Introduction

The pathogenesis of inflammatory bowel diseases has been linked to both the endognous cannabinoid system and its receptors (CB1, CB2) and nociceptin receptor (NOP). Considering the functional similarities between the CB and NOP systems along with the anti-inflammatory effects exerted by their ligands, we hypothesize that there is an interaction between CB1 and CB2 and NOP receptors during colitis.

Aims & Methods

Colitis was induced in mice by dextran sodium sulfate (DSS). NOP agonist (SCH 221510. 3 mg/kg), CB1 antagonist (AM 6545, 3mg/kg) or CB2 antagonist (AM 630, 5mg/kg) were given intraperitoneally, twice daily, alone or in combination. On day 8^th the mice were sacrificed, macroscopic scoring was performed and colonic samples were taken. The levels of IL-6, pERK and STAT3 was quantified using Western blot. The expression of genes encoding CB1 and CB2 receptors was determined in the mouse and human colonic samples collected from patients suffering from Crohn’s disease (CD) and ulcerative colitis (UC) and from healthy controls (HC) using real time RT-PCR. One-way ANOVA followed by the Tukey’s post-hoc test was used for analyses of multiple treatment means. Pearson correlation coefficient was used to evaluate the correlation between the CB1, CB2 and NOP receptors expression in healthy and inflamed colon.

Results

We observed increase in total colon damage score in DSS-treated mice as compared to control group (6.40±1.52 vs. 1.20±0.46), which decreased after treatment with SCH 221510 (4.10±0.50). Interestingly, addition of AM 6545, AM 630 or combination of both drugs significantly increased the total damage score as compared to mice treated with SCH 221510 alone (11.83±0.44 vs. 4.10±0.50, (P<0.01) for AM 6545, 9.12±1.54 vs. 4.10±0.50 (P<0.05) for AM 630 and 8.57±0.86 vs. 4.10±0.50 (P<0.05) for combination of both). Similar trend was found in comparison of myeloperoxidase activity in colon tissues (14.69±5.84 vs. 13.96±3.67, 18.97±2.40 vs. 13.96±3.67, and 17.60±3.31 vs. 13.96±3.67, respectively). We found a significant reduction in relative protein expression of IL-6 in the colon collected from SCH221510-treated mice, CB1 or CB2 antagonists-treated mice and SCH 221510- and both antagonists-treated mice as compared to DSS-treated group 7160471±3267620 (P<0.0001), 3675987±772660 (P<0.0001), 3379473±642716 (P<0.0001), and 25433153±11477866 (P<0.005) vs. 78758951±21976725 respectively. The relative protein levels of pERK and STAT3 tended to be elevated in groups treated with any ligand as compared to DSS-treated group and no difference between treated groups was noted. Real time RT-PCR analyses showed that combination of SCH 221510 and CB1 and CB2- antagonists increased the relative expression of CB1 and CB2 receptors as compared to mice treated with each drug solely. For CB1: 10819±4625, 20769±6775, 22558±8771 vs. 15044±6033, 3349±753.7 and 2660±546.4. For CB2: 15536±6860, 17696±7221, 16929±5452 vs. 9960±4043, 4406±2090, 3085±981.3. The relative expression of CB1 receptor in human samples was significantly lower in patients with CD and UC, as compared to HC 2284±1074 (P<0.01) and 1348±446.8 (P <0.05) vs. 6621±2935 respectively. Noteworthy, we discovered a strong, statistically significant positive correlation between the relative expression of CB1 and NOP receptors in CD (r= 0.71, P <0.05). These trends were not observed in UC patients, nor HC.

Conclusion

We obtained promising results suggesting the existence of cross-talk between the CB and NOP receptors. Fully elucidating this phenomenon could aid in developing novel treatment options for IBD patients.

Introduction

A compromised intestinal epithelial barrier plays a central role in inflammatory bowel diseases (IBD) by increasing intestinal permeability and promoting inflammation. Therefore, the restoration of intestinal barrier has become critical in IBD management ¹. Methylxanthine caffeine and its metabolites are widely consumed bioactive compounds that are known to influence various physiological processes ². However, their impact on gut health, particularly in modulating inflammation and epithelial permeability, remains underexplored.

Aims & Methods

The aim of this study was to investigate potential effects of caffeine (CAF) and its primary metabolites paraxanthine (PRX), theobromine (TBR) and theophylline (TPHL) on inflammatory responses and barrier integrity in colonic epithelial organoids, providing insights into their potential role in IBD management. Primary screening of different concentrations of CAF (1/2.5/5 mM) and its metabolites PRX (10/50/100 µM), TBR (10/20/50 µM) and TPHL (25/100/500 µM) was performed in Caco-2 cells. Selected metabolites and concentrations were further tested on inflamed 3D intestinal epithelial organoids derived from ulcerative colitis patient (n = 1) and non-IBD individual (n = 1). Both Caco-2 cells and organoids were pre-treated with metabolites for 1 hour before inflammation induction and were further co-treated with TNF-α/IFN-γ cytokine mix (10 ng/ml each) and metabolites for 24 hours. The effects on inflammation status and epithelial barrier integrity were tested through targeted gene expression analysis using RT-PCR and FITC-Dextran (4kDa) assay, while the effects on generation of reactive oxygen species were investigated by Flow cytometry and DHE assay. The study was approved by the Kaunas Regional Biomedical Research Ethics Committee (protocol no. BE-2-31).

Results

Primary screening revealed that both CAF and its metabolite PRX affected the expression of genes encoding inflammatory cytokines (TNF, CXCL9; p ≤ 0.05) and tight junction proteins (OCLN, TJP1, CLDN1; p ≤ 0.05) and were selected for further analysis in 3D intestinal organoids. Neither CAF, nor PRX reduced the expression of inflammatory cytokine-encoding genes in inflamed intestinal organoids. However, both CAF and PRX increased expression of tight junction proteins in inflamed intestinal organoids derived from control non-IBD (TJP1 – 3.16-fold by CAF and 1.33-fold by PRX (p ≤ 0.05); OCLN – 1.57-fold by CAF and 1.38-fold by PRX (p ≤ 0.05)) and ulcerative colitis (TJP1 – 1.32-fold by PRX; OCLN - 1.14-fold by CAF and 1.27-fold by PRX (p ≤ 0.05)) patient-derived organoids. Mean percentages of FITC-Dextran positive organoids were 43 %, 51 %, and 34 % in untreated, inflamed, and inflamed + PRX groups, respectively, revealing a trend in barrier restoration, though it did not reach statistical significance (p = 0.16 in inflamed vs. inflamed + PRX). A tendency of reduction in reactive oxygen species production was also observed after exposure to caffeine or paraxanthine (by 17 % and 11 %, respectively (p > 0.05)).

Conclusion

Methylxanthine paraxanthine might have a positive effect on gut epithelial barrier function through restoration of the expression of tight junction protein-coding genes.
The study was funded by the European Union and the State Secretariat for Education, Research and Innovation (SERI) (project: miGut-Health, grant no. 101095470).

References

1. Neurath MF et al. (2025) The intestinal barrier: a pivotal role in health, inflammation, and cancer. The Lancet Gastroeneterology & Hepatology, S2468-1253(24)00390-X, https://doi.org/10.1016/S2468-1253(24)00390-X
2. Reddy VS et al. (2024) Pharmacology of caffeine and its effects on the human body. European Journal of Medicinal Chemistry Reports, 10 (100138), https://doi.org/10.1016/j.ejmcr.2024.100138.

Introduction

Intestinal fibrosis is a common complication of inflammatory bowel diseases (IBD) characterized by increased production of extracellular matrix and crosslinking enzymes by intestinal mesenchymal cells. The advanced process of fibrosis leads to strictures often requiring surgical treatment. Therefore new methods of treatment of intestinal fibrosis are sought with endocannabinoid system (ES) as a potential therapeutic target. Cannabinoid receptors 1 and 2 (CB1 and CB2, respectively) are classic receptors of the ES involved in the regulation of the intestinal mucosa inflammation and permeability. Studies show that the regulation of ES activity can affect the process of fibrosis in various preclinical models of fibrosis.

Aims & Methods

The primary purpose of the study was to evaluate the effect of selective CB1 agonist, WIN 55, 212-2 (WIN) on expression of fibrosis regulatory proteins: alpha smooth muscle Actin 2 (Acta2), Collagen I (ColI), Fibronectin 2 (Fn2) and SMAD family member 3 (Smad3) in a mice model of Dextran sulfate sodium (DSS)-induced intestinal fibrosis. Moreover, the expression of the named proteins in colonic samples of patients with ulcerative colitis (UC) and healthy controls (HC) was compared.
Chronic colitis and fibrosis in mice model were induced by oral administration of 1.5% DSS in rotation with tap water for 3 weeks in three cycles. WIN was administrated starting from day 10th and continued every other day until day 77th, control mice were administered with saline. The mice were sacrificed on day 78th, their colon was excised and samples were taken for molecular analysis. Colonic samples from patients with UC and HC were collected and frozen for further exploration. The expression of genes encoding Acta2, Smad3, Col1, Fn2 in mouse and human material was assessed by Real Time RT-PCR. Statistical analysis was conducted using Student’s t-test to compare means of expressed molecules.

Results

We found lower relative expression of genes encoding assessed fibrosis regulatory proteins in mice treated with WIN than in inflamed mice: 4698±2323 vs. 9656±6032 for ACTA2, 4774±1073 vs 8510±2906 for COL1, 20993±7280 vs. 52148±17877 for FN1 and 373±31 vs. 478±77 for SMAD3. In human samples we noticed higher relative mean expression level of COL1 and SMAD3 in patients with UC compared to 7 HC 37422±6554 vs. 23816±2681 and 2134±261 vs. 1593±255, respectively.

Conclusion

We observed a decrease in relative expression of FN1, ACTA2, COL1, SMAD3 in samples from mice treated with CB1 agonist compared to inflamed mice. Also, a difference in expression of these proteins were found in intestinal samples from UC patients compared to HC, indicating a contribution of these molecules to intestinal fibrosis. Our results show that regulation of ECS activity play role in the inhibition of the intestinal fibrosis in IBD and appears as a potential therapeutic target in patients at high risk of developing strictures.

Introduction

Inflammatory bowel diseases (IBD), comprising two main disorders, Crohn’s disease (CD) and ulcerative colitis (UC) are chronic conditions characterized by a fluctuating disease course. Symptoms vary depending on disease activity, localization, and behavior. The etiopathogenesis of IBD is complex and environmental factors play a key role in developing intestinal inflammation. We hypothesized that seasonal changes may influence the severity and frequency of exacerbations.

Aims & Methods

The goal of the study was to determine the impact of seasonal changes on the onset and clinical presentation of IBD. As the normal distribution was violated the Kruskall-Wallis test was utilized. Results are presented as median with interquartile range (IQR). p-values <0.05 were considered statistically significant. We retrospectively assessed cases of IBD exacerbations requiring hospitalization in the Department of Digestive Tract Diseases, Medical University of Lodz, Lodz, Poland between 2010 and 2025. A total of 563 patients were analyzed (207 with CD and 365 with UC). Additional data, such as age of onset, disease duration, and disease severity were extracted from the medical records where possible.

Results

The median age was 43 years (IQR: 35.00-55.00) for CD patients and 43.5 years (IQR: 34.50-62.50) for UC patients. Median age at diagnosis was 27 years (IQR: 20.00-41.00) for CD patients and 29 years (IQR: 22.00-45.00) for UC patients. Median disease duration was 5 years (IQR: 1.00-11.00) for CD and 3 years (IQR: 0.00-9.00) for UC. No statistically significant seasonal variation in exacerbation rates was observed for either CD or UC patients (p = 0.509). There were also no significant seasonal differences in age (CD: p = 0.866; UC: p = 0.340), age at diagnosis (CD: p = 0.576; UC: p = 0.571), disease duration (CD: p = 0.434; UC: p = 0.244), or disease severity (Total Mayo Score: p = 0.619; Partial Mayo Score: p = 0.790; Harvey-Bradshaw Index: p = 0.540).

Conclusion

Despite anecdotal claims of seasonal patterns in IBD exacerbations, our 15-year retrospective analysis found no significant seasonal variation in hospitalization rates or clinical parameters. These findings challenge the presumed role of seasonal factors in IBD flare-ups. Further studies are warranted to validate these results in broader populations, including exacerbation managed in outpatient clinics.

Introduction

Micronutrient deficiency of amino acids display a hitherto underappreciated role in the pathophysiology of IBD. Serine is a nonessential amino acid and directly involved in the cellular redox balance. In the absence of sufficient external supply, serine can be synthesized via glycolysis through the rate-limiting enzyme phosphoglycerate dehydrogenase (PHGDH). Serine metabolism is upregulated in cancer and immune cells, maintaining survival and growth. However, its role in coordinating cellular function in the context of IBD is not known.

Aims & Methods

We hypothesized that serine metabolism provides a novel and underappreciated metabolic switch to maintain immunological homeostasis in the intestinal epithelium of IBD patients.We assessed the gene expression of the rate-limiting enzymes of de novo serine synthesis PHGDH in two cross-sectional IBD cohorts (1. N=67, 2. N=212 patients) and linked PHGDH expression with endoscopic or clinical disease activity. We used murine small intestinal epithelial cells (Mode-K cells) and murine small intestinal and colon organoids to understand the molecular regulator of de novo serine synthesis via PHGDH. To assess the potential role of PHGDH in coordinating innate immune response, Mode-K cells were treated with LPS or tunicamycin in the presence or absence of the Phgdh inhibitor BI-4916. The impact of proinflammatory cytokines Cxcl1 and Il6 was assessed through qPCR and ELISA. Upstream activation of the NFκB signaling pathway was assessed by WB. Seahorse assay was conducted to evaluate the impact of Phgdh on mitochondrial function.

Results

Compared with healthy control, serine metabolic enzyme PHGDH was significantly increased in both UC and CD patients. PHGDH expression correlated with the degree of inflammation in tissue biopsies of IBD patients correlated with ER stress marker (HSP90B1). Chemical induction of ER stress strongly increased the expression of Phgdh, Psat1, and Psph in Mode-K cells and intestinal organoids. To assess the regulatory role of serine metabolism on innate immune function, Mode-K cells were treated in serine/glycine-deprived medium. PHGDH function was inhibited using the specific inhibitor BI-4916. Treatment with tunicamycin or LPS resulted in strong induction of the proinflammatory cytokines Cxcl1 and Il6. Parallel Seahorse assay displayed significantly disrupted mitochondrial function (basal-, ATP linked- and maximum respiration). As serine feeds into the 1C-metabolism to provide mitochondrial-derived formate, we co-treated cells with glycine and formate to overcome the functional consequences of deprived de novo serine synthesis. We show that co-treatment of glycine and formate rescued mitochondrial dysfunction and inhibited Cxcl1 overactivation in intestinal epithelial cells.

Conclusion

Our data provide a novel link on how disrupted serine metabolism directly fuels epithelial-derived proinflammatory mechanisms via disruption of mitochondrial function. Our data suggest a so far underappreciated role of cellular serine metabolism on IBD pathophysiology.