Subacute Ruminal Acidosis Induces Colonic Inflammation and Barrier Damage Via Cgas-Sting Pathway in Hu Sheep Fed a High-Grain Diet.

High-grain diets widely applied in intensive ruminant farming frequently induce subacute ruminal acidosis, accompanied by intestinal inflammation and barrier damage, while the underlying molecular mechanisms remain unclear. Given the central role of the cGAS-STING cascade in inflammatory injury, this work explored its function in high-grain-induced colonic damage using lactating Hu sheep. In vivo, 12 healthy sheep were randomly divided into two equal groups, fed with either a low-grain (grain:forage=3:7, LG) or high-grain (grain:forage=7:3, HG) diet for 8 weeks, followed by comprehensive detection of ruminal pH, serum inflammatory profiles, colonic histopathological changes, and the expression of key molecules in the cGAS-STING pathway. To further validate the underlying mechanism, an in vitro inflammatory model was established by LPS stimulation of colonic epithelial cells. Mechanistically, ethidium bromide (EB)-mediated mtDNA depletion assay and specific cGAS/STING inhibitors were used to verify that mtDNA release acts as the essential upstream trigger for cGAS-STING pathway activation. This study revealed that HG diet reduced rumen pH below 5.6 for over 3 h daily, successfully establishing the subacute ruminal acidosis model. HG diet elevated LPS levels in rumen fluid, blood, and colon contents, and increased the cytoplasmic mtDNA/nDNA ratio in colon tissue, which further activated the cGAS‑STING/NF‑κB pathway and consequently triggered colonic inflammation and intestinal barrier dysfunction. In vitro assays further validated that EB or cGAS/STING inhibitors alleviated LPS-triggered inflammation and barrier impairment. Collectively, the cGAS-STING pathway is a key therapeutic target for maintaining ruminant intestinal homeostasis and preventing high-grain-related enteric disorders.