research article involvement of il-13 and tissue...

Research ArticleInvolvement of IL-13 and Tissue Transglutaminase in LiverGranuloma and Fibrosis after Schistosoma japonicum Infection

Juanjuan Tang1 Huayi Huang1 Xiaofang Ji1 Xunmin Zhu2 Yinyan Li2 Mingmin She1

Suikai Yan2 Mingchiu Fung3 and Zi Li1

1 Guangzhou Hoffmann Institute of Immunology School of Basic Sciences Guangzhou Medical University Guangzhou 510182 China2Morphology Department School of Basic Sciences Guangzhou Medical University Guangzhou 510182 China3 School of Life Sciences The Chinese University of Hong Kong Hong Kong

Correspondence should be addressed to Zi Li lizi1002gzhmueducn

Received 28 February 2014 Revised 20 May 2014 Accepted 5 June 2014 Published 3 July 2014

Academic Editor Yves Denizot

Copyright copy 2014 Juanjuan Tang 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

Schistosomiasis one of the most devastating parasitic diseases is caused by Schistosoma japonicum (Sj) infection resulting inserious liver fibrosis Interleukin- (IL-) 13 which is produced by TH2 cells is a critical profibrotic cytokine found in various organsincluding the liver Tissue transglutaminase (tTG) a group ofmultifunctional enzymes serves a central function in the pathogenesisof chronic liver diseases However the relationship between IL-13 tTG and liver fibrosis during Schistosoma infection has not beenestablished This study investigated the involvement of IL-13 and tTG in liver fibrogenesis during Sj infection in mice Five weeksafter Sj infection granuloma and fibrosis development in the liver coincided with an increase in IL-13 and tTG in the liver andthe upregulation of serum IL-13 in infected mice Administration of cystamine an inhibitor of tTG abrogated the increase in bothtTG and IL-13 in infected mice and ameliorated liver fibrogenesis and granuloma development This result establishes a novel linkamong IL-13 tTG and liver granuloma and fibrosis under Sj infection Based on their important functions in liver fibrosis inducedby Sj infection IL-13 and tTG could be promising potential drug targets against schistosomiasis

1 Introduction

Despite decades of effort Schistosoma japonicum (Sj) remainsto be a prevalent Schistosome in Asia and is the only Schis-tosome causing infection in China Although fundamentalcontrol has been achieved through potent governmentalmeasures Sj infection remains widespread and cannot beeffectively controlled in some lakes and marshland regionsthus inflicting massive social and economic burdens [1ndash3]Progressive liver fibrosis is one of the major hallmarks ofSj infections and accounts for the further development ofportal hypertension ascites hepatosplenomegaly or evenliver cirrhosis [4] However the mechanism behind schis-tosomal liver fibrogenesis and immune response remainspoorly understood

As Schistosome infection progresses into the chronicphase Schistosome eggs that are deposited in tissues beginto secrete soluble egg antigen (SEA) which suppresses

Th1-mediated response and promotes Th2-mediated inflam-matory reactions [4] Thus Th2-related cytokines areinferred to participate actively in fibrogenesis and have thusbeen extensively studied [5 6] Interleukin- (IL-) 13 is acytokine secreted by several cell types including eosinophilsmast cells basophils epithelial cells smooth muscle cellsfibroblasts macrophages and T cells especially Th2 cellsIL-13 participates in asthma tumorigenesis and parasiticdiseases In Schistosoma mansoni- (Sm-) infected mice IL-13mediated liver fibrosis as a promoter and sustainer [7] Liverfibrogenesis is severely decreased in Sm-infected IL-13-deficient mice as well as in wild-type animals treated withIL-13 antagonists whereas in TGF-1205731 and MMP-9 deficientmice such alleviation effect is not apparent [8ndash10] Sj haspathological patterns similar to those of Sm but no data haveshown that IL-13 serves a function in the upstream regulationmechanism of liver fibrogenesis caused by Sj infection

Hindawi Publishing CorporationMediators of InflammationVolume 2014 Article ID 753483 11 pageshttpdxdoiorg1011552014753483

2 Mediators of Inflammation

Tissue transglutaminase (tTG) as a multifunctional pro-tein performs a variety of intracellular and extracellular func-tions including hydrolyzing GTP transamidation activity asa catalyzing enzyme and cross-linking glutamine residuesand lysine residues of proteins These properties cause tTGto be involved in various physiological processes which ifregulated inappropriately can also lead to its involvementin a number of diseases such as metastatic cancer coeliacdisease and lung renal and liver fibrosis [11ndash13] tTG isoverexpressed in both the murine experimental liver fibrosismodel and hepatitis c virus-induced human liver fibrosisRNA interference can result in diminution of liver fibrosisand lesser aggregation of fibrotic tissue [14] Cystamine(CTM) can modify cysteine at the active site on tTG in adisulfide interchanging manner such that the activity of tTGis selectively inhibited [15] Inhibition of tTG activity by CTMresults in the diminution of liver fibrosis induced by CCl

4

[16] Meanwhile tTG knockout mice with CCl4intoxication

display high lethality as compared with wild-type controls[17] Therefore enhanced tTG level seems to protect theliver from acute and chronic injury but its net effect onfibrogenesis resulting from different causes requires furtherstudy

Several studies have indicated that tTG serves as animportant inflammatory and fibrogenetic factor [18] tTGmodulates inflammation exerting both pro- and anti-inflammatory effects such as positive feedback loop withTGF-1205731 [19] NF-120581B a key inflammation-associated factoris activated by tTG through the cross-linking of the C-terminal glutamine cluster of I120581Ba [20] Epithelial tTG caninduceTh17 differentiation and subsequent IL-17 productionand pulmonary fibrosis in bleomycin-treated mice More-over tTG reportedly regulates Th2 cytokine secretion andmediates in vitro and in vivo allergic inflammation [2021] tTG can also downregulate peroxisome proliferator-activated receptor 120574 (PPAR120574) as well as increasing the classicparameters of inflammation such as TNF-alpha tyrosinephosphorylation and MAPKs in a model of cystic fibrosis[22]

In this study we take an important step toward answeringthese questions by uncovering a novel connection amongtTG IL-13 and Sj-caused liver fibrosis We find that tTGand IL-13 levels are upregulated in mice with Sj infectionImportantly we further demonstrate that the treatment ofmice with Sj-induced liver fibrosis by using an inhibitorof the enzymatic activity of tTG inhibits the Sj-inducedupregulation of IL-13 and alleviates liver fibrosis To ourknowledge these findings show that tTG is involved in thedevelopment of Sj infection-induced liver fibrosis in miceand themechanismmay be associatedwith tTG-regulated IL-13 expression

2 Methods

21 Animal Grouping Parasite Infection and Cystamine Ad-ministration This study was performed in strict accordancewith the recommendations in the Guide for the Care and Useof Laboratory Animals of State Scientific and Technological

Commission The protocol was approved by the Committeeon the Ethics of Animal Experiments of Guangzhou MedicalUniversity All surgeries were performed under sodium pen-tobarbital anesthesia and every effort was made to minimizesuffering

Six- to eight-week-old female BALBCmice (Experimen-tal Animal Center of Sun Yat-Sen University GuangzhouChina) were infected percutaneously through the abdomenwith 20 plusmn 3 Sj cercariae The mice livers were collected anddetermined at weeks 5 6 8 and 12 after infection Concurrentcontrols were uninfected mice

Cystamine (CTM Sigma-Aldrich St Louis USA) is acommon tTG inhibitor [15 16 23 24] In this study for themice group with tTG inhibitor-CTM treatment starting atday 3 after infection mice were administered with 100120583Lof CTM (10minus2mM Sigma) in phosphate-buffered solution(PBS) through intraperitoneal injection once per day for7 d whereas the infection control group received PBS aloneTwo noninfected control mice groups were treated withCTM or PBS The number of mice in every group is 8Sera of mice of all groups were collected for underlyinganalysis

22 Calculation of Collagen Area Using Massonrsquos Trichrome(MT) Staining Fresh liver tissues were fixed in 4 parafor-maldehyde overnight and routinely paraffin-embedded Par-affin sections (4 120583m) were prepared from each liver tissuesample The liver tissue sections were stained by MT stainingto evaluate collagen content and distribution The collagenfibers were stained blue cell nuclei were stained black andthe background was stained red Percentage of total areaswith collagen positive color (blue) [percentage of collagenarea (positive blue color areatotal area)] was analyzedusing Image-Pro Plus 60 software Every stained samplewas evaluated in double-blind fashion by two independentinvestigators

23 RNA Isolation and RT-PCR and Quantitative-PCR TotalRNA was extracted from fresh liver tissue homogenizedin TRIZol reagent (Invitrogen) according to the manufac-turerrsquos protocol RNA purity and concentration were assessedby spectrophotometry Reverse transcriptase (RT) reactionsfor cDNA synthesis were performed using PrimeScriptRT Master Mix (TAKARA) Relative mRNA expressionlevel was determined by real-time quantitative polymerasechain reaction (Q-PCR) with SYBR Green I PCR Master(TAKARA) kit on ABI 7500 machine according to themanufacturerrsquos protocol The primers used were as followsmouse-IL-13 (forward CGGCAGCATGGTATGGAGTGreverse ATTGCAATTGGAGATGTTGGTCAG) mouse-tTG (forward 51015840-GTGAGCCGTGCTATCTGTCCTG-31015840reverse 51015840-ACTGCCTGCTTGGAACCTGAA-31015840) mouse-GAPDH (forward 51015840-TGTGTCCGTCGTGGATCTGA-31015840reverse 51015840-TTGCTGTTGAAGTCGCAGGAG-31015840) The Q-PCR results were expressed as fold amplification using the2minusΔΔCt method with mouse GAPDH as the internal controlEach experiment was repeated thrice

Mediators of Inflammation 3

24 Measurement of Mice Sera IL-13 and Hyaluronic Acidby ELISA Peripheral venous blood was collected by cut-ting the tail veins of mice After being incubated in roomtemperature for 1 h blood samples were centrifuged (1000 g4∘C) for 10ndash15min Supernatant was extracted to detectIL-13 (Sigma-Aldrich St Louis USA) and hyaluronic acid(Mouse Hyaluronic acid ELISA kit Shanghai Yueyang Bio-logical Technology Co Ltd) through ELISA measurementAbsorbance of the samples was measured at a 450 nmwavelength

25 Immunohistochemistry (IHC) Assay After sacrifice themouse livers were immediately fixed in 4paraformaldehydeand then paraffin-embedded Liver sections of 4120583m wereprepared Endogenous peroxidase was blocked with 3hydrogen peroxide (H

2O2) Anti-IL-13 (AF-413-NA RampD

Systems) antismoothmuscle actin alpha (120572-SMA) (BM0002BOSTER) or anti-tTG (sc-20621 Santa Cruz Biotechnology)primary antibody was diluted in 50-fold IL-13 120572-SMA andtTG expressions were detected using GTVision II DetectionSystemMoampRb (Gene Tech (Shanghai) Co Ltd) accordingto the manufacturerrsquos instructions

26 Western Blot Analysis Fresh mouse livers were groundinto powder in liquid nitrogen and moderate protein lysissolution (RIPA PMSF = 100 1) was added After incubationon ice for 30min tissue debris was removed by centrifugation(15min 4∘C) Protein concentrations were assayed by usingthe Bradford assay (BIO-RAD) Total protein was resolvedby SDS-PAGE and then transferred to a polyvinylidene flu-oride membrane (02 120583m Millipore) After blocking with 5skimmedmilk membranes were probedwith the appropriateantibody Protein bands were detected with ECL reagentsThe antibodies used were as follows anti-GAPDH Ab (CellSignaling Technology) anti-120572-SMA (BM0002 BOSTER)anti-120572-SMA (BM0002 BOSTER) and anti-tTG Ab (sc-20621 Santa Cruz Biotechnology)

27 Total Transglutaminase Activity in Lysates of Liver Tis-sue The transglutaminase (TGase) activity of liver tissuelysates was determined through a modified nonradioac-tive microtiter plate assay Briefly the microtiter plateswere coated with 100 120583L of NN1015840-dimethylcasein (Sigma10mgmL to 20mgmL) at 4∘C overnight and the wellswere blocked with nonfat dry milk (05 in 01M Tris-HClpH 85) for 30min The wells were then washed twice with350 120583L of 01M Tris-HCl (pH 85) The following reagentswere added to each well to obtain a total volume of 50 120583Lper well 5mM CaCl

2 10mM dithiothreitol 05mM 5-

(biotinamido)pentylamine 04 120583g lysates of liver tissue and01M Tris-HCl (pH 85) The microtiter plate was incubatedat 37∘C for 30min The liquid was then discarded andthe reaction was stopped by washing twice with 350 120583L of5mM of EDTA followed by washing twice with 350120583L of01MTris-HCl (pH85) Streptavidin-horseradish peroxidaseconjugates were diluted at a proportion of 1 200 with nonfatdry milk (05 in 01M Tris-HCl (pH 85)) prior to theaddition of 60120583L of solution per well to be incubated for

l h at room temperature The plate was washed once with350 120583L of 0001 Triton X-100 followed by washing for fourtimes with 350 120583L of 01M Tris-HCl (pH 85) Then 100 120583Lof substrate solution (TMB Sigma T0440) was added toeach well After incubation at room temperature for 10minthe reactions were stopped by the addition of 25120583L of3N HCl to each well and the proteins into which the 5-(biotinamido)pentylamine was incorporated were quantifiedby measuring the absorbance at 450 nm in a plate reader(BioTek) The relative TGase activity was determined usingthe absorbance at 450 nm (OD450)

28 Statistics All statistical analyses were performed usingSPSS130 software Statistical significance (119875 lt 005) betweenthe means of two groups was determined using Studentrsquos 119905-tests Statistical comparisons of the means of multiple (gt2)groups were determined using repeated-measure one-wayANOVA All data were expressed as means plusmn SD and allexperiments were repeated twice or thrice

3 Results

31 IL-13 Correlated with Hepatic Fibrosis after Sj InfectionBABLc mice were infected cutaneously with 20 plusmn 3 Sjcercariae of Chinese mainland strain and mice liver tissueswere collected at weeks 0 5 6 8 and 12 after infectionwith SjLiver granuloma and fibrosis were evaluated by monitoringthe liver manifestation area of collagen fiber (stained bluein MT staining) (Figure 1(a)) level of serum hyaluronic acid(Figure 1(b)) and strength of 120572-SMA-positive staining as amarker of hepatic stellate cell (HSC) activation (Figure 1(c))Sj liver granuloma began at week 5 and then fibrosisprogressed the most seriously at week 8 whereas chronicliver fibrosis appeared at week 12 To evaluate the cor-relation between IL-13 and liver fibrogenesis hepatic IL-13 expression profile in both Sj-infected mice and normalcontrol was detected by RT-PCR Gel analysis result showedthat IL-13 maintained a relatively low basal expression inthe liver of normal control mice but increased significantlyafter Sj infection (Figure 2(a)) This phenomenon was ver-ified further by Q-PCR using the same RNA specimens(Figure 2(b)) and by ELISA to test serum IL-13 levels(Figure 2(c)) These results showed that in response to Sjinfection IL-13 was highly expressed whether in liver tissueor peripheral venous blood IL-13 did not reach a highlevel of expression until the infection advanced into its latephase when egg-laying commenced [25] This finding wasin accordance with that of previous studies The mRNAlevel of IL-13 began to drastically increase since week 5and reached as high as fivefold in week 12 compared withthe noninfected control We also performed an IHC assayto monitor IL-13 expression in liver tissue (Figure 2(d)) Atweek 5 the extracellular matrix was distorted and cells inliver tissue became disorganized indicating the beginning ofgranuloma formation and hepatic fibrosis (Figure 1) Duringthis time IL-13 began to be detected in these damagedtissues (Figure 2(d)) This observation signified the possiblefunction of IL-13 in granuloma formation and in the early


0

10

20

30

40

50

Are

a per

cent

age o

f col

lage

n

Sj 5 6 8 12

(w)

5w

6w

8w

12w

lowastlowast

lowastlowast

(a)

0

1000

2000

3000

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5000

6000

7000

Sj 5 6 8 12(minus)

Seru

m h

yalu

roni

c aci

dco

ncen

trat

ion

(ng

mL)

(w)(b)

5w

6w6w

8w

12w

(minus)

120572-SMA

(c)

Figure 1 Extent of mouse hepatic fibrosis increased gradually after Sj infection BALBc mice were infected with 20 plusmn 3 infective cercariae ofSj for 5 6 8 and 12 weeks and noninfected mice served as negative control Liver tissues were fixed and stained with Masson trichrome oranti-120572-SMA antibody (original magnification 40x) Collagen deposition area percentage (Masson trichrome staining positive area percentageof each section) was calculated and shown in (a left panel) Representative liver granulomas stained with Massonrsquos trichrome staining areshown in (a right panel) at 20x magnification Concentration of hyaluronic acid in mouse liver serum is shown in (b) through ELISA assayExpression level of 120572-SMA protein in mouse liver tissues is shown in (c) through IHC assay Data are presented as mean plusmn SD from eightmice per group Experiments were performed twice lowastlowast119875 lt 001

stage of hepatic fibrogenesis An interesting finding was thata high level of IL-13 appeared around the granuloma in thearea of inflammatory cell infiltration especially at week 8after Sj infection but cells around the veins where Sj adultworms were located did not exhibit IL-13 positive staining(Figure 2(d)) Given the central function of granuloma inhepatic fibrosis these findings implied that IL-13 around theSj egg granuloma was induced by the host inflammatoryresponse to the stimulation of Sj SEA and thus acceleratedgranuloma formation and hepatic fibrosis in Sj infection

32 tTG Contributes to a High Extent of Hepatic Fibrosisafter Sj Infection The involvement of tTG in liver fibrosishas long been suggested [13 14 16 17] and our findings areconsistent with those of numerous past experiments Wesacrificed mice at weeks 0 5 6 8 and 12 after Sj infectionThe RT-PCR and Q-PCR results showed that tTG mRNAexpression levels were markedly increased in the liver after Sjinfection compared with uninfected mice (Figures 3(a) and3(b)) as well as tTG protein expression level (Figure 3(c))and total TGase activity (see Figure S1 in Supplementary


GAPDH

Sj 5 6 8 12

IL-13(minus)

(w)

(a)

0

1

2

3

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5

6

7

5 6 8 12(minus)

lowast

lowast

lowast

lowast

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Relat

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IL-13

mRN

A le

vel

(b)

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200

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600

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1400

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5 6 8 12Sj

Con

cent

ratio

n of

IL-13

in se

rum

(pg

mL)

(minus)

lowastlowastlowastlowast


(w)

(c)

5w 6w 8w 12wSj

IL-13

(minus)

(d)

Figure 2 IL-13 level was associated with hepatic fibrosis after Sj infection (a b) IL-13 mRNA level in BALBc mice liver is upregulated atweeks 5 6 8 and 12 after Sj infection as determined through RT-PCR and SYBR Green based Q-PCR GAPDH is detected as an internalcontrol Data are presented as mean plusmn SD from eight mice per group lowast119875 lt 005 and lowastlowast119875 lt 001 (c) Concentration of IL-13 in mice sera istested by ELISA Data are presented as mean plusmn SD from eight mice per group lowast119875 lt 005 and lowastlowast119875 lt 001 (d) Localization and expressionlevel of IL-13 protein in BALBc mice liver is shown through IHC assay at weeks 5 6 8 and 12 after Sj infection Representative IL-13 stainingsections are shown at primary 40x magnification

Material available online at httpdxdoiorg1011552014753483) Furthermore in Sj-infected mouse liver sectionstTG expression was found in the hepatic cells around thehepatic sinusoids where Sj adult worms were located oraround and in the liver granuloma and fibrosis areas whereSj eggs were deposited (Figure 3(d))

To provide further evidence that tTG affects the devel-opment of Sj-infected mouse liver fibrosis we treated theinfected mice by using an intraperitoneal injection of CTMonce per day for 7 d starting at day 3 after infectionConsidering that liver fibrosis was the most severe at week8 after infection (Figure 1) we sacrificed the CTM-treatedinfected mice and the infected mice without CTM treatment

at week 8 Liver fibrosis was evaluated by monitoring thearea of collagen deposition serum hyaluronic acid level andstrength of 120572-SMA-positive staining by IHC assay Comparedwith those of untreatedmice the liver tissues of CTM-treatedSj-infected mice showed significantly lower tTG expression(Figure 4(a)) and TGase activity (Figure S2) as well as 120572-SMA expression level (Figures 4(b) and 4(c)) the area sizeof collagen deposition (119875 lt 0001) (Figure 4(d)) and serumhyaluronicacid level (Figure 4(e)) The results indicated thattTG contributes to hepatic fibrosis after Sj infection

33 Cystamine Partially Downregulated the Level of IL-13in Response to Sj Infection CTM treatment can selectively


tTG

GAPDH

5w 6w 8w 12wSj infection in mice liver

(minus)

(a)

0

1

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6

Relat

ive m

ouse

tTG

mRN

A le

vel

lowast

lowast

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lowastlowast

lowastlowast

Sj(w)

(b)

GAPDH

tTG


tTGGAPDH 10 47 27 12 24

(minus)

(c)

tTG

5w 6w 8w 12wSj (minus)

(d)

Figure 3 tTG expression was upregulated progressively in mice liver after Sj infection (a b) tTG mRNA level in BABLc mice liver isupregulated at weeks 5 6 8 and 12 after Sj infection as determined through RT-PCR and Q-PCR GAPDH is detected as an internal controlData are presented as mean plusmn SD from eight mice per group lowast119875 lt 005 and lowastlowast119875 lt 001 compared with noninfected mice (minus) (c) tTG proteinlevel of mice liver homogenates is tested by Western blot analysis GAPDH is used as loading control (d) Different localization expressionlevels of tTG protein in BALBc mice liver are shown through IHC assay at weeks 5 6 8 and 12 after Sj infection Upper panel extent of tTGpositive staining in hepatic cell and liver tissue around the liver sinusoid Lower panel extent of tTG positive staining in the granuloma andnearby liver tissue after Sj egg deposition Representative tTG staining sections are shown at 40x magnification


+CT

M tr

eatm

ent

tTG

8w(minus)

(minus)

(a)

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CTM

trea

tmen

t

8w(minus)

(minus)

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(b)

+CTM

10 11 38 13

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(minus)Sj

120572-SMA

GAPDH

120572-SMAGAPDH

(c)

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5

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a per

cent

age o

f col

lage

n

Sj + 8wSj + 8w

lowastlowast CTM

trea

tmen

t

+

(minus)

+CTM (minus)

(d)

Figure 4 Continued


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m h

yalu

roni

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d le

vel (

ngm

L)8w8w(minus)(minus)

(minus) + (minus) +CTMSj

(e)

Figure 4 Cystamine as an inhibitor of tTG alleviated the extent of liver fibrosis tTG activity in BALBc mice is blocked by CTMintraperitoneal injection from day 3 to day 10 after Sj infection Mice are sacrificed at week 8 after infection Liver tissues are fixed and stainedwith anti-tTG (a) anti-120572-SMA antibody (b) (original magnification 40x) or Masson trichrome (d) (magnification 20x) Noninfected micewith or without CTM treatment acted as controls Collagen deposition area percentage (Masson trichrome staining positive area percentageof each section) is calculated and shown in (d) Level of 120572-SMA protein expression in mouse liver tissues is shown through Western blotanalysis and GAPDH is detected as an internal control (c) Concentration of hyaluronic acid in mouse liver serum is shown in (e) throughELISA assay Data are presented as mean plusmn SD from eight mice per group lowastlowast119875 lt 001

inhibit the extent of tTG and liver fibrosis Considering thatIL-13 and tTG are involved in liver fibrosis after Sj infectionwe tested the level change of IL-13 after CTM treatment CTMshowed amarked effect on IL-13mRNAexpressionWhile IL-13 was highly upregulated in Sj-infectedmice administrationof CTM significantly decreased the mRNA expression of IL-13 in Sj-infected mouse livers (Figure 5(a)) Meanwhile IL-13level in peripheral venous blood was also decreased by 216(Figure 5(b)) The alleviative effect was confirmed by IHCstaining (Figure 5(c)) These results showed that CTM candownregulate IL-13 in case of Sj infection at least partiallywhich provided a possibility that IL-13 expression can beupregulated by tTG

4 Discussion

Liver fibrosis caused by Sj is one of themost serious patholog-ical changes that may induce loss of liver function and livercancer [26] Owing to the early aggregate and massive egg-laying characteristics of Sj liver fibrosis symptoms causedby Sj are the most grievous among those of the prevalentschistosomiasis Hence finding effective measures to preventor even reverse liver fibrosis is critical in the battle against SjHowever safe drug intervention will be impossible before wetruly understand the immunopathogenesis and mechanismof liver fibrosis of this disease

Our data showed the association of IL-13 and tTG withliver fibrosis We elucidated the possible regulation of IL-13 expression by tTG as well IL-13 is thought to be the keymediator of liver fibrosis in Sm as antagonizing IL-13 or IL-13minusminus mouse liver fibrosis was abrogated [8 9] Although thecorrelation between IL-13 and liver fibrosis in Sm has beenfound more research is required for further assessment ofthe mechanisms by which IL-13 influences liver fibrogenesis

Nevertheless our findings serve as a basis for studying thefunction of IL-13 in infiltrating lymphocytes in case of Sjinfection

The function of tTG in liver fibrosis remains controver-sial While most studies report the positive involvement oftTG in liver fibrosis contradicting reports still exist Moststudies on the fibrogenesis function of tTG were performedon a carbon tetrachloride-induced liver fibrosismodel [16 17]and may thus fail to represent the fibrogenesis mechanismin many other pathological circumstances In this studywe provided several lines of evidence suggesting that tTGaccelerated Sj-induced liver fibrosis First tTG level wasconsistent with the extent of liver granuloma and fibrosis afterSj infection tTG was mainly overexpressed in hepatic cells atweek 5 and in theHSC at week 6 andmainly in the infiltratingcells around Sj eggs at weeks 8 and 12 (Figure 3) Second theblocking of tTG activity by CTM treatment from days 3 to10 after Sj infection alleviated liver fibrosis (Figure 4) Thedose and the duration of CTM treatment were referred toin the literature [27 28] We tried to treat the infected miceat week 5 or 6 with CTM once a day for 7 d or 14 d but theextent of fibrosis and also the level of tissue transglutaminaseexhibited no change (data not shown) The underlying roleandmechanisms of tTG involvement in the pathogenesis of Sjinfection are unclearThese data suggest that HSC activationas well as some inflammatory factors or secretory factors of Sjorigin may be the downstream targets of tTG However oncethe downstream pathways or cascades were initiated at theearly stage of Sj infection tTG inhibition could not reverse thepathologic changes Further studies are needed to determinethe detailed pathogenesis of tTG contribution to liver fibrosisafter Sj infection

The association of tTG with cytokines such as IL-6 IL-17[29] IL-33 [21] TGF-1205731 [30] tumor necrosis factor alpha and


0

02

04

06

08

1

12

14

CTM

Rela

tive I

L-13

mRN

A le

vel

++

GAPDH

Sj 8w8w

IL-13

(minus)(minus)(minus)(minus)

lowastlowast

(a)

0

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2000

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(minus)(minus)

lowast

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cent

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n of

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(pg

mL)

(b)

8w

(minus)

(minus)

CTM

trea

tmen

t

+

IL-13

Sj

(c)

Figure 5 CTM reduced IL-13 expression profile in the mice liver during Sj infection Mice are sacrificed at week 8 after Sj infection and theirliver or sera are collected (a) IL-13 mRNA level in mouse liver of normal or Sj-infected mice with or without CTM treatment is detected byRT-PCR andQ-PCR Upper panel Q-PCR lower panel RT-PCR GAPDH is detected as an internal control Data are presented asmean plusmn SDfrom eight mice per group lowastlowast119875 lt 001 (b) IL-13 concentration in mouse sera of normal or Sj-infected mice with or without CTM treatmentis detected through ELISA Data are presented as mean plusmn SD from 8 mice per group lowast119875 lt 005 (c) Mouse livers at indicated time points arefixed in paraformaldehyde embedded in paraffin sliced and IHC stained for IL-13 Representative slices of IHC staining for IL-13 are shownat original 40x magnification

interferon gamma had been reported [31] tTG is an impor-tant inflammatory and fibrogenetic factor involved in Th17[29] andTh2 responses [20]We found that tTG partially reg-ulates IL-13 in immunological response but the underlyingmechanisms of tTG regulation of IL-13 require further study

Therefore we propose a model to illustrate the possiblemechanisms linking tTG IL-13 and liver fibrosis Whenexposed to repetitive damage (viral parasitic toxic andmetabolic) the liver reacts with a chronic wound healingresponse that usually results in fibrosis Liver fibrogenesis ismainly driven by activated HSCs and an excess accumulationof extracellular matrix The high extent of tTG expression

and activity consequently activatesNF-120581B signalingThe acti-vated NF-120581B signaling pathway upregulates the productionof some inflammatory cytokines including IL-6 IL-17 [29]and IL-13 Meanwhile by activating Th2 response [21] tTGmay enhance the production of IL-13 by activating Th2 tTGinhibition through CTM reduces IL-13 and then suppressesliver fibrosis

These findings suggest that both tTG and IL-13 can bedrug targets for schistosomiasis liver fibrosis and that selec-tive tTG inhibitory drug and downstream inhibition reagentsmay abrogate the liver fibrogenesis possibly by regulatingIL-13 Further studies on the exact mechanisms of the tTG


regulation of IL-13 during Sj infection are needed Howeverwe have to consider that the IL-13 level in peripheral venousblood was only decreased by 2157 Some compensatorymechanisms can possibly increase the level of IL-13 in thebloodbut not in the liver or someother factors aside from tTGmay regulate IL-13 expression correlating with liver fibrosisafter Sj infection

5 Conclusion

Our data established a novel link among IL-13 tTG and livergranuloma and fibrosis The important function of IL-13 andtTG in Sj liver fibrosis makes them potential drug targets inpreventing liver fibrogenesis

Conflict of Interests

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

Authorsrsquo Contribution

Juanjuan Tang Huayi Huang and Xiaofang Ji have equallycontributed to this paper

Acknowledgments

This work was funded by the NSFC (no 30600516) Sci-ence amp Technology Planned Projects of Guangzhou (no2012J4100009) and Immunology as one of the Key Dis-ciplines of Guangzhou City (no B127007) Juanjuan Tangand Huayi Huang performed the research Zi Li and Juan-juan Tang designed the research Xunmin Zhu XiaofangJi Yinyan Li Mingmin She and Suikai Yan contributedessential reagents or tools Juanjuan Tang analyzed the dataHuayi Huang wrote the paper and Zi Li and Mingchiu Fungrevised the paper The authors appreciate Professor Tao Penrsquossuggestions during the revision of the paper

References

[1] X N Zhou L Y Wang M G Chen et al ldquoThe public healthsignificance and control of schistosomiasis in Chinamdashthen andnowrdquo Acta Tropica vol 96 no 2-3 pp 97ndash105 2005

[2] A G Ross R M Olveda L Acosta et al ldquoRoad to the elimina-tion of schistosomiasis fromAsia the journey is overrdquoMicrobesand Infection vol 15 no 13 pp 858ndash865 2013

[3] T Jia X Zhou X Wang J Utzinger P Steinmann and XWualdquoAssessment of the age-specific disability weight of chronicschistosomiasis japonicardquo Bulletin of the World Health Organ-ization vol 85 no 6 pp 458ndash465 2007

[4] M S Wilson MMMentink-Kane J T Pesce et al ldquoImmuno-pathology of schistosomiasisrdquo Immunology and Cell Biologyvol 85 no 2 pp 148ndash154 2007

[5] JM Grzych E Pearce A Cheever et al ldquoECGdeposition is themajor stimulus for the production of Th2 cytokines in murineschistomiasis mansonirdquo Journal of Immunology vol 146 no 4pp 1322ndash1327 1991

[6] X Xu X Wen Y Chi et al ldquoActivation-induced T helper celldeath contributes to Th1Th2 polarization following murineSchistosoma japonicum infectionrdquo Journal of Biomedicine andBiotechnology vol 2010 Article ID 202397 12 pages 2010

[7] T AWynn ldquoIL-13 effector functionsrdquoAnnual Review of Immu-nology vol 21 pp 425ndash456 2003

[8] T A Wynn R W Thompson A W Cheever and M MMentink-Kane ldquoImmunopathogenesis of schistosomiasisrdquo Im-munological Reviews vol 201 pp 156ndash167 2004

[9] M G Chiaramonte A W Cheever J D Malley D D Donald-son and T A Wynn ldquoStudies of murine schistosomiasis revealinterleukin-13 blockade as a treatment for established and pro-gressive liver fibrosisrdquo Hepatology vol 34 no 2 pp 273ndash2822001

[10] M Kaviratne M Hesse M Leusink et al ldquoIL-13 activates amechanism of tissue fibrosis that is completely TGF-beta inde-pendentrdquoThe Journal of Immunology vol 173 no 6 pp 4020ndash4029 2004

[11] K C Olsen R E Sapinoro R M Kottmann et al ldquoTransglu-taminase 2 its role in fibrosisrdquo American Journal of Respiratoryand Critical Care Medicine vol 184 no 6 pp 699ndash707 2011

[12] N Shweke N Boulos C Jouanneau et al ldquoTissue transglutam-inase contributes to interstitial renal fibrosis by favoring accu-mulation of fibrillar collagen through TGF-beta activation andcell infiltrationrdquoTheAmerican Journal of Pathology vol 173 no3 pp 631ndash642 2008

[13] A Mirza S L Liu E Frizell et al ldquoA role for tissue transglu-taminase in hepatic injury and fibrogenesis and its regulationbyNF-kappaBrdquoTheAmerican Journal of Physiology vol 272 pp281ndash288 1997

[14] G Zhao Z Q Zhang B Zhang M Luo Y Sun and Z WuldquoDown-regulation of tTG expression by RNAi inhibits HSCproliferation and attenuates liver fibrosisrdquo International Journalof Clinical and Experimental Pathology vol 4 no 5 pp 513ndash5202011

[15] A Martin V Gentile and G de Vivo ldquoPossible role of thetransglutaminases in the pathogenesis of alzheimerrsquos diseaseand other neurodegenerative diseasesrdquo International Journal ofAlzheimerrsquos Disease vol 2011 Article ID 865432 7 pages 2011

[16] J F Qiu Z Q Zhang W Chen and Z Y Wu ldquoCystamineameliorates liver fibrosis induced by carbon tetrachloride viainhibition of tissue transglutaminaserdquo World Journal of Gas-troenterology vol 13 no 32 pp 4328ndash4332 2007

[17] R Nardacci O Lo Iacono F Ciccosanti et al ldquoTransglutami-nase type II plays a protective role in hepatic injuryrdquo AmericanJournal of Pathology vol 162 no 4 pp 1293ndash1303 2003

[18] L Elli C M Bergamini M T Bardella and D SchuppanldquoTransglutaminases in inflammation and fibrosis of the gas-trointestinal tract and the liverrdquoDigestive and Liver Disease vol41 no 8 pp 541ndash550 2009

[19] I Nunes P E Gleizes C N Metz and D B Rifkin ldquoLa-tent transforming growth factor-120573 binding protein domainsinvolved in activation and transglutaminase-dependent cross-linking of latent transforming growth factor-120573rdquo The Journal ofCell Biology vol 136 no 5 pp 1151ndash1163 1997

[20] Y Kim S Eom K Kim et al ldquoTransglutaminase II interactswith rac1 regulates production of reactive oxygen speciesexpression of snail secretion of Th2 cytokines and mediates invitro and in vivo allergic inflammationrdquoMolecular Immunologyvol 47 no 5 pp 1010ndash1022 2010

[21] K OhMW Seo G Y Lee et al ldquoAirway epithelial cells initiatethe allergen response through transglutaminase 2 by inducing


IL-33 expression and a subsequent Th2 responserdquo RespiratoryResearch vol 14 no 1 article 35 2013

[22] L Maiuri A Luciani I Giardino et al ldquoTissue transglutam-inase activation modulates inflammation in cystic fibrosis viaPPAR120574 down-regulationrdquo Journal of Immunology vol 180 no11 pp 7697ndash7705 2008

[23] D Caccamo M Curro and R Ientile ldquoPotential of trans-glutaminase 2 as a therapeutic targetrdquo Expert Opinion onTherapeutic Targets vol 14 no 9 pp 989ndash1003 2010

[24] I B Suh DW Yoon OWo et al ldquoEffects of transglutaminase 2inhibition on ventilator-induced lung injuryrdquo Journal of KoreanMedical Science vol 29 no 4 pp 556ndash563 2014

[25] M L Burke M K Jones G N Gobert Y S Li M K Ellisand D P McManus ldquoImmunopathogenesis of human schistos-omiasisrdquo Parasite Immunology vol 31 no 4 pp 163ndash176 2009

[26] D C Qiu A E Hubbard B Zhong Y Zhang and R C SpearldquoA matched case-control study of the association betweenSchistosoma japonicum and liver and colon cancers in ruralChinardquo Annals of Tropical Medicine and Parasitology vol 99no 1 pp 47ndash52 2005

[27] M V Karpuj M W Becher J E Springer et al ldquoProlongedsurvival and decreased abnormal movements in transgenicmodel of Huntington disease with administration of the trans-glutaminase inhibitor cystaminerdquoNatureMedicine vol 8 no 2pp 143ndash149 2002

[28] B S Tzang T C Hsu T Y Chen C Huang S Li and SKao ldquoCystamine ameliorates ventricular hypertrophy associ-ated with modulation of IL-6-mediated signaling in lupus-prone micerdquo Life Sciences vol 92 no 12 pp 719ndash726 2013

[29] K Oh H Park O Byoun et al ldquoEpithelial transglutaminase 2is needed for T cell interleukin-17 production and subsequentpulmonary inflammation and fibrosis in bleomycin-treatedmicerdquo Journal of Experimental Medicine vol 208 no 18 pp1707ndash1719 2011

[30] D Telci R J Collighan H Basaga and M Griffin ldquoIncreasedTG2 expression can result in induction of transforming growthfactor 1205731 causing increased synthesis and deposition of matrixproteins which can be regulated by nitric oxiderdquoThe Journal ofBiological Chemistry vol 284 no 43 pp 29547ndash29558 2009

[31] M Bayardo F Punzi C Bondar N Chopita and F ChirdoldquoTransglutaminase 2 expression is enhanced synergistically byinterferon-120574 and tumour necrosis factor-120572 in human smallintestinerdquo Clinical and Experimental Immunology vol 168 no1 pp 95ndash104 2012

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


Tissue transglutaminase (tTG) as a multifunctional pro-tein performs a variety of intracellular and extracellular func-tions including hydrolyzing GTP transamidation activity asa catalyzing enzyme and cross-linking glutamine residuesand lysine residues of proteins These properties cause tTGto be involved in various physiological processes which ifregulated inappropriately can also lead to its involvementin a number of diseases such as metastatic cancer coeliacdisease and lung renal and liver fibrosis [11ndash13] tTG isoverexpressed in both the murine experimental liver fibrosismodel and hepatitis c virus-induced human liver fibrosisRNA interference can result in diminution of liver fibrosisand lesser aggregation of fibrotic tissue [14] Cystamine(CTM) can modify cysteine at the active site on tTG in adisulfide interchanging manner such that the activity of tTGis selectively inhibited [15] Inhibition of tTG activity by CTMresults in the diminution of liver fibrosis induced by CCl

4

[16] Meanwhile tTG knockout mice with CCl4intoxication

display high lethality as compared with wild-type controls[17] Therefore enhanced tTG level seems to protect theliver from acute and chronic injury but its net effect onfibrogenesis resulting from different causes requires furtherstudy

Several studies have indicated that tTG serves as animportant inflammatory and fibrogenetic factor [18] tTGmodulates inflammation exerting both pro- and anti-inflammatory effects such as positive feedback loop withTGF-1205731 [19] NF-120581B a key inflammation-associated factoris activated by tTG through the cross-linking of the C-terminal glutamine cluster of I120581Ba [20] Epithelial tTG caninduceTh17 differentiation and subsequent IL-17 productionand pulmonary fibrosis in bleomycin-treated mice More-over tTG reportedly regulates Th2 cytokine secretion andmediates in vitro and in vivo allergic inflammation [2021] tTG can also downregulate peroxisome proliferator-activated receptor 120574 (PPAR120574) as well as increasing the classicparameters of inflammation such as TNF-alpha tyrosinephosphorylation and MAPKs in a model of cystic fibrosis[22]

In this study we take an important step toward answeringthese questions by uncovering a novel connection amongtTG IL-13 and Sj-caused liver fibrosis We find that tTGand IL-13 levels are upregulated in mice with Sj infectionImportantly we further demonstrate that the treatment ofmice with Sj-induced liver fibrosis by using an inhibitorof the enzymatic activity of tTG inhibits the Sj-inducedupregulation of IL-13 and alleviates liver fibrosis To ourknowledge these findings show that tTG is involved in thedevelopment of Sj infection-induced liver fibrosis in miceand themechanismmay be associatedwith tTG-regulated IL-13 expression

2 Methods

21 Animal Grouping Parasite Infection and Cystamine Ad-ministration This study was performed in strict accordancewith the recommendations in the Guide for the Care and Useof Laboratory Animals of State Scientific and Technological

Commission The protocol was approved by the Committeeon the Ethics of Animal Experiments of Guangzhou MedicalUniversity All surgeries were performed under sodium pen-tobarbital anesthesia and every effort was made to minimizesuffering

Six- to eight-week-old female BALBCmice (Experimen-tal Animal Center of Sun Yat-Sen University GuangzhouChina) were infected percutaneously through the abdomenwith 20 plusmn 3 Sj cercariae The mice livers were collected anddetermined at weeks 5 6 8 and 12 after infection Concurrentcontrols were uninfected mice

Cystamine (CTM Sigma-Aldrich St Louis USA) is acommon tTG inhibitor [15 16 23 24] In this study for themice group with tTG inhibitor-CTM treatment starting atday 3 after infection mice were administered with 100120583Lof CTM (10minus2mM Sigma) in phosphate-buffered solution(PBS) through intraperitoneal injection once per day for7 d whereas the infection control group received PBS aloneTwo noninfected control mice groups were treated withCTM or PBS The number of mice in every group is 8Sera of mice of all groups were collected for underlyinganalysis

22 Calculation of Collagen Area Using Massonrsquos Trichrome(MT) Staining Fresh liver tissues were fixed in 4 parafor-maldehyde overnight and routinely paraffin-embedded Par-affin sections (4 120583m) were prepared from each liver tissuesample The liver tissue sections were stained by MT stainingto evaluate collagen content and distribution The collagenfibers were stained blue cell nuclei were stained black andthe background was stained red Percentage of total areaswith collagen positive color (blue) [percentage of collagenarea (positive blue color areatotal area)] was analyzedusing Image-Pro Plus 60 software Every stained samplewas evaluated in double-blind fashion by two independentinvestigators

23 RNA Isolation and RT-PCR and Quantitative-PCR TotalRNA was extracted from fresh liver tissue homogenizedin TRIZol reagent (Invitrogen) according to the manufac-turerrsquos protocol RNA purity and concentration were assessedby spectrophotometry Reverse transcriptase (RT) reactionsfor cDNA synthesis were performed using PrimeScriptRT Master Mix (TAKARA) Relative mRNA expressionlevel was determined by real-time quantitative polymerasechain reaction (Q-PCR) with SYBR Green I PCR Master(TAKARA) kit on ABI 7500 machine according to themanufacturerrsquos protocol The primers used were as followsmouse-IL-13 (forward CGGCAGCATGGTATGGAGTGreverse ATTGCAATTGGAGATGTTGGTCAG) mouse-tTG (forward 51015840-GTGAGCCGTGCTATCTGTCCTG-31015840reverse 51015840-ACTGCCTGCTTGGAACCTGAA-31015840) mouse-GAPDH (forward 51015840-TGTGTCCGTCGTGGATCTGA-31015840reverse 51015840-TTGCTGTTGAAGTCGCAGGAG-31015840) The Q-PCR results were expressed as fold amplification using the2minusΔΔCt method with mouse GAPDH as the internal controlEach experiment was repeated thrice












3 Results



0

10

20

30

40

50

Are

a per

cent

age o

f col

lage

n

Sj 5 6 8 12

(w)

5w

6w

8w

12w

lowastlowast

lowastlowast

(a)

0

1000

2000

3000

4000

5000

6000

7000

Sj 5 6 8 12(minus)

Seru

m h

yalu

roni

c aci

dco

ncen

trat

ion

(ng

mL)

(w)(b)

5w

6w6w

8w

12w

(minus)

120572-SMA

(c)





GAPDH

Sj 5 6 8 12

IL-13(minus)

(w)

(a)

0

1

2

3

4

5

6

7

5 6 8 12(minus)

lowast

lowast

lowast

lowast

lowastlowast

Sj(w)

Relat

ive l

iver

IL-13

mRN

A le

vel

(b)

0

200

400

600

800

1000

1200

1400

1600

1800

2000

5 6 8 12Sj

Con

cent

ratio

n of

IL-13

in se

rum

(pg

mL)

(minus)



(w)

(c)

5w 6w 8w 12wSj

IL-13

(minus)

(d)







tTG

GAPDH


(minus)

(a)

0

1

2

3

4

5

6

Relat

ive m

ouse

tTG

mRN

A le

vel

lowast

lowast

5 6 8 12(minus)

lowastlowast

lowastlowast

Sj(w)

(b)

GAPDH

tTG


tTGGAPDH 10 47 27 12 24

(minus)

(c)

tTG


(d)



+CT

M tr

eatm

ent

tTG

8w(minus)

(minus)

(a)

+

CTM

trea

tmen

t

8w(minus)

(minus)

120572-SMA

(b)

+CTM

10 11 38 13

8w(minus)+(minus)

(minus)Sj

120572-SMA

GAPDH

120572-SMAGAPDH

(c)

0

5

10

15

20

25

30

Are

a per

cent

age o

f col

lage

n

Sj + 8wSj + 8w

lowastlowast CTM

trea

tmen

t

+

(minus)

+CTM (minus)

(d)

Figure 4 Continued


0

1000

2000

3000

4000

5000

6000

7000

Seru

m h

yalu

roni

c aci

d le

vel (

ngm



(e)



4 Discussion







0

02

04

06

08

1

12

14

CTM

Rela

tive I

L-13

mRN

A le

vel

++

GAPDH

Sj 8w8w

IL-13


lowastlowast

(a)

0

200

400

600

800

1000

1200

1400

1600

1800

2000


(minus)(minus)

lowast

Con

cent

ratio

n of

IL-13

(pg

mL)

(b)

8w

(minus)

(minus)

CTM

trea

tmen

t

+

IL-13

Sj

(c)








5 Conclusion






Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























3 Results



0

10

20

30

40

50

Are

a per

cent

age o

f col

lage

n

Sj 5 6 8 12

(w)

5w

6w

8w

12w

lowastlowast

lowastlowast

(a)

0

1000

2000

3000

4000

5000

6000

7000

Sj 5 6 8 12(minus)

Seru

m h

yalu

roni

c aci

dco

ncen

trat

ion

(ng

mL)

(w)(b)

5w

6w6w

8w

12w

(minus)

120572-SMA

(c)





GAPDH

Sj 5 6 8 12

IL-13(minus)

(w)

(a)

0

1

2

3

4

5

6

7

5 6 8 12(minus)

lowast

lowast

lowast

lowast

lowastlowast

Sj(w)

Relat

ive l

iver

IL-13

mRN

A le

vel

(b)

0

200

400

600

800

1000

1200

1400

1600

1800

2000

5 6 8 12Sj

Con

cent

ratio

n of

IL-13

in se

rum

(pg

mL)

(minus)



(w)

(c)

5w 6w 8w 12wSj

IL-13

(minus)

(d)







tTG

GAPDH


(minus)

(a)

0

1

2

3

4

5

6

Relat

ive m

ouse

tTG

mRN

A le

vel

lowast

lowast

5 6 8 12(minus)

lowastlowast

lowastlowast

Sj(w)

(b)

GAPDH

tTG


tTGGAPDH 10 47 27 12 24

(minus)

(c)

tTG


(d)



+CT

M tr

eatm

ent

tTG

8w(minus)

(minus)

(a)

+

CTM

trea

tmen

t

8w(minus)

(minus)

120572-SMA

(b)

+CTM

10 11 38 13

8w(minus)+(minus)

(minus)Sj

120572-SMA

GAPDH

120572-SMAGAPDH

(c)

0

5

10

15

20

25

30

Are

a per

cent

age o

f col

lage

n

Sj + 8wSj + 8w

lowastlowast CTM

trea

tmen

t

+

(minus)

+CTM (minus)

(d)

Figure 4 Continued


0

1000

2000

3000

4000

5000

6000

7000

Seru

m h

yalu

roni

c aci

d le

vel (

ngm



(e)



4 Discussion







0

02

04

06

08

1

12

14

CTM

Rela

tive I

L-13

mRN

A le

vel

++

GAPDH

Sj 8w8w

IL-13


lowastlowast

(a)

0

200

400

600

800

1000

1200

1400

1600

1800

2000


(minus)(minus)

lowast

Con

cent

ratio

n of

IL-13

(pg

mL)

(b)

8w

(minus)

(minus)

CTM

trea

tmen

t

+

IL-13

Sj

(c)








5 Conclusion






Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

10

20

30

40

50

Are

a per

cent

age o

f col

lage

n

Sj 5 6 8 12

(w)

5w

6w

8w

12w

lowastlowast

lowastlowast

(a)

0

1000

2000

3000

4000

5000

6000

7000

Sj 5 6 8 12(minus)

Seru

m h

yalu

roni

c aci

dco

ncen

trat

ion

(ng

mL)

(w)(b)

5w

6w6w

8w

12w

(minus)

120572-SMA

(c)





GAPDH

Sj 5 6 8 12

IL-13(minus)

(w)

(a)

0

1

2

3

4

5

6

7

5 6 8 12(minus)

lowast

lowast

lowast

lowast

lowastlowast

Sj(w)

Relat

ive l

iver

IL-13

mRN

A le

vel

(b)

0

200

400

600

800

1000

1200

1400

1600

1800

2000

5 6 8 12Sj

Con

cent

ratio

n of

IL-13

in se

rum

(pg

mL)

(minus)



(w)

(c)

5w 6w 8w 12wSj

IL-13

(minus)

(d)







tTG

GAPDH


(minus)

(a)

0

1

2

3

4

5

6

Relat

ive m

ouse

tTG

mRN

A le

vel

lowast

lowast

5 6 8 12(minus)

lowastlowast

lowastlowast

Sj(w)

(b)

GAPDH

tTG


tTGGAPDH 10 47 27 12 24

(minus)

(c)

tTG


(d)



+CT

M tr

eatm

ent

tTG

8w(minus)

(minus)

(a)

+

CTM

trea

tmen

t

8w(minus)

(minus)

120572-SMA

(b)

+CTM

10 11 38 13

8w(minus)+(minus)

(minus)Sj

120572-SMA

GAPDH

120572-SMAGAPDH

(c)

0

5

10

15

20

25

30

Are

a per

cent

age o

f col

lage

n

Sj + 8wSj + 8w

lowastlowast CTM

trea

tmen

t

+

(minus)

+CTM (minus)

(d)

Figure 4 Continued


0

1000

2000

3000

4000

5000

6000

7000

Seru

m h

yalu

roni

c aci

d le

vel (

ngm



(e)



4 Discussion







0

02

04

06

08

1

12

14

CTM

Rela

tive I

L-13

mRN

A le

vel

++

GAPDH

Sj 8w8w

IL-13


lowastlowast

(a)

0

200

400

600

800

1000

1200

1400

1600

1800

2000


(minus)(minus)

lowast

Con

cent

ratio

n of

IL-13

(pg

mL)

(b)

8w

(minus)

(minus)

CTM

trea

tmen

t

+

IL-13

Sj

(c)








5 Conclusion






Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















GAPDH

Sj 5 6 8 12

IL-13(minus)

(w)

(a)

0

1

2

3

4

5

6

7

5 6 8 12(minus)

lowast

lowast

lowast

lowast

lowastlowast

Sj(w)

Relat

ive l

iver

IL-13

mRN

A le

vel

(b)

0

200

400

600

800

1000

1200

1400

1600

1800

2000

5 6 8 12Sj

Con

cent

ratio

n of

IL-13

in se

rum

(pg

mL)

(minus)



(w)

(c)

5w 6w 8w 12wSj

IL-13

(minus)

(d)







tTG

GAPDH


(minus)

(a)

0

1

2

3

4

5

6

Relat

ive m

ouse

tTG

mRN

A le

vel

lowast

lowast

5 6 8 12(minus)

lowastlowast

lowastlowast

Sj(w)

(b)

GAPDH

tTG


tTGGAPDH 10 47 27 12 24

(minus)

(c)

tTG


(d)



+CT

M tr

eatm

ent

tTG

8w(minus)

(minus)

(a)

+

CTM

trea

tmen

t

8w(minus)

(minus)

120572-SMA

(b)

+CTM

10 11 38 13

8w(minus)+(minus)

(minus)Sj

120572-SMA

GAPDH

120572-SMAGAPDH

(c)

0

5

10

15

20

25

30

Are

a per

cent

age o

f col

lage

n

Sj + 8wSj + 8w

lowastlowast CTM

trea

tmen

t

+

(minus)

+CTM (minus)

(d)

Figure 4 Continued


0

1000

2000

3000

4000

5000

6000

7000

Seru

m h

yalu

roni

c aci

d le

vel (

ngm



(e)



4 Discussion







0

02

04

06

08

1

12

14

CTM

Rela

tive I

L-13

mRN

A le

vel

++

GAPDH

Sj 8w8w

IL-13


lowastlowast

(a)

0

200

400

600

800

1000

1200

1400

1600

1800

2000


(minus)(minus)

lowast

Con

cent

ratio

n of

IL-13

(pg

mL)

(b)

8w

(minus)

(minus)

CTM

trea

tmen

t

+

IL-13

Sj

(c)








5 Conclusion






Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















tTG

GAPDH


(minus)

(a)

0

1

2

3

4

5

6

Relat

ive m

ouse

tTG

mRN

A le

vel

lowast

lowast

5 6 8 12(minus)

lowastlowast

lowastlowast

Sj(w)

(b)

GAPDH

tTG


tTGGAPDH 10 47 27 12 24

(minus)

(c)

tTG


(d)



+CT

M tr

eatm

ent

tTG

8w(minus)

(minus)

(a)

+

CTM

trea

tmen

t

8w(minus)

(minus)

120572-SMA

(b)

+CTM

10 11 38 13

8w(minus)+(minus)

(minus)Sj

120572-SMA

GAPDH

120572-SMAGAPDH

(c)

0

5

10

15

20

25

30

Are

a per

cent

age o

f col

lage

n

Sj + 8wSj + 8w

lowastlowast CTM

trea

tmen

t

+

(minus)

+CTM (minus)

(d)

Figure 4 Continued


0

1000

2000

3000

4000

5000

6000

7000

Seru

m h

yalu

roni

c aci

d le

vel (

ngm



(e)



4 Discussion







0

02

04

06

08

1

12

14

CTM

Rela

tive I

L-13

mRN

A le

vel

++

GAPDH

Sj 8w8w

IL-13


lowastlowast

(a)

0

200

400

600

800

1000

1200

1400

1600

1800

2000


(minus)(minus)

lowast

Con

cent

ratio

n of

IL-13

(pg

mL)

(b)

8w

(minus)

(minus)

CTM

trea

tmen

t

+

IL-13

Sj

(c)








5 Conclusion






Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















+CT

M tr

eatm

ent

tTG

8w(minus)

(minus)

(a)

+

CTM

trea

tmen

t

8w(minus)

(minus)

120572-SMA

(b)

+CTM

10 11 38 13

8w(minus)+(minus)

(minus)Sj

120572-SMA

GAPDH

120572-SMAGAPDH

(c)

0

5

10

15

20

25

30

Are

a per

cent

age o

f col

lage

n

Sj + 8wSj + 8w

lowastlowast CTM

trea

tmen

t

+

(minus)

+CTM (minus)

(d)

Figure 4 Continued


0

1000

2000

3000

4000

5000

6000

7000

Seru

m h

yalu

roni

c aci

d le

vel (

ngm



(e)



4 Discussion







0

02

04

06

08

1

12

14

CTM

Rela

tive I

L-13

mRN

A le

vel

++

GAPDH

Sj 8w8w

IL-13


lowastlowast

(a)

0

200

400

600

800

1000

1200

1400

1600

1800

2000


(minus)(minus)

lowast

Con

cent

ratio

n of

IL-13

(pg

mL)

(b)

8w

(minus)

(minus)

CTM

trea

tmen

t

+

IL-13

Sj

(c)








5 Conclusion






Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

1000

2000

3000

4000

5000

6000

7000

Seru

m h

yalu

roni

c aci

d le

vel (

ngm



(e)



4 Discussion







0

02

04

06

08

1

12

14

CTM

Rela

tive I

L-13

mRN

A le

vel

++

GAPDH

Sj 8w8w

IL-13


lowastlowast

(a)

0

200

400

600

800

1000

1200

1400

1600

1800

2000


(minus)(minus)

lowast

Con

cent

ratio

n of

IL-13

(pg

mL)

(b)

8w

(minus)

(minus)

CTM

trea

tmen

t

+

IL-13

Sj

(c)








5 Conclusion






Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

02

04

06

08

1

12

14

CTM

Rela

tive I

L-13

mRN

A le

vel

++

GAPDH

Sj 8w8w

IL-13


lowastlowast

(a)

0

200

400

600

800

1000

1200

1400

1600

1800

2000


(minus)(minus)

lowast

Con

cent

ratio

n of

IL-13

(pg

mL)

(b)

8w

(minus)

(minus)

CTM

trea

tmen

t

+

IL-13

Sj

(c)








5 Conclusion






Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















5 Conclusion






Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article involvement of il-13 and tissue...

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