antioxidants Review Update on the Effects of Antioxidants on Diabetic Retinopathy: In Vitro Experiments, Animal Studies and Clinical Trials Jose Javier Garcia-Medina 1,2,3,4,5, * , Elena Rubio-Velazquez 1 , Elisa Foulquie-Moreno 1 , Ricardo P Casaroli-Marano 5,6,7 , Maria Dolores Pinazo-Duran 3,4,5, * , Vicente Zanon-Moreno 8,† and Monica del-Rio-Vellosillo 9,† 1 Department of Ophthalmology, General University Hospital Morales Meseguer, 30007 Murcia, Spain; elena.rubio@carm.es (E.R.-V.); elisam.foulquie@carm.es (E.F.-M.) 2 Department of Ophthalmology and Optometry, University of Murcia, 30120 Murcia, Spain 3 Ophthalmic Research Unit Santiago Grisolia, 46017 Valencia, Spain 4 Cellular and Molecular Ophthalmolobiology Group, Surgery Department of the University of Valencia, 46010 Valencia, Spain 5 Red Temática de Investigación Cooperativa en Patología Ocular (OFTARED), Instituto de Salud Carlos III, 28029 Madrid, Spain; rcasaroli@ub.edu 6 Department of Surgery & Hospital Clinic de Barcelona (IDIBAPS), School of Medicine, University of Barcelona, E-08036 Barcelona, Spain 7 Institute of Biomedical Research (IIB-Sant Pau—SGR1113), Banc de Sang i Texits (BST), E-08041 Barcelona, Spain 8 Area of Health Sciences, Valencian International University, 46002 Valencia, Spain; vczanon@universidadviu.com 9 Department of Anaesthesiology, University Hospital Virgen de la Arrixaca, 30120 Murcia, Spain; monica.delrio@um.es * Correspondence: jj.garciamedina@um.es (J.J.G.-M.); dolores.pinazo@uv.es (M.D.P.-D.) † V.Z.-M. and M.d.-R.-V. shared last authorship as senior authors.  Received: 7 June 2020; Accepted: 23 June 2020; Published: 26 June 2020  Abstract: Current therapies for diabetic retinopathy (DR) incorporate blood glucose and blood pressure control, vitrectomy, photocoagulation, and intravitreal injections of anti-vascular endothelial growth factors or corticosteroids. Nonetheless, these techniques have not been demonstrated to completely stop the evolution of this disorder. The pathophysiology of DR is not fully known, but there is more and more evidence indicating that oxidative stress is an important mechanism in the progression of DR. In this sense, antioxidants have been suggested as a possible therapy to reduce the complications of DR. In this review we aim to assemble updated information in relation to in vitro experiments, animal studies and clinical trials dealing with the effect of the antioxidants on DR. Keywords: diabetic retinopathy; antioxidant; oxidative stress; retina; in vitro; cell; animal; clinical trial; human; patient 1. Introduction Diabetic retinopathy (DR) is one of the most frequent causes of blindness in the adult and elderly population worldwide [1]. Control of blood glucose, blood pressure and lipidemia have been shown to alleviate the appearance and evolution of DR [2]. Nonetheless, some patients might exhibit a progression of DR with appropriate blood glucose and pressure control. DR is primarily a microvascular disorder related to the loss of pericytes, endothelial cell proliferation, the disruption of tight junctions between endothelial cells, the thickening of the basement Antioxidants 2020, 9, 561; doi:10.3390/antiox9060561 www.mdpi.com/journal/antioxidants Antioxidants 2020, 9, 561 2 of 23 membrane, the leakage of fluid and macromolecules from the vessels, and neovascularization [3,4]. All these phenomena imply the damage of non-vascular cells of the retina such as neuronal and glial cells, with deleterious implications for visual function due to macular edema, vitreous hemorrhage or tractional retinal detachment. These events have been associated directly with oxidative stress [3,4]. Otherwise, standard therapies to treat DR (vitrectomy, photocoagulation, intraocular injections of anti-vascular endothelial growth factors (VEGFs) or corticosteroids) are not able to control DR progression in all cases. The difficulty to stop DR is related to the metabolic memory and this phenomenon has also been associated with oxidative stress [3]. These considerations about DR have led one to consider different therapeutic approaches, such as antioxidant supplementation, to rebalance the excess free radical production and/or the defect of antioxidant natural systems. In fact, a number of studies dealing with retinal cells cultured under hyperglycaemic conditions, animal models of DR and clinical trials with diabetic patients have been performed to test the effect of different antioxidants, alone or in combination, on the functional or structural alterations of DR. 2. In Vitro Experiments Evidence observed for in vitro approaches has allowed one to conclude reliable findings on the biological effects of the antioxidant active compounds. These mainly act to alleviate different related events (such as autophagy, inflammatory pathways, apoptosis, angiogenesis and oxidative disbalance) in the pathophysiology of diabetic retinopathy (DR) [4–6]. Several studies conclude that antioxidants are able to prevent or even ameliorate DR status, acting against inflammation and oxidative stress. These pathways play a major role in the appearance and clinical evolution of the disease [7–10]. An environment that mimics DR conditions is hard to emulate in vitro. The presence of multifactorial events with an inflammatory response and oxidative disbalance in the retina (capillary endothelial cells, pericytes, glia and neurosensory retinal cells) lead to a progressive cellular degeneration process with metabolic alterations of the retinal pigment epithelium (RPE) cells, and a dysfunction of the retinal blood barriers (internal and external). The final consequence is the loss of vision [8,11,12]. Hyperglycemia also induces mitochondrial dysfunction with an intracellular and extracellular augmentation of the reactive oxygen species (ROS). In this sense, several intracellular signalling pathways share the inflammatory and oxidative pathways in diabetes, such as polyol and protein kinase C (PKC), p38 MAPK and others [6]. Likewise, it has been observed that epigenetic adjustments are engaged with oxidative stress. The phenomenon, described as metabolic memory, is related to the deleterious effects on tissues caused by hyperglycaemia, even though there are strict glycemic controls. Thus, pathways related to regulatory role of microRNAs, histone modifications, DNA methylation and the sirtuins-histones function can act as epigenetic modifiers and modulators [4,13,14]. The metabolic status of the retina is normally isolated from the rest of the body by the blood–retinal barrier that is made up of an internal barrier by the endothelial capillaries and an external barrier by the RPE cells. In addition, these barriers maintain a state of “immune privilege”, with the ability to modulate the response to different external attacks independently [11,15]. In situations of prolonged hyperglycemia, the metabolic stress involves an insult to the retinal capillary network, its barrier systems, and the highly specialized neural connections of the retina, responsible for vision processes [16,17]. It is hard to emulate hyperglycemia experimentally under in vitro conditions. However, the approaches in which in vitro cell cultures are studied present an excellent efficiency and other various advantages, such as the precision of experimental conditions, and the possibility of mimicking alternative scenarios and concentrations of substances. Despite the additional difficulties of interpreting and extrapolating results in vitro, evidence related to antioxidant properties in settings similar to the DR have played a guiding role in the development of conclusive clinical trials. Antioxidants 2020, 9, 561 3 of 23 The results obtained so far have allowed us to conclude that several of the microvascular changes observed in DR can be prevented or mitigated by different antioxidant medicinal derivatives of plant origin. The properties of resveratrol—a powerful antioxidant, which is anti-inflammatory and neuroprotective—are widely known in in vitro studies, which have subsequently been confirmed in different clinical trials [5]. Other flavonoid polyphenols (e.g., naringin, anthocyanins) and non-flavonoids (e.g., curcumin), are characterised by a group of natural active compounds, of which the antioxidant and anti-inflammatory capacity was verified in human cell models of retinal endothelial cells (HREC) and retinal pigment epithelial cells (ARPE-19), under conditions of sustained high concentrations of glucose, mimicking a diabetic environment [18,19]. Non-provitamin A carotenoids (lutein, zeaxanthin, lycopene, and astaxanthin) were shown to be effective, not only in reducing in vitro inflammation, but also in modulating gene expression, eliminating ROS as a consequence [20]. Furthermore, there is strong evidence for xanthophyl carotenoids, such as lutein and zeaxanthin, which are selectively absorbed by RPE cells, and accumulate preferentially in the macular region, conferring protection on photoreceptors. Recently, it was found that this beneficial effect on the retina can be increased synergistically when lutein is administered in association with Ω-3 long-chain polyunsaturated fatty acids. An antioxidant and anti-inflammatory effect were found by reducing ROS production and inhibiting the expression of inflammatory mediators [21]. In this sense, the fish oil emulsion, rich in Ω-3 polyunsaturated fatty acids, has anti-inflammatory and antioxidant in vitro properties, with the capability to inhibit the production of pro-inflammatory growth factors and cytokines [22]. Table 1 summarizes the main in vitro studies on antioxidants and their results as possible therapeutic agents for DR treatment [22–50]. Antioxidants 2020, 9, 561 4 of 23 Table 1. In vitro studies on diabetic retinopathy (DR) and hyperglycemic conditions (↑ = increase of; ↓ = decrease of). Antioxidant(s) Studied. Culture Cell Line Type Main Outcomes Reference Modulation of endothelial proliferation. ALA ↓ Alpha-linolenic acid (ALA), zinc and linoleic reactive oxygen species (ROS) production and Choroid-retina endothelial cells (monkey) Shen et al. 2012 [43] acid vascular endothelial growth factors (VEGF) secretion, and ↑ superoxide dismutase (SOD) activity. AMG-487 Human endothelial cells ↓ oxidative and endoplasmic reticulum stress Wang et al. 2019 [48] Ascorbic acid, α-tocopherol and α-lipoic acid Bovine endothelial cells of the retina ↓ superoxide anion production Wu et al. 2012 [44] ↓ mitochondrial ROS. ↓ superoxide and hydrogen Astragaloside-IV Murine endothelial cells of the retina Qiau et al. 2017 [30] peroxide and β-carotene, lutein and lycopene Human retinal pigment epithelium (RPE) ↓ cell loss Gong et al. 2017 [23] BM-MSC (Bone-marrow mesenchymal stem ↑ defensive effect after alterations caused by H2O2, ↓ Murine ganglion cells of the retina Cui et al. 2017 [25] cells) cytokines, and ↑ neurotrophin secretion ↑ total antioxidant status (TAS) and ↓ Calcium dobesilate Human cultured veins Alda et al. 2011 [42] malondialdehyde ↑ NADH/NADPH, Nrf2, quinine oxidoreductase-1 dh404 (Nrf2 activator) Murine Müller cells Deliyanti et al. 2018 [32] and hemeoxygenase-1 EPA and DHA Human RPE ↓ ROS Dutot et al. 2011 [40] Epigallocatechin-3-gallate (EGCG) Human endothelial cells of the retina ↓ cytokines and apoptosis and Zhang et al. 2016 [31] Fidarestat (aldose reductase inhibitor) Bovine endothelial cells of the retina ↑ antioxidant defenses Obrosova et al. 2003 [36] ↑ antioxidant proprieties with ↓ pro-inflammatory Fish oil emulsion (FOE) U937 cell line (monocytes/macrophages) Laubertová et al. 2017 [22] cytokines, ↓ cellular damage ↑ activation of Nrf2, reverse ↓ expression of claudin-1 Galangin Human endothelial and RPE cells of the retina Zhang et al. 2019 [50] and occludin, and ↓ ROS formation ↓ endoplasmic reticulum stress; ↓ H2O2-induced He-Ying-Qing-Re Formula (HF) Murine retinal ganglion cell culture Zhang et al. 2018 [27] apoptosis; ↓ mitochondria-related proapoptotic factors KIOM-79 Murine pericyte cell culture ↓ apoptosis by ↓ ROS production Kim et al. 2010 [28] Lignans extract (Eucommia ulmoides) Choroid-retina endothelial cells (monkey) ↓ oxidant effects by regulating via Nrf2 pathway Liu et al. 2016 [29] MnTBAP Bovine endothelial cells of the retina ↓ mitochondrial DNA insult Madsen-Bouterse et al. 2010 [38] N-acetylcysteine (+ SS31, mitochondrial ↓ mitochondrial dysfunction, oxidative stress and Human RPE Yumnamcha et al. 2019 [47] antioxidant) mitophagic flux to lysosomes induced by Auranofin. Naringin Murine Müller cells ↓ inflammatory and pro-oxidant effects Liu et al. 2017 [26] Antioxidants 2020, 9, 561 5 of 23 Table 1. Cont. Antioxidant(s) Studied. Culture Cell Line Type Main Outcomes Reference ↑ glutathione peroxidase; apoptosis; Inhibition of PEDF Bovine pericytes of the retina Amano et al. 2005 [37] caspase-3 Selenium Human RPE ↓ glutathione peroxidase González De Vega et al. 2018 [24] SNJ-1945 Murine retinal ganglion cell culture ↓ apoptosis induced by high glucose environment Shanab et al. 2012 [45] SS31 Human endothelial cells of the retina ↓ ROS and caspase-3 Li et al. 2011 [41] ↓ TNF-α and IL-6 levels; ↑ GSH, SOD, and catalase Sulphoraphane Rat Müller cell line Li et al. 2019 [49] activities. Supplement combined (Vitamin C, Trolox, α-tocopherol acetate, N-acetyl cysteine, Bovine endothelial cell and pericyte culture ↓ caspase-3 Kowluru et al. 2002 [34] β-carotene, selenium) Supplement (ascorbic acid, Trolox, α-tocopherol acetate, N-acetyl cysteine, Bovine endothelial cell and pericyte culture ↓ NF-kB and nitric oxides and nitrotyrosine formation Kowluru et al. 2003 [35] β-carotene, selenium) ↓ TBARS, ROS; ↑ GSH-px, catalase and SOD activities Taurine Rat Müller cell line Zeng et al. 2010 [39] in relation to dose Trolox Cultured rat retina ↓ TBARS Ansari et al. 1998 [33] Vitamin D Human RPE ↓ ROS and caspase-3/7 activities Tohari et al. 2020 [46] Antioxidants 2020, 9, 561 6 of 23 3. Animal Studies There is an increasing body of evidence about the role of antioxidants in the control of diabetic retinopathy in animal models (Table 2) [51–109]. Vitamins have a determining task in the control of the oxidative cascade involved in the development of diabetic retinopathy [102–105]. Vitamins C (ascorbic acid) and E [α-tocopherol] have been shown to have the ability of avoiding abnormalities of ocular blood hemodynamics and leukostasis in streptozotocin-induced diabetic rats [102,103]. Moreover, they prevent the advancement of acellular capillaries [102–105]. Combinations of vitamins have a synergistic effect. In this manner, vitamin E + selenium and taurine diminish retinal conjugated dienes (CD) in the early stage of the diabetic disease. This combination can also reduce lipid hydroperoxides (LP), but with a 500 IU dose of vitamin E [97]. In addition, vitamins C and E, supplemented with other antioxidants (Trolox, N-acetyl cysteine, β-carotene and selenium), improve the survival of retinal cells more obviously. This combination prevents a decrease in SOD, glutathione reductase (GR) and catalase, and lowers the activation of retinal protein kinase C and diminishes lipid peroxidation indicators. [102,103]. Trolox is an analog of vitamin E that allows the regeneration of diminished pericytes in retinal vessels in diabetic mice [33]. Age-related eye disease study (AREDS)-based micronutrients (ascorbic acid, vitamin E, β-carotene, zinc, and copper) diminish oxidative and nitrative damage to retinas in diabetic rats and prevent the development of retinal acellular capillaries [52]. Nicanartine (also known as l-carnitine) is responsible for transporting fatty acids into the mitochondria. It also acts as an antioxidant and lipid-lowering compound. Nicarnitine slows down pericyte loss, but has no effect on the formation of capillaries or micro-aneurysms in diabetic rats [86]. Another study showed that l-carnitine can reduce glycemic levels. Administrated daily, it has an important role as an antioxidant factor and avoids protein degradation [77]. High levels of VEGF protein concentration are observed in diabetic rats if compared with control non diabetic rats. Taurine and α-lipoic acid alleviate VEGF levels in diabetic rats [36] and α-lipoic acid maintains a constant number of pericytes and foil the formation of acellular capillaries [54,55,57]. These molecules obtain the inhibition of lipid peroxidation, increase glutathione peroxidase and produce an activation of AMP-activated protein kinase (AMPK) [57]. The normalization of nuclear transcriptional factor and the restoration of antioxidant defences are observed in the retinas of diabetic rats [54–57]. Moreover, oral supplementation with lipoic acid allows one to re-establish the electroretinogram b-wave amplitude of diabetic animals, to control values [56]. It has been known for a long time that food plays a crucial function in the control of diabetes. However, it is not only necessary to avoid foods with a high glycemic index. Several studies are being carried out to prove the beneficial effect for the control of pathogenesis of structural damage caused by diabetes. The antioxidant agents present in commonly consumed food have shown beneficial effects. Some examples are blueberry anthocyanins [59], Litchi chinensis [78], Morus alba (white mulberry) [83] Crocin (Saffron) [67] and Sesamin [95]. Hydroxytyrosol is the main polyphenol compound present in olive oil. It has a neuroprotective effect on the retina. In diabetic rats treated with hydroxityroxol, the total retinal thickness and the cellular size were similar to non-diabetic animals. Retinal ganglion cell loss is smaller in rats treated with hydroxytyrosol, compared with non-treated diabetic rats [76]. Curcumin, a natural yellow pigment used commonly to color foods and cosmetics, decreases oxidative stress, diminishes levels of VEGF, NF- KB AND IL-1 β, and seems to have an anti-inflammatory effect on DR [68,69]. Resveratrol is present in the skin of grapes, blueberries, raspberries and blackberries. It is also found in high concentrations in red wine. Antioxidants 2020, 9, 561 7 of 23 Table 2. Results of antioxidant supplementation in animal models. Antioxidant Studied Outcomes in Treated Animals Reference Apocynin ameliorates (medicinal herb Regulate the inflammation through inhibition od TLR4/NF-kB pathway. Wang et al. [51] 2019 Picrorhiza kurroa) AREDS-based micronutrients Avoidance of oxidative and nitrative stress. Prevention of formation of ghost capillaries. Kowluru et al. [52] 2008 α-lipoic acid or taurine Improves levels of VEFG and reduces ROS biomarkers. Obrosova et al. [36] 2001 α-lipoic acid or D-α-tocopherol Frustrates the increase in leukostasis. Abiko et al. [53] 2003 α-lipoic acid Control of retinal lipid peroxidation. Stunting of capillary apoptosis and acellular capillaries. Kowluru et al. [54] 2004 Control of nuclear transcriptional factor and angiopoietin-2. Reduction of VEGF and ROS α-lipoic acid Lin et al. [55] 2006 species. Avoid pericyte ghost. Reestablishment of ERG b-wave amplitude. Avoidance of GSH depletion. Normalization α-lipoic acid Johnsen-Soriano et al. [56] 2008 of MDA. Inhibits cells death. Activation od AMP- activated protein kinase (AMPK). Inhibition of O- α-lipoic acid Kim et al. [57] 2018 linked–β-N acetylglucosamine transferase (GOT). Aster tataricus Preservation of vascular permeability. Attenuation of TNFa, IL10 and NF-kB. Du et al. [58] 2017 Blueberry anthocyanins Downregulation NRF2 pathway. Reestablishment of VEGF and IL-1β levels. Song et al. [59] 2016 Caffeic acid hexyl (CAF6) and dodecyl (CAF12) Increase superoxide dismutase (SOD) activity and iso prostaglandin F2 alpha. Decrease Fathalipour et al. [60] 2019 amide derivatives retinal oedema and improve neuronal survival signal. Calcium dobesilate Improvement of vascular tortuosity. Stunting of capillary apoptosis and acellular capillaries. Padilla et al. [61] 2005 Calcium dobesilate Avoids blood-retinal barrier breakdown and leukocyte adhesion to vessel wall. Leal et al. [62] 2010 Calcium dobesilate Inhibition NF-kB pathway. Reduction of TNF- α IL-6, and MPC-1. Bogdanov et al. [63] 2017 Increase of GFAP, attenuation of cytokine expression and increase in oxidised nitrotyrosine Calcium dobesilate Voabil et al. [64] 2018 and carbonyls. Cannabidiol (CBD) Diminution of TNF-α, VEGF, ICAM. Maintenance of vascular permeability. El-Remessy et al. [65] 2006 Vasoprotective effect. Induction of protective Het shock proteins in activated glial cells and Carnosine Pfister et al. [66] 2011 normalization of hyperglycemia-induced Ang-2. Crocin (saffron) Microglial activation. Neuroprotective. Yang et al. [67] 2017 Curcumin Improvement of oxidative stress biomarkers. Kowluru et al. [68] 2007 Curcumine Restoration of expression and function of DNAmethyltransfere (DNMT). Maugeri et al. [69] 2018 Restoration of ERG b-wave amplitude. Inhibition of lipid peroxidation and apoptosis DHA or lutein Arnal et al. [70] 2009 markers. Improvement of retinal thickness. Antioxidants 2020, 9, 561 8 of 23 Table 2. Cont. Antioxidant Studied Outcomes in Treated Animals Reference Ebselen or lutein Reduction of ROS species. Miranda et al. [71] 2004 Mitigation of retinal inflammation and plasma lipid peroxidation, Preservation of Eriodictyol Bucolo et al. [72] 2012 blood-retinal barrier. Fidarestat Inhibition aldose reductase pathway. Obrosova et al. [36] 2003 Green tea/Vitamin C-E Diminution of ghost pericytes and acellular capillaries. Lower superoxide capacity. Mustata et al. [73] 2005 Green Tea Lowering expression of proinflammatory mollecules (VEGF and TNF-α). Kumar et al. [74] 2012 Hesperetin Reduction of levels of cytokines. Inhibitory effect on caspase-3, GFAP and AQP4 expression. Kumar et al. [75] 2013 Neuroprotective effect. Slowing down on ganglion retina cell counts. Decrease of retinal Hydroxytyrosol [olive oil] Gonzalez-Correa et al. [76] 2018 thickness and cellular size. l carnitine Improvement of glucose levels. Inhibitory effect on protein degradation. Samir et al. [77] 2018 Lichi chinensis Downregulation of proteins carbonyl subproducts and aldose reductase. Kilari et al. [78] 2016 Lutein Avoidance of ganglion cell loss. Reduction of apoptosis markers like caspase-3. Sasaki et al. [79] 2010 Reduction od retinal nitrotyrosine and malondialdehyde levels, The vasomodulator Melatonin Ozdemir et al. [80] 2014 cytokines are decreased. Melatonin Depletion in concentrations of VEGF MMP9, and oxidation protein products (AOPP). Djordjevic et al. [81] 2018 Melatonin Decreased fluorescein retinal leakage, ROS and malondialdehyde levels. Mehrzadi et al. [82] 2018 Morus Alba Reduces glucose levels and VEGF levels. Inhibition polyol pathway. Mahmoud et al. [83] 2017 N-acetylcysteine Restoration VEGF and ICAM-1. Diminution of free radicals. Zhu et al. [84] 2012 Naringenin Controls glucose levels, increases insulin. and retinal glutathione. Al-Dosari et al. [85] 2017 Nicanartine Prevention of endothelial proliferation and pericyte loss. Hammes et al. [86] 1997 Obtisofplin Improvement of capillary cell apoptosis and the number of acellular capillaries in the retina. Hou et al. [87] 2014 Restoration of amplitudes of a- and b-wave of ERG; reduced retinal VEGF; reduction of PEDF retinal 8-hydroxydeoxyguanosine, a marker of oxidative stress. inhibition of retinal vascular Yoshida et al. [88] 2009 hyperpermeability. Resveratrol Inhibition of nitric oxide synthase in endothelial cells. Yar et al. [89] 2012 Strengthening of oxidative markers (lipid peroxidation index and oxidized to reduced Resveratrol Soufi et al. [90] 2012 glutathione ratio) and superoxide dismutase activity in blood and retina. Recovers insulin level. Improve paraoxonase 1 (PON1) gen activity, reducing vascular Resveratrol Chen et al. [91] 2018 permeability and of VEGF, TNF- α, MPC-1, IL-6 IL-1β, INFγ levels. Antioxidants 2020, 9, 561 9 of 23 Table 2. Cont. Antioxidant Studied Outcomes in Treated Animals Reference Reduces vascular lesion, NF-kB and TNF- α. Stimulates the expression of Ndf2 and Trans resveratrol Al Hussaini et al. [92] 2018 SIrT1 genes. Decrease expression of VEGF, TNF- α, MPC-1, ICAM 1, IL-6 and IL-1β. Restore balance Resveratrol coated gold nanoparticles Dong et al. [93] 2019 between inhibitors and stimulators of angiogenesis. Decrease of glutatione, brain-derived neurotrophic (BDNF), nerve growth factor Rutin Ola et al. [94] 2015 and caspase. Improves blood glucose levels and body weight. Reduces ROS levels and Sesamin Ahmad et al. [95] 2016 inflammatory biomarkers. Shikimic Acid (SA) (Artemisia absinthium) Reduces glucose and glycated hemoglobine levels. Decreases IL-1β and TNF- α. Al Malki et al. [96] 2019 Diminished conjugated dienes in retina at the early stage of diabetic retinopathy. Taurine/vitamin E+selenium Di Leo et al. [97] 2003 Reduced lipid hydroperoxides. Taxifolin Reduction of total glutathione level. Decrease MDA, IL-1β and TNF- α blood levels. Ahiskali et al. [98] 2019 Tempol Improvements in retinal microvascular hemodynamics and blood flow rates. Yadav et al. [99] 2011 Tempol Lowers oxidative stress, fibronectin and glial fibrillary acidic protein. Rosales et al. 2011 [100] Trigonella foenum Decreases of inflammatory and angiogenic markers (TNF-α, VEGF, IL1-β). Gupta et al. [101] 2014 Trolox Avoids pericyte loss. Ansari et al. 1998 [33] Vitamins C and E Less acellular capillaries and pericyte ghosts. Kowluru et al. 2001 [102] Vitamins C and E Prevent formation of acellular capillaries. Reduce pericyte ghost cells. Yatoh et al. [103] 2006 Vitamin C Suppression of leukocyte adhesion. Increase iris blow flow perfusion. Jariyapongskul et al. [104] 2007 Prevention of blood retinal barrier breakdown. Diminution of VEGF, ICAM1, TNF- α, SOD, Vitamin Kunisaki et al. [105] 1998 IL-1, IL-6 and aldose reductase. Zeaxanthin Reduction of Oxidative damage. Kowluru et al. [106] 2008 Antioxidants 2020, 9, 561 10 of 23 A daily dose of Resveratrol suppresses endothelial nitric oxide synthase production [89], the expression of oxidative biomarkers and superoxide dismutase capacity in the retina and blood of diabetic rats [90]. Furthermore, it improves the expression of the PON1 gene that regulates inflammatory response and microvascular complications in diabetes [91] and can restore the transcription of the proteins of the retinoic acid metabolism pathway [92]. Resveratrol coated gold nanoparticles redevelop the equilibrium between the stimulators and inhibitors of antiangiogenesis, increasing the retinal expression of PEDF and decreasing VEGF-1. Retinal expressions of TNFα, IL-1 β, MCP-1, ICAM-1 and IL-6 are also diminished [91–93]. Caffeic acid hexyl (CAF6) and Dodecyl (CAF12) amides also have a neuroprotective effect [57,60]. Another antioxidant, Calcium Dobesilate (Ca Dob), inhibits acellular capillaries and pericyte loss, and [61] protects the blood-retinal barrier by preserving tight junctions [62], and induces an important decrease of pro-oxidative markers [61–63]. Carotenoids, such as lutein or zeaxanthin, also seem to have protective effects on DR in diabetic rats. Zeaxanthin inhibits oxidative damage and performs anti-inflammatory action [106]. Plus, lutein normalizes the markers of retinal oxidative stress, avoids ganglion cell loss and decreases apoptosis markers (caspase-3) [79]. Furthermore, it attenuates the loss of ganglion cells and prevents impairment in ERGs [79]. Tempol, a superoxide dismutase (SOD) mimetic and pleiotropic intracellular antioxidant, can neutralize the excess of superoxide radical expression in DR. Tempol protects cells of the vessels, especially ameliorating hemodynamics in the retina of diabetic rats [100]. Moreover, tempol avoids the gathering of fibronectin and glial fibrillary acidic protein in a diabetic murine model [100]. N-acetylcysteine helps to regulate pro-oxidative markers (ROS, VEGF and ICAM-1) [84]. Apocyn ameliorates and downregulates the TLR4/NF- B pathway activity, prevents microglial activation and stops neuronal autophagy [51]. Eriodictyol, a flavonoid obtained from California Yerba Santa, preserves the blood-retinal barrier, decreasing pro-oxidative biomarkers as well [72]. Rutin has shown antiapoptotic activity (decreasing levels of caspase-3) [94]. It has activity at the systemic level, decreasing glucose levels and improving insulin concentration. Plus, other flavonoids with beneficial effects are naningerin [85] and taxifolin, a flavonone found in onion, milk thistle (carduus marianus), douglas fir bark and frech maritime (pinus pinaster) fir bark. Taxifolin is useful to prevent diabetic retinal damage. It can normalize the Il-6, TNF, IL1 and tGSH levels in blood serum [98]. Other flavonoids such as hesperetine [75] have been studied, obtaining promising results. Green tea, rich in polyphenols, decreases the number of pericyte ghosts and acellular capillaries and forces a lower concentration of VEFG and TNF-α in the retina of animal models [73,74]. Melatonin is a strong antioxidant naturally secreted by the pineal gland. When orally administered, it presents the amelioration of cytokine, nitrotyrosine, and malondialdehyde concentrations, [80] and depletion in concentrations of VEGF, MMP9, and oxidation protein products (AOPP) [81]. Merhazadi et al. also found that oral melatonin administered to diabetic rats decreased retinal cell size and the fluorescein leakage of retinal vessels [82]. 4. Clinical Studies There are not as many clinical studies as in vitro or animal investigations. There is an increased interest in antioxidant supplementation for human disorders, because they can be administered orally and they are easily available and affordable. Difficulties in analyzing these studies are that end-points and results are variables and, in some cases, controversial. Some studies focus their objectives on functional results (best-corrected visual acuity—BCVA—; contrast sensitivity; glare sensitivity), others in anatomical results (central macular thickness—CMT—), in analyzing oxidative stress status, or even in a combination of the anterior parameters. Plus, in the majority of these studies, the population studied include type 2 diabetic mellitus (T2DM) patients. Some studies deal with the first stages of the disease and other studies consider typical complications of late stages. Furthermore, as previously noted, tangled and not well-known mechanisms in the pathogenesis of diabetic retinopathy make early and new combined therapies of antioxidants desirable. The most relevant studies are listed in Table 3. Antioxidants 2020, 9, 561 11 of 23 Table 3. Results of human studies. CAT = combined antioxidant therapy. Oral Antioxidant Studied Recruited Patients [n] Outcomes in Treated Patients Follow-Up Reference No results preventing clinically significant macular Alpha lipoic acid 467 edema in T2DM. No improvement in best-corrected 2 years Haritoglou et al., 2011 [107] visual acuity. Antioxidant combination: Alpha lipoic All treated groups showed decreased blood TBARS 80 3 months Khahler et al., 1993 [108] acid or Selenium or Vit. E levels and urinary albumin excretion rates. No influence on the capillary resistance of Calcium Dobesilate 18 8 months Larsen et al., 1977 [109] diabetic retinopathy Calcium Dobesilate 42 No results in non-proliferative diabetic retinopathy 42 [6 months] 36 [1 year] Stamper et al., 1978 [110] Treated DM + glaucoma patients showed decrease of Calcium Dobesilate 50 capillary fragility, blood viscosity and microvascular 3 months Vojnikovic et al., 1984 [111] hyperpermeability. Decrease of whole blood viscosity and Calcium Dobesilate 37 3 months Benarroch et al., 1985 [112] capillary fragility. Both drugs improved exudates, Dexium® only in 1 Calcium Dobesilate [Dexium® ] or case. Both drugs, particularly Pycnogenol® , showed 32 6 months Leydhecker et al., 1986 [113] Pycnogenol® improvements on parameter of automated visual field. Non-insulin dependent diabetics showed reducing Calcium Dobesilate 79 of whole blood and plasma viscosity and 6 months Vojnikovic, 1991 [114] retinal hemorrhages. In T2DM, better improvement than placebo on Calcium Dobesilate 137 2 years Ribeiro et al., 2006 [115] microaneurysms, DR level and retinal hemorrhages. No effects reducing development of clinically Calcium Dobesilate 635 5 years Haritoglou et al., 2009 [116] significant macular edema in T2DM NPDR with macular edema received laser +placebo or laser + Ca Dob. This study showed no statistically Calcium Dobesilate 40 6 months Feghhi et al., 2014 [117] significant difference in macular thickness between Doxium and placebo. No effect on visual acuity. T2DM treated group show retardation of progression and maintainance of CAT formulation [Vitalux Forte® ] 105 5 years Garcia-Medina et al., 2011 [118] antioxidant plasma status level and decreased plasmatic MDA. Antioxidants 2020, 9, 561 12 of 23 Table 3. Cont. Oral Antioxidant Studied Recruited Patients [n] Outcomes in Treated Patients Follow-Up Reference Pre-retinopathic diabetics treated group showed CAT: Alpha lipoic acid 32 increases ERG oscillatory potencial values and 30 days Nebbioso et al., 2012 [119] +genistein+Vitamins C, E and B. plasma antioxidant levels. CAT: Coenzyme Q10, Pycnogenol® , Treated T2DM: significantly reduced free oxygen 68 6 months Domanico et al., 2015 [120] Vitamin E. radical test levels and CMT. CAT: DHA, glutathione, hydroxytyrosol, Reduced MDA, significant increased TAS in treated vitamins E, C, B1, B2, B3, B6, B9, B12, 208 18 months Roig Revert et al., 2015 [121] T2DM with diabetic retinopathy group. lutein, zeaxanthin, Se, Mn, Zn, Cu. Better visual function. No effect on macular CAT [DiVFuSS formula] 67 6 months Chous et al., 2016 [122] thickness. Lower ROS expression (LPO, nitrites/nitrates) and Rodriguez-Carrizalez. 2016 CAT or Coenzyme Q10 60 6 months augment antioxidant defences. [123] RD patients with macular edema treated with CAT: DHA, EPA, vitamins B, C, E 55 ranibizumab. Thinner macular in the 3 years Lafuente et al., 2018 [124] and Zeaxantin supplemented group. Patients with refractory DME divided in 3 groups: 5 mg crocin, 15 mg crocin and placebo. 15 mg crocin Crocin 60 group showed significant reduction of HbA1c and 9 months Sepahi S et al., 2018 [125] CMT and increase of BCVA compared with placebo group Decrease of MDA and fibrinogen levels. Improved Ginkgo biloba extract 25 blood parameters (viscosity, viscoelasticity, red blood 3 months Huang et al., 2004 [126] cell deformability, and retinal blood flow). Treated group showed improvement of visual acuity, Lutein + zeaxanthin 90 3 months Hu BJ et al., 2011 [127] contrast sensitivity and reduction of foveal thickness. Treated NPDR showed improvement at low spatial Lutein 31 9 months Zhang PC et al., 2017, [128] frequency in contrast sensitivity. T2DM NPDR patients 36 received L and 36 received Lutein + zeaxanthin 72 L+ Z. No significant differences in visual acuity, 4 months Yongo-bo et al., 2019, [129] contrast sensitivity and glare sensitivity. Treated group showed not worsening of retinal Pycnogenol® 30 2 months Spadea et al., 2001 [130] retinal function and visual acuity. Improvement of visual acuity, flow at the central Pycnogenol® 46 retinal artery. Reduction in retinal thickness. 3 months Steigerwalt et al. 2009 [131] In T2DM. Antioxidants 2020, 9, 561 13 of 23 Table 3. Cont. Oral Antioxidant Studied Recruited Patients [n] Outcomes in Treated Patients Follow-Up Reference T1DM showed significant decreased levels of MDA and increased total antioxidant capacity between Resveratrol 13 2 months Ali Movahed et al. 2020, [132] baseline and endpoint. There was no change in the serum levels of TNF- alpha and IL-1beta. Vitreous levels of vitamin C in PDR patients showed Vitamin C 40 a tenfold decrease, which was associated with degree Park SM et al., 2019, [133] of macular ischemia. Diabetic patients showed decreased retinal blood Vitamin E 45 flow that improved after treatment similarly to 8 months Bursell et al., 1999 [134] non-diabetic cases. Vitamin E 282 Decrease of MDA. 3 months Chatziralli et al., 2017 [135] Patients with retinopathy showed increasement of Zinc 18 plasmatic GSH-px activity. All patients showed 3 months Faure et al., 1995 [136] decrease of TBAR. DM patients had a negative correlation between Zinc 45 serum VEFG levels and Zinc. Treated group showed 3 months Kheirouri et al., 2019, [137] no changes in VEGF levels. Antioxidants 2020, 9, 561 14 of 23 Table 3 shows three studies with ALA. Single administrations in 467 T2DM patients over the course of two years found no results preventing diabetic macular edema (DME) or improving BCVA. [107,108]. ALA combined with genistein and vitamins C, E and B in 32 pre-retinophatic patients over the course of 30 days showed some increases in plasma antioxidant levels and eletroretinogram (ERG) oscillatory potential values [119]. Calcium dobesilate (Ca Dob) is a vasculoprotector agent that has been widely studied in the context of DR. [138]. However, the results of clinical trials in DR are controversial. Farsa [139] described some Ca Dob mechanism of actions, such as antioxidant and anti-ROS activity, defensive activity at the endothelial level, antiapoptoic properties and angiogenesis inhibition. Other mechanisms of Ca Dob improving microcirculation and reducing micro-vascular injury have been described recently [140]. There are nine studies listed with Ca Dob supplementation (Table 3). The study with the highest number of patients recruited (635) and the longest duration showed no effects on the reduction of the development of clinically significant diabetic macular edema (CSDME) in T2DM [116]. Likewise, another study with 40 non-proliferative DR (NPDR) patients with macular edema failed to find beneficial effects by adding Ca Dob to laser in the treatment of macular edema [117]. However, other studies have positive results analyzing microaneurisms and retinal hemorrhages [115], exudates [113], and blood viscosity [111,112]. There is an increasing interest in combined antioxidant therapy (CAT), as shown by the latest investigations. A long-lasting study (60 months) by Garcia-Medina et al. [118] evaluated antioxidant supplementation (lutein, VC, alpha-tocopherol, niacin, beta-carotene, Zn and Se) in 105 T2DM with NPDR. This long follow-up allowed one to demonstrate a retardation of the DR progression and the maintenance of antioxidant plasma status in the treated group. However, no effect on visual acuity was reported. In another work, the supplementation of lutein and zeaxanthin for three months in type 1 diabetic mellitus (T1DM) and T2DM showed an improvement of visual acuity, contrast sensivity and foveal thickness [127]. Likewise, Chous et al. reported an improvement in BCVA, but no changes in retinal thickness in his study. They administered a DiVFuSS formula that consists of vitamins C, D3 and E (d-α tocopherol), zinc oxide, eicosapentaenoic acid, docosahexaenoic acid, α-lipoic acid (racemic mixture), coenzyme Q10, mixed tocotrienols/tocopherols, zeaxanthin, lutein, benfotiamine, N-acetyl cysteine, grape seed extract, resveratrol, turmeric root extract, green tea leaf, and Pycnogenol [122]. Other studies focus their results on the interpretation of oxidative/nitrosative stress and antioxidant defenses, such as Rodriguez-Carrizalez in a trial with 60 T2DM patients over the course of months [123]. Furthermore, Roig Revert et al. studied 208 T2DM for 18 months and found a reduction of MDA and a significant increment of total antioxidant status (TAS), a reduction of MDA and a significant increase of TAS in the treated retinopathy group [121]. The association of CAT with the latest therapies such as anti-VEFG therapy is very interesting. Lafuente et al. [124] studied 55 T2DM for three years. They measured the effect of adding a combined antioxidant treatment to ranibizumab in the treatment of diabetic macular edema (DME). They reported lower macular thickness in the supplement group when compared to the control group. The antioxidants, anti-inflammatory and neuroprotective effects of crocin have also been studied by Sepahi et al., in 60 DM pattens with refractive DME showing promising results [125]. Ginkgo biloba supplementation has been tested by Huang in 25 T2DM patients for three months. They found a decrease in MDA and fibrinogen levels and an improvement in other blood parameters and retinal blood flow rate [126]. Pycnogenol® , a French maritime pine bark extract rich on flavonoids, has been dosed alone or combined since the 1980s, in several studies. Two studies administering this antioxidant alone (Table 3) are referenced. One is a study of 32 diabetic patients for six months, five of which displayed a reduction in exudates in both eyes [113]. The other study reported the functional and anatomic results of 46 T2DM for three months [131] Domanico et al. have studied patients with NPDR. Patients were divided into two groups: receiving supplementation or placebo. The treatment was made up of an Antioxidants 2020, 9, 561 15 of 23 antioxidant combination (pycnogenol, vitamin E and coenzyme Q10). Treated group showed that central macular thickness was significantly reduced during the study period (six months) [120]. In 2020, Ali et al. published an exploratory, two-month investigation, to evaluate the efficacy and safety of resveratrol in T1DM patients. A total of 13 patients were studied. Between the baseline and the endpoint resveratrol, treatment was associated with a significant decrease in the level of MDA and a significant increase in the level of total antioxidant capacity. There was no change in the serum levels of TNF-alpha and IL-1beta [132]. Tabatabaei-Malazy et al. reviewed 10 observational studies that reported lower vitamin C levels in subjects with DR compared to those without DR [141]. Park et al. studied the association of vitreous vitamin C depletion with diabetic macular ischemia in proliferative diabetic retinopathy (PDR). This is a study with 40 patients, 20 with PDR and 20 with idiopathic epiretinal membrane (control group), that underwent a pars plane vitrectomy. They found that a vitreous level of vitamin C in PDR patients showed a decrease, that was correlated with the degree of macular ischemia. Levels of vitamin C in the serum, aqueous humor and vitreous humor were lower in patients with PDR than in the control group. However, there was no correlation among the serum, aqueous humor and vitreous humor levels of vitamin C in the PDR group. These findings suggest that ocular factors could be relevant in the pathogenesis of PDR and open new strategies and routes of administration of the treatment [133]. Two clinical trials administering vitamin E (α-tocopherol) are included (Table 3). Bursell et al. studied high-dose vitamin E supplementation in a T1DM group and in a control group. The authors reported that diabetic patients showed a decreased retinal blood flow that improved after treatment, similarly to control cases [134]. Chatziralli et al. administered 300 mg of vitamin D to 282 insulin dependent T2DM with DR. They found a significant diminution of MDA in all DR stages [135]. Vitamin E supplementation was compared to ALA or selenium supplementation or placebo, in a study performed by Kähler in 80 diabetics during three months. They found that oxidative status was reduced in all treated groups compared to the placebo group [108]. Finally, a recent study by Kheirour analyzed serum VEFG levels with zinc supplementation. The authors did not observe any changes after three months of treatment [137]. 5. Conclusions The studies examined in this survey suggest that a number of antioxidants may prevent or alleviate the deleterious effects of hyperglycaemic environment in cultured tissues, or the complications of DR in animal models of diabetes. The results in human studies are more controversial, but some of them are quite promising, particularly when combinations of antioxidants are considered. It is necessary to keep in mind that studies in humans cannot be compared to those performed in cultured tissues with a perfect control of the environment or performed in laboratory animals in which diets and the rest of the living habits are supervised. In addition, metabolism, senescence rate or administered doses in animal models are not identical to those in human individuals [142]. The heterogenicity of the studies included in this review, in terms of antioxidant types, methods, doses and results, is remarkable. It is difficult to bring together all this information, but we can infer that there is a tendency to use antioxidants in combination. However, we have to keep in mind that pro-oxidative conditions and their consequences are still not well-known in a chronic disorder such as DR. Plus, a combination of antioxidant may interact not only with different metabolic pathways, but also among themselves. In this context, it is hard to define the exact effect of each antioxidant that may even change when administered in combination. Another important concern is the safety of administering antioxidants for years, when the clinical studies performed so far are not longer than five years. Despite the fact that encouraging outcomes have already been obtained in this field, future studies must be conducted considering the efficacy and safety of different doses of antioxidants, in order to get better results to prevent ocular damages in this blinding disease. Antioxidants 2020, 9, 561 16 of 23 Author Contributions: Conceptualization, J.J.G.-M., M.D.P.-D. and M.d.-R.-V.; methodology, J.J.G.-M., M.D.P.-D. and M.d.-R.-V.; data curation, E.R.-V., E.F.-M., R.P.C.-M., V.Z.-M.; writing—original draft preparation, J.J.G.-M., M.D.P.-D., E.R.-V., E.F.-M., R.P.C.-M., V.Z.-M. and M.d.-R.-V.; writing—review and editing, J.J.G.-M., M.D.P.-D., E.R.-V., E.F.-M., R.P.C.-M., V.Z.-M. and M.d.-R.-V.; supervision, J.J.G.-M. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest. References 1. Flaxman, S.R.; Bourne, R.R.; Resnikoff, S.; Ackland, P.; Braithwaite, T.; Cicinelli, M.V.; Das, A.; Jonas, J.B.; Keeffe, J.; Kempen, J.H.; et al. Global causes of blindness and distance vision impairment 1990–2020: A systematic review and meta-analysis. Lancet Glob. Health 2017, 5, e1221–e1234. [CrossRef] 2. 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