Deficient wound healing is frequently observed in patients diagnosed with diabetes, a clinical complication that compromises mobility and leads to limb amputation, decreasing patient autonomy and family lifestyle. Fibroblasts are crucial for secreting the extracellular matrix (ECM) to pave the wound site for endothelial and keratinocyte regeneration. The biosynthetic pathways involved in collagen production and crosslinking are intimately related to fibroblast redox homeostasis. In this study, two sets of human dermic fibroblasts were cultured in normal (5 mM) and high (25 mM)-glucose conditions in the presence of 1 µM selenium, as sodium selenite (inorganic) and the two selenium amino acids (organic), Se-cysteine and Se-methionine, for ten days. We investigated the ultrastructural changes in the secreted ECM induced by these conditions using scanning electron microscopy (SEM). In addition, we evaluated the redox impact of these three compounds by measuring the basal state and real-time responses of the thiol-based HyPer biosensor expressed in the cytoplasm of these fibroblasts. Our results indicate that selenium compound supplementation pushed the redox equilibrium towards a more oxidative tone in both sets of fibroblasts, and this effect was independent of the type of selenium. The kinetic analysis of biosensor responses allowed us to identify Se-cysteine as the only compound that simultaneously improved the sensitivity to oxidative stimuli and augmented the disulfide bond reduction rate in high-glucose-cultured fibroblasts. The redox response profiles showed no clear association with the ultrastructural changes observed in matrix fibers secreted by selenium-treated fibroblasts. However, we found that selenium supplementation improved the ECM secreted by high-glucose-cultured fibroblasts according to endothelial migration assessed with a wound healing assay. Direct application of sodium selenite and Se-cysteine on purified collagen fibers subjected to glycation also improved cellular migration, suggesting that these selenium compounds avoid the undesired effect of glycation.

Most of the biological impacts of Vitamin E, including the redox effects, have been raised from studies with α-tocopherol only, despite the fact that tocopherol-containing foods carry mixed tocopherol isomers. Here, we investigated the cellular mechanisms involved in the immediate antioxidant responses evoked by α-, γ- and δ-tocopherol in Caco-2 cells. In order to track the cytosolic redox impact, we performed imaging on cells expressing HyPer, a fluorescent redox biosensor, while cytosolic calcium fluctuations were monitored by means of Fura-2 dye and imaging. With this approach, we could observe fast cellular responses evoked by the addition of α-, γ- and δ-tocopherol at concentrations as low as 2.5 μM. Each isomer induced rapid and consistent increases in cytosolic calcium with fast kinetics, which were affected by chelation of extracellular Ca²⁺, suggesting that tocopherols promoted a calcium entry upon the contact with the plasma membrane. In terms of redox effects, δ-tocopherol was the only isomer that evoked a significant change in the HyPer signal at 5 μM. By mimicking Ca²⁺ entry with ionomycin and monensin, a decline in the HyPer signal was induced as well. Finally, by silencing calcium with 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), an intracellular Ca²⁺ chelator, none of the isomers were able to induce redox changes. Altogether, our data indicate that an elevation in cytoplasmic Ca²⁺ is necessary for the development of a tocopherol-induced antioxidant impact on the cytoplasm of Caco-2 cells reported by HyPer biosensor.

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.

Deficient wound healing is frequently observed in patients diagnosed with diabetes, a clinical complication that compromises mobility and leads to limb amputation, decreasing patient autonomy and family lifestyle. Fibroblasts are crucial for secreting the extracellular matrix to pave the wound site for endothelial and keratinocyte regeneration. The biosynthetic pathways for collagen production are one of the main components of the extracellular matrix, as the crosslinking of extracellular collagen fibers is intimately related to fibroblast redox homeostasis. In this study, human dermic fibroblasts were cultured with 1 mM sodium selenite (inorganic) and 1 mM of two selenium amino acids (organic), Se-cysteine and Se-methionine. This concentration was compatible with long-term exposure necessary for extracellular matrix production and allowed us to examine the impact of these three compounds on the state and response of the thiol-based HyPer biosensor expressed in the cytoplasm. In addition, the abundance of extracellular matrix and ultrastructural properties were evaluated by Picrus Sirius red staining and scanning electronic microscopy, respectively. We also evaluated whether these selenium compounds effectively ameliorated the perturbations induced by culturing fibroblasts at high glucose levels (25 mM) regarding cytoplasmic redox, extracellular matrix, and glycation on collagen fibers. Our results indicate that cytoplasm protein oxidation was sensitive to selenium compound supplementation. Selenium amino acids increased the sensitivity to protein oxidation, and only Se-cysteine increased the disulfide bond reduction in fibroblasts maintained in high glucose. Despite the lack of effect of selenium compounds on the glu-cose-induced increase in matrix fiber thickness, we found that endothelial migration improved in the matrix generated by high glucose-cultured fibroblasts.

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.