Betulinic acid ameliorates lipopolysaccharide-induced intestinal barrier dysfunction in broilers by remodeling gut microbiota homeostasis and inhibiting the LYG2/NOD1/NF-κB signaling axis.
Abstract
Betulinic acid (BA), a natural pentacyclic triterpenoid obtained from agroforestry by-products, possesses potent antioxidant, anti-inflammatory and intestinal-protective bioactivities. Our prior work verified that BA alleviated intestinal inflammatory damage in mouse and piglet models. Nevertheless, its protective effects and underlying molecular mechanisms against lipopolysaccharide (LPS)-evoked intestinal barrier dysfunction in broilers remain poorly understood. Under basal feeding conditions, dietary BA supplementation improved ileal mucosal integrity by elevating villus height and crypt depth, upregulating tight junction genes (Occludin, ZO-1) expression, boosting antioxidant capacity, and inhibiting pro-inflammatory gene transcription. In the LPS-challenged inflammatory model, BA pretreatment rescued LPS-reduced feed intake, restored goblet cell density and epithelial microvillus ultrastructure, decreased serum DAO and LPS leakage, and relieved intestinal inflammatory overactivation. Transcriptomics screened LYG2 as a pivotal hub gene modulated by BA. Follow-up RT-qPCR and immunofluorescence verified BA blunted LPS-triggered overactivation of the LYG2/NOD1/NF-κB cascade at transcriptional and protein levels. 16S rRNA sequencing illustrated BA reversed LPS-induced gut dysbiosis via enriching beneficial Ruminococcus_torques and Enterococcus. Correlation assays indicated these two taxa positively correlated with intestinal barrier markers and negatively correlated with inflammatory genes in the LYG2/NOD1/NF-κB axis. This study unveils a dual protective mechanism of BA against LPS-mediated intestinal barrier injury in broilers: BA remodels dysregulated gut microbiota and inhibits the LYG2/NOD1/NF-κB inflammatory cascade to sustain intestinal mucosal homeostasis. BA represents a safe, green phytogenic feed additive to alleviate intestinal inflammation and cut antibiotic usage in poultry farming. Our findings provide novel insights into dietary BA as a potential nutritional strategy to mitigate ileal injury in poultry, with implications for improving gut health and productivity in broilers.