Hydroxytyrosol Gut Microbial Metabolites Promote Tight Junction Protein Expression via the AhR-Nrf2 Pathway

• Hydroxytyrosol (HT) microbial metabolites promote intestinal barrier integrity by enhancing tight junction protein expression. • Five HT-derived microbial metabolites (IAA, I3A, skatole, kynurenine, and homovanillic acid) activate the AhR signaling pathway. • Activation of AhR leads to the upregulation of Cyp1a1 and Nrf2/HO-1 antioxidant pathway. • Inhibition of AhR attenuates the effects of HT metabolites on tight junction and antioxidant gene expression. • This study reveals an AhR-Nrf2 dependent mechanism by which HT microbial metabolites maintain epithelial barrier function. Hydroxytyrosol (HT), a major polyphenolic in olive oil, enhances intestinal mucosal barrier function by activating the Nrf2/HO-1 pathway and promoting the expression of tight junction (TJ) proteins such as occludin, claudin-1, and ZO-1. However, most HT is not absorbed in the upper gastrointestinal tract and instead reaches the colon, where it is metabolized by gut microbiota into various bioactive compounds. Among these microbial metabolites, five—including indole-3-acetic acid, indole-3-acetaldehyde, skatole, kynurenine, and homovanillic acid, exhibit a high affinity for AhR. It is speculated that these metabolites may potentially promote TJ protein expression by activating the AhR pathway. To investigate this, HT-29 cells were used to simulate the intestinal epithelial barrier environment and established an LPS-induced injury model. The AhR inhibitor CH223191 was applied to explore whether HT microbial metabolites promote TJ protein expression through the AhR-Nrf2 pathway. Results indicated that HT microbial metabolites significantly upregulated AhR and its downstream gene Cyp1a1, activated the Nrf2/HO-1 antioxidant pathway, and promoted the gene expression of TJ proteins occludin, claudin-1, and ZO-1. Under AhR inhibitor intervention, the promoting effects of HT microbial metabolites on Cyp1a1, Nrf2, and HO-1 were significantly attenuated, and the expression of TJ proteins decreased. These findings confirm that the gut microbiota metabolites of HT promote TJ protein expression by activating AhR and subsequently regulating the AhR-Nrf2 pathway, providing insights for developing dietary polyphenol-based strategies for gut health.