Role of TRPV1 receptor in inflammation and impairment of esophageal mucosal integrity in a murine model of nonerosive reflux disease

Abstract

Background
Microscopic inflammation and impairment of the esophageal epithelial barrier are considered relevant for perception of symptoms in patients with nonerosive reflux disease (NERD). In these patients, the receptor transient receptor potential vanilloid 1 (TRPV1) is overexpressed in the esophageal mucosa, but its role is not yet fully understood. We evaluated the role of TRPV1 in esophageal inflammation and mucosal barrier impairment in a murine model of NERD.

Methods
Nonerosive reflux disease was surgically induced in Swiss mice by pyloric substenosis and ligature of the gastric fundus, and the mice were killed 7 days post surgery. The experimental groups were: I, sham surgery (negative control); II, NERD untreated; III and IV, NERD + SB366791 or capsazepine (TRPV1 antagonists); and V, NERD + resiniferatoxin (for long‐term desensitization of TRPV1). The esophagus was collected for western blotting and histopathology and for evaluation of wet weight, myeloperoxidase (MPO), keratinocyte‐derived chemokine (KC), transepithelial electrical resistance (TEER), and basal permeability to fluorescein.

Key Results
Compared to sham, NERD mice had increased esophageal wet weight and MPO and KC levels. The mucosa had no ulcers but exhibited inflammation. NERD mice showed mucosal TRPV1 overexpression, a more pronounced decrease in TEER at pH 0.5 (containing pepsin and taurodeoxycholic acid), and increased basal permeability. Pharmacological modulation of TRPV1 prevented esophageal inflammation development, TEER changes by acidic exposure, and increase in esophageal permeability.

Conclusions & Inferences
The TRPV1 receptor has a critical role in esophageal inflammation and mucosal barrier impairment in NERD mice, suggesting that TRPV1 might be a pharmacological target in patients with NERD.

Full text

Neurogastroenterology & Motility. 2018;30:e133 40. wileyonlinelibrary.com/journal/nmo 
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https://doi.org/10.1111/nmo.13340
© 2018 John Wiley & Sons Ltd
Received:24September2017
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Accepted:15February2018
DOI : 10.1111 /nmo .133 40
ORIGINAL ARTICLE
Role of TRPV1 receptor in inflammation and impairment of
esophageal mucosal integrity in a murine model of nonerosive
reflux disease
R. O. Silva1 | R. D. Bingana1 | T. M. A. L. Sales2 | R. L. R. Moreira1 | D. V. S. Costa3 |
K. M. O. Sales2 | G. A. C. Brito3 | A. A. Santos1,2 | M. Â. N. Souza2 | P. M. G.
Soares1,3 | D. Sifrim4 | M. H. L. P. Souza1,2
Abbreviations:ANOVA,ana lysisofv arianc e;DIS,di latedint ercell ularspa ces;GER D,gastr oesop hagealr efluxdi sease;H &E,hem atoxyl inandeo sin;KC,ke ratino cyte -deriv edchemo kine;
KHB B,Kr ebs- Hen sele itbi car bona tebu ffe r;NE RD,n on-e rosi vere flux dise ase; TDC A,t auro deo xyc holi caci d;TEE R,t rans epi the liale lec tri calr esis tan ce;T RPV1 ,tra nsi entre cep torpo ten-
tial vanilloid member.
1Depar tmentofPhysiolog yand
Pharma cology,FederalUniversit yofCeará,
Fortaleza,Ceará,Brazil
2Depar tmentofClinicalM edicine,Federa l
UniversityofCeará,For talez a,Ceará,Brazil
3Depar tmentofMorphology,Federal
UniversityofCeará,For talez a,Ceará,Brazil
4Bart sandtheLondonSchoolofMedicine
andDent istry,QueenMar yUniver sityof
London,London,UK
Correspondence
MarcellusH.L.P.Souza ,Instituteof
Biomedicine,Facu ltyofMedicine,Fe deral
UniversityofCeará,For talez a,Ceará,Brazil.
Emails:souzamar@ufc.br;souzamar.ufc@
gmail.com
Funding information
Thisworkw assupportedbyNational
CouncilofTechnologicalandScientifi c
Develop ment(CNPq ,Brazil),grantnumber
400752/2013-1.
Abstract
Background:Microscopicinflammationandimpairmentoftheesophagealepithelial
barrierareconsideredrelevantforperceptionofsymptomsinpatientswithnonero-
siverefluxdisease(NERD).Inthesepatients,thereceptortransientreceptorpoten-
tialvanilloid1(TRPV1)isoverexpressedintheesophagealmucosa,butitsroleisnot
yetfully understood. We evaluatedthe role of TRPV1inesophageal inflammation
andmucosalbarrierimpairmentinamurinemodelofNERD.
Methods:NonerosiverefluxdiseasewassurgicallyinducedinSwissmicebypyloric
substenosis andligatureofthe gastricfundus,andthe micewerekilled7dayspost
surgery.Theexperimentalgroupswere:I,shamsurgery(negativecontrol);II,NERD
untreated;IIIandIV,NERD+SB366791orcapsazepine(TRPV1antagonists);andV,
NERD+resiniferatoxin(forlong-termdesensitizationofTRPV1).Theesophaguswas
collectedforwesternblottingandhistopathologyandforevaluationofwetweight,
myeloperoxidase(MPO),keratinocyte-derivedchemokine(KC),transepithelialelec-
tricalresistance(TEER),andbasalpermeabilitytofluorescein.
Key Results:Comparedtosham, NERD micehad increasedesophagealwet weight
andMPOandKClevels.Themucosahadnoulcersbutexhibitedinflammation.NERD
miceshowedmucosalTRPV1overexpression,amorepronounceddecreaseinTEERat
pH0.5(containingpepsinandtaurodeoxycholicacid),andincreasedbasalpermeabil-
ity.PharmacologicalmodulationofTRPV1preventedesophagealinflammationdevel-
opment,TEERchangesbyacidicexposure,andincreaseinesophagealpermeability.
Conclusions & Inferences:The TRPV1 receptor has acriticalrolein esophagealin-
flammation andmucosal barrierimpairmentinNERDmice,suggestingthat TRPV1
mightbeapharmacologicaltargetinpatientswithNERD.
KEYWORDS
inflammation,mucosalintegrity,non-erosiverefluxdisease,TRPV1

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1 | INTRODUCTION
Nonerosiverefluxdisease (NERD)isrecognized asthemostpreva-
lentphenotypeofgastroesophagealrefluxdisease(GERD).Itispri-
marily characterized bytypicalrefluxsymptoms (ie, heartburn) and
absence of m acroscopic da mage or visible in flammation in e ndos-
copy.1,2However,NERDpathogenesisisnotcompletelyelucidated.
Theesophagealmucosal basal layer ofpatients with NERD ischar-
acteri zed by dilated inte rcellular sp aces (DIS). Es ophageal m ucosal
integrityisalsofunctionallyimpairedinNERD.3Understandingthe
mechanism involved inim paired mucosal integrity in NERDis im-
port ant for developi ng new therap eutic strat egies to improve th e
clinicaloutcomesinrefractoryrefluxdiseases.
Recently, we est ablished a m urine mode l of NERD that repro -
ducedimportantaspect sof NERDpathophysiology,suchasmicro-
scopic inflammation without erosions, which are associated with
esophageal mucosal integrity impairment.4 In accordance with previ-
ousreports,3,5 we demonstrated that gastric secretion inhibition by
omeprazole,atleastpartially,decreases inflammationandmucosal
integrityimpairmentinourNERDmodel.4Themechanismunderly-
ingacid-inducedmucosalchangesinNERDisnotcompletelyclear.
One possibility is the involvement of acid-activated molecules,
such as transient receptor potential vanilloid 1 (TRPV1). TRPV1
overexpression is observed in esophageal biopsies from patients
with NERD.6,7 Ma etal., using human esophageal epithelial cells,
showed that HCl(pH 5) increases TRPV1 expression andcytokine
production.8 Moreover, gastric acid modulates esophageal inflam-
mationandTRPV1expressioninanimalmodelsofacuteesophagitis.
Fujinoreportedthat treatmentwitha TRPV1antagonistprevented
histological damageand myeloperoxidase (MPO)increasesin a rat
modeloferosiveesophagitis.9Thus,acidinducesTRPV1expression
and TRPV1 modulation decreases inflammationinesophagitis, but
itsroleinNERDpathogenesisisnotentirelyelucidated.
Previous studies involving preclinical models suggested that
TRPV1 plays akey role in esophagealhypersensitivit y and inflam-
mationassociatedwith acid-inducedesophagitis,astreatmentwith
aprotonpumpinhibitor(PPI)orareceptorantagonistblockedthese
responses.10,11However,theroleofTRPV1inesophagealmicroin-
flammat ion and impair ed mucosal inte grity, as obser ved in NERD,
hasnotbeeninvestigated.Thus,thisstudywasdesignedtoevaluate
therole ofTRPV1inesophagealinflammationandmucosal barrier
impairmentinamurinemodelofNERD.
2 | MATERIALS AND METHODS
2.1 | Animals
Female Swiss mice (25–30g; n=7–8) were obtained from the
DepartmentofPhysiologyandPharmacology,FederalUniversityof
Ceará,Ceará,Brazil.Theanimalsweredeprivedoffoodfor18hours
before thesurgicalprocedure, butwereprovided adlibitum access
toanoralrehydrationsolutioncontaining75mmol/LNa+,65mmol/L
Cl−,20mmol/L K+,10mmol/L citrate, and 75mmol/Lglucose. The
animalswere monitored throughout the experimentalp eriod.The
local et hics commi ttee (Proto col No. 108/14)ap proved this s tudy,
and all tre atments a nd surgical p rocedures w ere perfo rmed in ac-
cordancewiththeGuidefortheCareandUseofLaboratoryAnimals,
NationalInstitutesofHealth(Bethesda,MD,USA).
2.2 | Induction of NERD
Nonerosiverefluxdiseasewassurgicallyinducedinmiceasdescribed
previously.4Briefly,substenosiswasinducedbyplacingasiliconized
nontoxic ri ng (diameter, 3.25mm; wid th, 2.5mm; Embr amed, São
Paulo, Brazil)aroundtheduodenum near the pylorusandthetran-
sitionalregionbetweenthefundusandtheglandularportionofthe
stomach was ligated with a 4-0 nylon thread(Point suture, Ceará,
Brazil). Control mice underwent sham surgery. The animals were
killed 7days postsurgery.Theesophagus wasdissected,weighed,
and measu red. The esop hageal sample w as fixed in 10% forma lin
bufferforposteriorhistopathologicalanalysis.Esophagealsamples
werecollectedforwesternblotting,MPOactivit y,andkeratinocyte-
derivedchemokine(KC)assay.Themucosalintegritywasevaluated
inanUssingchamber bymeasuringtransepithelial electrical resist-
ance(TEER)andpermeabilitytofluoresceininthedistalesophagus.
2.3 | Western blotting for TRPV1
Esophageal samples were homogenized in RIPA lysis buffer
(25mmol/L Tris-HCl, pH 7.6; 150mmol/L NaCl; 5mmol/L EDTA;
1% NP-40; 1% Triton X-100; 1% sod ium deoxych olate; 0.1% SDS;
and RIPA). Th en, the samp les were centr ifuged at 1300 0rpm for
17minutes at 4°C, the supernatant was collected, and protein
concentrations were determined by the bicinchoninic acid assay
(Ther mo Fisher Sci entific, Walt ham, USA) accor ding to the manu-
facturer’s instructions. So diumdo decyl sulfate-polyacr ylamide gel
elect rophoresis ( 7.5%) was per formed usin g 50μg of protein. The
protein was transferred to a polyvinylidene fluoride membrane
(Bio-Rad,Hercules,CA, USA) for2hours, blockedwith 5%BSAfor
Key Points
• PatientswithNERDexhibitmicroscopicesophagealin-
flammation and impaired mucosa integrity, associated
with TRP V1ove rexpressi on. However, the role of t his
receptor has not been completely elucidated.
• AmurinemodelofNERDshowedincreased TRPV1ex-
pression, while pharmacologicalantagonism and long-
term desensitization of TRPV1 prevented the
development of esophageal inflammation and impair-
mentoftheesophagealmucosalbarrier.
• The TRPV1 receptor is a pharmacological target in
NERD, and an an tagonist may p otentially have cl inical
benefitsfortreatingthisdisorder.

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1hour, incubated overnight with a primar y antibody (rabbit anti-
TRPV1, 1:600;Abcam,Cambridge,UK )andthen with asecondary
antibod y (goat anti-rab bit, 1:1000; Invitr ogen, Carls bad, CA , USA)
for90minutes.ProteinswerevisualizedusingtheECLsystem(Bio-
Rad)accordingto manufacturer’sinstructions.Chemiluminescence
was detected using the ChemiDoc™ XRS system (Bio-Rad). The
membr an ewass tr ippedan dre-pro be dwi thana nti-β-actinantibody
(1:500; SantaCruzBiotechnology,Dallas,TX, USA) followedby in-
cubation w ith goat anti-mo use IgG antib ody (1:500; I nvitrogen) as
aloadingcontrol. Band densitieswerequantifiedusingImageLab™
software(Bio-Rad).
2.4 | Animal treatments
2.4.1 | Pharmacological antagonists
Mice were treated with TRPV1 antagonist, SB366791 (3mg/kg,
i.p.)12 or capsazepine (5mg/kg, i.p.)13 once a day beginning on the
day of surger y until 7days post sur gery, when the animals were
killed.
2.4.2 | Long- term desensitization of TRPV1
Toinducelong-term desensitizationofTRPV1, we used resinifera-
toxin(RTX)anultrapotentTRPV1agonist,asdescribedpreviously.14
RTXinducessystemicdesensitizationofTRPV1withlossofrespon-
siveness,whichrepresents functionaldenervationdueto acombi-
nation of receptordesensitization, neurondefunctionalization,and
neurotoxicity.15 Briefly, the mi ce received RTX i njections (s.c .) for
three consecutive days:day1, 30μg/k g; day 2, 70μg/k g, and day
3,100μg/kg.Toprotectagainstrespiratoryfailure,eachtoxininjec-
tionwasadministeredtogetherwithacocktailof0.2mg/kgterbuta-
line,0.2mg/k gatropine,and19.2mg/kgaminophylline (i.p.).Seven
daysafterthelastRTXinjection,allanimalsweresubmittedto the
eye-wiping test to confirm functional denervation.16 To this end,
micewereadministered0.1%capsaicininthecorneaandeye-wiping
wasmonitoredfor1minute.Completedener vationwasconsidered
wheneye-wipingmovementswereabsent.Then,thesurgicalproce-
durewasperformedandmicewerekilled7dayspostsurger y.
2.5 | Evaluation of interventions
2.5.1 | Esophagus wet weight
The esophagus was dissected, washedwith sterilesaline, weighed,
measured,andthewetweight/lengthratiowasusedasanindicator
ofe de ma.R es ul tsaree xp re ssedas mi ll igra mp ercentimeter( mg /cm).
2.5.2 | Histopathological analysis
The esop hagus was fixe d in 10% formalin fo r 18hours and tr ans-
ferred to 70 % alcohol until pro cessing. Then, t he esophagus was
embeddedin paraffin and sectioned(at5-μmthickness),deparaffi-
nized, and stained with hematox ylin and eosin (H&E). The slides
wereanalyzed bylightmicroscopyby apathologist(P.M.G.S.) with-
out knowl edge of the treatmen ts, according to cr iteria describe d
previously.4Briefly,basalcelllayerhyperplasia(scores,0–2),in-
traepithelialpolymorphonuclearcells(scores,0–2),erosions(scores,
0–1),edema(scores,0–4),andpolymorphonuclearcellsinthelamina
propria(scores,0–3)wereexamined.
2.5.3 | MPO activity
Esophagus samples were homogenized in PBS (pH 6.0) contain-
ing hexadecyltrimethyl-ammonium bromide and centrifuged at
4500rpmfor15minutes.MPOactivitywasmeasuredinaspectro-
photomete r at 450nm using o-dianisidine dihydrochloride and 1%
hydrogenperoxide.17Resultsareexpressedasunitspermilligramof
tissue(U/mgtissue).
2.5.4 | KC assay
A cytok ine kit (Bio- Ra d) was used acc ording to manu facture r’s in-
st ruc tions .T herea ction sw er er eadinth eL umine x™ Instrumentation
System (Bio-Rad) and analyzed using the Bio-Plex Manager™ 6.1
software. The Bradford method was used for protein quantifi ca-
tion.18 Results a re expresse d as picogram pe r milligram of p rotein
(pg/mgprotein).
2.6 | Protocols involving the Ussing chamber
For this exp eriment, th e esophagus w as dissected , stripped of it s
musclelayers,anddissectedina plate containing Krebs-Henseleit
bicarbonate buffer (KHBB; pH 7.4). Esophageal mucosa sections
were mounte d on an adapted Ussin g chamber (Mussle r Scientific
Instruments,Aachen,Germany)withanexposureareaof0.017cm2.4
TEERandpermeabilitytofluoresceinwere measuredinthesystem
withKHBB equilibrated at37°Candcontinuouslygassed with car-
bogen(95%O2and5%CO2).
2.6.1 | TEER evaluation
Basal TEERofthesolution (withouttissue)wasdetermined with
voltage deflections of 50μA for 200milliseconds every 6sec-
onds. Then, the tissueswere mountedin thechamberand asta-
ble TEER base line was obtaine d from an equilibr ated electri cal
system (30minutes).Next,therecording waspaused,and KHBB
solution in each esophageal mucosa was replaced with an acidic
solution(pH 0.5,containing 1mg/mLpepsin and 2mMtaurode-
oxycholicacid[TDCA])aspreviouslydescribed.4Thet issueswere
exposedtotheacidicsolution for60minutes,and theTEERwas
continuously measured.19Resultsareexpressedasthepercent-
agechange (%) in TEER from baselineat10,30,and 60minutes
postacidexposure.

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2.6.2 | Permeability assay
Esophagealmucosa was mountedina dif fusion chamber adapted for
small tissues.Fluorescein(376Da)was used as a marker of increased
permeability.Thesystemwasstabilized for30minutes inKHBBsolu-
tion.Then,thesol uti onintheluminalsid eoft hechamberwasreplaced
withKHBBcontaining 1mg/mLfluorescein.20 A sample(100μL)was
collectedfromtheserosalsideat1,2,and3hours.Themarkerconcen-
tration in thesample was measuredusinga fluorescence plate reader
(FLUOstarOmega;BMGLabtech,Ortenberg,Germany).Permeability
wasdeterminedfromastandardcurveplottedforeachanalysis.
2.7 | Drugs and reagents
RTX,capsazepine,SB366791,pepsin,TDCA,andfluoresceinwerepur-
chased fr om Sigma-Aldr ich (St. Louis , MO, USA). All oth er chemical s
wereofanal yticalgradeandwereobtainedfromcommercialsuppliers.
2.8 | Statistical analysis
Data are shown as the mean±SEM ormedian withminimum and
maximum values, as appropriate. Statistical analyses were per-
formed by one-way ANOVA followed by the Student-Newman-
Keuls test o r two-w ay ANOVA followed by Bonfe rroni’s test (for
parame tric data) and Kru skal-Wallis tes t followed by the Dunn’s
tests(fornonparametricdata).P<.05wereconsideredstatistically
significant.
3 | RESULTS
3.1 | Esophagus from NERD mice exhibits TRPV1
overexpression
Western blot analysis (Figure1) revealed that NERD mice
(3.33±0.66) exhibited a significant (P<.05) increase in TRPV1
protein expression in the esophagus as compared to sham animals
(1.00±0.07),asdemonstratedbyopticaldensityrelativetoβ-actin,
usedasareferenceprotein).
3.2 | Pharmacological antagonist and long- term
desensitization of TRPV1 prevent histological changes
in NERD mice
Figure2 and Table1 show that compared to sham animals
(Figure2A and F), NERD mice presented histological altera-
tions in the esophagus, including basal cell layer hyperplasia,
intraepithelial and lamina propriapolym orphonuclearinfiltrate,
and edema in the lamina propria (Figure2B and G). However,
there were no erosions in the esophageal mucosa. TRPV1
antagonist (SB366791 or capsazepine; Figure2C, D, H and
I, respectively) and long-term desensitization of TRPV1
FIGURE1 ExpressionofTRPV1inmiceesophagusasevaluated
bywesternblotting.Resultsarerepor tedastherelativedensityof
TRPV1/β-actinbands.*P<.05vssham,N=7-8
FIGURE2 Pharmacologicalblockadeandlong-termdesensitizationofTRPV1preventhistopathologicalchangesinamurinemodelof
NERD.(AandF)Shamgroupexhibitingnormalmorphologyoftheesophagus.(BandG)NERDgroupexhibitingbasalcelllayerhyperplasia
(greenarrow),edema(blackarrow),andpolymorphonuclearinfiltrateonthelaminapropria(redarrow).TreatmentwithSB366791(CandH),
capsazepine(DandI),orRTX(EandJ)at tenuatedthehistopathologicalchanges.PanelsA-D(magnification,100×)andE-H(magnification,4 00×)
(A) (B) (C) (D) (E)
(F) (G) (H) (I) (J)

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attenuated these esophageal histopathological changes
(Figure2EandJ).
3.3 | TRPV1 is involved in esophageal inflammation
in NERD mice
As demo nstrated in Table2, NER D mice showed signif icant in-
creases in esophagus wet weight (21.6±1 .5mg/cm) and MPO
activity (14.5±2.7U/mg tissue) compared to sham animals
(13.5±0.5mg/cm and 0.5±0.3U/mg tissue, respectively). On
the other hand, there were significant (P<.05) reductions in
these parameters after treatmentwith the TRPV1 antagonists,
SB366791 (wet weight: 12.2±0.6mg/cm, MPO: 0.5±0.4 U/
mg tissue) an d capsazepine (wet weight: 14.6±1.1mg/cm and
MPO: 7.8±1.5U/mgtissue)andafter long-term desensitization
of TRPV1 with RTX (wet weight: 14.6±0.5mg/cm and MPO:
1.4±1.6U/mgtissue).
Nonerosiverefluxdiseasemicepresentedasignificantincrease
in KC (515.4±100.3pg/mg prote in) compared to sham ani mals
(304.4±31.0pg/mgprotein;Table2).However,thisincreasewas
prevented by t reatment with t he TRPV1 antagon ists SB366791
(245.4±56.7pg/mg protein) and capsazepine (150.9±8.7pg/
mg protein) and TRPV1 depletion by RTX (264.2±32.6pg/mg
protein).
3.4 | Pharmacological antagonist and long- term
desensitization of TRPV1 attenuate the acid- induced
decrease in esophageal TEER and increased basal
permeability in NERD mice
The esophageal mucosa of NERD mice had a more pronounced
decreasein TEERwhen exposedtoacidicsolutioncontaining pep-
sin and TDC A (pH 0.5) for 30 and 60minutes (64.8±7.7% and
53.3±9.8%ofbasalresistance,respectively)thantheshamanimals
(30minute s, 94.4±6.2% and 60m inutes, 93.1±9.1%of b asal re-
sistance;Figure3A).Thesechangesweresignificantly(P<.05;n=8,
N=8;wherenis the numberof obser vations and Nisthenumber
ofanimals)preventedby TRPV1antagonistSB366791(60minutes,
75.8±7.3% of basal resistance) and by TRPV1 deplet ion by RTX
(60minutes,74.1±10.3%ofbasalresistance)(Figure3A).
In additi on, the esop hageal mucosa of N ERD mice exhibit ed a
significantincreasein basalpermeability tofluoresceinafter 2and
3hours(567.9±54.6μmo l/c m2 a nd 837.1 ± 128. 0 μmol/cm 2,respec-
tively)comparedtothatofshamanimals(2hours,192.9±33.1μmol/
cm2 and 3hours, 428.6±55.6μm ol /cm 2) (Figure3B). However,
treatment with the TRPV1 antagonist SB366791 (2hours,
272.9±43.9μmo l/cm2 and 3hours, 506.0±79.3μmol/cm 2) and
TRPV1 depletion by RTX (2hours, 300.9±37.5μmo l/c m2 and
3hours, 574.7±46.4μm ol/c m2) significantly prevented these
TABLE1 Effectsofpharmacologicalblockageandlong-termdesensitizationofTRPV1onesophagealhistopathologicalscoresina
murinemodelofNERD
Experimental groups
(N = 8)
Basal cell layer
hyperplasia (0–2)
Intraepithelial polymor-
phonuclear (0–2) Erosions (0–1)
Edema in lamina
propria (0–4)
Polymorphonuclear in
lamina propria (0–3)
Sham 0(0–1) 0(0–1) 0(0–0) 1(0–1) 1(1–1)
NERD 2(1–2)* 1(1–2)* 0(0–0) 2(2–4)* 2(1–3)*
SB366791+NERD 0(0–1)†0(0–1)†0(0–0) 1(0–2) 1(1–1)†
Capsazepine+
NERD
1(0–2)†0(0–1)†0(0–0) 1(1–2) 1(1–2)†
Resiniferatoxin+
NERD
0(0–1)†0(0–0)†0(0–0) 0(0–1)†0(0–1)†
Resultsareshownasmedianswithminimalandmaximumvalueinparentheses.
NERD,non-erosiverefluxdisease;TRPV1,tr ansientreceptorpotentialvanilloidmember1.
*P<.05vsshamgroup;†P<.05vsNERDgroup.
Experimental groups
(N = 8)
Esophagus wet
weight (mg/cm) MPO (U/mg tissue)
KC (pg/mg
protein)
Sham 13.5±0.5 0.5±0.3 304.4 ± 31.0
NERD 21.6±1.5* 14.5±2.7* 515.4±100.3*
SB366791+NERD 12.2±0.6†0.5±0.4†245.4±56.7†
Capsazepine+NERD 14.6±1.1†7.8±1.5†150.9±8.7†
Resiniferatoxin+NERD 14.6±0.5†1.4±1.6†264.2±32.6†
Resultsareshownasmean±SEM.
NERD, non-erosive refluxdisease; TRPV1,transientreceptor potential vanilloidmember 1; MPO,
myeloperoxidase;KC,keratinocyte-derivedchemokine.
*P<.05vsshamgroup;†P<.05vsNERDgroup.
TABLE2 Pharmacologicalblockage
andlong-termdesensitizationofTRPV1
preventesophagealinflammator y
responseinamurinemodelofNERD

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alterations in the esophageal mucosal permeability (P<.05; n=8,
N=8;wherenis the numberof obser vations and Nisthenumber
ofanimals).
4 | DISCUSSION
PreviousstudieshaveshownoverexpressionofTRPV1inhumanes-
ophageal b iopsies and a nimal model s of esophagit is. In this st udy,
weshowed,forthefirsttime,increasedTRPV1receptorexpression
in an animal m odel of NERD. Fur thermore , we demonst rated that
TRPV1isarelevant factorinesophageal inflammationand impair-
mentofepithelialbarrierintegrity,whichareconsideredkeyfactors
in NERD path ophysiolog y,usi ng experime nts involvin g pharmaco-
logical antagonism bySB366791orcapsazepineand long-termde-
sensitizationofTRPV1withRTX.
Transientreceptorpotentialvanilloid1is amolecular integrator
ofsignalingpathwaysformultipletypesofsensoryagents,including
chemical,thermal,andendogenousmediators.21Deregulationofthe
physiological function of this receptor,eg, throughoverexpression
oruncontrolledsensitization,iscriticalintheetiologyofGIdiseases,
includingGERD.22EsophagealTRPV1expressionhasbeenanalyzed
inpreclinicalandclinicalstudies.In patientswithreflux symptoms,
theacidexposureisassociatedwithincreasedTRPV1immunoreac-
tivityin biopsiesfromesophageal mucosa.6Guarino etal. reported
thatpatientswithNERDoverexpressTRPV1intheesophagealmu-
cosa as compared to healthy controls.7Usinganexvivoexperimen-
tal model,Cheng etal. demonstratedthat acid exposure promotes
TRP V1a ctivationinth ees ophag ealmucos aofcat sandi nduce ssy n-
thesis/release of sensory transmitters (ie, calcitonin gene-related
peptide andsubstanceP).23Similarly,wedemonstratedesophageal
TRPV1overexpressionintheNERDmodelcomparedtocontrolani-
mals.Therefore,wehypothesizethatTRPV1-mediatedsignalingmay
initiate and maintain acid-mediated esophageal inflammation and
epithelial barrier impairment.
Currently, patients are diagnosed as having NERD when vis-
ible macroscopic damage in the esophageal mucosa at endoscopy
is absent bu t microscopic a lterations , probably in re sponse to the
presen ce of reflux content (i e, acid, pepsin , and bile salt), a re ob-
served in the esophageal lumen.2 Our result s showed that NERD
miceexhibitedbasalcell layer hyperplasia, mild inflammation with
intraepithelialandlaminapropriapolymorphonuclearinfiltrate,and
edema. Zentilin et al. demonstrated a positive correlation bet ween
esophagealhistologicalalterationsandexposuretimebelowpH4.0
inpatients withNERD.24 Werecently demonstratedthat PPItreat-
mentpreventsmorphologicalalterationsintheesophagusofNERD
mice.4Takentogether,these findings strongly suggest that gastric
acidiscrucialinNERDpathogenesis.
Thus, we eva luated the effec t of pharmacological modul ation
of TRPV1, an ac id-ac tivated se nsorial mo lecule, on e sophageal in -
flammation in NERD mice. Pharmacological antagonism or long-
term desensitization of TRPV1 prevented histological alterations
in the eso phagus of NERD mice . TRPV1 is expres sed in spinal af-
ferentsandvagalafferentneuronsandprobablycontributestothe
esophagealinflammatory response.25 However,the genesisofmi-
croscopicinflammationinNERDisnotelucidated.Wehypothesized
that TRPV1-dependent signaling plays a role in the development
oflocal esophageal inflammation. Our results showed thatanimals
treated withaTRPV1antagonistorwith RTX exhibited decreased
esophaguswetweight,MPOactivity,andKClevelsascomparedto
NERDanimals.Similarly,FujinoshowedthatTRPV1-knockoutmice
develop le ss erosive eso phagitis in r esponse to ac id exposure a nd
exhibit lower MPO activity and histopathologicalscores than wild-
type mice.9
Ourresultsdidnotdemonstratetheprecise locationofTRPV1
intheesophagealmucosainNERDmice.Ithasbeensuggestedthat
TRPV1 receptors are expressed in afferentneuronal fibers,asob-
serve d in biopsies of patie nts with NERD,6,7 an d in non-n euronal
cells,suchasesophagealepithelialcells.8 ,26Arecentstudydemon-
stratedCGRP-positivenerves close totheesophageallumeninpa-
tientswithNERD,27whichcolocalizedwithTRPV1-immunoreactive
nervefibersinmouseesophagus.28Thus,fur therinvestigationsare
necessary.
Inthis study,weshowedthatNERD micemaintainedesophageal
mucosaintegrityintheabsenceofTRPV1ac tivationduetopharma-
cological blockade or dener vation, as demonstrated by functional
FIGURE3 Pharmacologicalblockadeandlong-term
desensitizationofTRPV1attenuatetheimpairmentofesophageal
mucosaintegrityinamurinemodelofNERD.(A)TEERevaluation
afterexposingtheesophagealmucosatoanacidicsolution
containingpepsinandTDCAatpH0.5for60minutes.(B)Basal
permeabilityoffluoresceinevaluatedfor3hours.Resultsare
expressedasmean±SEMandwereanalyzedbytwo-wayANOVA
followedbytheBonferronitest.*P<.05vsshamgroup,#P<.05vs
NERDgroup,n=8,N=8,wherenisthenumberofobservations
andNisthenumberofanimals

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SILVA et AL.
experiments involvingacidexposure-induced decrease in TEERand
basalpermeabilitytofluorescein.Moreover,previousinvitrostudies
havesuggestedakeyroleforTRPV1inpromotingbarrierfunctionim-
pairmentinseveraltissues.Isodaetal.demonstratedthattheTRPV1
agonistcapsaicinincreasesparacellularpermeabilityofintestinalcells
(Caco-2),aneffectrevertedbypre-incubationwiththeTRPV1antag-
onist capsazepine.29 In addition, TRPV1ac tivationdecreases TEER
and increases paracellular permeability in submandibular gland cells
(SMG-C6).30Xuetal.demonstratedthatnormalhumanbronchialep-
ithelialcells(16HBE)uponincubationinanacidicmedium(pH4.0or
5.0)exhibitedsignificantdecreasedinTEERandjunctionproteinex-
pression,whichwerereversedbypre-incubationwithcapsazepine.31
Despitedifferencesintheepithelialtissuetypesused intheseprevi-
ousstudies,thef indingscor roborateourresultsonthepivotalroleof
TRPV1intheimpairmentofesophagealmucosalintegrityinNERD.
It has been postulated that impaired esophageal mucosal integ-
rity is a significantfactor inGERD pathogenesis. Thealteration of
epithelialbarrierfunctioniswellelucidatedin patientswithEE,but
iscontroversial inpatients with NERD.32, 33 In contrast with studies
measuringinvivoimpedance,theexvivoevaluationofbiopsiesfrom
esophagealmucosainanUssingchambersystemrevealednodiffer-
enceinbasalTEERbetweenpatients with NERD and controls.32,3 4
Som efa ctorsmaye xplain,atleastinpa rt ,thisd isc rep ancy.Firs t,en-
dogenous fac tors (ie,bicarbonate, saliva secretion, and blood flow)
mayinfluencemucosalimpedance.Second,mediatorsreleasedfrom
sensor y nerve endings and immune cells may be present in the mu-
cosain vivo,butnotintheepitheliumexvivo.Third,thesizeofthe
sampleevaluatedinanUssingchamberismuchsmaller.33,34
The presentresults provide new insights into NERD pathophys-
iology. We demo nstrated that T RPV1 is critical f or the esophage al
inflammatoryresponseandimpairmentofesophagealmucosalinteg-
ri ty.Theca sc adeofe ve nt sisn ot ye tf ul lyel ucidated,bu twespe cu la te 
thati tco ul dbese co nd ar ytoth ep resenceofg as tr icconte ntref lu x(ie, 
acidandother ssubst ances)intheesop hageallum en,whi chp rom ote s
theactivationof esophageal epithelialcellsto initiatea complexsig-
nalingcascadewith production/secretionofchemoattractantcyto-
kines, suchasKC,inducingleukocyteinfiltration intotheesophageal
mucosa.A“positivefeedbackpathway”betweenTRPV1overexpres-
sionandstimulationoccurs, establishing the“vicious cycle,”thereby
contribu ting to the inflamma tory pathway net work. Therefore ,we
hypothe sized that the impair ment of esophageal m ucosal integrit y
observed in our study and previously reported in esophageal biopsies
fr om pa tient sw ithNERDoc cu rsbec auseofper si stentmi cr os copicin-
flammation.However,wecannot excludedirectTRPV1involvement
inthe impairmentof esophagealmucosalintegrity;this hypothesisis
currently under investigation. In addition, theimbalanceofesopha-
gealmucosahomeostasiswithincreasedpermeabilityreinforcedthis
cycle withpermeationofmore gastric contentsinthe mucosa,per-
petuatingtheneuronal/immune-mediatedresponse.
Our results suggested that the pharmacological modulation
ofTRPV1 may not be related, at not entirely, to data fromclini-
cal trials.Inarandomizedtrial in patients with NERD,the treat-
ment with a si ngle dose of AZD138 6, a TRPV1 antagon ist, was
ineffective in altering the pain threshold for various stimuli (ie,
heat,mechanical,orelectrical).35Inourview,long-ter mtreatment
withAZD1386isrequiredto decreaseesophagealinflammation.
In this reg ard, patients wit h PPI-resp onsive esophageal e osino-
philia exhibit improved esophageal barrier integrity associated
with histopathological response only after 8weeks of treatment
withhigh-doseesomeprazole.36Theautho rs su ggest ed th at re sto-
rationofthe esophagealmucosalintegritymightbedue todirect
anti-inflammatoryand/oranti-oxidanteffectsofPPIsandnotonly
duetoacidsuppression.Furthermore,althoughTRPV1activation
might be imp ortant for pa in perception in pat ients with NERD,
blockadewitha singledoseof AZD1386 is unableto desensitize
the nerve endings, which develop immediately around a ligand
agent,culminatinginpersistenthyperalgesia.37
Physiologically,TRPV1hasbeenimplicatedinthermoregulation,
as variou s TRPV1 antagoni sts caus ed hypert hermia in dogs , mice,
monkeys, and humans in preclinical and clinical studies.38 Thus,
theidentificationof brain-impermeable andperipherallyrestricted
TRPV1 anta gonists may ope n perspecti ves for differe ntiation be-
tweentherapeuticandthermoregulatoryeffectsofthesedrugs.39, 40
Inconclusion,thisstudydescribestheroleofTRPV1inamurine
model of NERD.Weshowed TRPV1 overexpression in the esoph-
ageal tissue of NERD mice,similar to previous findings in patients
withNERD.Moreover,wedemonstratedthatTRPV1isimportantin
esophagealinflammationandimpairmentoftheesophagealepithe-
lialbarrier.Therefore,wesuggestTRPV1asaneffectivepharmaco-
logical t arget for NER D, and TRPV1-blocker dr ugs may potenti ally
leadtonewtherapiesfor reflux-mediatedesophageal inflammation
and heartburn perception.
DISCLOSURE
Nocompetinginterestsdeclared.
AUTHOR CONTRIBUTIONS
ROSandMHLPScontributedtoconceptionanddesignofthestudy;
ROS,RDB,TMALS,KMOS,RLRM,andDVSperformedexperiments;
ROS, KMOS,GAB, DS, andMHLPS analyzed data;ROS, GAB,DS,
andM HLP Sinterp retedre sultsofexperiments;ROSan dPMGSpre-
paredfigures;ROSandMHLPSdraftedthemanuscript;ROS,PMGS,
MANS,A AS,DS,andMHLPSeditedandrevisedthemanuscript.All
authorsapprovedthefinalversionofthemanuscript.
ORCID
M. Â. N. Souza http://orcid.org/0000-0003-2376-3433
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How to cite this article:SilvaRO,BinganaRD,SalesTMAL,
etal.RoleofTRPV1receptorininflammationandimpairment
ofesophagealmucosalintegrityinamurinemodelof
nonerosiverefluxdisease.Neurogastroenterol Motil.
2018;30:e13340. ht tp s://doi.org/10.1111/nmo .133 40