b 1 and bFGF) in mouse embryonic fi broblast cell: In vitro study The effect of aloe vera on the expression of wound healing factors(TGF

Original article

The effect of aloe vera on the expression of wound healing factors (TGF b 1 and bFGF) in mouse embryonic fi broblast cell: In vitro study

Maryam Hormozi

^a,b

, Raheleh Assaei

^c

, Mandana Beigi Boroujeni

^d,

*

aRaziHerbalResearchesCenter,LorestanUniversityofMedicalSciences,Khoramabad,Iran

bDepartmentofBiochemistry,LorestanUniversityofMedicalScience,Khorramabad,Iran

cDepartmentofPhysiology,LorestanUniversityofMedicalScience,Khorramabad,Iran

dDepartmentofAnatomicalSciences,LorestanUniversityofMedicalScience,Khorramabad,Iran

ARTICLE INFO Articlehistory:

Received23October2016

Receivedinrevisedform11January2017 Accepted16January2017

Keywords:

Fibroblast Aloevera(A.v) bFGF TGFb¹

ABSTRACT

Background:Aloevera(A.v)havebeenusedtraditionallyfortopicaltreatmentofwoundsandburnsin differentcountriesforcenturies,butthemechanismofthiseffectisnotwellunderstood.Variousgrowth factorsareimplicatedintheprocessofwoundhealing.Amongthedifferentgrowthfactorsinvolvedin theprocess,TGFb^{1andbFGFarethemost}importantlyexpressedinfibroblastcells.Theaimofthisstudy wastoevaluatetheeffectofA.vontheexpressionofangiogenesisgrowthfactorsinmouseembryonic fibroblastcells.

Methods:WeexposedmouseembryonicfibroblastcellstodifferentconcentrationsofA.v(50,100and 150m^{g/ml)attwodifferenttimeof12and24h.FibroblastcellwithoutA.vtreatmentservesasthe} control.TheexpressionofTGFb^{1andbFGFwasmeasuredbyreal}time-polymerasechainreaction(real- time-PCR)andenzyme-linkedimmunosorbentassay(ELISA)atthelevelofgeneandprotein.

Results:WeobservedthatA.vgelatfirstup-regulatedtheexpressionofTGFb^{1andbFGF,but,thesegenes} werelaterrepressedafteraparticulartime.

Conclusion: Our results demonstrated that A.v was dose-dependent and time-dependent on the expression ofbFGFandTGFb^{1 in}fibroblastcell invitro.This mechanismcanbeemployedin the prospectivetreatmentofphysicallesion.

©2017ElsevierMassonSAS.Allrightsreserved.

1.Introduction

Woundhealingisahighlycomplicatedprocess,comprisingof cascadeof healingevents. Thenormal processof woundrepair consistsof fouroverlappingphaseswitha predictableseriesof biochemicalandcellularevent;hemostasis,inflammation,tissue formation(theproliferativephase),andfinallytissueremodeling [1–4]. These processes are affected by several factors such as cytokines,growthfactorsandlow-weightmolecularcompounds.

Angiogenesis is important in many processes such as wound healing, kidney function, fetal development, reproduction and fertilitypreservation[5,6].Woundhealingisaresponsetoinjured tissue,resultingintherestorationoftissueintegrity.

Severalinvestigationsshowthatgrowthfactorsplayacrucial roleincelldivision,migration,differentiation,enzymeproduction, andproteinexpression.Thesefactorscanberesponsibleforwound healing through the stimulation of angiogenesis and cellular

proliferation. This in-turn affects both the production and degradationof theextracellular matrix(ECM), and chemotactic fortherecruitmentofinflammatorycellsandfibroblasts[7].

Twokeygrowthfactorsareimplicatedintheprocessofwound healing, these are basic fibroblast growth factor (bFGF) and transforming growth factor

b

^1(TGF

b

^{1) [8]. The later control}

proliferationoffibroblast,transformationintomyofibroblasts,the production of ECM, stimulation of collagen production, elastin production, and fibronectin synthesis while inhibiting ECM degradation[9].bFGFisalsoimplicatedintheprocessofwound healing.Itiscapableofregulatingthereplicationandmigrationof epithelial, endothelial, and fibroblasts cells, which partake in collagen production, epithelialization and neovascularization respectively[10].

TheA.vplantisknownas“thehealingplant”.Thisplanthas beentraditionallyusedtotreatwoundsandburns[11].Theoral and topical administration of Av gel has been reported to be effectivebothinnormalanddiabeticwounds[12,13].Itcontains severalmedicinalpropertiessuchaswoundhealing,promotionof radiationdamagerepair,anti-inflammatoryeffects,anti-bacterial, anti-viral,anti-fungal,anti-diabeticandantineoplasticactivities,

*Correspondingauthor.

E-mailaddress:a-eatemadi@razi.tums.ac.ir(M.B.Boroujeni).

http://dx.doi.org/10.1016/j.biopha.2017.01.095

0753-3322/©2017ElsevierMassonSAS.Allrightsreserved.

Availableonlineat

ScienceDirect

www.sciencedirect.com

immunostimulation stimulation of hematopoiesis and anti-oxi- danteffects [14].StudiessuggestthattreatmentwitheitherA.v crude gel or its extracted components like

b

-sitosterol, ace- mannan, etc, resulted in faster wound healing through the stimulationofgrowthfactorproduction,angiogenesis,prolifera- tionoffibroblastandcollagendepositionandproductionofgrowth factors[3,15].A.vgelwasshowntoamelioratewoundhealingafter systemicandtopicaladministrationinseveralstudies,inspiteof allthestudiesonA.v,themechanismbywhichitcarriesoutits effectisyettobeunderstood[16].

Previous in vitro study by Jettanacheawchankit et al. was carriedoutonacemannan(polysaccharidefromA.v),todemon- strateitseffectontype Icollagen,keratinocytegrowth factor-1 (KGF-1)andVEGFproduction[17].InvivoinvestigationsusingA.v treatmentonwoundhealingmechanismhasbeencarriedoutas well.A.v

b

-sitosterol improves angiogenesistriggeredby VEGF, blood vessel matrix laminin,Von Willebrand factors and VEGF receptor[18,19].Yet,thesestudieswascarriedoutonchickembryo chorioallantoicmembraneassay.Inadditiontothis,anotherstudy by Atiba et al. [20] have demonstrated the effect of oral administration of A.v on cutaneous wound healing, analyzing VEGFandTGF-1

b

expression,inatype2diabeticratmodel.This was an in vivo study as well and bFGF expression was not determined.Inourstudy,weaimatinvestigatingtheeffectofA.v on the expression of bFGF and TGF

b

^{1 in mouse embryonic}

fibroblastcellsatthelevelofgeneandproteininvitro.

2.Materialandmethods

ThestudywasdesignedtocompareTGF

b

^{1andbFGFexpression}

in mouse embryonic fibroblasts cells, treated by different concentrationsof A.v.Theconcentrationofourextractin these studyare(50,100and150

m

^{g/ml),andthecontrolofourstudyis}

thefibroblastcellwithouttheactiveingredientat(12and 24h aftertreatment).

2.1.A.vgelseparation

Freshly harvested A.v leaves were thoroughly washed with sterilewater,anditsskinwaspeeledoffusingasterilecondition.

The inner gel was collected and frozen at 80C, and was subsequentlylyophilizedandstoredat 20Cuntilfurtheruse.

2.2.Isolationandcultureofmouseembryonicfibroblasts(MEFs)

In order to isolated the mouse embryonic fibroblast, we performed the following steps according to the protocol of Jozefczuketal.[21].Thefirststepwastoanesthetizeandsacrifice apregnantmouseat13or14d.p.c(daypost-coitum)bydislocating ofcervicalvertebrae.Afterthat,theuterinehornswasdissected out,brieflyrinsedin70%(v/v)ethanolandplacedinafalcontube alongwiththebufferPBSwithoutcalciumandmagnesiumions.

Thefollowingstepswereperformedunderasepticconditionswith sterileinstrumentsinatissueculturehood.First,theuterinehorns wasplacedinpetridish,andeachitsembryowasseparatedfrom itsembryonic sacand placenta.Headand thered organs were dissectedandwashedinPBSbuffer,then,allembryoswereplaced inasterilepetridish.Thetissueisthenmincedusinganintact razorbladeuntilwecouldpipetteit.Then,1mlof0.05%trpsin/

EDTA(Gibco,Invitrogen)containing100KunitsofDNaseIpereach embryowasadded.Afterthat,thetissuesweretransferredtoa 50mlfalcontube,incubatedfor15minattheroomtemperature.

Followingeach5minofincubationthecellsweredissociatedby pipetting thoroughly.In thenext stage,trypsinwas inactivated throughtheadditionof1voloffreshlypreparedMEFmedium;a culture medium (components to make 500ml of media, all

componentsand filterswasmixed) which consistsof 450ml of DMEM,50mlofFBS(10%(v/v)),5mlofPenicillin-streptomycin(1/

100(v/v))and5mlof200mML-glutamine(1/100(v/v)).Thecells are centrifuged with low speed (300g) for 5min. Then the supernatant was discarded, and cell pellet was in warm MEF medium. Afterthat, weplatedapproximatelya numberofcells equivalent to3–4 embryosineach T150(TPP) flask whichwas coatedwith0.2%ofbovinegelatin(Gelatinfrombovineskin,type B,Sigma)for2h.Atthistime,thefibroblasts(P0,passage0)were the onlycells which wereable toadheretothe gelatin-coated flasks.Ideally,thecellswereconfluentabout80–90%after24hand atthislevel,amajorityofP0cellswerefrozenforfutureusage.At theendoftheprocedure,theremainingT150flasksP0cellswere storedforfurtherstudy.

2.3.MTTassayforcellviabilitydetermination

ColorimetricMTTassaywasperformedtoassesscellviability.

Briefly,100

m

^l/wellfibroblastscells(10⁴cellpereachwell)were addedinto96-wellplatesandallowedtoadherefor24h.Itwas incubatedwithdifferentconcentrationsofA.vinwatermedium(0, 50,100 and 150

m

^{g/ml) for 24, 48and 72h. Atthe end of the}

treatment, 20

m

^{l of MTT (5mg/ml, Sigma) in PBS solutionwas}

added into each of the well, and then the plate was further incubatedfor4h.Theremainingsupernatantswereremovedand 200

m

^{lofDMSOwasaddedintoeachwellandthoroughlymixedto}

dissolvetheformedcrystalformazan.Afterincubatingfor15min toensureallcrystalshavebeendissolved,thelightabsorptionwas measured by using an enzyme-linked immunosorbent assay (ELISA) reader. Viability was expressed as a percentage of absorbancevaluesintreatedcellstothatincontrolcells.

2.4.AnalysisofTGF

b

^{1andbFGFgeneexpressionbyrealtime-PCR}

Mouseembryonicfibroblastcellsweregrownin6-wellplates (10⁵cellpereachwell).Cellswereexposedtodifferentconcen- trationsofA.v(0,50, 100and150

m

^{g/ml)andcellswerecollectedat}

12and 24hbyprocessoftrypsin/EDTA.TotalRNAwas isolated fromthecellsusingtheTotalRNAPurificationKit(JenaBioscience, Germany)accordingtotheinstructionofthemanufacturer’s.All RNApreparationandhandlingstepsweredoneunderRNAse-free conditions.TheconcentrationandpurityofRNAwasdetermined using biophotometer (Eppendorf, Hamburg, Germany). The concentrationandqualityoftheRNAsampleswerelaterconfirmed by electrophoresis on denaturated 1% agarose gel. cDNA was synthesized from 1

m

^{gtotal RNAusing the cDNASynthesis Kit}

(Roche Diagnostics GmbH, Mannheim, Germany). The house keeping gene for normalization was HPRT. The oligonucleotide sequencesoftheprimersarepresentedinTable1.

Real-time quantitative PCR was performed with Rotor-Gene 6000 real time PCR system and SYBR-Green quantitative PCR (qPCR)kit(JenaBioscience).TheqPCRreactionwaspreparedina totalvolumeof20

m

^{lcontaining10}

m

^{lof2XSYBRGreenmaster}

Table1

SequencesofprimersforReal-timequantitativePCR.

Gene Primer Productsize Tm

HPRT Sense:CCTCCTCAGACCGCTTTTT 91 79.5

Antisense:AACCTGGTTCATCATCGCTAA

FGF2 Sense:AACGGCGGCTTCTTCCTG 133 78.9

Antisense:TGGCACACACTCCCTTGATAG

TGFb1 Sense:ATTCCTGGCGTTACCTTGG 117 76.9

Antisense:CCTGTATTCCGTCTCCTTGG

mix,1.6

m

^{lofthecDNAtemplate,0.6}

m

^{lofeachprimer(10pmol/}

m

^l)and7.2

m

^{lofdeionizedwater.Anegativecontrolwasusedby}

replacing the cDNA template with deionized water. Primer sequencesused in this study and their annealing temperature areshowninTable1.The PCRamplification startedwithinitial denaturation and polymerase activation at 95C for 2min, followedby40cyclesofdenaturationat95Cfor15s,annealing at60Cfor40sandextensionat72Cfor30s.Thespecificityof PCRproductswasverifiedbymeltingcurvesandelectrophoresis through1%agarosegel.

2.5.AnalysisofTGF

b

^{1andbFGFproteinexpressionbyELISA}

Mouseembryonicfibroblastcellsweregrownin6-wellplates (10⁵cellpereachwell),cellswereexposedtoA.vat0,50,100and 150

m

^{g/ml, and its} supernatant were collected at 12 and 24h.

Sampleswerefreezedat 20C.TGF-

b

^{1andbFGFlevelsfromcell}

culturesupernatantsweremeasuredbyELISAkit(TGF-

b

^1ELISA

kit,eBiosciences,bFGFELISAkit,RayBiotech,Inc)accordingtothe instructionofthemanufacturer’s.

TomeasureTGF-

b

^1levels,theReady-Set-GOTGF-

b

^1cytokine

ELISAkitfromeBioscience(SanDiego,CA,USA)Human/MouseTGF beta1 ELISA Ready-SET-Go (Catalog Number: 88–8350) were utilized.Briefly, itwasfilledona96-wellplatesovernight with a suitable dilution for capturing antibody in 0.2M sodium phosphatebufferwithpH6.5.Theplatewaswashedtreetimes withPBS plus 0.05%Tween-20 (PBS-T),and then blocked with AssayDiluentat21Cfor1h.InordertoinactivatelatentTGF-

b

^1,

thesamplesweretreatedwith20

m

^{lsolutionof1NHClper100}

m

^l

ofsamplefor 10minand afterthatneutralizedwith1N NaOH.

Then,100

m

^{lofsampleorcytokinespreparedinassaydiluentwere}

addedandincubatedat21Cfor2h.Afterfivetimesofwashing withPBS-T,100

m

^{lofassaydiluentcontaining}biotin-conjugated detectingantibodyandavidin-horseradishperoxidasereagentat thesuitabledilutionswereaddedandincubatedat21Cfor1h.

AfterseventimesofwashingwithPBS-T,100

m

^{lofthesubstrate}

solution was added to each well plate. Following 30min of incubationindarkcondition,50

m

^{lofthestopsolution(2NH}2SO₄) wereadded.TheELISAresultwerereadwithin30minbyusingan enzyme-linked immunosorbent assay (ELISA) reader. The final calculationwasperformedusingadilutionfactorof1.4inorderto investigateacidactivation/neutralization.

Mouse bFGF release in the supernatant was detected with mousebasicFGFELISAkit(Catno.ELH-bFGF-001;RayBiotech,Inc., St.Louis,MO).Asabriefdescription, 100

m

^{lwereaddedtoeachcell}

culturesupernatantintoappropriatewells.Thewellswerecovered andincubatedatroomtemperaturefor2.5h.Then, thesolution wasdiscardedandwashed4timeswith200

m

^{lofwashsolution}

(1) for each well. Then, 100

m

^{l of 1x prepared} biotinylated antibodywereaddedintoeachwell.Theplatewasincubatedatthe room temperature for one hour. Repeatedly, the solution was discardedandwashed4timeswith1200

m

^{lofwashsolutionfor}

each well. After the washing, we added 100

m

^{l of prepared}

streptavidin solution into each well and waited for 45min for incubationattheroomtemperature.Thesolutionwasdiscarded onceagain,andwashedfor5timeswith1200

m

^{lofsashsolution}

for each well. Then after, we added 100

m

^{l of TMB One-Step}

Substrate Reagent to each well and incubated at the room temperature for 30min in dark condition. Finally we added 50

m

^{l ofstop solutionand read} immediately atthe absorbance 450nm.TheresultswereexpressedasbFGFlevelinpgpermlof cellculturesupernatant.

2.6.BlockingofTGF

b

^1activity

TosystemicallyinhibitTGF

b

^{1function,samplesfromanalysis}

of TGF

b

^{1 gene and proteinexpression by RealTime-PCR. They}

weretreatedusingamonoclonalantibody(mab)againstmouse TGF

b

^{1(TGFmab;5mg/kgdilutedin150}

m

^{lofPBS),clone1D11}

(Bio-x-cell,West Lebanon,NH) after24hof A.vadministration.

Thisantibodyhasbeenemployedinseveralstudiesandhasbeen showntobeefficientinblockingsignalinginvivoofallTGF

b

¹

isoforms[22].Thesampleinthecontrolgroupweretreatedwitha similardoseof isotypecontrolmab.Theexpression ofTGF

b

¹

proteinwasre-analysedaccordingtothepreviousprotocol.

2.7.Statisticalanalysis

Our result are fromdifferent independent experiments, the total expressionratio ofthegenesof interestatthree different concentrations was compared between Av and control groups usingarandomizationtestimplementedintherelativeexpression software tool (REST), which is an Excel-based application for comparisonandstatisticalanalysisofrelativeexpressionresultsin qRT-PCR[23].Proteinexpressioninthetwogroupswerecompared

Fig.1.EffectofvariousconcentrationsofA.vonmouseembryonicfibroblastcellviability.CellsweretreatedwithdifferentconcentrationofA.vfor24,48and72handcell viabilitywasmeasuredbyMTTassay.

bytheStatisticalPackagefortheSocialScience(SPSS,Cary,NC).

DifferenceswereconsideredsignificantatP<0.05.

3.Results

3.1.EffectofA.vonmouseembryonicfibroblastcellviability

TheresultsofMTTassayshowedthatA.vgelwasnon-toxicto thecells(Fig.1).SomestudieshaveshowedthatA.vhadnotoxic effectonmacrophagesandperipheralbloodmononuclearcellsup toaconcentrationof100

m

^g/ml.

3.2.TGF

b

^{1andbFGFgeneexpression}

AnalysisofTGF

b

^{1geneexpressionbyreal-timeRT-PCRshowed}

an opposite patterns at the two study time. There was no significantdifferencebetween0

m

^g/mland50

m

^{g/mlatbothtime}

point, however,there wasa significantincrease in this geneat different concentrations of 100 and 150

m

^{g/ml at 12h after}

treatmentbyA.vascomparedtothecontrol(P=0.001,P=0.001 and P=0.001 respectively). But the expression of TGF

b

^{1 gene}

decreased at 24h after treatment which was significant at a concentrationof150

m

^{g/ml(Fig.2,Table2),showingnosigni}ficant differenceateverypoint.

Gene expression of bFGF in cells treated with A.v showed similar patterns with TGF

b

^{1 gene} expression. There was no significantdifferencebetween0

m

^g/mland50

m

^{g/mlatbothtime}

point, however,there wasa significantincrease in this geneat different concentrations of 100 and 150

m

^{g/ml at 12h after}

treatmentbyA.vascomparedtothecontrol(P=0.001,P=0.001 and P=0.001 respectively). But the expression of bFGF gene decreased at 24h after treatment which was significant at a concentrationof150

m

^{g/ml,showingnosigni}ficantdifferenceat everypoint(Fig.3,Table3).

3.3.TGF

b

^{1andbFGFproteinexpression}

ThepatternofTGF

b

^{1andbFGFprotein}expression,weresimilar tothoseofthegenesexpression,butnostrictconsistencyingene expression. The increase in TGF

b

^{1 protein expression was}

significantat the three concentrations studied at 12h, but the decreaseinproteinexpression24haftertreatmentwassignificant

at 100 and 150

m

^{g/ml (Fig. 4). bFGF protein expression was}

increasedsignificantly12haftertreatmentatallconcentrationsof studybutdecreaseofproteinexpression24haftertreatmentwas significantatallconcentrationsofstudy(Fig.5).

3.4.TGF

b

^{1blockingandexpression}

TheblockingofTGF

b

^{1forgeneandproteinexpressionat24h}

wasshownin(Fig.6),itwasabletoinhibitoverproduction.From thegraph,itwasobservedthat,theantibodyblockingwasableto reverse the inhibition of the expression at 24h which was significant at the concentrations studied. The decrease of TGF

b

^{1at24hwasduetofeedbackmechanism(P=0.001).}

4.Discussion

The present investigation shows the effect of A.v on the expressionofbFGFandTGF

b

^{1inmouseembryonic}fibroblastcells atthelevelofgeneandproteininvitro,whichcanbeasaresultof improvedfibroblastsandendothelialcellsstimulatedbybFGFand TGF-

b

^1migration.

Previous studies suggests that A.v active components like acemannan can bind special ligands to mannose receptors presentedonthecellsurfaceoffibroblastsandmacrophages,this binding results in cells triggering the growth factors and proliferation[24].

Inthepresentstudy,MTTanalysisofA.vshowedthattheplant was not toxic atthe analysedconcentrations in the study,this result is similar to that reviewed by Reynolds et al., [25] and researchstudybyGopakumaretal.,[26].

ThemainfindingofourstudywasthatA.vgelatfirst(12h)up- regulatedtheexpressionofTGF

b

^{1andbFGF,butaftersometime}

(24h), the expression of these genes were down-regulated.

Analysis of gene and protein expression by real-time RT-PCR showedasignificantlyincreasesatdifferentconcentrationsforthe first12haftertreatmentwithA.vascomparedwiththecontrol.

However, theseexpressionwas decreased 24haftertreatment, whichwassignificantatsomeconcentrationsoftreatmentwithA.

v for both TGF

b

^{1 and bFGF. The mechanism of by which A.v}

stimulatestheproductionofgrowthfactorandfibroblastis still unclear.However,inthisstudy,thedown-regulationofTGF

b

^1at

24h might be due to feedback mechanism inhibiting

** ** ** **

**

-1 0 1 2 3 4 5

Control 50μg/ml 100μg/ml 150μg/ml

Relave expression

Aloe vera concentraon

12h 24h

Fig.2.RelativeexpressionofTGFb^{1geneinmouseembryonicfibroblastcell,treatedwithvarious}concentrationsofA.vascomparedwiththecontrolgroupatdifferenttime intervals(12and24h).MouseembryonicfibroblastcellsweretreatedwithA.vextracts(50,100and150mg/ml)andtheexpressionofTGFb1genewasassessedby quantitativerealtimePCR.Allcomparisonsanalysedwiththecontrolgroup.P<0.05,P<0.01.

Table2

TheeffectofdifferentconcentrationsofAloeveraonexpressionofTGFb1gene.

12haftertreatment 24haftertreatment

10mg/ml 20mg/ml 30mg/ml 10mg/ml 20mg/ml 30mg/ml

Relativeexpression 2.72 2.84 3.12 1.36 0.70 0.64

Standarderror 0.48 0.53 0.67 0.36 0.26 0.17

P-value 0.001 0.001 0.001 0.32 0.32 0.001

Foldincrease/decrease +2.72 +2.84 +3.12 +1.36 1.43 1.56

Fig.3.RelativeexpressionofbFGFgeneinmouseembryonicfibroblastcellstreatedwithvariousconcentrationsofA.vascomparedwiththecontrolgroup,atdifferenttime intervals(12and24h).MouseembryonicfibroblastcellsweretreatedwithA.v(50, 100and150m^{g/ml)andtheexpressionofbFGFgenewasassessedby}quantitativerealtime PCR.Allcomparisonsweremadewiththecontrolgroup.P<0.05,P<0.01.

Table3

TheeffectofdifferentconcentrationsofAloeveraonexpressionofbFGFgene.

12haftertreatment 24haftertreatment

10mg/ml 20mg/ml 30mg/ml 10mg/ml 20mg/ml 30mg/ml

Relativeexpression 3.12 3.96 4.46 0.64 0.50 0.45

Standarderror 0.45 0.53 0.68 0.21 0.17 0.18

P-value 0.001 0.001 0.001 0.32 0.001 0.001

Foldincrease/decrease +3.12 +3.96 +4.46 1.56 1.98 2.23

Fig.4.EffectofvariousconcentrationsofA.vontheexpressionofTGFb^{1proteininmouseembryonicfibroblastcellculture}supernatants.Cellsweretreatedwithdifferent concentrationofA.v(50,100and150mg/ml)atdifferenttimeintervalstreatment(12and24h)andtheexpressionofTGFb1proteinwasassessedbyELISA.P<0.05,P<0.01.

overproductionofthesefactors,andtheresultofourexperimentin Fig.6showsthat,blockingofTGF

b

¹over-productionreversedthe inhibitionandshowsasharpexpressionofthegeneparticularlyat thehighestconcentration.

Despitetheimportantroleoffibroblasts,TGF

b

^1andbFGFin

woundhealing,ithasbeenshownthattheup-regulationofTGF-

b

¹consistentlyleadtofibroticdisease,blockingitsbioactivitymay inhibittheproductionofmatrixandmodulatethefibroticprocess [27].TGF

b

^{1doesnotonlyincrease}transcriptionofcollagen,but also decreases its degradation through inhibition of collagen activitybyincreasingproductionofMMPinhibitorlikeplasmino- gen activator inhibitor and TIMP. A similar results were also obtainedfromotherstudiesinhumanperitonealfibroblasts[28].

TheresultsofthisstudyshowedthatA.Veracanincreasethe productionofTGF

b

^{1andbFGF,whichwillstimulatethecollagen}

deposition, fibroblast proliferation, and angiogenesis, but after that,down-regulationofthesefactorscaninhibitoverproduction and accumulationof matrix proteins which causehypertrophic scar.

5.Conclusion

OurstudydemonstratedA.vwouldprovideasystemiceffecton fibroblast cells in vitro via growth factors production and angiogenesis.Our investigationopensanavenueforprospective research and application of this plant extract on ameliorating delayedwoundhealingproblems.However,furtherstudystillis requiredtoconfirmthisspeculation.

Conflictofinterest

Thereisnoconflictofinterestinthispaper.

Acknowledgment

ThisworkwassupportedbyagrantfromLorestanUniversityof MedicalScience,Korramabad,Iranwithgrantnumber100/394242.

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**

**

**

* * *

-10 0 10 20 30 40 50 60

Control 50μg/ml 100μg/ml 150μg/ml

bFGF (pg/ml)

Aloe vera concentraon 12h 24h

Fig.5.EffectofvariousconcentrationsofA.vontheexpressionofbFGFproteininmouseembryonicfibroblastcellculturesupernatants.Cellsweretreatedwithdifferent concentrationofA.v(50,100and150m^{g/ml)atdifferenttimeintervalstreatment(12and24h)andtheexpressionofbFGFproteinwasassessedbyELISA.P}<0.05,P<0.01.

*

**

** **

0 5 10 15 20 25 30 35 40 45 50

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Aloe vera concentraon

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