Involvement Of Symbiotic/Dysbiotic Gut Microbiota On The Expression Of Intestinal Reg ? And GLP-1

Aims: Gut microbiota interacts with innate and adaptive immune system,playing a pivotal role in the maintenance of gut immunity.On the other hand,gut microbiota plays important roles in host energy and metabolism regulation by modulation of hormonal secretion.A balanced composition of symbiotic microbiota is thought to stimulate host homeostatic responses,while dysbiotic microbiota composition that causes a drastic imbalance between the beneficial and potentially pathogenic bacteria,is thought to disturb homeostatic of gut and associated with inflammatory disorders of the gut,and associated with metabolic diseases.Regenerating gene ?(Reg ?)protein plays pivotal role in host immune system by acting as an anti-microbial peptide in the intestinal mucosa.Glucagon-like peptide 1(GLP-1),an incretin hormone produced by intestinal endocrine cells is crucial regulators of processes that contribute to whole-body energy metabolism,including satiety,gut motility,insulin secretion,and glucose uptake.Therefore,in the present study we aimed to examine the effect of symbiotic/dysbiotic microbiota on the expression of Reg ? and related intestinal immunity,and on the expression of GLP-1 and related functions.Part 1 Regulation of Reg ? expression in relation to gut microbiota and mucosal immunityMethods: To investigate the effect of symbiotic gut microbiota on Reg ? expression and intestinal immunity,germ free mice was inoculated with a fecal bacterial suspension(fecal transplantation,FT)from SPF mice.To establish dysbiotic condition of gut microbiota,SPF mice were orally administered antibiotic(vancomycin;0.1 mg/ml)for 7 days.Gastrointestinal tissues were obtained from those experimental mice and examined in histopathology.The expression of Reg ? and cytokines was examined by immunohistochemistry and real time RT-PCR.In vitro,the expression of human Reg ? was evaluated in human colon carcinoma cell line(Caco-2)stimulated by related cytokines.Results: 1.Under the symbiotic microbiota condition induced by four weeks FT to GF mice,Reg ?g expression was significantly increased in the intestine of GF mice with FT compared with GF mice without FT.On the other hand,under the dysbiotic microbiota condition induced by seven days antibiotic treatment,Reg ?g expression was significantly decreased in the intestine and of the SPF mice with vancomycin treatment compared with the control SPF mice.2.Under the symbiotic microbiota condition induced by four weeks FT to GF mice,Th17 related cytokines(IL-6,IL-17 A,IL-22)m RNA expression were significantly increased in the intestine of GF mice with FT compared with GF mice without FT.On the other hand,under the disbiotic microbiota condition induced by seven days vancomycin treatment,Th17 related cytokines(IL-6,IL-17 A,IL-22)m RNA expression were significantly decreased in the intestine of the SPF mice with vancomycin treatment compared with the control SPF mice.3.The expression of Reg ? g m RNA in mice intestine tissues was positively correlated with that of Th17 related cytokines(IL-6,IL-17 A,IL-22).4.Th17 effective cytokines(IL-17 A and IL-22)induced human Reg ? expression in Caco-2 cell line,might through STAT3 signaling.Part 2 Effect of dysbiotic gut microbiota on GLP-1 expression and related functionsMethods: To establish dysbiotic condition of gut microbiota,SPF mice were orally administered different dose of vancomycin(0.1 mg/ml?0.2 mg/ml and 0.5 mg/ml)for5 weeks.Germ free mice was inoculated with a fecal dysbiotic bacterial suspension from a SPF mice with vancomycin(0.2 mg/ml)treatment,as control,fecal suspensions from SPF mice fecal were orally administered to GF mice.Animal body weight,food intake,GITT were monitored,serum glucose and Serum GLP-1 were evaluated.The expression of GLP-1 and GPR43 were examined by immunohistochemistry and real time RT-PCR.Results: 1.The observed food intake?body weight and GITT increased significantly in vancomycin(0.2mg/ml)treated mice compared with control SPF mice.Similarly,the observed food intake?body weight and GITT increased significantly in GF mice after five weeks FT with vancomycin treated fecal compared with the control GF mice with normal FT.2.The serum glucose and GLP-1 levels were significantly elevated in the SPF mice after 5 weeks vancomycin treatment compared with control SPF mice.Similarly,the serum glucose and GLP-1 levels were significantly elevated in GF mice after five weeks FT with vancomycin treated fecal compared with the control GF mice with normal FT.3.Proglucagon m RNA expression and GLP-1 postive cells were significantly increased in colon of SPF mice after 5 weeks vancomycin treatment compared with control SPF mice.Similarly,Proglucagon m RNA expression and GLP-1 postive cells were significantly increased in colon of GF mice after five weeks FT with vancomycin treated fecal compared with the control GF mice with normal FT.4.GPR43 expression was significantly increased in colon of SPF mice after 5 weeks vancomycin treatment compared with control SPF mice.Similarly,GPR43 expression was significantly increased in colon of GF mice after five weeks FT with vancomycin treated fecal compared with the control GF mice with normal FT.Conclusions:1.Gut microbiota affects the expression of intestinal Reg ?.The regulation of Reg ? by gut microbiota,might be induced through intestinal Th17 effective cytokines(IL-17 A and IL-22).2.Dysbiotic gut microbiota induced by vancomycin,affects the expression of intestinal GLP-1.Dysbiotic gut microbiota plays role in the intestinal motility and energy metabolism functions,might through GPR43/GLP-1 signaling.