Vollstädt ML et al. (2024), Cinnamic Acid and Caffeic Acid Effects on Gastr...

XB-ART-60605

Membranes (Basel) 2024 Feb 01;142:. doi: 10.3390/membranes14020040.

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Cinnamic Acid and Caffeic Acid Effects on Gastric Tight Junction Proteins Analyzed in Xenopus laevis Oocytes.

Vollstädt ML , Stein L , Brunner N , Amasheh S .

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Analysis of secondary plant compounds for the development of novel therapies is a common focus of experimental biomedicine. Currently, multiple health-supporting properties of plant-derived molecules are known but still information on many mechanisms is scarce. Cinnamic acid and caffeic acid are two of the most abundant polyphenols in human dietary fruits and vegetables. In this study, we investigated cinnamic acid and caffeic acid effects on the gastric barrier, which is primarily provided by members of the transmembrane tight junction protein family of claudins. The Xenopus laevis oocyte has been established, in recent years, as a heterologous expression system for analysis of transmembrane tight junction protein interactions, by performing paired oocyte experiments to identify an effect on protein-protein interactions, in vitro. In our current study, human gastric claudin-4, -5, and -18.2. were expressed and detected in the oocyte plasma membrane by freeze fracture electron microscopy and immunoblotting. Oocytes were paired and incubated with 100 µM or 200 µM cinnamic acid or caffeic acid, or Ringer's solution, respectively. Caffeic acid showed no effect on the contact area strength of paired oocytes but led to an increased contact area size. In contrast, cinnamic acid-incubated paired oocytes revealed a reduced contact area and a strengthening effect on the contact area was identified. These results may indicate that caffeic acid and cinnamic acid both show an effect on gastric barrier integrity via direct effects on tight junction proteins.

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Species referenced: Xenopus laevis
Genes referenced: cldn18 cldn4 cldn5 ctrl
GO keywords: tight junction organization

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	Figure 1. Flow chart: experimental procedures from oocyte isolation to functional analyses (day 0–6, DOC: double orbital challenge).
	Figure 2. Freeze fracture electron microscopy showing formation of TJ strands in oocytes expressing cldn 4, cldn 5, or cldn 18.2, respectively (arrows). (A) Freeze fracture electron microscopy reveals TJ protein cldn 4 as an intermittent network of connected square strands; (B) freeze fracture electron microscopy reveals TJ protein cldn 5 as angular fibrils in Xenopus laevis oocytes; (C) freeze fracture electron microscopy of oocytes expressing cldn 18.2 reveals a meshwork of undulating strands with intersections; (D) water-injected control oocytes with a smooth surface, representative images of oocytes derived from three animals. Scale bar: 250 nm.
	Figure 3. Immunoblots of heterologous co-expression of CLDN4/5/18. Claudins were detected to locate protein expression within the oocyte membrane. In contrast, no endogenous signals for the tight junction proteins were detected in controls (ctrl, representative images).
	Figure 4. Contact areas of paired oocytes expressing cldn 4, cldn 5, and cldn 18.2. After 24 h there is no significant caffeic acid effect on the contact area size but after 48 h there is an increase in the contact area. In contrast, cinnamic acid caused a decrease in the contact area after 24 h. Data presented in means ± SEM (n = 13–21, * p < 0.05, ** p < 0.01.
	Figure 5. Change of contact area size before and after DOC (A) and visualization and quantification by transmitted light microscopy (B). Oocytes co-expressing cldn 4, cldn 5, and cldn 18.2 treated with 200 µM cinnamic acid retained a significantly higher contact area size after DOC compared to the ctrl. Similarly, 100 µM cinnamic acid led to a larger contact area but the difference compared to the ctrl was not significant. Both concentrations of caffeic acid did not affect the contact area size after DOC compared to the ctrl. (n = 11–16, Kruskal–Wallis test followed by Dunn–Bonferroni correction, * p < 0.05, representative images, scale bars = 200 µm).