The Punicalagin Metabolites Ellagic Acid and Urolithin A Exert Different Strengthening and Anti-Inflammatory Effects on Tight Junction-Mediated Intestinal Barrier Function In Vitro

Scope: Ellagitannins are polyphenols found in numerous fruits, nuts and seeds. The elagitannin punicalagin and its bioactive metabolites ellagic acid and urolithins are discussed to comprise a high potential for therapeutically or preventive medical application such as in intestinal diseases. The present study characterizes effects of punicalagin, ellagic acid and urolithin A on intestinal barrier function in the absence or presence of the proinflammatory cytokine tumor necrosis factor-alpha (TNF alpha). Methods and Results: Transepithelial resistance (TER), fluorescein and ion permeability, tight junction protein expression and signalling pathways were examined in Caco-2 and HT-29/B6 intestinal epithelial cell models. Punicalagin had less or no effects on barrier function in both cell models. Ellagic acid was most effective in ileum-like Caco-2 cells, where it increased TER and reduced fluorescein and sodium permeabilities. This was paralleled by myosin light chain kinase two mediated expression down-regulation of claudin-4, -7 and -15. Urolithin A impeded the TNF alpha-induced barrier loss by inhibition of claudin-1 and -2 protein expression upregulation and claudin-1 delocalization in HT-29/B6. Conclusion: Ellagic acid and urolithin A affect intestinal barrier function in distinct ways. Ellagic acid acts preventive by strengthening the barrier per se, while urolithin A protects against inflammation-induced barrier dysfunction

Anti-Diarrheal Mechanism of the Traditional Remedy Uzara via Reduction of Active Chloride Secretion

BACKGROUND AND PURPOSE: The root extract of the African Uzara plant is used in traditional medicine as anti-diarrheal drug. It is known to act via inhibition of intestinal motility, but malabsorptive or antisecretory mechanisms are unknown yet. EXPERIMENTAL APPROACH: HT-29/B6 cells and human colonic biopsies were studied in Ussing experiments in vitro. Uzara was tested on basal as well as on forskolin- or cholera toxin-induced Cl(-) secretion by measuring short-circuit current (I(SC)) and tracer fluxes of (22)Na(+) and (36)Cl(-). Para- and transcellular resistances were determined by two-path impedance spectroscopy. Enzymatic activity of the Na(+)/K(+)-ATPase and intracellular cAMP levels (ELISA) were measured. KEY RESULTS: In HT-29/B6 cells, Uzara inhibited forskolin- as well as cholera toxin-induced I(SC) within 60 minutes indicating reduced active chloride secretion. Similar results were obtained in human colonic biopsies pre-stimulated with forskolin. In HT-29/B6, the effect of Uzara on the forskolin-induced I(SC) was time- and dose-dependent. Analyses of the cellular mechanisms of this Uzara effect revealed inhibition of the Na(+)/K(+)-ATPase, a decrease in forskolin-induced cAMP production and a decrease in paracellular resistance. Tracer flux experiments indicate that the dominant effect is the inhibition of the Na(+)/K(+)-ATPase. CONCLUSION AND IMPLICATIONS: Uzara exerts anti-diarrheal effects via inhibition of active chloride secretion. This inhibition is mainly due to an inhibition of the Na(+)/K(+)-ATPase and to a lesser extent to a decrease in intracellular cAMP responses and paracellular resistance. The results imply that Uzara is suitable for treating acute secretory diarrhea

Effects of Uzara, ouabain and forskolin on R^para and R^trans in HT-29/B6 cells.

<p>The influence of Uzara and ouabain on R^para and R^trans with and without forskolin was determined by two-path impedance spectroscopy in HT-29/B6 cell monolayers mounted in Ussing chambers. Forskolin reduced R^trans, whereas R^para remained unaltered. The co-incubation of Uzara with forskolin caused no change of R^trans compared to forskolin alone, whereas R^para was clearly reduced in the co-incubation group. Similarly, compared to forskolin alone, ouabain together with forskolin had no effect on R^trans. The slight reduction of R^para observed under these conditions did not reach significance. Data are expressed as mean ± s.e.m. (n = 5-6). Untreated control values set as 1. Asterisk indicates a difference compared to forskolin: **P<0.01. Hash keys mean no statistical significance compared to forskolin alone.</p

Campylobacter jejuni enters gut epithelial cells and impairs intestinal barrier function through cleavage of occludin by serine protease HtrA

Abstract Campylobacter jejuni secretes HtrA (high temperature requirement protein A), a serine protease that is involved in virulence. Here, we investigated the interaction of HtrA with the host protein occludin, a tight junction strand component. Immunofluorescence studies demonstrated that infection of polarized intestinal Caco-2 cells with C. jejuni strain 81–176 resulted in a redistribution of occludin away from the tight junctions into the cytoplasm, an effect that was also observed in human biopsies during acute campylobacteriosis. Occludin knockout Caco-2 cells were generated by CRISPR/Cas9 technology. Inactivation of this gene affected the polarization of the cells in monolayers and transepithelial electrical resistance (TER) was reduced, compared to wild-type Caco-2 cells. Although tight junctions were still being formed, occludin deficiency resulted in a slight decrease of the tight junction plaque protein ZO-1, which was redistributed off the tight junction into the lateral plasma membrane. Adherence of C. jejuni to Caco-2 cell monolayers was similar between the occludin knockout compared to wild-type cells, but invasion was enhanced, indicating that deletion of occludin allowed larger numbers of bacteria to pass the tight junctions and to reach basal membranes to target the fibronectin receptor followed by cell entry. Finally, we discovered that purified C. jejuni HtrA cleaves recombinant occludin in vitro to release a 37 kDa carboxy-terminal fragment. The same cleavage fragment was observed in Western blots upon infection of polarized Caco-2 cells with wild-type C. jejuni, but not with isogenic ΔhtrA mutants. HtrA cleavage was mapped to the second extracellular loop of occludin, and a putative cleavage site was identified. In conclusion, HtrA functions as a secreted protease targeting the tight junctions, which enables the bacteria by cleaving occludin and subcellular redistribution of other tight junction proteins to transmigrate using a paracellular mechanism and subsequently invade epithelial cells

Inhibitory effect of Uzara on the Na⁺/K⁺-ATPase enzymatic activity in membrane preparations from HT-29/B6 cells.

<p>In cell culture experiments, monolayers were treated with 50 µ µg/ml Uzara for 90 min. Then, cells were homogenized and a crude membrane fraction was prepared. Herein, the Na⁺/K⁺-ATPase activity was measured (n = 5). In a further set of experiments, membrane fractions of untreated HT-29/B6 cell monolayers were prepared and subsequently treated with 50 µg/ml Uzara which was added for 1 h to the enzyme activity assay (n = 8). Both conditions revealed a direct inhibitory activity on the ion pump. Values are expressed as percentage of control activity. Basal activity of the Na⁺/K⁺-ATPase was between 36 and 73 nmol P_i/(min·mg protein). Data are expressed as mean ± s.e.m. Asterisks indicate difference versus control: ***P<0.001.</p

Effects of DIDS on forskolin-induced I_SC in the absence and presence of Uzara or ouabain.

<p>Experiment in HCO₃⁻ free solutions equilibrated with 100% O₂. Application of 10 µM forskolin induced an I_SC that was reduced in the presence of Uzara (50 µg/ml) and ouabain (100 µM). Under all three conditions, application of DIDS (100 µ µM) caused a pronounced transient increase in I_SC, presumably due to an increase in intracellular Ca²⁺. Similar to the carbachol-induced I_SC, the current amplitude was reduced in the presence of Uzara and ouabain. I_SC in the presence of DIDS then rapidly decreased, if preparations were pre-incubated with Uzara (light blue) or ouabain (pink). This effect is attributed to additional Na⁺ loading of the cells. Application of DIDS before the addition of Uzara (blue) had no such accelerating effect.</p

Influence of Uzara and forskolin on Na⁺ and Cl⁻ fluxes in HT-29/B6 cells.

<p>J_Na and J_Cl, unidirectional Na⁺ and Cl⁻ fluxes; ^ms, from mucosal to serosal side; ^sm, from serosal to mucosal side; all fluxes are expressed as µmol·h⁻¹·cm⁻².</p><p>J^net, net fluxes (J^ms-J^sm).</p><p>J_res, residual fluxes (I_SC-J_Na^net+J_Cl^net, usually assumed to reflect bicarbonate secretion).</p><p>I_SC, short-circuit current (for comparison expressed in µmol·h⁻¹·cm⁻² of monovalent cations).</p><p>R^t, transepithelial resistance (Ω·cm²).</p><p>All values represent means ± SEM.</p><p>*, P versus control <0.05.</p><p>#, P versus control <0.001.</p><p>§, P versus forskolin alone <0.001.</p

Inhibitory effect of Uzara on the forskolin-induced I_SC rise in HT-29/B6 cells.

<p>I_SC was measured on HT-29/B6 monolayers mounted in Ussing chambers. In A and B, Uzara (50 µg/ml) was added on the basolateral side either 30 minutes prior (A) or 30 minutes after (B) basolateral addition of 10 µM forskolin. In C, different Uzara concentrations were applied basolaterally 30 minutes after forskolin stimulation. The percentage of the inhibitory activity of Uzara to the forskolin-induced I_SC rise was calculated after an incubation time of 1 hour. An IC₅₀ of 17 µg/ml and a Hill coefficient of 1.45 were determined from a Hill plot diagram and used to calculate the continuous line. All values are expressed as mean ± s.e.m. (n = 5-7). D: Transepithelial resistance measurements over 48 hours after application of either ethanol (control: final concentration 0.05%) or 50 µg/ml Uzara (final ethanol concentration 0.054%). With the exception of the resistances 24 hours after the application of uzara, none of the resistance values in the presence of uzara differed significantly from the corresponding control value. (means ± s.e.m. n = 12).</p