Tumor necrosis factor alpha (TNFα) has been shown to impair the intestinal barrier, inducing and maintaining inflammatory states of the intestine. The aim of the current study was to analyze functional, molecular and regulatory effects of TNFα in a newly established non-transformed jejunal enterocyte model, namely IPEC-J2 monolayers. Incubation with 1000 U/mL TNFα induced a marked decrease in transepithelial electrical resistance (TEER), and an increase in permeability for the paracellular flux marker [³H]-D-mannitol compared to controls. Immunoblots revealed a significant decrease in tight junction (TJ) proteins occludin, claudin-1 and claudin-3. Moreover, a dose-dependent increase in the TNF receptor (TNFR)-1 was detected, explaining the exponential nature of pro-inflammatory effects, while TNFR-2 remained unchanged. Recovery experiments revealed reversible effects after the removal of the cytokine, excluding apoptosis as a reason for the observed changes. Furthermore, TNFα signaling could be inhibited by the specific myosin light chain kinase (MLCK) blocker ML-7. Results of confocal laser scanning immunofluorescence microscopy were in accordance with all quantitative changes. This study explains the self-enhancing effects of TNFα mediated by MLCK, leading to a differential regulation of TJ proteins resulting in barrier impairment in the intestinal epithelium.

Cannabis-based biomaterials have the potential to deliver anti-inflammatory therapeutics specifically to desired cells, tissues, and organs, enhancing drug delivery and the effectiveness of anti-inflammatory treatment while minimizing toxicity. As a major component of Cannabis, Cannabidiol (CBD) has gained major attention in recent years because of its potential therapeutic properties, e.g., for restoring a disturbed barrier resulting from inflammatory conditions. The aim of this study was to test the hypothesis that CBD has beneficial effects under normal and inflammatory conditions in the established non-transformed intestinal epithelial cell model IPEC-J2. CBD induced a significant increase in transepithelial electrical resistance (TER) values and a decrease in the paracellular permeability of [³H]-D-Mannitol, indicating a strengthening effect on the barrier. Under inflammatory conditions induced by tumor necrosis factor alpha (TNFα), CBD stabilized the TER and mitigated the increase in paracellular permeability. Additionally, CBD prevented the barrier-disrupting effects of TNFα on the distribution and localization of sealing TJ proteins. CBD also affected the expression of TNF receptors. These findings demonstrate the potential of CBD as a component of Cannabis-based biomaterials used in the development of novel therapeutic approaches against inflammatory pathogenesis.

Peyer’s patches (PPs) are part of the gut-associated lymphatic tissue (GALT) and represent the first line of the intestinal immunological defense. They consist of follicles with lymphocytes and an overlying subepithelial dome with dendritic cells and macrophages, and they are covered by the follicle-associated epithelium (FAE). A sealed paracellular pathway in the FAE is crucial for the controlled uptake of luminal antigens. Quercetin is the most abundant plant flavonoid and has a barrier-strengthening effect on tight junctions (TJs), a protein complex that regulates the paracellular pathway. In this study, we aimed to analyze the effect of quercetin on porcine PPs and the surrounding villus epithelium (VE). We incubated both tissue types for 4 h in Ussing chambers, recorded the transepithelial electrical resistance (TEER), and measured the unidirectional tracer flux of [³H]-mannitol. Subsequently, we analyzed the expression, protein amount, and localization of three TJ proteins, claudin 1, claudin 2, and claudin 4. In the PPs, we could not detect an effect of quercetin after 4 h, neither on TEER nor on the [³H]-mannitol flux. In the VE, quercetin led to a higher TEER value, while the [³H]-mannitol flux was unchanged. The pore-forming claudin 2 was decreased while the barrier-forming claudin 4 was increased and the expression was upregulated. Claudin 1 was unchanged and all claudins could be located in the paracellular membrane by immunofluorescence microscopy. Our study shows the barrier-strengthening effect of quercetin in porcine VE by claudin 4 upregulation and a claudin 2 decrease. Moreover, it underlines the different barrier properties of PPs compared to the VE.

The plant alkaloid berberine has been shown to have many beneficial effects on human health. This has led to its use as a treatment for various cancer types, obesity, and diabetes. Moreover, a described barrier‐strengthening effect in human cancer cell lines indicates that it might be useful for the treatment of inflammatory bowel disease. Detailed information regarding its effects on intestinal epithelium remains limited. In our current study, we describe the impact of berberine on a non‐transformed porcine small intestinal epithelial cell model, IPEC‐J2. Incubation of IPEC‐J2 monolayers with berberine revealed dose‐ and time‐dependent effects on barrier properties. A viability assay confirmed the specific effect of berberine on the apoptotic pathway, paralleled by the internalization of the sealing tight‐junction (TJ) proteins claudin‐1, claudin‐3, and occludin within 6 h. Hence, the barrier function of the cells was reduced, as shown by the reduced transepithelial electrical resistance and the increased [³H]‐D‐Mannitol flux. A decrease of claudin‐1, claudin‐3, and occludin expression was also observed after 24 h, whereas ZO‐1 expression was not significantly changed. These data indicate an early effect on both cell viability and barrier integrity, followed by a general effect on TJ architecture. The intracellular co‐localization of claudin‐1 and occludin or claudin‐3 and occludin points to an initial induction of apoptosis accompanied by the internalization of sealing TJ proteins. Although barrier strengthening has been reported in cancerogenic epithelial models, our results show a barrier‐weakening action, which represents a new aspect of the effect of berberine on epithelia. These results agree with the known toxic potential of plant alkaloids in general and show that berberine is also capable of exerting adverse effects in the intestinal epithelium.

Colon cancer is accompanied by a decrease of epithelial barrier properties, which are determined by tight junction (TJ) proteins between adjacent epithelial cells. The aim of the current study was to analyze the expression of TJ proteins in a rat model of 1,2-dimethylhydrazine (DMH)-induced colorectal cancer, as well as the barrier properties and TJ protein expression of IPEC-J2 cell monolayers after incubation with DMH. Transepithelial electrical resistance and paracellular permeability for sodium fluorescein of IPEC-J2 were examined by an epithelial volt/ohm meter and spectrophotometry. The expression and localization of TJ proteins were analyzed by immunoblotting and immunohistochemistry. In the colonic tumors of rats with DMH-induced carcinogenesis, the expression of claudin-3 and -4 was significantly increased compared to controls. The transepithelial electrical resistance of IPEC-J2 cells increased, while paracellular permeability for sodium fluorescein decreased, accompanied by an increased expression of claudin-4. The increase of claudin-4 in rat colon after chronic DMH exposure was consistent with the acute effect of DMH on IPEC-J2 cells, which may indicate an essential role of this protein in colorectal cancer development.

Cannabidiol (CBD), a major non-psychoactive cannabinoid of the Cannabis sativa L. plant, has demonstrated anti-inflammatory effects in various studies. However, the therapeutic use of CBD is still limited. Despite its potential, little is known about the molecular mechanisms of CBD on epithelial integrity, particularly concerning effects in native intestinal tissue. To accomplish this, our study aimed to investigate the effects of CBD ex vivo on the follicle-associated epithelium of Peyer’s Patches (PP) and villus epithelium (VE) from porcine intestine. To measure the epithelial barrier, the Ussing chamber technique was employed, followed by immunoblotting and confocal laser-scanning immunofluorescence microscopy of tight junction proteins and specific receptors. The results revealed that CBD significantly strengthens the epithelial barrier of PP by upregulation of sealing tight junction proteins, including occludin, claudin-1, -3, and -7. Additionally, the study showed the potential of CBD to decrease the expression of Tumor necrosis factor alpha (TNFɑ) receptor 1 (TNFR-1) in PP that plays a key role in chronic inflammatory diseases. The study highlights the potential of CBD in the prevention of inflammatory conditions and underlines the important role of PP as a target for bioactive compounds.