Grape seed-derived procyanidin alleviates gliadin p31–43-induced intestinal epithelial stress associated with ATF4 and autophagy–iron homeostasis

Gluten-derived gliadin p31–43 is a digestion-resistant peptide implicated in intestinal epithelial stress, but its mechanism of intestinal epithelial injury remains unclear. This study investigated the relationships among p31–43-induced endoplasmic reticulum (ER)–mitochondrial stress responses, autophagic flux impairment, iron dyshomeostasis, ferroptosis-related injury, and epithelial barrier-related alterations, and evaluated the protective effects of grape seed-derived procyanidin (PC) and their association with activating transcription factor 4 (ATF4)-related signaling. In Caco-2 cells, p31–43 decreased mitochondrial membrane potential, increased mitochondrial reactive oxygen species(mtROS) and Fe²⁺ accumulation, activated protein kinase R-like endoplasmic reticulum kinase (PERK)–ATF4–C/EBP homologous protein (CHOP) signaling, impaired autophagic flux, disrupted iron homeostasis, and enhanced lipid peroxidation. MitoTEMPO or 4-phenylbutyric acid (4-PBA) partially reversed these abnormalities. ATF4 knockdown aggravated epithelial injury, whereas ATF4 overexpression alleviated it. PC attenuated p31–43-induced cellular stress, improved barrier-related markers, and increased ATF4 expression, while these protective effects were markedly weakened after ATF4 knockdown. In mice, PC improved gliadin-induced intestinal mucosal injury. These findings support PC as a food-derived polyphenol for maintaining gluten-related epithelial barrier homeostasis.