Involvement Of Mucosal Flora And Enterochromaffin Cell Of Cecum And Descending Colon In Diarrhea-Predominant Irritable Bowel Syndrome

Background:Irritable bowel syndrome(IBS)is one of functional gastrointestinal disorders and severely impacts the quality of life.IBS is characterized by diverse symptoms like abdominal pain or distension,constipation or diarrhea.According to clinical manifestations,IBS is divided into diarrhea-predominant type(IBS-D),constipation-predominant type,mixed type and unsubtyped IBS.However,Current treatment approaches of IBS are dissatisfactory because of the undefined pathogenesis which is considered to be multifactorial.The dysbiosis of gut microbiota was deemed to be associated with IBS and previous studies indicated that probiotic therapies were beneficial for IBS patients.A systematic review summarized previous studies and suggested lack of consistency among studies,mucosal microflora assessment in particular.The intestinal flora has heterogeneous microbial communities in different segments of gut.It was reported that there were higher concentrations of short chain fatty acids(SCFAs)in the cecum of mice,the metabolic products of gut microbiota,which can promote production of 5-hydroxytryptamine(5-HT)by increasing the expression of tryptophan hydroxylase 1(TPH1),the rate-limiting enzyme of 5-HT synthesis.As an important neurotransmitter,5-HT is associated with altered motility and visceral discomfort.The vast majority of 5-HT is synthesized by enterochromaffin(EC)cell The bacterial abundance of cecum contents was 100 times more than that of the terminal ileum contents in People.Comparing the contents of human cecum and feces,there were significant differences in the bacterial abundance.The colonic flora is relatively stable among individuals due to the stable physical and chemical conditions.In addition,the colonic EC cell expresses a variety of receptors,which can directly sense the stimulation from the metabolites of intestinal flora.Therefore,colonic mucosa is also a good choice for the microbiota researches.However,there were little studies on the comparison between cecal mucosa and colonic mucosa in IBS patients and much unknown need to study.Aims1.To evaluate the differences of mucosal-associated flora between IBS-D patients and Health Controls(HCs);2.To evaluate the number of EC cells and TPH1 expression in IBS-D patients and HCs;3.To assess the EC cells number and bacterial abundance between the cecum and descending colon;4.To evaluate the correlation between intestinal flora and clinical parameters in IBS-D patients.Methods1.Collecting basic information of subjectsIBS-D patients and HCs were recruited in the Gastroenterology Outpatient Department at Qilu Hospital,Shandong University from Mar.2019 to Jul.2019.The information of subjects was collected by completing questionnaires.Biopsies from descending colon respectively were obtained using endoscopic biopsy forceps during colonoscopy withdrawal.2.Counting the number of EC cells by immunofluorescenceThe sections were incubated in rabbit anti-chromogranin A antibody and observed using fluorescence microscope.A total of 5 nonoverlapping high-power fields(final magnification 200 x)were chosen to count the number of EC cells.3.Evaluating the expression of TPH1 by Real-time Fluorescent Quantitative Polymerase Chain Reaction(RT-PCR)and western blottingTotal RNA and protein were extracted using AllPrep DNA/RNA/Protein Mini Kit according to manufacturer's protocol.RT-PCR reactions were made to evaluate the expression of TPH1 in transcriptional level.Polyvinylidenedifluoride membranes with protein were exposed to rabbit anti-TPH1 antibody and enhanced chemiluminescence kit was used to reveal the reaction products.4.Mucosal microbiota assessmentThe V3-V4 hypervariable regions of the bacteria 16S rRNA gene were amplified by the thermocycler PCR system and relative abundance was analyzed between groups.The correlation among EC cell number,Degree of Abdominal Pain,Frequency of Abdominal Pain,Frequency of Defecation,Hamilton Anxiety Scale(HAM-A),Hamilton Depression Scale(HAM-D),IBS Severity Scores(IBS-SSS)and bacterial relative abundance were assessed by using Spearman correlation analysis.Results1.A total of 22 HCs and 22 IBS-D patients were recruited in this study.There was no difference in terms of sex,age and BMI(P>0.05)between two groups.However,the HAM-A and HAM-D in IBS-D patients were higher than that in HCs and the frequency of defecation increased in IBS-D patients(P<0.0001).2.The number of EC cells in cecum of IBS-D patients was more than that in HCs(P<0.05)and the number of EC cells of IBS-D patients tended to increase in descending colon(P=0.08).However,there was no difference between cecum and descending colon.3.Transcript level of TPH1 in IBS-D patients was higher than that of HCs in both descending colon and cecum(P<0.05).In addition,the results of western blotting revealed that the expression of TPH1 of IBS-D patients was also higher than HCs(P<0.05).However,the comparison between cecum and descending colon,there was no significant difference.4.The results of mucosal microbiota assessment showed no difference between IBS-D patients and HCs both in microbiota richness and microbiota variability.The community composition analysis showed no statistical difference between IBS-D patients and HCs on the level of phylum.Among bacterial preponderant genera,the relative abundances of Ruminococcus_torques_group and Dorea in descending colon of IBS-D patients increased compared with that of HCs(5.94%vs.2.29%,P=0.04183;2.68%vs.1.14%,P=0.04962),while Fusobacterium decreased in IBS-D patients(1.52%vs.1.89%,P=0.0345).Compared with microbiota of cecum in HCs,the relative abundance of Ruminococcus torques_group in IBS-D patients increased(4.91%vs.2.20%,P=0.04763)and the relative abundances of Fusobacterium and Raoultella decreased(1.58%vs.1.76%,P=0.03117;0.12%vs.1.66%,P=0.01892).However,there were no difference of gut microbiota between descending colon and cecum both in IBS-D patients and HCs.The Spearman correlation analysis showed that Ruminococcus_torques_group were positively associated with HAM-A(r=0.66,P=0.004),HAM-D(r=0.61,P-0.009),EC cell number(r=0.49,P=0.047),IBS-SSS(r=0.65,P=0.004),Degree of Abdominal Pain(r=0.63,P=0.007),Frequency of Abdominal Pain(r=0.63,P=0.007),Frequency of Defecation(r=0.60,P=0.011)The abundance of Dorea were positively associated with EC cell number(r=0.57,P=0.018),IBS-SSS(r=0.52,P=0.034),HAM-A(r=0.72,P=0.001),HAM-D(r=0.59,P=0.012),Frequency of Abdominal Pain(r=0.67,P=0.003).Conclusions:EC cell increased in cecum of IBS-D patients and the expression of TPH1 was higher than HCs both in cecum and descending colon.In addition,Ruminococcus_torques_group increased in IBS-D patients both in cecum and descending colon and Dorea increased in IBS-D patients in descending colon,which had a positive relation with the number of EC cell,the symptom of IBS-D,HAM-A,HAM-D.Therefore,the microbiota may be potential targets for the treatment of IBS-D patients but still need further studies.