Caffeic acid phenethyl ester (CAPE) is a hydrophobic phytochemical typically found in propolis that acts as an antioxidant, anti-inflammatory and cardiovascular protector, among several other properties. However, the molecular entity responsible for recognising CAPE is unknown, and whether that molecular interaction is involved in developing an antioxidant response in the target cells remains an unanswered question. Herein, we hypothesized that a subfamily of TRP ion channels works as the molecular entity that recognizes CAPE at the plasma membrane and allows a fast shift in the antioxidant capacity of intact endothelial cells (EC). By monitoring cytoplasmic Ca²⁺ in a microvascular EC model, we compared the calcium responses evoked by three structurally related compounds: caffeic acid phenethyl ester, neochlorogenic acid and caffeic acid. Only CAPE induced rapid and transient calcium responses at nanomolar concentrations together with a gradual increase in cytoplasmic sodium levels, suggesting the activation of a non-selective cationic permeation at the plasma membrane. Electrophysiological as well as pharmacological, and RNA silencing assays confirmed the involvement of TRPV1 in the recognition of CAPE by ECs. Finally, we demonstrated that Ca²⁺ influx by TRPV1 was necessary for recording CAPE-induced cytoplasmic redox changes, a phenomenon captured in real-time in ECs expressing the HyPer biosensor. Our data depict a molecular mechanism behind the antioxidant effect of CAPE in endothelial cells, connecting the activation of TRPV1 ion channels, cytoplasmic calcium increase, and a reduction of disulfide bonds on a redox biosensor. This phenomenon occurs within seconds to minutes and contributes to a better understanding of the mechanisms underlying the vasodilatory effect of CAPE and other compounds that interact with TRPV1 in the vascular bed.

The association between dietary fiber and phenolic compounds allows the latter to reach the colon, where most polysaccharides undergo fermentation. This bioprocessing weakens the matrix and promotes the release of the phenolic compounds, which then exert beneficial effects on intestinal function. Although this notion is widely accepted, supporting evidence remains scarce. In this study, we subjected grape pomace skin to in vitro digestion to obtain an indigestible fraction suitable for SHIME bioreactors. Throughout these stages, we observed a sequential increase in the release of phenolic compounds, with colonic fermentation playing an important role. Although we did not observe an increase in short-chain fatty acid (SCFA) production by the gut microbiota, we performed a repeated-challenge design on differentiated Caco-2 monolayers. With this approach, we found that the phenolic-rich ferment prevented the transepithelial electrical resistance (TEER) drop on the second challenge and modulated the transcriptomic profile assessed by RNA-seq. Our findings indicate that the Caco-2 cellular responses mentioned above were SCFA-independent and likely due to the differential impact of phenolic compound load after colonic fermentation of grape pomace skin.

Dietary fiber usually comes associated with phenolic compounds either trapped in a network formed by indigestible polysaccharides or, in some cases, bound to this matrix by covalent bonds. This natural association protects some phenolic compounds during their journey through the gastrointestinal tract, reaching the colon, where most polysaccharides undergo fermentation, weakening the matrix and promoting the release of these compounds to exert benefits by modulating intestinal function. Unfortunately, there are still difficulties in investigating the impact of the human microbiota on the release of phenolic compounds from the indigestible fraction and on their effects on the intestinal epithelium. In this study, grape pomace skin was subjected to in vitro digestion to obtain an indigestible fraction, which was further incorporated into SHIME bioreactors with human colonic microbiota. Throughout these stages, we monitored the release of phenolic compounds using HPLC-DAD. We determined the impact of a phenolic-rich indigestible fraction on the fermentative capacity of human microbiota by measuring the short-chain fatty acids (SCFA) levels and the effect of the resulting colonic ferments on the barrier properties of differentiated monolayers of Caco-2 cells by monitoring the transepithelial electrical resistance and the transcriptomic profile by RNA sequencing. Our results indicated that digestive processing sequentially increases the release of phenolic compounds and that colonic fermentation releases even more phenolic compounds trapped in the polysaccharide network. Unexpectedly, neither the phenolic-rich nor the depleted fractions increased SCFA levels in the colonic microbiota. Despite the lack of effect on the microbial community, the phenolic-rich ferments had a positive impact in the manner that monolayers of Caco-2 cells adapted better to repeated exposures with the ferments compared with phenolic-depleted ferments. In addition to his subtle difference, Caco-2 cells exposed to phenolic-rich ferments showed a remarkable increase in proteins associated with tight junctions, like claudin-3, -4, -15 and − 19, other proteins that connect the cytoskeleton with tight junctions, such as VASP (Ena/vasodilator-stimulated phosphoprotein) and CGNL1 (cingulin), supporting the idea that phenolic-rich ferments strengthened intercellular communication. In addition to this finding, we observed significant decreases in the transcription of genes encoding apical ion channels, such as CFTR, and the basolateral Na ⁺ /K ⁺ /Cl ⁻ cotransporter, both of which are relevant to intestinal chloride secretion and therefore may reduce fluid loss by the gut. In conclusion, our data support the positive impact of a subset of phenolic compounds that reach the colon, independent of SCFA levels. To achieve this finding in a biologically relevant context, we had to process grape pomace through physiological processes of digestion and fermentation. The latter stage was performed in a realistic colonic environment with human microbiota. The transcriptomic profile induced by the phenolic-rich ferments was observed following repeated exposures, a characteristic that warrants exploration of experimental designs to simulate dietary habits.