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Resveratrol improves diabetic retinopathy
possibly through oxidative stress –
nuclear factor kB – apoptosis pathway
Farhad Ghadiri Soufi1, Daryoush Mohammad-nejad2, Hamid Ahmadieh3
1Department of Physiology, Faculty of Medicine, Hormozgan University of Medical Sciences, Bandar-Abbas, Iran
2Department of Histology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
3Ophthalmic Research Center, Labbafinejad Medical Center, Shahid Beheshti University of Medical Sciences,
Tehran, Iran
Correspondence: Hamid Ahmadieh, e-mail: [email protected]
Abstract:
Background: This study was designed to investigate the possible effectiveness of resveratrol (trans-3,5,4’-trihydroxystilbene) ad-
ministration on oxidative stress, nuclear factor kB (NF-kB) activity and apoptosis rate in streptozotocin-nicotinamide-induced dia-
betic retinopathy.
Methods: Male Wistar rats were divided into four groups: normal control, diabetic control, normal rats treated with resveratrol, and
diabetic rats treated with resveratrol. Diabetes was induced by injection of streptozotocin (50 mg/kg; ip), 15 min after the prescrip-
tion of nicotinamide (110 mg/kg; ip) in 12 h fasted rats.
Results: Four-month oral resveratrol administration (5 mg/kg/day) significantly alleviated hyperglycemia, weight loss, enhance-
ment of oxidative markers (lipid peroxidation index and oxidized to reduced glutathione ratio) and superoxide dismutase activity in
both blood and retinas of the diabetic rats. Moreover, resveratrol administration to diabetic rats improved the elevated levels of reti-
nas NF-kB activity and apoptosis rate. On the other hand, four months resveratrol administration prevented from disarrangement and
reduction in thickness of retinal layers.
Conclusion: These beneficial antidiabetic observations suggest that resveratrol may be considered as a therapeutic supplement to
prevent from diabetic retinopathy.
Key words:
diabetes, antioxidant, hyperglycemia, redox status, cell death
Abbreviations: DR – diabetic retinopathy, GSH – glutathione
(reduced), GSSG – oxidized glutathione, HbA1c – glycosy-
lated hemoglobin, NAD – nicotinamide adenine dinucleotide,
NF-kB – nuclear factor kB, NIDDM – non insulin dependent
diabetic mellitus, ROS – reactive oxygen species, SOD – su-
peroxide dismutase, STZ – streptozotocin
Introduction
Diabetic retinopathy (DR) is a common problem in
both type of diabetes and is a leading cause of ac-
Pharmacological Reports, 2012, 64, 1505�1514 1505
Pharmacological Reports2012, 64, 1505�1514ISSN 1734-1140
Copyright © 2012by Institute of PharmacologyPolish Academy of Sciences
quired blindness among the people of occupational
age [33, 44]. One of the early manifestations of DR is
persistent apoptosis of vascular and neural cells in the
retinal tissue [3, 9]. Breakdown of blood retinal bar-
rier, retinal edema, neovascularization and detach-
ment and finally loss of vision are further consequents
which occur during DR [9]. Although the precise
mechanism of DR is unclear, it widely has been ac-
cepted that hyperglycemia-induced oxidative stress,
an imbalance between production of reactive oxygen
species (ROS) and neutralization of ROS by antioxi-
dants, plays an important role in the pathogenesis of
DR [3, 15].
Diabetes-related hyperglycemia can induce oxida-
tive stress via several mechanisms including enhance-
ment in glucose oxidation, advanced glycation end
products, protein kinase C and hexosamine and polyol
pathways fluxes [44]. Peroxidation or glycation of
lipids, proteins and DNA, reduction of antioxidants
defenses and progression of tissues inflammations are
some disturbances, which are induced by oxidative
stress [16, 35]. Many of above hyperglycemia-
induced pathways converge to activate nuclear factor
kB (NF-kB), an inducible transcription factor, which
in turn contributes to further enhancement in pro-
inflammatory cytokines, oxidative stress worsening
and finally propelling to programmed cell death,
apoptosis [10, 40]. The retinal cells are more suscepti-
ble to oxidative stress relative to the any other tissue
because they are rich in membranes polyunsaturated
fatty acids and have the highest oxygen consumption
and glucose oxidation [16, 26].
During the past decades, some approaches (such as
partial and pan-retinal photocoagulation, laser therapy
and vitrectomy) have provided to diminish loss of vi-
sion. Due to the relative effectiveness and adverse
side effects of these approaches, to date, intensive
glycemic control remains as one of the most effective
options which is recommended to prevent DR [9, 16].
In spite of numerous researches and several medical
interventions, DR has remained difficult to prevent
and treat and vision loss still occurs at an alarming
rate [16, 45]. Thus, providing additional strategies
with negligible adverse effects, which would pre-
vent/improve DR, is an urgent issue.
Resveratrol (trans-3,5,4’-trihydroxystilbene), a poly-
phenolic phytoalexin found in different plants such as
grapes, peanuts and berries, has several beneficial
properties such as lifespan extending, antioxidant,
anti-inflammatory, anticancer, anticoagulant, cardio-
protective and vasoprotective effects [5, 21, 42]. In re-
gard to the central role of oxidative stress and
proinflammatory cytokines in the pathogenesis of dia-
betes, in recent years, numerous investigations have
focused on the role of resveratrol in prevention or
treatment of diabetes complications [5, 39, 42]. In this
context, it has been reported that treatment with res-
veratrol has been beneficial antidiabetic effects,
mainly via reduction in blood glucose, lipid peroxida-
tion, circulatory proinflammatory cytokines and apop-
tosis levels with concomitant enhancement of antioxi-
dants defenses [21, 29, 38, 39, 47]. On the other hand,
to date, no serious adverse side effects were reported
for long-term resveratrol treatment in healthy subjects
during in vitro and in vivo studies [6].
Retinal protective effect of resveratrol on oxygen-
and antibody-induced retinopathy [2, 12, 22], retinal
neovascularization in very low density lipoprotein
mutant mice [8], hyperglycemia-induced inflamma-
tion in pigment epithelial cells [23] and light-induced
retinal degeneration [17] have recently been reported.
There are few investigations focusing on the effect of
resveratrol on DR in which, Kim et al. have reported
that four weeks resveratrol administration (20 mg/kg)
inhibited neuronal apoptosis and elevated calcium/
calmodulin dependent protein kinase II activity, as
well as it blocked early vascular lesions and vascular
endothelial growth factor (VEGF) induction in dia-
betic mouse retina [13, 14]. Also, Khan et al. have
shown that resveratrol can inhibit pathological angio-
genesis in DR through inhibition of the proliferation
and migration of vascular endothelial cells in vitro
and in vivo [11].
Currently, resveratrol has become available in pill
forms as a dietary supplement and based on previous
studies, it seems that prescription of resveratrol is use-
ful, safe and well tolerated. Given that hyperglyce-
mia-induced oxidative stress plays a key role in the
pathogenesis of DR, we conducted the present study
to evaluate the anti-hyperglycemia and antioxidant ef-
fects of oral resveratrol ingestion on experimental
model of type 2 DR. In this study we measured the ef-
fect of resveratrol on the retinal morphology, apopto-
sis rate and NF-kB activity. For assessment of redox
state we measured the levels of 8-isoprostane, oxi-
dized to reduced glutathione ratio (GSSG/GSH), and
superoxide dismutase (SOD) activity in the retina and
blood samples. We also measured blood glycosylated
hemoglobin (HbA1C) and insulin concentrations.
1506 Pharmacological Reports, 2012, 64, 1505�1514
Materials and Methods
Experimental design
Male Wistar rats (Razi Institute, Tehran, Iran) weigh-
ing 320–350 g were housed at room temperature
(22–25°C) with 12:12-h light/dark cycles and free ac-
cess to food and water. Rats were randomly divided
into four groups (12 in each): normal control (NC),
diabetic control (DC), normal control treated with res-
veratrol (NTR), and diabetic treated with resveratrol
(DTR). The study protocol was designed in accor-
dance with NIH guidelines for the care and use of
laboratory animals and based on the method of Pal-
samy and Subramanian [31]. Diabetes was induced by
injection of STZ (50 mg/kg; ip) dissolved in 0.1 M of
citrate buffer (pH 4.5), 15 min after the administration
of nicotinamide (110 mg/kg; ip) in 12 h fasted rats.
Citrate buffer was injected alone in control rats.
Nicotinamide preserves the pancreatic b-cells (up to
40%) from STZ cytotoxicity and produces NIDDM
similar to human NIDDM [25]. To prevent from the
fatal hypoglycemic effect of pancreatic insulin re-
lease, 10% glucose solution, was provided for the rats
6 h after STZ injection for the next 24 h. After 48 h
blood glucose levels were measured using glucometer
(Arkray, Kyoto, Japan) and the rats with fasting blood
glucose levels higher than 250 mg/dl were included to
the protocol as diabetic rats. Body weight and fasting
blood glucose were measured monthly for four
months. Resveratrol treatment (5 mg/kg; minimum
dose with antihyperglycemic and non-toxic effects)
was carried out orally in aqueous solution for four
months [31]. The dosage was regulated every week.
At the end of experimental period, fasted rats were
anesthetized with ketamine (80 mg/kg) and blood
samples (5 ml/rat) were collected from retro-orbital
sinus. After cervical decapitation, the eyes were
quickly removed. The right eyes of 6 rats were har-
vested for morphological studies and were fixed in
4% paraformaldehyde and the left eyes were collected
for NF-kB activity and apoptosis rate measurements.
The right eyes of the other six rats were considered
for determination of redox status. All manipulations
were held in the morning. All above chemicals (ex-
cept resveratrol) were purchased from Sigma (St.
Louis, MO, USA). Resveratrol was obtained from
Cayman Chemicals (Ann Arbor, MI, USA).
Retinal morphologic study
Glass slides contained 4 µm sections of eyes deparaf-
finized in xylene and serially treated with 100, 96 and
70% ethanol. Slides were stained with hematoxylin
and eosin for light microscopy. Retinal thickness was
measured as the length (µm) from outer limiting
membrane to inner limiting membrane, 1 mm away
from the optic nerve on superior side [13].
Cytoplasmic and nuclear extraction
According to the manufacturer’s instructions for nu-
clear extraction kit (Cayman Chemicals, Ann Arbor,
MI, USA), each fresh isolated retina was homogenized
in 200 µl of ice-cold hypotonic buffer (10 mM NaCl,
2 mM MgCl2, 10 mM HEPES, 20% glycerol, 0.1%
Triton X-100, 1 mM dithiothreitol, 3 µl of 1 M of
10% P-40, complete protease inhibitor cocktail, pH
7.4) for 15 min, was centrifuged at 14,000 × g for
10 min at 4°C. The supernatant containing the cyto-
plasmic protein fraction was used for determination of
cell death detection, SOD activity, 8-isoprostane level
and GSSG/GSH ratio. The remaining nuclear pellet
was resuspended in 50 µl of ice-cold extract buffer
(hypotonic buffer, 39.8 µl of 5 M NaCl and 5 µl of
10 mM dithiothreitol) for 10 min and was centrifuged
at 14,000 × g for 10 min at 4°C. The supernatant con-
taining the nuclear fraction was used for quantifica-
tion of NF-kB activity. Cayman protein determination
kit (Item No. 704002) was used to quantitate protein
concentrations in cytoplasmic and nuclear extracts.
Quantification of NF-kB activity
NF-kB activity was determined using NF-kB p65
transcription factor assay kit (Cayman Chemicals,
Ann Arbor, MI, USA) according to the procedures pro-
vided by the manufacturer. Briefly, 2 µg of retinal nu-
clear extracts were incubated with an oligonucleotide
containing the NF-kB consensus site, and then were
incubated with monoclonal and secondary antibody
directed against the NF-kB p65 subunit. Reaction was
quantified at 450 nm.
Quantification of apoptosis
Cell death detection ELISA kit (1544675, Roche,
Germany) was used to quantitatively detect the cyto-
solic histone-associated DNA fragmentation, based on
Pharmacological Reports, 2012, 64, 1505�1514 1507
Resveratrol and diabetic retinopathyFarhad Ghadiri Soufi et al.
the manufacturer’s instructions. Retinal cytoplasmic
extracts (25 µl) were used as an antigen source in
a sandwich ELISA. The data were reported as an
apoptotic index (OD405/mg protein) to indicate the
level of cell death.
Estimation of redox status in retinas and blood
samples
SOD activity, GSSG/GSH ratio and 8-isoprostane
level were estimated using colorimetric assay kits
(Cayman Chemicals, Ann Arbor, MI, USA) according
to the procedures provided by the manufacturer and
were calculated from plotted standard curves at
450 nm.
Assessment of blood insulin and HbA1c
concentrations
The plasma insulin was determined with rat ELISA
kit (Cayman Chemicals, Ann Arbor, MI, USA; Cat.
No.: 589501) and HbA1c concentration was measured
spectrophotometrically by the Zistshimi lab Kit (Zist-
shimi Lab., Tehran, Iran) in accordance to manufac-
turer’s instructions.
Data analysis
Data were expressed as the mean ± SD and were ana-
lyzed by repeated measure ANOVA (for analysis of
the changes in body weights and blood glucose levels)
1508 Pharmacological Reports, 2012, 64, 1505�1514
Tab. 1. Changes in body weight, HbA1c and the levels of fasting blood glucose and insulin during the experimental period
Month(s) after induction of diabetes
1 2 3 4
Body weight (g)
NC 350.17 ± 3.11 359.11 ± 4.92 367.32 ± 4.77 376.41 ± 5.61
DC 280.41 ± 5.19* 263.82 ± 5.56* 248.39 ± 2.91* 229.91 ± 4.76*
NTR 333.18 ± 3.41 339.21 ± 4.03 347.19 ± 2.71 352.63 ± 6.08
DTR 303.08 ± 5.13*## 289.72 ± 7.44*# 277.26 ± 4.48*# 272.59 ± 8.19*#
Blood glucose (mg/dl)
NC 98.18 ± 3.49 96.21 ± 5.18 101.43 ± 4.70 104.29 ± 3.44
DC 285.51 ± 4.91* 313.93 ± 4.13* 346.61 ± 3.98* 421.58 ± 5.01*
NTR 99.26 ± 4.51 101.19 ± 3.87 98.59 ± 4.66 97.91 ± 4.96
DTR 280.92 ± 5.17*# 283.32 ± 4.83*# 303.87 ± 3.07*# 356.17 ± 5.29*#
HbA1c (% Hb)
NC – – – 7.46 ± 0.12
DC – – – 16.61 ± 0.20*
NTR – – – 8.71 ± 0.17
DTR – – – 12.17 ± 0.14*#
Insulin (ng/ml)
NC – – – 16.43 ± 0.19
DC – – – 6.76 ± 0.86*
NTR – – – 15.91 ± 0.55
DTR – – – 8.02 ± 0.37*
The values represent the mean ± SD of 6 animals per group; * p < 0.01 vs. normal control group (NC), # p < 0.01 vs. diabetic control group(DC). NTR – normal rats treated with resveratrol, and DTR – diabetic rats treated with resveratrol
and one-way ANOVA (for other parameters), using
SPSS 18 software. When a significant p-value was
obtained, the Tukey post-hoc test was employed to
determine the differences between groups. A level of
p < 0.05 was considered statistically significant.
Results
Body weight, glucose, HbA1c and insulin
concentrations
The changes of body weights during the experimental
period are presented in Table 1. Although significant
weight loss occurred in both diabetic groups (p < 0.01
for both), but this weight loss in DRT group was
lower than DC group (p < 0.01). We did not see sig-
nificant changes in body weights between NTR and
NC groups.
In comparison to the NC group, blood glucose con-
centration progressively increased during the protocol
period in DC and DRT groups (p < 0.01 for both);
however, its level in DRT group was significantly
lower than DC group (p < 0.05). Four-month treat-
ments with resveratrol have not statistically signifi-
cant effect on the nondiabetic rats blood glucose level
(Tab. 1).
Insulin concentration in both diabetic groups (DC
and DRT) was significantly lower than NC group (p <
0.01 for both); and resveratrol treatment did not
change statistically their levels in diabetic and nondia-
betic rats (Tab. 1).
The level of HbA1c was higher in both diabetic
groups (p < 0.01 for both) when compared with NC
group (Tab. 1). Four-month treatments with resvera-
trol statistically reduced HbA1c level in DRT group
when compared with DC group (p < 0.01).
Redox status
Table 2 represents the effect of chronic resveratrol
treatment on the levels of retinas and blood oxidative
stress markers and SOD activity. In comparison to the
normal controls, treatment with resveratrol enhanced
blood SOD activity (p < 0.05) and reduced blood
8-isoprostane level in NTR group (p < 0.01), while it
has no effect on retinal tissue of NTR group. We ob-
served reduced SOD activity with concomitant
enhancement in GSSG/GSH ratio and the level of
8-isoprostane in the blood of both diabetic groups
(p < 0.01 for all comparisons) and increase in
GSSG/GSH ratio and 8-isoprostane level in the reti-
nas of DC group (p < 0.05 for all comparisons). These
changes were markedly attenuated by chronic res-
veratrol administration.
NF-kB activity and apoptosis rate
Figure 1 depicts that the NF-kB activity and apoptosis
rate significantly increased in the retinas of DC and
DTR groups as compared with normal groups (p <
0.01 for DC group and p < 0.05 for DTR group com-
parisons). Treatment with resveratrol reduced these
enhancements statistically (p < 0.05 for both). There
was no significant difference in NF-kB activity or
apoptosis rate between NTR and NC groups.
Pharmacological Reports, 2012, 64, 1505�1514 1509
Resveratrol and diabetic retinopathyFarhad Ghadiri Soufi et al.
Tab. 2. The effects of chronic resveratrol treatment on the levels of retinas and blood oxidative stress markers and SOD activity
Blood Retina
SOD(U/ml)
8-Isoprostane(ng/ml)
GSSG/GSH(%)
SOD(U/mg protein)
8-Isoprostane(ng/mg protein)
GSSG/GSH(%)
NC 3.97 ± 0.32 0.947 ± 0.03 0.33 ± 0.05 6.76 ± 0.51 2.624 ± 0.09 0.19 ± 0.03
DC 1.51 ± 0.38* 1.433 ± 0.02* 0.69 ± 0.04* 5.09 ± 0.44** 1.574 ± 0.07* 0.47 ± 0.03*
NTR 5.89 ± 0.56* 0.817 ± 0.01* 0.38 ± 0.04 7.08 ± 0.39 2.449 ± 0.11 0.20 ± 0.04
DTR 2.73 ± 0.29*# 1.098 ± 0.03*# 0.51 ± 0.05*# 6.32 ± 0.58## 1.924 ± 0.05*## 0.34 ± 0.05**##
The values represent the mean ± SD of 6 animals per group; * p < 0.01, ** p < 0.05 vs. normal control group (NC), # p < 0.01, ##p < 0.05 vs. dia-betic control group (DC). NTR – normal rats treated with resveratrol, and DTR – diabetic rats treated with resveratrol
Retinal morphology
In hematoxylin and eosin staining, retinas of NC and
NTR groups were highly organized, with intact layers
(Fig. 2a and 2b). The retinas of DC group were disor-
ganized with impaired layers (Fig. 2c). Treatment
with resveratrol improved layer disorganization as
compared with DC group (Fig. 2d).
In comparison to NC group, retinal thickness was
significantly reduced (by 17.6%) four months after
diabetes induction (p < 0.01) and resveratrol treat-
ment attenuated this impairment by 8.3% (p < 0.05;
Fig. 2e).
Discussion
Alkylating property of STZ (1-methyl-1-nitrosourea
derivative) causes DNA strand breaks, activation of
poly-ADP-ribose synthtetase, depletion of nicotina-
mide adenine dinucleotide (NAD) and finally b cell
necrosis in many animal models [31]. Prescription of
nicotinamide (a poly-ADP-ribose synthtetase inhibi-
tor) preserves the pancreatic islets (up to 40%) from
STZ cytotoxicity by protecting the decrease in the
levels of NAD and produces a model of diabetes simi-
lar to human NIDDM [31]. Hence, in the present
study, STZ-nicotinamide-induced diabetes has been
used as the animal model of type 2 diabetes.
Insufficient secretion or action of insulin causes
hyperglycemia, mainly via an enhanced release of
glucose by the liver and reduced utilization of glucose
in peripheral tissues. In this situation, the body has to
provide itself energy by degradation of proteins and
lipids from their reservoirs, which ultimately accounts
for weight loss [29, 36]. Treatment with resveratrol
attenuated weight loss process hyperglycemia in our
1510 Pharmacological Reports, 2012, 64, 1505�1514
Fig. 2. Presentation of retinal morphology and thickness in rats 4months after induction of diabetes and resveratrol treatment. The reti-nas of NC and NTR groups (a and b, respectively) were highly organ-ized, with intact layers. The retinas of DC group were disorganizedwith impaired layers (c). Four months treatment with resveratrol im-proved layer disorganization (d). Retinal thickness significantly re-duced by 17.6% four months after diabetes induction and resveratroltreatment attenuated this impairment by 8.3% (e). The values repre-sent the mean ± SD of 6 animals per group. Scale bar, 10 µm. For ab-breviations and symbols see Table 2
Fig. 1. Effect of four months resveratrol administration on the retinalNF-kB activity (a) and apoptosis rate (b) in diabetic rats. The valuesrepresent the mean ± SD of 6 animals per group. For abbreviationsand symbols see Table 2
diabetic rats, suggesting possible improvement in energy
metabolism. This observation is in agreement with previ-
ously published literature [29, 31]. Our results showed
that long-term treatment with resveratrol reduces
diabetes-induced hyperglycemia and HbA1c level with-
out any enhancement in blood insulin concentration.
One of the mechanisms involved in the pathogene-
sis of diabetes complications is oxidation of glucose
[24]. Glucose oxidation enhances glycation of some
proteins such as hemoglobin (and produces HbA1c)
and antioxidant enzymes which in turn, can reduce
their activities for detoxification of ROS and lead to
lipids, proteins and DNA peroxidation and finally
programmed cell death [35]. The concentration of
HbA1c is considered as a good marker for diagnosis
and prognosis of diabetes complications. There is
strong correlation between HbA1c and risk of diabetic
retinopathy, nephropathy and neuropathy [7]. It has
been reported that reduction of HbA1c by only 1 unit
(8 to 7%) can reduce the risk of retinopathy by over
30% [16]. Reduction of HbA1c level in diabetic rats
after 4-month resveratrol supplementation is in line
with previous studies and indicates that resveratrol
has beneficial effects in attenuation of diabetic com-
plications, especially DR.
Unchanged insulin concentration after 4-month res-
veratrol prescription suggests that the beneficial effects
of resveratrol may be resulted from reducing the blood
glucose release by the liver [4, 30, 32] or enhancing
glucose utilization by the tissues through potentiation
of insulin signaling [34] or its direct action [41]. This
proposed mechanism has been depicted in Figure 3.
8-Isoprostane (8-iso-prostaglandin F2a), a member
of eicosanoids family produced by the oxidation of
tissue phospholipids by oxygen radicals, has been
proposed as a marker of oxidative stress and antioxi-
dant deficiency [27]. It has been shown that plasma
concentration of 8-isoprostane increases with dia-
betes-induced lipid peroxidation and oxidative stress
[28, 37]. Moreover, Kimura et al. have shown that
retinal 8-isoprostane level increased during hypoxia-
induced retinopathy [15]. Reducing of 8-isoprostane
concentrations in blood and retinal tissue of the nor-
mal and diabetic rats after the 4-month period of res-
veratrol treatment shows that resveratrol has a strong
antioxidant effect and attenuates oxidative stress.
Under normal conditions, moderate amounts of re-
active oxygen species (ROS) including superoxide
(O2��), hydrogen peroxide (H2O2), and hydroxyl radi-
cal (OH�), which mainly are produced by mitochon-
dria and NAD(PH) oxidase, contribute in some phy-
siological processes. Dismutation of O2�� (the most
abundant reactive oxygen radical producing in the
cells) to H2O2 by SOD is the first step in detoxifica-
tion of ROS. Then, H2O2 is metabolized into water by
the activities of catalase and glutathione peroxidase.
Moreover, GSH, a co-substrate for glutathione peroxi-
dase activity, is another major intra- and extracellular
antioxidant molecule and acts as a direct free radical
scavenger by conversion to GSSG. GSSG to GSH ra-
tio is a good marker for estimation of redox state [16,
29, 44]. Overproduction of ROS or reduction in the
ability of the endogenous antioxidants to neutralize
ROS, results in oxidative stress which in turn, can
lead to damages of lipids, proteins and DNA. This
situation is traceable by measurement of lipid peroxi-
dation, protein oxidation and DNA fragmentation or
cell death [3, 16, 26, 44].
Pharmacological Reports, 2012, 64, 1505�1514 1511
Resveratrol and diabetic retinopathyFarhad Ghadiri Soufi et al.
Fig. 3. A proposed schematic diagram for the mechanisms of thehyperglycemia-induced retinal damage and the alleviating effects ofresveratrol. AGEs: advanced glycation end products, PKC: proteinkinase C
It has been reported that chronic hyperglycemia re-
sults in oxidative stress either by the direct generation
of ROS or by altering the redox balance via increased
polyol pathway flux (which reduces NADPH reser-
voirs and reduces conversion of GSSG to GSH), in-
creased formation of advanced glycation end prod-
ucts, activation of protein kinase C and overproduc-
tion of superoxide by the mitochondrial electron
transport chain [16, 35, 44]. In this context, our data
are completely in agreement with the previous studies,
in which our diabetic control rats experienced chronic
hyperglycemia with enhancement in oxidative stress
markers (including lipid peroxidation and GSSG to
GSH ratio) with concomitant reduction in SOD activ-
ity in both blood and retina.
Many of above mentioned hyperglycemia-induced
pathways converge to elevate NF-kB, a proinflamma-
tory master switch, which activates proinflammatory
cytokines gene expressions and apoptosis cascade
[10, 29]. Our data also are in line with previous stud-
ies, in which the retinal NF-kB activity and apoptosis
rates in DC and DTR groups were significantly higher
than normal controls [3, 10].
Among beneficial antidiabetic actions of resvera-
trol, its antioxidant effect seems to be best docu-
mented. A variety of animal and human studies have
shown that resveratrol administration for diabetic sub-
jects, could enhance the antioxidant defense, reduce
lipid and protein oxidation and decrease apoptosis
rate [13, 19, 29, 40]. The antioxidant property of res-
veratrol may be accomplished directly by modulating
the antioxidants gene expression and its free radical
quenching action or indirectly by reducing of hyper-
glycemia (summarized pathways have been presented
in Fig. 3) [1, 36, 46]. It has been documented that
short-term treatment with resveratrol in diabetic sub-
jects has inhibited the activation of NF-kB at tran-
scriptional or post-transcriptional levels [20, 47]. In
this regard, Kubota et al. have previously depicted
that resveratrol attenuates the inflammatory process
through reducing both oxidative damage and NF-kB
activity [18]. Our data are in accordance with the re-
sults of these papers in which 4-months oral resvera-
trol prescription increased SOD activity, reduced oxi-
dative stress markers (GSSG/GSH ratio and lipid per-
oxidation) and NF-kB activity and finally decreased
tissues apoptosis rates.
It has been reported that retinal damage could re-
sult from a decrease in retinal thickness, and struc-
tural disarrangement in diabetes might contribute to
the progression of DR, leading to vision loss [13, 43].
Prevention from structural disorganization of the ret-
ina in diabetic rats, which was observed by resveratrol
administration in this study, may be another certificate
to support the beneficial potential of resveratrol in
preventing DR. This result is in line with the results
obtained by Kim et al., which recently reported that
4-week resveratrol administration (20 mg/kg) blocked
vessel leakage and early vascular lesions in diabetic
mouse retina [14]. Furthermore, Khan et al. have
shown that resveratrol prevents from retinal patho-
logical neovascularization and eliminates abnormal
blood vessels that already had begun to develop [11].
In conclusion, our results depict that chronic res-
veratrol administration has an anti-hyperglycemic ef-
fect. Moreover, it reduces antioxidant machinery im-
pairment and reverts back the activation of NF-kB
and the rate of apoptosis in the retinas of diabetic rats.
Since good glycemic control can reduce development
of DR, and given a key role of oxidative stress in the
progression of DR, the observed antidiabetic proper-
ties of resveratrol make it candidate as a therapeutic
supplement to reduce diabetic-related complications.
Acknowledgments:
The grant for this study was provided by cooperation of
Ophthalmology Research Center, Shahid Beheshti University
of Medical Sciences, Tehran, Iran (60%) and Faculty of Medicine,
Tabriz University of Medical Sciences, Tabriz, Iran (40%).
Conflict of interest:
The authors have declared that there is no conflict of interest.
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Received: January 21, 2012; in the revised form: July 28, 2012;
accepted: August 13, 2012.
1514 Pharmacological Reports, 2012, 64, 1505�1514