Increasing the Hindgut Carbohydrate/Protein Ratio by Cecal Infusion of Corn Starch or Casein Hydrolysate Drives Gut Microbiota-Related Bile Acid Metabolism To Stimulate Colonic Barrier Function

Dietary high protein and low carbohydrate levels compromise colonic microbiota and bile acid metabolism, which underlies a detrimental gut environment. However, it remains unclear if the diet-induced changes in colonic health are due to a change in hindgut nutrient availability and what key intermediates link the microbe-epithelium dialogue. To specifically alter the hindgut nutrient substrate availability, here we used a cecally cannulated pig model to infuse corn starch and casein hydrolysate directly into the cecum to generate a stepwise change of carbohydrate/nitrogenous compound (C/N) ratio. Pigs were cecally infused daily with either saline (Control), corn starch (Starch), or casein hydrolysate (Casein) (n = 8 per group), respectively, for 19 days. After infusion, C/N ratios in colonic digesta were 16.33, 12.56, and 8.54 for the starch, control, and casein groups, respectively (P < 0.05). Relative to the control group, casein infusion showed greater abundance of the bacteria (Eubacterium) capable of bile acid 7α-dehydroxylation (baiJ), higher levels of expression of bacterial genes encoding the baiJ enzyme, and higher levels of secondary bile acid (deoxycholic acid [DCA] and lithocholic acid [LCA]), while the starch infusion showed the opposite effect. Correspondingly, casein infusion downregulated expression of genes encoding tight junction proteins (ZO-1 and OCLD) and upregulated expression of genes encoding epidermal growth factor receptor (EGFR). The ratio of C/N was linearly related with the concentrations of DCA and LCA and gene expression levels of ZO-1, occludin, and EGFR. Caco-2 cell experiments further showed that DCA and LCA downregulated expression of genes involved in barrier function (ZO-1 and OCLD) and upregulated the gene expression of EGFR and Src. Inhibition of EGFR and Src could abolish DCA- and LCA-induced downregulation of ZO-1, indicating that DCA and LCA impair gut barrier function via enhancing the EGFR-Src pathway. These results suggest that the ratio of C/N in the large intestine is an important determinant of microbial metabolism and gut barrier function in the colon. The findings provide evidence that microbe-related secondary bile acid metabolism may mediate the interplay between microbes and gut barrier function. IMPORTANCE High-fiber or high-protein diets could alter gut microbiota and health in the large intestine, but factors involved in the effects remain unclear. The present study for the first time demonstrates that the starch- and casein-induced C/N ratio in the hindgut is an important factor. Using the cannulated pig model, we found that the distinct C/N ratio induced by cecal infusion of corn starch or casein hydrolysate was linearly correlated with microbial metabolites (secondary bile acids) and tight junction proteins (ZO-1 and OCLD). Cell culture study further demonstrates that the gut microbial metabolites (DCA and LCA) could impair the intestinal barrier function via the EGFR-Src pathway. These suggest that DCA and LCA were key metabolites mediating microbe-epithelium dialogue when the hindgut C/N ratios were altered by cecal infusion of corn starch or casein hydrolysate. These findings provide new insight into the impact of C/N ratio in the large intestine on colonic health and provide a new framework for therapeutic strategy in gut health through targeted manipulation of hindgut microbiota by increasing the carbohydrate level in the large intestine.