diethylcarbamazine reduces chronic inflammation and fibrosis in

Research ArticleDiethylcarbamazine Reduces Chronic Inflammationand Fibrosis in Carbon Tetrachloride- (CCl4-) InducedLiver Injury in Mice

Sura Wanessa Santos Rocha12 Maria Eduarda Rocha de Franccedila1

Gabriel Barros Rodrigues1 Karla Patriacutecia Sousa Barbosa1 Ana Karolina Santana Nunes1

Andreacute Filipe Pastor3 Anne Gabrielle Vasconcelos Oliveira4 Wilma Helena Oliveira1

Rayana Leal Almeida Luna1 and Christina Alves Peixoto1

1 Laboratory of Ultrastructure Aggeu Magalhaes Research Center (CPqAM) Avenida Professor Moraes Rego snCidade Universitaria 50740-465 Recife PE Brazil

2 Department of Entomology Aggeu Magalhaes Research Center (CPqAM) Avenida Professor Moraes Rego snCidade Universitaria 50740-465 Recife PE Brazil

3 Laboratory of Virology and Experimental Therapy Aggeu Magalhaes Research Center (CPqAM) Avenida Professor Moraes Regosn Cidade Universitaria 50740-465 Recife PE Brazil

4 Laboratory of Microscopy and Microanalisis Northeastern Center for Strategic Technologies (CETENE)Avenida Professor Luiz Freire Cidade Universitaria 50740-540 Recife PE Brazil

Correspondence should be addressed to Sura Wanessa Santos Rocha surawanessagmailcom

Received 15 June 2014 Revised 15 August 2014 Accepted 27 August 2014 Published 13 October 2014

Academic Editor Magdalena Klink

Copyright copy 2014 Sura Wanessa Santos Rocha et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

This study investigated the anti-inflammatory effects of DEC on the CCl4-induced hepatotoxicity in C57BL6 mice Chronic

inflammation was induced by ip administration of CCl405 120583Lg of body weight through two injections a week for 6 weeks

DEC (50mgkg) was administered by gavage for 12 days before finishing the CCl4induction Histological analyses of the

DEC-treated group exhibited reduced inflammatory process and prevented liver necrosis and fibrosis Immunohistochemicaland immunofluorescence analyses of the DEC-treated group showed reduced COX-2 IL1120573 MDA TGF-120573 and 120572SMAimmunopositivity besides exhibiting decreased IL1120573 COX-2 NF120581B IFN120574 and TGF120573 expressions in the western blot analysisTheDEC group enhanced significantly the IL-10 expression The reduction of hepatic injury in the DEC-treated group was confirmedby the COX-2 and iNOS mRNA expression levels Based on the results of the present study DEC can be used as a potential anti-inflammatory drug for chronic hepatic inflammation

1 Introduction

Chronic liver disease leads to several complications withhighmorbidity andmortality rates Aworldwide health prob-lem chronic liver inflammation is a progressive disease witha slow stage causing liver dysfunction characterized by theformation of fibrosis [1] The chronicity of inflammation aswell as the type of inflammation (ieTh2 versusTh1) is oftenimportant in many types of liver disease as is the interplaybetween inflammation and environmentalmetabolicgenetic

factors [2] Excessive consumption of alcohol and viral hep-atitis are among the most common causes of liver disease [3]The liver is the main organ responsible for the metabolism ofdrugs and toxic chemicals and consequently it is the primarytarget organ for nearly all toxic chemicals [4]

Carbon tetrachloride (CCl4) is a well-known hepatotoxin

that is widely used to induce toxic hepatic injuries in exper-imental animals models [5 6] Hepatotoxicity is believed toresult from two events the first involves CCl

4metabolism

by cytochrome P-450 to the trichloromethyl radical (CCl3

∙)

Hindawi Publishing CorporationMediators of InflammationVolume 2014 Article ID 696383 15 pageshttpdxdoiorg1011552014696383

2 Mediators of Inflammation

part of which generates the trichloromethyl peroxyl radical(OOCCl

3

∙) [7 8] which leads to lipid peroxidation [9]the second involves the activation of Kupffer cells which isaccompanied by the production of proinflammatory media-tors [10] Carbon tetrachloride (CCl

4) should be an effective

stimulus for prostaglandin synthesis in hepatocytes since itis believed to release arachidonic acid in the liver through theactivation of phospholipase A2 [11 12]

Progression of the disease involves various proinflamma-tory molecules such as interleukins cytokines and nuclearfactor-120581B (NF-120581B) [13ndash15] The activated NF-120581B if translo-cated into the nucleus will facilitate the transcription ofmanydownstream genes including inducible nitric oxide syn-thase (iNOS) and cyclooxygenase-2 (COX-2) which are keymediators in the recruitment of inflammatory cells [16 17]The iNOS-derived nitric oxide production activated down-stream of NF-120581B is followed by the generation of reactiveoxygen species and other free radicals that are detrimental tocells Cellular lipids are easily attacked by free radicals result-ing in an intracellular accumulation of malondialdehyde(MDA) [18] Although the relationships between oxidativestress cytotoxic cytokines and liver cell injuries are not fullyunderstood NF-120581B is considered to play an important rolein liver cell injuries Studies have shown an increased acti-vation of NF-120581B through oxidative stress induced by carbontetrachloride in liver injuries [19 20]

After a chronic liver injury of any etiology the damagedhepatocytes their membrane components metabolites oftoxic agents and infiltrating inflammatory cells activateKupffer cells releasing soluble agents including cytokinessuch as platelet-derived growth factor (PDGF) transforminggrowth factor-1205731 (TGF-120573) tumor necrosis factor-a (TNF-120572)and endothelin-1 (ET-1) as well as reactive oxygen species(ROS) [21] These factors act on the hepatic stellate cells(HSCs) which undergo a response known as activation andstart expressing new receptors such as PDGF and TGF-120573receptors and new proteins such as a-smooth muscle actin(120572-SMA) The activation of the HSCs triggers the expressionof 120572-SMA and increases cellular proliferation and extracellu-lar matrix accumulation [22] Activated HSCs are consideredto play a role in the excessive deposition of ECM proteinssuch as type I collagen (Col-1) and in the disruption ofnormal parenchyma during the hepatic fibrogenesis processChronic inflammation and the repeated sustenance of liverinjuries such as those associated with hepatitis and alcoholicliver disease usually progress to liver cirrhosis and hepato-carcinogenesis [23]

The inhibition of proinflammatory cytokines enzymestranscription nuclear factor and free radicals could offer anew therapeutic strategy against inflammatory liver disease

Diethylcarbamazine (DEC) has anti-inflammatory prop-erties as a result of its interference with the arachidonicacid metabolism which involves lipoxygenase (LOX) andcyclooxygenase (COX) enzymes [24ndash27] DEC led to reduc-tions in lung injury PMNs migration the formation of NOproduction and the release of proinflammatory cytokinesand COX-2 in a model of acute inflammation induced bycarrageenan [28] DEC is a potential drug for the treatment ofacute lung inflammation for Queto et al demonstrated

that DEC plays an important role in blocking pulmonaryeosinophilic inflammation in mice sensitized with ovalbu-min effectively preventing the effects of subsequent air-way resistance Th1Th2 cytokine production pulmonaryeosinophil accumulation and eosinophilopoiesis in vivo andex vivo [29] DEC effects have contributed to the reduction ofhepatic abnormalities caused by malnutrition [30] In addi-tion DEC has decreased liver injuries in an experimentalalcoholismmodel and revealed a clinical potential for thera-peutic anti-inflammatory applications [15]

The aim of the present study was to investigate the pro-tective effects of DEC against chronic CCl

4-induced damage

and a possible mechanism for its anti-inflammatory andhepatoprotective activity

2 Materials and Methods

21 Animals Forty male 5-week-old C57BL6 mice weight-ing 15-16 g were used in all experiments The mice wereexamined to determine their health status and acclimatedto the laboratory environment of 23-24∘C They were keptin a 1212 h daynight cycle photoperiod The animals werehoused in metal cages and fed a standard diet with water adlibitumThe animal studies Ethics Committee of the OswaldoCruz Institute approved all the experiments reported hereinunder protocol number 112010

22 Diethylcarbamazine Solutions The solutions were com-posed of distilled water and DEC (FarmanguinhosFIOCRUZ Brazil) and were adjusted in accordance with thebody weight of each animal They were administered bygavage (02mL)The control group (C) only received distilledwater via the same administration route [15 30 31]

23 Experimental Design Chronic inflammation was indu-ced by ip administration of CCl

405120583Lg of body weight

(Sigma-Aldrich St Louis MO USA) dissolved in olive oil(final volume 01mL per mouse) [32] There were two injec-tions a week for 6 weeks DEC (50mgkg) was administeredby gavage for 12 days before finishing the liver injury Thecontrol groups received distilled water via the same routeThe C57BL6 mice were separated into four groups (119899 = 10)(1) the control group (C) which received just distilled water(2) the DEC-treated group (DEC) (3) the CCl

4group (CCl

4)

and (4) the CCl4plus DEC group (CCl

4+ DEC)

24 Histological Analysis Liver fragments were fixed in 10formalin for 24 hours before being processed and embeddedin paraffin [30] Five sections of 4-5 120583m from each groupwerecut and mounted on glass slides The slices were stained withhematoxylin-eosin and examined by an inverted microscope(Observer Z1 Zeiss MicroImaging GmbH) equipped with acamera and 474 image analysis software (AxionCam MRmZeiss) at a magnification of 400x The fibrosis areas werequantified in five random fields on each slide using GIMP 26imaging software [15]

Mediators of Inflammation 3

25 Measurement of Hepatic Collagen Content The hepaticcollagen content was also assessed by the Sirius-red stainingof five paraffin-embedded sections Sirius-red positive areaswere analyzed in five random fields (magnification times400) oneach slide and quantified using GIMP 26 imaging software[15]

26 Electron Transmission Microscopy Assays The fragmentsof liver were fixed in a solution containing 25 glutaralde-hyde and 4 formaldehyde in 01M cacodylate buffer Afterfixation the samples were washed twice in the same bufferand then postfixed in a solution containing 1 osmiumtetroxide 2mM calcium chloride and 08 potassium fer-ricyanide in 01M cacodylate buffer with a pH of 72 dehy-drated in acetone and embedded in Epon 812 resin (SigmaCompany St Louis MO) Polymerization was carried out at60∘C for 2 days Ultrathin sections were collected on 300-mesh copper grids counterstained with uranyl acetate andlead citrate and examined with a Morgani FEI transmissionelectron microscope [15]

27 Immunohistochemistry (IHC) Five sections (5 120583m inthickness) from each group were cut and adhered to slidestreated with 3-amino-propyl-triethoxy-silane (APES (SigmaUSA)) The sections were deparaffinized with xylene andrehydrated in graded ethanol (100 to 70) To increaseepitope exposure the sections were heated for 30 minutesin a sodium citrate buffer (001M pH60) To minimizeendogenous peroxidase activity the slides were treated with03 (vv) H

2O2in water for five minutes The sections were

washed with 001M PBS (pH 72) and then blocked with 1BSA and 02 Tween 20 in PBS for 1 h at room temperatureThe sections were then incubated for 12 hours at 4∘C with amonoclonal antibody anti-cyclooxygenase (COX-2 Abcamab15191) an antibody against transforming growth factor-beta (TGF-120573 Abcam ab66043) and anti-mouse interleukin1 (IL-1120573 Abcam ab9722) The optimal concentration usedwas 1 100 for these antibodiesThe antigen-antibody reactionwas visualizedwith avidin-biotin peroxidase (DakoUniversalLSAB + Kit Peroxidase) using 33-diaminobenzidine as thechromogen The slides were counterstained in hematoxylinPositive staining resulted in a brown reaction product Neg-ative controls were treated as above with the exception ofthe first antibody which was omitted Five pictures at thesamemagnificationwere quantitatively analyzed usingGIMP26 software (GNU Image Manipulation Program UNIXplatforms)

28 Immunofluorescence (IF) After anesthesia the animalswere euthanized and fragments of liver were fixed in asolution of 4paraformaldehyde (Sigma-Aldrich) (40mL) in01M phosphate (sodium phosphate monobasic and dibasicheptahydrate Sigma-Aldrich) buffered saline (PBS) with apH of 72 for two hours The livers were immersed in 15sucrose overnight followed by 30 sucrose for a second night(36 hours total)The specimenswere then embedded inOCT-Tissue Tek compound (Sakura Finetek Torrance CA USA)frozen in n-hexane (Dinamica Sao Paulo SP Brazil) and

cooled with liquid nitrogen Cryosections (8120583m thick) werepermeabilized (03 Triton X-100) and incubated for 1 hwith blocking solution (3 BSA plus 02 Tween 20 in Trisbuffered saline) Subsequently the sections were incubatedwith antibodies for COX-2 (Abcam ab15191) 120572SMA (Abcamab66043) and IL-1120573 (Abcam ab9722) (both 1 100) The sec-tions were incubated with primary antibodies overnight andthen incubated with polyclonal Cy3-conjugated secondaryantibodies (Jackson cat no 705-165-147) against rabbitimmunoglobulin (1 200) for 1 h The slides were washedand mounted in fluorescent Prolong Gold Antifade medium(Life Technologies cat no P36930) for observation underan inverted fluorescence microscope (Zeiss MicroImagingGmbH) which was equipped with a camera (Zeiss AxioCamMRM) and Release 472 image analysis software

29 Western Blot The livers were quickly dissected andthen homogenized in a Wheaton Overhead Stirrer (no903475) using the following extraction cocktail 10mMethylenediamine tetraacetic acid (EDTA) 2mM phenyl-methylsulfonyl fluoride (PMSF) 100mM sodium fluoride10mM sodium pyrophosphate 10mM sodium orthovana-date (NaVO

4) 10mg aprotinin and 100mM Tris (hydrox-

ymethyl)aminomethane (pH 74) Homogenates were cen-trifuged at 3000timesg for 10min and the supernatant wascollected and stored atminus70∘Cuntil its use in the immunoblot-ting Protein levels were determined using the Bradfordmethod with bovine serum albumin as the standard [33]The proteins (40mg) were separated with 10 (IL10 IL-1120573 NF-120581B-p65 COX-2 IFN120574 and TGF-120573) sodium dodecylsulfate-polyacrylamide by gel electrophoresis under reducedconditions and were electrophoretically transferred ontonitrocellulose membranes (Bio Rad CA USA Ref 162-0115)After blocking overnight at 4∘Cwith 5nonfatmilk in TBS-T(Tris buffered saline 01 plus 005 Tween 20 pH 74) themembraneswere incubated at room temperature for 3 hwithrabbit polyclonal antibodies anti-IL10 and anti-IL-1120573 (both1 1000 dilution Abcam USA) anti-IFN120574 and anti-TGF-120573(both 1 2000 dilution Abcam CA USA) and with rabbitpolyclonal antibodies anti-NF-120581B-p65 and anti-COX-2 (both1 1500 dilution Abcam CA USA) diluted in buffer solu-tion TBS-T containing 3 nonfat milk After washing (sixtimes 10min each) in TBS-T the membranes were furtherreacted with horseradish peroxidase-conjugated anti-rabbitsecondary antibody (1 8000 (Ref A6154) dilution SigmaUSA) diluted in TBS-T with 1 nonfat milk for 1 h 30minat room temperature An enhanced chemiluminescencereagent (Super Signal Pierce Ref 34080) was used to visu-alize the labeled protein bands and the blots were developedon X-ray film (Fuji Medical Kodak Ref Z358487-50EA)For quantification the density of pixels of each bandwas determined by the Image J 138 program (avail-able at httprsbwebnihgovijdownloadhtml developed byWayne Rasband NIH Bethesda MD) The results wereconfirmed in three sets of experiments for each protein inves-tigatedThe immunoblot for 120573-actin was used as a control forthe above protein blots After protein blot visualization withenhanced chemiluminescence the protein antibodies were


stripped from the membranes which were reprobed withmonoclonal anti-120573-actin antibody (1 1000 dilution SigmaUSA) Protein densitometry was subsequently carried out

210 RNA Isolation RT-PCR and Real-Time QuantitativePCR Total RNA from mouse tissues was isolated usingTrizol reagent (Invitrogen Carlsbad CA USA) The RNAwas treated with RNase-free DNase I and amplified witholigo (dT) primer using the SuperScript First-Strand Syn-thesis System for RTPCR (Invitrogen) Then 1 120583g of totalRNA was reverse-transcribed using the QuantiTec ReverseTranscription kit (Qiagen Hilden Germany) using randomhexameric primers according to the manufacturerrsquos instruc-tions Quantitative real-time PCR was performed withthe SYBR Green PCR system (Applied Biosystems FosterCity CA USA) using GAPDH as an internal control fornormalization RTqPCR was carried out with an ABI PRISM7500 instrument (Applied Biosystems CA USA) The for-ward and reverse primers used for each gene were as fol-lows 51015840-TGAGCAACTATTCCAAACCAGC-31015840 and 51015840-GCA-CGTAGTCTTCGATCACTATC-31015840 forCOX-2 51015840-GTTCTC-AGCCCAACAATACAAGA-31015840 and 51015840-GTGGACGGGTCG-ATGTCAC-31015840 for iNOS and 51015840-AGGTCGGTGTGAACG-GATTTG-31015840 and 51015840-TGTAGACCATGTAGTTGAGGTCA-31015840 for GAPDH (endogenous control) All reactions wereperformed in triplicate and included the following 1 120583L ofcDNA 5 120583Mof each primer 2x SYBRGreen PCRMasterMix(Applied Biosystems) and water added to a final volume of25 120583L The relative amount of mRNA was determined usingthe comparative threshold (Ct)method by normalizing targetcDNA Ct values to that of GAPDH Fold increase ratios werecalculated relative to the control (basal conditions) for eachgroup using the formula 2119890 minus ΔΔCt

211 Determination of Serum Oxide Nitric (NO) Activity TheNO2

minus levels in serum were determined by a method basedon the Griess reaction [34] The Griess colorimetric reactionwas used to measure nitric oxide which involved detectingnitrite (NO

2

minus) and the oxidation of NO in the plasma Induplicate 50120583L of the plasma was added to a 96-well ELISAplate followed by the same volume of Griess reagent which iscomposed of 1 sulfanilamide diluted in 25 H

3PO4(solu-

tion A) and N-1-naphtyl-ethtylenodiamina also diluted in25 H

3PO4(solution B) A solution of sodium nitrite in an

initial concentration of 100 120583Mwas serially diluted in PBS toprepare the standard curve After incubation for 10minutes inthe dark a reading was performed by the spectrophotometerat 490 nmThe absorbance of different sampleswas comparedwith the standard curve and the results were expressed asthe mean plusmn standard error of the duplicate using GraphPadPrism software (v 50)

212 Statistical Analyses GraphPad Prism software (version5) was used for statistical analyses Data were expressed usingmean plusmn standard deviation Differences between the controland treated groups were analyzed by analysis of variance

(ANOVA) followed by Dunnettrsquos test Tukeyrsquos test or the 119905-test as post hoc tests Probability values less than 005 wereconsidered significant

3 Results

31 Histopathology Histological analysis of the control groupmice (Figure 1(a)) showed normal liver architecture Animalsfrom the DEC group exhibited similar patterns (Figure 1(b))In the CCl

4group several histological characteristics of a

persistent inflammatory process were found particularlypericentral necrosis and fibrosis vacuolar fatty change mildinflammatory cell infiltration cytoplasmic degeneration andnuclear disorganization (Figure 1(c)) DEC treatment of ani-mals exposed toCCl

4completely prevented liver necrosis and

fibrosis It also attenuated the inflammatory infiltrates withminimal hepatic damage (Figure 1(d))

32 Collagen Analysis Hepatic fibrosis was assessed bySirius-red staining (marking type I and type III collagenfibers) The control and DEC groups did not exhibit signif-icant Sirius-red positive areas (Figures 2(a) and 2(b)) TheCCl4group exhibited significant collagen deposition around

the portal spaces and in fibrotic areas (Figure 2(c)) Howeverthe CCl

4+ DEC group exhibited a reduction in collagen

deposition along the hepatic tissue as observed in the controlgroup (Figure 2(d)) Positive Sirius-red areas were quanti-fied by GIMP 26 imaging software and are illustrated inFigure 2(e)

33 Ultrastructural Assays The ultrastructural analysis ofhepatocytes of the control group exhibited typical morpho-logical patterns such as rough endoplasmic reticulum mito-chondria glycogen granules and euchromatin in the nucleus(Figure 3(a)) Ultrastructural analysis of the CCl

4group

revealed chronic cell injury characterized by the presenceof numerous swollen mitochondria and peroxisomes dilatedrER and several vacuoles including autophagosomes as wellas condensed chromatin with areas of nucleic acid lyse thatare characteristic of a necrotic process (Figures 3(b) 3(c) and3(d)) The hepatocytes of the CCl

4+ DEC group exhibited

typical morphology with well-preserved organelles euchro-matic nuclei and numerous mitochondria in the cytoplasmand many cisterns of rER similar to those observed in thecontrol group (Figures 3(e) and 3(f))

34 Immunohistochemistry and Immunofluorescence Therelease of inflammatory cytokines can activate other hepaticcells (endothelial cells stellate cells and hepatocytes) andinduce the expression of chemokines cytokines and enzymesthat attract and activate inflammatory cells from the circula-tion [35 36]

The administration of CCl4produced severe liver dam-

age as indicated by a marked increase in malondialde-hyde (MDA) (Figure 4(a)) Immunohistochemical stainingshowed that theMDA in the control andDEC groups was notimmunopositive On the other hand MDA was highly accu-mulated in the hepatic tissue especially in perivenular areas


Control100120583m

(a)

DEC

(b)

CCl4100 120583m

(c)

CCl4 + DEC100120583m

(d)

0

100

200

300

400

500

c

Fibr

otic

area

()

a b d

Control DEC CCl4 CCl4 + DEC

(e)

Figure 1 Micrograph of hepatocytes (a) Control group showing typical morphology (b) group treated with 50mgkg DEC (DEC) (c) CCl4

group (CCl4) and (d) CCl

4plus 50mgkg DEC group (CCl

4+ DEC) Magnification = 100 120583m HampE stain (e) Quantification fibrosis area

(mean plusmn SD 119899 = 5) a119875 lt 005 when compared with control group b119875 lt 005 when compared with DEC group c119875 lt 001 when comparedwith CCl

4group d119875 lt 005 when compared with CCl

4+ DEC group Fibrotic areas (arrows)

of the CCl4group In contrast MDA was depleted in the

CCl4+ DEC group Immunostaining quantitation of MDA

was performed using GIMP 26 image softwareTGF-120573 has been characterized as an important cytokine

which mediates hepatic fibrogenesis Kupffer cells and hep-atic stellate cells (HSCs) are the major producers of theextracellular matrix in the fibrotic liver and are fundamentalin liver fibrogenesis [23 37] TGF-120573 immunohistochemistry(Figure 4(b)) in the control and DEC groups confirmed noimmunopositivity for TGF-120573 However the CCl

4group

exhibited a significant increase in TGF-120573 mainly in fibroticareas and in theHSCsA significant reduction of this cytokinewas observed in the CCl

4+ DEC group suggesting that DEC

is involved in the decrease of hepatic fibrosis

COX-2 expression is increased in inflammatory condi-tions which results in the induction of different stimuliincluding proinflammatory cytokines such as TNF-120572 and IL-1120573 [34] COX-2 inhibition has a hepatoprotective effect onliver injuries induced by carbon tetrachloride [38]

COX-2 physiological levels were observed in the controlgroup and the DEC group (Figures 4(c) and 4(d)) Howeversignificantly increased expression levels of this enzyme werefound in the CCl

4group COX-2 immunoreactivity was

mainly observed in fibrotic areas with a predominanceof pericentral staining COX-2 expression was significantlyreduced in the CCl

4+ DEC group Immunostaining quanti-

tation immunohistochemistry and immunofluorescence for


Control

(a)

DEC

(b)

CCl4

(c)

CCl4 + DEC

(d)

0

2000

4000

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10000a b d

c

Siriu

s-re

d qu

antifi

catio

n

Control DEC 50 CCl4 CCl4 + DEC

(e)



4plus DEC group (CCl

4+ DEC) Magnification = 100 120583m Sirius-red staining (e) Quantification of fibrosis area



4+ DEC group Collagen depositions (arrow)

COX-2 were performed using GIMP 26 imaging softwareand are illustrated in Figures 4(c) and 4(d) respectively

In the immunofluorescence analysis the control andDECgroups were not immunopositive to IL-1120573 (Figure 4(e))However in the CCl

4group a high expression of the proin-

flammatory cytokine IL-1120573was observedThis expressionwassignificantly lower in the CCl

4+DEC group than in the CCl

4

group (Figure 4(e))

Activated HSCs are transformed into myofibroblastsproducing 120572-SMA and increasing the secretion of collagenfibers which results in the deposition of fibrotic matrixconstituents [39]

In the present study the activation of HSCs was identifiedby the increased expression of 120572-SMA in the CCl

4group

(Figure 4(f)) while the expression of120572-SMAwas significantlyreduced in theCCl

4+DECgroup (Figure 4(f))This indicates


Control

N

M

rER

G

Nu

(a)

NM

VV

CCl4

(b)

rER

M

CCl4

(c)

M

M

CCl4

lowast

(d)

Nu

M

GN

CCl4 + DEC

(e)

M

rER

G

NNu

CCl4 + DEC

(f)

Figure 3Ultrathin sections of hepatocytes (a) Control group (b) (c) and (d)CCl4group (e) and (f)DEC+CCl

4groupNote that the chronic

cell injury exhibits swollenmitochondria several vacuoles (V) and lyses of chromatin characteristic of a necrosis process (head arrows) Notealso the collagen fibers (asterisk) rER dilatation (black arrows) and an autophagosome containing mitochondria (star) Mitochondria (M)glycogen (G) nucleus (N) nucleolus (Nu) rough endoplasmic reticulum (rER) and peroxisomes (white arrows) Bar 1 and 2 120583m

that DEC may reduce this activation by preventing theinitiation of the HSC fibrous process This finding correlateswith the Sirius-red staining results (Figure 2)

35 Expression Analysis (Western Blot) of the Pro- and Anti-Inflammatory Markers IL10 IL-1120573 NF-120581B COX-2 IFN120574 andTGF-120573 The ability of IL-10 (18 kDa) to modulate the inflam-matory response and limit hepatotoxicity has been demon-strated in several models of liver injury [40] Accordingto the results of the present study the CCl

4+ DEC group

showed a significant increase in the expression of anti-inflammatory cytokines when compared to the other groups(Figure 5(a))

Interleukin IL-1120573 (17 kDa) showed a significant increasein expression in the CCl

4group In the CCl

4+ DEC group

the expression of this cytokine was significantly decreased(Figure 5(b))

Similarly low expression of p65-NF-120581B (60 kDa) wasobserved in the control and DEC groups In contrast signifi-cantly elevated levels of expression ofNF-120581Bwere observed inthe CCl

4group whereas NF-120581B was significantly reduced in



a b d

c0

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A q

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(pix

els)

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ixel

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(c)

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(e)

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120572-S

MA

120572-SMA

quan

tifica

tion

(pix

els)

(f)

Figure 4 Immunohistochemistry (IHC) and immunofluorescence (IF) for (a) MDA (b) TGF-120573 (c) and (d) COX-2 (e) IL-1120573 and (f) 120572-SMA Control group 50mgkg DEC-treated group CCl

4group DEC + CCl

4group Black arrows indicate IHC labeling White arrows show

IF staining IHC and IF quantification (mean plusmn SD 119899 = 5) a119875 lt 005 when compared with control group b119875 lt 005 when compared withDEC group c119875 lt 001 when compared with CCl


4+ DEC group


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(f)

Figure 5 Markers of liver injury (a) IL-10 (b) IL-1120573 (c) NF-120581B (d) COX-2 (e) IFN120574 and (f) TGF-120573 expressions Each value represents themean plusmn SD for 5 animals per group Data were analyzed using one-way ANOVA followed by Dunnettrsquos test and Tukeyrsquos test a119875 lt 005 whencompared with control group b119875 lt 001 when compared with DEC group c119875 lt 001 when compared with CCl

4group d119875 lt 001 when

compared with CCl4+ DEC group The results were confirmed in three sets of experiments


0

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OX-

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lowast

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)


lowast

(b)

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lowast

(NO

2minus)

nitr

ites s

erum

leve

l


(c)

Figure 6 (a) Relative expression of mRNA COX-2 (b) mRNA expression of iNOS CCl4group significantly increased mRNA COX-2 and

iNOS expressions when compared with other groups An asterisk represents a significant difference of the CCl4group between groups (c)

Assessment of NO in serum through the measure of total nitrite metabolites Quantification of NO (mean plusmn SD 119899 = 6) 119875 lt 005 whencompared with control group lowast119875 lt 002 when compared with CCl

4group

theCCl4+DECgroupwhen compared to the inflamed group

(Figure 5(c))The DEC group did not alter COX-2 (42 kDa) expression

when compared to the control group The levels of COX-2 inthe CCl

4group increased when compared to the control and

DEC groups However the CCl4+ DEC group significantly

reduced the expression of this enzyme indicating an anti-inflammatory effect against chronic liver inflammation (Fig-ure 5(d)) thereby confirming the results obtained in theimmunohistochemistry and immunofluorescence (Figures4(c) and 4(d))

IFN120574 expressionwas not observed in the control andDECgroups However a significant increase was observed in theCCl4group The CCl

4+ DEC group decreased IFN120574 levels

significantly (Figure 5(e))TGF-120573 was detected in all groups An increase was

observed in the CCl4group due to the liver injury caused by

CCl4(Figure 5(f)) However a reduction in expression was

observed in the CCl4+ DEC group suggesting a beneficial

DEC effect against chronic inflammation as well as the anti-fibrotic effect confirming the data obtained in the immuno-histochemistry (Figure 4(b))

36 Analysis of the mRNA Expression of COX-2 and iNOSThe levels of COX-2 and iNOS mRNA expression increased

46-fold and 97-fold over the CCl4group respectively when

compared with the control group COX-2 and iNOS mRNAexpression were significantly attenuated in the CCl

4+ DEC

group (Figures 6(a) and 6(b))

37 Nitric Oxide Levels With regard to the level of NO2

minus

in the serum a significant increase was detected in the NOmetabolites in the CCl

4group (1109 plusmn 1823) when com-

pared to the other groups In the CCl4+ DEC group (6422 plusmn

696) a significant decrease was observed when compared tothe CCl

4group (Figure 6(c))

4 Discussion

Chronic inflammation leads to continuous hepatocyte dam-age and subsequently hepatic fibrosis [23 36 41]

The response is generalized with features common tomultiple organ systems In the liver a variety of different typesof injury lead to inflammation and fibrogenesis implyinga common pathogenesis The recruitment and migration ofKupffer cells and HSCs are critical events in the developmentof liver inflammation and fibrosis [32]

Although a number of specific therapies for patientswith different liver diseases have been successfully developedincluding antiviral therapies for patients with hepatitis B


and hepatitis C infections specific and effective hepaticanti-inflammatory and antifibrotic therapy remains elusiveElucidation of these mechanisms has been of fundamentalimportance in highlighting new potential therapies [2]

According to Rocha et al [15] treatment of alcoholicanimals with DEC 50mgkg prevented lipid accumulationand promoted a reduction of the inflammatory infiltrates andnecrosis areas caused by alcoholism In addition DEC at 25and 50mgkg was used to treat malnourished animals andprevented lipid accumulation while promoting a reductionin the damage caused by malnutrition [30]

A CCl4-induced hepatic injury is a widely used exper-

imental model for anti-inflammatory and hepatoprotectivedrug screening promoting hepatic pathology similar to thatobserved in humans [42ndash45] The CCl

4exerts effects on

immunity cells such asT-cellsNKcellsmacrophages phago-cytes and lymphatic organs besides increasing cytokinesinflammatory (IL-2 TGF-beta) [46 47] The present paperused the CCl

4-injury hepatic model to analyze the DEC

therapeutic potential in preventing chronic hepatotoxicity byattenuating the inflammatory response and fibrosis in theliver

Gonzalez et al [48] showed that rats with liver inflamma-tion induced by CCl

4had less liver damage after DEC treat-

ment at 25 and 50mgkg The animals had well-preservedorganelles and a membrane system of hepatocytes showingthat DEC had a protective effect In the present study theCCl4group (CCl

4) exhibited an evident inflammatory pro-

cess as well as necrosis and fibrosis However a reduction ofinflammatory infiltrates liver necrosis and fibrosis wasobserved in theCCl

4+DECgroupThereforeDEC treatment

caused an evident reduction of hepatic damage corroborat-ing the results of previous studies

Several studies have shown that chronic liver inflamma-tion and fibrosis is related to the chronic phase of Kupffercell accumulation HSC activation and collagen deposition[49ndash51] The present study demonstrated that there was asignificant increase in collagen fiber deposition in liver tissuein the CCl

4group It has been shown that hepatic fibrosis is

largely the result of disorder in the homeostasis of the syn-thesis deposition degeneration and absorption of collagensIn hepatic fibrosis myofibroblasts characteristically assumethe ability to remodel the extracellular matrix (ECM) via theproduction of ECM proteins in the liver [52 53] After DECtreatment a significant decrease of fiber collagen depositionswas assessed by Sirius-red staining Therefore the presentstudy suggests that DEC has an antifibrotic effect

Nitric oxide (NO) is known to react with superoxideradical forming peroxynitrite an evenmore potent oxidizingagent [54 55] Peroxynitrite can react directly with sulfhydrylresidues in cell membranes and DNA leading to lipid peroxi-dation and cytotoxicity [54 56] The present study illustratedthat there was an elevation in serum nitric oxide levels inCCl4-treated mice and that DEC treatment was capable of

attenuated serum NO levels This may be related to thereduction of iNOS levels in hepatic tissue

The iNOS enzyme is expressed in hepatocytes and inflam-matory cells during the development of acute and chronicdiseases [57 58] but the role of NO∙ in tissue damage is

still controversial Although a hepatoprotective function ofiNOS has been observed in different types of liver injuryincluding chronic CCl

4-intoxication [59 60] hepatotoxic

effects of iNOS have also been reported [61] The iNOS geneis expressed by hepatocytes in a number of physiological andpathophysiological conditions affecting the liver includingseptic hemorrhagic shock and alcoholism [62] It seems thatiNOS-derived NO∙ could contribute to nitrosative stress [63]but also regulate proinflammatory gene expression therebycontributing to inflammatory liver injuries [45] Rocha et al[15] demonstrated that DEC suppresses the activation ofNF-120581B and downstream proinflammatory mediators includ-ing iNOS The results of the present study suggest antini-trosative and anti-inflammatory effects of DEC in CCl

4-

induced liver injuriesIt is known that different types of cells and cytokines

involved in inflammation participate in chronic hepaticinjuries These cytokines such as extracellular stimulantsregulate the genetic expression of various factors throughinteractions with transcription-modulating factors NF-120581Bhas been considered a modulating factor which regulatesimmunologic reactions and apoptosis mediating acute andchronic inflammatory reactions [64] NF-120581B reportedly playsan important role in chronic liver injuries resulting from theaction of CCl

4[65]Themain action ofNF-120581B in liver injuries

is to mediate the release of cytotoxic cytokines and inflam-matory cytotoxins The present study provides additionalfindings supporting the activity of DEC in modulating theseverity of inflammation through several key transcriptionfactors such as NF-120581B

Several studies have demonstrated that certain nons-teroidal anti-inflammatory drugs (NSAIDs) such as sodiumsalicylate sulindac ibuprofen and flurbiprofen exhibit anti-inflammatory effects independent of the cyclooxygenasepathways Much interest has focused on NF-120581B as a potentialtarget for certain NSAIDS [66ndash68] Similarly DEC alsoexhibits anti-inflammatory effects while decreasing NF-120581Bexpression

Over one hundred target genes forNF-120581Bhave been iden-tified during the past few years One of them is the COX-2gene It is known that COX-2 is responsible for inflammatoryreactions and carcinogenesis of hepatocellular carcinoma[69] COX is another keymolecule in the inflammatory path-way and it is induced by several stimuli including cytokinesand mitogens COX-2 expression results in the release ofprostaglandin at the site of inflammation In the presentstudy the levels of COX-2 protein and mRNA expressionincreased in the CCl

4group and DEC markedly attenuated

these increases High levels of iNOS and COX-2 cause theproduction of high concentrations of NO and eicosanoidsthrough the initiation of the COX-prostanoid pathway [2670] which leads to cellular inflammation necrosis andfibrosis Taken together the results of the present studysuggest that DEC largely regulates the production of iNOSand COX-2 on a transcriptional level

Although the results of liver injuries are associated withthe CCl

4metabolism secondary damage occurs due to the

inflammatory process initiated by Kupffer cell activation[9 71] Kupffer cells activate the release of inflammatory


mediators including TNF-120572 and IL-1120573 Interleukin-1120573 isa potent inflammatory cytokine that is involved in thesynthesis of prostaglandins macrophage activation and theinduction of neutrophil infiltration as well as several aspectsof inflammation [13 72]The present study showed that DECtreatment effectively reduced CCl

4-induced liver injuries

through the inhibition of IL-1120573The results of the present study demonstrated that there

was a significant elevation in MDA content in the liver tissueof CCl

4-treatedmiceThis enhanced lipid peroxidation led to

tissue damage and the failure of antioxidant defense mecha-nisms In the present study there was a significant elevationin liver MDA content in the CCl

4group and a significant

reduction in the group treated with DEC which suggestsenhanced lipid peroxidation leading to tissue damage and thefailure of antioxidant defense mechanisms to prevent the for-mation of excessive free radicals [73] Therefore since DECtreatment reducedMDA levels it probably exhibits an antiox-idant action

IFN120574 plays a crucial role in modulating immuneresponses Previous studies have reported that the level ofIFN120574 was positively correlated with serologic markers ofhepatic injury and fibrogenesis [74] The reason for elevatedlevels of IFN120574 in the development of hepatic injuries andfibrogenesis remains unclear [75] The present study showedthat IFN120574 expression decreased afterDEC treatment suggest-ing an advantageous response against the CCl

4-caused injury

in the animalsCertain immunomodulatory cytokines can contribute to

the establishment of an anti-inflammatory state in the liverand ameliorate injuries One such example is IL-10 whichexhibits potent anti-inflammatory and immunosuppressiveproperties decreasing the production of proinflammatorycytokines including TNF-120572 and IL-1120573 [76] The ability of IL-10 to modulate the inflammatory response and to limit hep-atotoxicity has been shown in several models of liver injury[77ndash79] IL-10 may also have antifibrogenic properties linkedto the downregulation of profibrogenic cytokines such asTGF-120573 [80] Increased endogenous IL-10 expression amelio-rates chronic inflammatory burst and subsequent liver fibro-sis after repeated stimulation with CCl

4[40] Similarly the

results of the present study showed that DEC increased IL-10expression during CCl

4intoxication and decreased inflam-

mation and the subsequent fibrogenic response in the liverChronic damage such as a liver inflammatory response

results in fibrosis in conjunction with the accumulationof ECM proteins characteristic of most types of chronicliver disease [81 82] HSCs are the primary ECM-producingcells in an injured liver [83] HSCs that are activated ortransdifferentiated into myofibroblast-like cells acquire con-tractile proinflammatory and fibrogenic properties [49 8485] Key cytokines are involved in liver fibrosis regulatingthe inflammatory response to injury and modulating hep-atic fibrogenesis TGF-120573 and 120572SMA appear to be crucialmediators in human fibrogenesis [86] The stimulation ofactivatedHSCs by TGF-120573 is believed to be a crucial fibrogenicresponse in liver fibrosis for the following reasons higherTGF-120573 expression in activated HSC potency of TGF-120573 toupregulate ECM expression higher expression of TGF-120573

receptors in relation to HSCs and increased expressionof TIMP-1TGF-120573 liver fibrosis induced [87 88] Strategiesaimed at disrupting TGF-120573 synthesis andor reducing thesignaling pathways decrease fibrosis in experimental models[89] The immunohistochemistry and immunofluorescenceanalysis showed that TGF-120573 increased significantly in theCCl4group compared to the others groups in contrast with

CCl4+ DEC which significantly reduced TGF-120573 expression

confirming previous observationsAlpha smooth muscle actin (120572-SMA) is a protein that

participates in the processes of tissue repair present at aparticular stage of fibroblast differentiation These cells wereshown to be responsible for the contraction of granulation tis-sue and fibrotic lesions [90] Activated HSCs are responsiblefor liver damage producing type I collagen in hepatic fibrosisand also expressing 120572-SMA filamentsThe activation of HSCsis an essential characteristic of hepatic fibrosis and thisactivation is indicated by the expression of 120572-SMA althoughit can be expressed in other cell types [91] A significantlyhigher labeling for 120572-SMA was observed in the CCl

4group

than in the control group On the other hand low levels of120572-SMAwere observed in theCCl

4+DECgroupThese results

indicate that DEC can inhibit TGF-120573 and 120572-SMA expressionconsequently reducing the impact of the proliferation andactivation of HSCs These results are in agreement withprevious observations that DEC treatment significantlyreduced fiber collagen depositions confirming that DEC hasan antifibrotic effect

In summary the present study demonstrated for the firsttime thatDEC can protect against CCl

4-induced chronic hep-

atotoxicity and exerts an antifibrotic effect through reductionof inflammatory markers as TGF-120573 120572-SMA and Sirius-redstaining revealing a clinical potential of DEC for therapeuticantifibrotic applications

Conflict of Interests

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

Acknowledgments

This study was supported by PAPESFIOCRUZCNPq Insti-tuto Nacional de Biologia Estrutural e Bioimagem (INBEB)Fundacao de Amparo a Ciencia e Tecnologia do Estado dePernambuco (FACEPE) and Aggeu Magalhaes ResearchCenter of the Oswaldo Cruz Foundation in Recife Brazil(CPqAMFIOCRUZ)

References

[1] D P da Rosa S Bona D Simonetto C Zettler C A Marroniand N P Marroni ldquoMelatonin protects the liver and erythro-cytes against oxidative stress in cirrhotic ratsrdquo Arquivos deGastroenterologia vol 47 no 1 pp 72ndash78 2010

[2] D C Rockey ldquoCurrent and future anti-fibrotic therapies forchronic liver diseaserdquo Clinics in Liver Disease vol 12 no 4 pp939ndash962 2008


[3] J I Tsui M J Pletcher E Vittinghoff K Seal and R GonzalesldquoHepatitis C and hospital outcomes in patients admitted withalcohol-related problemsrdquo Journal of Hepatology vol 44 no 2pp 262ndash266 2006

[4] D Montgomery Bissell G J Gores D L Laskin and J HHoofnagle ldquoDrug-induced liver injury mechanisms and testsystemsrdquo Hepatology vol 33 no 4 pp 1009ndash1013 2001

[5] T-Y Lee H-H Chang J-H Chen M-L Hsueh and J-JKuo ldquoHerb medicine Yin-Chen-Hao-Tang ameliorates hepaticfibrosis in bile duct ligation ratsrdquo Journal of Ethnopharmacologyvol 109 no 2 pp 318ndash324 2007

[6] G Pereira-Filho C Ferreira A Schwengber C Marroni CZettler and N Marroni ldquoRole of N-acetylcysteine on fibrosisand oxidative stress in cirrhotic ratsrdquo Arquivos de Gastroen-terologia vol 45 no 2 pp 156ndash162 2008

[7] P M Amalia M N Possa M C Augusto and L S FranciscaldquoQuercetin prevents oxidative stress in cirrhotic ratsrdquo DigestiveDiseases and Sciences vol 52 no 10 pp 2616ndash2621 2007

[8] J L Poyer P B McCay E K Lai and E G Janzen Davis ldquoCon-firmation of assignment of the trichloromethyl radical spinadduct detected by spin trapping during 13C-carbon tetra-chloride metabolism in vitro and in vivordquo Biochemical andBiophysical Research Communications vol 94 no 4 pp 1154ndash1160 1980

[9] M J Edwards B J Keller F C Kauffman and R G ThurmanldquoThe involvement of Kupffer cells in carbon tetrachloridetoxicityrdquo Toxicology and Applied Pharmacology vol 119 no 2pp 275ndash279 1993

[10] K Nakahira T Takahashi H Shimizu et al ldquoProtective role ofheme oxygenase-1 induction in carbon tetrachloride-inducedhepatotoxicityrdquo Biochemical Pharmacology vol 66 no 6 pp1091ndash1105 2003

[11] E A Glende Jr andC K Pushpendran ldquoActivation of phospho-lipase A

2by carbon tetrachloride in isolated rat hepatocytesrdquo

Biochemical Pharmacology vol 35 no 19 pp 3301ndash3307 1986[12] D E Johnston and C Kroening ldquoStimulation of prostaglandin

synthesis in cultured liver cells by CCl4rdquoHepatology vol 24 no3 pp 677ndash684 1996

[13] R N Achur W M Freeman and K E Vrana ldquoCirculatingcytokines as biomarkers of alcohol abuse and alcoholismrdquoJournal of Neuroimmune Pharmacology vol 5 no 1 pp 83ndash912010

[14] Z K Ballas R T Cook M R Shey and R A Coleman ldquoAdynamic flux in natural killer cell subsets as a function of theduration of alcohol ingestionrdquo Alcoholism Clinical and Experi-mental Research vol 36 no 5 pp 826ndash834 2013

[15] S W S Rocha B S Silva F O D S Gomes et al ldquoEffect ofdiethylcarbamazine on chronic hepatic inflammation inducedby alcohol in C57BL6 micerdquo European Journal of Pharmacol-ogy vol 689 no 1ndash3 pp 194ndash203 2012

[16] N Bhaskaran S Shukla J K Srivastava and S GuptaldquoChamomile an anti-inflammatory agent inhibits induciblenitric oxide synthase expression by blocking RelAp65 activityrdquoInternational Journal of Molecular Medicine vol 26 no 6 pp935ndash940 2010

[17] DArias-Salvatierra E K Silbergeld L CAcosta-Saavedra andE S Calderon-Aranda ldquoRole of nitric oxide produced by iNOSthrough NF-120581B pathway in migration of cerebellar granuleneurons induced by Lipopolysacchariderdquo Cellular Signallingvol 23 no 2 pp 425ndash435 2011

[18] B Y Chen D P C Lin C Y Wu et al ldquoDietary zerumboneprevents mouse cornea from UVB-induced photokeratitis

through inhibition of NF-120581B iNOS and TNF-120572 expression andreduction of MDA accumulationrdquoMolecular Vision vol 17 pp854ndash863 2011

[19] K S Lee S J Lee H J Park et al ldquoOxidative stress effect on theactivation of hepatic stellate cellsrdquo Yonsei Medical Journal vol42 no 1 pp 1ndash8 2001

[20] G M Campo A Avenoso S Campo et al ldquoThe antioxidantactivity of chondroitin-4-sulphate in carbon tetrachloride-induced acute hepatitis in mice involves NF-120581B and caspaseactivationrdquo British Journal of Pharmacology vol 155 no 6 pp945ndash956 2008

[21] E Gabele D A Brenner andR A Rippe ldquoLiver fibrosis signalsleading to the amplification of the fibrogenic hepatic stellatecellrdquo Frontiers in Bioscience vol 8 pp d69ndashd77 2003

[22] C J ParsonsM Takashima and R A Rippe ldquoMolecularmech-anisms of hepatic fibrogenesisrdquo Journal of Gastroenterology andHepatology vol 22 supplement 1 pp S79ndashS84 2007

[23] S L Friedman ldquoMechanisms of hepatic fibrogenesisrdquoGastroen-terology vol 134 no 6 pp 1655ndash1669 2008

[24] R M Maizels and D A Denham ldquoDiethylcarbamazine (DEC)immunopharmacological interactions of an anti-filarial drugrdquoParasitology vol 105 pp S49ndashS60 1992

[25] H F McGarry L D Plant and M J Taylor ldquoDiethylcarba-mazine activity against Brugia malayi microfilariae is depen-dent on inducible nitric-oxide synthase and the cyclooxygenasepathwayrdquo Filaria Journal vol 4 article 4 2005

[26] SW S Rocha and C PeixotoArachidonic Acid Cyclooxygenaseand Hepatic Inflammation Nova Science Publishers 2014

[27] C A Peixoto and B S Silva ldquoAnti-inflammatory effects ofdiethylcarbamazine a reviewrdquo European Journal of Pharmacol-ogy vol 734 no 1 pp 35ndash41 2014

[28] E L Ribeiro K P D S Barbosa I T Fragoso et al ldquoDiethylcar-bamazine attenuates the development of Carrageenan-inducedlung injury in micerdquo Mediators of Inflammation vol 2014Article ID 105120 12 pages 2014

[29] T Queto P Xavier-Elsas M A Gardel et al ldquoInducible nitricoxide synthaseCD95L-dependent suppression of pulmonaryand bone marrow eosinophilia by diethylcarbamazinerdquo TheAmerican Journal of Respiratory and Critical Care Medicine vol181 no 5 pp 429ndash437 2010

[30] S W S Rocha A C O D Santos B D S Silva et al ldquoEffectsof diethylcarbamazine (DEC) on hepatocytes of C57BL6J micesubmitted to protein malnutritionrdquo Journal of Food and DrugAnalysis vol 20 no 2 pp 524ndash558 2012

[31] K L A Saraiva V A Silva Jr E S F Dias and C A PeixotoldquoMorphological changes in the testis induced by diethylcarba-mazinerdquo Reproductive Toxicology vol 22 no 4 pp 754ndash7592006

[32] E Seki S deMinicis S Inokuchi et al ldquoCCR2 promotes hepaticfibrosis in micerdquo Hepatology vol 50 no 1 pp 185ndash197 2009

[33] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[34] L C Green D A Wagner J Glogowski P L Skipper J SWishnok and S R Tannenbaum ldquoAnalysis of nitrate nitriteand [15N]nitrate in biological fluidsrdquo Analytical Biochemistryvol 126 no 1 pp 131ndash138 1982

[35] L Bujanda EHijona LHijona and J I Arenas ldquoInflammatorymediators of hepatic steatosisrdquo Mediators of Inflammation vol2010 Article ID 837419 7 pages 2010


[36] D L Laskin andK J Pendino ldquoMacrophages and inflammatorymediators in tissue injuryrdquoAnnual Review of Pharmacology andToxicology vol 35 pp 655ndash677 1995

[37] R Bataller and D A Brenner ldquoScience in medicine liverfibrosisrdquo News Letters de Medicina Interna vol 115 2005

[38] P Akarasereenont J AMitchell Y S Bakhle CThiemermannand J R Vane ldquoComparison of the induction of cyclooxygenaseand nitric oxide synthase by endotoxin in endothelial cells andmacrophagesrdquo European Journal of Pharmacology vol 273 no1-2 pp 121ndash128 1995

[39] C M Chu W C Shyu and Y F Liaw ldquoComparative studies onexpression of 120572-smooth muscle actin in hepatic stellate cells inchronic hepatitis B and Crdquo Digestive Diseases and Sciences vol53 no 5 pp 1364ndash1369 2008

[40] H Louis J-L Van Laethem W Wu et al ldquoInterleukin-10controls neutrophilic infiltration hepatocyte proliferation andliver fibrosis induced by carbon tetrachloride in micerdquoHepatol-ogy vol 28 no 6 pp 1607ndash1615 1998

[41] K M Jumaa Z A Ahmed I T Numan and S A R Hus-sain ldquoDose-dependent anti-inflammatory effect of silymarin inexperimental animal model of chronic inflammationrdquo AfricanJournal of Pharmacy and Pharmacology vol 3 no 5 pp 242ndash247 2009

[42] R P Tamayo ldquoIs cirrhosis of the liver experimentally producedby CCl4 an adequate model of human cirrhosisrdquo Hepatologyvol 3 no 1 pp 112ndash120 1983

[43] H Tsukamoto M Matsuoka and S W French ldquoExperimentalmodels of hepatic fibrosis a reviewrdquo Seminars in Liver Diseasevol 10 no 1 pp 56ndash65 1990

[44] J JMaher andR FMcGuire ldquoExtracellularmatrix gene expres-sion increases preferentially in rat lipocytes and sinusoidalendothelial cells during hepatic fibrosis in vivordquo Journal ofClinical Investigation vol 86 no 5 pp 1641ndash1648 1990

[45] G L Tipoe T M Leung E C Liong T Y H Lau M L Fungand A A Nanji ldquoEpigallocatechin-3-gallate (EGCG) reducesliver inflammation oxidative stress and fibrosis in carbontetrachloride (CCl

4)-induced liver injury in micerdquo Toxicology

vol 273 no 1ndash3 pp 45ndash52 2010[46] B Delaney S C Strom S Collins andN E Kaminski ldquoCarbon

tetrachloride suppresses T-cell-dependent immune responsesby induction of transforming growth factor-1205731rdquo Toxicology andApplied Pharmacology vol 126 no 1 pp 98ndash107 1994

[47] Y K Ahn and J H Kim ldquoPreventive effects of diphenyldimethyl dicarboxylate on the immunotoxicity of carbon tetra-chloride in ICR micerdquo Journal of Toxicological Sciences vol 18no 3 pp 185ndash195 1993

[48] R Gonzalez O Ancheta M Marquez and S RodriguezldquoHepatoprotective effects of diethylcarbamazine in acute liverdamage induced by carbon tetrachloride in ratsrdquo Zhongguo YaoLi Xue Bao vol 15 no 6 pp 495ndash497 1994

[49] F Marra ldquoChemokines in liver inflammation and fibrosisrdquoFrontiers in Bioscience vol 7 pp d1899ndashd1914 2002

[50] J P Iredale ldquoModels of liver fibrosis exploring the dynamicnature of inflammation and repair in a solid organrdquo Journal ofClinical Investigation vol 117 no 3 pp 539ndash548 2007

[51] P Jarcuska M Janicko E Veselıny P Jarcuska and L SkladanyldquoCirculating markers of liver fibrosis progressionrdquo ClinicaChimica Acta vol 411 pp 1009ndash1017 2010

[52] R Domitrovic and H Jakovac ldquoAntifibrotic activity of antho-cyanidin delphinidin in carbon tetrachloride-induced hepato-toxicity in micerdquo Toxicology vol 272 no 1ndash3 pp 1ndash10 2010

[53] M Ghazwani Y Zhang X Gao J Fan J Li and S LildquoAnti-fibrotic effect of thymoquinone on hepatic stellate cellsrdquoPhytomedicine vol 21 no 3 pp 254ndash260 2014

[54] H Ischiropoulos L Zhu and J S Beckman ldquoPeroxynitriteformation from macrophage-derived nitric oxiderdquo Archives ofBiochemistry and Biophysics vol 298 no 2 pp 446ndash451 1992

[55] A Pautz J Art S Hahn S Nowag C Voss and H KleinertldquoRegulation of the expression of inducible nitric oxide syn-thaserdquo Nitric OxidemdashBiology and Chemistry vol 23 no 2 pp75ndash93 2010

[56] R Radi J S Beckman K M Bush and B A FreemanldquoPeroxynitrite oxidation of sulfhydryls the cytotoxic potentialof superoxide and nitric oxiderdquo Journal of Biological Chemistryvol 266 no 7 pp 4244ndash4250 1991

[57] A K Nussler and T R Billiar ldquoInflammation immunoregula-tion and inducible nitric oxide synthaserdquo Journal of LeukocyteBiology vol 54 no 2 pp 171ndash178 1993

[58] C Hierholzer B Harbrecht J M Menezes et al ldquoEssential roleof induced nitric oxide in the initiation of the inflammatoryresponse after hemorrhagic shockrdquo Journal of ExperimentalMedicine vol 187 no 6 pp 917ndash928 1998

[59] M G Moreno and P Muriel ldquoInducible nitric oxide synthaseis not essential for the development of fibrosis and liver damageinduced by CCl4 inmicerdquo Journal of Applied Toxicology vol 26no 4 pp 326ndash332 2006

[60] W Hu W Han C Huang and M-H Wang ldquoProtectiveeffect of the methanolic extract from Duchesnea indica againstoxidative stress in vitro and in vivordquo Environmental Toxicologyand Pharmacology vol 31 no 1 pp 42ndash50 2011

[61] C H Lee S W Park Y S Kim et al ldquoProtective mechanism ofglycyrrhizin on acute liver injury induced by carbon tetrachlo-ride inmicerdquo Biological and Pharmaceutical Bulletin vol 30 no10 pp 1898ndash1904 2007

[62] B S Taylor L H Alarcon and T R Billiar ldquoInducible nitricoxide synthase in the liver regulation and functionrdquo Biochem-istry vol 63 no 7 pp 766ndash781 1998

[63] R Domitrovic H Jakovac and G Blagojevic ldquoHepatoprotec-tive activity of berberine is mediated by inhibition of TNF-120572 COX-2 and iNOS expression in CCl

4-intoxicated micerdquo

Toxicology vol 280 no 1-2 pp 33ndash43 2011[64] S H Kim H J Chu D H Kang et al ldquoNF-kappa B binding

activity and cyclooxygenase-2 expression in persistent CCl4-treated rat liver injuryrdquo Journal of Korean Medical Science vol17 no 2 pp 193ndash200 2002

[65] M T Pritchard J I Cohen S Roychowdhury B T Pratt andL E Nagy ldquoEarly growth response-1 attenuates liver injury andpromotes hepatoprotection after carbon tetrachloride exposurein micerdquo Journal of Hepatology vol 53 no 4 pp 655ndash662 2010

[66] I Tegeder J Pfeilschifter and G Geisslinger ldquoCyclooxygenase-independent actions of cyclooxygenase inhibitorsrdquo The FASEBJournal vol 15 no 12 pp 2057ndash2072 2001

[67] Y Bayon A Alonso and M Sanchez Crespo ldquo4-Triflu-oromethyl derivatives of salicylate triflusal and its main meta-bolite 2-hydroxy-4-trifluoromethylbenzoic acid are potentinhibitors of nuclear factor 120581B activationrdquo British Journal ofPharmacology vol 126 no 6 pp 1359ndash1366 1999

[68] E Kopp and S Ghosh ldquoInhibition of NF-120581B by sodiumsalicylate and aspirinrdquo Science vol 265 no 5174 pp 956ndash9591994

[69] A A Nanji L Miao P Thomas et al ldquoEnhanced cyclooxyg-enase-2 gene expression in alcoholic liver disease in the ratrdquoGastroenterology vol 112 no 3 pp 943ndash951 1997


[70] J Li and T R Billiar ldquoNitric oxide IV Determinants of nitricoxide protection and toxicity in liverrdquo American Journal ofPhysiologymdashGastrointestinal and Liver Physiology vol 276 no5 pp G1069ndashG1073 1999

[71] H Ishiyama M Sato K Matsumura M Sento K Ogino andT Hobara ldquoProliferation of hepatocytes and attenuation fromcarbon tetrachloride hepatotoxicity by gadolinium chloride inratsrdquo Pharmacology and Toxicology vol 77 no 4 pp 293ndash2981995

[72] S K Durum J A Schmidt and J J Oppenheim ldquoInterleukin 1an immunological perspectiverdquo Annual Review of Immunologyvol 3 pp 263ndash287 1985

[73] K Ashok Shenoy S N Somayaji and K L Bairy ldquoHepato-protective effects of Ginkgo biloba against carbon tetrachlorideinduced hepatic injury in ratsrdquo Indian Journal of Pharmacologyvol 33 no 4 pp 260ndash266 2001

[74] N Hyodo M Tajimi T Ugajin I Nakamura and M ImawarildquoFrequencies of interferon-120574 and interleukin-10 secreting cellsin peripheral blood mononuclear cells and liver infiltratinglymphocytes in chronic hepatitis B virus infectionrdquo HepatologyResearch vol 27 no 2 pp 109ndash116 2003

[75] C Peralta M B Jimenez-Castro and J Gracia-Sancho ldquoHep-atic ischemia and reperfusion injury effects on the liversinusoidalmilieurdquo Journal ofHepatology vol 59 no 5 pp 1094ndash1106 2013

[76] J J OrsquoShea and P J Murray ldquoCytokine signaling modules ininflammatory responsesrdquo Immunity vol 28 no 4 pp 477ndash4872008

[77] T Arai K Hiromatsu N Kobayashi et al ldquoIL-10 is involved inthe protective effect of dibutyryl cyclic adenosine monophos-phate on endotoxin-lnduced inflammatory liver injuryrdquo Journalof Immunology vol 155 no 12 pp 5743ndash5749 1995

[78] H Louis O LeMoineM-O Peny et al ldquoHepatoprotective roleof interleukin 10 in galactosaminelipopolysaccharide mouseliver injuryrdquo Gastroenterology vol 112 no 3 pp 935ndash942 1997

[79] L J Su C C Chang C H Yang et al ldquoGraptopetalum para-guayense ameliorates chemical-induced rat hepatic fibrosis invivo and inactivates stellate cells andKupffer cells in vitrordquo PLoSONE vol 8 no 1 Article ID e53988 2013

[80] P van Vlasselaer B Borremans U van Gorp J R Dasch andR de Waal-Malefyt ldquoInterleukin 10 inhibits transforminggrowth factor-120573 (TGF-120573) synthesis required for osteogeniccommitment of mouse bone marrow cellsrdquo Journal of CellBiology vol 124 no 4 pp 569ndash577 1994

[81] S Martın-Vılchez P Sanz-Cameno Y Rodrıguez-Munoz et alldquoThe hepatitis B virus X protein induces paracrine activation ofhuman hepatic stellate cellsrdquoHepatology vol 47 no 6 pp 1872ndash1883 2008

[82] C-M ChuW-C Shyu andY-F Liaw ldquoComparative studies onexpression of 120572-smooth muscle actin in hepatic stellate cells inchronic hepatitis B and Crdquo Digestive Diseases and Sciences vol53 no 5 pp 1364ndash1369 2008

[83] M B Bansal K Kovalovich R Gupta et al ldquoInterleukin-6 pro-tects hepatocytes from CCl4-mediated necrosis and apoptosisin mice by reducing MMP-2 expressionrdquo Journal of Hepatologyvol 42 no 4 pp 548ndash556 2005

[84] SMilani H Herbst D Schuppan K Y Kim E O Riecken andH Stein ldquoProcollagen expression by nonparenchymal rat livercells in experimental biliary fibrosisrdquo Gastroenterology vol 98no 1 pp 175ndash184 1990

[85] S Bisht M A Khan M Bekhit et al ldquoA polymeric nanopar-ticle formulation of curcumin (NanoCurc) ameliorates CCl

4-

induced hepatic injury and fibrosis through reduction of pro-inflammatory cytokines and stellate cell activationrdquo LaboratoryInvestigation vol 91 no 9 pp 1383ndash1395 2011

[86] A M Gressner R Weiskirchen K Breitkopf and S DooleyldquoRoles of TGF-beta in hepatic fibrosisrdquo Front Biosci vol 7 ppd793ndashd807 2002

[87] T Knittel D Kobold F Piscaglia et al ldquoLocalization of livermyofibroblasts and hepatic stellate cells in normal and diseasedrat livers Distinct roles of (myo-)fibroblast subpopulations inhepatic tissue repairrdquo Histochemistry and Cell Biology vol 112no 5 pp 387ndash401 1999

[88] F W Shek and R C Benyon ldquoHow can transforming growthfactor beta be targeted usefully to combat liver fibrosisrdquoEuropean Journal of Gastroenterology amp Hepatology vol 16 no2 pp 123ndash126 2004

[89] X Fan Q Zhang S Li et al ldquoAttenuation of CCl4-inducedhepatic fibrosis in mice by vaccinating against TGF-1205731rdquo PLoSONE vol 8 no 12 Article ID e82190 2013

[90] N Akpolat S Yahsi A Godekmerdan M Yalniz and KDemirbag ldquoThe value of 120572-SMA in the evaluation of hepaticfibrosis severity in hepatitis B infection and cirrhosis devel-opment a histopathological and immunohistochemical studyrdquoHistopathology vol 47 no 3 pp 276ndash280 2005

[91] J Li Y PanM Kan et al ldquoHepatoprotective effects of berberineon liver fibrosis via activation of AMP-activated protein kinaserdquoLife Sciences vol 98 no 1 pp 24ndash30 2014

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


part of which generates the trichloromethyl peroxyl radical(OOCCl

3

∙) [7 8] which leads to lipid peroxidation [9]the second involves the activation of Kupffer cells which isaccompanied by the production of proinflammatory media-tors [10] Carbon tetrachloride (CCl

4) should be an effective

stimulus for prostaglandin synthesis in hepatocytes since itis believed to release arachidonic acid in the liver through theactivation of phospholipase A2 [11 12]

Progression of the disease involves various proinflamma-tory molecules such as interleukins cytokines and nuclearfactor-120581B (NF-120581B) [13ndash15] The activated NF-120581B if translo-cated into the nucleus will facilitate the transcription ofmanydownstream genes including inducible nitric oxide syn-thase (iNOS) and cyclooxygenase-2 (COX-2) which are keymediators in the recruitment of inflammatory cells [16 17]The iNOS-derived nitric oxide production activated down-stream of NF-120581B is followed by the generation of reactiveoxygen species and other free radicals that are detrimental tocells Cellular lipids are easily attacked by free radicals result-ing in an intracellular accumulation of malondialdehyde(MDA) [18] Although the relationships between oxidativestress cytotoxic cytokines and liver cell injuries are not fullyunderstood NF-120581B is considered to play an important rolein liver cell injuries Studies have shown an increased acti-vation of NF-120581B through oxidative stress induced by carbontetrachloride in liver injuries [19 20]

After a chronic liver injury of any etiology the damagedhepatocytes their membrane components metabolites oftoxic agents and infiltrating inflammatory cells activateKupffer cells releasing soluble agents including cytokinessuch as platelet-derived growth factor (PDGF) transforminggrowth factor-1205731 (TGF-120573) tumor necrosis factor-a (TNF-120572)and endothelin-1 (ET-1) as well as reactive oxygen species(ROS) [21] These factors act on the hepatic stellate cells(HSCs) which undergo a response known as activation andstart expressing new receptors such as PDGF and TGF-120573receptors and new proteins such as a-smooth muscle actin(120572-SMA) The activation of the HSCs triggers the expressionof 120572-SMA and increases cellular proliferation and extracellu-lar matrix accumulation [22] Activated HSCs are consideredto play a role in the excessive deposition of ECM proteinssuch as type I collagen (Col-1) and in the disruption ofnormal parenchyma during the hepatic fibrogenesis processChronic inflammation and the repeated sustenance of liverinjuries such as those associated with hepatitis and alcoholicliver disease usually progress to liver cirrhosis and hepato-carcinogenesis [23]

The inhibition of proinflammatory cytokines enzymestranscription nuclear factor and free radicals could offer anew therapeutic strategy against inflammatory liver disease

Diethylcarbamazine (DEC) has anti-inflammatory prop-erties as a result of its interference with the arachidonicacid metabolism which involves lipoxygenase (LOX) andcyclooxygenase (COX) enzymes [24ndash27] DEC led to reduc-tions in lung injury PMNs migration the formation of NOproduction and the release of proinflammatory cytokinesand COX-2 in a model of acute inflammation induced bycarrageenan [28] DEC is a potential drug for the treatment ofacute lung inflammation for Queto et al demonstrated

that DEC plays an important role in blocking pulmonaryeosinophilic inflammation in mice sensitized with ovalbu-min effectively preventing the effects of subsequent air-way resistance Th1Th2 cytokine production pulmonaryeosinophil accumulation and eosinophilopoiesis in vivo andex vivo [29] DEC effects have contributed to the reduction ofhepatic abnormalities caused by malnutrition [30] In addi-tion DEC has decreased liver injuries in an experimentalalcoholismmodel and revealed a clinical potential for thera-peutic anti-inflammatory applications [15]

The aim of the present study was to investigate the pro-tective effects of DEC against chronic CCl

4-induced damage

and a possible mechanism for its anti-inflammatory andhepatoprotective activity

2 Materials and Methods

21 Animals Forty male 5-week-old C57BL6 mice weight-ing 15-16 g were used in all experiments The mice wereexamined to determine their health status and acclimatedto the laboratory environment of 23-24∘C They were keptin a 1212 h daynight cycle photoperiod The animals werehoused in metal cages and fed a standard diet with water adlibitumThe animal studies Ethics Committee of the OswaldoCruz Institute approved all the experiments reported hereinunder protocol number 112010

22 Diethylcarbamazine Solutions The solutions were com-posed of distilled water and DEC (FarmanguinhosFIOCRUZ Brazil) and were adjusted in accordance with thebody weight of each animal They were administered bygavage (02mL)The control group (C) only received distilledwater via the same administration route [15 30 31]

23 Experimental Design Chronic inflammation was indu-ced by ip administration of CCl

405120583Lg of body weight

(Sigma-Aldrich St Louis MO USA) dissolved in olive oil(final volume 01mL per mouse) [32] There were two injec-tions a week for 6 weeks DEC (50mgkg) was administeredby gavage for 12 days before finishing the liver injury Thecontrol groups received distilled water via the same routeThe C57BL6 mice were separated into four groups (119899 = 10)(1) the control group (C) which received just distilled water(2) the DEC-treated group (DEC) (3) the CCl

4group (CCl

4)

and (4) the CCl4plus DEC group (CCl

4+ DEC)

24 Histological Analysis Liver fragments were fixed in 10formalin for 24 hours before being processed and embeddedin paraffin [30] Five sections of 4-5 120583m from each groupwerecut and mounted on glass slides The slices were stained withhematoxylin-eosin and examined by an inverted microscope(Observer Z1 Zeiss MicroImaging GmbH) equipped with acamera and 474 image analysis software (AxionCam MRmZeiss) at a magnification of 400x The fibrosis areas werequantified in five random fields on each slide using GIMP 26imaging software [15]

















2


3PO4(solu-






3 Results











4group








Control100120583m

(a)

DEC

(b)

CCl4100 120583m

(c)

CCl4 + DEC100120583m

(d)

0

100

200

300

400

500

c

Fibr

otic

area

()

a b d


(e)












4group











Control

(a)

DEC

(b)

CCl4

(c)

CCl4 + DEC

(d)

0

2000

4000

6000

8000

10000a b d

c

Siriu

s-re

d qu

antifi

catio

n


(e)













4

group (Figure 4(e))



4group




Control

N

M

rER

G

Nu

(a)

NM

VV

CCl4

(b)

rER

M

CCl4

(c)

M

M

CCl4

lowast

(d)

Nu

M

GN

CCl4 + DEC

(e)

M

rER

G

NNu

CCl4 + DEC

(f)






4+ DEC group



4group In the CCl

4+ DEC group






a b d

c0

2000

4000

6000

8000

MD

A q

uant

ifica

tion

(pix

els)


(a)

a b d

c

0

5000

10000

15000

20000


TGF-120573

TGF-120573

quan

tifica

tion

(pix

els)


(b)

00

2000000

4000000

6000000

8000000

c

a b d

COX-

2 qu

antifi

catio

n (p

ixel

s)



(c)

c

a b d

0

50000

100000

150000

200000

COX-

2 qu

antifi

catio

n (p

ixel

s)

COX-2



(d)

0

100000

200000

300000

a b d

c


IL-1120573

quan

tifica

tion

(pix

els)


IL-1120573

(e)

0

10000

20000

30000

40000

50000a b d

c



120572-S

MA

120572-SMA

quan

tifica

tion

(pix

els)

(f)


4group DEC + CCl




4+ DEC group


0

10000

20000

30000

40000a b c

IL-1

0 qu

antifi

catio

n (p

ixel

s)

d


(a)

0

100000

200000

300000

a b d

c

IL-1120573

quan

tifica

tion

(pix

els)


(b)

0

5000

10000

15000

20000

25000 a b d

c

NF-120581

B qu

antifi

catio

n (p

ixel

s)


(c)

0

5000

10000

15000

20000 a b d

c

COX-

2 qu

antifi

catio

n (p

ixel

s)


(d)

0

10000

20000

30000

40000a b d

c

IFN120574

quan

tifica

tion

(pix

els)


(e)

0

10000

20000

30000

40000b d

c

TGF-120573

quan

tifica

tion

(pix

els)


(f)





0

2

4

6

mRN

A re

lativ

e exp

ress

ion

of C

OX-

2 (fo

ld)


lowast

(a)

0

5

10

15

mRN

A re

lativ

e exp

ress

ion

of iN

OS

(fold

)


lowast

(b)

0

50

100

150

lowast

(NO

2minus)

nitr

ites s

erum

leve

l


(c)




4group

















4+ DEC



minus






4 Discussion









4exerts effects on




















4-






















4-caused injury



4[40] Similarly the









4group









Acknowledgments


References
































































































4-













of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
































2


3PO4(solu-






3 Results











4group








Control100120583m

(a)

DEC

(b)

CCl4100 120583m

(c)

CCl4 + DEC100120583m

(d)

0

100

200

300

400

500

c

Fibr

otic

area

()

a b d


(e)












4group











Control

(a)

DEC

(b)

CCl4

(c)

CCl4 + DEC

(d)

0

2000

4000

6000

8000

10000a b d

c

Siriu

s-re

d qu

antifi

catio

n


(e)













4

group (Figure 4(e))



4group




Control

N

M

rER

G

Nu

(a)

NM

VV

CCl4

(b)

rER

M

CCl4

(c)

M

M

CCl4

lowast

(d)

Nu

M

GN

CCl4 + DEC

(e)

M

rER

G

NNu

CCl4 + DEC

(f)






4+ DEC group



4group In the CCl

4+ DEC group






a b d

c0

2000

4000

6000

8000

MD

A q

uant

ifica

tion

(pix

els)


(a)

a b d

c

0

5000

10000

15000

20000


TGF-120573

TGF-120573

quan

tifica

tion

(pix

els)


(b)

00

2000000

4000000

6000000

8000000

c

a b d

COX-

2 qu

antifi

catio

n (p

ixel

s)



(c)

c

a b d

0

50000

100000

150000

200000

COX-

2 qu

antifi

catio

n (p

ixel

s)

COX-2



(d)

0

100000

200000

300000

a b d

c


IL-1120573

quan

tifica

tion

(pix

els)


IL-1120573

(e)

0

10000

20000

30000

40000

50000a b d

c



120572-S

MA

120572-SMA

quan

tifica

tion

(pix

els)

(f)


4group DEC + CCl




4+ DEC group


0

10000

20000

30000

40000a b c

IL-1

0 qu

antifi

catio

n (p

ixel

s)

d


(a)

0

100000

200000

300000

a b d

c

IL-1120573

quan

tifica

tion

(pix

els)


(b)

0

5000

10000

15000

20000

25000 a b d

c

NF-120581

B qu

antifi

catio

n (p

ixel

s)


(c)

0

5000

10000

15000

20000 a b d

c

COX-

2 qu

antifi

catio

n (p

ixel

s)


(d)

0

10000

20000

30000

40000a b d

c

IFN120574

quan

tifica

tion

(pix

els)


(e)

0

10000

20000

30000

40000b d

c

TGF-120573

quan

tifica

tion

(pix

els)


(f)





0

2

4

6

mRN

A re

lativ

e exp

ress

ion

of C

OX-

2 (fo

ld)


lowast

(a)

0

5

10

15

mRN

A re

lativ

e exp

ress

ion

of iN

OS

(fold

)


lowast

(b)

0

50

100

150

lowast

(NO

2minus)

nitr

ites s

erum

leve

l


(c)




4group

















4+ DEC



minus






4 Discussion









4exerts effects on




















4-






















4-caused injury



4[40] Similarly the









4group









Acknowledgments


References
































































































4-













of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control100120583m

(a)

DEC

(b)

CCl4100 120583m

(c)

CCl4 + DEC100120583m

(d)

0

100

200

300

400

500

c

Fibr

otic

area

()

a b d


(e)












4group











Control

(a)

DEC

(b)

CCl4

(c)

CCl4 + DEC

(d)

0

2000

4000

6000

8000

10000a b d

c

Siriu

s-re

d qu

antifi

catio

n


(e)













4

group (Figure 4(e))



4group




Control

N

M

rER

G

Nu

(a)

NM

VV

CCl4

(b)

rER

M

CCl4

(c)

M

M

CCl4

lowast

(d)

Nu

M

GN

CCl4 + DEC

(e)

M

rER

G

NNu

CCl4 + DEC

(f)






4+ DEC group



4group In the CCl

4+ DEC group






a b d

c0

2000

4000

6000

8000

MD

A q

uant

ifica

tion

(pix

els)


(a)

a b d

c

0

5000

10000

15000

20000


TGF-120573

TGF-120573

quan

tifica

tion

(pix

els)


(b)

00

2000000

4000000

6000000

8000000

c

a b d

COX-

2 qu

antifi

catio

n (p

ixel

s)



(c)

c

a b d

0

50000

100000

150000

200000

COX-

2 qu

antifi

catio

n (p

ixel

s)

COX-2



(d)

0

100000

200000

300000

a b d

c


IL-1120573

quan

tifica

tion

(pix

els)


IL-1120573

(e)

0

10000

20000

30000

40000

50000a b d

c



120572-S

MA

120572-SMA

quan

tifica

tion

(pix

els)

(f)


4group DEC + CCl




4+ DEC group


0

10000

20000

30000

40000a b c

IL-1

0 qu

antifi

catio

n (p

ixel

s)

d


(a)

0

100000

200000

300000

a b d

c

IL-1120573

quan

tifica

tion

(pix

els)


(b)

0

5000

10000

15000

20000

25000 a b d

c

NF-120581

B qu

antifi

catio

n (p

ixel

s)


(c)

0

5000

10000

15000

20000 a b d

c

COX-

2 qu

antifi

catio

n (p

ixel

s)


(d)

0

10000

20000

30000

40000a b d

c

IFN120574

quan

tifica

tion

(pix

els)


(e)

0

10000

20000

30000

40000b d

c

TGF-120573

quan

tifica

tion

(pix

els)


(f)





0

2

4

6

mRN

A re

lativ

e exp

ress

ion

of C

OX-

2 (fo

ld)


lowast

(a)

0

5

10

15

mRN

A re

lativ

e exp

ress

ion

of iN

OS

(fold

)


lowast

(b)

0

50

100

150

lowast

(NO

2minus)

nitr

ites s

erum

leve

l


(c)




4group

















4+ DEC



minus






4 Discussion









4exerts effects on




















4-






















4-caused injury



4[40] Similarly the









4group









Acknowledgments


References
































































































4-













of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control

(a)

DEC

(b)

CCl4

(c)

CCl4 + DEC

(d)

0

2000

4000

6000

8000

10000a b d

c

Siriu

s-re

d qu

antifi

catio

n


(e)













4

group (Figure 4(e))



4group




Control

N

M

rER

G

Nu

(a)

NM

VV

CCl4

(b)

rER

M

CCl4

(c)

M

M

CCl4

lowast

(d)

Nu

M

GN

CCl4 + DEC

(e)

M

rER

G

NNu

CCl4 + DEC

(f)






4+ DEC group



4group In the CCl

4+ DEC group






a b d

c0

2000

4000

6000

8000

MD

A q

uant

ifica

tion

(pix

els)


(a)

a b d

c

0

5000

10000

15000

20000


TGF-120573

TGF-120573

quan

tifica

tion

(pix

els)


(b)

00

2000000

4000000

6000000

8000000

c

a b d

COX-

2 qu

antifi

catio

n (p

ixel

s)



(c)

c

a b d

0

50000

100000

150000

200000

COX-

2 qu

antifi

catio

n (p

ixel

s)

COX-2



(d)

0

100000

200000

300000

a b d

c


IL-1120573

quan

tifica

tion

(pix

els)


IL-1120573

(e)

0

10000

20000

30000

40000

50000a b d

c



120572-S

MA

120572-SMA

quan

tifica

tion

(pix

els)

(f)


4group DEC + CCl




4+ DEC group


0

10000

20000

30000

40000a b c

IL-1

0 qu

antifi

catio

n (p

ixel

s)

d


(a)

0

100000

200000

300000

a b d

c

IL-1120573

quan

tifica

tion

(pix

els)


(b)

0

5000

10000

15000

20000

25000 a b d

c

NF-120581

B qu

antifi

catio

n (p

ixel

s)


(c)

0

5000

10000

15000

20000 a b d

c

COX-

2 qu

antifi

catio

n (p

ixel

s)


(d)

0

10000

20000

30000

40000a b d

c

IFN120574

quan

tifica

tion

(pix

els)


(e)

0

10000

20000

30000

40000b d

c

TGF-120573

quan

tifica

tion

(pix

els)


(f)





0

2

4

6

mRN

A re

lativ

e exp

ress

ion

of C

OX-

2 (fo

ld)


lowast

(a)

0

5

10

15

mRN

A re

lativ

e exp

ress

ion

of iN

OS

(fold

)


lowast

(b)

0

50

100

150

lowast

(NO

2minus)

nitr

ites s

erum

leve

l


(c)




4group

















4+ DEC



minus






4 Discussion









4exerts effects on




















4-






















4-caused injury



4[40] Similarly the









4group









Acknowledgments


References
































































































4-













of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control

N

M

rER

G

Nu

(a)

NM

VV

CCl4

(b)

rER

M

CCl4

(c)

M

M

CCl4

lowast

(d)

Nu

M

GN

CCl4 + DEC

(e)

M

rER

G

NNu

CCl4 + DEC

(f)






4+ DEC group



4group In the CCl

4+ DEC group






a b d

c0

2000

4000

6000

8000

MD

A q

uant

ifica

tion

(pix

els)


(a)

a b d

c

0

5000

10000

15000

20000


TGF-120573

TGF-120573

quan

tifica

tion

(pix

els)


(b)

00

2000000

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