Research On The Pathogenesis Of Chronic Spontaneous Urticaria Based On Microbiomics And Metabonomics

Objective:Using high-throughput sequencing technology and non-targeted metabolomics technology to study the composition of intestinal microflora and the characteristics of serum metabolites in patients with chronic spontaneous urticaria,and further analyze the correlation between differential microflora and metabolites.To expound the possible pathogenesis of chronic spontaneous urticaria.Methods:A total of 100 CSU patients and 100 healthy individuals(18-75 years of age)were enrolled in this study between June 2018 and June 2019.A questionnaire was completed for each participant to conduct single factor and multiple factor Logistic regression analysis of CSU risk factors.We randomly selected 10 patients with CSU and 10 healthy individuals after matching for age and gender,collected their fecal and serum samples for further 16 S rRNA gene sequencing and non targeted metabolomics analyses.Finally,the correlation analysis method of Spearman was used to analyze the selected differential microorganisms and differential metabolites.Results:1.The results of epidemiological investigation showed that there was no significant differences in age or gender existed between the two groups.The results of single factor analysis showed that the place of residence,the use of antibiotics in pregnancy,the way of production,the way of feeding,the eating habits,the use of antibiotics in the last year,factors such as surgery and mental trauma in the past six months,and alcohol drinkingwas significantly associated with chronic spontaneous Urticaria(P<0.05).Multiple factor Logistic regression analysis showed that the use of antibiotics during pregnancy,caesarean section,high fat and low carbohydrate,use of antibiotics in the last year,and drinking alcohol were independent risk factors for the onset of chronic spontaneous urticaria.2.16 S rRNA gene sequencing: The results of Alpha diversity showed that the species richness and diversity of intestinal flora in CSU patients were significantly lower than that in healthy people(P < 0.05).The results of Metastat analysis showed that there were statistically significant differences in Firmicutes at the phylum level and Bacteroides,unidentified Enterobacteriaceae,Faecalibacterium,Bifidobacterium,and unidentified Ruminococcaceae at the level of genus.LEfSe analysis showed that the CSU patients were characterized by a higher abundance of unidentified Enterobacteriaceae,whereas the healthy controls primarily showed higher enrichment with Bacteroides,Firmicutes and Bifidobacteriales.Tax4 Fun functional prediction mainly indicated that modifications of the gut microbiota may lead to changes in related metabolic pathways,such as nucleotide metabolism.3.Non-targeted metabolomics analysis: The heatmap shows that there are significant differences between serum metabolites in CSU patients and healthy people;The number of differential metabolites in the two groups was 154 in the positive ion mode and 56 were in the negative ion mode.These metabolites included amino acids(glutamic acid and succinic acid),fatty acids(docosahexaenoic acid and arachidonic acid),carbohydrates(sucrose),and purines.The main metabolic pathways involved in differential metabolites are: under the positive ion mode,including Biosynthesis of unsaturated fatty acids,Histidine metabolism,Inflammatory mediator regulation of TRP channels,etc.Under the negative ion mode,including Butanoate metabolism,phenylalanine metabolism,Alanine,aspartate and glutamate metabolism,Arginine and proline metabolism,etc.4.Spearman correlation analysis showed that docosahexaenoic acid and arachidonic acid had a positive correlation with Bacteroides.L-glutamic acid had a negative correlation with unidentified Enterobacteriaceae and a positive correlation with Bifidobacterium.Succinic acid showed a positive correlation with Faecalibacterium.Conclusion:First of all,we found that antibiotics used in pregnancy,caesarean section,high fat and low carbohydrate,antibiotics used in the past year and alcohol consumption may affect the incidence of CSU by changing the structure and composition of intestinal microflora.Secondly,the abundance and diversity of intestinal microflora in CSU patients were lower than those in the normal control group,and the number of unidentified Enterobacteriaceae in CSU patients increased,while the number of Firmicutes,Bacteroides,Faecalibacterium,Bifidobacterium,and unidentified Ruminococcaceae decreased.Finally,by metabonomics and correlation analysis,Our study revealed profound changes in the composition of gut microbes-metabolites and the corresponding relationship,including Bacteroides associated with docosahexaenoic acid and arachidonic acid,Bifidobacterium,Faecalibacterium,and unidentified Enterobacteriaceae associated with L-glutamic acid and succinic acid.Taken together,we speculated that Bacteroides*unidentified Enterobacteriaceae,Faecalibacterium and Bifidobacterium caused the changes of docosahexaenoic acid,arachidonic acid and L-glutamic acid and succinic acid in serum,respectively,which may contribute to exacerbated inflammatory responses and dysregulated immune function with or without Treg cell dependence in the pathogenesis of CSU.