resveratrol improves diabetic retinopathy possibly · pdf fileresveratrol improves diabetic...

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


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)


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.



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


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].



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.


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