Study On Intestinal Microecological Changes In The Process Of Type 2 Diabetes Mellitus In Mice

?Objective?Our study aim to the change of bacterial abundance and immunological changes in type 2 diabetes mellitus.16Sr RNA high throughput sequencing was used to analyze the changes in fecal flora abundance,ELISA and flow cytometry were used to observe the changes of intestinal immunity,humoral immunity and cell immunity in the process of diabetes.?Methods?(1)Type 2 diabetes mouse model.Male C57BL/6J mice(SPF grade)were randomly divided into 6 groups:0 days(group A),20 days(group B),40 days(group C),60 days group(group D),80 days group(group E),105 days group(group F),16 mice in each group.A group is a normal group with no treatment.The group B-E was fed with high-fat and high-sugar diets,and the group F was fed with high-fat and high-sugar diets combining with small doses of multiple injections of STZ from the 81st day.Fasting blood glucose(FBG)was used to detect the blood glucose every 2weeks.Glucose intolerance were measured by intraperitoneal glucose tolerance trial(IPGTT),insulin tolerance test(ITT)and the area under curve(AUC)for 15 weeks.At the corresponding time point,mice were sacrificed and various tissues were collected as follow:feces,blood,intestines,islets,and liver.(2)HE staining was used to observe the pathological changes of pancreas,liver,ileum and other pathological changes in the pathogenesis of T2DM.Insulin content in pancreas was tested with an anti-insulin antibody for immunohistochemistry.(3)Diabetes associated biochemical indicators(CRE,FBG,FFA,CHO,HDL,LDL,TG)were detected by biochemical analyzer.(4)16S rRNA high-throughput second-generation sequencing was used to detect the abundance of intestinal flora.(5)Serum levels of interleukin-6(IL-6),tumor necrosis factor-?(TNF-?)and secretory immunoglobulin A(SIgA)in ileum were measured by enzyme-linked immunosorbent assay(ELISA).The level of hypersensitive c-reactive protein(hs-CRP)was detected by turbidimetric method.(6)The percentage of CD3~+,CD4~+and CD8~+cells in T lymphocyte was measured by flow cytometry.?Results?(1)In the development of T2DM,the abdominal fat of mice in each experimental group increased significantly.FBG levels increased gradually from group A to F and there was a statistical significance between the experimental groups(P<0.05).IPGTT and ITT data showed that glucose tolerance gradually declined with the progression of diabetes.Compared to group A,the AUC in group C-F increased in the development of diabetes.(2)Histopathological results showed that the acinar of pancreas was clearly in group A.In group C,pancreas was mildly atrophic and fibrotic.The pancreatic islet was severely atrophied in the F group.Pancreatic?-cell appeared hypertrophy and revealed a large amount of transparent fat and vacuolated cells.The pathogenesis of T2DM was accompanied with pathological changes in liver and ileum.(3)Biochemical indicators in the experimental groups were as follows:Compared with group A,FBG?FFA?CRE?TG?CHO?HDL?LDL?UA in group C and D showed significant differences(P<0.05);There was no obvious difference in UREA between the group E and F(P>0.05).TG,CHO,HDL,UA in group F were significantly higher than those in group A(P<0.05).(4)Illumina sequencing technology was used to sequence the 16S r RNA V4 region PCR products from feces samples to study bacterial population diversity.The top 5 of the fecal flora in each experimental group were Firmicutes,Bacteroidetes,Proteobacteria,Actinobacteria,Spirochaetes.The level of Proteobacteria was significantly higher in group C than in other groups with the development of diabetes;the level of Firmicutes in group F was significantly higher than that other groups.In the progression of T2DM,the intestinal microbial populations in each experimental group showed dysregulation at different degree,suggesting that the intestinal microbiota may be related to the occurrence of type 2 diabetes.(5)Serum Hs-CRP,IL-6,TNF-?and SIgA in ileum:Our results showed that Hs-CRP,IL-6,and TNF-?in group E and F were higher than group A(P<0.05);SIg A level decreased gradually(group A:22.3 pg/m L,group D:20.1 pg/m L,group E:18.3 pg/mL,and group F:17.6 pg/m L).SIgA level in group F and had statistically significant difference from group A(P<0.05).(6)Flow cytometry data showed that CD3~+CD8~+and CD3~+CD4~+in group C,E,and F had statistically significant compared to group A(P<0.05).CD3~+in group F had statistically significant compared to group A(P<0.05).There was no significant difference between group A and B,C,and D(P>0.05).?Conclusion?(1)In the present study,we constructed the T2DM model in different stage.We found that insulin resistance exists in the pre-diabetic stage,and insulin resistance aggravated gradually accompanying with varying degree of injury in liver,islet,and ileum.(2)The development of type 2 diabetes was accompanied with a change in the abundance of intestinal microflora,especially in the abundance of populations:the bacterial flora of the phylum Streptomyces,Clostridium,Proteobacteria,and Cyanobacteria,etc.These changes may cause chronic inflammation resulting in insulin resistance.(3)The imbalance of intestinal flora abundance during the development of diabetes caused a disturbance in intestinal immunity,cellular immunity and humoral immunity,which suggested abnormal of immune indices and T lymphocyte subsets were related to diabetes.