**Background:** Oxidative stress is a key driver of intestinal inflammation, causing cellular damage and tissue injury. Natural antioxidants from agro-industrial by-products offer potential alternatives to conventional drugs. Grape seed meal (GSM), a winery by-product rich in polyphenols (catechins, epicatechin, caffeic acid) and polyunsaturated fatty acids, has documented anti-inflammatory and antioxidant properties. This study evaluated GSM's capacity to counteract oxidative stress induced by E. coli lipopolysaccharide (LPS) in IPEC-1 cells and by dextran sulphate sodium (DSS) in weaned piglets.
**Methods:** For the in vitro study, IPEC-1 cells were treated with LPS (5 μg/mL) for 4 h followed by GSM extract (50 μg polyphenols/mL) or EGCG (50 μM) for 24 h. ROS were quantified by flow cytometry. Gene expression of CAT, SOD, GPx, eNOS, iNOS, Nrf2, Keap1, NQO1, and HO1 was measured by qPCR. CAT and SOD enzyme activities and total antioxidant capacity (TAC) were assessed spectrophotometrically. For the in vivo study, 20 weaned piglets (21 days old) were assigned to four groups (n=5): Control diet, Control + DSS (1 g/b.w./day), 8% GSM diet, and 8% GSM + DSS. DSS was administered orally in two 5-day cycles (days 1–5 and 21–25). On day 30, colon and mesenteric lymph nodes were collected. ROS, TBARS, DNA/RNA oxidative damage (8-OHdG/8-OHG), and protein carbonyl content were measured. CAT, SOD, GPx activities and TAC were determined. Gene expression of antioxidant enzymes and Nrf2 pathway markers was analyzed by qPCR. Nrf2 protein expression (total, cytoplasmic, and nuclear) was assessed by Western blotting.
**Key Results:** GSM extract contained 3915 mg total polyphenols/100g dry matter, with catechin (104.70 mg/100g) as the most abundant polyphenol. In vitro, LPS increased ROS-positive cells 50-fold vs Control (p=0.000018). GSM and EGCG reduced ROS-positive cells by 31- and 23-fold, respectively (p=0.001 and p=0.043 vs LPS). LPS downregulated CAT (0.3±0.1 Fc, p=0.018), SOD (0.6±0.0 Fc, p=0.029), and GPx (0.4±0.0 Fc, p=0.008) gene expression, while upregulating eNOS (3.8±0.2 Fc, p=0.005) and iNOS (2.1±0.1 Fc, p=0.012). GSM restored all these genes to Control levels. CAT activity decreased 42% (p=0.050) and SOD activity decreased 23% (p=0.005) with LPS; GSM restored both. LPS increased Keap1 expression (2.5±0.2 Fc, p=0.021) and decreased Nrf2 (0.4±0.1 Fc, p=0.031), NQO1 (0.2±0.1 Fc, p=0.025), and HO1 (0.2±0.1 Fc, p=0.013); GSM reversed these effects. In vivo, DSS increased ROS 1.5-fold in colon and 2-fold in lymph nodes (p=0.050). TBARS increased +48% in colon (p=0.0000003) and +127% in lymph nodes (p=0.0000002). DNA oxidative damage increased +65% in colon (p=0.041) and +74% in lymph nodes (p=0.017). Protein carbonyl increased +48% in colon (p=0.045) and +30% in lymph nodes (p=0.003). GSM diet restored all these markers toward Control levels. DSS downregulated CAT and GPx gene expression and enzyme activities in both organs, and upregulated eNOS and iNOS; GSM restored these. TAC decreased -16% in colon (p=0.030) and -22% in lymph nodes (p=0.005) with DSS; GSM restored TAC. DSS increased Keap1 expression (+116% in colon, p=0.043; +65% in lymph nodes, p=0.004) and decreased NQO1 (-57% in colon, p=0.001; -60% in lymph nodes, p=0.005) and HO1 (-80% in colon, p=0.002; -65% in lymph nodes, p=0.007). GSM restored Keap1 and NQO1 in colon and Keap1 in lymph nodes but not HO1 or NQO1 in lymph nodes. Nrf2 mRNA decreased -72% in colon (p=0.044) and -62% in lymph nodes (p=0.049) with DSS; Nrf2 protein decreased -49% in colon (p=0.020) and -44% in lymph nodes (p=0.0004). DSS increased cytoplasmic Nrf2 sequestration (colon: 56.88% vs 42.92%, p=0.012; lymph nodes: 52.32% vs 45.58%, p=0.013). GSM restored nuclear/cytoplasmic Nrf2 distribution to Control levels.
**Clinical Implications:** GSM demonstrates potent antioxidant capacity against LPS- and DSS-induced oxidative stress in both intestinal epithelial cells and weaned piglets, acting through Nrf2 pathway activation. Dietary inclusion of 8% GSM could serve as a natural nutritional strategy to manage post-weaning oxidative stress and intestinal inflammation in pigs, potentially reducing reliance on conventional drugs. The parallel in vitro and in vivo results support IPEC-1 cells as a reliable model for screening antioxidant compounds. Given the similarity between porcine and human intestinal physiology, these findings may also have translational relevance for human intestinal inflammatory conditions.