The Effects Of Obesity On Intestinal Micro-ecology And Short-chain Fatty Acids

Objective: Intestine, the largest mammalian digestive organ, settled with a large number of microbial communities involved in the digestion and absorption. Short-chain fatty acids(SCFAs),a kind of metabolites of microbial communities, play an important role in maintaining the intestinal health and normal function. Intestinal epithelial cells(IECs), the first protective barrier of the gut, contact with the intestinal environment directly. In this study, we demonstrated the changes of microbial communities and SCFAs in intestine tract and discussed the effects of butyrate acid on IECs.Methods:(1) For the study of the changes of intestinal microbial communities, C57BL/6 mice were divided into obese group(HFD) and normal group(LFD). We determined the change of the intestinal microbial communities through Miseq high-throughput sequencing technologies.(2) To establish a rapid method to detect fecal SCFAs and determine the stability of the test results, feces from mature normal mice,mature normal rats, healthy people were collected, using gas chromatography mass spectrometry(GC-MS), to determine the contents of SCFAs of these samples and explore the way of existence of SCFAs in normal conditions for making sure of the feasibility and stability of this kind of method.(3) The colon contents from obesity and normal control mice separately were collected to determine the level of SCFAs in these samples by GC-MS for exploring the changes of SCFAs in obesity condition.(4) For researching the mode of existence of fecal SCFAs in the case of intestinal diseases such as inflammatory bowel diseases, C57BL/6 mice were divided into inflammatory bowel disease group(DSS) and normal group(NC). After 7 days, the intestinal contents were collected to detecting the change of SCFAs by GC-MS.(5) In vitro, in order to study the effect of butyrate acid on intestinal epithelial cells,SW480 cells were divided into 6 kinds of groups: NC Group, LPS Group, BU Group, BU+LPS Group, TSA Group and TSA+LPS Group. First, the influence of butyrate acid and TSA on cell growth condition and the cell proliferation were observed. Next, TLR4 expression was determined by FCM, western blot and quantitative PCR when cells treated with butyrate acid and TSA, and then the TLR4 expression was tested by western blot after different densities of butyrate acid and TSA treatment. Finally, the effects of butyrate acid and TSA on TLR4 downstream signal pathways activation were studied by western blot.Results:(1) After detecting bacterial 16 s r RNA by Miseq high-throughput sequencing technologies, we found that the richness and diversity of intestinal microbial community and the number of S24-7 and Bacteroides were decreased, whereas the Desulfovibrio was increased.(2) To detect the SCFAs from mature normal mice, mature normal rats, healthy people by GC-MS, we found that the SCFAs in feces were mainly acetic acid, propionic acid, butyric acid and valeric acid in normal conditions. The content of acetic acid was the highest, and then the butyric acid, propionic acid and the valeric acid content were relatively low, and all the SCFAs had s similar form of existence in different mammalian.(3) After detecting the SCFAs in feces from obese mice by GC-MS, we found that all SCFAs, in addition to acetic acid, had a different degree of decrease, and butyrate acid decreased more significantly.(4) During detecting the SCFAs from mice with colitis by GC-MS, the results showed that the change of SCFAs was similar to that in obesity cases under the condition of colitis. Except the acetic acid, the content of propionic acid, butyrate acid and valeric acid had a marked decline, especially the butyrate acid.(5) In vitro experiment, after the SW480 cells treated with butyrate acid and TSA, we found that the proliferation of SW480 cells was inhibited after butyrate acid and TSA treatment, and the expression of TLR4 was up regulated.Conclusions:(1) Miseq high-throughput sequencing technologies can be implemented for the detection and identification of intestinal microbial community, and significant changes have taken place when obesity occurs compared with normal conditions.(2) GC-MS can detect the short-chain fatty acids in faces quickly, and have potential clinical applications such as the auxiliary diagnosis of intestinal diseases.(3) The contents of SCFAs in feces have been down regulated in the condition of obesity and colitis.(4) Butyrate acid can increase the expression of TLR4, and regulate its downstream signal transduction.