Effects Of Dominant Microflora On Glucose Metabolism In Type 2 Diabetes Mellitus And Study On The Biological Activity Of Enterococci (DM9112)

Objective:To explore the effects of intestinal dominant flora on glucose metabolism in type 2 diabetic mice（T2DM）and Study on the biological activity of enterococci（DM9112）.Methods:1.Effects of intestinal dominant flora and Enterococci on glucose Metabolism in T2DM miceSix-week-old C57/BL6J male mice were divided into normal group（C）,T2DM group（D）,T2DM dominant bacteria reperfused group（DP）and T2DM enterococcal（DM9112）reperfused group（DE）.Isolation and identification of dominant intestinal microflora in mice,and making a scale of dominant bacteria reperfused bacteria.According to the amount of dominant bacteria reperfused,T2DM mice were reinjected once a day for 4weeks,while the control group was replaced by the same volume of normal saline.During the experiment,the body weight,random blood glucose,food intake and water intake of mice were recorded.PCR-DGGE method was used to analyze the fecal samples of mice every week during the experiment.At the end of 4 weeks of the experiment,the HOMA-IR values of mice in each group were measured,and the jejunum and colon tissues were stained with HE,and then the results were observed.2.Study on the Biological activity of Enterococci（DM9112）2.1 Biological characteristics of EnterococcusEnterococcus faecalis（DM9112）is a subspecies of human Enterococcus faecalis with multiple metabolic activities isolated in our laboratory,named DM9112.From the beginning of self-culture of enterococci,the doubling time was measured and calculated every 2 hours,and the bacterial growth curve was drawn.2.2 Biochemical characteristics of metabolites of EnterococciThe p H value of enterococci was measured every 2 hours.DNA hydrolase was added to the metabolites to identify the properties of nucleic acids by agarose gel electrophoresis and the calculated concentration was measured.The components and contents of fatty acids in metabolites were determined by chromatography every 4 hours,and the curves of nucleic acid concentration and fatty acid content were drawn.2.3 Study on the safety of enterococciA total of 20±2g Balb/c mice,half male and half female,were randomly divided into enterococci（DM9112）injection group and enterococci（DM9112）metabolite injection group.Each group was randomly divided into 5 concentration groups.The mice were injected intraperitoneally with different concentrations of bacterial injection and metabolite injection for one week,and the death of mice was recorded.Results:1.Effects of intestinal dominant flora and Enterococci on glucose Metabolism in T2DM mice1.1 In the 4-week experiment,it was found that compared with the normal group,the T2DM dominant bacteria group had higher food intake and water intake and lower body weight,and the T2DM dominant bacteria group had lower food intake and water intake and higher body weight compared with the T2DM group.Compared with the normal group,the food intake and water intake of T2DM enterococci reperfusion group were always higher,and the body weight was significantly lower in the second and third week（P<0.05）.Compared with the T2DM group,T2DM enterococci had higher food intake,lower water intake and lower body weight all the time.Compared with the enterococcal perfusion group,the food intake and drinking water of the T2DM dominant bacteria group were always lower,and the body weight was always higher（P>0.05）.1.2 In the 4-week experiment,it was found that compared with the normal group,the T2DM dominant bacteria group and enterococcal reperfusion group were always higher（P<0.001）.Compared with the T2DM group,the random blood glucose in the dominant bacteria group was always lower in four weeks,and significantly lower in the second and third week（P<0.05,P<0.01）,and significantly lower in the first 3 weeks and significantly lower in the third week（P<0.05）.The random blood glucose in the T2DM dominant bacteria group was always lower than that in the T2DM enterococci group within 4 weeks.1.3 In the 4-week experiment,it was found that compared with the normal group,the HOMA-IR of T2DM dominant bacteria group and T2DM enterococci reperfusion group was significantly higher,the HOMA-IR of T2DM dominant bacteria group was significantly lower than that of T2DM group,the HOMA-IR of T2DM enterococcus group was also lower than that of T2DM enterococci group,and the HOMA-IR of dominant bacteria group was significantly lower than that of T2DM enterococci group.1.4 In the 4-week experiment,it was found that there was no significant change in Shannon index in T2DM group,T2DM enterococcal perfusion group and T2DM dominant bacteria group,but the richness was significantly lower than that in normal group.According to the results of UPGMA similarity cluster analysis,after 2 weeks of experiment,the fecal microflora of T2DM dominant flora reperfused group clustered into a single cluster,which was different from that of normal group and T2DM group.However,after 4 weeks of experiment,the fecal flora of the T2DM enterococcal perfusion group still did not gather into a single cluster,which was still similar to that of the T2DM group,but different from the normal group.1.5 In the 4-week experiment,it was found that compared with the normal group,the results of HE staining in T2DM group showed that the epithelial villi of jejunal mucosa were broken and atrophied,and the result of colonic HE had no significant change.Compared with the T2DM group,HE staining showed that the rupture and atrophy of jejunal mucosal villi were improved in the T2DM dominant bacteria group,but there was no change in the T2DM enterococcus group.2.Study on the Biological activity of Enterococci（DM9112）2.1 Enterococci（DM9112）has a multiple growth period in 4-8 hours.The fatty acids in its metabolites are mainly lactic acid and acetic acid,and there are nucleic acids in the metabolites.The type is RNA.2.2 The median lethal dose(LD₅₀)of enterococci（DM9112）was 4.12x10¹⁰CFU/kg.There was no death in mice in the LD₅₀test of its metabolites.Therefore,enterococci（DM9112）and its metabolites belong to non-toxic substances.Conclusion:1.The recirculation of dominant bacteria could improve the symptoms of"three more and one less"in T2DM mice,but enterococci was not obvious.2.The recirculation of dominant bacteria could reduce the random blood glucose of T2DM mice,but enterococci was not obvious3.Recirculation of dominant bacteria could significantly improve insulin resistance in T2DM mice,but enterococci was not obvious.4.The structure of intestinal flora in T2DM mice was significantly changed by recirculation of dominant bacteria,but not by enterococci.