The Role Of Colonic Glucagon-like Peptide-1 And Intestinal Mucosal Barrier In Patients With Functional Constipation

Part Ⅰ Evaluation of clinical symptoms,psychological state,colonic motility,and rectal sensitivity in patients with functional constipationBackground:Psychological state,colonic motility and rectal sensitivity play important roles in the pathogenesis of functional constipation(FC).However,few studies comprehensively explored associations between psychological state,colonic motility,rectal sensitivity and clinical symptoms in patients with FC,and correlations among the three.Aim:To comprehensively evaluate clinical symptoms,psychological state,colonic motility and rectal sensitivity of FC patients,and to preliminarily explore how psychological state,colonic motility and rectal sensitivity are involved in the pathogenesis of FC.Methods:Clinical symptoms and psychological state were assessed using standardized questionnaires,colonic motility was evaluated using gastrointestinal transit time(GITT)and the Bristol Stool Form Scale(BSFS),and rectal sensitivity was examined using rectal balloon distention.These indicators were compared between FC patients and healthy controls,and associations between psychological state,colonic motility,rectal sensitivity and clinical symptoms in FC patients,as well as correlations among psychological state,colonic motility,rectal sensitivity in all subjects were analyzed.Results:Sixty FC patients who fulfilled the Rome IV criteria and 54 healthy controls were included in the present study.The median duration of constipation in patients was 15 years accompanied with obvious constipation-related symptoms(infrequent bowel movements,dry and hard stools,a sense of rectal obstruction,abdominal distension)and impaired quality of life.Compared to healthy controls,scores in anxiety,depression and sleep quality in FC patients were significantly increased;the BSFS score was significantly decreased and was significantly positively correlated with GITT(48h discharge rate of barium bars);the initial sensation threshold and defecation sensation threshold were significantly elevated.Correlation analysis showed that psychological scores(anxiety,depression and sleep quality)in FC patients were significantly positively correlated with the Patient Assessment of Constipation-Symptom Questionnaire(PAC-SYM),the Constipation Scoring System(CSS)and the Patient Assessment of Constipation-Quality of Life(PAC-QOL),and the BSFS score was significantly negatively correlated with PACSYM.Psychological scores(anxiety,depression and sleep quality)in all subjects were significantly negatively correlated with the BSFS score,and the initial sensory threshold was significantly positively correlated with the depression score and negatively correlated with the BSFS score.Conclusions:FC patients exhibited psychological disorders,slow colonic transit and rectal hyposensitivity,which correlated with clinical symptoms and might interact with each other through the brain-gut interaction to jointly participate in the pathogenesis of FC.Part Ⅱ Study on the role of colonic glucagon-like peptide-1 in functional constipationBackground:Colonic dysmotility and rectal hyposensitivity are considered as important pathogenic mechanisms of FC.Glucagon-like peptide-1(GLP-1)secreted by the intestine can regulate gastrointestinal motility and sensation and participate in the occurrence and development of several digestive system diseases.However,no studies explored the role and underlying mechanisms of colonic mucosal GLP-1 in patients with FC.Aim:To evaluate the expression of GLP-1 in the colonic mucosa in FC patients,and to preliminarily explore the effect of colonic GLP-1 on gut motility and rectal sensitivity in FC patients as well as possible mechanisms.Methods:The BSFS was used to evaluate colonic motility,and rectal balloon distention was used to test rectal sensitivity.Mucosal tissues from the rectosigmoid junction were collected in subjects for immunohistochemistry and quantitative real-time polymerase chain reaction(qRT-PCR)to evaluate the expression of colonic mucosal GLP-1 and GLP1 receptor(GLP-1 R),the colocalization of GLP-1 R and 5-hydroxytryptamine(5-HT)in the colonic mucosa was observed using immunofluorescence double staining;the densities of colonic mucosal L cells and enterochromaffin cells(ECCs)were calculated and the expression level of 5-HT was evaluated;correlations between colonic mucosal GLP-1 and colonic motility,rectal sensitivity,ECCs and 5-HT in FC patients were also analyzed.Results:Thirty FC patients and 21 healthy controls were included in the present study.Compared to healthy controls,FC patients showed slow colonic motility and rectal hyposensitivity;the protein and mRNA expression levels of GLP-1 and the protein expression of GLP-1R in the colonic mucosa were significantly higher.GLP-1R coexpressed with 5-HT in the colonic mucosa.The densities of colonic mucosal L cells and ECCs in FC patients were significantly higher than that of healthy controls.Correlation analysis displayed that the protein expression of colonic mucosal GLP-1 in FC patients was positively correlated with the desire to defecate threshold,GLP-1 mRNA expression,densities of L cell and ECCs;GLP-1 mRNA level was positively correlated with the desire to defecate threshold,GLP-1 R mRNA expression and L cell density.Conclusions:Increased GLP-1 in the colonic mucosa in FC patients is associated with rectal hyposensitivity.Colonic GLP-1 may interact with adjacent ECCs through a paracrine pathway,participating in the regulation of gut sensation.Part Ⅲ Evaluation of the structure and function of intestinal mucosal barrier in patients with functional constipationBackground:The intestinal mucosal barrier has selective absorption and secretory functions,and it is the first line of defense against harmful substances in the intestinal lumen,which contribute to the maintenance of inner homeostasis.Recent studies showed that the intestinal mucosal barrier impairment was involved in the occurrence and development of several intestinal diseases.Patients with FC exhibited slow colonic motility and rectal hyposensitivity,resulting in prolonged exposure of the intestinal mucosa to intestinal contents.However,no study comprehensively evaluated the intestinal mucosal barrier in FC patients.Aim:To comprehensively assess the structure and function of the intestinal mucosal barrier in FC patients,including the mucus barrier,intercellular junctional structures in the intestinal epithelium,mucosal immune cells,and gut permeability.Methods:Serum and colonic mucosal tissue samples were collected in subjects.Goblet cells were distinguished and counted using Alcian Blue/Periodic acid Schiff(AB/PAS)staining,the ultrastructure of intercellular junctional complexes in the colonic epithelium was observed under a transmission electron microscope,occludin and zonula occludens-1(ZO-1)in the colonic mucosa were located and quantified using immunohistochemistry,occludin and ZO-1 mRNA expression levels were examined using qRT-PCR,colonic mucosal CD3+ intraepithelial lymphocytes(IELs)and CD3+ lymphocytes in the lamina propria were identified and counted using immunofluorescence.Serum D-lactic acid levels were measured using enzyme-linked immunosorbent assay(ELISA).Results:Forty FC patients and 24 healthy controls were recruited in the present study.Compared to healthy controls,the number of goblet cells in the colonic mucosa was significantly increased in FC patients,and the mucus staining was darker.Intercellular junctional complexes in the colonic epithelium were continuous and integral in FC patients,and the localization,the expression of protein and mRNA in occludin and ZO-1 were not significantly altered.No statistical differences were observed in the number of colonic mucosal CD3+ IELs and CD3+ lamina propria lymphocytes between the two groups.FC patients did not have higher D-lactic acid levels than healthy controls.Conclusions:The intestinal mucosal barrier in FC patients showed a compensatory increase in the number of goblet cells as well as continuity and integrity of intercellular junctional structures in the colonic epithelium,and no obvious activation of mucosal immunity or increased gut permeability was found.