Study On The Unbalanced Pattern Of Intestinal Flora And Serum Mass Spectrometry Analysis In Type 2 Diabetes Mellitus And Diabetic Nephropathy

Diabetes mellitus（DM）is a set of long-term high blood sugar as the main features of the metabolic syndrome,which is due to a lack of insulin and（or）insulin biological function disorder lead to glucose metabolic disorder.DM,at the same time,is accompanied by fat、protein、water、electrolyte metabolic disorders,and can concurrent chronic damaging disease of organs such as eyes、kidney、nerve cardiovascular.According to the latest diabetes map released by the international diabetes federation（IDF）in 2017,the number of people with diabetes in China has reached 114 million,ranking the first place in the world.Type 2 diabetes mellitus（T₂DM）,also known as adult-onset diabetes mellitus,is a polygenic genetic disease,whose pathogenesis involves genetic and environmental factors.Its pathogenesis is insulin resistance and insufficient secretion,and its onset age is usually after 35 years old.Diabetic nephropathy（Diabetic nephropathy,DN）is one of the three major clinical common complications of diabetes.It has become the second cause of end-stage renal disease after glomerulonephritis.The incidence of diabetic nephropathy is also rising in China.Diabetic nephropathy usually occurs in patients with type 1 diabetes mellitus for about 10 to 15 years.While diabetic nephropathy occurs in patients with type 2 diabetes mellitus for a short period,which may be related to aging and many other basic diseases at the same time.Recent years,of intestinal flora got more and more attention,a lot of evidences show that gut bacteria in a variety of complex diseases play an important role in occurrence and development.Large number of scientific studies have shown that health and environmental factors associated with the structure and function changes of intestinal flora.The modification of intestinal flora may contribute to the prevention and treatment of disease.Intestinal flora closely associated with diabetes.Gut bacteria can affect body’s absorption of sugar and energy,promote the synthesis and storage of fat.The intestinal barrier dysfunction for bacterial endotoxin invasion of the body’s blood provides in a possible way,which could induce low-grade inflammation and insulin resistance.Chinese population studies have shown that people with diabetes intestinal flora significant change compared with healthy people.The intestinal flora associated with blood glucose metabolism,characterized that good bacteria ratio is reduced and the decrease of species diversity and potential pathogens.Time of flight mass spectrometry is a kind of a variety of complex technology,which can analyze the serum,urine,saliva,and organize.With mature of technology and decreasing analysis cost,time of flight mass spectrometry is widely used in aspects such as genotyping analysis;compose a biomarker identification,and pathogen identification.The development of mass spectrometry provides an effective way for our further study on diabetes and diabetic nephropathy in patients with changes in proteomics and polypeptide comics.Research purpose and significanceType 2 diabetes mellitus（T₂DM）and Diabetic nephropathy（Diabetic nephropathy,DN）patients compared to healthy controls group intestinal flora imbalance pattern and function change.We are looking for two kinds of disease with intestinal flora consistent and specific changes in the signal and analyze intestinal flora and the relationship between serum inflammation indexes.At the same time,we explore the Type 2 diabetes mellitus and Diabetic nephropathy patients serum protein and peptide groups learning characteristics.Its significance is that we are trying to find out the key dangerous strains which prompt progress for patients with type 2 diabetes mellitus to diabetic nephropathy.At the same time we set up through the intestinal flora to distinguish classification algorithm model of patients with type 2 diabetes mellitus and diabetic nephropathy.Through the network analysis,we also explore the most influential groups of the ecosystems in patients with type 2 diabetes mellitus and diabetic nephropathy.Furthermore,through the analysis of serum mass spectrometry,the diagnosis model and differential diagnosis model of type 2 diabetes mellitus and diabetic nephropathy were established.The research methods1.We selected from the inpatient department of Zhujiang hospital of southern medical university as the study subjects.There are 30 patients with type 2 diabetes mellitus and 25 patients with diabetic nephropathy.Meanwhile,30 healthy subjects were selected as the control group to collect venous blood and fecal specimens within 48 hours after admission.2.We used illumina high-throughput sequencing technology to sequence the 16S rdna-v4 region of all bacteria in fecal samples.QIIME（v1.9.1）was used to process the original sequence.Compared with the Greengenes（v13₈）database,the operational taxonomic unit（OTU）was used to cluster to analyze the diversity and flora structure.3.We statistically analyze the differences between the two disease groups and the control group.Serum IL-6 was determined by Roch E601 analyzer and supporting reagents.Serum CRP was determined by SIEMENS BNP analyzer and supporting reagents.4.The m/z values in the range of 1000-10000 of the serum samples after protein removal were detected by the Quan TOF mass spectrometer（Intelligene Biosystems of QingDao）.We analyzed the data of each group by principal component analysis（PCoA）,and then the random forest model was established.Statistical methodsWe use software R 3.3.2 and SPSS20.0 to do the data statistics.Two independent sample t tests were used for age comparison between the two groups,and chi-square test was used for gender composition ratio.Wilcoxon rank-sum test was used for nonparametric test of the two groups.Kruskal-waills test was used for nonparametric data greater than that of the two groups,and Adonis was used for PCoA statistical analysis.We also use R for drawing.P<0.05 was considered statistically significant.ResultsThe intestinal flora diversity of T₂DM and DN patients is significantly different from the healthy control group（P<0.05）.T₂DM and DN patients harbored lots of similar changes.For example,there is a significant decrease in Lachnospira、Faecalibacterium、Roseburia and Coprococcus（P<0.05）.However,there is also a disease-specific pattern of imbalance between the two diseases.There is a significant increase in Bacteroides in T₂DM patients.However,DN patients have significant increasing in Lactobacillus,Slackia,Anaerotruncus,Haemophilus and Enterococcus.Functional prediction is also confirmed that T₂DM and DN patients had more consistent changes.Serum inflammatory markers of T₂DM and DN are significantly higher than those of healthy controls,which might caused by the decrease of beneficial bacteria in T₂DM and DN.Serum mass spectrometry analysis reveals that the serum mass spectrometry of patients with type 2 diabetes mellitus and diabetic nephropathy is different from that of healthy controls,and the serum mass spectrometry of patients with type 2 diabetes mellitus and diabetic nephropathy is also different.ConclusionT₂DM and DN patients have similar changes in intestinal flora----a decrease of bacteria producing butyrate.However,there is also a disease-specific change between the two diseases.The analysis of serum mass spectrometry suggests that it is feasible to distinguish type 2 diabetes mellitus,diabetic nephropathy and healthy control by using the method of mass spectrometry analysis.It also provides data basis for further study to evaluate the risk of diabetic nephropathy by intestinal flora and serum mass spectrometry.