White Wine Pomace Mitigates Hypoxia in 3D SH-SY5Y Model ^†

Abstract

Hypoxia-induced reactive oxygen species (ROS) contribute to neuronal death and play a major role in various neurodegenerative diseases. The use of food by-products with antioxidant and anti-inflammatory properties, such as white wine pomace products (wWPPs), could be valuable not only allows for their revalorization but also for their potential in disease prevention. The aim of this study was to evaluate the neuroprotective effect of bioaccessible wWPP against hypoxia in 3D models of the SH-SY5Y human neuroblastoma cell line Cells were treated with 1.5 μg GAE/mL of bioaccessible wWPP and then subjected to hypoxia induced by CoCl₂. Cell viability, ROS levels, and gene expression were assessed. Hypoxia significantly increased the expression of the hypoxia-inducible factor gene(HIF1), cell deathand ROS levels, while pretreatment with bioaccessible wWPP mitigated these effects. Hypoxia also altered the mRNA expression of Nrf2, NF-kB, and Nrf2 inhibitor (Keap1), resulting in increased NF-kB and Keap1 expression and decreased Nrf2 levels. Bioaccessible wWPP fractions were able to reverse these changes, restoring mRNA expression to control levels and upregulating antioxidant enzymes like SOD2. These results suggest a potential neuroprotective effect of wine pomace and highlight the relevance of using natural products from the food industry in disease prevention.

1. Introduction

A constant flow of oxygen to the brain is necessary for its correct function, and it is highly sensitive to oxygen deprivation. A restriction oxygenaccess, also known as hypoxia, causes neurotoxicity, leadingto cell death and the development of neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease [1].

Hypoxia-inducible factor 1 (HIF1) is the nuclear factor characteristic of the hypoxic condition; it is activated under oxygen deprivation and regulatedgenes to promote cell survival, angiogenesis, or glucose uptake. In addition, neuronal reoxygenation leads to the production of reactive oxygen species (ROS), which can promote neurotoxicity.

Recent studies have shown the potential of antioxidant molecules to protect against the damaging hypoxia effects through the regulation of HIF1, a reduction in intracellular ROS, and the modulation of the transcriptional pathways involved in the antioxidant response [2]. Wine pomace, a by-product of winemaking, has been studied in recent years for its potential use as functional ingredient. It has a high content of polyphenols and dietary fiber, which exhibit high antioxidant and anti-inflammatory activity [3,4]. The polyphenol content and their biological activities are not affected by the digestion process [5]. Indeed, previous studies have shown a reduction in ROS production and the modulation of the antioxidant pathways and enzymes by the bioaccessible fractions in several cell lines [5,6]. These protective effects have been studied in 2D in vitro models but have not yet been explored in 3D models. As a key innovation of this study, a 3D spheroid model was used, as it more closely mimics in vivo conditions.

In view of the above, the aim of this study was to determine the potential protective effect of the bioaccessible fractions of white wine pomace against hypoxia in a cellular 3D model.

2. Materials and Methods

2.1. In Vitro Gastrointestinal Digestion and Colonic Fermentation of White Wine Pomace Product

The white wine pomace product (wWPP) was prepared at the University of Burgos from seedless Vitis vinifera L. cv. Verdejo provided by different wineries of the region. It was dehydrated, milled, and subjected to a heat treatment to avoid contamination following a patented method (ES2524870 B2 Spain).

To obtain the bioaccessible fractions, the wWPP was submitted to an in vitro gastrointestinal digestion mimicking human conditions according to the method described by Minekus et al. (2014) [7]. After this in vitro digestion, the potential bioaccessible gastrointestinal fraction (WPGI) was obtained. The non-bioaccessible fraction was subjected to an in vitro colonic fermentation following the same method [7] using the caecal content from healthy rats to mimic human conditions in the large intestine to obtain the bioaccessible fermented fraction (WPF). Experimentation with live animals was approved by the Ethics Committee for Experimental Animal Care at the University of Burgos (SCEBA012021) and carried out in accordance with Spanish and European laws.

2.2. Cell Culture and Treatment

Human neuroblastoma cell line SH-SY5Y (ATCC-CRL2266™) was cultured in a DMEM/F12 medium with amphotericin B 0.2%, fetal bovine serum (FBS) 10%, and penicillin–streptomycin 1% at 5% CO₂, 37 °C, and 80% humidity. The medium was changed every 2–3 days and the cells were subcultured at 90% density. For the development of the in vitro model, a 3D model was created by the method of forced flotation [8]. Briefly, the spheroids were formed with an initial cell density of 9000 cells per spheroid with 4% Methocel^® A4M in a low-adhesion U-shaped 96-well plate and differentiated with retinoic acid at 5 µM for the first two days and PMA at 80 nM for the next two days. The cells were treated with the bioaccessible fractions WPGI and WPF (1.5 µg GAE/mL) for 24 h. Then, hypoxia was induced using CoCl₂ (200 mM 24 h). Treated and non-treated spheroids in normoxia and hypoxia were collected in PBS and stored at −80 °C for the expression analysis.

2.3. Cell Death Assessment

To determine cell death, the spheroids were incubated with propidium iodide (PI) (50 µg/mL) for 5 min in the dark at 37 °C; afterwards, the medium was replaced with DMEM/F12 without FBS and nuclear damage was detected using a fluorescent Leica CTR6000 microscope and LAS AF Version:4.0.0.11706 Software (Leica Microsystems, Wetzlar, Germany). A Z-Stack of the spheroid planes was obtained to form one image, and PI fluorescence intensity was analyzed using FIJI 1.52b Software (NIH, Bethesda, MD, USA). Differences in cell death were determined by comparing the fluorescence intensity of the samples to the non-treated normoxia control.

2.4. Intracellular Reactive Oxygen Species (ROS) Assessment

To determine ROS production, the spheroids were incubated with a 2′,7′-dichloro-dihydro-fluorescein diacetate (H₂DCF-DA) probe (20 µM) for 30 min in the dark at 37 °C; DCF was detected using a fluorescent Leica CTR6000 microscope and LAS AF Software (Leica 177 Microsystems, Wetzlar, Germany). A Z-Stack of the spheroid planes was obtained to form one image, and DCF fluorescence intensity was analyzed using FIJI 1.52b Software (NIH, Bethesda, MD, USA). Differences in ROS production were determined by comparing the fluorescence intensity of the samples to the non-treated normoxia control.

2.5. Quantitative Real-Time PCR Analysis (qPCR)

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

2.6. Statistical Analysis

Data are expressed as mean ± standard deviation of independent experiments (n = 3). Statistical analysis was performed using Statgraphics Centurion 18.1.13 (Statpoint Technologies Inc., Warrenton, Virginia, USA). One-way analysis of variance (ANOVA), using Fisher’s least significant difference (LSD) test, was performed to determine significant differences (p < 0.05) between the treatments.

3. Results and Discussion

3.1. Hypoxia-Induced Cell Death and ROS Production

Hypoxia-induced cell death is a well-documented process in SH-SY5Y cells in both 2D and 3D models [9,10,11]. The hypoxia generated by CoCl₂ results in a significant increase in cell death (150%), and both fractions were able to significantly decrease it (95 and 100%, respectively) as shown in Figure 1.

Hypoxia-induced ROS production [12,13] and bioactive compounds, such as polyphenols, that provide protection against ROS-induced cell death, have been established in SH-SY5Y 2D and 3D models [14,15]. However, since no specific literature about the effect of grape pomaces’ bioaccessible polyphenols in hypoxia-mediated cell death in SH-SY5Y 3D models was found, the ROS intracellular levels were analyzed to determine if the bioaccessible fractions effect in reducing cell death was due to ROS production suppression (Figure 2). The treatment of neuronal cells with CoCl₂ to induce hypoxia significantly increased (34%) ROS intracellular production in the spheroids, and both the WPGI and WPF fractions were able to significantly reduce the production to normoxic levels. These results agree with the prior-mentioned studies and with the wine pomace protective effects against oxidative stress observed in other cell lines and in vivo studies [5,6,16].

3.2. wWPP Modulation of Molecular Pathways in Hypoxia Conditions

To confirm hypoxia in our model, the mRNA expression of hypoxia-inducible factor 1 (HIF-1) was analyzed via qPCR since its production and accumulation have been established as markers of hypoxia in SH-SY5Y [17]. A 152% significant increase in HIF-1 expression was observed in hypoxia compared to the control, which was significantly decreased with the bioaccessible fractions (90 and 93%, respectively) to lower levels than the normoxic control (Figure 3A). These results are in agreement with previous studies that showed an increase in HIF-1 expression and protein activity by hypoxia and a decrease with polyphenol treatment [18].

However, other studies showed that some polyphenols induce HIF-1 protein stability since this transcription factor induces an early cell response to hypoxia through the induction of enzymes like HO-1 and SOD1 [19,20,21]. Therefore, to determine the pathways activated by the bioactive wWPP fractions to reduce hypoxia and ROS production, the mRNA expression of proteins involved in the Nrf2 and NF-κB pathways was analyzed. Studies have shown that hypoxia and oxidative stress reduce the expression of the nuclear factor Nrf2, which is a known promotor of the antioxidant response, in 2D and 3D models [2,21,22]. Our study also showed a significant 48% reduction in Nrf2 mRNA expression (Figure 3B), and bioaccessible fractions did not increase their expression to the control levels We analyzed two proteins associated with the Nrf2 pathway, one upstream (Keap1) (Figure 3C) and one downstream (SOD2) (Figure 3D), to determine other possible effects of the bioaccessible fractions in this pathway. Keap1, a known suppressor of the Nrf2 pathwayby promoting the degradation of Nrf2 and preventing its translocation to the nucleus [23], was not significantly upregulated by hypoxia, but both bioaccessible samples were able to significantly decrease the Keap1 expression levels. SOD2 is an antioxidant enzyme that can be regulated by Nrf2 [24]; in our study, the SOD2 expression levels were significantly reduced in hypoxia, and WPGI and WPF were able to significantly increase them to normoxia levels. These results indicate that the bioactive compounds of wWPPs could act as modulators of Nrf2 activity, by the disruption of Keap1 interactions, independently of Nrf2 gene expression. On the other hand, NF-κB, a nuclear factor involved in inflammation and pro-oxidant responses [25], was significantly upregulated in hypoxia compared to the normoxic control (Figure 3E), in agreement with the increase in ROS production. Both bioaccessible fractions were able to significantly decrease NF-κB to lower levels than normoxia, in agreement with previous studies analyzing resveratrol’s effect on the NF-κB pathway in 3D neuroblastoma models [26].

4. Conclusions

In conclusion, the present study shows that wine pomace products offerpromising health benefits against hypoxiaby reducing cell death, HIF-1 expression, and ROS production. Both fractions showed similar effects and were also able to reduce the hypoxia-mediated activation of the NF-κB pathway, as well as regulate the Nfr2 pathway upstream, resulting in an increased expression of antioxidant enzymes.

From our perspective, these findings emphasize the potential of wine pomace, an already available winery by-product, as a functional ingredient with health properties to mitigate the damage caused by hypoxia in neuroblast cells. Additionally, the use of 3D models provides a more accurate model of hypoxia. Further s in vitro and in vivo studies are neccesaryto better define the potential of wine pomace as a nutraceutical and to characterize its potential health benefits.