Mediterranean countries account for approximately 70% of all global olive oil (OO) production, which is mainly undertaken by Spain, Turkey, Greece, Italy, Morocco, and Tunisia [1
]. Not only have the beneficial effects of OO (Olea europaea
L.) been evaluated in the context of the so-called Mediterranean diet [2
] but also due to its largely recognized bioactivity [3
]. Thus, the regular consumption of OO is currently associated with beneficial effects on health due to its specifically nutritional components.
A large number of physical, chemical and organoleptic characteristics is routinely used to define and classify an olive oil in different categories, following European Commission Regulation (ECC) No. 2568/91 [4
]. Despite all the possible parameters to classify olive oils, the consensus is based on the maximum percentage values of free acidity, thus distinguishing between extra virgin olive oil (EVOO, ≤ 0,8%), virgin olive oil (VOO, ≤ 2%) and ordinary olive oil (OO, > 2%) [5
Among the health benefits of EVOO, antioxidant, antitumoral and anti-inflammatory properties have been attributed to different components. Triacylglycerols are the main constituents of EVOO, followed by free fatty acids, glycerol, phosphatides, pigments, flavor compounds and sterols. In addition, the high proportion of unsaturated fats, mainly monounsaturated, in contrast to a low proportion of saturated fats, designs its characteristic biochemical profile [7
]. In this way, oleic acid (C18:1) is the most abundant monounsaturated fatty acid (70–80%), and one of the most studied in terms of healthy effects [8
]. Between 4% and 20% of polyunsaturated fatty acids (PUFAs) are represented by linoleic (C18:2) and α-linolenic (C18:3) acids, while saturated fatty acids (SFA) only account for 8–14% [9
]. However, significant differences in minor components also result in diverse varieties of olive oils, which differ in quality and nutritional attributes.
Concerning minor constituents, triterpenic and phenolic compounds, tocopherols and sterols contained in EVOO have been involved in a variety of biological activities, including the activation of different signaling pathways related to redox state, homeostasis, inflammation [10
] and epigenetics modifications of the chromatin [12
]. As a consequence, these compounds isolated from olive oil have been recognized as powerful nutraceutical tools for the prevention and management of cardiovascular, cancer and degenerative diseases [14
]; specifically, hydroxytyrosol (HT) and its derivates, tyrosol, oleocanthal and oleuropein, have been proven as the more remarkable compounds in this regard [16
]. Other authors claim that additional minor components with still unknown bioactivity might contribute to the beneficial effects of these phenolic compounds.
The well-known PREDIMED study, a randomized controlled trial, showed the importance of EVOO in the Mediterranean diet for reducing the risk of cardiovascular disease and cardiovascular mortality, in comparison with a standard diet [17
]. Moreover, neurodegenerative diseases [18
] and cancer [19
] presented with lower incidence in the context of the Mediterranean diet, which was in part attributed to the regular consumption of EVOO.
Unfortunately, reports on the beneficial effects of EVOO consumption against ocular diseases are still scarce. The rich fatty acid composition of ocular tissues indicates EVOO as an adequate supplement for the treatment of eye diseases. The Mediterranean diet has been linked to a lower onset and progression of age-related macular degeneration (AMD) [20
], where EVOO might have an important role. In this sense, the Alienor study, a population study based on eye diseases in the elderly, suggested a protective role for EVOO in AMD [22
], and in vitro studies showed that HT might be one of the actors responsible for this beneficial effect [23
]. In addition, studies in rats demonstrated the neuroprotective effect of HT in the context of diabetic retinopathy (DR) [25
]. Interestingly, neuroprotective properties of EVOO in DR have also been recently associated with other components of EVOO, such as oleuropein [26
]. Nonetheless, the uncertain mechanisms related to the beneficial properties of EVOO consumption in ocular pathologies certainly warrants further research on this topic.
The wild olive tree (Olea europaea var. sylvestris)—also known as acebuche (ACE) when referring to the Spanish ancient specimens—is a variety of cultivated olive tree (Olea europaea var. europaea) mainly restricted to Mediterranean countries, with remarkable presence in areas such as Andalusia in Southern Spain. In spite of the copious evidence regarding the composition and beneficial effects of EVOO, very little is known about the chemical composition and/or therapeutic effects of ACE oil. Lower antigenic and allergenic capacities have been attributed to ACE in comparison with its cultivated counterpart, and preliminary studies have shown that ACE oil has a higher proportion of tocopherols (vitamin E) and sterols than EVOO [27
At present, a consensus exists on the important role of oxidative stress in the pathogenesis of various systemic and retinal diseases, including AMD [29
], glaucoma, retinitis pigmentosa [30
] and different types of retinopathies such as DR [31
] or hypertensive retinopathy [32
]. In this sense, arterial hypertension (AH) has been associated with the excessive release of reactive oxygen species (ROS) through diverse molecular mechanisms, where the NADPH oxidase system and superoxide anions (O2•−
) seem to be the pivotal agents [33
]. Seven isoforms of the NADPH oxidase (NOX) system (namely NOX1-5 and Duox1-2) have been characterized so far, although the predominant NOXes in vascular cells with the highest relevance in AH development are NOX1, 2, 4 and 5 [35
Nitric oxide (NO) metabolism is closely related to the NADPH system because excess O2•−
production can induce uncoupling of endothelial nitric oxide synthase (eNOS); this might result in endothelial dysfunction and neovascularization eventually, since NO helps maintain ocular hemodynamics by protecting the endothelial cells of vascular beds and nerve fibers against pathogenic processes, e.g., diabetes and glaucoma [36
]. Considering that NO is a key mediator in blood pressure regulation and that NO deficiency results in AH, it seems plausible that this system participates in the development of oxidative imbalance in hypertensive retinas. However, and despite the reported relationship between AH and retinal damage [38
], the precise mechanisms involved in this regard remain unclear.
The aim of this study was to explore the beneficial, antioxidant effects of a diet enriched with ACE oil, based upon its capacity to counteract ocular (retina/choroid) damage, in a rodent model of AH induced with NG-nitro-L-arginine-methyl-ester (L-NAME). To this end, blood pressure monitoring and morphometric analyses were carried out in hypertensive mice under ACE oil- or EVOO-enriched diets (for comparison purposes). Determinations of oxidative stress-related parameters in ocular layers included: estimation of reactive oxygen species (ROS) levels by dihydroethidium fluorescence; H2O2, nitrotyrosine and NO levels; activity, gene/protein expression and immunohistofluorescence of NADPH oxidase isoforms; eNOS activation and eNOS/inducible (iNOS)/arginase 1-2 expression; and quantification of antioxidant enzymes. In addition, glial fibrillary acidic protein (GFAP, as an oxidative/inflammatory marker of gliosis) and transcription factors nuclear factor kappa-B (NF-kB) and nuclear factor erythroid-2 (Nrf-2) (related to oxidative stress pathways) were also quantified.
Despite the well-known healthy effects of the Mediterranean diet and EVOO in particular, very little is known about the properties of other varieties of OOs, such as ACE (wild olive) oil. The chemical composition profile of the oils used in this study, i.e., ACE oil and EVOO (which were obtained from the same geographic area and processed following equal protocols) revealed a similar fatty acid composition, but interesting differences at the level of minor components. Thus, the unsaponifiable fraction from ACE oil was richer than that of EVOO in sterols, tocopherols, triterpene acids, alcohols and secoiridoids. Both sterols and tocopherols are well-known essential micronutrients in the diet of all mammals, with potent hypolipidemic and antioxidant capacities [49
]. Furthermore, the proportion of triterpene acids in ACE is remarkable, considering the antioxidant and neuroprotective effects associated with maslinic acid, among others [52
Despite the fact that the phenol content is similar in both oils, they showed an inverted ratio ortodiphenol/secoiridoids, the latter being the main polyphenols in ACE oil. A variety of pharmacological effects has been reported for these compounds against different pathologies related to inflammatory and oxidant events, due to their antidiabetic, antioxidant, anti-inflammatory, immunosuppressive, neuroprotective, anticancer, and anti-obesity properties [54
]. Therefore, our findings on the chemical composition of ACE oil and EVOO suggest a different behavior in terms of health protection.
Experimental treatment with L-NAME is a well-established model of AH. The administration of oil-enriched diets and/or L-NAME did not affect food/water intake nor weight gain throughout the 6-week experimental period. As expected, a significant and sustained elevation of SBP and DBP was found in the L-NAME group [45
]. Interestingly, hypertension was alleviated in L-NAME-treated animals subjected to simultaneous administration of ACE oil- and EVOO-enriched diets, but a clearly higher depletion of blood pressure was observed in the former. Olive polyphenols have been associated with positive blood pressure outcomes [58
]; additional experiments carried out in our lab showed an improvement in endothelial function, vascular remodeling and hypertrophy in aortas from hypertensive animals upon administration of an ACE oil-enriched diet (unpublished results). Since no changes were found in total phenol content between both oils, the higher hypotensive effect observed for the ACE oil diet might perhaps be attributable to its elevated secoiridoid compound content.
No morphological changes in retinal layers nor signs of hypertension-induced cellular infiltration were revealed by hematoxylin–eosin staining. However, the morphometric analysis evidenced thinner GCL, OS and RPE/CH layers in the L-NAME group compared with normotensive animals. Similar results were reported in hypertensive patients without previous ocular abnormalities, which was associated with likely arterial sclerosis and vascular contraction due to a high intravascular pressure in the choroid [59
], and a decrease in retinal blood flow [60
]. These modifications in hypertensive eyes were reversed by the simultaneous administration of ACE oil and EVOO, suggesting a positive modulation of vascular sclerosis and retinal blood flow. Surprisingly, EVOO administration to normotensive animals also resulted in a decrease in OS and RPE/CH thickness when compared with control mice.
Oxidative stress is highly related to ocular pathologies, including AMD [61
] or DR [31
]; however, the origin of this oxidative imbalance and the pathways involved in the subsequent development of ocular damage are still under research. Preliminary experiments in our lab brought out an increase in ROS production and NADPH oxidase activity in retinas from L-NAME hypertensive Wistar rats (unpublished observations). In the current study, designed using L-NAME hypertensive C57B/6J mice, similar alterations were observed in both the retina and choroid layers of the eye. The increase in ROS generation in hypertensive mice could be reversed in all retinal layers by the simultaneous administration of ACE oil, whereas the EVOO-enriched diet only mitigated ROS overproduction in ONL and OS layers. These results might be attributable, at least in part, to the higher activity of the enzyme NADPH oxidase, an alteration that was also blocked in retina and choroid homogenates from the L-NAME+ACE group, but not in those from the L-NAME+EVOO group. Previous studies demonstrated a decrease in ROS production in LPS-induced murine peritoneal macrophages incubated with oleocanthal, one of the major secoiridoids present in OOs [62
]. Therefore, the changes between ACE oil and EVOO might be due to a higher amount of secoiridoids in the former. Other bioactive minor compounds, such as tocopherols (also elevated in ACE oil over EVOO), might have contributed to better antioxidant outcomes for the ACE oil-enriched diet.
Studies using NOX inhibitors demonstrated a preferential role for the NOX2 isoform of NADPH oxidase in ROS production and NADPH oxidase activity in the retina and choroid of hypertensive animals, because the inhibitory action of GKT136901 and ML171 (affecting NOX1/NOX4) was much lower than that of VAS2870 (which can also inhibit NOX2). These results were confirmed by additional experiments on the gene and protein expression of NOXes, which revealed a significant rise in all three isoforms in the retinas of hypertensive mice, with NOX2 showing the highest upregulation. In this case, the simultaneous administration of both ACE oil and EVOO to L-NAME-treated animals brought the values back to levels observed in control animals. These results did not match those observed for NADPH oxidase activity, where the L-NAME+EVOO group retained the elevated values found in hypertensive mice; on the other hand, the increase reported in the L-NAME group for H2O2 production was reversed by the administration of either oils. Carnevale et al. [63
] reported that EVOO downregulates NOX2 via H2O2, which is in agreement with the reduction in H2O2 found in the retinas of hypertensive mice fed either ACE oil or EVOO. Furthermore, it has been demonstrated that polyphenols from EVOO have effects on ROS levels and NOX expression [64
]. Overall, these findings suggest that both oils (especially ACE oil) are able to counteract the hyperactivation of NADPH oxidase system with the subsequent regulation of ROS production, which eventually results in an improvement in AH-related retinal oxidative stress. This recovery of the oxidative balance was confirmed by the normalization of the protein expression of GFAP and nitrotyrosine as markers of microglial activation and oxidative stress in the retina. The downregulation of both parameters might indicate a better prognosis for some retinopathies, as previously reported for AMD and DR [61
]. The effects of the oils in this regard might be attributed to minor components including hydroxytyrosol, as previously described [67
Excessive ROS production and diminished NO bioavailability are associated with ocular pathologies [68
]. The alterations observed in NO concentration and in the activation/expression of T-eNOS and iNOS in the retinas of hypertensive mice in our studies confirm the role of NO metabolism in eye diseases. Furthermore, the protein expression of arginase, an enzyme that competes with NOS for the use of the common substrate L-arginine, was significantly enhanced in L-NAME-treated animals. All these alterations were corrected by dietary administration of ACE oil and EVOO. The beneficial effects of the oils on NO metabolism might be due to the action of triterpene acids such as maslinic and oleanolic acids, which can activate eNOS via Ser1177 phosphorylation and increase NO production [70
], and also to oleocanthal, which has previously been reported to downregulate iNOS expression [62
]. eNOS activation is considered beneficial in some retinopathies [72
], whereas iNOS is usually considered a biomarker of oxidative stress and inflammation in retinopathies such as AMD [73
] or DR [74
]. In the current study, both ACE oil and EVOO were able to reduce iNOS expression in hypertensive animals back to normal values, thus suggesting an additional protective effect against AH-associated retinal damage.
There is a great deal of evidence concerning the role of arginase in the regulation of NOS system, especially in the context of hypertension [75
]. Two isoforms of arginase enzymes (Arg-1 and, in a major proportion, Arg-2) are expressed in the retina [76
], whose regulation could be crucial in the development of some retinopathies such as DR [76
] or retinal ischemia [77
]. An upregulation of these isoforms correlates positively with the development of ocular pathologies, while Arg-2 depletion showed neuroprotective effects in optic nerve trauma [78
] and in hyperoxia-induced retinal vascular degeneration [79
]. In fact, the inhibition of Arg-1 activity has been suggested as a possible therapeutic strategy to alleviate DR [77
Rojas et al. [80
] showed that diabetes-induced endothelial cell senescence was due to NOX2 activation and subsequent ROS production, leading to an increase in arginase expression/activity that, in turn, led to a reduction in NO in the retina and favored eNOS uncoupling. Retinas from our animal model of L-NAME-induced hypertension seem to follow a mechanism similar to that described in diabetic retinas. Therefore, the association between NOX2 and arginase downregulation found in the retina after the administration of ACE oil and EVOO confers to these oils a retinoprotective effect in the hypertensive context.
Concerning the protein and gene expression of antioxidant enzymes, SOD-1 and GSH-Red were upregulated in retinas from L-NAME-treated animals, whereas the opposite pattern was observed for the GSH-Px enzyme. Simultaneous administration with oils reversed these alterations and led to values similar to those observed in normotensive mice. Interestingly, a higher protein expression of SOD-1 was found in ACE oil groups when compared with the equivalent EVOO groups. We hypothesize that the strong effect of ACE oil on SOD expression favors the conversion of O2•−
to H2O2, and excess H2O2 is then reduced by GSH-Px into H2
O and O2
at the expense of GSH-Red. This hypothesis is confirmed by the above results, showing a reduction in O2•−
and nitrotyrosine levels in ACE oil-fed hypertensive mice. Our results are in agreement with previous studies reporting that the secoiridoid oleuropein, a major component of olive polyphenols, increased the levels of SOD and GSH-Px in gentamicin-induced renal toxicity and in cisplatin-induced renal injury models [62
], as well as in the kidneys of rats with unilateral ureteral obstruction [83
]. However, due to the complexity of the biochemical pathways involved and the relevance of antioxidant enzymes in the retina, their implication in the eye is currently unpredictable [84
], and different antioxidant profiles are found depending on the specific retinopathy, as reported in AMD [85
] and DR [86
], among others.
In addition to studying the NADPH oxidase system, NO metabolism, GFAP expression and antioxidant enzyme profile, we finally explore the modulation of transcription factors NF-κB and Nrf-2 by ACE oil and EVOO. NF-κB is a well-known regulator of inflammation and oxidative processes surrounding cardiovascular diseases and hypertension [87
]; it has been implicated in different retinopathies, such as hypertensive retinopathy induced by angiotensin [88
], AMD [89
] and DR [90
]. Our data demonstrated an overexpression of NF-κB in hypertensive L-NAME retinas that was reverted by ACE oil- and EVOO-enriched diets. EVOO has classically been attributed as having anti-inflammatory actions [91
], mainly due to its minor polyphenol constituents [11
], which can affect the expression of NF-κB [90
]. The high content of triterpene acids in ACE oil might also be implicated in this effect. Additionally, Ampofo et al. [92
] described a maslinic acid-dependent downregulation of NF-κB in endothelial cells, paralleled by increased eNOS expression and reduced oxidative DNA damage. Experiments carried out in human umbilical vein endothelial cells (HUVEC) also demonstrated NF-κB downregulation by maslinic acid [93
]. Furthermore, ursolic and oleanolic acids have also been described as promising anti-inflammatory compounds via NF-κB inactivation [94
Nrf-2 is a transcription factor that activates important cellular defense mechanisms against oxidative stress and is associated with neuroprotective mechanisms [95
]. Several authors have focused on the role of Nrf-2 in retinal diseases in which this factor modulates antioxidant pathways, such as in AMD [96
], DR [97
], or ischemic retinopathy, where a novel therapy based on Nrf-2 activation was able to counteract the oxidative retinal damage [98
]. In this sense, our results demonstrated that ACE oil and EVOO upregulated retinal Nrf-2 expression, with a preferential effect in favor of the former. Several authors have attributed to OO the ability to modulate Nrf-2 [99
], where polyphenols such as secoiridoids seem to be positively implicated in this effect [62
], as these are also associated with a reduction in NF-κB levels [100
]. In the same way, maslinic [93
] and ursolic acids [101
] are also positive modulators of Nrf-2 with beneficial effects against oxidative/inflammatory damage in different situations.
Despite the fact that our data point out the retinoprotective effect of ACE oil and EVOO on hypertensive mice based upon their antioxidant capacity, the wide variability of components obtained from any oil extraction warrants further studies using more specific components, in order to clarify their role in the different molecular pathways affected by oil diets. Moreover, specific chemical analysis should be considered with oil extracts obtained from different geographic areas and subjected to additional extraction methods. Finally, due to the limited number of animals used in the current study, subsequent in vivo and in vitro experiments will certainly shed light on the intracellular pathways modulated by ACE oil and eventually help explore its function in human trials.