Ovotransferrin alleviates ulcerative colitis by mediating gut microbiota involvement in secondary bile acid biosynthesis

Abstract Ovotransferrin (OVT) is a functionally active protein that has been demonstrated to alleviate ulcerative colitis (UC), yet its underlying mechanism of action has not yet been fully elucidated. In this context, this study employed a DSS-induced UC mouse model to confirm that OVT alleviates UC, as evidenced by reduced disease activity index (DAI; 40.63%), histopathological score (42.50%), and expression of TNF-α (38.50%), IL-1β (41.73%), and IL-6 (41.73%). However, in the pseudo-germ-free mouse model, OVT failed to alleviate UC. Subsequently, after fecal microbiota transplantation, the alleviation of OVT on UC was restored, including reducing the DAI (46.38%), histopathological score (38.46%), and the expression of TNF-α (30.97%), IL-1β (28.95%), and IL-6 (42.40%). These results indicated that OVT alleviates UC in a gut microbiota-dependent manner. The 16S rRNA gene sequencing was employed to investigate the changes in the gut microbiota of mice, and it was found that g__Lactobacillus might be a potential genus (highest LDA score: 4.77). Subsequently, broad-target metabolomics identified 206 differential metabolites (DMs) in the intestinal contents of the OVT vs DSS group. KEGG pathway analysis of the DMs revealed significant enrichment in the secondary bile acid biosynthesis pathway. Furthermore, 6 differential bile acids (DBAs) were identified, including ursodeoxycholic acid (UDCA), 3-epideoxycholic acid (3β-DCA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), tauroursodeoxycholic acid (TUDCA) and isochodeoxycholic acid (isoCDCA), which reduced paracellular flux of FITC-Dextran (16.80%-31.16%) and Lucifer yellow (31.61%-47.09%), the expressions of TNF-α (36.19%-59.30%), IL-1β (41.30%-54.12%), and IL-6 (31.40%-53.15%), thereby exhibiting anti-inflammatory properties. Notably, UDCA, isoCDCA, and TUDCA exhibited stronger anti-inflammatory effects. In summary, this study established the connection among OVT, the gut microbiota, and bile acids, and elucidated the mechanism by which OVT alleviates UC through modulation of the gut microbiota and its involvement in secondary bile acid biosynthesis.