Zebrafish Model Insights into Mediterranean Diet Liquids: Olive Oil and Wine

Olive oil is a crucial liquid food for the MD as it is the primary source of fat and offers numerous health benefits. EVOO is especially beneficial as it retains its organoleptic and nutritional properties after extraction and contains high levels of MUFA, tocopherols, and polyphenols. From a chemical point of view, 98–99% of the total weight of EVOO is represented by fatty acids, especially MUFA such as oleic acid. Tocopherols, polyphenols, and other minor constituents represent the remaining 1–2% which have antioxidant properties that contribute to the health effects of EVOO. EVOO has been linked to various preventive and therapeutic health benefits for different pathologies, including cardiovascular and inflammatory diseases, obesity, cancer, and neurodegenerative alterations associated with aging [61,62].

Several observational studies have shown that the minor constituents of olive oil mediate its health benefits, attenuate CVD prevalence, reduce the risk of aging-associated diseases, decrease weight in obese patients, and increase longevity [62,63,64]. Over the last decade, positive feedback from the scientific community has motivated researchers to further investigate the role of the minor constituents of olive oil in the treatment of various global pathologies. Olive oil has been attributed to its antioxidant, anti-inflammatory, antiatherogenic, antithrombotic, antiaging, neuroprotective, and antimutagenic properties, with many yet to be discovered [63,65,66]. Several in vitro studies have demonstrated the beneficial effects of olive oil. However, simple and suitable animal models are required in order to determine their nutraceutical advantages.

Wine is a notable source of polyphenols, some of which have antibacterial, antifungal, antiviral, antineoplastic, and anti-inflammatory properties. Furthermore, it has been reported that their therapeutic use is beneficial in several diseases, including CVDs and other diseases associated with aging. These pharmacological effects are primarily associated with their antioxidant capacity [67]. Owing to its greater content of antioxidant substances released from the grape skin and seeds, red wine is considered to have a more protective effect. In a bottle of red wine, the total polyphenol content is around 1.8 g/L, whereas in a bottle of white wine, it ranges from only 0.2 to 0.3 g/L of polyphenols. During the process of producing white wine, the skin and seeds are promptly removed from the must, which is then left for fermentation. Since in vitro antioxidant capacity is closely linked to total polyphenol content, white wines exhibit approximately five to ten times lower antioxidant activity than red wines. White wine also contains significant amounts of hydroxycinnamic acids, tyrosol, and hydroxytyrosol, which are known for their antioxidant properties [65,68]. One noteworthy aspect of this diversity is the variation in antioxidant content among the different types of wine. The specific antioxidants and their levels can vary depending on the grape variety, terroir, and winemaking technique. In recent years, there has been a growing interest in organic winemaking, including its subsets, such as biodynamic, natural, and clean wine. Organic winemaking emphasizes environmentally friendly practices and avoids the use of synthetic chemicals. This approach benefits not only the environment, but also the antioxidants present in wine [69]. Understanding the intricacies of winemaking methods is crucial. For example, biodynamic winemaking incorporates holistic farming principles, enhancing the overall vitality of the vineyard and potentially leading to unique antioxidants in grapes. Natural wines are crafted with minimal intervention, allowing grapes to express themselves fully and potentially preserving a broader range of antioxidants [69]. Additionally, the rise of clean wine, which focuses on transparency regarding what goes into the bottle, ensures that consumers receive wines free from additives and unnecessary processing, retaining the natural antioxidants present in grapes. In this evolving landscape, wine enthusiasts seek not only exquisite tastes but also health-conscious choices, exploring the diverse world of antioxidants in different types of wine, especially those emerging from organic winemaking and its subsets [69].

As previously mentioned, numerous observational studies have been conducted over the past few decades to investigate the health effects of the MD, including moderate alcohol consumption (mainly wine). Moderate alcohol intake is associated with reduced CVDs risk and overall mortality in both healthy and unhealthy individuals. Observational studies examining the correlation between alcohol consumption and mortality have demonstrated that individuals who refrain from consuming alcohol have a greater likelihood of death and cardiovascular incidents [70,71]. In the ATTICA study, an interesting pattern emerged regarding the relationship between low wine or beer consumption and the risk of developing CVD. When individuals consumed less than one glass per week, a distinct and striking correlation was observed, indicating a decreased risk of CVD for wine (hazard ratio: 0.40, 95% CI: 0.17–0.98) and beer (hazard ratio: 0.43, 95% CI: 0.20–0.93) compared to individuals who did not consume alcohol. However, no significant relationship was observed between abstainers and individuals who consumed more than one glass per week. Nonetheless, compared with subjects who consumed less than two grams of ethanol per day, those who consumed between 2 and 10 g, 10 and 20 g, and more than 20 g of ethanol per day had a higher hazard ratio [72]. The Moli-sani study, a large-scale cohort study, examined the association between alcohol consumption and the onset of HF and ventricular fibrillation. The study revealed that moderate alcohol consumption, ranging from 10 to 20 g/day, was associated with a reduced risk of HF; however, it did not exhibit a comparable effect on AF when compared to individuals who abstained from alcohol. In addition, this study examined certain biochemical parameters, including HDL and total cholesterol, in relation to alcohol consumption. Those who consumed more than 48 g of alcohol per day had the highest HDL levels (63 mg/dL). Nonetheless, their total cholesterol levels were the highest in the entire study cohort, averaging 226 mg/dL. In contrast, non-drinkers had HDL levels of 54 mg/dL, and occasional drinkers had total cholesterol levels of 207 mg/dL. The authors found that the mean reduction in the risk of total mortality associated with alcohol consumption was 11%. Additionally, alcohol consumption of >20 g/day was linked to a 13% reduction in the risk of total mortality. Considering the deaths attributed to cardiovascular causes alone, these trends were consistent. Notably, the correlation between alcohol consumption and mortality was similar between men and women. Furthermore, the observed benefits were more pronounced in individuals who favored wine, suggesting that the observed benefits might be attributed to components other than ethanol [73]. In summary, observational studies have demonstrated a protective effect of moderate alcohol consumption on the occurrence of cardiovascular events; however, the precise mechanisms involved are still not sufficiently understood.

Animal trials to study the antioxidant effects of specific foods such as olive oil and wine offer numerous advantages. It helps to identify active components, provides experimental control, establishes dose–response relationships, offers mechanistic insights, has practical applications, and can be cost-efficient, reducing the expense of sociosanitary treatment of determinate pathologies. These insights can contribute to a deeper understanding of the health benefits associated with the components of the MD and can inform dietary recommendations. Wine and olive oils were selected for studies on the antioxidant effects of the MD because of their prominence in the diet, distinct antioxidant profiles, existing scientific interest, ease of isolation and control, consumer relevance, and documented positive health outcomes. Studying these specific components allows researchers to explore the mechanisms responsible for the potential health benefits of the diet.

Traditionally, mammalian models, especially rats and mice, have been the primary models used for research in food science, although their complexity and relatively slow rate of development often compromise the rapid progress in resolving fundamental nutritional questions. However, new animal models such as zebrafish and frogs, whose genomes are fully sequenced and very similar to humans, have emerged [98]; 70% of human genes have at least one zebrafish ortholog. Zebrafish have several advantages as model organisms because of their short life cycle, strong reproductive ability, easy rearing, and low cost (Figure 1). Therefore, the use of zebrafish in nutritional studies provides a valuable reference for researchers in the field of food science [99].

Zebrafish are emerging as very high-throughput and genetically tractable vertebrate models for replicating the developmental environment of disease progression in CVD pathophysiology. A diverse range of gene editing tools have been developed to propagate the modified contractile apparatus or CVD pathophysiology in multiple zebrafish generations, thereby enabling the production of multiple, stable zebrafish generations for the purpose of studying mutagenesis [100]. Zebrafish have a great potential for cardiac function disorders. Their heart rate is similar to that of humans compared to other model organisms. Electrocardiogram (ECG) analysis of adult zebrafish underscores the high similarity in electrophysiology between the adult zebrafish heart and that of humans. Although zebrafish hearts are small, techniques have been developed to measure ECG profiles in embryos. These methods have identified similar ECG features in embryos as early as three days post-fertilization (dpf), which mirrors those observed in adult zebrafish (Figure 1) [101]. Atherosclerosis (AS) is a multifaceted chronic disease that poses a significant threat to human health, and it is a fundamental factor underlying numerous CVDs. In clinical practice, one of the most prevalent diagnostic indicators of AS is the presence of abnormal blood lipid levels, which are frequently observed in patients with AS due to disturbances in lipid levels. Typically, cholesterol is transported to peripheral tissues via LDL and subsequently returned to the liver via HDL through its cholesterol reverse-transport function, ultimately leading to its elimination. However, when exposed to oxidative stress, LDL is frequently oxidized into ox-LDL, which is avidly engulfed by macrophages, giving rise to foam cells. This disrupts normal cholesterol metabolism. These foam cells actively contribute to the formation of atherosclerotic plaques and rupture can precipitate ischemic heart disease or stroke. Ox-LDL also has detrimental effects on AS by damaging the vascular endothelium, stimulating the migration and proliferation of smooth muscle cells, and activating platelets. Consequently, inhibition of LDL oxidation has emerged as a promising avenue for enhancing lipid metabolism, preventing the onset of AS (Figure 1) [102]. Hyperlipidemia can be effectively induced in zebrafish through both dietary and genetic strategies, thus providing valuable models for screening novel lipid-lowering compounds. Currently, there are two established zebrafish genetic models of hyperlipidemia: apolipoprotein C-II (apoc2) and LDL receptor (ldlr) mutants [103,104]. In accordance with the clinical phenotype observed in human patients with APOC2 deficiency, zebrafish mutants deficient in Apoc2 exhibit hypertriglyceridemia when fed a standard diet. Apoc2-deficient zebrafish larvae exhibit lipid accumulation and lipid-laden macrophages within their vasculature, similar to the early atherosclerotic lesions observed in humans and mice [103]. In contrast, ldlr mutants develop moderate hypercholesterolemia when fed a standard diet. This hypercholesterolemic state is further aggravated following a short-term, 5-day high cholesterol diet (HCD) regimen initiated 4.5 days post-fertilization (dpf) (Figure 1) [104]. Feeding zebrafish a high-cholesterol diet (HCD) closely mimics certain processes observed in the early AS stages, including hypercholesterolemia, lipoprotein oxidation, vascular lipid accumulation, and recruitment of myeloid cells to the vasculature [105]. It was observed that feeding zebrafish an HCD for up to 10 days, beginning at 5 dpf, resulted in increased expression of inflammatory markers, such as tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β). Additionally, there is a reduction in the expression of the gene encoding the anti-inflammatory protein peroxisome proliferator-activated receptor-γ in the endothelium before myeloid cell accumulation and lipid deposition occur [106]. Numerous studies have demonstrated that telomeres are shorter in human arterial tissue located in vascular beds that are susceptible to AS than in those located in sites that are resistant to AS. Furthermore, the telomere length of white blood cells is shorter in individuals with conditions such as hypertension, diabetes, and coronary artery disease than in unaffected controls (Figure 1) [107]. The regulation of telomere length in zebrafish resembles that in mammalian organisms, making it an ideal model for investigating the link between telomeres and vascular aging (Figure 1) [108]. In conclusion, the zebrafish model has emerged as a valuable tool for advancing our understanding of CVDs, particularly AS and hyperlipidemia. This versatile model combines the practical advantages of in vitro systems and the physiological relevance of mammalian models. Zebrafish share a substantial degree of genetic and functional similarity with humans, making them an ideal platform for investigating the intricate mechanisms underlying these diseases.

Zebrafish present an exceptionally promising model organism, exhibiting significant potential for unraveling the mechanisms associated with ROS in crucial human diseases. Studies have demonstrated their ability to evaluate antioxidant responses to oxidative stress in vivo, thus making them a rapid and straightforward experimental model. Researchers have explored the effects of oxidative stress on disease progression in both transgenic and wild-type zebrafish. Remarkably, zebrafish embryos are invaluable for conducting in vivo experiments and devising protocols for measuring the oxidative stress in living organisms. Recent advancements in zebrafish research include the generation of homozygous null mutants using a gene-targeted approach. This innovation allows for precise study of oxidative stress-mediated toxicity [107]. Zebrafish are also used as a model with which to evaluate the antioxidant efficacy of polyphenols, including those found in olive oil and wine. Resveratrol (5 µM), an important wine polyphenol, was shown to prevent oxidative stress and mitochondrial damage in zebrafish embryos (Figure 2) [109]. Administration of quercetin (1 µg/L) significantly enhanced the activities of SOD, GPX, and CAT, as well as the expression of the respective genes. Moreover, the expression of inflammatory genes in the liver and intestine of zebrafish is reduced (Figure 2) [110]. In addition, a recent study showed that zebrafish fed a diet supplemented with 200 mg/kg of hydroxytyrosol, the main polyphenol present in EVOO, alleviates fat accumulation, oxidative stress, and mitochondrial dysfunction (Figure 2) [111]. In a previous study, quercetin (50 and 100 mg/kg; administered intravenously) was found to inhibit lipopolysaccharide-induced behavioral changes, neuronal disruption, and levels of TNF-α, IL-1β, lipid peroxidation, and acetylcholinesterase in adult zebrafish (Figure 2) [112]. Administration of gallic acid resulted in a reduction in the neutrophil inflammation index in a zebrafish inflammatory model induced by a high-cholesterol diet (Figure 2) [113]. Arteaga et al. (2021) evaluated the in vivo protective effects of six phenolic compounds (naringenin, apigenin, rutin, oleuropein, chlorogenic acid, and curcumin) and three carotenoids (lycopene B, β-carotene, and astaxanthin) that are naturally present in foods using a zebrafish embryo model. They found that each compound, except β-carotene, had a protective effect against oxidative stress-induced lethality (Figure 2) [114].