamed colon Our observations of decreased cNOS andincreased iNOS activity in the sedentary colitis group are alsoconsistent with previous findings We do not know to whatextent the two constitutive isoforms (nNOS and eNOS) areresponsible for the reduction of cNOS activity Experimentswith eNOS deficient mice show the important role of theother constitutive NOS isoform in limiting intestinal injuryin IBD [35] In IBD nNOS is downregulated while iNOSexpression increases cyclically during the active phase ofinflammation Excess NO produced by iNOS can generatethe prooxidant peroxynitrite [36] This overproduction ofNO by upregulation of iNOS seems to be responsible for theobserved intestinal hypomotility and subsequent bacterialovergrowth [18]

We presented that rat colonic cNOS activity increasedwhile iNOS activity remained unchanged with exercise com-pared to the sedentary control group Together with theobserved expression changes induced by TNBS it appearsthat prior exercise is able to mitigate colonic inflamma-tion and macroscopic damage by maintaining cNOS andattenuating iNOS expression in IBD These observations arealso in agreement with previous data showing that regularphysical activity induces increased eNOSgene expression and


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2

64

0125025

051

48

1632

Relat

ive g

ene e

xpre

ssio

nC

olon

00625

8

4

2

1

05

025

0125

lowastlowast

lowastlowastlowast

lowast

lowast lowast

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

fold

chan

ge (L

og2)

(b)

Figure 5 The relative gene expression of selected pro- and anti-inflammatory mediators in peripheral blood mononuclear cell (PBMC) (a)and colon (b)The expression pattern of TNF-120572 IL-1120573 CXCL1 and IL-10 from sedentary and running groupswith or without TNBS treatmentwas determined with qPCR and compared to sedentary nontreated animals (absolute controls) Results are shown as data points and median(lowast119875 lt 005 119899 = 6ndash20)

0

100

200

300

400

500

600

700 +

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

TNF-120572

conc

entr

atio

n (p

gm

L)

(a)

1

2

3

4

5

6

7

8

+++

+++

HO

activ

ity(n

M b

iliru

bin

hm

g pr

otei

n)

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

(b)

Figure 6 (a) Concentration of TNF-120572 in 1 g tissue in sedentary and running groups with or without TNBS treatment Results are shown asmean plusmn SD (+119875 lt 005 119899 = 4ndash10) (b) Effects of 6 weeks of running on colonic HO activity either in the control group or in TNBS-inducedcolitis after 72 hrs Results are shown as mean plusmn SEM (+++119875 lt 0001 compared to the sedentary nontreated control group 119899 = 8ndash16)


0

100

200

300

400

500+

+++

cNO

S ac

tivity

(pm

olm

inm

g pr

otei

n)

lowastlowastlowast

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

(a)

0

50

100

150

200

250

++

iNO

S ac

tivity

(pm

olm

inm

g pr

otei

n)

lowast

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

(b)

Figure 7 Effects of 6 weeks of running on colonic cNOS activity (a) and iNOS activity (b) either in the control group or in TNBS-inducedcolitis after 72 hrs Results are shown as mean plusmn SEM (+119875 lt 005 ++119875 lt 001 and +++119875 lt 0001 compared to the sedentary control groupand lowast119875 lt 005 lowastlowastlowast119875 lt 0001 compared to the sedentary TNBS group 119899 = 6ndash15)

reduces the expression of iNOS in CD34(+) PBMCs [37]Moreover NO-releasing derivative of aspirin can acceleratethe healing of colonic inflammation in an animal model[38] Our results are in agreement with earlier studiesabout naturally occurring compounds and drugs such asnaringenin a flavonoid in citrus grapefruits and tomatoes[39] 34-oxo-isopropylidene-shikimic acid [40] which hasanti-inflammatory effect on experimental colitis throughreduction of the expression of inflammatory cytokines andmediators such as iNOS Therefore it appears that the NOpathway plays a central role in the resolution of inflammationin IBD

Exercise has been speculated to be protective against theonset of IBD but the literature is inconsistent Preliminarystudies reveal that exercise training (relatively low intensity)may be beneficial to reduce stress and symptoms of IBD Cur-rent research also recommends exercise to help counteractsome IBD-specific complications by improving bone min-eral density immunological response psychological healthweight loss and stress management ability However theliterature advises that some patients with IBD may havelimitations to the amount and intensity of exercise that theycan perform [2]

It has become clear that physical activity is not harmfulfor patients with inflammatory bowel disease despite acuteexercise related responses such as increased serum mal-ondialdehyde levels and activated neutrophils In additionphysical activity may reduce disease activity and improvephysical health general well-being and perceived stress Insummary exercise may be beneficial to IBD patients butfurther research is required to make a convincing conclusionregarding its role in the management of IBD and to help

establish exercise regimens that can account for each IBDpatientrsquos unique presentation [2] In conclusion in the presentstudy we aimed to investigate young adult rats which areage-matched population to the incidence of IBD [41] Ourresults suggest that exercise may be protective against theonset of IBD through the modulation of pro- and anti-inflammatory and antioxidant mediators with HO and NOSenzymes playing a central role in these effects Along theselines regular exercise can promote healthy aging via the samemechanisms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the Janos Bolyai ResearchScholarship of the Hungarian Academy of Sciences (to AnikoPosa) by the Hungarian National Development AgencyTAMOP 422-081-2008-0006 and TAMOP 422B-101-2010-0012 (to Zita Szalai) research grants This research wasrealized in the frames of TAMOP 424 A2-11-1-2012-0001(toAniko Posa KedvesneKrisztinaKupai andRenata Szabo)National Excellence Programmdashelaborating and operatingan inland student and researcher personal support systemThe project was subsidized by the European Union andcofinanced by the European Social Fund


References

[1] K van Wijck K Lenaerts J Grootjans et al ldquoPhysiology andpathophysiology of splanchnic hypoperfusion and intestinalinjury during exercise strategies for evaluation and preventionrdquoThe American Journal of PhysiologymdashGastrointestinal and LiverPhysiology vol 303 no 3 pp G155ndashG168 2012

[2] NNarula andRN Fedorak ldquoExercise and inflammatory boweldiseaserdquo The Canadian Journal of Gastroenterology vol 22 no5 pp 497ndash504 2008

[3] H P F Peters W R de Vries G P Vanberge-Henegouwen andLM A Akkermans ldquoPotential benefits and hazards of physicalactivity and exercise on the gastrointestinal tractrdquo Gut vol 48no 3 pp 435ndash439 2001

[4] I L Huibregtse A U van Lent and S J H van DeventerldquoImmunopathogenesis of IBD insufficient suppressor functionin the gutrdquo Gut vol 56 no 4 pp 584ndash592 2007

[5] B Khor A Gardet and R J Xavier ldquoGenetics and pathogenesisof inflammatory bowel diseaserdquo Nature vol 474 no 7351 pp307ndash317 2011

[6] I Arijs G deHertogh KMachiels et al ldquoMucosal gene expres-sion of cell adhesion molecules chemokines and chemokinereceptors in patients with inflammatory bowel disease beforeand after infliximab treatmentrdquo The American Journal of Gas-troenterology vol 106 no 4 pp 748ndash761 2011

[7] L Kruidenier I Kuiper C B H W Lamers and H WVerspaget ldquoIntestinal oxidative damage in inflammatory boweldisease semi-quantification localization and association withmucosal antioxidantsrdquo The Journal of Pathology vol 201 no 1pp 28ndash36 2003

[8] B Juhasz P Der P Szodoray et al ldquoAdrenocorticotropehormone fragment (4ndash10) attenuates the ischemiareperfusion-induced cardiac injury in isolated rat heartsrdquo Antioxidants andRedox Signaling vol 9 no 11 pp 1851ndash1861 2007

[9] B Juhasz B Varga A Czompa et al ldquoPostischemic cardiacrecovery in heme oxygenase-1 transgenic ischemicreperfusedmouse myocardiumrdquo Journal of Cellular and MolecularMedicine vol 15 no 9 pp 1973ndash1982 2011

[10] A Kertesz M Bombicz D Priksz et al ldquoAdverse impactof diet-induced hypercholesterolemia on cardiovascular tissuehomeostasis in a rabbit model time-dependent changes incardiac parametersrdquo International Journal ofMolecular Sciencesvol 14 no 9 pp 19086ndash19108 2013

[11] N G Abraham and A Kappas ldquoPharmacological and clinicalaspects of heme oxygenaserdquo Pharmacological Reviews vol 60no 1 pp 79ndash127 2008

[12] M P Soares and F H Bach ldquoHeme oxygenase-1 from biologyto therapeutic potentialrdquo Trends in Molecular Medicine vol 15no 2 pp 50ndash58 2009

[13] C Varga F Laszlo P Fritz et al ldquoModulation by heme and zincprotoporphyrin of colonic heme oxygenase-1 and experimentalinflammatory bowel disease in the ratrdquoThe European Journal ofPharmacology vol 561 no 1ndash3 pp 164ndash171 2007

[14] K Horvath C Varga A Berko A Posa F Laszlo and B J RWhittle ldquoThe involvement of heme oxygenase-1 activity in thetherapeutic actions of 5-aminosalicylic acid in rat colitisrdquo TheEuropean Journal of Pharmacology vol 581 no 3 pp 315ndash3232008

[15] S Adamopoulos J Parissis D Karatzas et al ldquoPhysicaltraining modulates proinflammatory cytokines and the solubleFassoluble Fas ligand system in patients with chronic heart

failurerdquo Journal of the American College of Cardiology vol 39no 4 pp 653ndash663 2002

[16] M Thirunavukkarasu B Juhasz L Zhan et al ldquoVEGFR1 (Flt-1+minus) gene knockout leads to the disruption of VEGF-mediatedsignaling through the nitric oxideheme oxygenase pathwayin ischemic preconditioned myocardiumrdquo Free Radical Biologyand Medicine vol 42 no 10 pp 1487ndash1495 2007

[17] G Kolios V Valatas and S G Ward ldquoNitric oxide in inflam-matory bowel disease a universal messenger in an unsolvedpuzzlerdquo Immunology vol 113 no 4 pp 427ndash437 2004

[18] M Porras M T Martın R Torres and P Vergara ldquoCyclicalupregulated iNOS and long-term downregulated nNOS are thebases for relapse and quiescent phases in a rat model of IBDrdquoThe American Journal of PhysiologymdashGastrointestinal and LiverPhysiology vol 290 no 3 pp G423ndashG430 2006

[19] M Harpavat D J Keljo and M D Regueiro ldquoMetabolicbone disease in inflammatory bowel diseaserdquo Journal of ClinicalGastroenterology vol 38 no 3 pp 218ndash224 2004

[20] N Lee G Radford-Smith and D R Taaffe ldquoBone loss inCrohnrsquos disease exercise as a potential countermeasurerdquo Inflam-matory Bowel Diseases vol 11 no 12 pp 1108ndash1118 2005

[21] G P Morris P L Beck M S Herridge W T Depew M RSzewczuk and J LWallace ldquoHapten-inducedmodel of chronicinflammation and ulceration in the rat colonrdquoGastroenterologyvol 96 no 3 pp 795ndash803 1989

[22] P P Bradley D A Priebat R D Christensen and G RothsteinldquoMeasurement of cutaneous inflammation estimation of neu-trophil content with an enzyme markerrdquo Journal of InvestigativeDermatology vol 78 no 3 pp 206ndash209 1982

[23] R TenhunenH SMarver andR Schmid ldquoThe enzymatic con-version of heme to bilirubin by microsomal heme oxygenaserdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 61 no 2 pp 748ndash755 1968

[24] N K Boughton-Smith S M Evans F Laszlo B J R Whittleand S Moncada ldquoThe induction of nitric oxide synthase andintestinal vascular permeability by endotoxin in the ratrdquo TheBritish Journal of Pharmacology vol 110 no 3 pp 1189ndash11951993

[25] H H Tong Y Chen X Liu and T F DeMaria ldquoDifferentialexpression of cytokine genes and iNOS induced by nonviablenontypeableHaemophilus influenzae or its LOSmutants duringacute otitis media in the ratrdquo International Journal of PediatricOtorhinolaryngology vol 72 no 8 pp 1183ndash1191 2008

[26] X-Z Shi J H Winston and S K Sarna ldquoDifferentialimmune and genetic responses in rat models of Crohnrsquos colitisand ulcerative colitisrdquo The American Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 300 no 1 pp G41ndashG51 2011

[27] R J Xavier and D K Podolsky ldquoUnravelling the pathogenesisof inflammatory bowel diseaserdquo Nature vol 448 no 7152 pp427ndash434 2007

[28] S Ghosh S Golbidi I Werner B C Verchere and I LaherldquoSelecting exercise regimens and strains to modify obesity anddiabetes in rodents an overviewrdquo Clinical Science vol 119 no2 pp 57ndash74 2010

[29] A Saxena E Fletcher B Larsen M S Baliga J L Durstineand R Fayad ldquoEffect of exercise on chemically-induced colitisin adiponectin deficient micerdquo Journal of Inflammation vol 9no 1 article 30 2012


[30] A Mekinian R Tamouza S Pavy et al ldquoFunctional study ofTNF-120572 promoter polymorphisms literature review and meta-analysisrdquoThe European Cytokine Network vol 22 no 2 pp 88ndash102 2011

[31] S K Powers and M J Jackson ldquoExercise-induced oxidativestress cellularmechanisms and impact onmuscle force produc-tionrdquo Physiological Reviews vol 88 no 4 pp 1243ndash1276 2008

[32] S V Brooks A Vasilaki L M Larkin A McArdle and M JJackson ldquoRepeated bouts of aerobic exercise lead to reductionsin skeletal muscle free radical generation and nuclear factor 120581BactivationrdquoThe Journal of Physiology vol 586 no 16 pp 3979ndash3990 2008

[33] L George M F Lokhandwala and M Asghar ldquoExerciseactivates redox-sensitive transcription factors and restores renalD1 receptor function in old ratsrdquo The American Journal ofPhysiologymdashRenal Physiology vol 297 no 5 pp F1174ndashF11802009

[34] O Kasimay E Guzel A Gemici et al ldquoColitis-induced oxida-tive damage of the colon and skeletal muscle is amelioratedby regular exercise in rats the anxiolytic role of exerciserdquoExperimental Physiology vol 91 no 5 pp 897ndash906 2006

[35] M Sasaki S Bharwani P Jordan et al ldquoIncreased diseaseactivity in eNOS-deficient mice in experimental colitisrdquo FreeRadical Biology and Medicine vol 35 no 12 pp 1679ndash16872003

[36] W Aoi Y Naito T Takagi et al ldquoRegular exercise reducescolon tumorigenesis associated with suppression of iNOSrdquoBiochemical and Biophysical Research Communications vol 399no 1 pp 14ndash19 2010

[37] N T Jenkins R Q Landers S J Prior N Soni E ESpangenburg and J M Hagberg ldquoEffects of acute and chronicendurance exercise on intracellular nitric oxide and superoxidein circulating CD34+ and CD34minus cellsrdquo Journal of AppliedPhysiology vol 111 no 3 pp 929ndash937 2011

[38] M Zwolinska-Wcislo T Brzozowski A Ptak-Belowska et alldquoNitric oxide-releasing aspirin but not conventional aspirinimproves healing of experimental colitisrdquo World Journal ofGastroenterology vol 17 no 36 pp 4076ndash4089 2011

[39] W Dou J Zhang A Sun et al ldquoProtective effect of naringeninagainst experimental colitis via suppression of Toll-like receptor4NF-120581B signallingrdquoTheBritish Journal of Nutrition vol 110 no4 pp 599ndash608 2013

[40] J Xing C You K Dong et al ldquoAmeliorative effects of 34-oxo-isopropylidene-shikimic acid on experimental colitis and theirmechanisms in ratsrdquo International Immunopharmacology vol15 no 3 pp 524ndash531 2013

[41] A Sonnenberg ldquoAge distribution of IBD hospitalizationrdquoInflammatory Bowel Diseases vol 16 no 3 pp 452ndash457 2010

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


proinflammatory mediators such as tumour necrosis factor-120572 (TNF-120572) interleukin-1120573 (IL-1120573) and chemokine ligand1 (CXCL1) The importance of anti- and proinflammatorybalance is reflected in the efficacy of anti-TNF-120572 therapy inthe treatment of IBD [5] TNF-120572 inhibitors have proven suc-cessful in inducing and maintaining remission of moderate-to-severe IBD In addition CXCL1 is a chemoattractant forneutrophils and plays a role in inflammation increased ilealand colonic CXCL1 gene expression was also reported in IBDpatients [6]

IBD is associated with enhanced production of reactivemetabolites of oxygen and nitrogen (RONS) Moreoveroxidative stress plays an important role in the pathogen-esis of this disease [7] Heme oxygenase-1 (HO-1) theinducible isoform of heme oxygenase enzymes is a stress-responsive enzyme which can be induced by oxidative stressinflammatory cytokines and many other molecules HO-1 is thought to play an important role in the protectionof tissues from oxidative injuries and inflammation [8ndash10]These beneficial effects of HO-1 are mediated in part by theantioxidant and anti-inflammatory end products of hemebreakdown namely CO and bilirubin which can inhibitthe expression of proinflammatory cytokines such as TNF-120572 and IL-1120573 Several molecules such as interleukin-10 (IL-10) function through an HO-1 dependent mechanism [11 12]Pharmacological studies suggest a beneficial role for HO-1in modulating colitis Upregulation of HO-1 by heme andcadmium chloride can ameliorate while inhibition of theenzyme by tin protoporphyrin can aggravate experimentalinflammation in the colon [13] 5-Aminosalicylic-acid aclinically approved anticolitic agent can exert beneficialeffects in vivo partially through the induction or activationof HO-1 [14]

Another enzyme involved in intestinal inflammation andoxidative stress is nitric oxide synthase [15] The constitutiveproduction of NO derived from the constitutive isoforms ofthe enzyme namely neuronal NOS (nNOS) and endothelialNOS (eNOS) plays a role in various physiological processesin the GI mucosa such as the regulation of microvascularand epithelial permeability and the maintenance of adequateperfusion [16] On the other hand the effects of iNOS-produced [15]NO can be beneficial or detrimental dependingon the amount duration and anatomical site of synthesis[16 17] In experimental colitis the downregulation of nNOSand the overproduction of NO by iNOS are observed [18]For example proinflammatory cytokines such as TNF-120572 caninduceNOproduction and iNOS activity in colonic epithelialcells [17]

Earlier studies have focusedmainly on the role of exercisein the treatment of extraintestinal manifestations of IBD [1920] However the effects of physical exercise on the inflamedgut are still inconsistent Therefore the aims of the presentstudy were to investigate the effects of recreational physicalexercise (1) on the colonic damage in a TNBS rat model(damage score lesion) (2) on the gene expression (TNF-120572IL-1120573 CXCL1 and IL-10) and protein concentration (TNF-120572)of inflammatory mediators in experimental acute colitis and(3) on the activity of oxidativeantioxidative enzymes (MPOHO andNOS) in the inflamed colon of ratsWe hypothesized

that voluntary exercise prior to TNBS challenge will reducecolonic damage and oxidative and inflammatory mediatorgene and protein expressions

2 Materials and Methods

21 Animals Male Wistar rats (180ndash220 g) were housed ingroups Food was withdrawn overnight before induction ofcolitis otherwise the animals had access to food anddrinkingwater ad libitum throughout the experiments The animalcare and research protocols were in accordance with theguidelines of the University of Szeged

22 Experimental Design The animals were randomlydivided into four groups 1 sedentary control (nonrunningnon-colitis-induced 119899 = 13) 2 running control (runningnon-colitis-induced 119899 = 18) 3 sedentary TNBS (119899 = 14)and 4 running TNBS groups (119899 = 18) (Figure 1) Rats of therunning groups were placed in cages with a running wheel(Acellabor Ltd Budapest Hungary) Before the beginningof experimental period rats were allowed to accommodateto the running wheel for a week The activity of the rats onthe running wheel was monitored by a bicycle computerattached to each wheel After 6 weeks of self-administeredphysical exercise colitis was induced by 246-trinitrobenzenesulphonic acid (TNBS once 10mg in 025mL of 50ethanol wv) The intracolonic administration of TNBSwas performed with an 8 cm long plastic catheter undertransient ether anaesthesia (the method originally describedby Morris et al [21]) The control groups did not receiveany treatment Physical activity was continued followingTNBS treatment Rats were weighed twice a week untilthe induction of colitis and then daily following the TNBSchallenge 72 hours after the induction of colitis the animalswere sacrificed and the distal 8 cm portion of the colonwas dissected longitudinally opened gently rinsed withice-cold physiological saline and photographed (PanasonicLumix DMC-TZ6 digital camera) for the determination ofmacroscopic colonic inflammatory damage The colon wasweighed and divided into longitudinal segments to be usedfor the following molecular and biochemical analyses

23 Damage Score and Lesion Measurement The extentof macroscopically apparent inflammation ulceration andtissue disruption was determined in a randomized mannerfrom the colour images using a proprietary computerizedplanimetry software developed in our laboratory (Stat 2 1 1)The area of macroscopically visible mucosal involvement wascalculated and expressed as the percentage of the total colonicsegment area under study

The degree of colonic inflammation was scored on a 0ndash11scale in a randomized blinded fashion The criteria has beenadapted from what has been used previously 0 = no damage1 = focal hyperemia and no ulcers 2 = ulceration withouthyperemia or bowel wall thickening 3 = ulceration withinflammation at 1 site 4 = more than 2 sites of ulceration andinflammation 5 = more than 2 major sites of ulceration andinflammation or 1 site of ulcerationinflammation extending


Sedentary control

Sedentary TNBS

TNBS challenge

Accommodation

Running control

Running TNBS

Weekly measuring of

the body weight


Sacrifce

Measuring of


Te concentration of

Timeline

Sedentary Running

Week 0


Week 7

Experimental design

Groups

TNF-120572TNF-120572IL-1120573

CXCL1IL-10


∙ MPO














2PO4 10mM


2 10mMDTT 10mM








119879method


3 Results



1 11 21 31 41

100

125

150

175

200


Days

Body

wei

ght c

hang

e (

)

lowast

(a)

0 1 2 380

90

100

110


Days

Body

wei

ght c

hang

e (

)

(b)













0

2

4

6

8

10

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS


Dam

age s

core

(sca

le0

ndash11

)

(a)

0

10

20

30

40

50

60

70

Lesio

nTo

tal c

olon

ic se

gmen

t (

)

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

lowastlowast

(b)

Sedentary control

Running control

Sedentary TNBS

Running TNBS

(c)





4 Discussion




0

250

500

750

1000

MPO

activ

ity (m

Um

g pr

otei

n)

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

lowastlowast













IL-1120573TNF-120572 CXCL1 IL-10

00625006250062500625Relat

ive g

ene e

xpre

ssio

nPB

MCs

8

4

2

1

05

025

0125

4

2

1

05

025

0125

4

2

1

05

025

0125

2

1

05

025

0125

lowast


lowast

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

fold

chan

ge (L

og2)

(a)

00625

2

64

0125025

051

48

1632

00625

2

64

0125025

051

48

1632

00625

2

64

0125025

051

48

1632

Relat

ive g

ene e

xpre

ssio

nC

olon

00625

8

4

2

1

05

025

0125

lowastlowast

lowastlowastlowast

lowast

lowast lowast

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

fold

chan

ge (L

og2)

(b)


0

100

200

300

400

500

600

700 +

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

TNF-120572

conc

entr

atio

n (p

gm

L)

(a)

1

2

3

4

5

6

7

8

+++

+++

HO

activ

ity(n

M b

iliru

bin

hm

g pr

otei

n)

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

(b)



0

100

200

300

400

500+

+++

cNO

S ac

tivity

(pm

olm

inm

g pr

otei

n)

lowastlowastlowast

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

(a)

0

50

100

150

200

250

++

iNO

S ac

tivity

(pm

olm

inm

g pr

otei

n)

lowast

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

(b)








Acknowledgments



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Sedentary control

Sedentary TNBS

TNBS challenge

Accommodation

Running control

Running TNBS

Weekly measuring of

the body weight


Sacrifce

Measuring of


Te concentration of

Timeline

Sedentary Running

Week 0


Week 7

Experimental design

Groups

TNF-120572TNF-120572IL-1120573

CXCL1IL-10


∙ MPO














2PO4 10mM


2 10mMDTT 10mM








119879method


3 Results



1 11 21 31 41

100

125

150

175

200


Days

Body

wei

ght c

hang

e (

)

lowast

(a)

0 1 2 380

90

100

110


Days

Body

wei

ght c

hang

e (

)

(b)













0

2

4

6

8

10

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS


Dam

age s

core

(sca

le0

ndash11

)

(a)

0

10

20

30

40

50

60

70

Lesio

nTo

tal c

olon

ic se

gmen

t (

)

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

lowastlowast

(b)

Sedentary control

Running control

Sedentary TNBS

Running TNBS

(c)





4 Discussion




0

250

500

750

1000

MPO

activ

ity (m

Um

g pr

otei

n)

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

lowastlowast













IL-1120573TNF-120572 CXCL1 IL-10

00625006250062500625Relat

ive g

ene e

xpre

ssio

nPB

MCs

8

4

2

1

05

025

0125

4

2

1

05

025

0125

4

2

1

05

025

0125

2

1

05

025

0125

lowast


lowast

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

fold

chan

ge (L

og2)

(a)

00625

2

64

0125025

051

48

1632

00625

2

64

0125025

051

48

1632

00625

2

64

0125025

051

48

1632

Relat

ive g

ene e

xpre

ssio

nC

olon

00625

8

4

2

1

05

025

0125

lowastlowast

lowastlowastlowast

lowast

lowast lowast

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

fold

chan

ge (L

og2)

(b)


0

100

200

300

400

500

600

700 +

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

TNF-120572

conc

entr

atio

n (p

gm

L)

(a)

1

2

3

4

5

6

7

8

+++

+++

HO

activ

ity(n

M b

iliru

bin

hm

g pr

otei

n)

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

(b)



0

100

200

300

400

500+

+++

cNO

S ac

tivity

(pm

olm

inm

g pr

otei

n)

lowastlowastlowast

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

(a)

0

50

100

150

200

250

++

iNO

S ac

tivity

(pm

olm

inm

g pr

otei

n)

lowast

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

(b)








Acknowledgments



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















2PO4 10mM


2 10mMDTT 10mM








119879method


3 Results



1 11 21 31 41

100

125

150

175

200


Days

Body

wei

ght c

hang

e (

)

lowast

(a)

0 1 2 380

90

100

110


Days

Body

wei

ght c

hang

e (

)

(b)













0

2

4

6

8

10

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS


Dam

age s

core

(sca

le0

ndash11

)

(a)

0

10

20

30

40

50

60

70

Lesio

nTo

tal c

olon

ic se

gmen

t (

)

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

lowastlowast

(b)

Sedentary control

Running control

Sedentary TNBS

Running TNBS

(c)





4 Discussion




0

250

500

750

1000

MPO

activ

ity (m

Um

g pr

otei

n)

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

lowastlowast













IL-1120573TNF-120572 CXCL1 IL-10

00625006250062500625Relat

ive g

ene e

xpre

ssio

nPB

MCs

8

4

2

1

05

025

0125

4

2

1

05

025

0125

4

2

1

05

025

0125

2

1

05

025

0125

lowast


lowast

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

fold

chan

ge (L

og2)

(a)

00625

2

64

0125025

051

48

1632

00625

2

64

0125025

051

48

1632

00625

2

64

0125025

051

48

1632

Relat

ive g

ene e

xpre

ssio

nC

olon

00625

8

4

2

1

05

025

0125

lowastlowast

lowastlowastlowast

lowast

lowast lowast

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

fold

chan

ge (L

og2)

(b)


0

100

200

300

400

500

600

700 +

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

TNF-120572

conc

entr

atio

n (p

gm

L)

(a)

1

2

3

4

5

6

7

8

+++

+++

HO

activ

ity(n

M b

iliru

bin

hm

g pr

otei

n)

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

(b)



0

100

200

300

400

500+

+++

cNO

S ac

tivity

(pm

olm

inm

g pr

otei

n)

lowastlowastlowast

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

(a)

0

50

100

150

200

250

++

iNO

S ac

tivity

(pm

olm

inm

g pr

otei

n)

lowast

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

(b)








Acknowledgments



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















1 11 21 31 41

100

125

150

175

200


Days

Body

wei

ght c

hang

e (

)

lowast

(a)

0 1 2 380

90

100

110


Days

Body

wei

ght c

hang

e (

)

(b)













0

2

4

6

8

10

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS


Dam

age s

core

(sca

le0

ndash11

)

(a)

0

10

20

30

40

50

60

70

Lesio

nTo

tal c

olon

ic se

gmen

t (

)

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

lowastlowast

(b)

Sedentary control

Running control

Sedentary TNBS

Running TNBS

(c)





4 Discussion




0

250

500

750

1000

MPO

activ

ity (m

Um

g pr

otei

n)

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

lowastlowast













IL-1120573TNF-120572 CXCL1 IL-10

00625006250062500625Relat

ive g

ene e

xpre

ssio

nPB

MCs

8

4

2

1

05

025

0125

4

2

1

05

025

0125

4

2

1

05

025

0125

2

1

05

025

0125

lowast


lowast

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

fold

chan

ge (L

og2)

(a)

00625

2

64

0125025

051

48

1632

00625

2

64

0125025

051

48

1632

00625

2

64

0125025

051

48

1632

Relat

ive g

ene e

xpre

ssio

nC

olon

00625

8

4

2

1

05

025

0125

lowastlowast

lowastlowastlowast

lowast

lowast lowast

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

fold

chan

ge (L

og2)

(b)


0

100

200

300

400

500

600

700 +

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

TNF-120572

conc

entr

atio

n (p

gm

L)

(a)

1

2

3

4

5

6

7

8

+++

+++

HO

activ

ity(n

M b

iliru

bin

hm

g pr

otei

n)

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

(b)



0

100

200

300

400

500+

+++

cNO

S ac

tivity

(pm

olm

inm

g pr

otei

n)

lowastlowastlowast

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

(a)

0

50

100

150

200

250

++

iNO

S ac

tivity

(pm

olm

inm

g pr

otei

n)

lowast

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

(b)








Acknowledgments



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

2

4

6

8

10

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS


Dam

age s

core

(sca

le0

ndash11

)

(a)

0

10

20

30

40

50

60

70

Lesio

nTo

tal c

olon

ic se

gmen

t (

)

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

lowastlowast

(b)

Sedentary control

Running control

Sedentary TNBS

Running TNBS

(c)





4 Discussion




0

250

500

750

1000

MPO

activ

ity (m

Um

g pr

otei

n)

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

lowastlowast













IL-1120573TNF-120572 CXCL1 IL-10

00625006250062500625Relat

ive g

ene e

xpre

ssio

nPB

MCs

8

4

2

1

05

025

0125

4

2

1

05

025

0125

4

2

1

05

025

0125

2

1

05

025

0125

lowast


lowast

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

fold

chan

ge (L

og2)

(a)

00625

2

64

0125025

051

48

1632

00625

2

64

0125025

051

48

1632

00625

2

64

0125025

051

48

1632

Relat

ive g

ene e

xpre

ssio

nC

olon

00625

8

4

2

1

05

025

0125

lowastlowast

lowastlowastlowast

lowast

lowast lowast

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

fold

chan

ge (L

og2)

(b)


0

100

200

300

400

500

600

700 +

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

TNF-120572

conc

entr

atio

n (p

gm

L)

(a)

1

2

3

4

5

6

7

8

+++

+++

HO

activ

ity(n

M b

iliru

bin

hm

g pr

otei

n)

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

(b)



0

100

200

300

400

500+

+++

cNO

S ac

tivity

(pm

olm

inm

g pr

otei

n)

lowastlowastlowast

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

(a)

0

50

100

150

200

250

++

iNO

S ac

tivity

(pm

olm

inm

g pr

otei

n)

lowast

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

(b)








Acknowledgments



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

250

500

750

1000

MPO

activ

ity (m

Um

g pr

otei

n)

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

lowastlowast













IL-1120573TNF-120572 CXCL1 IL-10

00625006250062500625Relat

ive g

ene e

xpre

ssio

nPB

MCs

8

4

2

1

05

025

0125

4

2

1

05

025

0125

4

2

1

05

025

0125

2

1

05

025

0125

lowast


lowast

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

fold

chan

ge (L

og2)

(a)

00625

2

64

0125025

051

48

1632

00625

2

64

0125025

051

48

1632

00625

2

64

0125025

051

48

1632

Relat

ive g

ene e

xpre

ssio

nC

olon

00625

8

4

2

1

05

025

0125

lowastlowast

lowastlowastlowast

lowast

lowast lowast

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

fold

chan

ge (L

og2)

(b)


0

100

200

300

400

500

600

700 +

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

TNF-120572

conc

entr

atio

n (p

gm

L)

(a)

1

2

3

4

5

6

7

8

+++

+++

HO

activ

ity(n

M b

iliru

bin

hm

g pr

otei

n)

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

(b)



0

100

200

300

400

500+

+++

cNO

S ac

tivity

(pm

olm

inm

g pr

otei

n)

lowastlowastlowast

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

(a)

0

50

100

150

200

250

++

iNO

S ac

tivity

(pm

olm

inm

g pr

otei

n)

lowast

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

(b)








Acknowledgments



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















IL-1120573TNF-120572 CXCL1 IL-10

00625006250062500625Relat

ive g

ene e

xpre

ssio

nPB

MCs

8

4

2

1

05

025

0125

4

2

1

05

025

0125

4

2

1

05

025

0125

2

1

05

025

0125

lowast


lowast

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

fold

chan

ge (L

og2)

(a)

00625

2

64

0125025

051

48

1632

00625

2

64

0125025

051

48

1632

00625

2

64

0125025

051

48

1632

Relat

ive g

ene e

xpre

ssio

nC

olon

00625

8

4

2

1

05

025

0125

lowastlowast

lowastlowastlowast

lowast

lowast lowast

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

fold

chan

ge (L

og2)

(b)


0

100

200

300

400

500

600

700 +

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

TNF-120572

conc

entr

atio

n (p

gm

L)

(a)

1

2

3

4

5

6

7

8

+++

+++

HO

activ

ity(n

M b

iliru

bin

hm

g pr

otei

n)

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

(b)



0

100

200

300

400

500+

+++

cNO

S ac

tivity

(pm

olm

inm

g pr

otei

n)

lowastlowastlowast

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

(a)

0

50

100

150

200

250

++

iNO

S ac

tivity

(pm

olm

inm

g pr

otei

n)

lowast

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

(b)








Acknowledgments



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

100

200

300

400

500+

+++

cNO

S ac

tivity

(pm

olm

inm

g pr

otei

n)

lowastlowastlowast

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

(a)

0

50

100

150

200

250

++

iNO

S ac

tivity

(pm

olm

inm

g pr

otei

n)

lowast

Sede

ntar

y co

ntro

l

Runn

ing

cont

rol

Sede

ntar

y TN

BS

Runn

ing

TNBS

(b)








Acknowledgments



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















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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