| ObjectiveDiabetes?Diabetes Mellitus,DM?is one of the most commom chronic non-communicable diseases in the world.Type 2 diabetes?Type 2 Diabetes Mellitus,T2DM?is based on insulin resistance and insufficient insulin secretion.China is now home to the most cases worldwide[1].Since the widespread application of hypoglycemic agents and insulin,hyperglycemia caused by DM and its acute complications such as ketoacidosis,hyperosmolar coma,and lactic acidosis have been well prevented and treated.However,the occurrence and development of chronic complications of DM can still seriously affect their living quality,even disability.Among them,cardiovascular complications are based on atherosclerosis?AS?[2],which is the main cause of death in patients with DM.Considering that,the prevention and treatment of DM has become a global public health issue.Many domestic and foreign scholars have devoted themselves to the research on prevention and treatment of DM.However,the pathogenesis of DM and its complications is not very clear,many elements such as genetic factors and environmental factors play an important role during the development.In recent years,with the development of high-throughput sequencing technology,research on the role of intestinal flora in regulating host metabolism has grown exponentially.Sequencing results of fecal specimens from DM patients and animal models revealed the differences in flora and the correlation between metabolites and diabetes.However,fecal specimens could be easily affected by environmental and dietary factors.Considering that,if the sequencing results can be obtained from the colon intestinal lavage fluid after the intestinal cleansing process,external influence factors might be greatly excluded.This study intends to obtain gut microbiota information from intestinal lavage fluid,select certain relevant indicators from three levels of bacterial metabolite,intestinal inflammatory factors and intestinal barrier to reveal the relationship between flora and intestinal inflammation.Based on the results of high LPS in the intestinal fluid,use the in-vitro experiments to determine the relationship between gut microbiota and T2DM cardiovascular complications,then provide experimental evidences for intervention treatment and diagnosis.Methods1.Specimen collection and sequencing:The experiment was divided into two groups,the T2DM patient group?group D?and the non-DM control group?ND group?.According to T2DM diagnostic criteria,17 patients in group D and 10 patients in ND group were finally selected.Intestinal lavage fluid was collected during intestinal endoscopy,and the sediment was collected after centrifugation to extract sample genomic DNA.The V3-V4 region of the 16SrDNA gene was extracted from all the samples by PCR to compare the intestinal flora structure,abundance,and diversity of the two groups.2.Enzyme Linked Immuno Sorbent Assay?ELISA?was used to determine the level of lipopolysaccharide?LPS?,Interleukin-4,Interleuki-6 and Zonulin in the supernatants from two groups.3.In vitro cell experiments:Stimulate primary human aortic smooth muscle cells?HASMCs?with appropriate concentrations of LPS,extract cellular RNA at different time points to detect the expression of the proinflammatory cytokine IL-6.4.Statistical analysis:SPSS software?version 20.0?was used.Measurement data of normal distribution are expressed as meanąstandard deviation,t test was used to compare the means between two groups.Non-normally distributed data are expressed as median and interquartile range?QR?.Nonparametric tests were used for the comparison between two groups.The analysis of significant differences in flora was performed by Wilcoxon rank-sum test,P<0.05 indicated that the difference was statistically significant.The correlation between the factors was analyzed by Spearman correlation.The difference was statistically significant at P<0.05.Results1.The sequencing depths of group D and ND have reached an enough level.Compared the Sobs index,Simpson index,Chao index,Ace index,Shannon index between the two groups,there was no significant difference,indicating that the community richness and diversity are similar.PCo A analysis can clearly separate the samples of group D and ND,indicating that there is a certain difference in the flora composition between the two groups.The results of composition analysis showed that at phylum level,two groups were both mainly composed of Firmicutes,Bacteroidetes,Proteobacteria,Fusobacteria,and Actinobacteria with no significant difference in relative abundance.At genus level,the relative abundance ratios of main bacteria between the two groups are different.The highest relative abundance of group D is Prevotella,while the ND group is Bacteroides.Significant tests were conducted to find that the level of Prevotella?P=0.044<0.05?and Haemophilus?P=0.047<0.05?in group D was higher than that of group ND,the level of Bacteroides of group D was lower than that of group ND?P=0.047<0.05?.2.ELISA results showed that the level of LPS in intestine of group D was higher than that of group ND?P=0.005<0.01?.The level of Zonulin?P=0.035<0.05?and IL-6?P=0.013<0.05?of group D was higher than that of group ND.The level of IL-4?P=0.006<0.01?of group D was lower then that of group ND.3.The Real-time PCR results showed that LPS could significantly increase the expression of inflammatory factors IL-6 over time?P<0.05?.4.Spearman correlation analysis was used and then find that in gut,LPS level were positively correlated with IL-6 levels?r=0.4634?and Zonulin levels?r=0.45?,IL-6 levels were positively correlated with Zonulin levels?r=0.49?.There were also a large number of correlations between gut microbiota and related indexes.Conclusion1.Although there is no significant difference about the gut microbiota diversity between T2DM patients and the non-DM controls,but some differences of their composition and proportion actually exist,which may be involved in the progression of T2DM.2.The intestinal permeability of T2DM is increased with a certain inflammatory state,which may be caused by the high level of LPS in gut and the changes of some bacteria abundance.The high level of LPS in the intestine is closely related to the expression of IL-6,which is involved in the process of inflammation.Intestinal epithelial cells and vascular damage caused by the inflammatory state may be the basis for LPS and others entering into the blood circulation even leading to cardiovascular complications. |
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