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Membranes (Basel)
2024 Jan 08;141:. doi: 10.3390/membranes14010018.
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Cannabidiol Strengthening of Gastric Tight Junction Complexes Analyzed in an Improved Xenopus Oocyte Assay.
Stein L, Vollstaedt ML, Amasheh S.
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Cannabidiol (CBD), the non-psychoactive compound derived from the cannabis plant, has gained attention in recent years as a remedy against gastrointestinal disorders ranging from nausea and inflammation to abdominal pain. Recent advances demonstrated an effect on inflammatory pathways and barrier proteins. However, information on possible direct effects is scarce and needs to be addressed, as applications are currently increasing in popularity. To accomplish this, we have employed Xenopus laevis oocytes as a heterologous expression system for analysis of the direct effects on stomach-specific claudins and further developed tight junction (TJ) protein interaction assays. Human claudin-4, claudin-5, and claudin-18.2 were expressed in Xenopus oocytes, clustered in pairs to form contact areas, and analyzed in a two-cell model approach, including measurement of the contact area and contact strength. CLDN4/5/18 + CLDN4/5/18 oocyte pairs were incubated with 20 µM CBD or with 40 µM CBD and were compared to cells without CBD treatment (ctrl). For interaction analysis, the contact area was measured after 24 h and 48 h. Whereas CBD did not affect the size of the protein interaction area, Double Orbital Challenge experiments revealed an increased contact strength after 24 h incubation with CBD. In addition, the Xenopus oocyte experiments were accompanied by an analysis of claudin-4, -5, and -18 expression in gastric epithelium by immunoblotting and immunohistochemistry. Claudin-4, -5, and -18 were strongly expressed, indicating a major role for gastric epithelial barrier function. In summary, our study shows direct effects of 40 µM CBD on Xenopus oocytes heterologously expressing a stomach-specific claudin combination, indicating a supportive and beneficial effect of CBD on gastric TJ proteins.
Figure 1. Schematic depiction of the Double Orbital Challenge (DOC), a standardized adhesion assay. The DOC was carried out with oocyte pairs placed in a 24-well plate for 120 s using a plate reader to apply constant shear stress (lettering of supplier; arrow: direction of double orbital shaking treatment).
Figure 2. (A) Immunoblots of claudin-4, -5, -18 expression in porcine gastric tissue. (B) Localization of claudin-4, -5, -18 by confocal laser scanning immunofluorescence microscopy and (C–E) z-stacks of the respective claudin combinations; location of sections indicated by horizontal green and vertical red lines, respectively. Representative images (scale bars: 20 μm).
Figure 3. (A) Immunoblots of heterologous co-expression of claudin-4, claudin-5, and claudin-18.2 and (B) confocal laser scanning immunofluorescence microscopy to locate protein accumulation within the oocyte membrane. Representative images (scale bars: 20 μm).
Figure 4. Contact areas size analysis of the paired oocyte assay after 24 h and 48 h. CBD revealed no changes in contact areas of claudin 4/5/18 expressing oocyte pairs (CLDN4/5/18 + CLDN4/5/18). Data are presented in mean ± SEM (N = 6, ctrl: n = 18, 20 µM CBD: n = 28, 40 µM CBD: n = 24, p > 0.05, Kruskal–Wallis test followed by a Dunn–Bonferroni correction).
Figure 5. Contact strength analysis using the Double Orbital Challenge (DOC) (A) in box plots and (B) visualization of paired oocytes (CLDN4/5/18 + CLDN4/5/18) by transmitted light optical microscopy before and after carrying out DOC. The Δ contact area after incubation with 40 µM CBD was significantly larger than in control oocytes (N = 3, n = 11, * p = 0.0472, Kruskal–Wallis test followed by a Dunn–Bonferroni correction). In contrast, the contact area difference of the oocyte pairs incubated with 20 µM CBD and ctrl was not significantly altered. Representative images (scale bars = 100 µm).
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