Glycyrrhizinic Acid and Isatidis folium extracts combined with Bacillus subtilis protect against dextran sulfate sodi-um-induced colitis in Hu sheep

Weaning stress caused by maternal separation and dietary changes often leads to intestinal disorders such as diarrhea and enteritis in young animals, and the development of antibiotic-free, safe preparations to manage these conditions is therefore a promising strategy. The optimal proportion of each component in the formulation was determined in our preliminary study using orthogonal experimental design and range analysis. This study first evaluated the effects of glycyrrhizic acid and Isatidis folium extract on the growth of Bacillus subtilis ( B. subtilis ), and then examined the effects of the compound preparation (CP) composed of glycyrrhizic acid, Isatidis folium extract, and B. subtilis on in vitro antibacterial activity, growth performance, immune modulation, and the amelioration of dextran sulfate sodium (DSS)-induced colitis in Hu sheep. Twenty-seven 45-day-old healthy male Hu lambs (body weight = 15.83 ± 0.41 kg) were randomly assigned to 3 dietary groups, each dietary group consisted of 9 animals, were housed in 3 pens with 3 sheeps in each. The lambs were fed with the basal diets (control group), supplemented with 25 g (compound preparation group 1, CP1), 30 g (compound preparation group 2, CP2) CP per kg diet, respectively. Experimental design of protective effects of CP on lamb colitis was eighteen 45-days-old healthy male Hu lambs (body weight = 16.24 ± 0.86 kg) were randomly assigned to three groups (n = 6 per group), the lambs were fed with the basal diets (control group); lambs were fed with the basal diets for the first 7 days and were then given a single daily oral gavage of DSS at 4 g/kg body weight (DSS group); lambs were fed with the basal diet supplemented with 25 g CP per kg diet for the first 7 days and were then given a single daily oral gavage of DSS at 4 g/kg body weight (CP-DSS). The results showed that glycyrrhizic acid and Isatidis folium extract did not affect the growth of B. subtilis , while the CP significantly inhibited the growth of Escherichia coli ( E. coli ) and Staphylococcus aureus ( S. aureus ). CP1 significantly increased daily weight gain; CP1 and CP2 significantly increased serum immunoglobulin G (IgG) and colonic mucosal secretory immunoglobulin A (SIgA) levels, as well as the relative mRNA expression of interferon γ (IFN-γ), interleukin-1β (IL-1β), interleukin-4 (IL-4), interleukin-6 (IL-6) and interleukin-10 (IL-10). Supplementation with 25 g CP per kg diet significantly downregulated the relative mRNA expression of proinflammatory cytokines (IFN-γ, IL-1β, IL-6) and upregulated the relative mRNA expression of anti-inflammatory cytokines (IL-4, IL-10) and intestinal barrier factors (ZO-1, occludin, Muc2, α-catenin), alleviating colonic damage. These findings support its potential as a safe and effective feed additives for promoting intestinal health in sheep.