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
Alpha-linolenic acid (ALA), zinc and linoleic
acid Choroid-retina endothelial cells (monkey)
Modulation of endothelial proliferation. ALA ↓
reactive oxygen species (ROS) production and
vascular endothelial growth factors (VEGF) secretion,
and ↑ superoxide dismutase (SOD) activity.
Shen et al. 2012 [43]
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]
Astragaloside-IV Murine endothelial cells of the retina ↓ mitochondrial ROS. ↓ superoxide and hydrogenperoxide and Qiau et al. 2017 [30]
β-carotene, lutein and lycopene Human retinal pigment epithelium (RPE) ↓ cell loss Gong et al. 2017 [23]
BM-MSC (Bone-marrow mesenchymal stem
cells) Murine ganglion cells of the retina
↑ defensive effect after alterations caused by H2O2, ↓
cytokines, and ↑ neurotrophin secretion Cui et al. 2017 [25]
Calcium dobesilate Human cultured veins ↑ total antioxidant status (TAS) and ↓malondialdehyde Alda et al. 2011 [42]
dh404 (Nrf2 activator) Murine Müller cells ↑ NADH/NADPH, Nrf2, quinine oxidoreductase-1and hemeoxygenase-1 Deliyanti et al. 2018 [32]
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]
Fish oil emulsion (FOE) U937 cell line (monocytes/macrophages) ↑ antioxidant proprieties with ↓ pro-inflammatorycytokines, ↓ cellular damage Laubertová et al. 2017 [22]
Galangin Human endothelial and RPE cells of the retina ↑ activation of Nrf2, reverse ↓ expression of claudin-1and occludin, and ↓ ROS formation Zhang et al. 2019 [50]
He-Ying-Qing-Re Formula (HF) Murine retinal ganglion cell culture ↓ endoplasmic reticulum stress; ↓ H2O2-inducedapoptosis; ↓ mitochondria-related proapoptotic factors Zhang et al. 2018 [27]
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
antioxidant) Human RPE
↓ mitochondrial dysfunction, oxidative stress and
mitophagic flux to lysosomes induced by Auranofin. Yumnamcha et al. 2019 [47]
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
PEDF Bovine pericytes of the retina ↑ glutathione peroxidase; apoptosis; Inhibition ofcaspase-3 Amano et al. 2005 [37]
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]
Sulphoraphane Rat Müller cell line ↓ TNF-α and IL-6 levels; ↑ GSH, SOD, and catalaseactivities. Li et al. 2019 [49]
Supplement combined (Vitamin C, Trolox,
α-tocopherol acetate, N-acetyl cysteine,
β-carotene, selenium)
Bovine endothelial cell and pericyte culture ↓ caspase-3 Kowluru et al. 2002 [34]
Supplement (ascorbic acid, Trolox,
α-tocopherol acetate, N-acetyl cysteine,
β-carotene, selenium)
Bovine endothelial cell and pericyte culture ↓ NF-kB and nitric oxides and nitrotyrosine formation Kowluru et al. 2003 [35]
Taurine Rat Müller cell line ↓ TBARS, ROS; ↑ GSH-px, catalase and SOD activitiesin relation to dose Zeng et al. 2010 [39]
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-1β, 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
Picrorhiza kurroa) Regulate the inflammation through inhibition od TLR4/NF-kB pathway. Wang et al. [51] 2019
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
α-lipoic acid Control of nuclear transcriptional factor and angiopoietin-2. Reduction of VEGF and ROSspecies. Avoid pericyte ghost. Lin et al. [55] 2006
α-lipoic acid Reestablishment of ERG b-wave amplitude. Avoidance of GSH depletion. Normalizationof MDA. Johnsen-Soriano et al. [56] 2008
α-lipoic acid Inhibits cells death. Activation od AMP- activated protein kinase (AMPK). Inhibition of O-linked–β-N acetylglucosamine transferase (GOT). Kim et al. [57] 2018
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)
amide derivatives
Increase superoxide dismutase (SOD) activity and iso prostaglandin F2 alpha. Decrease
retinal oedema and improve neuronal survival signal. Fathalipour et al. [60] 2019
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
Calcium dobesilate Increase of GFAP, attenuation of cytokine expression and increase in oxidised nitrotyrosineand carbonyls. Voabil et al. [64] 2018
Cannabidiol (CBD) Diminution of TNF-α, VEGF, ICAM. Maintenance of vascular permeability. El-Remessy et al. [65] 2006
Carnosine Vasoprotective effect. Induction of protective Het shock proteins in activated glial cells andnormalization of hyperglycemia-induced Ang-2. Pfister et al. [66] 2011
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
DHA or lutein Restoration of ERG b-wave amplitude. Inhibition of lipid peroxidation and apoptosismarkers. Improvement of retinal thickness. Arnal et al. [70] 2009
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
Eriodictyol Mitigation of retinal inflammation and plasma lipid peroxidation, Preservation ofblood-retinal barrier. Bucolo et al. [72] 2012
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
Hydroxytyrosol [olive oil] Neuroprotective effect. Slowing down on ganglion retina cell counts. Decrease of retinalthickness and cellular size. Gonzalez-Correa et al. [76] 2018
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
Melatonin Reduction od retinal nitrotyrosine and malondialdehyde levels, The vasomodulatorcytokines are decreased. Ozdemir et al. [80] 2014
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
PEDF
Restoration of amplitudes of a- and b-wave of ERG; reduced retinal VEGF; reduction of
retinal 8-hydroxydeoxyguanosine, a marker of oxidative stress. inhibition of retinal vascular
hyperpermeability.
Yoshida et al. [88] 2009
Resveratrol Inhibition of nitric oxide synthase in endothelial cells. Yar et al. [89] 2012
Resveratrol Strengthening of oxidative markers (lipid peroxidation index and oxidized to reducedglutathione ratio) and superoxide dismutase activity in blood and retina. Soufi et al. [90] 2012
Resveratrol Recovers insulin level. Improve paraoxonase 1 (PON1) gen activity, reducing vascularpermeability and of VEGF, TNF- α, MPC-1, IL-6 IL-1β, INFγ levels. Chen et al. [91] 2018
Antioxidants 2020, 9, 561 9 of 23
Table 2. Cont.
Antioxidant Studied Outcomes in Treated Animals Reference
Trans resveratrol Reduces vascular lesion, NF-kB and TNF- α. Stimulates the expression of Ndf2 andSIrT1 genes. Al Hussaini et al. [92] 2018
Resveratrol coated gold nanoparticles Decrease expression of VEGF, TNF- α, MPC-1, ICAM 1, IL-6 and IL-1β. Restore balancebetween inhibitors and stimulators of angiogenesis. Dong et al. [93] 2019
Rutin Decrease of glutatione, brain-derived neurotrophic (BDNF), nerve growth factorand caspase. Ola et al. [94] 2015
Sesamin Improves blood glucose levels and body weight. Reduces ROS levels andinflammatory biomarkers. Ahmad et al. [95] 2016
Shikimic Acid (SA) (Artemisia absinthium) Reduces glucose and glycated hemoglobine levels. Decreases IL-1β and TNF- α. Al Malki et al. [96] 2019
Taurine/vitamin E+selenium Diminished conjugated dienes in retina at the early stage of diabetic retinopathy.Reduced lipid hydroperoxides. Di Leo et al. [97] 2003
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
Vitamin Prevention of blood retinal barrier breakdown. Diminution of VEGF, ICAM1, TNF- α, SOD,IL-1, IL-6 and aldose reductase. Kunisaki et al. [105] 1998
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
Alpha lipoic acid 467
No results preventing clinically significant macular
edema in T2DM. No improvement in best-corrected
visual acuity.
2 years Haritoglou et al., 2011 [107]
Antioxidant combination: Alpha lipoic
acid or Selenium or Vit. E 80
All treated groups showed decreased blood TBARS
levels and urinary albumin excretion rates. 3 months Khahler et al., 1993 [108]
Calcium Dobesilate 18 No influence on the capillary resistance ofdiabetic retinopathy 8 months Larsen et al., 1977 [109]
Calcium Dobesilate 42 No results in non-proliferative diabetic retinopathy 42 [6 months] 36 [1 year] Stamper et al., 1978 [110]
Calcium Dobesilate 50
Treated DM + glaucoma patients showed decrease of
capillary fragility, blood viscosity and microvascular
hyperpermeability.
3 months Vojnikovic et al., 1984 [111]
Calcium Dobesilate 37 Decrease of whole blood viscosity andcapillary fragility. 3 months Benarroch et al., 1985 [112]
Calcium Dobesilate [Dexium®] or
Pycnogenol®
32
Both drugs improved exudates, Dexium® only in 1
case. Both drugs, particularly Pycnogenol®, showed
improvements on parameter of automated
visual field.
6 months Leydhecker et al., 1986 [113]
Calcium Dobesilate 79
Non-insulin dependent diabetics showed reducing
of whole blood and plasma viscosity and
retinal hemorrhages.
6 months Vojnikovic, 1991 [114]
Calcium Dobesilate 137 In T2DM, better improvement than placebo onmicroaneurysms, DR level and retinal hemorrhages. 2 years Ribeiro et al., 2006 [115]
Calcium Dobesilate 635 No effects reducing development of clinicallysignificant macular edema in T2DM 5 years Haritoglou et al., 2009 [116]
Calcium Dobesilate 40
NPDR with macular edema received laser +placebo
or laser + Ca Dob. This study showed no statistically
significant difference in macular thickness between
Doxium and placebo.
6 months Feghhi et al., 2014 [117]
CAT formulation [Vitalux Forte®] 105
No effect on visual acuity. T2DM treated group show
retardation of progression and maintainance of
antioxidant plasma status level and decreased
plasmatic MDA.
5 years Garcia-Medina et al., 2011 [118]
Antioxidants 2020, 9, 561 12 of 23
Table 3. Cont.
Oral Antioxidant Studied Recruited Patients [n] Outcomes in Treated Patients Follow-Up Reference
CAT: Alpha lipoic acid
+genistein+Vitamins C, E and B. 32
Pre-retinopathic diabetics treated group showed
increases ERG oscillatory potencial values and
plasma antioxidant levels.
30 days Nebbioso et al., 2012 [119]
CAT: Coenzyme Q10, Pycnogenol®,
Vitamin E.
68 Treated T2DM: significantly reduced free oxygenradical test levels and CMT. 6 months Domanico et al., 2015 [120]
CAT: DHA, glutathione, hydroxytyrosol,
vitamins E, C, B1, B2, B3, B6, B9, B12,
lutein, zeaxanthin, Se, Mn, Zn, Cu.
208 Reduced MDA, significant increased TAS in treatedT2DM with diabetic retinopathy group. 18 months Roig Revert et al., 2015 [121]
CAT [DiVFuSS formula] 67 Better visual function. No effect on macularthickness. 6 months Chous et al., 2016 [122]
CAT or Coenzyme Q10 60 Lower ROS expression (LPO, nitrites/nitrates) andaugment antioxidant defences. 6 months
Rodriguez-Carrizalez. 2016
[123]
CAT: DHA, EPA, vitamins B, C, E
and Zeaxantin 55
RD patients with macular edema treated with
ranibizumab. Thinner macular in the
supplemented group.
3 years Lafuente et al., 2018 [124]
Crocin 60
Patients with refractory DME divided in 3 groups: 5
mg crocin, 15 mg crocin and placebo. 15 mg crocin
group showed significant reduction of HbA1c and
CMT and increase of BCVA compared with
placebo group
9 months Sepahi S et al., 2018 [125]
Ginkgo biloba extract 25
Decrease of MDA and fibrinogen levels. Improved
blood parameters (viscosity, viscoelasticity, red blood
cell deformability, and retinal blood flow).
3 months Huang et al., 2004 [126]
Lutein + zeaxanthin 90 Treated group showed improvement of visual acuity,contrast sensitivity and reduction of foveal thickness. 3 months Hu BJ et al., 2011 [127]
Lutein 31 Treated NPDR showed improvement at low spatialfrequency in contrast sensitivity. 9 months Zhang PC et al., 2017, [128]
Lutein + zeaxanthin 72
T2DM NPDR patients 36 received L and 36 received
L+ Z. No significant differences in visual acuity,
contrast sensitivity and glare sensitivity.
4 months Yongo-bo et al., 2019, [129]
Pycnogenol® 30
Treated group showed not worsening of retinal
retinal function and visual acuity. 2 months Spadea et al., 2001 [130]
Pycnogenol® 46
Improvement of visual acuity, flow at the central
retinal artery. Reduction in retinal thickness.
In T2DM.
3 months Steigerwalt et al. 2009 [131]
Antioxidants 2020, 9, 561 13 of 23
Table 3. Cont.
Oral Antioxidant Studied Recruited Patients [n] Outcomes in Treated Patients Follow-Up Reference
Resveratrol 13
T1DM showed significant decreased levels of MDA
and increased total antioxidant capacity between
baseline and endpoint. There was no change in the
serum levels of TNF- alpha and IL-1beta.
2 months Ali Movahed et al. 2020, [132]
Vitamin C 40
Vitreous levels of vitamin C in PDR patients showed
a tenfold decrease, which was associated with degree
of macular ischemia.
Park SM et al., 2019, [133]
Vitamin E 45
Diabetic patients showed decreased retinal blood
flow that improved after treatment similarly to
non-diabetic cases.
8 months Bursell et al., 1999 [134]
Vitamin E 282 Decrease of MDA. 3 months Chatziralli et al., 2017 [135]
Zinc 18
Patients with retinopathy showed increasement of
plasmatic GSH-px activity. All patients showed
decrease of TBAR.
3 months Faure et al., 1995 [136]
Zinc 45
DM patients had a negative correlation between
serum VEFG levels and Zinc. Treated group showed
no changes in VEGF levels.
3 months Kheirouri et al., 2019, [137]
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.
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