Involvement And Mechanism Of Fecal Microbiota Transplantation On Gastrointestinal Motility In GF Mice

Aims Gut microbiota has been known to interact with not only the immune but also the neuroendocrine systems,involving in host physiology in the gastrointestinal(GI)tract.Gut hormones also play a pivotal role in the neuroendocrine system in GI tissues,and indeed affect the secretion,metabolism and motility of GI tract.Furthermore,since gut microbiota are possible to modify the attitudes of gut hormones,those effects may play pivotal roles in the fine-tuning of GI motility;however,this hypothesis has not been fully tested.Serotonin(5-hydroxytryptamine;5-HT),as a neurotransmitter and/or local hormone,may be a key player for not only GI motility but also GI immune system.Glucagon-like peptide 1(GLP-1),an incretin hormone,which produced by intestinal endocrine cells,is involved in metabolism as well as GI motility.Here,we first examined how gut microbiota affects the gastrointestinal motility.Then we tend to clarify the association among microbiota,5-HT expression and macrophage behavior in the GI tract,and how microbiota affects the link between GLP-1/GLP-1 receptor(GLP-1R)expression and motility of the GI tract.Part 1 Effect of fecal microbiota transplantation on gastrointestinal motility in germ free miceMethods Six weeks old wild-type ICR mice and germ free ICR mice were randomized into two groups: wild type control group(ordinary drinking water)and fecal microbiota transplantation(FMT)group(orally administered a fecal bacterial suspension prepared from specific pathogen-free mice).After fecal microbiota transplantation,GI tissues were obtained from the GF mice at various time points(0week,1week,2weeks and 4weeks).Morphological changes were investigated by microscope and HE staining.Gastrointestinal transit time(GITT)was measured by administration of carmine red solution.Gastric emptying and immune-related molecular were determined at 4weeks after FMT.Results The thickness of gastric,small intestinal and colonic mucosa was significantly thicker than that of 6-week-old and age matched germ free mice after 4 weeks of FMT,but no significant compare to the wide type group.The length of small intestine and colon in the FMT group was tend to be higher than that in the 6-week-old and age matched germ free mice,but no significant compare to the wide type group.The gastrointestinal transit time(GITT)was significantly longer in germ free mice than in wild type mice.The GITT was significantly shorter in FMT mice than in the 6-week-germ free mice and age-matched germ free mice,but no significant compare to the wide type group.The gastric emptying was lower in germ free mice than the group of wild type mice.The gastric emptying was higher in the FMT mice than in age matched germ free mice.The proportion of CD4 + T cells,the level of IL22 factor and the expression of Reg proteins was increased in FMT mice compare to the age matched germ free mice.Part 2 Involvement of gut microbiota in the association between gastrointestinal motility and 5-HT expression/M2 macrophage behavior in the gastrointestinal tractMethods Germ-free(GF)mice(6 weeks old)were orally administered a fecal bacterial suspension prepared from specific pathogen-free mice and sacrificed at 4 weeks later to obtain GI tissues.The expression of 5-HT and mannose receptor(MR)was examined by immunohistochemistry,and gastrointestinal transit time(GITT)was measured by administration of carmine red solution.The level of TPH,the rate-limiting enzyme in 5-HT synthesis was detected by realtime-PCR.Results The numbers of 5-HT-positive endocrine cells and muscularis MR-positive macropahges were significantly increase in upper GI and colon in GF mice with FMT than in GF mice without gut microbiota reconstitution.The GITT was significantly shorter in GF mice with fecal microbiota transplantation(FMT)than in control GF mice without FMT,and negatively correlated with the number of both 5-HT-positive cells in the upper GI and muscularis MR-positive macropahges throughout the GI tract.The number of 5-HT-positive endocrine cells and muscularis MR-positive macropahges was significantly correlated throughout the GI tract.Part 3 Involvement of gut microbiota in the association between GLP-1/GLP-1 receptor expression and gastrointestinal motilityMethods Germ-free(GF)mice(6 weeks old)were orally administered a fecal bacterial suspension prepared from specific pathogen-free(SPF)mice,and then after fecal mocrobiota transplantation(FMT)GI tissues were obtained from the GF mice at various time points.The change of GLP-1 and its receptor was examined by immunohistochemistry and Realtime PCR,and gastrointestinal transit time(GITT)was measured by administration of carmine red solution.Results GLP-1 was expressed in endocrine cells in the colonic mucosa,and GLP-1R was expressed in myenteric neural cells throughout the GI wall.GLP-1R-positive cells throughout the GI wall were significantly fewer in GF mice with FT than in GF mice without gut microbiota reconstitution.The GITT was significantly shorter in GF mice with FT than in control GF mice without FT,and correlated with the number of GLP-1R-positive cells throughout the GI wall.The GITT was significantly longer in GF controls than in SPF mice.When those mice were treated with GLP-1 agonist extendin4,GITT was significantly longer in GF mice group.Conclusions Fecal microbiota transplantation can promote gastrointestinal motility in germ free mice and play a key role by interacting with the immune and neuroendocrine system in the gastrointestinal tract.Gut microbiota A: is involved in the link between the accelerated GI motility and induction of 5-HT/muscularis macropahges axis in the GI tract,B: may accelerate or at least modify GI motility while suppressing GLP-1R expression in myenteric neural cells throughout the GI tract.