Bacteroides fragilis alleviates necrotizing enterocolitis through restoring bile acid metabolism balance using bile salt hydrolase and inhibiting FXR-NLRP3 signaling pathway

Abstract

Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in premature infants with no specific treatments available. We aimed to identify the molecular mechanisms underlying NEC and investigate the therapeutic effects of Bacteroides fragilis on NEC. Clinical samples of infant feces, bile acid-targeted metabolomics, pathological staining, bioinformatics analysis, NEC rat model, and co-immunoprecipitation were used to explore the pathogenesis of NEC. Taxonomic characterization of the bile salt hydrolase (bsh) gene, enzyme activity assays, 16S rRNA sequencing, and organoids were used to explore the therapeutic effects of B. fragilis on NEC-related intestinal damage. Clinical samples, NEC rat models, and in vitro experiments revealed that total bile acid increased in the blood but decreased in feces. Moreover, the levels of FXR and other bile acid metabolism-related genes were abnormal, resulting in disordered bile acid metabolism in NEC. Taurochenodeoxycholic acid accelerated NEC pathogenesis and taurodeoxycholate alleviated NEC. B. fragilis displayed bsh genes and enzyme activity and alleviated intestinal damage by restoring gut microbiota dysbiosis and bile acid metabolism abnormalities by inhibiting the FXR-NLRP3 signaling pathway. Our results provide valuable insights into the therapeutic role of B. fragilis in NEC. Administering B. fragilis may substantially alleviate intestinal damage in NEC.

Keywords: B. fragilis; BSH; FXR; NLRP3; Necrotizing enterocolitis; Organoids; bile acid metabolism.

Figure 1.

Differences in bile acid levels in feces between NEC patients (n = 9) and healthy infants (n = 10). (a). PCA of bile acids. (b). The heatmap displays concentrations of bile acids, and the data was transformed with Log10. (c). Volcano plot of differential bile acids. (d). Concentration of TDCA in feces of healthy infants and infants with NEC. (e). Concentration of TBA in the feces of healthy infants and infants with NEC. (f). The ratio of secondary bile acids/primary bile acids. (g). The ratio of free bile acids/conjugated bile acids. *p < 0.05; **p < 0.01.

Figure 2.

TCDCA promoted NEC pathogenesis. (a). The grouping and flowchart of the rat experiment (n = 10 per group). (b). Images of the small intestine in different groups and HE staining of the rat ileum. (c). The length of the small intestine in different groups. (d). Inflammatory scores of HE staining in different groups. (e). Relative mRNA expression of Il-6 in the ileum. (f). Images of ileum organoids obtained under a light microscope. (g). Cell death in the ileum organoids in each group (n = 30 per group). *p < 0.05; **p < 0.01.

Figure 3.

Bile acid metabolism was disorderly in NEC. (a). Enrichment plot of KEGG pathways using GSEA. (b). IHC staining of FXR in the ileum and liver. (c). IHC staining scores of FXR in the ileum and liver. (d). The protein levels of FXR and FGF19 in the ileum and comparison of grayscale values of Western blot bands. (e). The protein levels of FXR, OATP, NTCP, CYP7A1, and BSEP in the liver and comparison of grayscale values of Western blot bands. (f). The grouping and flowchart of the rat experiment (n = 10 per group). (g). Images of the small intestine in different groups and HE staining of the rat ileum. (h). The length of the small intestine in different groups. (i). Inflammatory scores of HE staining in different groups. (j). Relative mRNA expression of Il-6 in the ileum. *p < 0.05; **p < 0.01.

Figure 4.

NLRP3 interacted with FXR. (a). Enrichment plot of KEGG pathways in the GSE46619 and GSE64801. (b). Co-IP identified the interaction between NLRP3 and FXR.

Figure 5.

bsh in bacteria strains and B. fragilis had efficient BSH enzyme activity. (a). Number of strains in top 10 genera with bsh. (b). Pie chart of strains with one bsh gene. (c). Pie chart of strains with two or three paralogous bsh genes. (d). Phylogenetic analysis of 1577 bsh genes. (e). Enzyme activity of BSH in five probiotics and B. fragilis knockout bsh strain. (f). Determining the amount of amino acids liberated from TDCA and GDCA to show the enzyme activity of BSH. *p < 0.05; **p < 0.01.

Figure 6.

The therapeutic effects of B. fragilis in the NEC model. (a). The grouping and flowchart of the rat experiment (n = 10 per group). (b). Images of the small intestine in different groups and HE staining of the rat ileum. (c). The length of the small intestine in different groups. (d). Inflammatory scores of HE staining in different groups. (e). Relative mRNA expression of Il-6 in the ileum. (f). PcoA of the gut microbiota in different groups. (g). Shannon index of the gut microbiota. (h). Relative abundance of microbiota at the phylum level. (i). Relative abundance of microbiota at the genus level. (j). Lefse analysis of microbiota, with LDA score ≥ 2. *p < 0.05; **p < 0.01.

Figure 7.

The effects of B. fragilis on bile acid metabolism (n = 3 per group). (a). PCA of the bile acids in different groups. (b). The heatmap displays concentrations of bile acids, and the data was transformed with Log₁₀. (c). Concentration of TCDCA in feces of rats in different groups. (d). The ratio of secondary bile acids/primary bile acids. (e). The ratio of free bile acids/conjugated bile acids. *p < 0.05; **p < 0.01.

Figure 8.

B. fragilis inhibited the FXR-NLRP3 signaling pathway and alleviated NEC. (a). IHC staining of FXR in the ileum and liver. (b). IHC staining scores of FXR in the ileum and liver. (c). The concentration of IL-1β in the ileum. (d). The protein levels of FXR and FGF19 in the ileum and comparison of grayscale values of Western blot bands. (e). The protein levels of FXR, OATP, NTCP, CYP7A1, and BSEP in the liver and comparison of grayscale values of Western blot bands. (f). Images of ileum organoids taken under a light microscope. (g). Cell death in the ileum organoids in each group (n = 30 per group). *p < 0.05; **p < 0.01.