Alfalfa leaf meal reprograms macrophage polarization via the gut microbiota-lanosterol-TLR4 axis to maintain intestinal epithelial homeostasis

Intestinal inflammatory dysfunction characterized by persistent mucosal inflammation and impaired epithelial barrier integrity causes substantial economic losses in livestock production. Nutritional interventions that modulate the gut microbiota–innate immune axis represent a promising adjunct to pharmaceutical treatments, yet the complete mechanistic cascade underlying the protective effects of plant-derived feed additives remains poorly defined. This study employed rabbits with diarrhea as a transformation model and fed them alfalfa leaf meal (ALM) rich in bioactive plant proteins. Utilising 16S rRNA sequencing, non-targeted metabolomics, and fecal microbiota transplantation techniques, we investigated whether ALM could alleviate Intestinal inflammatory. Results revealed that ALM supplementation was associated with improved growth performance, reduced intestinal damage, altered microbiota composition (reduced potentially harmful taxa, increased beneficial bacterial ratios), and elevated colonic lanosterol levels. In vitro experiments demonstrated that lanosterol suppressed TLR4 expression and promoted an anti-inflammatory macrophage phenotype under LPS challenge. Diarrhoeic rabbits transplanted with fecal microbiota from ALM-fed donor rabbits experienced reduced diarrhoea severity. Notably, LPS-treated macrophages exhibited a pro-inflammatory phenotype, which shifted toward an anti-inflammatory M2-like phenotype upon lanosterol supplementation in vitro. Concurrently, lanosterol suppressed TLR4 expression in macrophages and attenuated LPS-mediated pro-inflammatory responses. Molecular docking predicted a potential interaction between lanosterol and TLR4. The Caco2-macrophage co-culture model demonstrated that lanosterol enhances intestinal barrier function and alleviates LPS-induced Caco2 stress responses by inhibiting TLR4, downregulating the M1-like marker CD11C, and upregulating the M2-like marker CD206. This work identifies a tentative nutrition-regulated, microbe-mediated anti-inflammatory cascade: the ALM–microbiota–lanosterol axis provides a feed-applicable preclinical strategy to alleviate weaning-associated intestinal dysfunction in rabbits by restraining TLR4 signaling, balancing macrophage activation states, and reinforcing intestinal epithelial barriers.