| Backgroud:The incidence of T1DM was increasing in recent years, whereas thepathogenesis has not been understood completely. Recent studies have indicated that an imbalance of gut microbiota is associated with the development of type 1 diabetes mellitus (T1DM),yet there is no literature reporting that in Chinese children. The aim of this study was to evaluate the alteration of gut microbiota between the newly diagnosed T1DM children and the healthy controls, and to determine if gut microbiota could partly explain the etiology of the disease.Methods:A case-control study was carried out with 15 children with T1DM and 15 healthy children. The fast peripheral blood and fresh stool samples were collected and stored in -80℃ refregerator within 2 hours for further tests and analyses. The fecal bacteria composition was investigated by high-throughput sequencing based on the V3-4 region of the 16S rDNA gene and analyzed by the estimators of community richness (Chao) indexes. The genotype of HLA-DRB1/DQB1 were detected by using PCR-SBT. The serum concentration of IL-6, IL-10, IL-17A and TNF-α were measured by allergic electrochemical luminescence method.Results:There were no statistical differences between the groups in gender, age,birth way, birth weight, breast feeding, blood pressure, body mass index, Triglycerides and total cholesterol. The levels of glucose and HbAlc were significantly higher in the children with T1DM than healthy controls (P<0.01). The T1DM patients were diagnosed at 10.93±3.03 years.11 of T1DM patients had above 2 autoantibodies, amount to 74.37% of total patients. The serum concentration of IL-17A, IL-6 were significantly higher in theT1DM patients than healthy controls (2.47±1.76 vs.1.49±0.80,1.15±0.98 vs.0.59±0.43), whereas the IL-10 level was lower than the controls (0.55±0.27 vs. 0.37±0.15, P=0.012). The serum level of TNF-α was lower in T1DM patients than the controls (2.42±0.91 vs.3.19±1.46),with no statistical difference (P=0.052). The HLA allele frequencies of DRB1*0901 and DRB1*0405 were 53.3% and 40% in T1DM patients, higher than healthy controls, and so did the frequencies of DQB1*0201 and DQB1*0302.The pyrosequencing provided 402857 reads in total and 13428 reads per stool sample, with an average lenth 252bps per read. As a result,389 optional taxonamy units (OTU) were determined by using the Uparse software with a similarity of 97% as a cutoff shreshold. Alpha-diversity analysis showed that refaction curves of all the samples had a slowing tendency at the point of 2000 sequence. There was a notable lower richness (Chao index) of fecal bacteria in T1DM group than the controls (156.53±36.96 vs.130.0±32.85,p<0.05), the OTU numbers and Shannon index were similar between the two groups. Principal Coordinate Analysis (PCoA) showed that 30 samples were devided into two clusters at PC1-PC3 axis, and a few overlaps were seen between the two kind of the samples. By using UPGMA software, clustering analysis was performed to show the similarities of the gut bacteria composition among all the samples. The overall tendency was that the samples were first clustered within the T1DM or the control group and then further clustered with other samples or clusters, some overlaps were found between the two groups. LEfSe analysis showed there were statistical differences between the groups in the famliy, genus and species level. At the genus level, the composition of Blautia was increased in theT1DM group than the control group, whereas the compositon of Haemophilus, Lachnospira, Dialister and Acidaminococcus were decreased.Spearman correlation analysis showed that that the Blautia percentage correlated positively with HbA1c (ρ= 0.40, P = 0.031), the numbers of TIDM autoantibodies (P =0.42, P=0.023), the titers of IA-2 (ρ=0.8214, P=0.0002) and the serum level of IL-6 (ρ=0.362, P=0.049), yet there was no correlation between the Blautia percentage and the level of FBG. The percentage of Haemophilus had negative correlation with FBG, HbA1c, and the numbers of T1DM autoantibodies.Conclusion:There was a notable lower richness of fecal bacteria in T1DM group than the controls.The gut microbiota structure was different between the two groups, showing that the abundanceof Haemophilus, Lachnospira, Dialister and Acidaminococcus were higher in T1DM children yet the abundance of Blautia were reduced in T1DM group. We found that the percentage of Blautia and Haemophilus were correlated with the numbers of T1DM autoantibodies and the titers of IA-2. In addition, there was a positive relation between the blautia and the serum level of IL-6. It is indicated that the alteration of gut microbiota may take part in the pathogenesis of T1DM via the regulation of autoimmunity. |
PDF Full Text Request