The Structural Characteristics Of Gut Microbiota In Mongolian Gestational Diabetes Mellitus Patients And The Relationship Between It And Metabolism、Inflammation

Gestational diabetes mellitus(GDM)is a type of diabetes.It is increased year by year.Currently.it is believed that it may be related to genetic,environmental,and immunological factors.In recent years,a large number of studies have shown that gut microbiota may be involved in the occurrence of diabetes as an environmental factor.Researchers used genome sequencing to reveal the composition and abundance of gut microbiota in diabetes patients,validated them in animals,and identified disease-related bacterial functions.Reviewing a large number of literatures,we found that compared with normal people,the structure of gut microbiota in patients with type 2 diabetes(T2D)was changed significantly.T2D strains were mostly derived from conditional pathogens,such as Bacteroides genus,Clostridium heewayi,Clostridium multifrequens,Clostridium communis,Streptomyces agglomerans and Escherichia coli.The distribution of gut microbiota in children with type 1 diabetes(T1D)was unbalanced.Actinomyces/Firmicutes,Firmicutes/Bacteroides was reduced,and some beneficial bacteria such as butyrate-producing bacteria were reduced.However,there was few data on the gut microbiota structure of GDM patients.The study showed that the prevalence of diabetes in the Mongolian population was significantly lower than the national prevalence rate.Meanwhile,the gut microbiota is related to race.Therefore,this paper will combine race and GDM,focusing on the study of changes in the structural characteristics of gut microbiota in Mongolian GDM patients.In addition,GDM was often accompanied by inflammatory processes and metabolic abnormalities.As a pro-inflammatory factor,Interleukin-6(IL-6)and Tumor necrosis factor-alpha(TNF-a)played a regulatory role in the development of GDM and insulin resistance(IR).The sugar,lipid metabolism factors and the inflammatory factors were closely related to GDM.Therefore,we will detecte multiple inflammatory factors and metabolic markers in Mongolian GDM serum and placenta,which will lay the foundation for its correlation research with gut microbiota.Lactobacillus was positively correlated with fasting blood glucose and glycated hemoglobin.There was a negative correlation between Clostridium and fasting blood glucose(FBG),glycated hemoglobin(HbA1C),insulin,C-peptide and triglyceride(TG),and a positive correlation with adiponectin and high-density lipoprotein(HDL).Therefore,we will selecte the statistically significant different metabolic indicators and inflammatory factors of Mongolian GDM patients.Then the relevance of them and gut microbiota will be studied.The latest three-generation sequencing technology,PacBio’s single-molecule real-time(SMRT)sequencing platform is used in this paper.16SrRNA fragments with a read length of 1500 bp are completely detected,no data assembly is required,and the error rate is low.So the experimental results obtained are more reliable.In addition,The third-generation PacBio SMRT sequencing technology,by using its long read length function,can accurately classify and identify bacterial populations in the sample at the genus and species level,and the number of species detected is greater than the second-generation sequencing technology.So the advantages are significant.Part 1Analysis of 16S rRNA sequencing of the gut microbiota in Mongolian and Han nationality GDMpatientsObjective The changes of gut microbiota in GDM patients of Mongolian and Han nationality were studied in this part.And by comparing the species with abundance differences,it is clear whether it is related to the low prevalence of Mongol diabetes.Then,the Mongolian and Han nationality GDM were merged into the disease group and compared with the healthy control group to determine whether the gut microbiota structure changed.Methods A total of 114 subjects were included in the study and divided into 4 groups:Group 1(Mongolian GDM group)18 cases,Group 2(Han GDM group)29 cases,and Group 3(Mongolian pregnancy healthy group)32 cases And Group 4(Han pregnancy healthy group)35 cases.Collect fresh stool samples from the study subjects.The fecal metagenomic DNA was extracted and its 16S rRNA full-length fragment was amplified.The third-generation sequencing method(PacBio SMRT sequencing platform)was used to analyze the gut microbiota structure.Finally,bioinformatics analysis was used to determine the characteristics of the intestinal flora.Results Demographic data and clinical data display that there was no significant difference between the group 1 and group 2 in age,body mass index(BMI),triglyceride(TG),fasting blood glucose(FBG),and glycosylated hemoglobin(HbA1C)(P>0.05).Gut microbiota analysis:A total of 82 fecal samples were included in the study.Through the PacBio SMRT sequencing,a total of 387699 effective sequences were obtained.The average number of sequences per sample was 4,728.Using the Uparse software clustering sequences with a similarity greater than 98.65%,17218 Operational Taxonomic Units(OTUs)were obtained.A total of 3102 was used for flattening,and the total number of flattened OTUs was 15,780.The average number of OTUs per sample was 192.The dilution curve in the Alpha diversity analysis showed that the dilution curves of the four groups of samples tend to be flatter as the sequencing amount increases;the numbers of OTUs,chao1 index,and shannon index of group 1 were significantly higher than those of the group 2,and the statistical difference was significant.The numbers of OTUs and chaol index in group 1 were also significantly higher than those in group 3.There was a statistically significant difference(P<0.05).The principal CO-ordinates analysis(PCoA)in the beta diversity analysis shows that in the PC1-PC2 coordinate system representing the scatter plot of 82 samples,the four groups of samples mostly overlap and are not clearly distinguished.Cluster analysis showed that some of the samples were first clustered in its sample,and then further merged with other samples or clusters,and the two groups had different degrees of overlap.The LEFSee analysis in the taxonomic analysis showed that the gut microbiota in group 1 and group 2 were different at the levels of phylum,class,order,familie,and specie.The abundances of Verrucomicrobia.Verrucomicrobiae、Verrucomicrobiales and Verrucomicrobiaceae were all significantly increased in the group 1 than those in the group 2(P<0.05),while the abundance of Ruminococcus sp.YE78 was significantly decreased(P = 0.029).In comparison between the 1,2 combined group(GDM group)and the 3,4 combined group(normal pregnancy group),it was found that there were differences in the levels of phylum,genus and species in the gut microbiota.Among the three species,the abundances of bacteroides fragilis,paraclostridium bifermentans and sutterella wadsworthensis in the GDM group were all higher than those in the normal pregnancy group(P = 0.0441,0.0229,0.0450).Conclusions1.The gut microbiota of Mongolian GDM patients is superior to Han GDM,and most of them are not susceptible to diabetes.It is speculated that the superiority of the gut microbiota in the Mongolian population may be one of the reasons for the low incidence of diabetes.However,due to the controversy of the role of rumenococcus,and the microecology of gut microbiota is too complex,it cannot be determined by only one or two genera(species),so it needs further study.2.There is an imbalance of gut microbiota in patients with GDM,and there are more pathogenic bacteria in the condition.3.The difference in strains between the experimental group and the control group was found at the species level,showing the advantages of the third generation sequencing technology.Part 2Study on Metabolism and Inflammation in MongolianGDM PatientsObjective Serum inflammatory factors and metabolic indicators were measured in Mongolian GDM patients to study the mechanism of Mongolian GDM and to lay the foundation for further research on the relationship between inflammatory factors,metabolic indicators and gut microbiota.Methods Blood samples were collected from 60 Mongolian GDM women and 60 Mongolian normal pregnant women(Including 18 cases of Mongolian GDM patients in the first part).Metabolic indicators were tested at the Laboratory of Hohhot First Hospital.The inflammatory IL-1β,IL-6,IL-10,and TNF-α were detected using the Bio Plex ProTM kit.Results In terms of clinical characteristics,compared with healthy pregnant women,Mongolian GDM patients are older,have a higher BMI index,have the first child,have a family history of type 2 diabetes,or have a history of GDM(p<0.05).The differences of the serum inflammatory factors:IL-6,IL-6/IL-10 ratio between the two groups of pregnant women were statistically significant(P<0.05).The differences of the metabolic indicators:fasting plasma glucose(FBG),fasting insulin(FINS),insulin resistance index(HOMA-IR)and islet beta cell function index(HOMA-β)between the two groups were also statistically significant(P<0.05).Conclusions There are abnormal expressions of inflammatory factors,glucose and lipid metabolism indicators in serum of Mongolian GDM patients.Abnormal inflammation,glucose and lipid metabolism are closely related to the Mongolian GDM patients.The relationship between them deserves further study.Part 3Study on the Relationship between gut microbiota,Metabolism and Inflammation in Mongolian GDMPatientsObjective Through the experimental results of part 1 and part 2,we select the 18 Mongolian GDM differential strains in part 1 and their serum metabolic indicators,inflammatory factors in part 1 and part 2 for correlation studies.Different strains were selected:Verrucomicrobia and ruminococcs sp.YE78.Metabolism and inflammatory factors were selected:fasting plasma glucose(FBG),fasting insulin(FINs),triglyceride(TG)and interleukin-6(IL-6)and Tumor necrosis factor-α(TNF-α).Methods Using SPSS22.0 statistical softwareThe spearman correlation analysis was performed on the differential genotypes and serum metabolic indicators,inflammatory factors in Mongolian GDM.P<0.05 was considered statistically significant.Results There was a negative correlation between the abundance of Verrucomicrobia and FINs、TG、HbA1C in the Mongolian GDM patients.The correlation coefficients were-0.870,-0.732,and-0.686 and were statistically significant.(P=0.000,0.001,0.002).No significant correlation with FBG,IL-6,TNF-α(P>0.05).The abundance of Ruminococcus sp.YE78 was positively correlated with FBG,and the correlation coefficients were 0.666(P=0.004).There was no significant correlation with FINs,TG,HbA1C,IL-6 and TNF-α(P>0.05).Conclusions The abundance of Verrucomicrobia which was significant increased in Mongolian GDM group was negatively correlated with FINs,HbA1C and TG.The abundance of significantly reduced Ruminococcus sp.YE78 strain was positively correlated with FBG.The conclusion that the gut microbiota structure of MongolianGDM patients is better than Han nationality GDM is further supported.It is speculated that the superiority of gut microbiota in Mongolian population may be one of the reasons for the low incidence of diabetes.However,because the microecology of the gut microbiota is too complex,it cannot be concluded by only one or two genera(species),so it needs further study.