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

Abstract Background Irritable bowel syndrome (IBS) is a common disorder worldwide. It is characterized by abdominal pain/discomfort and changes in bowel habits. Due to the multifactorial pathophysiology and the heterogeneity of IBS patients, appropriate treatment of IBS is still a challenge. Spascupreel (SP‐11), as a multicomponent medication, has the potential to modulate multiple pathophysiological pathways simultaneously. Therefore, the objective of the current study was to investigate the effects of oral SP‐11 treatment on stress‐induced changes of peripheral and central functions in a rat model mimicking human IBS. Methods Naïve Wistar rats were treated with SP‐11 (0.9 tab/kg) or NaCl 0.9% by oral gavage for 4 days before 2‐hour partial restraint stress (PRS) procedure. Twenty minutes after PRS, central and peripheral stress‐induced changes affecting IBS were assessed. These include the hypothalamic‐pituitary‐adrenal (HPA) axis response through plasma ACTH and corticosterone measurements, visceral pain in response to colorectal distension, gut permeability, colonic mast cell number, and sensitization as well as gut transit time. Results Treatment with SP‐11 reduced the HPA axis activation in response to PRS. At the gut level, a reduction in colonic hypersensitivity to colorectal distension, a normalization of gut transit time acceleration, a reduced mast cell sensitization, and a trend toward reduced gut hyperpermeability were observed. Conclusions These data suggest that stress‐induced IBS signs can be reduced using SP‐11 in rats. The observed effects and the good tolerability of the drug make SP‐11 an innovative candidate in the management of IBS.


| INTRODUC TI ON
and represent a significant socioeconomic burden. 4 In the absence of a clear identification of organic features and lack of reliablebiomarkers,theIBSpathophysiologyisstillnotcompletelyunderstood.IBSsymptomsmayoriginatefromseveralperipheraland/ orcentralmechanisms.Amongthem,dysfunctionalgut-brainaxis, 5 low-gradeintestinalinflammation, 6 increaseinmucosalmastcells, 7 intestinalmicrobiotadysbiosis, 8 and impaired intestinal barrier function 9 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, and impaired intestinal barrier function, have been developed. 13,14 Indeed,inratsacuterestraintstressisreflectedbybothanincrease inadrenocorticotropichormone(ACTH)andcorticosteroneplasma concentrations. 15 This is associated with visceral hypersensitivity to colorectal distension (CRD), a central release of corticotrophin releasing 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 functional gastrointestinal disorders. 18 Inrats,acutestressincreasesthe colonicmastcellhistaminecontent,aperipheraleffect,mediatedby thereleaseinthecascadeofinterleukin-1(IL-1)andCRF. 19 Regarding the multifactorial pathophysiology and the heterogeneity of the IBS population, appropriate treatment of IBS is still a challenge. Therapeutic strategies are often limited to the treatment of symptoms with drugs focusing on motor/sensory abnormalities.

Inthefirstseries,visceralsensitivitytocolorectaldistensionwas
assessed by electromyography (EMG) recording in the same animals before (basal condition) and 20 minutes after the PRS session ( Figure 1A). The second series was performed to assess the
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).

| PRS procedure
Allstresssessionswereperformedatthesametimerangeofthe day(between10am and 12 pm)tominimizeanyinfluenceofcircadianrhythms.Stresseffectswerestudiedusingasingle2-hourses-sionofPRSwhichisconsideredasamildnon-ulcerogenicmodel. 24 Underlightanesthesiawithethylether,upperforelimbsweretaped up to the thoracic trunk in order to constrain animal body movements. Rats were then replaced in their home cages for 2 hours.

| Colonic mast cell numbers and RMCPII expression
Distal colon specimens were collected from male rats and fixed in 4%bufferedformalinandincubated24hoursin30%sucroseat4°C.   AvalueofP<.05wasconsideredasstatisticallysignificant,anda value between P=<.05toP < .1 was considered as a trend toward statistical significance.

| D ISCUSS I ON
This study shows that a short-term pretreatment (4 days prior to stressexposure)withSP-11reducedtheHPAaxisresponsetoacute Therearenopreviouslypublishedstudiesexploringthemodeof actionofSP-11,althoughafewclinicalreportsdocumentitsusefor gastrointestinal symptoms with referencing an expected action on the smooth musculature. 23,27 We speculate that the mode of action will be a rather complex "network pharmacology" 28  Later,otherclinicalstudiesconfirmedthattheincreasedpainperception 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,mucosalmastcellactivation,anddysbiosis)arediscussed. 32 Avisceralhypersensitivity in response to colorectal distension was also reported in animal models of stress. 13,16 Thecentraloriginofthestress-inducedvisceral hypersensitivity was attributed to CRF and the peripheral one to colonic mast cell degranulation and increased gut permeability. 13 in gut permeability was also described 16,37 resulting from colonocytemyosinlightchain(MLC)phosphorylationleadingtocytoskeletoncontractionandsubsequenttightjunctionopeningaswellas fromoccludinandJAM-Adown-regulation. 38 Further,acause-effect relationship between stress-induced gut hyperpermeability and visceral hypersensitivity was shown in rats. 16 Interestingly, in IBS patients a positive correlation between increased intestinal permeability and visceral pain was also reported. 39 The epithelial barrier impairment observed in both humans and animals mayresultfromanuptakeofluminalcontents(antigens,microbial patterns, etc) able to activate the mucosal immunity and release ofmediators,which,inturn,sensitizeafferentneuronsleadingto visceral hypersensitivity. 40 Another major player able to be acti- The peripheral manifestation of stress includes also gut motilitydisturbances.IBSpatientsexhibitanincreasedcolonicandsmall intestine motor response to stress when compared to healthy subjects. 49 Inrodentmodels,theabnormalgutmotorpatternwasalso documented several years ago. 50

ACK N OWLED G M ENTS
The authors gratefully acknowledge the EZOP animal facility for animalcare.ThismanuscriptisdedicatedtoLBwhodiedafterthe experimentswerecompleted.ThestudywasfundedbyHeelGmbH, Baden-Baden,Germany.Thefunderwasneitherinvolvedintheconduct of the study nor in data analysis.

CO N FLI C T S O F I NTE R E S T
YB,AM,andBSareemployeesofHeel.

AUTH O R CO NTR I B UTI O N S
VT drafted the manuscript; CB, SY, and GF performed the experimentsandanalyzedthedata;YBcontributedtotheinterpretation of the results; AM critically reviewed and revised the manuscript; BSdesignedthestudyandcriticallyreviewedandrevisedthemanuscript;LBdesignedthestudyandinterpretedthedata,andHEanalyzed and interpreted the data and drafted the manuscript.