The multicomponent medication Spascupreel attenuates stress-induced gut dysfunction in rats

HAL Id: hal-02623594

https://hal.inrae.fr/hal-02623594

Submitted on 26 May 2020

HAL is a multi-disciplinary open access

archive for the deposit and dissemination of

sci-entific research documents, whether they are

pub-lished or not. The documents may come from

teaching and research institutions in France or

abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est

destinée au dépôt et à la diffusion de documents

scientifiques de niveau recherche, publiés ou non,

émanant des établissements d’enseignement et de

recherche français ou étrangers, des laboratoires

publics ou privés.

Distributed under a Creative Commons Attribution| 4.0 International License

The multicomponent medication Spascupreel attenuates

stress-induced gut dysfunction in rats

Vassilia Theodorou, Catherine Beaufrand, Sophie Yvon, Guylaine Laforge,

Yvonne Burmeister, Andrea Mueller, Bernd Seilheimer, Lionel Bueno, Hélène

Eutamène

To cite this version:

Vassilia Theodorou, Catherine Beaufrand, Sophie Yvon, Guylaine Laforge, Yvonne Burmeister, et

al.. The multicomponent medication Spascupreel attenuates stress-induced gut dysfunction in rats.

Journal of Neurogastroenterology and Motility, Korean Society of Neurogastroenterology and Motility,

2020, 9 p. �10.1111/nmo.13798�. �hal-02623594�

Neurogastroenterology & Motility. 2020;00:e13798. wileyonlinelibrary.com/journal/nmo 

|

 1 of 9 https://doi.org/10.1111/nmo.13798

Received:11September2019 

|

  Revised:13December2019 

|

  Accepted:23December2019 DOI:10.1111/nmo.13798

O R I G I N A L A R T I C L E

The multicomponent medication Spascupreel attenuates

stress-induced gut dysfunction in rats

Vassilia Theodorou

1

_{| Catherine Beaufrand}

1

_{| Sophie Yvon}

1

_{| Guylaine Laforge}

1

_|

Yvonne Burmeister

2

_{| Andrea Müller}

2

_{| Bernd Seilheimer}

2

_{| Lionel Bueno}

†

_|

Helene Eutamene

1

ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsuse,distributionandreproductioninanymedium, provided the original work is properly cited.

©TheAuthors.Neurogastroenterology & MotilitypublishedbyJohnWiley&SonsLtd

†_Deceased. 1 INRA,ToxAlim,UMR1331,Neuro-GastroenterologyandNutritionGroup, ENVT,INP-Purpan,UPS,Universitéde Toulouse,Toulouse,France 2_{HeelGmbH,Baden-Baden,Germany} Correspondence HeleneEutamene,INRA,ToxAlim,UMR 1331,Neuro-Gastroenterologyand NutritionGroup,UniversitédeToulouse, ENVT,INP-Purpan,UPS,180Cheminde Tournefeuille,31027Toulouse,France. Email: [email protected] Funding information HeelGmbH,Baden-Baden,Germany

Abstract

Background: Irritablebowelsyndrome(IBS)isacommondisorderworldwide.Itis

characterized by abdominal pain/discomfort and changes in bowel habits. Due to themultifactorialpathophysiologyandtheheterogeneityofIBSpatients,appropri-atetreatmentofIBSisstillachallenge.Spascupreel(SP-11),asamulticomponent medication,hasthepotentialtomodulatemultiplepathophysiologicalpathwayssi-multaneously.Therefore,theobjectiveofthecurrentstudywastoinvestigatethe effectsoforalSP-11treatmentonstress-inducedchangesofperipheralandcentral functionsinaratmodelmimickinghumanIBS. Methods: NaïveWistarratsweretreatedwithSP-11(0.9tab/kg)orNaCl0.9%by oralgavagefor4daysbefore2-hourpartialrestraintstress(PRS)procedure.Twenty minutesafterPRS,centralandperipheralstress-inducedchangesaffectingIBSwere assessed. These include the hypothalamic-pituitary-adrenal (HPA) axis response throughplasmaACTHandcorticosteronemeasurements,visceralpaininresponse tocolorectaldistension,gutpermeability,colonicmastcellnumber,andsensitization as well as gut transit time.

Results: TreatmentwithSP-11reducedtheHPAaxisactivationinresponsetoPRS.

Atthegutlevel,areductionincolonichypersensitivitytocolorectaldistension,a normalizationofguttransittimeacceleration,areducedmastcellsensitization,anda trend toward reduced gut hyperpermeability were observed.

Conclusions: Thesedatasuggestthatstress-inducedIBSsignscanbereducedusing SP-11inrats.TheobservedeffectsandthegoodtolerabilityofthedrugmakeSP-11 aninnovativecandidateinthemanagementofIBS. K E Y W O R D S gut-brainaxis,irritablebowelsyndrome,multicomponentmedication,partialrestraintstress, Spascupreel,SP-11

2 of 9 

|

     THEODOROU ETal.

1 | INTRODUCTION

Irritablebowelsyndrome(IBS)isoneofthemostcommonconditions thataphysicianfacesinthegastrointestinalclinic.Accordingtothe RomecriteriaIV,5%-7%ofthegeneralpopulationsuffersfromIBS symptoms.1_{ Abdominal pain is a cardinal IBS symptom associated}

withchangesinbowelhabits(diarrheaand/orconstipation)(forre-viewEncketal,20162_{).IBSpatientsarestratifiedintofoursubtypes}

according to the predominant changes in bowel habits: diarrhea-predominant (IBS-D), constipation-diarrhea-predominant (IBS-C), both diar-rhea and constipation (IBS-M) and unclassified (IBS-U) (for review Encketal,20162_{).Thesymptomsstronglyaffectthepatient'squality}

of life3 _{and represent a significant socioeconomic burden.}4_{ In the}

absence of a clear identification of organic features and lack of reli- ablebiomarkers,theIBSpathophysiologyisstillnotcompletelyun-derstood.IBSsymptomsmayoriginatefromseveralperipheraland/ orcentralmechanisms.Amongthem,dysfunctionalgut-brainaxis,5

low-gradeintestinalinflammation,6_{increaseinmucosalmastcells,}7

intestinalmicrobiotadysbiosis,8 _{and impaired intestinal barrier}

func-tion9_{havebeenreportedintheliterature.Forinstance,alterations}

inthecentralnervoussystem(CNS),causedbyanxietyandstressful psychologicalstimuli,triggeredabnormalgastrointestinalmotility,10

heightenedvisceralsensations,11 _{and increased gut permeability.}12

In order to understand the IBS pathophysiology, acute and chronicstressanimalmodelsmimickingIBSfeatures,suchaschanges in visceral sensitivity, gut transit alterations, mast cell infiltration, andimpairedintestinalbarrierfunction,havebeendeveloped.13,14

Indeed,inratsacuterestraintstressisreflectedbybothanincrease inadrenocorticotropichormone(ACTH)andcorticosteroneplasma concentrations.15 _{This is associated with visceral hypersensitivity to}

colorectaldistension(CRD),acentralreleaseofcorticotrophinre-leasing factor (CRF),13 _{and an increase in gut permeability.}16_{ Mast}

cells and their products play an important role in the pathophysiol-ogyofIBS.17_{Uncontrolledordysregulatedmastcellactivationmay}

interfere with gut homeostasis, generate tissue dysfunction, and promote inflammation in diverse gastrointestinal diseases and func-tional gastrointestinal disorders.18_{Inrats,acutestressincreasesthe}

colonicmastcellhistaminecontent,aperipheraleffect,mediatedby thereleaseinthecascadeofinterleukin-1(IL-1)andCRF.19

Regarding the multifactorial pathophysiology and the heteroge-neity of theIBS population,appropriatetreatment of IBS is stilla challenge. Therapeutic strategies are often limited to the treatment of symptoms with drugs focusing on motor/sensory abnormalities. Forpatientswithpsychologicalcomorbidity,psychopharmacological agentscanalsobeusefulinthetreatmentofIBS.20

To target multiple pathophysiological pathways associated with IBSsimultaneously,amulticomponent/multitarget-basedtreatment might be suitable. SP-11 is a multicomponent medicinal product, which was previously investigated in several clinical studies in con-ditions such as gastrointestinal cramps or spasmodic gastritis21-23_;

however,itsmodeofactionisstillpoorlyunderstood.Inthisstudy, weaimedtoinvestigatetheeffectsofSP-11onthemultifactorial pathophysiologicalmechanismsofIBS.

2 | MATERIALS AND METHODS

2.1 | Test item

Spascupreel (SP-11) tablets were manufactured by Heel GmbH, Germany,accordingtoGMPstandards.Thestudymedicationwas packaged,shipped,andlabeledbyHeelGmbH,Germany.Onetablet containsCitrulluscolocynthisD430mg,AmmoniumbromatumD4 30mg,AtropinumsulfuricumD630mg,VeratrumalbumD630mg, MagnesiumphosphoricumD630mg,GelsemiumsempervirensD6 30mg,PassifloraincarnataD215mg,AmanitamuscariaD415mg, Matricaria recutita D3 15 mg, Cuprum sulfuricum D6 15 mg, and AconitumnapellusD660mg(Dstandsfor10×dilutionofthemother tincture).Thetabletsweredissolvedinwaterandadministeredata dose of 0.9 tab/kg once daily at 9 amfor4daysbyoralgavagein1mL

ofwaterusingagastro-esophagealcannula.

2.2 | Animals

Accordingtotheparameterstudied,maleorfemaleWistarrats(175-225g;JanvierSA)wereused.Animalswereindividuallyhousedina temperature-controlledroom(21±1°C)andmaintainedina12/12-hour light/dark cycle. Rats had free access to water and were fed ad libitum with laboratory pellets (Envigo, Teklad Global Diet®). TheLocalAnimalCareandUseCommitteeofInstitutNationalde laRechercheAgronomiqueapprovedallexperimentalprotocols(n° MP/01/53/10/11).

2.3 | Experimental design

Threeseriesofexperimentswereconducted.Inallofthem,SP-11 (0.9 tab/kg) or NaCl 0.9% (1 mL/rat) were administered per oral gavage4daysbefore2-hourpartialrestraintstress(PRS)session. TwentyminutesafterPRS,ACTH,corticosterone,visceralhyper-sensitivity,gutpermeability,guttransittime,mastcellnumber,and rat mast cell protease II (RMCPII) expression were investigated. Inthefirstseries,visceralsensitivitytocolorectaldistensionwas assessed by electromyography (EMG) recording in the same ani-mals before (basal condition) and 20 minutes after the PRS ses-sion(Figure1A).Thesecondserieswasperformedtoassessthe Key Points • SP-11positivelymodulatedthegutfunction. • SP-11reducedtheHPAaxisactivation. • SP-11reducedIBSsignsinaratmodel. • SP-11beneficiallytargetedmultiplepathophysiological pathwaysassociatedwithIBS.

physiological stress response via measuring plasma corticosterone and ACTH concentrations. In addition, after removal of colonic segments, paracellular permeability was assessed, and mast cell number and RMCPII expression were measured (Figure 1B). The third series aimed to investigate gut motility by measuring the transittime(Figure1C).

2.4 | PRS procedure

Allstresssessionswereperformedatthesametimerangeofthe day(between10am and 12 pm

)tominimizeanyinfluenceofcirca- dianrhythms.Stresseffectswerestudiedusingasingle2-hourses-sionofPRSwhichisconsideredasamildnon-ulcerogenicmodel.24

Underlightanesthesiawithethylether,upperforelimbsweretaped up to the thoracic trunk in order to constrain animal body move-ments. Rats were then replaced in their home cages for 2 hours.

2.5 | Visceral sensitivity in response to CRD

To evaluate abdominal contractions, an index of visceral sensitiv-ity,femaleratsweresurgicallypreparedforEMGandequippedwith threegroupsofNiCrwireelectrodes(Sandvik).Thesewereimplanted intotheabdominalexternalobliquemuscle5dayspriortotheexperi-ment.ThemyoelectricalactivitywasrecordedbyEMGaspreviously describedbyMorteauetal,1994.25 _{Rats were placed in a}

polypro-pylene tunnel and a balloon consisting of an arterial embolectomy catheter (Fogarty®; Edwards Laboratories Inc) was slowly placed 4 cm into the rectum and taped at the base of the tail. The balloon wasprogressivelyinflatedbystepsof0.4mL,from0to1.2mL,each

steplasting5minutes.Dataarepresentedasthenumberofabdominal contractions/5minutes.

2.6 | Gut permeability

Twenty minutes after the stress procedure, male rats were le-thally anaesthetized (pentobarbital sodium 100 mg/kg ip (Ceva)). Immediatelyaftersacrifice,sectionsofthecolon,cutalongthemes-entericborder,weremountedinUssingchambers(Easymount).All segmentswereproperlyadjustedtofittheentireexposedtissuesur-faceareacorrespondingto0.5cm2_{.Chamberswerefilledwith5mL}

ofKrebs-Henseleitsolution(Sigma)maintainedat37°Candcontinu-ouslygassedwith95%O2/5%CO2.Transepithelialresistance(TER)

wasmonitoredthroughouttheexperimenttoassesstheviabilityof the tissue.26_{ Paracellular permeability was assessed by measuring}

mucosal-to-serosalfluxoffluoresceinisothiocyanate(FITC)-labeled 4kDadextran(Sigma).After10-minuteequilibriumperiod,500µL oftheKrebs-Henseleitsolutionwasreplacedby500µLoftheFITC-labeled4kDadextran(2.2mg/mLasthefinalconcentration)inthe mucosalside.After1hour,fluorescencewasmeasuredintheserosal buffersidebyafluorimeter(wavelengthof540nm,Tecan).Results wereexpressedasthefluxofdextrancrossingtheepithelialbarrier (nmol/cm2_/h).

2.7 | Transit time

Under general anesthesia (acepromazine 0.6 mg/kg ip (Calmivet, Vetoquinol)andketamine120mg/kgip(Imalgene,RhoneMerieux)), maleratswereequippedwithanintracoloniccatheter(Folioplast)and

F I G U R E 1   Experimentaldesigns.A,In

thefirstexperiment,theeffectofSP-11 onpartialrestraintstress(PRS)-induced visceral hypersensitivity to colorectal distensionwasevaluated.B,Thesecond experimentaimedtoinvestigatetheeffect ofSP-11onstress-associatedchangesof plasmaACTHandcorticosteronelevelsas wellasgutpermeability,mastcellnumber, andactivation(RMCPIIexpression).C,In thelastexperiment,theeffectofSP-11on PRS-inducedguttransittimeacceleration was analyzed

4 of 9 

|

     THEODOROU ETal. accustomedtoeat3hoursperdayduring15days.Then,theanimals received1µCiof51_CrO 4Na2(PerkinElmerLifeSciences)dissolvedin 0.1mLofsalinebyintracolonicrouteandplacedonaconveyorbelt supportingcollectortubeschangedevery60minutes.Theexcretion of 51_CrO

4Na2 was collected for each hour during 24 hours and was

analyzedwithagammacounter(CobraII;Packard,Meriden,CT,USA). The mean retention time (MRT) was calculated using the following formula:

t5,t15....t95correspondstotimesforwhich5,15...0.95%ofthe

markerwasexcretedinthefeces.

2.8 | Colonic mast cell numbers and

RMCPII expression

Distalcolonspecimenswerecollectedfrommaleratsandfixedin 4%bufferedformalinandincubated24hoursin30%sucroseat4°C. SampleswereembeddedinNeg50medium(Microm)andfrozenin isopentaneat−45°C.Cryostatsections(7µm)werepostfixedwith acetone(10minutes,−20°C)andhydratedinPBS-Tween.Afterin- cubationinblockingsolution(PBScontaining1%bovineserumal-buminand2%donkeyserum),sectionswereincubatedwithsheep anti-RMCPII antibodies (overnight, 4°C, 1/500) (Moredun) fol-lowedbyincubationwithAlexaFluor®594donkeyantisheepIgG (1hour30minutes,roomtemperature,1/2000)(LifeTechnologies). After each incubation period, sections were rinsed in PBS-Tween. Sections were mounted in ProLong® Gold Antifade Mountant (Life Technologies) and examined under a Nikon 90i fluorescence microscope(Nikon).Mastcellnumberpermm2 _{of mucosa and the}

intensityofRMCPIIwithinmastcellswerequantifiedusingNikon-Elements-Ar software. For each animal, five fields of view were counted.

2.9 | Plasma ACTH and corticosterone levels

Aftersacrifice(pentobarbitalsodium100mg/kgip),bloodforACTH and corticosterone levels was collected from the abdominal aorta into EDTA-containingVacutainer®tubes(Sigma).Thefreshlydrawnblood was centrifuged at 2000 gfor15minutesat4°C.Plasmawascollected andsubsequentlystoredat−80°CuntilusedforACTHandcorticos-teronedetermination.QuantificationofplasmaACTHwasperformed usingtheACTH(mouse/rat)ELISAkit(Tebu-bio),andcorticosterone wasevaluatedusingtheIDScorticosteroneEIAkit(Immunodiagnostic System)accordingtothemanufacturer'sinstructions.Plasmacorticos-teroneisexpressedinµg/mLandplasmaACTHisshowninng/mL.

2.10 | Statistical analysis

Foreachparameterstudied,datawereexpressedasmean±SEM. Forstatisticalanalysis,Prism4.0(GraphPad)wasused.Comparisons between the different groups were performed using an analysis of variance (one-way ANOVA) followed by a Bonferroni's post-test. AvalueofP<.05wasconsideredasstatisticallysignificant,anda value between P=<.05toP < .1 was considered as a trend toward statistical significance.

3 | RESULTS

3.1 | Visceral sensitivity

Colorectaldistensionincreasedthefrequencyofabdominalcon-tractionsper5minutesinavolume-dependentmannerinallrats. Comparing non-stressed (basal conditions) and stressed animals treatedwithsaline,thefirstvolumeofdistensionthatsignificantly increased the number of abdominal contractions was 0.8 mL, indicating a visceral hypersensitive response (16.45 ± 1.33 vs

MRT (h) =(t_{5+ t15+ … … … … .t95) ∕100.}

F I G U R E 2   InfluenceofSP-11onstress-inducedvisceralhypersensitivitytocolorectaldistension.A,Partialrestraintstress(PRS)

increasedtheabdominalcontractionsincomparisonwithnon-stressedsaline-treatedanimals(salinegroup:n=11andsaline+PRSgroup: n=10).SP-11hadnoeffectonvisceralsensitivityinbasalconditions(SP-11group:n=9),butsignificantlyreducedthePRS-inducedvisceral hypersensitivity(SP-11+PRSgroup:n=9).B,InhibitoryeffectofSP-11onvisceralhypersensitivityinducedbystressatthedistending volumeof0.8mL.++_{P<.01comparingnon-stressedandstressedsaline-treatedrats.}##_{P < .01 comparing stressed animals treated with}

24.70 ± 2.00 number of abdominal contractions/5 minutes, re-spectively,P<.01;Figure2A).Therefore,0.8mLwasconsidered asareferencevolumetoevaluatetheeffectofSP-11onvisceral sensitivity to CRD. SP-11 induced a significant inhibitory effect onstress-inducedvisceralhypersensitivityresponse(24.70±2.00 vs 14.00 ± 4.29 number of abdominal contractions/5 minutes, respectively, P < .01), but had no impact on visceral sensitiv-ityinresponsetoCRDinnon-stressedanimals(16.45±1.33vs 16.67 ± 2.58 number of abdominal contractions/5 minutes, re-spectively;Figure2B).

3.2 | Intestinal permeability

PartialrestraintstresssignificantlyincreasedtheFITC-dextranper- meabilityincolonicsegmentscomparedwithnon-stressedsaline-treatedrats(0.35±0.07vs0.95±0.05nmol/cm2_{/h,respectively,} P<.05).SP-11showedatrendtowardattenuatingthecolonichyper-permeabilityinducedbyPRS(0.95±0.05vs0.71±0.11nmol/cm2_/h, respectively,P=.07;Figure3).

3.3 | Gut transit

Afterstressinduction,colonicmeanretentiontimesignificantlyde-creasedincomparisonwithnon-stressedanimalstreatedwithsaline (9.85±0.65vs3.75±0.52hours,respectively,P<.01).Pretreatment with SP-11 significantly suppressed stress-induced colonic transit acceleration(3.75±0.52vs9.89±0.91hours,respectively,P < .01; Figure4).

3.4 | Mast cells

Compared with non-stressed saline-treated animals, PRS significantly increased RMCPII expression in mast cells (941676.93±120476.49vs1244611.35±238132.58totalmast cells/mm2_{, respectively, P < .05), without affecting the colonic}

mastcellnumber(50.23±4.08vs57.17±4.45mastcellnumber/ mm2_{, respectively). Pretreatment with SP-11 had also no}

influ-enceoncolonicmastcellnumbers(57.17±4.45vs49.78±3.22 mast cell number/mm2_{, respectively), but significantly reduced}

the RMCPII levels in stressed rats (1 244 611.35 ± 238 132.58 vs 830 665.39 ± 169 482.81 total mast cells/mm2_{, respectively,}

P<.05;Figure5A,B).

3.5 | Corticosterone and ACTH levels

Partialrestraintstressledtoasignificantincreaseinplasmacorti-costerone levels compared with non-stressed saline-treated rats (202.69±8.43vs448.76±28.30µmol/mL,respectively,P<.01). SP-11 showed a trend toward reduced corticosterone levels

(448.76±28.30vs355.78±29.58µmol/mL,respectively,P=.05; Figure6A).

PRS also resulted in an increase of plasma ACTH levels in com-parison with non-stressed saline-treated rats (46.66 ± 6.76 vs 167.43±23.56nmol/mL,respectively,P<.01).SP-11significantlyre-ducedthestress-inducedincreaseinplasmaACTHlevel(167.43±23.56 vs98.52±20.72nmol/mL,respectively,P<.01;Figure6B).

4 | DISCUSSION

This study shows that a short-term pretreatment (4 days prior to stressexposure)withSP-11reducedtheHPAaxisresponsetoacute F I G U R E 3   EffectofSP-11onintestinalhyperpermeability inducedbystress.Partialrestraintstress(PRS)increasedintestinal FITC-dextranpermeabilityincomparisonwithnon-stressedanimals treatedwithsaline(salinegroup:n=8andsaline+PRSgroup: n=9,*P<.05).SP-11treatmentshowedatrendtowardreduced guthyperpermeabilityofstressedanimals(SP-11+salinegroup: n=8,P=.07) F I G U R E 4   EffectofSP-11ongutintestinaltransit.Partial restraintstress(PRS)acceleratedintestinaltransitincomparison withsaline-treatednon-stressedrats(salineandsaline+PRS group:n=10,**P<.01).SP-11reducedstress-inducedguttransit acceleration(SP-11+PRSgroup:n=10,**P<.01)

6 of 9 

|

     THEODOROU ETal.

stress in rats as demonstrated by a significant decrease in plasma ACTH and a trend toward reduced corticosterone levels. Further, at the peripheral level, SP-11 significantly reduced stress-induced activationofmastcells,asindicatedbythedecreaseinintracellular RMCPIIexpression,significantlyreducedthevisceralhypersensitiv-ityaswellasguttransitacceleration,andshowedatrendtoward reduced gut hyperpermeability.

Therearenopreviouslypublishedstudiesexploringthemodeof actionofSP-11,althoughafewclinicalreportsdocumentitsusefor gastrointestinalsymptomswithreferencinganexpectedactionon the smooth musculature.23,27 _{We speculate that the mode of action}

will be a rather complex “network pharmacology”28 _{of potentially}

hundreds of targets covering immune, endocrine, smooth muscle and neural molecular interactions. The present study is the first at-tempt to characterize this medicinal product comprehensively in a modelofIBS.

The effects of SP-11 were first assessed by focusing on the two major features of IBS pathophysiology, visceral hypersensi-tivity, and impaired intestinal barrier function. SP-11 showed a trend toward reduced stress-induced gut hyperpermeability and significantly reduced visceral pain. The visceral hypersensitivity

observedinIBSisdefinedasanenhancedperceptionofmechani-caltriggers(pressuresofvolumes)appliedtothegut.Thepioneer workofRitchie(1973)29 _{reported for the first time the enhanced}

painfulresponseofIBSsufferersresultingfromrectaldistension. Later,otherclinicalstudiesconfirmedthattheincreasedpainper-ception or discomfort in response to rectal distension is an import-antclinicalfeatureinthemajorityofIBSpatients.30,31 _{The origin}

ofvisceralhypersensitivityisnotfullyunderstoodyet,butcentral (stress, anxiety, depression) and/or peripheral factors (increased gutpermeability,low-gradeinflammation,mucosalmastcellacti-vation,anddysbiosis)arediscussed.32_{Avisceralhypersensitivity}

in response to colorectal distension was also reported in animal models of stress.13,16

Thecentraloriginofthestress-inducedvis-ceral hypersensitivity was attributed to CRF and the peripheral one to colonic mast cell degranulation and increased gut permea-bility.13,16

Despitethedebateattributingtheincreaseingutper-meabilitymostlytoIBS-Dpatients,33_{Picheetal(2009)}9 _showed

that increased intestinal paracellular permeability is a common characteristic of all IBS subtypes. This increased gut permeabil-ityisreflectedbyalterationsinthetightjunctioncomplex.InIBS patientsincreasedclaudin2proteinexpression,34 _{downregulation} F I G U R E 5   InfluenceofSP-11onmastcellnumberandactivation.A,Partialrestraintstress(PRS)didnotaffectmastcellnumber(saline andsaline+PRSgroup:n=10andSP-11+PRSgroup:n=9);B,butinducedanincreaseinintracellularRMCPIIexpressioncomparedwith saline-treatednon-stressedrats(salineandsaline+PRSgroup:n=7,*P<.05).SP-11significantlyreducedtheRCMPIIexpression(SP-11+PRSgroup:n=7,*P<.05) F I G U R E 6   EffectofSP-11onplasmacorticosteroneandACTHlevels.AandB,Partialrestraintstress(PRS)increasedboth corticosteroneandACTHplasmalevelscomparedwithnon-stressedanimalstreatedwithsaline(salineandsaline+PRSgroup:n=10, **P<.01).SP-11treatmentshowedatrendtowardreducedplasmacorticosteronelevelsandsignificantlyreducedtheplasmaACTH concentration(SP-11+PRSgroup:n=10forACTH,**_{P<.01andn=9forcorticosterone,P=.05)}

ofZO-19_{andoccludinexpression}35,36 _{as well as redistribution of}

claudin 136_{wereobserved.Inanimalmodelsofstress,anincrease}

in gut permeability was also described16,37 _{resulting from}

colono- cytemyosinlightchain(MLC)phosphorylationleadingtocytoskel-etoncontractionandsubsequenttightjunctionopeningaswellas fromoccludinandJAM-Adown-regulation.38

Further,acause-ef-fect relationship between stress-induced gut hyperpermeability and visceral hypersensitivity was shown in rats.16_{ Interestingly,}

in IBS patients a positive correlation between increased intesti-nal permeability and visceral pain was also reported.39 _The

epi-thelial barrier impairment observed in both humans and animals mayresultfromanuptakeofluminalcontents(antigens,microbial patterns,etc)abletoactivatethemucosalimmunityandrelease ofmediators,which,inturn,sensitizeafferentneuronsleadingto visceral hypersensitivity.40

Anothermajorplayerabletobeacti- vatedbystressand,inturn,releasemediatorsimpairingtheepi-thelialbarrierintegrityisamastcell.ThestudyfromVanuytselet al(2014)12 _{in humans clearly showed that increased small bowel}

permeability induced by acute psychological stress or CHR ad- ministrationwaspreventedbyco-administrationofdisodiumcro-moglycate(amastcellstabilizer).Besidestheeffectofmastcell mediatorsonintestinalepithelialbarrier,theymayalsoplayarole in visceral sensitivity. For example, Wang et al (2014)41 _showed

thathistamineandmastcellproteaseIIreleasedbymastcellsmay diffuse in a paracrine manner and sensitize enteric nerve termi-nalsleadingtoenhancedsensitivityofspinalafferents.Similarly, Barbaraetal(2007)42

demonstratedthatmediatorsfromIBSpa-tientsstronglyexciteratnociceptivesensorynervesinthedorsal rootganglia.SP-11didnotmodifycolonicmastcellnumbersbut significantly inhibited mast cell activation as reflected by the re-duction in the RMCPII immunostaining. This finding is in agree-ment with a previous study showing that an acute stress in rats induces activation of colonic mast cells as reflected by intracellular increase in histamine content without modification of the mucosal mast cell number.19_{Interestingly,accordingtothesedata,SP-11}

reducedtheHPAaxisresponsetostressbysignificantlyreducing plasmalevelsofACTHandshowingatrendtowardreducedcor-ticosterone levels. Further, SP-11 also reduced peripheral stress manifestations such as colonic mast cell activation by reducing theircontentinproteases(RMCPII),welldescribedasapronoci-ceptivemediator,43_{andguttransit.Alltheseresultsclearlyshow} thattheSP-11treatmentaffectsthegut-brainaxisregulationin themodelofstress-inducedIBSusedherein.Dysregulationofthe gut-brainaxisinIBSisknowntodependonseveralfactors.Among thesefactors,stressplaysacrucialrole.44,45_{Inthestress-exposed} gut-brainaxis,themaineffectoristheHPAaxisandtheactiva-tion of the autonomic nervous system, which is responsible for theperipheralmanifestationsofstress.ConcerningtheHPAaxis responsetostress,IBSpatientsinfusedbyCRHrespondedwith greaterincreaseinbothACTHandcortisolsuggestingahyperre-sponsivenessofthestimulatedHPAaxis.44

Inanimals,similarob-servationsweredocumented.Indeed,rodentssubmittedtoacute or chronic stress show an increase in corticosterone and ACTH

plasma levels and central CRH-positive neurons46,47 _{as well as}

peripheralincreaseinCRFreceptorexpression,48 _{confirming the}

relevance of animal models of stress in the investigation of IBS pathophysiology.

The peripheral manifestation of stress includes also gut motil-itydisturbances.IBSpatientsexhibitanincreasedcolonicandsmall intestine motor response to stress when compared to healthy sub-jects.49_{Inrodentmodels,theabnormalgutmotorpatternwasalso}

documented several years ago.50

Indeed,anaccelerationofgastro-intestinal transit was shown using the restraint stress model51 _and

confirmedlaterbyothersusingothermodelsofstress(wateravoid-ancestress,earlylifetrauma)52,53

orbyCRFadministrationmimick-ing the stress effects.54_{Inthisstudy,thestress-inducedacceleration}

oftheguttransitwassignificantlyreducedbySP-11.

Takenthesefindingsaltogether,thefollowingmodeofaction isproposed:SP-11reducesthestress-inducedHPAaxisactivation and dampens stress-induced mast cell activation, preventing in turn intestinal epithelial barrier disruption and visceral hypersen-sitivity.ThereductionintheHPAaxisresponsetostressbySP-11 probably leads also to a reduced autonomic nervous system acti-vation resulting in a normalization of gut transit acceleration. The prevention of visceral hypersensitivity may result from a reduced direct activation of sensory nerve endings by mast cell mediators or by an indirect effect on the sensory nerve sensitization related toreducedluminalcontentuploadandpro-inflammatorymediator releasebythemucosalimmunity.Viceversa,thereducedHPAaxis activationcouldalsobearesultofaneffectofSP-11atthegut level,asthebrainandgutcommunicateinacomplexbidirectional way. Inconclusion,thesedatademonstratethepotentialforSP-11in thetreatmentofIBS.Afteroraladministration,theHPAaxisshowed a significantly reduced activation in terms of ACTH and a trend toward reduced corticosterone levels. Moreover, a significantly reduced visceral sensitivity to colorectal distension and mast cell activation,asignificantnormalizationoftheguttransittime,anda trend toward reduced gut permeability were shown. The observed effects and the good tolerability of the drug23

makeSP-11aninno-vativecandidateinthemanagementofIBS.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the EZOP animal facility for animalcare.ThismanuscriptisdedicatedtoLBwhodiedafterthe experimentswerecompleted.ThestudywasfundedbyHeelGmbH, Baden-Baden,Germany.Thefunderwasneitherinvolvedinthecon-duct of the study nor in data analysis.

CONFLIC TS OF INTEREST

YB,AM,andBSareemployeesofHeel.

AUTHOR CONTRIBUTIONS

VTdraftedthemanuscript;CB,SY,andGFperformedtheexperi-mentsandanalyzedthedata;YBcontributedtotheinterpretation of the results; AM critically reviewed and revised the manuscript;

8 of 9 

|

     THEODOROU ETal.

BSdesignedthestudyandcriticallyreviewedandrevisedthemanu- script;LBdesignedthestudyandinterpretedthedata,andHEana-lyzed and interpreted the data and drafted the manuscript.

ORCID

Helene Eutamene https://orcid.org/0000-0002-2983-1938

REFERENCES

 1. WhiteheadWE,PalssonOS,SimrenM.Irritablebowelsyndrome: whatdothenewRomeIVdiagnosticguidelinesmeanforpatient management? Expert Rev Gastroenterol Hepatol.2017;11:281-283.  2. EnckP,AzizQ,BarbaraG,etal.Irritablebowelsyndrome.Nat Rev

Dis Primers.2016;2:16014.

 3. DeanBB,AguilarD,BarghoutV,etal.Impairmentinworkproduc-tivity and health-relatedquality of lifein patients with IBS. Am J

Manag Care.2005;11:S17-26.

 4. Peery AF, Dellon ES, Lund J, et al. Burden of gastrointestinal disease in the United States: 2012 update. Gastroenterology. 2012;143:1179-1187.

 5. FichnaJ,StorrMA.Brain-GutInteractionsinIBS.Front Pharmacol. 2012;3:127.

 6. Barbara G, De Giorgio R, Stanghellini V, Cremon C, Corinaldesi R. A role for inflammation in irritable bowel syndrome? Gut. 2002;51(Suppl1):i41-44.

 7. GuilarteM,SantosJ,deTorresI,etal.Diarrhoea-predominantIBS patients show mast cell activation and hyperplasia in the jejunum.

Gut.2007;56:203-209.

 8. Rajilić-StojanovićM,JonkersDM,SalonenA,etal.Intestinalmicro-biotaanddietinIBS:causes,consequences,orepiphenomena?Am

J Gastroenterol.2015;110:278-287.

 9. PicheT,BarbaraG,AubertP,etal.Impairedintestinalbarrierinteg-rity in the colon of patients with irritable bowel syndrome: involve-ment of soluble mediators. Gut.2009;58:196-201.

10. Monnikes H, Tebbe JJ, Hildebrandt M, et al. Role of stress in functional gastrointestinal disorders. Evidence for stress-induced alterations in gastrointestinal motility and sensitivity. Dig Dis. 2001;19:201-211.

11. Greenwood-VanMeerveldB,MoloneyRD,JohnsonAC,VicarioM. Mechanismsofstress-inducedvisceralpain:implicationsinirritable Bowelsyndrome.J Neuroendocrinol;2016:28,1-10.

12. VanuytselT,vanWanrooyS,VanheelH,etal.Psychologicalstress and corticotropin-releasing hormone increase intestinal per-meability in humans by a mast cell-dependent mechanism. Gut. 2014;63:1293-1299.

13. GueM,DelRio-LachezeC,EutameneH,TheodorouV,Fioramonti J, Bueno L. Stress-induced visceral hypersensitivity to rectal dis-tensioninrats:roleofCRFandmastcells.Neurogastroenterol Motil. 1997;9:271-279.

14. SchwetzI,BradesiS,McRobertsJA,etal.Delayedstress-induced colonichypersensitivityinmaleWistarrats:roleofneurokinin-1and corticotropin-releasingfactor-1receptors.Am J Physiol Gastrointest

Liver Physiol.2004;286:G683-691.

15. Sun Y, Liu FL, Song GQ, Qian W, Hou XH. Effects of acute and chronic restraint stress on visceral sensitivity and neuroendocrine hormones in rats. Chin J Dig Dis.2006;7:149-155.

16. Ait-Belgnaoui A, Bradesi S, Fioramonti J, Theodorou V, Bueno L. Acute stress-induced hypersensitivity to colonic distension de-pends upon increase in paracellular permeability: role of myosin light chain kinase. Pain.2005;113:141-147.

17. WoutersMM,VicarioM,SantosJ.Theroleofmastcellsinfunc-tionalGIdisorders.Gut.2016;65:155-168.

18. BischoffSC,KramerS.Humanmastcells,bacteria,andintestinal immunity. Immunol Rev.2007;217:329-337.

19. Eutamene H, Theodorou V, Fioramonti J, Bueno L. Acute stress modulates the histamine content of mast cells in the gastrointes-tinaltractthroughinterleukin-1andcorticotropin-releasingfactor release in rats. J Physiol.2003;553:959-966.

20. JembrekMJ,AuteriM,SerioR,VlainicJ.GABAergicSysteminac-tion: connection to gastrointestinal stress-related disorders. Curr

Pharm Des.2017;23:4003-4011.

21. FraseW.GastricumeelundSpascupreel-analternativetherapyin the treatment of spasmodic gastritis. Biol Ther.1986;4:62-64. 22. WeiserM,ReusV.TreatingspasmodicconditionswithSpascupreel:

results of a prospective study. Biologische Medizin.2000;29:14-17. 23. Müller-Krampe B, Oberbaum M, Klein P, Weiser M. Effects of

Spascupreel versus hyoscine butylbromide for gastrointestinal cramps in children. Pediatr Int.2007;49:328-334.

24. Williams CL, Peterson JM, Villar RG, Burks TF. Corticotropin-releasing factor directly mediates colonic responses to stress. Am

J Physiol.1987;253:G582-586.

25. MorteauO,HachetT,CaussetteM,BuenoL.Experimentalcolitis alters visceromotor response to colorectal distension in awake rats.

Dig Dis Sci.1994;39:1239-1248.

26. Westerhout J, Wortelboer H, Verhoeckx K. Ussing Chamber. In: Verhoeckx K, Cotter P, Lopez-ExpositoI, et al. eds. The Impact of

Food Bioactives on Health: In vitro and Ex vivo Models.Cham(CH):Sp

ringer;2015:263–273.

27. Müller-KrampeB,KleinP,WeiserM.BehandlungvonSpasmenbei Kindern. JATROS PÄD.2004;10:20-22.

28. Li S, Zhang B. Traditional Chinese medicine network pharma-cology: theory, methodology and application. Chin J Nat Med. 2013;11:110-120.

29. RitchieJ.Painfromdistensionofthepelviccolonbyinflatingabal-loon in the irritable colon syndrome. Gut.1973;14:125-132. 30.

BouinM,MeunierP,Riberdy-PoitrasM,PoitrasP.Painhypersen-sitivity in patients with functional gastrointestinal disorders: a gas-trointestinal-specificdefectorageneralsystemiccondition?Dig Dis

Sci.2001;46:2542-2548.

31. LudidiS,ConchilloJM,KeszthelyiD,etal.Rectalhypersensitivityas hallmark for irritable bowel syndrome: defining the optimal cutoff.

Neurogastroenterol Motil.2012;24(729–733):e345-726.

32. SinagraE,MorrealeGC,MohammadianG,etal.Newtherapeutic perspectivesinirritablebowelsyndrome:Targetinglow-gradein-flammation, immuno-neuroendocrine axis, motility, secretion and beyond. World J Gastroenterol.2017;23:6593-6627.

33. DunlopSP,HebdenJ,CampbellE,etal.Abnormalintestinalperme-ability in subgroups of diarrhea-predominant irritable bowel syn-dromes. Am J Gastroenterol.2006;101:1288-1294.

34. MartínezC,LoboB,PigrauM,etal.Diarrhoea-predominantirrita-ble bowel syndrome: an organic disorder with structural abnormal-ities in the jejunal epithelial barrier. Gut.2013;62:1160-1168. 35. Coëffier M, Gloro R, Boukhettala N, et al. Increased

protea-some-mediated degradation of occludin in irritable bowel syn-drome. Am J Gastroenterol.2010;105:1181-1188.

36. Bertiaux-Vandaële N, Youmba SB, Belmonte L, et al. The ex-pression and the cellular distribution of the tight junction pro-teins are altered in irritable bowel syndrome patients with differences according to the disease subtype. Am J Gastroenterol. 2011;106:2165-2173.

37. Eutamene Hélène, Lamine F, Chabo C, et al. Synergy between Lactobacillus paracasei and its bacterial products to counteract stress-induced gut permeability and sensitivity increase in rats. J

Nutr.2007;137:1901-1907.

38. AgostiniS,GoubernM,TondereauV,etal.Amarketedfermented dairy product containing Bifidobacterium lactis CNCM I-2494 suppresses gut hypersensitivity and colonic barrier disrup-tion induced by acute stress in rats. Neurogastroenterol Motil. 2012;24:376-e172.

39. Zhou Q, Zhang B, Verne GN. Intestinal membrane permeabil-ity and hypersensitivpermeabil-ity in the irritable bowel syndrome. Pain. 2009;146:41-46.

40. HylandNP,QuigleyEM,BrintE.Microbiota-hostinteractionsinir-ritablebowelsyndrome:epithelialbarrier,immuneregulationand brain-gutinteractions.World J Gastroenterol.2014;20:8859-8866. 41. WangG-D,WangX-Y,LiuS,etal.Innervationofentericmastcells

by primary spinal afferents in guinea pig and human small intestine.

Am J Physiol Gastrointest Liver Physiol.2014;307:G719-731.

42. BarbaraG,WangB,StanghelliniV,etal.Mastcell-dependentex-citationofvisceral-nociceptivesensoryneuronsinirritablebowel syndrome. Gastroenterology.2007;132:26-37.

43. CenacN,AndrewsCN,HolzhausenM,etal.Roleforproteaseac-tivity in visceral pain in irritable bowel syndrome. J Clin Investig. 2007;117:636-647.

44. Dinan TG, Quigley EMM, Ahmed SMM, et al. Hypothalamic-pituitary-gut axis dysregulation in irritable bowel syndrome: plasma cytokines as a potential biomarker? Gastroenterology. 2006;130:304-311.

45. Drossman DA. Do psychosocial factors define symptom sever-ity and patient status in irritable bowel syndrome? Am J Med. 1999;107:41s-50s.

46. Ait-BelgnaouiA,DurandH,CartierC,etal.Preventionofgutleak-inessbyaprobiotictreatmentleadstoattenuatedHPAresponse to an acute psychological stress in rats. Psychoneuroendocrinology. 2012;37:1885-1895.

47. Ait-Belgnaoui A, Eutamene H, Houdeau E, Bueno L, Fioramonti J, Theodorou V. Lactobacillus farciminis treatment attenuates stress-inducedoverexpressionofFosproteininspinalandsupra-spinal sites after colorectal distension in rats. Neurogastroenterol

Motil.2009;21(567–573):e518-569.

48. VicarioM,AlonsoC,GuilarteM,etal.Chronicpsychosocialstressin-ducesreversiblemitochondrialdamageandcorticotropin-releasing

factorreceptortype-1upregulationintheratintestineandIBS-like gut dysfunction. Psychoneuroendocrinology.2012;37:65-77. 49. DuPontAW,JiangZ-D,HaroldSA,etal.Motilityabnormalitiesin

irritable bowel syndrome. Digestion.2014;89:119-123.

50. BuenoL.InvolvementofbrainCCKintheadaptationofgutmo-tility to digestive status and stress: a review. J Physiol Paris. 1993;87:301-306.

51. Williams CL, Villar RG, Peterson JM, Burks TF. Stress-induced changes in intestinal transit in the rat: a model for irritable bowel syndrome. Gastroenterology.1988;94:611-621.

52. DaSilvaS,Robbe-MasselotC,Ait-BelgnaouiA,etal.Stressdisrupts intestinal mucus barrier in rats via mucin O-glycosylation shift: prevention by a probiotic treatment. Am J Physiol Gastrointest Liver

Physiol.2014;307:G420-429.

53. RibaA,OlierM,Lacroix-LamandéS,etal.Panethcelldefectsin-duce microbiota dysbiosis in mice and promote visceral hypersensi-tivity. Gastroenterology.2017;153:1594-1606.

54. MillionM,GrigoriadisDE,SullivanS,etal.Anovelwater-solublese-lectiveCRF1receptorantagonist,NBI35965,bluntsstress-induced visceral hyperalgesia and colonic motor function in rats. Brain Res. 2003;985:32-42.

How to cite this article:TheodorouV,BeaufrandC,YvonS,

et al. The multicomponent medication Spascupreel attenuatesstress-inducedgutdysfunctioninrats. Neurogastroenterol Motil. 2020;00:e13798. https ://doi. org/10.1111/nmo.13798