The Research Of Bacillus Subtilis, Enterococcus Faecalis And Enterococcus Faecium Supernatants Regulating The Serotonin Transporter Expression In Intestinal Epithelial Cells

Objects: Irritable Bowel Syndrome(IBS)is a common functional gastrointestinal disease characterized by symptoms such as abdominal pain,bloating and abdominal discomfort and has a relatively high prevalence worldwide.IBS severely lowers the quality of life of IBS patients and takes up medical resources excessively.However,the pathogenesis of IBS remains unclear over the decades.Further research is to be conducted to find more effective treatment.It has been indicated that the occurrence of IBS has an intimate relationship with gastrointestinal motility.Serotonin(5-hydroxytryptamine,5-HT)and serotonin transporter(SERT)are key substance in regulating gastrointestinal motility.It was demonstrated that the level of SERT is affected by gut microbiota.Numerous basic and clinic trails have shown the efficacy of probiotics in the treatment of IBS.Nevertheless,the mechanism underlying is ambiguous.Previous experiment showed that Lactobacillus rhamnosus GG supernatant can up-regulate SERT expression in intestinal epithelial cells and intestinal tissues of post-infectious IBS rats.Bacillus subtilis,Enterococcus faecalis and Enterococcus faecium are three common probiotics,but whether they influence SERT expression of enterocytes is unknown.Thus,the present study aims to confirm if they can influence SERT expression and to provide clue for the action of mechanism of probiotics in treating IBS.Methods: HT-29 cells and Caco-2 cells were treated respectively with Bacillus subtilis,Enterococcus faecalis and Enterococcus faecium supernatant for 12 hours and 24 hours.SERT mRNA and protein levels were detected by Real-Time PCR and Western-blot respectively.Data were collected and analyzed by statistical analysis of SPSS statistical software.Data were expressed as mean±standard deviation.Statistically,difference was only considered significant when P <0.05.Results: 1.Bacillus subtilis supernatant showed concentration dependent regulation manner to SERT mRNA and protein levels of Caco-2 cells,and the degree of upregulation decreased during the latter period.While Bacillus subtilis supernatant up-regulated SERT mRNA and protein levels of HT-29 at a earlier time in a concentration dependent regulation manner but the regulation has declined at 24 h.2.Enterococcus faecalis supernatant up-regulated SERT mRNA and protein levels of Caco-2 cells with a large degree at first and small at last.The regulation of SERT protein expression in Caco-2 cells was concentration dependent.Enterococcus faecalis supernatant up-regulated SERT mRNA and protein levels of HT-29 cells.There might be two climaxes during the period and up-regulation was concentration dependent.3.Enterococcus faecium up-regulated SERT mRNA and protein levels of Caco-2 cells and the up-regulation was time and concentration dependent.However,the up-regulation of Enterococcus faecium supernatant towards HT-29 cells emerged earlier than 12 h according to the inference,and did not follow concentration dependent manner.But there might be another climax of up-regulation after 24 hours in HT-29 cells treated with 1:20 dilution of Enterococcus faecium supernatant.4.The mixture of Bacillus subtilis and Enterococcus faecium(1:1)supernatant could also up-regulate SERT level in HT-29 and Caco-2 cells but was different from that when acting alone.Conclusions: Bacillus subtilis,Enterococcus faecalis and Enterococcus faecium supernatant can up-regulate the expression of SERT mRNA and protein,but does not share the same pattern.The regulation model of the mixture of Bacillus subtilis and Enterococcus faecium(1:1)supernatant on SERT mRNA and protein is more complex than that of Bacillus subtilis supernatant or Enterococcus faecium supernatant alone.