Introduction

Epithelial barrier dysfunction is a hallmark of inflammatory bowel diseases (IBD), contributing to increased permeability, microbial translocation, and sustained mucosal inflammation^1,2. 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

Introduction

Inflammatory bowel disease (IBD) is characterised by inflammatory flares with excruciating pain, for which effective analgesics are lacking. Cocaine- and amphetamine-regulated transcript (CART) peptide regulates peripheral pain in mice.¹ However, its role in visceral nociception as well as the underlying molecular signalling mechanism in primary sensory neurons remains elusive. CART is highly expressed in TRPV1-positive colon-projecting sensory neurons.² In addition, the enzyme prohormone convertase 1/3, which is involved in processing CART into its bioactive fragment 55-102, is upregulated in colonic biopsies of IBD patients (internal data), suggesting a potential role for the peptide in colonic pain.

Aims & Methods

Here, we aimed to test if CART would affect firing of the colon-innervating lumbar splanchnic nerve (LSN) as model of visceral nociception and characterise its effect and signalling pathway in sensory neurons innervating the colon. Studies were performed using tissue from male and female C57BL/6J mice euthanised by cervical dislocation and exsanguination in accordance with Schedule 1 of the Animals (Scientific Procedures) Act 1986. Thoracolumbar dorsal root ganglia (DRG) neurons were dissociated and cultured overnight for Ca²⁺ imaging using Fluo-4 AM as previously described.³ Cells were perfused with extracellular solution (ECS) alone or supplemented with either rat CART 55-102, (20-300nM, ECS), thapsigargin (10mM, DMSO), xestospongin C (1mM, ECS), edelfosine (10mM, ECS), capsaicin (10mM, ethanol) or KCl solution (60mM, ddH₂O) for respective challenge or pre-treatment. All conditions were blinded and randomised. Colorectum with attached lumbar splanchnic nerve was carefully removed and mounted on a tissue bath superfused with Krebs buffer as previously described.³ Rat CART 55-102 was superfused at 5ml/min, resulting in a final concentration of 200nM. Statistical analysis was performed using Student´s t test or ANOVA and Dunnett´s multiple comparisons test. Data is displayed as mean ± SEM.

Results

CART elicited a concentration-dependent increase in intracellular Ca²⁺ in DRG neurons, which at the highest concentration tested (300nM) induced a significant increase in intracellular Ca²⁺ in 29.2 ± 2.98% of DRG neurons relative to 8.35 ± 1.25% of vehicle responders (p < 0.0001). 55.05% of CART-responsive neurons were putative nociceptors due to co-sensitivity to capsaicin. Further investigation revealed a statistically significant reduction in the proportion of CART-sensitive neurons compared to CART (300nM) alone following pre-treatment with edelfosine, a phospholipase C inhibitor (10.5 ± 3.88%, p = 0.006), thapsigargin, a SERCA pump inhibitor (7.25 ± 1.78%, p = 0.001), or xestospongin C, an inositol triphosphate receptor inhibitor (6.74± 1.87%, p = 0.006). Furthermore, tissue bath application of CART (200nM) elicited a marked transient increase in ongoing LSN discharge, which increased from a baseline of 12.8 ± 3.9 (spikes/s) to a peak firing of 19.7 ± 6.1 spikes/s (p = 0.0385).

Conclusion

CART stimulates intracellular Ca²⁺ release in capsaicin-sensitive neurons consistent with a role in pain processing. This response was mediated by the release of Ca²⁺ from intracellular stores such as the endoplasmic reticulum and attenuated by inhibitors of the G_q pathway, suggesting activation of a G_q-coupled GPCR. The pronociceptive activity of CART on nerve firing suggests a potential role in colonic pain. Further research is necessary to identify the role of CART in IBD.

References

1. Ohsawa M, Dun SL, Tseng LF, Chang J, Dun NJ. Decrease of hindpaw withdrawal latency by cocaine- and amphetamine-regulated transcript peptide to the mouse spinal cord. Eur J Pharmacol. Jul 7 2000;399(2-3):165-9. doi:10.1016/s0014-2999(00)00374-5
2. McGuire C, Boundouki G, Hockley JRF, et al. Ex vivo study of human visceral nociceptors. Gut. Jan 2018;67(1):86-96. doi:10.1136/gutjnl-2016-311629
3. Higham JP, Bhebhe CN, Gupta RA, et al. Transcriptomic profiling reveals a pronociceptive role for angiotensin II in inflammatory bowel disease. Pain. Jan 29 2024;doi:10.1097/j.pain.0000000000003159

Disclosure

The PhD research is partially funded by Metrion Biosciences.

Introduction

Endoscopic submucosal dissection (ESD) is an effective treatment for superficial esophageal cancer, but postoperative stenosis remains a significant concern. Particularly, probability of the stenosis is less investigated in cases with a 50%-75% circumferential mucosal defect.

Aims & Methods

This study aimed to identify influential factors for postoperative stenosis after the moderate circumferential resection in esophageal ESD. This multi-center retrospective observational study analyzed data from 513 patients across 19 institutions who underwent esophageal ESD from April 2018 to March 2022 and showed 50% to 75% of the circumferential mucosal defect. We firstly overviewed the current status on the postoperative stenosis, the steroid use, and postoperative adverse events in these patients. Subsequently, we identified influential factors for postoperative stenosis. Patients were categorized into stenosis and no stenosis groups, and univariate and multivariate analyses were performed for each parameter. We further investigated influential factors of stenosis in two cohorts: steroid and no steroid groups. Finally, the parameters were compared between the steroid and no steroid groups to assess the tendency of the steroid use in general practice.

Results

Postoperative stenosis was observed in 28 cases (5%). Univariate analysis comparing the stenosis group (n = 28) and no stenosis group (n = 485) revealed significant differences in the prior endoscopic treatment near the lesion (p = 0.02), cervical esophageal location (p = 0.04), defect circumference (p =0.01), and procedure duration (p = 0.02). There was no significant difference in the steroid use between two groups. Multivariate analyses identified history of prior endoscopic treatment close to the lesion (p = 0.01, odds ratio [OR], 5.10), cervical esophageal lesions (p = 0.03, OR, 3.97), large circumferential defect (p <0.01, OR, 3.78), and longer procedure duration (p <0.01, OR, 3.83) as independent risk factors for stenosis formation. Among the steroid group, significant associations of the postoperative stenosis were revealed with the prior endoscopic treatment near the lesion (p = 0.02), cervical esophageal location (p = 0.04), and >5/8 of defect circumference (p = 0.02). In the no steroid group, only the procedure duration (p = 0.01) showed a significant difference between the stenosis and non-stenosis groups. Comparison between the steroid (n = 223) and no steroid groups (n = 290) revealed significant differences in the prior endoscopic treatment near the lesion (p <0.01), cervical esophageal lesions (p <0.01), and resected specimen dimensions (p <0.01). The stenosis rates were 7% in the steroid group and 4% in the no steroid group, without a significant difference. No significant difference in adverse events was observed between the two groups.

Conclusion

In esophageal ESD with a 50%-75% circumferential mucosal defect, the steroid was used in over 40% of patients, and postoperative stenosis still occurred in 5%. Because wider circumference, location of the cervical esophagus, prior endoscopic treatment close to the lesion, and prolonged operative time were identified as independent risk factors on the stenosis, steroid therapy should be preferred in these cases. Particularly, lesions with a history of endoscopic treatment, on cervical esophagus, and with >5/8 of defect circumference should require more aggressive countermeasures than currently applied.