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Current Neurovascular Research, 2011, 8, 00-00 1
1567-2026/11 $58.00+.00 © 2011 Bentham Science Publishers
Nrf2 and NF-κB Modulation by Sulforaphane Counteracts Multiple
Manifestations of Diabetic Neuropathy in Rats and High Glucose-Induced
Geeta Negi, Ashutosh Kumar and Shyam S. Sharma
Molecular Neuropharmacology Laboratory, Department of Pharmacology and Toxicology, National Institute of
Pharmaceutical Education and Research (NIPER), Punjab, India
Abstract: High glucose driven reactive oxygen intermediates production and inflammatory damage are recognized
contributors of nerve dysfunction and subsequent damage in diabetic neuropathy. Sulforaphane, a known
chemotherapeutic agent holds a promise for diabetic neuropathy because of its dual antioxidant and anti-inflammatory
activities. The present study investigated the effect of sulforaphane in streptozotocin (STZ) induced diabetic neuropathy in
rats. For in vitro experiments neuro2a cells were incubated with sulforaphane in the presence of normal (5.5 mM) and
high glucose (30 mM). For in vivo studies, sulforaphane (0.5 and 1 mg/kg) was administered six weeks post diabetes
induction for two weeks. Motor nerve conduction velocity (MNCV), nerve blood flow (NBF) and pain behavior was
improved and malondialdehyde (MDA) level was reduced by sulforaphane. Antioxidant effect of sulforaphane is derived
from nuclear erythroid 2-related factor 2 (Nrf2) activation as demonstrated by increased expression of Nrf2 and
downstream targets hemeoxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO-1) in neuro2a cells and
sciatic nerve of diabetic animals. Nuclear factor-kappa B (NF-κB) inhibition seemed to be responsible for anti-
inflammatory activity of sulforaphane as there was reduction in NF-κB expression and IκB kinase (IKK) phosphorylation
along with abrogation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression and tumor
necrosis factor-α (TNF-α) and interleukine-6 (IL-6) levels. Here in this study we provide an evidence that sulforaphane is
effective in reversing the various deficits in experimental diabetic neuropathy. This study supports the defensive role of
Nrf2 in neurons under conditions of oxidative stress and also suggests that the NF-κB pathway is an important modulator
of inflammatory damage in diabetic neuropathy.
Keywords: Diabetic neuropathy, inflammation, NF-κB, Nrf2, oxidative stress, sulforaphane.
Oxidative stress and inflammation have long been consi-
dered as cornerstones of pathogenesis of diabetes and dia-
betic neuropathy. Numerous studies have correlated oxida-
tive stress to the inability of endogenous oxidant defense
machinery handle the amplified reactive oxygen species
(ROS) production. Many studies have concluded that this
disparity arises from decreased expression of Nrf2, a trans-
cription factor demonstrated to regulate the genes encoding
antioxidant proteins and phase 2 detoxifying enzymes. Nrf2
interacts with specific DNA binding domain (antioxidant
response element, ARE) to augment the expression of
antioxidant enzymes (HO-1; glutamylcysteine synthetase, γ-
GCS and thioredoxin reductase 1) and phase 2 detoxifying
enzymes (NQO1; glutathione S-transferases, GST; UDP-
glucuronyltransferases) [1-4]. Protective effects of Nrf2 acti-
vators have been reported in experimental models for various
diseases like diabetes, cerebral ischemia, cancer, neuro-
degeneration, atherosclerosis and numerous other inflamma-
tory conditions [5-9]. Nrf2 activators hold a potential for
*Address correspondence to this author at the Molecular
Neuropharmacology Laboratory, Department of Pharmacology and
Toxicology, National Institute of Pharmaceutical Education and Research
(NIPER), Sec-67, S.A.S.Nagar, Mohali, Punjab-160062, India; Tel: +91-
172-2214683-87; Fax: +91-172-2214692; E-mail:
Received: March 15, 2011 Revised: May 30, 2011 Accepted: June 27, 2011
protection of peripheral nerves from a miscellany of stresses
that originate from hyperglycemia [10].
Numerous studies have been carried out in the past few
decades on the transcription factor NF-κB and the molecular
mechanisms of its activation. NF-κB seems to play pivotal
role in acute and chronic inflammatory diseases, auto-
immune diseases and different type of cancers [11]. NF-κB
regulates the transcription of many genes like iNOS, COX,
endothelin-1 and lipoxygenase. As far as diabetes associated
metabolic changes are concerned NF-κB activation has been
well implicated [12-14]. It forms a central mechanism in the
etiology of the early neurovascular changes seen in diabetic
neuropathy. Many major pathways which are involved in the
pathophysiology of diabetic neuropathy are related to NF-κB
inflammatory cascade [15, 16].
Sulforaphane, a major isothiocyanate present in broccoli
is a potent inducer of enzymes under Nrf2 regulation.
Besides its direct effect on Nrf2, sulforaphane also regulates
inflammatory responses through its effect on NF-κB. It has
been found to protect pancreatic β -cell damage induced by
IL-1β, interferon-γ and STZ through inhibition of NF-κB and
its downstream signaling pathway [17]. The role of trans-
cription factors Nrf2 and NF-κB has been explicitly accepted
in cancer, but their involvement in the pathophysiology of
diabetes and related complications is yet to evolve. Thus, in
the present study we investigated the effect of sulforaphane
on high glucose induced changes in Nrf2 and NF-κB
signaling cascades in neuro2a cells. We further explored the
2 Current Neurovascular Research, 2011, Vol. 8, No. 4 Negi et al.
effect of Nrf2 activation and NF-κB inhibition by sulfora-
phane on STZ induced experimental diabetic neuropathy in
Sprauge Dawley rats.
Unless otherwise stated, all chemicals were of reagent
grade and were purchased from Sigma (St Louis, Missouri,
In-Vitro (Cell Culture) Studies
Experimental Protocol
Neuro2a cells (CLL-131) were obtained from National
Centre for Cell Science, Pune, India. Cells were maintained
in DMEM supplemented with 10% FBS and antibiotics
(penicillin 100 IU/ml, streptomycin 100 µg/ml) in 5% CO
C. Medium was replaced after every second day. For
differentiation, cells were switched to serum-starvation
medium (containing 0.1 % FBS). Cells were then cultured in
either low (5.5 mM) or high (30 mM) glucose DMEM
medium in the presence or the absence of the drug for 24 h.
NF-κB was immunocytochemically detected on the cells
according to the method described previously with minor
modifications [18]. Briefly, after incubation with drug,
media was aspirated and cells were washed with PBS, fixed
in ice-cold 95% ethanol for 15 min at 2C twice. Then,
cells were permeabilized in 0.2% (v/v) Triton X 100 in
phosphate buffered saline (PBS) for 15 min, washed, and
incubated with goat serum for 30 min at room temperature.
The cells were first treated with NF-κ B (p65 subunit) rabbit
polyclonal antibody, (Cell Signaling Technology,
Massachusetts, USA) at a dilution of 1:400 at 4
C for 12 and
subsequently incubated with Fluorescein isothiocyanate
(FITC)-labeled anti-rabbit IgG for 30 min at 37°C. Sections
were then mounted with mounting media containing 4',6-
diamidino-2-phenylindole (DAPI). The image analysis was
made by investigator who was unaware of the cell identity.
Preparation of Cell Lysates
Neuro2a cells were harvested in cell lysis buffer (1%
Triton X-100, 150 mM NaCl, 1 mM EDTA, 2.5 mM sodium
pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium
orthovandate, 2 mM phenylmethylsulphonyl fluoride, 1
µg/mL leupeptin, 1 µg/mL aprotinin and 20 mM Tris, pH 7.5
at 4°C for 30 min). Cell lysates were centrifuged at 16,000×g
for 15 min at C, sonicated and then stored at -80°C. The
lysate was used for western blotting as described later.
Induction of Diabetes and Experimental Design
The experiments were performed in accordance with
regulations specified by the Institute Animal Ethics Com-
mittee (IAEC), NIPER, SAS Nagar. Male Sprague Dawley
rats (250-270 g) were used and fed on standard rat diet and
water ad libitum. Diabetes was induced by single dose of
STZ (55 mg/kg, i.p.). Blood samples were collected 48 hours
after STZ administration. Rats with plasma glucose level
more than 250 mg/dl were considered as diabetics and were
further considered for the study. The experimental groups
comprised of non diabetic control rats (ND), diabetic control
rats (STZ-D) and diabetic rats treated with sulforaphane
(D+SFN 0.5 and D+SFN 1 respectively for 0.5 and 1 mg/kg,
i.p.). Each groups contained 6-8 animals. The treatment was
started 6 weeks after diabetes induction and was continued
for two weeks. The functional experiments were performed
and nerves were isolated for biochemical and protein
expression studies 24 hours after administration of last dose.
Nerve Function Studies
Motor Nerve Conduction Velocity (MNCV)
MNCV was determined in the sciatic-posterior tibial
conducting system using Power Lab 8sp system
(ADInstruments, Bellaviata, NSW, Australia) as previously
described [19]. Briefly, animals were anesthetized by 4%
halothane in a mixture of nitrous oxide and oxygen and
anesthesia was maintained with 1% halothane, using gaseous
anesthesia system (Harvard apparatus, Kent, UK). Body
temperature was monitored using a rectal probe and main-
tained with homeothermic blanket throughout the experi-
ment. Sciatic nerve was stimulated with 3V proximally at
sciatic notch and distally at ankle via bipolar electrodes.
Receiving electrodes were placed on the foot muscle. The
latencies of the compound muscle action potentials were
recorded via bipolar electrodes from the first interosseous
muscle of the hind paw and measured from the stimulus
artifact to the onset of the negative M-wave deflection.
MNCV was calculated by dividing the distance between the
stimulating and recording electrode with the difference
obtained by subtracting the distal latency from the proximal
latency and expressed in m/s.
Nerve Blood Flow (NBF)
NBF was measured using LASER Doppler system
(Perimed, Jarfalla, Sweden)[20]. Sciatic nerve was exposed
by giving incision on the left flank and the LASER Doppler
probe was applied just in contact with an area of sciatic trunk
free from epi- or perineurial blood vessels. Flux measure-
ment was obtained from the same part of nerve and for the
same time period (over a 10 minute period). The blood flow
was reported in arbitrary perfusion units (PU).
2.4. Behavioral Studies
Thermal Hyperalgesia
Thermal hyperalgesia to both hot (45ºC) and cold (10ºC)
stimuli were studied. The tail flick latency was taken as an
end point in the tail immersion test. 15 s cut-off time was
kept for both tests. Three consecutive readings were taken at
an interval of ~30 min [21].
Mechanical Hyperalgesia
Sensitivity to noxious mechanical stimuli was determined
by quantifying the withdrawal threshold of the hind paw in
response to mechanical stimulation using a von Frey anes-
thesiometer and Randall Selitto callipers (IITC Life Science,
California, USA) as described [22]. The force causing the
withdrawal response was recorded in grams. The test was
repeated four to five times at ~5-min intervals on each
animal, and the mean value was calculated.
Sulforaphane and Diabetic Neuropathy Current Neurovascular Research, 2011, Vol. 8, No. 4 3
Biochemical Parameters
Blood was collected from tail vein and plasma was
separated at 5000 rpm for 5 min at 4°C. Plasma glucose level
was estimated using GOD–POD kit from Accurex, Mumbai,
India as per manufacturer’s instructions.
For estimation of lipid peroxidation sciatic nerve was
homogenized in phosphate buffer saline (PBS, pH 7.4).
Malondialdehyde (MDA) levels were measured as per
method of Ohkawa et al. [23] with slight modification. The
reaction was initialized by adding 100 µl of plasma/buffer/
standard to a reaction mixture that consist of 750 µl of 0.8%
w/v thiobarbituric acid, 750 µl of 20% v/v acetic acid pH 3.4
and 100 µl of 10% w/v Sodium dodecyl sulphate. The mix-
ture was heated for 60 min in boiling water. Then the
contents were centrifuged and the absorbance of supernatant
was taken at 532 nm.
For TNF-α and IL-6 estimations, nerve was homogenized
in PBS containing phenylmethanesulfonylfluoride and pro-
tease inhibitor cocktail. Homogenate was kept in ice cold
water for 30 min and then sonicated. Then the homogenate
was centrifuged at 14000 rpm at 4°C. Supernatant fraction
was used for assaying TNF-α and IL-6 proteins by using
commercially available ELISA kits (eBiosciences, San
Diego, California, USA). Absorbance was taken at 450 nm.
TNF-α and IL-6 levels were expressed as pg/mg of protein
Sciatic nerves were fixed in 10 % buffered formaldehyde,
dehydrated, embedded in paraffin, and sectioned. Sections (5
µm thick) were mounted on slides, cleared, hydrated and
treated with a buffered blocking solution (3% BSA) for 15
min. Then, sections were co-incubated with primary anti-
bodies for NF-κB, p65 and COX -2 (rabbit polyclonal, Cell
Signaling Technology, Massachusetts, USA) at a dilution of
1:400 at 4
C for 12 h. Sections were washed with PBS and
incubated with secondary antibody, at room temperature for
1 hr. Thereafter, sections were washed, co-incubated with a
3,3-o-diaminobenzidine, counterstained with hematoxylin
and observed under light microscope (Leica, Solms,
Germany) [22].
Western Blotting
Protein lysates were obtained by homogenizing sciatic
nerves with lysis buffer (composition described under pre-
paration of cell lysates). Equal amounts of proteins were
separated by SDS–PAGE and transferred to a nitrocellulose
membrane (Pall Life Sciences, Florida, USA). After
blocking with 3 % bovine serum albumin, membranes were
incubated with primary rabbit polyclonal IgG NF-κB, IKKβ,
phospho- IKKβ, iNOS, HO-1 (Cell Signaling Technology,
Massachusetts, USA) (1:1000), Nrf-2, COX-2 and NQO1
(Santa Cruz Biotechnologies, California, USA) (1:400) for
12 hours at 4°C. After washing, membranes were incubated
with horse radish peroxidase/alkaline phosphatase conju-
gated secondary antibody (1:2000) and bound antibody was
visualized by enhanced chemilumniscence or by using a
colored reaction with 5-Bromo-4-chloro-3-indolyl phosphate
(BCIP) and Nitro blue tetrazolium (NBT). The relative band
densities were quantified by densitometry. Equal loading of
protein was confirmed by measuring β-actin expression [16,
Statistical Analysis
Data were expressed as mean ± SEM. For comparing the
differences between the two groups Student t-test was used.
For multiple comparisons analysis of variance (ANOVA)
was used. If ANOVA test showed significant difference post
hoc Tukey or Dunnet test was applied. Significant was
defined as p<0.05. All statistical analysis was performed
using Jandel Sigma Stat 2, statistical software (Jandel
Scientific, Erkrath, Germany).
Effect of Sulforaphane on Body Weight, Plasma Glucose
and Nerve Lipid Peroxidation Levels
Body weight of diabetic animals was significantly
(p<0.001) lower than non diabetic animals (ND: 392 ± 6 Vs
STZ-D: 209±7 g). The final body weights were similarly low
in untreated and sulforaphane-treated diabetic rats compared
with the control group (D+SFN 0.5: 215 ± 9; D+SFN 1: 219
± 9 g). Plasma glucose level was significantly (p<0.001)
higher in untreated and sulforaphane-treated diabetic rats
Fig. (1). Effect of sulforaphane on nerve functions motor nerve conduction velocity (MNCV) and nerve blood flow (NBF) in diabetic
rats. Effect of sulforaphane on nerve functions, MNCV and NBF, in treated diabetic animals was measured in sciatic-posterior tibial
conducting system using Power Lab 8sp system and LASER Doppler system respectively. Sulforaphane treatment resulted in a significant
reversal of nerve conduction and perfusion deficits suggesting its beneficial effects in diabetic neuropathy. ND: non diabetic; STZ-D:
Diabetic; D+SFN 0.5, D+SFN 1: Diabetic group treated with sulforaphane 0.5 and 1 mg/kg respectively. ND+SFN 1: Non-diabetic group
treated with sulforaphane 1 mg/kg. Results are expressed as Mean ± SEM; ### p<0.001 Vs ND; ** p<0.01 and *** p<0.001 Vs STZ-D (n=
4 Current Neurovascular Research, 2011, Vol. 8, No. 4 Negi et al.
than in non-diabetic animals (ND: 109 ± 4; STZ-D: 403 ±
12; D+SFN 0.5: 414 ± 7; D+SFN 1: 392 ± 8 mg/dl). Thus,
sulforaphane did not alter body weight or plasma glucose
levels of treated animals.
Diabetic animals showed an increased oxidative stress
which was evident from increased levels of MDA (ND: 7.3 ±
2.8 Vs STZ-D 21.9 ± 2.1 µM/mg of protein). Sulforaphane
treatment showed decreased oxidative stress as manifested
by decreased MDA levels (D + SFN 0.5: 13.3 ± 3.1 Vs D +
SFN 1: 10.3 ± 2.7 µM/mg of protein).
Effect of Sulforaphane on Nerve Function
MNCV and NBF were significantly reduced in diabetic
animals as compared to non-diabetic animals (p<0.001).
Treatment with sulforaphane produced a significant reversal
of nerve conduction and perfusion deficits at the doses of 0.5
mg/kg (p<0.01) and 1 mg/kg (p<0.001) (Fig. 1). Treatment
with sulforaphane at 1mg/kg did not affect the MNCV or
NBF in per se group.
Effect of Sulforaphane on Nociception
Diabetic animals also presented with significant reduc-
tion in tail flick latency and paw withdrawal pressure as
compared to ND animals (p<0.001). Both the doses of sulfo-
raphane, 0.5 mg/kg (p<0.01) and 1 mg/kg (p<0.001) resulted
in a significant alleviation of mechanical hyperalgesia evi-
denced by correction in paw withdrawal pressure. Thermal
hyperalgesia, however, was corrected only with higher dose
of sulforaphane (1 mg/kg) (p<0.01) (Fig. 2).
Fig. (2). Effect of sulforaphane on thermal (A) and mechanical (B) hyperalgesia in diabetic rats. Thermal hyperalgesia to both hot
(45ºC) and cold (10ºC) stimuli were studied using tail immersion test and for quantifying mechanical hyperalgesia von Frey anesthesiometer
and Randall Selitto callipers were employed. Sulforaphane resulted in a significant alleviation of mechanical hyperalgesia at both dose
levels. However, thermal hyperalgesia, was corrected only with higher dose of sulforaphane i.e. 1 mg/kg. ND: non diabetic; STZ-D:
Diabetic; D+SFN 0.5 and D+SFN 1: Diabetic group treated with sulforaphane 0.5 and 1 mg/kg respectively. Results are expressed as Mean ±
SEM; ### p<0.001 Vs ND; ** p<0.01 and *** p<0.001 Vs STZ-D (n= 6).
Fig. (3). Effect of sulforaphane on Nrf2 expression. Nrf2 protein levels were measured by Western blotting in diabetic rats sciatic nerve
(A) and in neuro2a cells (B) and equal loading was confirmed by β-Actin. After eight weeks of diabetes in rats or after 24 h incubation of
neuro2a cells with high glucose, there was a significant reduction in the Nrf2 protein levels. Both, in vitro and in vivo experiments
demonstrated that sulforaphane increased the expression of Nrf2 thereby conferring protection against high glucose led oxidative stress. ND:
non diabetic; STZ-D: Diabetic; D+SFN 0.5 and D+SFN 1: Diabetic group treated with sulforaphane 0.5 and 1 mg/kg respectively. Results
are Mean ± SEM of three independent experiments. (A) ### p<0.001 Vs ND; * p<0.05 and ** p<0.01 Vs STZ-D. (B) # p<0.05 Vs NG; *
p<0.05 and ** p<0.01 Vs HG.
Sulforaphane and Diabetic Neuropathy Current Neurovascular Research, 2011, Vol. 8, No. 4 5
Effect of Sulforaphane on Nrf2 Expression
Diabetic animals showed the decreased expression of
Nrf2 (p<0.001). Expression of Nrf2 proteins showed a
significant rise in groups treated at both dose levels (0.5 and
1 mg/kg) of sulforaphane (Fig. 3A). In vitro studies of
sulforaphane supported the findings of in vivo experiments.
Upon culturing neuro2a cells in the presence of 30 mM
glucose, pronounced decrease in Nrf2 expression was obser-
ved. Neuro2a cells were incubated for 24 h with different
concentrations of Nrf2 activator, sulforaphane. Treatment
with sulforaphane (2.5–20 µM) resulted in substantial
concentration-dependent protection against oxidative stress,
with increased expression of Nrf2 (Fig. 3B).
Effect of Sulforaphane on HO-1 and NQO1 Expression
In order to determine the downstream effect of Nrf2
activation, levels of protein under transcriptional regulation
of Nrf2 were analyzed. HO-1 and NQO1 were reduced in
diabetic nerves to significantly low levels in comparison to
normal animals (p<0.001). A significant rise in HO-1 and
NQO1 protein level was observed in diabetic animals treated
with sulforaphane 0.5 mg/kg (p<0.05) and 1 mg/kg (p<0.01)
(Fig. 4A). Neuro2a cells incubated with high glucose showed
a substantial reduction in both these proteins as compared to
cells in 5.5 mM glucose whereas cells incubated with
sulforaphane for 24 hrs in the presence of high glucose
demonstrated improved levels of HO-1 and NQO-1 (Fig.
Effect of Sulforaphane on NF-κB Expression
Nrf2 is proposed to down regulate the expression of NF-
κB, hence levels of NF-κB were determined after activating
Nrf2 with sulforaphane. Immunofluorescent analysis reveals
that increase in levels of NF-κB was induced when neuro2a
cells were incubated with 30 mM glucose which was
counteracted by sulforaphane (Fig. 5A). A similar elevation
in NF-κB was seen in sciatic nerve of untreated diabetic
animals. In-vivo treatment with sulforaphane produced a
significant dose dependant decrease in NF-κB expression
(Fig. 5B). Findings of protein expression studies were
perpetuated by immunohistochemical analysis of sciatic
nerve micro-sections where the number of NF-κB (p65
subunit) immunopositive cells was more pronounced in
diabetic rats than in control rats (p<0.001). Sulforaphane 0.5
mg/kg (p<0.05) and 1 mg/kg (p<0.001) produced a
significantly reduced NF-κB (p65 subunit) immunopositive
cells in treated diabetic rats (Fig. 5C).
Effect of Sulforaphane on IKKβ Phosphorylation
Diabetes did not produce any change in the expression of
IKKβ as diabetic and non-diabetic groups’ sciatic nerve.
Since phosphorylation is a critical step in IKKβ activation,
we determined IKKβ phosphorylation. Diabetic animals
displayed a significantly augmented expression of phospho-
rylated IKKβ (2-3 folds, p<0.001) without any effect on total
IKK protein. Treatment with sulforaphane inhibited the
phosphorylation of IKKβ indicating that sulforaphane also
has an inhibitory effect on activation of NF-κB (Fig. 6).
Fig. (4). Effect of sulforaphane on HO-1and NQO-1 expression. HO-1and NQO-1 protein levels were measured by Western blotting in
diabetic rats sciatic nerve (A) and in neuro2a cells (B) and equal loading was confirmed by β-Actin. Since HO-1and NQO-1 are under
transcriptional regulation of Nrf2, their levels were also were analyzed in diabetic condition. Similar to Nrf2, these proteins were decreased
significantly in diabetic rats and in neuro2a cells incubated in high glucose media. Treatment with sulforaphane demonstrated improved
levels of HO-1 and NQO-1 in in vitro and in vivo studies. ND: non diabetic; STZ-D: Diabetic; D+SFN 0.5 and D+SFN 1: Diabetic group
treated with sulforaphane 0.5 and 1 mg/kg respectively; NG and HG: neuro2a cells incubated in 5.5 and 30 mM glucose respectively;
NG+SFN and HG+SFN: neuro2a cells treated with sulforaphane 10 µM in the presence of 5.5 and 30 mM glucose respectively. Results are
Mean ± SEM of three independent experiments. (A) ## p<0.01 and ### p<0.001 Vs ND; * p<0.05 and ** p<0.01 Vs STZ-D. (B) ###
p<0.001 Vs NG; *** p<0.001 Vs HG.
6 Current Neurovascular Research, 2011, Vol. 8, No. 4 Negi et al.
Effect of Sulforaphane on TNF-α and IL-6 Levels
TNF-α and IL-6 play a central role in the inflammatory
response in acute and chronic inflammation. Nerve homo-
genate from diabetic animals showed 2 fold increase in TNF-
α and IL-6 levels when compared to normoglycemic animals
(Fig. 7). A significant reduction in both these parameters was
observed with sulforaphane at both doses (0.5 mg/kg and 1
Fig. (5). Effect of sulforaphane on NF-κB expression. (A) NF-κB expression in neuro2a cell was measured by immunocytochemistry Left
panel (FITC) is showing NF-κB positive cells, middle panel (DAPI) is showing total number of cells in the corresponding section and right
panel is showing superimposed images. Increased green fluorescence indicates elevated NF-κB levels in neuro2a cells incubated with 30 mM
glucose. This increase was abrogated by sulforaphane 10 µM. Next, we assessed the effect of sulforaphane on NF-κ B in diabetic rats’ sciatic
nerve by western blotting (B) as well as by immunohistochemistry (C). The photographs were taken at 40 X. The arrowheads represent NF-
κB immunopositive nuclei. Both protein expression studies and immunohistochemical analysis of sciatic nerve micro-sections revealed that
NF-κB expression was more pronounced in diabetic rats than in control rats which was brought down by treatment. Thus sulforaphane not
only mitigates the oxidative damage occurring in hyperglycaemic conditions, but also dampens the inflammatory response mediated by NF-
κB. NG and HG: neuro2a cells incubated in 5.5 and 30 mM glucose respectively; HG+SFN: neuro2a cells treated with sulforaphane 10 µM
in the presence 30 mM glucose; ND: non diabetic; STZ-D: Diabetic; D+SFN 0.5 and D+SFN 1: Diabetic group treated with sulforaphane 0.5
and 1 mg/kg respectively. Results are Mean ± SEM of three independent experiments. ### p<0.001 Vs ND; * p<0.05 and ***p<0.001Vs
Sulforaphane and Diabetic Neuropathy Current Neurovascular Research, 2011, Vol. 8, No. 4 7
Effect of Sulforaphane on iNOS and COX-2 Expression
Eight week duration of diabetes induced increase in
iNOS and COX-2 protein expression (p<0.001). This effect
was completely abrogated by sulforaphane treatment.
Sulforaphane reduced the expression of iNOS (p<0.01
D+SFN 0.5 and p<0.001 D+SFN 1 Vs STZ-D) and COX-2
(p<0.05 D+SFN 0.5 and p<0.001 D+SFN 1 Vs STZ-D),
blunting the damage caused by inflammatory stress in
diabetes (Fig. 8).
Oxidative stress and inflammation seem to occupy a
centre-stage in the development and progression of diabetic
neuropathy although there are various other acceptable
pathogenetic factors which explain for the observed deficits.
With tremendous advance in the field of molecular research,
it has surfaced that these two pathogenetic pathways are
orchestrated through transcription factors namely, Nrf2 and
NF-κB. Sulforaphane is a natural isothiocyanate abundant in
cruciferous vegetables which has shown its potential in
targeting both these transcription factors. The focal outcome
of the present study is that protective effect of sulforaphane
in diabetic neuropathy arises from induction of antioxidant
enzymes through Nrf2 driven transcription system and
reduction in the production of inflammatory mediators
through NF-κB pathway inhibition.
In the in vivo studies, STZ-treated animals developed
diabetic neuropathy illustrated by reduction in MNCV and
NBF. Moreover, diabetic animals also demonstrated altered
sensorimotor perceptions and increased oxidative stress as
characterized by increased lipid preoxidation. These markers
of experimental diabetic neuropathy closely mimic those of
diabetic neuropathy in clinical condition. Sulforaphane treat-
ment reduced the diabetes-induced neuronal dysfunction,
verified by the attenuation in the aforementioned markers of
nerve dysfunction. This indicates that sulforaphane is
effective in diabetic neuropathy, the mechanisms for which
were explored in this study.
Nrf-2 belongs to a family of transcription factors named
cap ’n collar basic region leucine zipper (CNC-bZIP) [25].
When cell is in homeostasis Nrf2 is sequestered in the
cytosol via two molecules of Kelch-like ECH associated
Fig. (6). Effect of sulforaphane on expression of phosphorylated IKK. Phosphorylation state of IKK was measured by Western blotting in
diabetic rats’ sciatic nerve and equal loading was confirmed by β-Actin. IKK is one of the key enzymes in NF-κB cascade which when
phosphorylated, activates NF-κB. We found that diabetes led to phosphorylation of IKKβ. Sulforaphane inhibited the phosphorylation of
IKKβ indicating that it acts upstream in NF-κB cascade to inhibit the activation of NF-κB. ND: non diabetic; STZ-D: Diabetic; D+SFN 0.5
and D+SFN 1: Diabetic group treated with sulforaphane 0.5 and 1 mg/kg respectively. Results are Mean ± SEM of three independent
experiments. ### p<0.001 Vs ND; * p<0.05 and ** p<0.01 Vs STZ-D.
Fig. (7). Effect of sulforaphane on TNF-α and IL-6 levels. TNF-α and IL-6 levels were measured in sciatic nerve homogenate by ELISA.
After eight weeks of diabetes there was a 2 fold increase in TNF-α and IL-6 levels in sciatic nerve homogenates. Both doses of sulforaphane
produced a significant reduction in TNF-α and IL-6 levels. This points towards the anti-inflammatory actions of sulforaphane which may be
a direct action of sulforaphane or may be mediated through NF-κB inhibition. ND: non diabetic; STZ-D: Diabetic; D+SFN 0.5 and D+SFN
1: Diabetic group treated with sulforaphane 0.5 and 1 mg/kg respectively. Results are Mean ± SEM of three independent experiments. ###
p<0.001 Vs ND; * p<0.05, ** p<0.01 and *** p<0.001 Vs STZ-D.
8 Current Neurovascular Research, 2011, Vol. 8, No. 4 Negi et al.
protein 1 (Keap1) [26-29]. However, whenever there is
excessive generation of reactive species, oxidation of cys-
teine thiol groups of Keap1 occurs and brings in confor-
mational changes resulting in the dissociation of Nrf2 from
Keap1, allowing nuclear translocation of Nrf2. Interactions
of Nrf2 with specific DNA-recognition sequence ARE result
in the increased expression of antioxidant enzymes and
phase 2 detoxifying enzymes [1-3, 30]. Sulforaphane acts on
the cytosolic complex of Nrf2-Keap1 via its isothiocyanates
moiety and disrupts this complex by modifying the cysteine
thiol residues of Keap1. This allows the nuclear translocation
of Nrf2 and induction of ARE gene transcription. It has been
reported that sulforaphane protects kidney and brain against
ischemic-reperfusion injury and abrogates ROS production
in human microvascular endothelial cells cultured in high
glucose conditions through Nrf2 [31-33]. Pertinent to pre-
vious findings, here also we found that in neuro2a cell
exposed to high glucose and sciatic nerves isolated from
diabetic animals expressed lower levels Nrf2. Sulforaphane
increased Nrf2 protein in both neuro2a cells and diabetic
sciatic nerves.
Western blot studies also revealed that sulforaphane
increased the expression of HO-1 and NQO-1 in neuro2a
cells incubated in high glucose and sciatic nerve of diabetic
animals. This correlates with increased expression of Nrf2.
HO-1 is a microsomal enzyme induced by oxidative stress
that cleaves the α-methene goup of heme generating carbon
monoxide, bilirubin, and iron. Recently considerable em-
phasis has been given to delineate the role of the HO-1 in
diabetes. In many animal models, induction of HO-1 imp-
roved insulin sensitivity and reduced adiposity [34, 35].
Another antioxidant enzyme controlled by Nrf2, NQO-1
although a metabolic detoxifying enzyme also possesses
cytoprotective and antioxidant activities [36]. In a study in
rat liver epithelial cells sulforaphane increased the trans-
criptional activation of NQO-1 [37, 38]. We found that
reduction in HO-1 and NQO-1 levels are among the conse-
quences of hyperglycemia that contribute to prevailing
oxidative stress condition in peripheral nerves. Sulforaphane
treatment normalized the levels of these enzynmes thus
affording neuroprotection in diabetic neuropathy.
Beside its antioxidant effects sulforaphane has been re-
ported to be effective in conditions related to inflammation.
A common denominator of more or less, all inflammatory
responses is NF-κB activation. NF-κB seems to play pivotal
role in chronic and acute inflammatory diseases, auto-
immune diseases, and different type of cancers [11]. In
normal condition, NF-κB is retained in the cytoplasm by an
inhibitory protein IκB Stimulation by proinflammatory
cytokines activates IKK which rapidly phosphorylates IκB
leading to its degradation which frees p65/p50 dimer [39,
40]. This free dimer can get translocated into the nucleus
where it interacts with the promoter region of the target
genes and can initiate the expression of various proinflam-
matory mediators and inducible enzymes [41]. From the
review of scientific literature and from our previous work it
is well documented that inhibition of NF-κ B contributes to
neuroprotection in diabetic neuropathy. With sulforaphane,
we showed that it efficiently blocks activation of NF-κB
both in sciatic nerve of diabetic animals. However it was
unclear that inhibition of NF-κB by sulforaphane is a direct
action on NF-κB signaling or indirectly through Nrf2/HO-1
upregulation. Previous studies findings show that HO-1
inhibits the nuclear translocation of p65 (a subunit of NF-
Fig. (8). Effect of sulforaphane on iNOS and COX-2 expression. Protein levels of iNOS and COX-2 in diabetic rats’ sciatic nerve were
measured by Western blotting and equal loading was confirmed by β-Actin. Sulforaphane reduced the expression of two inflammation
related enzymes i.e. iNOS and COX-2, thus blunting the damage caused by inflammatory stress in diabetes. ND: non diabetic; STZ-D:
Diabetic; D+SFN 0.5 and D+SFN 1: Diabetic group treated with sulforaphane 0.5 and 1 mg/kg respectively. Results are Mean ± SEM of
three independent experiments. ### p<0.001 Vs ND; * p<0.05, ** p<0.01 and *** p<0.001 Vs STZ-D.
Sulforaphane and Diabetic Neuropathy Current Neurovascular Research, 2011, Vol. 8, No. 4 9
κB). In the recent years emphasis has been laid on the poten-
tial cross-talk between Nrf2 and NF-κB signaling pathways.
Thus, whether the decrease in NF-κB level by sulforaphane
is a direct action or through HO-1, needs further investi-
gation. Moreover, phosphorylation of IKK was abrogated by
sulforaphane. Although IKK is not essential for IκB
degradation but it is indispensible for cytokine-induced
transcription of NF-κB target genes as it is required for
histone modification through phosphorylation [42, 43]. Thus
inhibiting phosphorylation of IKK prevents its activation and
thus serves to abolish the transcriptional functions of NF-κB.
Besides corroborating that sulforaphane abolished in-
creased NF-κB level, our findings also show that increase in
the levels of proinflammatory cytokines is brought to the
normal level by sulforaphane. Nuclear transport of NF-κB
results in the translation of many proteins, including TNF-α
and IL-6. These cytokines exert pleiotropic effects on peri-
pheral nerve and are essential for the maintenance of their
homeostasis as well as for nerve degeneration and regene-
ration [44, 45]. However under conditions of hyperglycemia
over production of these cytokines result in exaggerated
inflammatory response which end up in nerve damage and
neuropathy [46-48]. IL-6 and TNF- α have also been impli-
cated in low grade inflammation that underlies insulin resis-
tance and diabetes [49]. Thus sulforaphane holds promise in
preventing the development of not only diabetic complica-
tion but also diabetes and insulin resistance if employed
Outcome of various earlier studies suggest that iNOS is
NF-κB target gene that regulates inflammation and plays a
role in neurodegeneration by generating a highly reactive
peroxynitrite [50]. A study by Gilad et al. concluded that
Fig. (9). Nrf2 pathway showing the various steps involved in the activation and translocation of Nrf2. Dissociation from Keap1 is one
of the critical steps leading to liberation of Nrf2 and its interaction with DNA, resulting in the expression of the antioxidant and phases 2
detoxifying enzymes. Sulforaphane disrupts cytosolic complex of Nrf2-Keap1 allowing the nuclear translocation of Nrf2 and induction of
ARE gene transcription. It also acts on NF-κB to inhibit the production of inflammation related proteins such as TNF-α and IL-6. Thus it
combats oxidative stress by strengthening the antioxidant response and also reduces inflammatory insult in diabetic nerves.
10 Current Neurovascular Research, 2011, Vol. 8, No. 4 Negi et al.
melatonin a known Nrf2 activator, reduces iNOS mRNA
levels and protein expression which was probably through
inhibition of NF-κB by melatonin [51]. COX-2, another NF-
κB target, has been shown to be a key enzyme in nerve
injury in diabetes. Matsunaga et al. observed that intrathecal
administrations of the COX-2 inhibitors SC-58125 and NS-
398 exert anti-nociceptive effects in diabetic rats [52]. The
work of Heiss et al. and Lin et al. substantiate the key role of
iNOS and COX-2 in inflammation as they demonstrated that
sulforaphane suppressed inflammation and reduced the
expression of iNOS, COX-2 and TNF-α in macrophages [53,
54]. On western blot analysis, we found that iNOS and
COX-2 expression in sciatic nerve was significantly reduced
by sulforaphane [55]. As thought in other previous studies,
we also are in idea that decrease in the protein expression of
iNOS and COX-2 might be due to NF-κB down-regulation
by sulforaphane
In conclusion, sulforaphane emerges to provide two
neuroprotective mechanisms, i.e. activation of Nrf2 and inhi-
bition of NF-κB, which may contribute to its putative favo-
rable effects in reversing the various deficits in peripheral
nerve occurring during diabetes (Fig. 9).
This study was financially supported by the Department
of Pharmaceuticals, Ministry of Chemicals and Fertilizers,
Government of India to Dr. S.S. Sharma. Ms Geeta Negi is a
recipient of CSIR-NET research fellowship.
All the authors have no competing interests.
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Full-text available
Sulforaphane is an activator of transcription factor NF-E2-related factor-2 (nrf2) that regulates gene expression through the promoter antioxidant response element (ARE). Nrf2 regulates the transcription of a battery of protective and metabolic enzymes. The aim of this study was to assess whether activation of nrf2 by sulforaphane in human microvascular endothelial cells prevents metabolic dysfunction in hyperglycemia. Human microvascular HMEC-1 endothelial cells were incubated in low and high glucose concentrations (5 and 30 mmol/l, respectively), and activation of nrf2 was assessed by nuclear translocation. The effects of sulforaphane on multiple pathways of biochemical dysfunction, increased reactive oxygen species (ROS) formation, hexosamine pathway, protein kinase C (PKC) pathway, and increased formation of methylglyoxal were assessed. Activation of nrf2 by sulforaphane induced nuclear translocation of nrf2 and increased ARE-linked gene expression, for example, three- to fivefold increased expression of transketolase and glutathione reductase. Hyperglycemia increased the formation of ROS-an effect linked to mitochondrial dysfunction and prevented by sulforaphane. ROS formation was increased further by knockdown of nrf2 and transketolase expression. This also abolished the counteracting effect of sulforaphane, suggesting mediation by nrf2 and related increase of transketolase expression. Sulforaphane also prevented hyperglycemia-induced activation of the hexosamine and PKC pathways and prevented increased cellular accumulation and excretion of the glycating agent methylglyoxal. We conclude that activation of nrf2 may prevent biochemical dysfunction and related functional responses of endothelial cells induced by hyperglycemia in which increased expression of transketolase has a pivotal role.
Full-text available
Nrf2 regulates expression of genes containing antioxidant-respons(iv)e element (ARE) in their promoters and plays a pivotal role among all redox-sensitive transcription factors. Nrf2 is constitutively controlled by repressor protein Keap1, which acts as a molecular sensor of disturbances in cellular homeostasis. These molecular patterns are in close interconnection and function as parts of the integrated redox-sensitive signaling system Nrf2/Keap1/ARE. Depending on cellular redox balance, activity of this signaling system changes at the levels of transcription, translation, posttranslational modification, nuclear translocation of transcription factor, and its binding to ARE-driven gene promoters. This review summarizes current conceptions of Nrf2/Keap1/ARE induction and inactivation.
Although Radix clematidis has commonly been used in Chinese medicine for the treatment of arthralgia, the anti-diabetic effects of Radix clematidis have not yet been reported. In the present study, we demonstrated that Radix clematidis extract (RCE) could prevent cytokine-induced β-cell damage and streptozotocin (STZ)-induced diabetes in mice. Treatment of RINm5F insulinoma cells with interleukin-1β and interferon-y reduced cell viability; however, RCE protected the cells from this cytokine-mediated viability reduction in a concentration-dependent manner. Additionally, incubation with RCE resulted in a significant suppression of cytokine-induced nitric oxide (NO) production, which was correlated with reduced levels of the inducible form of NO synthase (iNOS) mRNA and protein. The molecular mechanism by which RCE inhibited iNOS gene expression appeared to involve inhibition of NF-κB activation. Furthermore, RCE abolished the cytokine-induced increases in NF-κB binding activity and p65 subunit levels in the nucleus, as well as IκBα degradation in the cytosol when compared to unstimulated cells. The protective effect of RCE was further demonstrated by the observed suppression of NF-KB-dependent iNOS expression and normal insulin secreting responses to glucose in cytokines-treated islets. The anti-diabetic effect of RCE was even more striking in vivo, where nearly complete protection against STZ-induced diabetes was observed. Treatment of mice with STZ resulted in hyperglycemia and hypoinsulinemia, which was further evidenced by immunohistochemical staining; however, pretreatment of mice with RCE blocked the destruction of STZ-induced islets and the development of type 1 diabetes.
The induction of phase II detoxifying enzymes is an important defense mechanism against intake of xenobiotics. While this group of enzymes is believed to be under the transcriptional control of antioxidant response elements (AREs), this contention is experimentally unconfirmed. Since the ARE resembles the binding sequence of erythroid transcription factor NF-E2, we investigated the possibility that the phase II enzyme genes might be regulated by transcription factors that also bind to the NF-E2 sequence. The expression profiles of a number of transcription factors suggest that an Nrf2/small Maf heterodimer is the most likely candidate to fulfill this rolein vivo.To directly test these questions, we disrupted the murinenrf2 genein vivo.While the expression of phase II enzymes (e.g., glutathione S-transferase and NAD(P)H: quinone oxidoreductase) was markedly induced by a phenolic antioxidantin vivoin both wild type and heterozygous mutant mice, the induction was largely eliminated in the liver and intestine of homozygousnrf2-mutant mice. Nrf2 was found to bind to the ARE with high affinity only as a heterodimer with a small Maf protein, suggesting that Nrf2/small Maf activates gene expression directly through the ARE. These results demonstrate that Nrf2 is essential for the transcriptional induction of phase II enzymes and the presence of a coordinate transcriptional regulatory mechanism for phase II enzyme genes. Thenrf2-deficient mice may prove to be a very useful model for thein vivoanalysis of chemical carcinogenesis and resistance to anti-cancer drugs.
Metabolic disorders including obesity, type 2 diabetes, and atherosclerosis have been viewed historically as lipid storage disorders brought about by overnutrition. It is now widely appreciated that chronic low-grade inflammation plays a key role in the initiation, propagation, and development of metabolic diseases. Consistent with its central role in coordinating inflammatory responses, numerous recent studies have implicated the transcription factor NF-kappaB in the development of such diseases, thereby further establishing inflammation as a critical factor in their etiology and offering hope for the development of new therapeutic approaches for their treatment.
Enhanced levels of nuclear factor (NF)-κB-inducing kinase (NIK), an upstream kinase in the NF-κB pathway, have been implicated in the pathogenesis of chronic inflammation in diabetes. We investigated whether increased levels of NIK could induce skeletal muscle insulin resistance. Six obese subjects with metabolic syndrome underwent skeletal muscle biopsies before and six months after gastric bypass surgery to quantitate NIK protein levels. L6 skeletal myotubes, transfected with NIK wild-type or NIK kinase-dead dominant negative plasmids, were treated with insulin alone or with adiponectin and insulin. Effects of NIK overexpression on insulin-stimulated glucose uptake were estimated using tritiated 2-deoxyglucose uptake. NF-κB activation (EMSA), phosphatidylinositol 3 (PI3) kinase activity, and phosphorylation of inhibitor κB kinase β and serine-threonine kinase (Akt) were measured. After weight loss, skeletal muscle NIK protein was significantly reduced in association with increased plasma adiponectin and enhanced AMP kinase phosphorylation and insulin sensitivity in obese subjects. Enhanced NIK expression in cultured L6 myotubes induced a dose-dependent decrease in insulin-stimulated glucose uptake. The decrease in insulin-stimulated glucose uptake was associated with a significant decrease in PI3 kinase activity and protein kinase B/Akt phosphorylation. Overexpression of NIK kinase-dead dominant negative did not affect insulin-stimulated glucose uptake. Adiponectin treatment inhibited NIK-induced NF-κB activation and restored insulin sensitivity by restoring PI3 kinase activation and subsequent Akt phosphorylation. These results indicate that NIK induces insulin resistance and further indicate that adiponectin exerts its insulin-sensitizing effect by suppressing NIK-induced skeletal muscle inflammation. These observations suggest that NIK could be an important therapeutic target for the treatment of insulin resistance associated with inflammation in obesity and type 2 diabetes.
Nuclear factor-kappa B (NF-κB) being reported to play an important role in the pathogenesis of diabetic neuropathy is believed to be a central mechanism involved in the genesis and promulgation of inflammatory insult. Here we have targeted the nuclear translocation of NF-κB using JSH-23 to elucidate its role in diabetic neuropathy. JSH-23 (1 and 3 mg/kg) was administered for 2 weeks in diabetic rats, after 6 weeks of diabetes induction using streptozotocin (55 mg/kg) as diabetogenic agent. Functional (motor nerve conduction velocity and blood flow), behavioural (mechanical hyperalgesia), biochemical [malondialdehyde, glutathione, tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels] and NF-κB translocation studies (western blot technique) were then undertaken. JSH-23 treatment significantly reversed the nerve conduction and nerve blood flow deficits seen in diabetic animals. Reduction in mechanical pain threshold was also partially corrected by the treatment. Protein expression studies showed that nuclear translocation of p65/p50 subunit was inhibited by JSH-23 treatment in the sciatic nerve. The treatment also lowered the elevated IL-6, TNF-α, cyclo-oxygenase (COX-2) and inducible nitric oxide synthase (iNOS) levels/expression, indicating reduction in the inflammatory damage of the sciatic nerve. Apart from these effects, JSH-23 also increased Nrf2 and hemeoxygenase-1 (HO-1) levels which could imply its potential in increasing the strength of antioxidant defence. We observed that NF-κB inhibition partially reversed functional, behavioural and biochemical deficits with JSH-23 treatment. This study substantiates the role of NF-κB activation in the aetiology of diabetic neuropathy and protection afforded by inhibition of NF-κB by JSH-23, which can be attributed to its effect on neuroinflammation and oxidative stress.
Long-standing diabetes and complications thereof particularly, neuropathy stands for one of the major causes of morbidity across the globe. It is postulated that excessive production of reactive oxygen species is a key component in the development and progression of diabetic neuropathy. Oxidative damage is the most common concluding pathway for various pathogenetic mechanisms of neuronal injury in diabetic neuropathy. However despite optimistic preclinical data, it is still very ambiguous that why antioxidants have failed to demonstrate significant neuroprotection in humans. A growing body of evidences now suggests that strategies utilizing a more targeted approach like focusing on Nrf2 (a transcription factor modulating oxidative stress) may provide an enthralling avenue to optimize neuroprotection in diabetes and diabetic neuropathy. This review presents an emerging concept of Nrf2 in diabetic neuropathy; thus looking forward to newer strategies for combating the oxidant induced damage.
Reactive oxygen species and their detrimental effects on the brain after transient ischemia have been implicated in the pathogenesis of the ischemic injury. The Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) system is currently recognized as the major cellular defense mechanism under oxidative stress, but the involvement of the Keap1-Nrf2 system in the ischemic brain injuries has not been fully investigated to date. In the present study, we investigated temporal changes of Keap1, Nrf2, and their downstream antioxidative proteins in post-ischemic mice brains with respect to spacial differences between the peri-infarct regions and the regions destined to infarct. In the peri-infarct regions, a steady level of Keap1 showed a decremental expression started at 2h of reperfusion after 60 min of transient middle cerebral artery occlusion (tMCAO). In contrast, Nrf2 began to show a significant increase at 2h with a peak at 8h of reperfusion after tMCAO. Both Keap1 and Nrf2 are mainly expressed in neuronal cells but not in glial cells. In the same peri-infarct region, downstream antioxidative proteins such as thioredoxin, glutathione, and heme oxygenase-1 showed significant increases at later time-points of 24-72 h of reperfusion after tMCAO. In the regions destined to infarct, a similar trend of expression changes to those in the peri-infarct regions was observed in Keap1, Nrf2, and 3 downstream antioxidative proteins with much less reactions. The changes found in this study suggest that the induced antioxidative stress proteins after cerebral ischemia may play an important endogenous neuroprotective response under oxidative stress after ischemic stroke.