The Role And Mechanism Of Fructo-oligosaccharide In Visceral Hypersensitivity And Intestinal Inflammation In An IBS Mouse Model

Irritable bowel syndrome(IBS)is a functional gastrointestinal disorder characterized by chronic abdominal pain associated with changes in bowel habit and frequency affecting more than tenth of the general population.Many factors contribute to the development of IBS including altered visceral sensitivity,low-grade intestinal inflammation,changes in microbiota,and psychosocial factors.The complex pathophysiology of IBS has posed challenges to developing effective interventions,and the poor effect of medical treatment has brought great financial and psychological burden to IBS patients.Importance of dietary factors in triggering symptoms is increasingly being recognized in patients with IBS.Specifically,poorly absorbed,fermentable carbohydrates categorized as Fermentable Oligosaccharides,Disaccharides,Monosaccharides,and Polyols(FODMAPs)have been studied.Consumption of food content high in FODMAPs triggers abdominal pain,bloating,and flatulence in patients with IBS.Furthermore several randomized trials have demonstrated that low FODMAPs diet reduces gastrointestinal symptoms in patients with IBS.Although largely unexplored,the accumulation of intestinal fluid from osmotic load of poorly digested carbohydrates and excessive colonic gas production associated with ingestion of FODMAPs has been proposed as a mechanism for the development of gastrointestinal symptoms.Intestinal dysmotility,visceral hypersensitivity,altered microbiota,and changes in metabolic output also likely contribute to the pathophysiology of gastrointestinal symptoms associated with ingestion of FODMAPs in IBS patients.In addition,the production of short-chain fatty acids(SCFAs),such as acetic,propionic,and butyric acids,may also be important in the development of symptoms in IBS.Fructo-oligosaccharide(FOS)is one of the most frequently consumed FODMAPs components in the general diet.The aim of our study was to investigate the effects of FOS on visceral sensitivity,intestinal SCFAs production,and intestinal inflammation in Water avoidance stress(WAS)-induced IBS mouse model.Part I.To Explore the Intestinal Immune Activation and Production of SCFAs in a Stress-Induced IBS Mouse ModelAimsTo investigate the differences of intestinal mucosal immune activation and production of SCFAs between control and stress-induced IBS mouse modelMethodFemale C57BL/6 mice were subjected to either WAS or sham-WAS for 10 consecutive days.Body weight and food intake were recorded every day.Abdominal withdrawal reflex(AWR)scores,pain thresholds,and volume thresholds were used to evaluate visceral sensitivity.SCFAs production was quantified by using gas chromatography.Mice were sacrificed by cervical dislocation,and intestines were harvested for histological evaluation.Intestinal cytokines expression such as TNF-a,interleukin(IL)-6,IL-23,IL-10 and IL-1? in ileal and colonic tissues were detected by quantitative real-time PCR.Intestinal mucosal mast cells were estimated by immunohistochemistry and the number of mucosal mast cell was counted under a light microscope by two independent observers.ResultsDuring the 10 days of modeling,mice receiving WAS had lower rate of weight gain compared to mice receiving sham-WAS(P<0.05),but there was no significant difference in the daily food intake between two groups(P>0.05).Mice subjected to WAS had higher mean abdominal withdrawal reflex scores at 20mmHg(3(2.5-3)vs.1.7(1.7-1.7),P<0.01),40mmHg(3.5(3.3-4.0)vs.2(2-2),P<0.01),60mmHg(4(3.8-4)vs.2.5(2.3-2.7),P<0.01)and 80mmHg(4(4-4)vs.3.2(3-3.3),P<0.01)pressure of colorectal distention,lower pain(33.2±18.0 mmHg vs.70.8±4.57mmHg,P<0.01)and volume thresholds(44.6±19.2mmHg vs.90.2±5.12mmHg,P<0.01)compared to mice subjected to sham-WAS.No difference of acetic acid,propionic acid,butyric acid and total SCFAs in the cecum was found between mice subjected to WAS and sham-WAS(P>0.05).No difference in histologic score were observed between WAS and sham-WAS groups both in ileal and colonic tissues(P>0.05).Mice subjected to WAS had higher expressions of IL-6(8.25±3.95 vs.1.86±1.66,P<0.01)and TNF-a(2.05±1.73 vs.0.56±0.28,P<0.05)mRNA in the ileal specimen,as well as,higher IL-6(1.60±1.10 vs.0.46±0.29,P<0.05)and IL-1p(0.88±0.53 vs.0.34±0.35,P<0.05)mRNA expression in the colonic specimen compared to those that received sham-WAS.Mice subjected to WAS had higher mean mast cell counts in the ileum(8.3±3.6 per HPF vs.4.9±1.4 per HPF,P<0.05)and colon(3.4±1.2 per HPF vs.1.8±1.5 per HPF,P<0.05)compared to those subjected to sham-WAS.ConclusionMice subjected to WAS established an IBS mouse model which simulating the subclinical malnutrition and visceral hypersensitivity of IBS patients.Mice subjected to WAS exhibited low-grade inflammation demonstrated by higher mucosal expressions of pro-inflammatory cytokines and increased number of intestinal mast cells but without difference in histologic score.No difference of SCFAs in the cecum was found between IBS mice and control mice.Part II.The Role and Mechanism of Fructo-oligosaccharide Intensifing Visceral Hypersensitivity and Intestinal Inflammation in an IBS Mouse ModelAimsTo determine whether FOS affects visceral sensitivity,inflammation,and SCFAs production in water avoidance stress induced IBS mouse model.MethodTo evaluate the effects of FOS on WAS induced-visceral hypersensitivity and intestinal inflammation,thirty-two C57BL/6 mice were randomly divided into four groups of eight mice(sham-WAS+saline administration,sham-WAS+FOS administration,WAS+saline administration,and WAS+FOS administration).Mice were randomly assigned to daily oral gavage of saline solution with or without FOS for 14 days.Mice were further assigned to receive either daily one-hour water avoidance stress(WAS)or sham-WAS for the first 10 days.After 2 weeks,visceral sensitivity was measured by abdominal withdrawal reflex in response to colorectal distension.Gas chromatography used to quantify cecal concentrations of SCFAs.Intestinal histology score,reverse transcription,and immunohistochemistry assays were used to evaluate mucosal inflammation,intestinal cytokine expression,and number of intestinal mast cells per high-power field(HPF),respectively.ResultsAmong mice subjected to WAS,FOS administration increased abdominal withdrawal reflex scores(4(3.5-4)vs.3(2.5-3),P<0.01)at the pressure of 20mmHg compared to saline administration,decreased pain(12.6±6.0 mmHg vs.33.2±18.0 mmHg,P<0.01)and volume thresholds(21.6±8.1 mmHg vs.44.6±19.2 mmHg,P<0.01)compared to saline administration.FOS administration also led to higher cecal concentrations of acetic acid(2.49±0.63 mmol/L vs.1.49±0.72 mmol/L,P<0.05),propionic acid(0.48±0.09 mmol/L vs.0.36±0.05 mmol/L,P<0.01),butyric acid(0.28±0.09 mmol/L vs.0.19±0.003 mmol/L,P<0.05),as well as total SCFA(3.62±0.87 mmol/L vs.2.27±0.75 mmol/L,P<0.01)compared to saline administration.FOS also increased ileal interleukin(IL)-23 mRNA(4.71±4.16 vs.1.00±0.99,P<0.05)and colonic IL-1? mRNA(2.15±1.68 vs.0.88±0.53,P<0.05)expressions as well as increased mean mast cell counts in the ileum(12.3±2.6 per HPF vs.8.3±3.6 per HPF,P<0.05)and colon(6.3±3.2 per HPF vs.3.4±1.2 per HPF,P<0.05)compared to saline administration in mice subjected to WAS.No difference in visceral sensitivity,intestinal inflammation,and cecal SCFA levels were detected with or without FOS administration in mice subjected to sham-WAS.ConclusionIn conclusion,administration of FOS,a component of FODMAPs,intensified visceral hypersensitivity and gut inflammation in stressed induced-IBS mice,but not in control mice.A parallel increased production of intestinal SCFAs was also observed with FOS administration in IBS mice but not in control mice.Our findings suggest a mechanism of FODMAPs-induced gastrointestinal symptoms specific to IBS.