Yigongsan alleviates diarrhea in laying hens through cecal microbiota modulation and restoration of intestinal barrier related responses via EGFR PI3K AKT signaling

Diarrhea in laying hens impairs productive performance and intestinal health. Yigongsan (YGS), a classical traditional Chinese medicine formula, has been used clinically for gastrointestinal disorders; however, its therapeutic effects and underlying mechanisms in diarrheal laying hens remain unclear. This study evaluated the efficacy of YGS at different doses and explored its potential mechanisms in naturally diarrheal laying hens. A clinical intervention trial was conducted in laying hens with diarrhea. Hens received low-dose YGS (0.53 g/kg) or high-dose YGS (2.12 g/kg) for 7 d. Productive performance, ileal histopathology, inflammatory cytokines, oxidative stress-related indices, barrier-related markers, EGFR/PI3K/AKT pathway-related genes and proteins, and cecal microbiota composition were assessed using an integrated strategy including network pharmacology, histological analysis, gene and protein expression assays, and 16S rRNA gene sequencing. High-dose YGS showed broader and more consistent beneficial effects than low-dose YGS. It improved diarrhea-associated phenotypes, including body weight and egg production, alleviated ileal mucosal injury, and increased the villus height/crypt depth ratio. High-dose YGS also increased the mRNA expression of TJP1 and Occludin, reduced inflammatory responses and oxidative stress in serum and ileal tissues, and was associated with modulation of EGFR/PI3K/AKT pathway-related gene expression, increased PTEN expression, decreased PI3K and AKT phosphorylation, and normalization of the Bcl-2/Bax balance. In contrast, the low-dose treatment produced only partial improvements in several indices. Microbiota analysis showed no significant changes in α-diversity, but suggested shifts in β-diversity, microbial composition, and predicted microbial functions after YGS treatment, particularly in the high-dose group.In conclusion, YGS, especially at the high dose, alleviated diarrhea-associated pathological changes in laying hens and improved intestinal homeostasis. These effects may be associated with attenuation of inflammation and oxidative stress, restoration of barrier-related responses, modulation of EGFR/PI3K/AKT signaling, and restructuring of cecal microbiota composition.