Squalene alleviates DSS-induced colitis by restoring Nrf2-dependent redox balance and gut microbiota homeostasis.

Background: Ulcerative colitis (UC) is a chronic inflammatory disorder characterized by excessive oxidative stress, intestinal epithelial barrier dysfunction, and gut microbiota dysbiosis; despite expanding therapeutic options, achieving durable disease control remains challenging. Squalene (SQ), a naturally occurring triterpene with antioxidant and anti-inflammatory properties, has emerged as a promising bioactive compound. Although the protective potential of SQ in experimental colitis has been previously reported, the molecular dependence of this effect and the functional contribution of the gut microbiota remain incompletely defined. This study investigated the protective effects of SQ against DSS-induced experimental colitis and the roles of Nrf2 signaling and the gut microbiota in mediating these effects. Methods: DSS-induced colitis was established in mice to evaluate the protective effects of SQ. Disease severity, histopathological injury, oxidative stress, inflammatory responses, intestinal barrier integrity, and gut microbiota composition were assessed. Nrf2-deficient mice, pharmacological inhibition of Nrf2, antibiotic-mediated microbiota depletion, fecal microbiota transplantation (FMT), 16S rRNA sequencing, cellular thermal shift assay (CETSA), and lipopolysaccharide-stimulated Caco-2 cells were employed to investigate the underlying mechanisms. Results: SQ markedly alleviated experimental colitis by reducing disease activity, preventing colon shortening, and improving histopathological injury. Mechanistically, SQ activated Nrf2-dependent antioxidant defense, attenuated oxidative stress, suppressed NF-κB-mediated inflammation, and restored intestinal barrier integrity by enhancing tight junction protein expression. These protective effects were substantially diminished in Nrf2-deficient mice, identifying Nrf2 as a critical mediator of SQ activity. SQ also reshaped gut microbial composition and partially restored microbial community structure. Antibiotic-mediated microbiota depletion markedly weakened the beneficial effects of SQ, whereas fecal microbiota transplantation from SQ-treated donors transferred protection against DSS-induced colitis, supporting a functional contribution of microbiota remodeling to SQ-mediated protection. Consistently, SQ suppressed inflammatory responses and restored barrier-related protein expression in lipopolysaccharide-stimulated Caco-2 cells, effects that were attenuated by pharmacological inhibition of Nrf2. CETSA further demonstrated enhanced thermal stability of Nrf2 following SQ treatment, suggesting potential target engagement between SQ and Nrf2. Conclusion: SQ exerts protective effects against experimental colitis primarily through an Nrf2-dependent host molecular axis, with gut microbiota remodeling providing an important complementary functional contribution. These findings identify Nrf2 as the principal molecular mediator of SQ activity and position microbiota remodeling as a complementary component of its protective effects, supporting SQ as a promising bioactive candidate for intestinal inflammatory disorders.