| Objective: Functional Constipation(FC)is one of the most common gastrointestinal diseases in clinical practice,with a global incidence rate of 14-17%.The incidence rate significantly increases with age,and the incidence rate in females is about 1.5 times higher than that in males.The specific pathogenesis is still unclear.In order to explore the pathogenesis of FC in Chinese female patients,we used multiple omics methods such as metagenomics,targeted metabolomics,and Genome-Wide Association Studies(GWAS)to analyze the differences in intestinal microbial structure and metabolites,as well as single nucleotide polymorphisms(SNPs)and genes associated with FC among low-risk,high-risk,and patient populations.This study provides a theoretical basis for the early clinical warning and treatment of high-risk FC populations.Methods: 1.We recruited 461 postmenopausal Chinese women from Guangzhou and divided them into four groups according to FC diagnostic criteria(Group 1(n=38)and Group 2(n=361)were FC-low-risk groups,Group 3(n=52)was a FC-high-risk group,and Group 4(n=10)were patients),and evaluated the gut microbiota structure,serum short chain fatty acid(SCFA)concentrations,and GWAS analysis of the population using metagenomic sequencing,whole-genome sequencing,and targeted metabolomics.2.We recruited 12 premenopausal women from Huaihua,Hunan,collected samples for metagenomic sequencing and serum targeted metabolomics testing,and analyzed the differences in gut microbiota structure and serum short-chain fatty acid concentrations in this population to verify whether the results were consistent with those of the first part of the study.3.To explore the specific function of butyric acid in the development of intestinal neurons,we isolated and cultured primary mouse intestinal neurons in vitro,and detected the neuronal proliferation with different concentrations of butyric acid salt(0m M,0.1m M,0.5m M,1m M,2.5m M)treatment for 72 h,and used transcriptome sequencing to detect the gene expression of mouse intestinal neurons in the three butyric acid treatment groups(0m M,0.5m M,and 2.5m M).Results: 1.By comparing the intestinal microbiota structure of the FC-low-risk,FC-high-risk,and patient groups,we found that the butyrate-producing bacterium Fusobacterium was a key differential bacterium between the groups.Its relative abundance was negatively correlated with the risk of FC in the population,but serum butyric acid concentration was the opposite.These results were verified in an independent cohort.2.In vitro experiments with different concentrations of butyric acid treatment of mouse neuronal cells found that only the 0.5m M butyric acid treatment group promoted the proliferation and development of mouse intestinal neuronal cells.The GO,KEGG,and GSEA enrichment analysis of transcriptome sequencing showed significant upregulation and downregulation in cell cycle,oxidative phosphorylation,and other signaling pathways compared with other concentration treatment groups.3.GWAS analysis identified 19 SNPs associated with FC-high-risk populations,including rs9311526 on the CACNA2D3 gene and rs1021834201 on the CACNA1 A gene,both of which belong to the CACNA family,closely related to neuronal cell signaling,and may be associated with the disease process of functional constipation.Conclusion: This study conducted multi-omics analysis including metagenomics,whole-genome sequencing,and targeted serum metabolomics on 473 Chinese female population and found that the difference in abundance distribution of butyrate-producing bacteria led to a reduction in butyrate production,which affected the development of intestinal nerve cells and the insufficient energy supply of intestinal cells,thus affecting intestinal function.In addition,combined with the genetic background of SNPs and genes such as rs9311526,rs1021834201 and CACNA2D3 related to neural transmission in the population,this may be the cause of the eventual development of FC high-risk populations into patients. |
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