Red Ginseng-Derived 20(S)-Ginsenoside Rg3 and Compound K Restore Redox Homeostasis and Alleviate LPS-Induced Barrier Damage in Bovine Intestinal Epithelial Cells

This study aimed to determine whether 20(S)-ginsenoside Rg3 (S-Rg3) and compound K (CK), two representative ginsenosides retained in red ginseng processing by-products, could restore redox homeostasis and alleviate lipopolysaccharide (LPS)-induced injury in bovine intestinal epithelial cells, and to clarify the associated autophagy–lysosome mechanisms. Ginsenoside constituents were characterized by LC-MS. Network pharmacology, machine learning-assisted target screening, molecular docking, and molecular dynamics simulations were integrated to predict compound–target interactions. For in vitro validation, bovine small intestinal epithelial cells were pretreated with 8 μM S-Rg3, 8 μM CK, or their combination for 24 h, followed by a 24 h challenge with 50 mg/L LPS. Computationally, S-Rg3 and CK showed favorable predicted binding with Keap1, TLR4, NKIRAS2 p65, and iNOS. Experimentally, S-Rg3, CK, and particularly their combined pretreatment significantly improved cell viability, reduced reactive oxygen species and nitric oxide accumulation, restored antioxidant enzyme activities and Nrf2/HO-1-associated antioxidant defense, suppressed inflammatory activation, and preserved tight junction protein expression compared with the LPS group. The treatments further alleviated LPS-induced autophagy–lysosome dysfunction, endoplasmic reticulum stress, mitochondrial injury, and apoptosis. Multi-omics integration linked these protective effects to inflammatory lipid mediator, arginine/nitric oxide, amino acid, and energy metabolism, together with redox-related signaling pathways. Collectively, S-Rg3 and CK protected bovine intestinal epithelial cells from LPS-induced injury primarily by restoring redox homeostasis and supporting autophagy–lysosome function. These findings provide mechanistic evidence supporting the valorization of red ginseng processing by-products as potential bioactive resources for improving intestinal epithelial resilience in animal production.