| Objective1.To compare the structure of vaginal and gut microbiome between patients with unexplained spontaneous abortion and normal early pregnancy women and their relationship with the immune balance of Treg/Th17 at the maternal-fetal interface.2.To explore the possible mechanism of Modified Shoutai Wan through gut microbiome-metabolite-immune regulation.Methods? Clinical Study1.Collect vaginal and stool samples from patients with unexplained SA and normal pregnancy for 16 S r RNA gene sequencing.The composition and diversity of the vaginal and gut microbiome of the two groups were compared.LEf Se analysis screened the differential microbiome and using PICURSTs to predict its function,and studied the biological pathways that may be involved in the functional genes of the differential bacteria.2.Collect unexplained SA and NP decidual tissues and detect the expression of Foxp3 and ROR?t protein by WB,and associate them with vaginal and gut microbiome,to preliminarily explore the relationship between the bacteria and the Treg/Th17 immune balance of the maternal-fetal interface.? Experimental research1.The abortion model rats with mifepristone were constructed,and the Modified Shoutai Wan was used for intervention.Effects were evaluated by calculating the abortion rate and observing the embryo morphology.Collect rat stool samples for 16 S r RNA gene sequencing,LEf Se analysis to screen differential bacteria,and use PICURSTs software to predict the biological pathways of differential bacteria functional genes.2.HPLC-MS technology detects non-targeted metabolites in feces,screens differential metabolites,and correlates with different gut bacteria to clarify the effect of Modified Shoutai Wan on fecal metabolites.3.Flow cytometry to detect the percentage of Treg and Th17 cells in the peripheral blood of rats;WB to detect the expression of Foxp3 and ROR?t protein in decidua;ELISA to detect serum TGF-?,IL-6,IL-10,and IL-17 concentrations.The correlation of relevant indicators with different bacteria and different metabolites,to initially explore the effect of Modified Shoutai Wan on intestinal microbiome-metaboliteimmune regulation.Results? Clinical Study1.Vaginal microbiome(1)The composition of the vaginal microbiome of SA and NP groups is similar at the phylum and genus level.(2)Compared to SA group,the chao1 and observed species indexes were significantly lower in the NP group(P<0.05).(3)The similarity,dispersion,abundance,and evolutionary relationship between the two groups calculated using Binary Jaccard,Bray Curtis,Euclidean,and Unweighted Unifrac distances are significantly different(P<0.05).(4)LEf Se analysis found that the significantly different species in the SA group was Shuttleworthia;the significantly different species in the NP group were:Bifidobacteriaceae,Bifidobacteriales,Scardovia,Gammaproteobacteria,etc.(5)PICRUSt function prediction shows: NP group metabolic pathway is mainly enriched in the "bacterial invasion of epithelial cells";SA group metabolic pathway is enriched in "biosynthesis of type II polyketide backbone","cytokine receptor","cytokine-cytokine receptor interaction" and so on.2.Gut microbiome(1)The composition of the gut microbiome of the two groups is similar at the phylum and genus level.(2)There were no differences in chao1,shannon,observed species,simpson,and PD whole tree indexes between women in the NP group and SA group(P>0.05).(3)The difference in the ANOSIM analysis of the gut microbiome between the two groups using Binary Jaccard and Unweighted Unifrac distance was statistically significant(P<0.05).(4)LEf Se analysis found that the significantly different species in the SA group were Alphaproteobacteria,SAR11?clade,etc.;the significantly different species in the NP group were Rikenellaceae,Alitipes,etc.(5)PICRUSt function prediction shows that the metabolic pathways of the NP group are mainly enriched in "toluene degradation";the metabolic pathways of the SA group are enriched in "cytokine receptor","cytokine-cytokine receptor interaction",etc.3.Immune balance of unexplained SA maternal-fetal interface and its association with different bacteria(1)The expression of Foxp3 protein in decidua tissue of SA group was significantly lower than that of NP group(P<0.05),while the expression of ROR?t protein was significantly higher than that of NP group(P<0.05).Compared with NP group,the expression of Foxp3/ROR?t in SA group was obvious decrease(P<0.05).(2)The expression of Foxp3 protein was negatively correlated with the relative abundance of gut Clade?III,Eubacterium ruminantium group,and SAR11?clade(P<0.05),and positively correlated with vaginal Escherichia coli-Shigella,Scardovia,etc.(P<0.05).The expression of ROR?t protein is positively correlated with the relative abundance of gut SAR11?clade and Clade?III,and is related to the vaginal Bifidobacteriaceae,Bifidobacteriales,Escherichia coli-Shigella,etc.(P<0.05).The relationship between Foxp3/ROR?t and different bacteria has a similar trend with Foxp3.? Experimental research1.The effect of Modified Shoutai Wan on the gut microbiome of rats with mifepristone abortion model(1)Drug efficacy(1)The abortion rate of the model control group was 50.54%,which was significantly higher than that of the negative control group(P<0.05).The abortion rates of the positive control group and the Modified Shoutai Wan group were 30.17%and 33.09%,respectively,which decreased by 20.37% and 17.28% compared with the model control group(P<0.05).(2)Comparing the negative control group,positive control group,Modified Shoutai Wan group and the model control group,there was no significant difference in the weight of uterus and embryos(P>0.05).(3)The appearance of uterus and embryos in the negative control group,positive control group and Modified Shoutai Wan group was better than that of the model control group.(2)Structure of gut microbiome(1)The composition of the gut microbiome in the 4 groups of rats was similar at the level of phylum and genus.(2)Compared with the model control group,there is no difference in chao1,shannon,observed species,simpson,and PD whole tree index in the negative control group,positive control group,and Modified Shoutai Wan group(P>0.05).(3)The difference in ANOSIM analysis based on the five distances of Binary Jaccard,Bray Curtis,Euclidean,Unweighted Unifrac,and Weighted Unifra is significant(P<0.05).(4)LEf Se analysis found that: the characteristic species of the negative control group included Clostridiales,Clostridia,Ruminococcaceae,etc.;the characteristic species of the model control group included Prevotella?9,Bdellovibrio,Bdellovibrionaceae,etc.The characteristic species of the positive control group include Ruminococcus?gnavus?group,Lysinibacillus,etc.;the characteristic species of the Modified Shoutai Wan group include: Bacteroidaceae,Bacteroides,Proteobacteria,Gammaproteobacteria and so on.(5)PICURSt function prediction found that the metabolic pathways of the model control group were mainly enriched in the "pantothenate and Co A biosynthesis","fatty acid biosynthesis",etc.;the metabolic pathways of the Modified Shoutai Wan group were mainly enriched in "primary bile acid biosynthesis","flavonoid and flavonol synthesis ","secondary bile acid biosynthesis",etc.2.The effect of Modified Shoutai Wan on fecal metabolites in abortion model rats(1)PCA analysis indicates that the samples of the negative control group and the Modified Shoutai Wan group and the model control group cannot be separated well,but the positive control group and the model control group have a good degree of discrimination;based on the PLS-DA and OPLS-DA analysis,compared with the model control group,the other three group both showed a significant difference.(2)Differential metabolites were screened based on the VIP value and statistical hypothesis testing method.A total of 273 differential metabolites were screened in the model control group and the Modified Shoutai Wan group.(3)Comparing the quantitative results of bile acid related to the bile acid metabolism pathway in each group,it was found that compared with the model group,LCA,UCD and MCA were significantly increased in the Modified Shoutai Wan group(P<0.05),and SCDCA was significantly increased in the positive control group(P<0.05).(4)The content of UDCA is positively correlated with intestinal Holdmania,Proteobacteria,Bacteroides,etc.(P<0.05),and negatively correlated with Rothia,etc.(P<0.05);LCA is positive correlation with Holdemania,Proteobacteria,etc.(P<0.05),and negative correlation with the genus of Ruminococcaceae?NK4A214?group and Rothia(P<0.05);MCA is positive correlation with Holdmania,etc.(P<0.05),negatively correlated with Rothia,etc.(P<0.05).3.The effect of Modified Shoutai Wan on the immune balance of the maternal-fetal interface in abortion model rats(1)Flow cytometry showed that the negative control group had significantly higher Treg cells in peripheral blood,while the percentage of Th17 cells was significantly reduced(P<0.05).The use of Modified Shoutai Wan can significantly increase the Treg of abortion model rats Cells,reduce Th17 cells.(2)The expression of Foxp3 protein in the decidua tissue of the negative control group,the positive control group and the Modified Shoutai Wan group was significantly increased compared with the model control group(P<0.05);The expression of ROR?t protein in negative control group and the Modified Shoutai Wan group was significantly lower than that of the model control group(P<0.05).(3)Modified Shoutai Wan can increase serum TGF-? and IL-6 content,and decrease IL-10 and IL-17 content(P>0.05).(4)The results of correlation analysis showed that UDCA,MCA,and LCA were negatively correlated with IL6,ROR?t,and IL-17,and positively correlated with IL-10,Foxp3,Foxp3/ROR?t,and TGF-?;Bacteroides and Bacteroides were related to ROR?t,respectively,IL-17 is negatively correlated,positively correlated with IL-10,Foxp3,Foxp3/ROR?t,and not correlated with IL-6 and TGF-?;Proteobacteria and ?-Proteobacteria are positively correlated with TGF-?,Foxp3/ROR?t,Foxp3,IL-10 and negatively correlated with IL6,ROR?t,IL-17.Conclusion1.There are differences in the structure of vaginal and gut microbiome in unexplained SA and NP populations.Corresponding different bacteria may be interacted with host through biological pathways such as cytokine-cytokine receptor interactio.2.There is an imbalance of Treg/Th17 cells in the decidua tissue of SA patients with unexplained reasons,which may be one of the reasons for the occurrence of SA.3.Modified Shoutai Wan may mediate intestinal Bacteroides,Proteobacteria,etc.through the link of gut microbiomea-metabolites-immune regulation,increase the content of LCA,MCA,UDCA,and correct the Treg/Th17 immune imbalance at the maternal-fetal interface to the role of tonifying the kidney and anti-fetus. |
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