Lactobacillus Rhamnosus GG Promotes Defecation Function Via Upregulating Serotonin Receptor 4 In Mice Colon

AimStudies have shown that the probiotic Lactobacillus rhamnosus GG(LGG)can regulate the contraction of intestinal smooth muscle and improve symptoms in patients with constipation.Serotonin receptor 4(5-HT4R)is closely related to intestinal motility and the agonists of 5-HT4 R are effective in treating constipation.However,Tegaserod,one of the agonists of 5-HT4 R,has been withdrew because of the cardiovascular side effects.It is reported in a latest study that activation of 5-HT4 R can increase the release of mucin in goblet cells.As is known to all,goblet cells are responsible for forming the intestinal mucus layer.Many factors can disrupt the mucus layer by influencing function of goblet cells.For example,intestinal microorganisms,microbial toxins and certain cytokines can stimulate or inhibit the synthesis and secretion of mucin in goblet cells.What’s more,the decrease of goblet cells and change of mucus layer is crucial for the development of constipation.Therefore,this study aims to explore the regulatory effect of Lactobacillus rhamnosus GG supernatant(LGGs)on 5-HT4 R expression in colon,gut microbiota,intestinal barrier function and defecation function of mice,then provide experimental basis for probiotics in defecation function improvement.Methods40 male C57BL/6 mice 6-8 weeks in age were randomly divided into 3 groups: MRS group(n=10),Tegaserod group(n=15)and LGGs group(n=15).Intragastric administration of MRS,distilled water solution of Tegaserod and LGG supernatant(1ml/d)was respectively given to these 3 groups of mice for 7 days.The weight was recorded before and after the intragastric administration.Defecation parameter including number of pellets in 2 hours,fecal weight,fecal dry weight and fecal water content were detected.5 mice in each group were randomly selected for(gastrointestinal transit time)GITT test.Finally,all the mice were sacrificed and the colon tissue were collected for later use.The m RNA and protein levels of 5-HT4 R and MUC2 were detected by real-time PCR and Western blotting.Immunohistochemistry was used to detect the changes of 5-HT4 R and MUC2 expression in mice colon.Periodic Acid-Schiff(PAS)and Alcian Blue Periodic acid Schiff(AB-PAS)staining staining were used to evaluate number of goblet cells and mucin secretion in mice colon.Analysis of short chain fatty acids(SCFAs)and microbiota compostion were done by gas chromatography(GC)method and 16 S r DNA pyrosequencing analysis.To explore the possible upstream regulation mechanism of 5-HT4 R,Western blotting was used to measure level of S100A10.ResultsLGGs can upregulate the m RNA and protein level of 5-HT4 R,meanwhile,m RNA level of MUC2 was also upregulated by LGGs in mice colon tissue.Besides,the number of defecation pellets in 2 h,the fecal weight,fecal dry weight and fecal water content in the Tegaserod group and LGGs group are increased than in MRS group.PAS staining showed that the average number of MUC2-positive cells in the colon in Tegaserod group and LGGs group are increased than in MRS group.AB-PAS staining results indicated that agonist of 5-HT4R(Tegaserod)could increase mucin secretion especially in LGGs group when compared with MRS group and antagonist of 5-HT4R(Piboserd)could decrease secretion of mucin in mice goblet cells.Microbiota analysis indicated that LGGs altered the diversity among these groups.In genus level,LGGs increased abundance of Alistipes,Alobaculum and Desulfovibrioin which are related with gut barrier and motility.On the contrary,Tegaserod increased relative abundance of inflammatory bacteria and opportunistic pathogens,disorganize the normal composition of gut microbiota of mice.In addition,the level of S100A10 was increased in Tegaserod and LGGs groups compared with MRS group.Finally,our data indicated that no differences were found in total SCFAs among three groups.ConclusionsLGGs can increase mucin secretion to improve intestinal barrier function and defecation function via upregulating 5-HT4 R expression in mice colon.Meanwhile,compared with Tegaserod,LGGs can help to maintain the normal microbiota composition.