Introduction
Epithelial barrier dysfunction is a hallmark of inflammatory bowel diseases (IBD), contributing to increased permeability, microbial translocation, and sustained mucosal inflammation1,2. Targeting barrier restoration has emerged as a promising therapeutic strategy3. Gallic acid, a dietary polyphenol with known anti-inflammatory and antioxidative properties, has shown potential in modulating mucosal responses4. To investigate its impact on epithelial integrity under inflammatory stress, we utilized intestinal organoids as a physiologically relevant ex vivo model system.
Aims & Methods
The aim of this study was to investigate the potential of gallic acid to restore intestinal epithelial barrier function under inflammatory conditions, using colonic epithelial organoids and integrating transcriptomic and permeability analyses.
The study was approved by the Kaunas Regional Biomedical Research Ethics Committee (protocol no. BE-2-31). Inflamed differentiated colonic epithelial organoids were established from a patient with ulcerative colitis (UC) and non-IBD control individual by exogenous stimulation with TNF-α and IFN-γ (10 ng/mL each) for 24 hours. For gallic acid (GA) screening, organoids were pre-treated with metabolite (100 μM) for 1 hour prior to inflammation induction and co-treated during the induction period. Subsequently, organoids were either processed for mRNA sequencing (1×75 bp, NextSeq 550, Illumina) or subjected to a FITC-Dextran (4kDa) assay. RNA-seq data was processed, analyzed, and visualized using nf-core/RNAseq pipeline and RStudio. Images for permeability assay analysis were acquired using an inverted fluorescence microscope (Zeiss Axio Observer 7), processed with ImageJ, and analyzed and visualized in RStudio.
Results
Principal component analysis of transcriptomic profiles from both UC and non-IBD organoids revealed clear sample clustering driven by stimulation. Differential gene expression and Gene set enrichment (GSEA) analyses indicated a more pronounced cell response to both treatments in UC organoids compared to non-IBD organoids. Inflammation activated immune (CXCL9/10/11, CX3CL1), interferon (IFI44, SOCS1, NOS2), and defense genes (GBP4, GBP5, OAS2), while downregulating metabolic genes (FABP6, CFTR, HMGCS2) in both groups. Gene Ontology-based GSEA confirmed immune pathway enrichment and lipid metabolism suppression in inflamed cultures. In total, 3,148 and 1,479 DEGs were identified in inflamed UC and non-IBD organoids, respectively. GA treatment modulated 2,144 and 384 genes (e.g., LYPD8, MKI67, MT-ATP6, CXCL14, CCL22) in these groups, suggesting potential to rebalance inflammation and metabolism. GA co-treatment enhanced epithelial integrity in UC organoids via upregulation of cell junction, peptide hormone processing, and immune-related pathways. In non-IBD organoids, GA promoted cell adhesion pathways, supporting epithelial cohesion. Functional permeability analysis confirmed GA’s protective role, as supported by transcriptomic data. Mean percentages of FITC-Dextran-permeable organoids were 16.02% (untreated), 86.08% (inflamed), and 42.58% (inflamed + GA). A trend toward statistical significance (Wilcoxon p = 0.125) was observed in both untreated vs. inflamed and inflamed vs. inflamed + GA comparisons.
Conclusion
In conclusion, gallic acid modulates inflammatory and metabolic pathways during colonic inflammation and enhances epithelial barrier integrity.
Study was funded by the European Union and the State Secretariat for Education, Research and Innovation (SERI) (project: miGut-Health, grant no. 101095470).
References
1Jostins, L., et al. (2012). Host–microbe interactions have shaped the genetic architecture of inflammatory bowel disease. Nature, 491(7422), 119–124. https://doi.org/10.1038/nature11582
2Cleynen, I., et al. (2016). Inherited determinants of Crohn’s disease and ulcerative colitis phenotypes: A genetic association study. The Lancet, 387(10014), 156–167. https://doi.org/10.1016/S0140-6736(15)00465-1
3Vieujean, S., et al. (2025). Understanding the therapeutic toolkit for inflammatory bowel disease. Nature Reviews Gastroenterology & Hepatology. Online ahead of print. https://doi.org/10.1038/s41575-024-01035-7
4Tian, B., Ye, P., Zhou, X., Hu, J., Wang, P., Cai, M., Yang, K., Sun, P., & Zou, X. (2025). Gallic acid ameliorated chronic DSS-induced colitis through gut microbiota modulation, intestinal barrier improvement, and inflammation. Molecular Nutrition & Food Research. Online ahead of print. https://doi.org/10.1002/mnfr.70024