White Wine Pomace Mitigates Hyperglycemia-Induced Cell Damage and Oxidative Stress in Caco-2 Cells †

: Hyperglycemia is a significant risk factor in metabolic syndrome, contributing to the development of cardiovascular diseases and diabetes mellitus. Hyperglycemia increases ROS (reactive oxygen species) production by glucose oxidation and protein glycosylation, leading to cell damage. Our previous studies have highlighted the antioxidant properties of wine pomace products (wWPP), a co-product of winemaking, and their ability to modulate oxidative stress. The objective of this study was to evaluate the protective effect of wWPP against oxidative stress in hyperglycemic Caco-2 cells. They were treated with 1.5 μg GAE/mL of wWPP bioaccesible fractions, obtained from gastrointestinal digestion (WPGI) and colonic fermentation (WPF), under normoglycemic or hyper-glycemic (35 mM glucose) conditions. After 24 h of treatment, cell viability, oxidative stress biomarkers and the expression of transcription factors and enzymes involved in cellular oxidation balance were evaluated. Hyperglycemia induced a 30% reduction in cell viability, which was restored to normoglycemic levels by WPF treatment. The bioaccessible fractions were able to counter-act hyperglycemia induced oxidative stress in intestinal cells, as evidenced by significant decreases in carbonyl groups and MDA levels (10 and 40% respectively). Furthermore, hyperglycemia induced NF-κB overexpression was also significantly reduced by WPGI and WPF pre-treatment (be-tween 15 and 53%), modulating the redox activity. In conclusion, the bioaccessible fractions of wWPP, particularly WPF, demonstrated significant potential in mitigating hyperglycemia-induced oxidative stress and enhancing cell viability in Caco-2 cells.


Introduction
The metabolic syndrome is characterized by different metabolic and vascular disorders, including obesity, hypertension, and hyperglycemia, that can lead to high mortality diseases like cardiovascular diseases and diabetes mellitus type II (Kuntz et al., 2015).
Persistent hyperglycemia can result in a series of chronic complications, with the gastrointestinal tract being one of the target organs (Bellastella et al., 2018;Palanissami and Paul, 2018).Hyperglycemia plays a role in these diseases through the production of reactive oxygen species (ROS) by glucose oxidation and protein glycosylation, causing low grade inflammation and interfering with homeostatic epithelial integrity (Bellastella et al., 2018;Morresi et al., 2019;Sharma et al., 2020).Indeed, mechanisms of redox signaling are involved in the development of oxidative stress associated to hyperglycemia by modulating the expression of the transcription factors NF-κB, thus activating the transcription of pro-inflammatory genes (Gerardi et al., 2019).The regulation of ROS production plays an important role in the prevention of epithelial functions dysregulation associated with hyperglycemia.Different studies suggested that antioxidant and anti-inflammatory bioactive compounds present in food could be used in the maintenance of epithelial functions.Wine pomace is the main co-product of winemaking industry and has a high content in polyphenols and dietary fiber (García-Lomillo and González-SanJosé, 2017;Gerardi et al., 2021).Our previous studies highlighted the antioxidant properties of wine pomace products (wWPP), a by-product of winemaking, and their ability to modulate oxidative stress associated to hyperglycemia in endothelial cells (Gerardi et al., 2021).
In view of the above the aim of this study was to determine the potential protective effect of the bioaccesible fractions of white wine pomace against oxidative stress in hyperglycemic intestinal epithelial cells, similar effects might be caused by a high-glucose diet, which may induce alterations of epithelial functions implicated in multitude of disorders.

White Wine Pomace Product (wWPP) and In Vitro Digested Fractions
The white wine pomace product (wWPP) was prepared at the University of Burgos from seedless white wine pomace from the winemaking of Vitis vinífera L. cv.Verdejo following a previously patented method (García-Lomillo et al., 2014).The wWPP obtained product was submitted to an in vitro gastrointestinal digestion (Del Pino-García et al., 2016) with oral, gastric and intestinal phases with solutions containing α-amylase, pepsin and pancreatin and bile salts respectively.At the end, a soluble digested bioaccesible fraction (WPGI) was obtained.In vitro colonic fermentation was performed in non-bioaccesible fraction (Del Pino-García et al., 2016) with caecal content of healthy rats in a sterile anaerobic environment to mimic the human microbiota (Experimentation with live animals was approved by the Ethics Committee for Experimental Animal Care at the University Hospital of Burgos (ref.CEBA 13) and it was carried out in accordance with the Spanish and European laws).After centrifugation, the supernatant obtained represents the soluble fermented bioaccesible fraction (WPF).WPGI and WPF were freeze dried and stored.

Characterization of the Bioaccesible Pomace Fractions WPGI and WPF
The antioxidant capacity of the fractions was assessed with Q-ABTS and Q-FRAP assays following previously described QUENCHER methods (Del Pino-García et al., 2015). 1 mg of the samples were analyzed at 734 and 593 nm respectively after incubation in the dark for 30 min.with ABTS •+ radical solution or FRAP reagent.Results were expressed as Trolox equivalents (TE)/ 100 g sample or mmol FE (II) E/100 g sample respectively.Total polyphenol content was assessed incubating 1 mg of the samples with 0,1 mL of Folin-Ciocalteu reagent, and after 2 min, 2 mL of Na2CO3 75 g/L solution and Milli-Q water up to 5 mL (Del Pino-García et al., 2015).The supernatant absorbance was determined at 750 nm and gallic acid was used for calibration.The results were expressed as gallic acid equivalents (GAE)/100 g sample.

Cell Culture and Treatment
Human colon adenocarcinoma cell line Caco-2 (ATCC ® HTB-37™) was purchased from the American Type Culture Collection (ATCC, Barcelona, Spain).Cells were cultured as a monolayer using Minimum Essential Medium Eagle (MEM) supplemented with 20% (v/v) heat inactivated Fetal Bovine Serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 1% non-essential amino acids, and 0,5 μg/mL amphotericin B. Cells were incubated at 90% humidity, 37 °C and 5% CO2 atmosphere.Normoglycemic cells were incubated with MEM 5 mM and hyperglycemic cells with the medium high in glucose (35 mM D-glucose) for 48 h in normo or hyperglycemic conditions, afterwards the WPGI and WPF fractions were added at 1.5 μg GAE/mL.

Cell Viability Assessement
Cell viability was measured using the MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium) method according to previous studies (Gutierrez-Gonzalez et al., 2023).Caco-2 cells were cultured at a density of 10 4 cells in 150 μL per well on a 96-well plate, then the cells were incubated with the bioaccessible fractions of wWPP at concentration of 1.5 μg GAE/mL for 24 h.Subsequently, 40 μL of MTT solution (5 mg/mL) was added and cells were incubated for 2 h at 37 °C.After incubation MTT formazan crystals were dissolved in 100 μL of DMSO and the optical density was measured at 570 nm.The results were expressed as % cell viability with respect to normoglycemic control cells.

Assessement of Oxidative Stress Biomarkers
Malondialdehyde (MDA) levels were analyzed to determine lipid oxidation in the normoglycemic and hyperglycemic cell sonicated solution.The samples were incubated with 15 μL of 3M NaOH, for 30 min at 60 °C.After, 75 μL of 6% (v/v) H3PO4 and 75 μL of 0.8% (w/v) thiobarbituric acid (TBA) were added and incubated at 90 °C for 45 min.The MDA levels were measured by injected 50 μL into an Agilent 1100 Series HPLC systems (Agilent Technologies, Inc., Palo Alto, CA, USA) equipped with a diode array detector for its detection.Absorbance was measured at 534 nm and a calibration curve of 1,1,3,3-tetramethoxypropane (TMP) was performed to determine results, which were expressed as μM of MDA equivalents.
Carbonyl groups (CGs) were determined in 50 μL of the cell suspension that was mixed with 250 μL of 2,4-dinitrophenylhydrazine (DNPH) 0.2% and incubated 1 h at room temperature.Then, 250 μL of 20% trichloroacetic acid (TCA) was added and incubated for 15 min at 4 °C.It was washed with ethyl acetate and ethanol and resuspended in 200 μL of acidized guanidine.Samples were measured at 373 nm and results were expressed as nmol of GCs/ mg of protein.

Quantitative Real Time PCR Analysis (qPCR)
Total RNA was extracted from the frozen Caco-2 suspensions using TRI Reagent (Applied Biosystems, Foster City, CA, USA).After quantification by NanoDrop (BioTek, Winooski, VT, USA), 3 μg were treated with DNase I (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and the RNA was reverse-transcribed using a First Strand cDNA Synthesis kit (Thermo Fisher Scientific).qPCRs were performed using specific primers (Supplementary Table S1) and SYBR Green q PCR Master Mix (EURx Sp. z. o. o., Gdansk, Poland) with ROX.
qPCR was carried out with a Quant Studio 5 Real-time PCR instrument (Applied Biosystems, Thermo Fisher Scientific Inc. Results were calculated using the efficiency ∆∆Ct method, with GADPH as the housekeeping gene.Results were expressed as folds of change compared to the normoglycemic non-treated cells.

Statistical Analysis
Statistical analysis was performed using StatGraphics ® Centurion 18.1.13(Statpoint Technologies Inc., Warrenton, VA , USA).Data was expressed as means ± standard deviation of independent experiments performed in triplicate and One-way analysis of variance (ANOVA) to determine significant differences (p < 0.05) between data.

Antioxidant Activity and Total Polyphenol Content of the wWPP Fractions
The wWPP was subjected to an in vitro gastrointestinal digestion and colonic fermentation to obtain the fractions that would be bioaccessible in the small and large intestine respectively.The antioxidant capacity and the polyphenol content are shown in Table 1.The analysis of the bioaccesible fractions showed a high phenolic content and an antioxidant activity.The WPF showed a significantly higher antioxidant capacity compared with the WPGI, when were analyzed by both Q-ABTS and Q-FRAP.The higher antioxidant activity in the WPF may be associated to a higher content of phenolic acids as a consequence of intestinal microorganism actions that release and modify more complex polyphenols (Saura-Calixto et al., 2007).These results show that the high antioxidant activity and phenolic composition of the wWPP is not altered by the digestion and fermentation process, and in fact is enhanced during the digestions, especially the antioxidant activity (Gerardi et al., 2020).

Effects of Hyperglycemia in Cell Viability and Oxidative Stress Biomarkers
The hyperglycemic effect on epithelial cells is well known and is characterized by a decrease in cell proliferation and an increase in apoptosis due to an increase in oxidative stress (Chen et al., 2021;Sharma et al., 2020).Caco-2 cells viability significantly decreased in hyperglycemic conditions compared to normoglycemia (Figure 1).However, this effect was regulated by the WPF fractions, which increased the cell viability of the hyperglycemic cells to the level of the normoglycemic control.MDA levels, an indicator of lipid damage and peroxidation were increased by 20% with hyperglycemia, this increase was reduced by both bioaccesible fractions, and the WPGI fraction could significantly reduce it to normoglycemic levels.The lipid peroxidation, as well as the carbohydrates oxidation, can cause the production of reactive carbonyl species, which can be introduced as carbonyl groups into proteins damaging their structure (Hecker and Wagner, 2018).This agrees with our results showing a 50% increase in carbonyl groups content in hyperglycemia compared to normoglycemic conditions.Both WPGI and WPF fractions were able to reduce it significantly to normoglycemic levels.This results are in line with other studies showing that polyphenols were able to reduce the formation of Schiff bases and dicarbonyl groups by oxidative stress (González et al., 2020).
The effect of wWPP in reducing biomarkers of oxidative damage is consistent with previous studies on endothelial cells exposed to hyperglycemia and in vivo models of oxidative stress-related diseases (Gerardi et al., 2020; González et al., 2020; Pino-García et al., 2016) and may result from the modulation of several cellular pathways by the phenolic compounds present in wine pomace products, such as hydroxycinnamic acids and resveratrol.In this regard, we evaluated the NF-κB transcription factor, a mechanism involved in the hyperglycemic-induced oxidative stress.
Under hyperglycemic conditions a significant increase was observed in the mRNA levels of NF-κB by almost two-fold (Figure 3).The increase in the NF-kB could to explain the increase in the biomarkers of oxidative stress observed and associated to inflammation process in hyperglycemia (Palanissami and Paul, 2018).The treatment with WPGI fractions decreased the expression of NF-κB to not treated values.in line with the observed for oxidative stress biomarkers.

Conclusions
In conclusion, our study has demonstrated that wine pomace products offer promising benefits in attenuating hyperglycemic related disorders through their capacity to ameliorate intestinal inflammation and oxidative stress.Both the gastrointestinal and fermented bioaccessible fractions of wine pomace have shown the ability to reduce lipid and protein oxidation and inhibit the proinflammatory transcriptional factor NF-κB.
From our perspective, these findings suggest the potential of wine pomace as functional ingredient, obtained from a winery co-product, with substantial health-promoting properties for preventing hyperglycemia-associated complications.Further studies are needed to develop this product as a nutraceutical or to explore its applications as a functional food.

Supplementary Materials:
Author Contributions: Funding: Institutional Review Board Statement: Experimentation with animals was approved by the Ethics Committee for Experimental Animal Care at the University of Burgos (PEAUBU012018) and was carried out in accordance with Spanish and European laws (RD53/2013 of Spanish Ministry and European Directive (2010/63/EU).

Figure 1 .
Figure 1.Cell viability in normoglycemic and hyperglycemic Caco-2 cells treated with WPGI and WPF fractions.Values represent mean (n = 3) ± SD.Significant differences between the non-treated and the treated fractions for both conditions are indicated by Latin letters (a, b, c).ANOVA Variance test (p < 0.05).NG: normoglycemic; HG: hyperglycemic; NT: non-treated with pomace fractions; WPGI: bioaccesible digested fraction of the white wine pomace product; WPF bioaccesible fermented fraction of the white wine pomace product.Chronic hyperglycemia increases the expression of NOX4, ROS, apoptosis-related proteins and inflammatory factors in intestinal epithelial cells (Chen et al., 2021).To determine the protective effect of bioaccessible fractions on intestinal epithelial cells in hyperglycemic conditions, we evaluated the malondialdehyde levels and carbonyl groups content as biomarker of oxidative stress.Both biomarkers significantly increased in hyperglycemia (Figure 2A,B).

Figure 2 .
Figure 2. Cell biomarkers of oxidative stress in normoglycemic and hyperglycemic Caco-2 cells treated with WPGI and WPF fractions.(A) carbonyl groups (GCs) levels and (B) malondialdehyde (MDA) levels.Values represent mean (n = 3) ± SD.Significant differences between the non-treated and the treated fractions for both conditions are indicated by Latin letters (a, b, c).ANOVA Variance test (p < 0.05).NG: normoglycemic; HG: hyperglycemic; NT: non-treated with pomace fractions; WPGI: bioaccesible digested fraction of the white wine pomace product; WPF bioaccesible fermented fraction of the white wine pomace product.

Figure 3 .
Figure 3. Nf-κB gene expression.Values represent mean (n = 3) ± SD.Significant differences between the non-treated and the treated fractions for normoglycemic and hiperglycemic conditions are indicated by Latin letters (a, b, c).ANOVA Variance test (p < 0.05) was performed.NT: non-

Table 1 .
Total antioxidant capacity and polyphenol content of the bioaccessible gastrointestinal (WPGI) and the bioaccessible fermented (WPF) fractions.