The Effect Of PM_2.5 Exposure-gut Microbiota Axis In The Occurrence And Development Of Gestational Diabetes Mellitus

ObjectiveTo analyze the relationship between PM_2.5、gut microbiota and gestational diabetes mellitus（GDM）,to clarify the characteristics of gut microbiota in pregnant women with GDM,to explore whether gut microbiota moderate the effect of PM_2.5exposure on GDM,to analyze the relationship between gut microbiota and GDM-related plasma metabolites and circ RNA,and to explore the potential mechanism of PM_2.5 exposure and gut microbiota affecting the risk of GDM,so as to provide theoretical basis for the prevention and treatment of GDM.MethodsPartⅠ:A prospective cohort study was used.A total of 1248 pregnant women who entered the study cohort of pregnancy metabolic diseases and adverse pregnancy outcomes（PMDAPO）from January 2017 to February 2020 and who underwent routine antenatal examination and delivered in Guangzhou Women and Children Medical Center were selected as subjects.The demographic data,clinical information and air pollutant exposure data of subjects pre-pregnancy 3 months to delivery were collected.To analyze the effect of PM_2.5 exposure level on the risk of GDM and its correlation with blood glucose level.PartⅡ:In the first part of this study,672 pregnant women were selected as the subjects,and the fecal samples of 13-28 weeks of pregnancy were collected.The gut microbiota was detected by 16Sr RNA high-throughput sequencing.The relative abundance and diversity of gut microbiota in pregnant women with GDM were compared with those in control pregnant women,and differential bacteria were screened.Analysis of the correlation between blood glucose levels and gut microbiota and gut microbiota alpha diversity and beta diversity.PartⅢ:672 pregnant women in the second part of this study were selected as subjects.Combined with PM_2.5 exposure data and gut microbiota data,the correlation between PM_2.5exposure and gut microbiota was analyzed.Analysis of the correlation between microflora which significantly related to PM_2.5 and blood glucose level of OGTT.Modulation effect analysis was used to explore the modulation role of PM_2.5 exposure-related gut microbiota in the effect of PM_2.5 exposure on blood glucose level.PartⅣ:A 1:1 matched nested case-control study was conducted.Metabonomics and RT-q PCR experiment selected 30 pairs of pregnant women enrolled in the PMDAPO cohort from November 2014 to January 2016 respectively.The serum differential metabolites related to GDM were screened by liquid chromatography-mass spectrometry（LC-MS）,find the related metabolic pathways,and construct the metabonomic biological network to predict the targets.The expression profile of circ RNA in peripheral blood of pregnant women with GDM was analyzed by gene chip technology,and the differential expression of circ RNA was verified by RT-q PCR.The correlation between gut microbiota and GDM differentially expressed metabolites and circ RNA was analyzed.ResultsPartⅠ:Correlation between PM_2.5 and GDM1.A total of 1248 subjects were included in this study,the incidence of GDM was19.15%.2.The results of single pollutant logistic regression model showed that when PM_2.5exposure level increased by 10μg/m³,the risk of GDM increased by 36%（RR=1.36,95%CI:1.05,1.77）,46%（RR=1.46,95%CI:1.14,1.88）and 67%（RR=1.67,95%CI:1.22,2.29）,in pre-pregnancy 3 months,the first trimester and pre-pregnancy 3 months to 13 gestational weeks,respectively.After the inclusion of SO₂ and NO₂,the results of the multi-pollutant model showed that for every 10μg/m³ increase in PM_2.5 exposure,the risk of GDM in the first trimester,pre-pregnancy 3 months to 13 gestational weeks and pre-pregnancy 3 months to 27 gestational weeks increased by 104%（RR=2.04,95%CI:1.47,2.84）,149%（RR=2.49,95%CI:1.65,3.75）and 290%（RR=3.90,95%CI:2.12,7.18）,respectively.3.The results of multiple linear regression model of single pollutant showed that when the exposure level of PM_2.5 increased by 10μg/m³,1-hour postprandial blood glucose in the first trimester,the second trimester and pre-pregnancy 3 months to 27 gestational weeks increased 0.35（95%CI:0.20,0.49）mmol/L,0.01（95%CI:0.16,0.18）mmol/L and 0.58（95%CI:0.34,0.82）mmol/L,respectively.After the inclusion of SO₂ and NO₂,the results of the multi-pollutant model showed that for every 10μg/m³ increase in PM_2.5 exposure,fasting blood glucose in the first trimester,pre-pregnancy 3 months to 13 gestational weeks and pre-pregnancy 3 months to 27 gestational weeks increased 0.05（95%CI:0.01,0.10）mmol/L,0.05（95%CI:0.00,0.11）mmol/L and 0.11（95%CI:0.03,0.19）mmol/L,respectively.In the first trimester,the second trimester,pre-pregnancy 3 months to 13gestational weeks and pre-pregnancy 3 months to 27 gestational weeks,1-hour postprandial blood glucose increased 0.35（95%CI:0.16,0.54）mmol/L,0.37（95%CI:0.17,0.57）mmol/L,0.59（95%CI:0.34,0.84）mmol/L and 0.79（95%CI:0.42,1.16）mmol/L for every10μg/m³ increase in PM_2.5 exposure,respectively.2-hour postprandial blood glucose increased 0.36（95%CI:0.18,0.53）mmol/L and 0.32（95%CI:0.11,0.54）mmol/L in the first trimester and pre-pregnancy 3 months to 13 gestational weeks for every 10μg/m³increase in PM_2.5 exposure.PartⅡ:Correlation between gut microbiota and GDM1.A total of 672 subjects were included in this study,the incidence of GDM was18.90%.2.There was no significant difference inαandβdiversity of gut microbiota between pregnant women with GDM and control pregnant women（P>0.05）.3.The results of Wilcoxon test showed that there were significant differences in the relative abundance of 10 genera between pregnant women with GDM and control pregnant women.the abundance of Faecalitalea,Bacteroides＿pectinophilus＿group and Angelakisella in pregnant women with GDM increased,while the abundance of Turicibacter,Lactobacillus,Catabacter,Clostridium＿sensu＿stricto＿1,Fusicatenibacter,g＿Oscillospirales＿UCG-010 and Romboutsia decreased.4.LEf Se analysis showed that（LDA>2）there were 15 different bacteria between the pregnant women with GDM and control pregnant women.Bacilli,Clostridiales,Lactobacillales,Lactobacillaceae,Clostridiaceae,Turicibacter,Lactobacillus,Catabacter,Clostridium＿sensu＿stricto＿1,Fusicatenibacter and Romboutsia were enriched in the control pregnant women.f＿Oscillospirales＿UCG-010,Bacteroides＿pectinophilus＿group,g＿Oscillospirales＿UCG-010 and Angelakisella were enriched in pregnant women with GDM.After adjusting for demographic data,differences in Lactobacillus and Bacteroides＿pectinophilus＿group remained significant.5.There was no significant correlation between blood glucose level andαdiversity index of gut microbiota（P>0.05）.Fasting blood glucose and 1-hour postprandial blood glucose were positively correlated with gut microbiotaβdiversity（P<0.05）.The pro-inflammatory bacteria Eggerthella,Bacteroides,Colidextribacter and SCFAs-producing Rothia,Odoribacter,Ruminococcus＿torques＿group,Lachnospiraceae＿UCG-008,Streptococcus,Parabacteroides,Anaerostipes and Turicibacter,etc.37 genera were significantly associated with maternal blood glucose levels（P<0.05）.PartⅢ:Study on the moderating effects of gut microbiota on PM_2.5 on blood glucose level1.There was no significant correlation between PM_2.5 exposure level andαdiversity index of gut microbiota（P>0.05）,but there was a significant positive correlation between PM_2.5 exposure level and gut microbiotaβdiversity（P<0.05）.2.PM_2.5 was significantly correlated with 42 bacteria genera（P>0.05）,among which11 bacteria were significantly correlated with blood glucose level.Fasting blood glucose was negatively correlated with Rothia（r=-0.09）,Raoultibacter（r=-0.10）,Eggerthella（r=-0.16）,Candidatus＿Stoquefichus（r=-0.09）,Streptococcus（r=-0.09）and Ruminococcaceae＿UBA1819（r=-0.11）（P<0.05）,but positively correlated with Bacteroides（r=0.08）,Lachnospiraceae＿UCG-008（r=0.11）,Colidextribacter（r=0.08）and Oscillospiraceae＿UCG-003（r=0.09）.The 2-hour postprandial blood glucose was negatively correlated with Odoribacter（r=-0.11）（P<0.05）.3.The results of moderating effect analysis showed that a total of 7 bacteria could moderate the level of blood glucose affected by PM_2.5 exposure.PM_2.5 has a positive effect on blood glucose.With the increase of the relative abundance of gut microbiota,Solobacterium and Escherichia＿Shigella had a positive moderate effect on the correlation between PM_2.5 and fasting blood glucose,while Fusicatenibacter and Ruminococcaceae＿UBA1819 had a negative moderate effect on the correlation between PM_2.5 and 1-hour postprandial blood glucose,and Raoultibacter,Anaerofustis,Ruminococcaceae＿UBA1819 and Phascolarctobacterium had a negative moderate effect on the correlation between PM_2.5 and 2-hour postprandial blood glucose.PartⅣ:Correlation between gut microbiota and GDM-related differences in blood metabolites and circ RNA1.The results of peripheral serum metabonomics analysis between pregnant women with GDM and controls showed that 28 differential metabolites such as Sphinganine 1-phosphate,sphingomyelin,Lactosylceramide,phosphatidylcholine,Dolichylβ-D-glucosyl phosphate and Citicoline were screened.Glycerophospholipid metabolism and Sphingolipid metabolism were the main metabolic pathways of differential metabolites of GDM.2.The results of the metabolomic biological network analysis showed that PLD1,PLD2,EHHADH and HADHA are the key target genes for the development of GDM and can be used as potential therapeutic targets for GDM.3.RT-q PCR was found that in the peripheral blood of pregnant women with GDM,the expression level of hsa＿circ＿0042852,hsa＿circ＿0004001 and hsa＿circ＿0006936 were lower than that of the control group,while the expression level of hsa＿circ＿0001946,hsa＿circ＿0000154,hsa＿circ＿0001016,hsa＿circ＿0006732 and hsa＿circ＿0001439 were higher than that of the control group.Differentially expressed circ RNA is involved in the regulation of insulin signaling pathway.4.A total of 8 GDM differential bacteria and 5 PM_2.5 effect on blood glucose levels were significantly associated with 17 differential metabolites,and 5 GDM differential bacteria and 3 PM_2.5 effect on blood glucose levels were significantly associated with 7differentially expressed circ RNAs.Among them,the significantly associated differential metabolites Sphinganine 1-phosphate,Sphingomyelin（d18:0/26:1（17Z））and Sphingomyelin（d18:0/12:0）were involved in the Glycerophospholipid metabolism;the significantly associated differential metabolites PE（24:1（15Z）/24:1（15Z））,Citicoline,PA（16:0/16:0）,PE（24:1（15Z）/22:0）and PC（24:0/24:1（15Z））are involved in the Sphingolipid metabolism;significantly associated differential expressions hsa＿circ＿0006732 and hsa＿circ＿0001439 are involved in the Insulin signaling pathway.It is suggested that PM_2.5 and gut microbiota may influence the development of GDM through Glycerophospholipid metabolism,Sphingolipid metabolism and Insulin signaling pathway.Conclusions1.PM_2.5 exposure can increase the incidence of GDM and the level of blood glucose.The sensitive exposure period is pre-pregnancy 3 months to 27 gestational weeks.can increase the incidence of GDM and the level of blood glucose.2.GDM pregnant women have gut microbiota imbalance during 13-28 weeks of gestation.Gut microbiota was significantly associated with blood glucose levels in pregnant women.3.Gut microbiota can moderate the effect of PM_2.5 on blood glucose level.Solobacterium and Escherichia＿Shigella had a positive moderate effect on the correlation between PM_2.5 and fasting blood glucose,while Fusicatenibacter,Ruminococcaceae＿UBA1819,Raoultibacter,Anaerofustis and Phascolarctobacterium had a negative moderate effect on the correlation between PM_2.5 and 2-hour postprandial blood glucose.It is suggested that PM_2.5 exposure causes an increase in the abundance of pro-inflammatory bacteria or a decrease in the abundance of SCFAs-producing bacteria,which in turn causes inflammation and insulin resistance,leading to the occurrence of GDM.4.PLD1,PLD2,EHHADH and HADHA in lipid metabolism can be used as potential targets for GDM therapy.PM_2.5 and gut microbiota may influence the development of GDM through Glycerophospholipid metabolism,Sphingolipid metabolism and Insulin signaling pathway.