Establishment And Application Of In Vitro Culture Model Of Gut Bacteria From Prediabetic Mice

The gut microbiota is considered to be a new "organ" of the human body and plays an important role in the host’s metabolism and the development of disease.The in vitro evaluation model of gut bacteria has the characteristics of high throughput and ability to clarify the influence of different factors,so it has bright prospects of applying.However,the current in vitro evaluation model neither optimized the culture conditions nor characterized the difference between the gut microbiota after culturing and the gut microbiota in stool,which could affect the accuracy in studies.In response to the above problems,this study has carried out the following research:Firstly,an in vitro culture model of gut microbiota of prediabetic mice was constructed.Based on the similarity between the gut microbiota after culturing and the gut microbiota in stool,the GAM medium with the best culture effect was selected to be the initial medium and optimized to increase the similarity between the gut microbiota after culturing and the gut microbiota in stool from the media such as YCFA,MPYG and GAM.The factors that significantly affect the similarity were determined through the PB experiment,the interval of the optimal similarity were determined through the steepest climbing experiment,and the optimal similarity was predicted by the Box-Behnken Design.Therefore,optimal media composition was obtained.The culture condition is 37℃,200 r/min anaerobic culture for 24 h.The similarity between the gut microbiota after culturing and the gut microbiota in stool was49.4%,which was an increase of 30.7% compared with the pre-optimized conditions.It is reported that the combination of acarbose and baicalein in vivo could lower blood sugar and improve the side effects of acarbose,but its mechanism of action was still unclear.The constructed in vitro culture model was used to investigated the effects of acarbose alone,baicalein alone,their combination,and the starch hydrolysate after their treatment on gut microbiota,and the gas production and short chain fatty acid content and other indicators related to drug efficacy and side effects.The results showed that acarbose and the hydrolysate after treatment with acarbose significantly increase gas production,and unhydrolyzed starch was the main reason for acarbose to lead to flatulence,and the higher the starch content,the higher the gas production,while baicalein could significantly reduce gas production.In addition to high-dose baicalein,acarbose,low-dose baicalein and starch hydrolysate could significantly increase the production of short chain fatty acids.The analysis of the diversity of gut microbiota showed that acarbose and baicalein could increase the diversity of gut microbiota.Acarbose and starch hydrolysate after the treatment of acarbose significantly increased the relative abundance of Parabacteroides,Clostridiales,Dorea,Coprococcus,Enterobacter,etc.,which had positive correlations with gas production,decreased the relative abundance of Lactobacillus,Bifidobacterium,which were had negative correlations with gas production.Thereby increasing gas production and the ratio of hydrogen,leading the side effect of flatulence.And baicalein could increase the abundance negatively correlated with gas production and reduce the side effects of acarbose by inhibiting the relative abundance of gas producing bacteria.To varying degrees,acarbose,baicalein and their starch hydrolysate could increase the abundance of SCFA-producing bacteria such as Roseburia,Clostridium,Akkermanisia,etc..Acetic acid can promote the development of peripheral tissues,propionic acid can promote the growth of hepatocytes,butyric acid can promote the growth of intestinal epithelial tissues.SCFA can promote the normal maturation of intestinal cells and differentiation.In addition,SCFA can also provide energy for the host,act as a signal molecule to participate in the host’s metabolism,reduce inflammation,improve insulin sensitivity,and thereby improve diabetes.This study successfully constructed an in vitro culture model of gut microbiota,and clarified the mechanism of that acarbose and baicalein regulate the gut microbiota to reduce the side effects of flatulence and increase insulin sensitivity.It provides a basis for the use of in vitro models of gut bacteria to evaluate the research of foods and medicines which could regulate the gut microbiota.