| Objective: Recent years, more and more researches found that there is a close relationship between intestinal microbiota and diabetes. The change of intestinal microbiota influence the permeability of the intestinal inflammation and then influence the permeability of the intestine, thus causing the chronic inflammatory process in the body. The processes above all participate in the development of diabetes. Our studu choose OLETF rats as the research objece, because the rats can experience the whole process of diabetes, including the hyperinsulinemia, impared glucose tolerance and diabetes. The aim of the study is to explore the change of the intestinal microbiota, the level of lipopolysaccharide(LPS) in the intestinal and serum, the expression of the TLR-4 and NFκb in the gut, the pathological change of the gut and the change of the intestinal permeability in the whole process of the biabetes.Method: We selected OLETF rats and its normal contral rats as research objects. At the different stages of the progression(to be specific is, normal glycemia,hyperinsulinemia, impaired glucose tolerance, diabetes mellitus), we collcted blood samples and faeces, and then execute appropriate number of rats to get the intestinal tissue segments. We use biochemical method detection for basic biochemical indicators,and use faeces for DNA extraction for analysis with 454 pyrosequencing for which diversity and representatives of primitive community of microbes at different classifications. We use ELISA method to detect the LPS level in serum and feces, as well as the level of DAO and zonulin in serum as the biomarker of intestinal permeability. We use intestinal tissue segments for pathological histology obvervation and western blot technology for the detection of the local inflammation indicators of intestinal tissues, in specific, the expression of TLR-4 and NFκB. Analysis the indexes above all in the whole duration of the disease.Results:(1) There is no difference in the phylum of the gut microbiota between the LETO group and the OLETF group in the normal glycemia stage, but the Lactobacillus and Phascholarctobacterium is lower in the OLETF rats of the normal glycemia stage. There is no difference between the two groups in the level of LPS in the serum and fecal, the expression of TLR4 and NFκB in the gut, and the level of DAO and zonulin in the serum.(2) there is an obvious difference in the diversity among the different groups, such as the normal glycemia group, the hyperinsulinemia group, the impaired glucose tolerance group and the diabetes group. The level of microbiota diversity from lowest to highest is hyperinsulinemia group, the impaired glucose tolerance group, the normal control group and the diabetes group. The changes of the diversity of microbiota indicates that the gut bacteria may be involved in the development of diabetes.(3) The dominant phylums of the intestinal microbiota in each group are Bacteroidetes, Firmicutes, Proteobacteria and there is a big difference in the proportion of the microbiota among the different groups.(4) The comparison of the relative abundance of different phylums among different groups. Firmicutes: the normal control group is the highest compared with the other three groups(P <0.05) and it is a decreasing trend in hyperinsulinemia group, impaired glucose tolerance group and the diabetes group, but thisdifference is not obvious. Bacteroidetes: the relative abundance in hyperinsulinemia group is obvious increased than the other three groups(P <0.05) and the impaired glucose tolerance group has an increasing trend than the normal control group, but the difference is not obvious. Proteobacteria: the normal control group and the hyperinsulinemia group is not different obviously, but the relative abundance of the impaired glucose group is obvious increased than the hyperinsulinemia group(P <0.05), with the course progressing to the diabetes, the relatively abundance is increasing more significantly(P <0.05).(5) The level of LPS in serum and feces is an increasing trend in NC group, hyperinsulinemia group, IGT group and the DM group and hyperinsulinemia group is increasing obviously than NC group(P <0.05), at the same time, the DM group is increasing significantly than the other three groups(P <0.05).(6) The expression of TLR-4 and NFκB in intestinal tissue is increasing in NC group, hyperinsulinemia group, IGT group and DM group. The level of the inflammatory biomarker in hyperinsulinemia group and IGT group is obviously increased than the NC group(P <0.05) and the DM group is increasing significantly than the other three groups(P <0.05).(7) The pathology result shows that at the hyperinsulinemia stage, the integrity of the intestinal mucosa is impaired and the tight junction as well as the intermediate junction is changed. When it is progressing to the diabetes, these changes become more and more obviously.(8) The zonulin level in serum is gradually increased in NC group, hyperinsulinemia group, IGT group and DM group, but the difference is not significant. The same trend can be seen for the DAO level in serum in the four groups, but the hyperinsulinemia group and DM group is increased significantly than the NC group(P <0.05).Conclusion:(1) There is a difference in the intestinal microbiota between the LETO group and the OLETF group in the normal glycemia stage. This is related to the difference of the gene of the different rats and the intestinal microbiota participated in the development of diabetes of the OLETF rats.(2) There is a big difference in the intestinal microbiota at different groups, and the change of the intestinal microbiota participate in the course of diabetes. The change of the intestinal microbiota can effect the LPS level in the intestine, thus effect the intestinal inflammation, and intestinal inflammation can impair the barrier function of the intestinal mucosa. Next the harmful material in the intestine can enter blood through the impaired intestinal mucosa and the LPS level in the blood is increased.The changes above all can course the chronic and low level inflammation in body and participate in the development of diabetes. |
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