European Journal of Pharmacology 771 (2016) 84–92

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European Journal of Pharmacology

journal homepage: www.elsevier.com/locate/ejphar

Neuropharmacology and analgesia

Rutin ameliorates diabetic neuropathy by lowering plasma glucose and

decreasing oxidative stress via Nrf2 signaling pathway in rats
Ruifeng Tian a,1, Wenqing Yang b,1, Qiang Xue c,1, Liang Gao d, Junli Huo f, Dongqing Ren e,n,
Xiaoyan Chen f,n
a
Department of Infectious Disease, Xijing Hospital, The Fourth Military Medical University, Xi'an 710032, China
b
Lin-tong Sanitarium of Land Force, Lanzhou Military Region of PLA, Xi'an 710600, China
c
Department of Cardiology, Xijing Hospital, The Fourth Military Medical University, Xi'an 710032, China
d
Department of Ultrasonography, Xijing Hospital, The Fourth Military Medical University, Xi'an 710032, China
e
Department of Radiation Medicine and the Ministry of Education Key Lab of Hazard Assessment and Control in Special Operational Environment, School of
Public Health, Fourth Military Medical University, Xi'an, Shaanxi 710032, China
f
Department of Neurosurgery, Xijing Hospital, The Fourth Military Medical University, Xi'an 710032, China

art ic l e i nf o a b s t r a c t

Article history: Rutin exhibits antidiabetic, antioxidant and anti-inflammatory properties, which makes rutin an at-
Received 8 June 2015 tractive candidate for diabetic complications. The present study was designed to investigate the potential
Received in revised form effect of rutin on diabetic neuropathy. After induction of diabetic neuropathy, rutin (5 mg/kg, 25 mg/kg
8 November 2015
and 50 mg/kg) were daily given to the diabetic rats for 2 weeks. At the end of rutin administration, rutin
Accepted 9 December 2015
produced a significant inhibition of mechanical hyperalgesia, thermal hyperalgesia and cold allodynia, as
Available online 10 December 2015
well as partial restoration of nerve conduction velocities in diabetic rats. Furthermore, rutin significantly
Keywords: increased Na þ , K þ -ATPase activities in sciatic nerves and decreased caspase-3 expression in dorsal root
Rutin ganglions (DRG). In addition, rutin significantly decreased plasma glucose, attenuated oxidative stress
Diabetic neuropathy
and neuroinflammation. Further studies showed that rutin significantly increased hydrogen sulfide (H2S)
Oxidative stress
level, up-regulated the expression of nuclear factor-E2-related factor-2 (Nrf2) and heme oxygenase-1
Hydrogen sulfide
Nrf2 (HO-1) in DRG. The evidences suggest the beneficial effect of rutin on diabetic neuropathy. Additionally,
Neuroinflammation insulin (2 IU) and BG-12 (15 mg/kg) were used to investigate the mechanisms underlying the beneficial
effect of rutin on diabetic neuropathy. Insulin achieved lower plasma glucose and BG-12 achieved
comparable Nrf2 expression than/to rutin (50 mg/kg), respectively. In contrast, the beneficial effect of
insulin and BG-12 was inferior to that of rutin (50 mg/kg), suggesting that both lowered plasma glucose
and Nrf2 signaling contribute to the beneficial effect of rutin on diabetic neuropathy. In conclusion, rutin
produces significant protection in diabetic neuropathy, which makes it an attractive candidate for the
treatment of diabetic neuropathy.
& 2015 Elsevier B.V. All rights reserved.

1. Introduction
Diabetes mellitus is a serious global health problem and its
prevalence is estimated to be 366 million worldwide by the year of
2025 (Wild et al., 2004). Diabetic neuropathy affects more than
50% of diabetic patients and is a major cause of disability. The
patients with diabetic neuropathy often suffer from severe pain,
hyperalgesia and allodynia, which can be disabling and devastat-
ing (Didangelos et al., 2014). Thus far, no approved therapy has
been proposed for diabetic neuropathy. Therefore, it is imperative
n
Corresponding authors.
E-mail addresses: dongqingren@163.com (D. Ren),
xiaoyanchenfmmu@163.com (X. Chen).
1
These authors contributed equally to this work.
to look for effective drugs for the management of painful diabetic
neuropathy.
Chronic hyperglycemia is known to activate both oxidative
stress and inflammatory pathways, which are two key factors
contributing to nerve tissue damage and leading to the neuro-
pathic pain in diabetes mellitus (Sandireddy et al., 2014). Hy-
perglycemia results in the production of reactive oxygen species
(ROS, a molecular signature of oxidative stress) by directly acti-
vating the polyol pathway, protein kinase C (PKC) pathway, for-
mation of advanced glycation end products (AGE) pathway, and
hexosamine pathway etc (Brownlee 2001; Sandireddy et al., 2014).
The oxidative stress results in oxidative damage to dorsal root
ganglion (DRG) neurons and peripheral nerves, which has been
recognized as one of the vital mechanisms in the pathogenesis of
painful diabetic neuropath (Sandireddy et al., 2014). In addition,

http://dx.doi.org/10.1016/j.ejphar.2015.12.021
0014-2999/& 2015 Elsevier B.V. All rights reserved.
 R. Tian et al. / European Journal of Pharmacology 771 (2016) 84–92
persistent hyperglycemia is able to initiate neuroinflammation,
leading to the neuropathic pain in diabetes mellitus. The hy-
perglycemia-associated classical pathways like polyol pathway,
PKC pathway, AGE pathway could directly or indirectly initiate and
progress the inflammatory process in nerve tissues (Sandireddy
et al., 2014; Singh et al., 2014 ). In addition, oxidative stress and
these classical pathways in combination activate transcription
factors such as nuclear factor kappa enhancer of B cells (NF-κB),
resulting in neuroinflammation, which contributes to nerve da-
mage in painful diabetic neuropathy (Sandireddy et al., 2014). Thus
far, oxidative stress and neuroinflammation are identified as pi-
votal pathophysiological triggers in diabetic neuropathy. Those
two factors interact at multiple levels, which complicate the hy-
perglycemia-mediated neuronal damage. Oxidative stress has
been shown to activate NF-κB to initiate inflammation (Poli et al.,
2004). In turn, NF-κB activation could suppress the expression of
antioxidant genes and thus indirectly weakening the innate anti-
oxidant defense (Ganesh Yerra et al., 2013). The cross-talk between
oxidative stress and neuroinflammation made us realized that
pharmacological targeting both oxidative stress and neuroin-
flammation simultaneously might produce a better therapeutic
response than targeting them individually.
Rutin (3, 30, 40, 5, 7-pentahydroxyflavone-3-rhamnoglucoside)
is one of the most common native flavonoids present in foods
including onions, apples, tea and red wine (Hosseinzadeh and
Nassiri-Asl, 2014). Rutin exhibits multiple pharmacological activ-
ities including antidiabetic (Kamalakkannan and Prince, 2006;
Kamalakkannan and Stanley Mainzen Prince, 2006), antioxidant
(Kamalakkannan and Stanley Mainzen Prince, 2006), and anti-in-
flammatory (Koda et al., 2009; Mascaraque et al., 2014) in different
models of rodents. It has been shown that rutin, by its ability to
scavenge free radicals and to inhibit lipid peroxidation, prevents
streptozotocin (STZ)-induced oxidative damage and protects pan-
creatic β cells to increase insulin secretion and decrease blood
glucose levels (Kamalakkannan and Prince, 2006; Kamalakkannan
and Stanley Mainzen Prince, 2006). In addition, rutin is capable of
increasing enzymic and non-enzymic antioxidants in liver, kidney
and brain in STZ-induced experimental diabetes, suggesting its
antioxidant capability in diabetes mellitus (Kamalakkannan and
Stanley Mainzen Prince, 2006). Further studies showed that rutin
is capable of inhibiting oxaliplatin-induced painful peripheral
neuropathy by partially attenuating oxidative stress-induced da-
mage in dorsal horn neurons (Azevedo et al., 2013). Those evi-
dences showed the potent antioxidant activities of rutin, raising
the possibility that rutin might attenuate hyperglycemia-induced
oxidative damage to neurons and nerve tissues in diabetic neu-
ropathy. In addition, rutin has anti-inflammatory activity, which is
evidenced by its ability in suppressing the microglia activation and
release of pro-inflammatory cytokines (Koda et al., 2009). The
antioxidant and anti-inflammatory activities of rutin make it an
attractive candidate for diabetic neuropathy. However, the role of
rutin in diabetic neuropathy has not been investigated thus far.
The present study was designed to investigate the potential ther-
apeutic effect of rutin on experimental diabetic neuropathy and to
explore its underlying mechanism.
2. Materials and methods
2.1. Animal model of painful peripheral diabetic neuropathy
All experimental procedures were performed under a protocol
which was approved by the Institutional Ethical Committee of
Fourth Military Medical University. Adult male Sprague–Dawley
rats (Laboratory Animal Center of the Fourth Military Medical
University, Xi'an, China), weighing from 200 g to 240 g, were used
85
for the experiments. Streptozotocin (STZ, Sigma, St. Louis, MO,
USA) was intraperitoneally injected (55 mg/kg) to induce painful
diabetic neuropathy (n ¼72). Normal control rats (n ¼12) received
the same volume/kg saline. Three weeks after administration,
Plasma glucose was determined using a commercial glucose kit
(Sigma-Aldrich, St. Louis, MO, USA), and rats with plasma glucose
levels above 13.89 mmol/L (250 mg/dl) were considered as dia-
betic. In addition, Neuropathy with hyperalgesia and allodynia was
found in STZ-treated animals at 3 weeks after STZ (Messinger
et al., 2009). Then the STZ-treated rats were randomly divided into
6 groups (n ¼12 in each group), STZ control group, low-dose rutin
(Sigma Chemical Co., St. Louis, MO, USA) group (5 mg/kg), mid-
dose rutin group (25 mg/kg), high-dose rutin group (50 mg/kg),
insulin replacement group, BG-12 (dimethylfumarate, tecfidera, a
potent Nrf-2 activator) group. Three weeks after STZ injection,
rutin was dissolved in 3 ml of saline, which was daily injected
intraperitoneally for 2 weeks. For the normal and STZ-treated
control groups, the animals received daily normal saline (3 ml) for
2 weeks. For insulin replacement group, insulin of 2 international
units [IU] was daily injected for 2 weeks, which can reliable low-
ered blood glucose. For BG-12 group, BG-12 at 15 mg/kg was dis-
solved in 3 ml of saline, which was daily injected intraperitoneally
for 2 weeks. At the end of drug administrations, behavioral, elec-
trophysiological, biochemical and protein analysis was performed.
Plasma glucose was assayed using commercial kits (Sigma-Aldrich,
St. Louis, MO, USA).
2.2. Behavioral assessments
The behavioral assessments were performed before STZ treat-
ment, every 3 days after STZ treatment and 2 weeks after drug
treatment. In addition, the effect of rutin on mechanical, heat and
cold sensation was recorded at 0, 2, 4, 6, 8, 10 and 12 h after final
rutin administration.
2.2.1. Mechanical sensitivity
The mechanical sensitivity was measured using von Frey fila-
ments (Vogelaar et al., 2004). Briefly, the animals were placed in a
cage with wire-mesh-bottom. A von Frey filament (diameter:
0.5 mm, bending force: 13.5 g), which has been shown to elicit
nociceptive responses (Messinger et al., 2009), was applied 10
times to the foot pads in the plantar aspect of the hind paw and
the number of positive responses was recorded. Care was taken
not to stimulate the same point twice in succession. Abrupt paw
withdrawal, licking and shaking were taken to be positive re-
sponses. A significant increase in the frequency of foot with-
drawals in response to mechanical stimulation was interpreted as
mechanical hyperalgesia.
2.2.2. Heat nociception
The heat nociception was measured according to the procedure
described in a previous study (Hargreaves et al., 1988). Briefly, a
paw thermal stimulation system was used to measure the noci-
ceptive response to heat. A radiant heat source was used to sti-
mulate the plantar side of the hind paw (46–48 °C). Then the paw
withdrawal latency in seconds was recorded when the rats with-
draw the paw. If the animals donot withdraw the paw for 20 s, the
heat stimulation would be stopped to prevent thermal injury. All
the tests were repeated for 3 times at intervals of 5 min between
each application.
2.2.3. Cold allodynia
The cold allodynia was assessed according to the procedure as
described previously (Sayyed et al., 2006). Briefly, to apply cold
stimulation, rats were placed on a cold stainless steel plate (model
35100, Ugo Basile) maintained at 10 °C. The latency (s) to the first
86 R. Tian et al. / European Journal of Pharmacology 771 (2016) 84–92
lifting or shaking of the hind paws was measured. If the animals
donot withdraw the paw for 30 s, the cold stimulation would be
stopped to prevent tissue injury. The cold stimulation was re-
peated twice at intervals of 5 min between each application.
2.3. Sciatic nerve conduction velocities
After behavioral assessments, the animals were anesthetized
by intraperitoneal injection of 1.0 wt% sodium pentobarbital so-
lution (40 mg/kg body weight). The motor or sensory nerve con-
duction velocities were recorded as described previously (Sayyed
et al., 2006). Immediately after electrophysiological analysis, the
sciatic nerves and dorsal root ganglions (DRG, L4-L6) of the ani-
mals were harvested and stored at  80 °C for further
investigations.
2.4. Na þ , K þ -ATPase activity
Crude homogenate was prepared by immersing sciatic nerves
in prechilled solution of 10 mM Tris–HCl (pH 7.5). The Na þ ,
K þ -ATPase activity in sciatic nerves was measured using a coupled
enzymatic method as previously described (Liu, et al., 2010). The
activity of Na þ , K þ -ATPase was expressed as micromole of NADH
oxidized per hour, which was normalized to the protein content of
the membrane-enriched fraction.
2.5. Measurement of reactive oxygen species (ROS)
The ROS was measured in sciatic nerve tissue homogenates as
described previously (Padiya et al., 2014). In brief, 100 μM of 2,7-
dichlorofluorescein diacetate and the nerve homogenate was in-
cubated at room temperature in dark for 30 min. The volume was
adjusted with phosphate buffer saline (0.1 M, pH 7.4) and the
fluorescence was detected by excitation wavelength at 488 nm and
emission wavelength at 525 nm. The data were expressed as re-
lative fluorescence units.
2.6. Oxidative stress
Oxidative stress was evaluated by assessing the contents of
malondialdehyde (MDA), superoxide-dismutase (SOD), catalase,
glutathione peroxidase (GPx) and glutathione-S-transferase (GST)
in sciatic nerves. The levels of MDA, catalase, GPx, GST and SOD
were measure by standard biomechanical methods according the
standard sassy kits (Sigma-Aldrich, St. Louis, MO, USA). The results
were expressed as μmol/mg protein and U/mg protein,
respectively.
2.7. Hydrogen sulfide (H2S) measurement
The concentration of H2S in DRG neurons was measured as
described previously (Padiya et al., 2014). In brief, 0.1 ml of DRG
supernatant was added into a tube containing 0.125 ml of zinc
acetate (1.0%) and 0.15 ml of double distilled water. Subsequently,
0.067 ml of N,N-dimethyl-phenylene diamine dihydrochloride
(20 mM) in 7.2 M HCl was then added. Then 0.067 ml of FeCl3
(30 mM) was added to the tube. The absorbance of the resulting
solution was measured by a wavelength at 670 nm. The con-
centration of H2S was calculated and data were expressed as H2S
concentration in μmol/mg protein.
2.8. Western blotting
The harvested DRGs were washed with PBS and lysed with lysis
buffer containing protease inhibitors (Promega, US). The primary
antibodies were rabbit anti-rat nuclear factor-E2-related factor-2
(Nrf2) antibody, mouse anti-rat heme oxygenase-1(HO-1) anti-
body, rabbit anti-rat caspase-3 antibody, rabbit anti-rat NF-κB
antibody, rabbit anti-rat IκBα antibody, rabbit anti-rat phos-
phorylated-IκBα (p-IκBα) antibody. All antibodies were purchased
from Sigma (St. Louis, MO, USA) and were used at a dilution of
1:1000. Rabbit anti-rat GAPDH polyclonal antibody (1:10,000;
Sigma- Aldrich, US) was used as an internal control.
2.9. ELISA analysis for inflammatory cytokine measurements
The DRG homogenates were used to determine the amount of
interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). The DRG
homogenates were assayed using ELISA kits following the manu-
facturer instructions (Antibodies Online, Aachen, Germany). The
plates were read at 450 nm and analyzed using a microELISA
reader (Multiscan MK3, Thermo Labsystems, Helsinki, Finland).
2.10. Statistical analysis
All data in the study were expressed as the mean 7 standard
error of mean. One-way analysis of variance (ANOVA) was applied
to compare the data using SPSS13.0 software (SPSS Inc., Chicago,
IL, USA). Tukey post hoc test was applied for comparisons between
groups. Values of P o0.05 were considered as statistically
significant.
3. Results
3.1. The effect of rutin on plasma glucose
Rutin significantly decreased plasma glucose in diabetic rats,
which is in line with previous studies (Kamalakkannan and Prince,
2006; Kamalakkannan and Stanley Mainzen Prince, 2006). The
glucose level in diabetic control rats was 23.17 71.82 mmol/l,
which was significantly higher than that in normal control
(3.92 70.26 mmol/l). Two weeks after rutin administration, The
glucose level was decreased to 18.25 70.63 mmol/l (5 mg/kg),
14.82 70.41 mmol/l (25 mg/kg) and 12.96 70.55 mmol/l (50 mg/
kg), which were significantly lower than that in diabetic control
rats (Po 0.05). The glucose level in insulin-treated rats was sig-
nificantly decreased to 6.82 7 0.43 mmol/l, which was significantly
lower than that in rutin-treated rats (P o0.05). BG-12 has little
effect on plasma glucose in diabetic rats. The glucose level in BG-
12-treated rats was 22.64 72.15 mmol/l.
3.2. Rutin ameliorates hyperalgesia and allodynia in diabetic rats
Significant mechanical hyperalgesia, heat hyperalgesia and cold
allodynia were induced from the 9th day of STZ treatment
(Fig. 1A–C). Three weeks after STZ treatment, the number of paw
withdrawal responses to mechanical stimuli (bending force:
13.5 g), was significantly higher compared to that in the normal
group, suggesting the establishment of mechanical hyperalgesia
(P o0.05, Fig. 1A). In addition, the paw withdrawal latency to heat
and cold stimuli in the STZ group was significantly lower than that
in normal group (P o0.05, Fig. 1B,C), suggesting the establishment
of heat hyperalgesia and cold allodynia in diabetic rats.
Rutin is capable of attenuating mechanical hyperalgesia, heat
hyperalgesia and cold allodynia in diabetic rats. The increased
number of paw withdrawal responses to mechanical stimuli in
diabetic rats was inhibited by rutin dose dependently, which las-
ted for at least 8 h, suggesting that rutin is capable of attenuating
mechanical hyperalgesia in diabetic rats (Po 0.05, Fig. 1D). In ad-
dition, the decreased paw withdrawal latency to heat and cold
stimuli in the STZ-treated rats was partially reversed by rutin in a
 R. Tian et al. / European Journal of Pharmacology 771 (2016) 84–92 87

Fig. 1. Rutin alleviates diabetes-induced mechanical hyperalgesia, heat hyperalgesia and cold allodynia (n ¼12 in each group). STZ significantly increased the number of paw
withdrawal responses to mechanical stimuli (A), shortened the paw withdrawal latency to heat (B) and cold stimuli (C), suggesting the establishment of diabetic neuropathy.
*P o0.05 for the comparison to the normal rats. Rutin significantly decreased the number of paw withdrawal responses to mechanical stimuli (D), increased the paw
withdrawal latency to heat (E) and cold stimuli (F) in diabetic rats. Insulin and BG12 significantly decreased the number of paw withdrawal responses to mechanical stimuli
(G), as well as increased the paw withdrawal latency to heat (H) and cold stimuli (I) in diabetic rats at 4 h after drugs administration. In contrast, the number of paw
withdrawal responses to mechanical stimuli was significantly higher, and the withdrawal latency to heat and cold stimuli was significantly lower in insulin or BG-12
treatments than those receiving rutin at 50 mg/kg. *Po 0.05 for the comparison to the normal rats, # Po 0.05 for the comparison to the vehicle-treated diabetic rats, &
Po 0.05 for the comparison to diabetic rats treated with rutin at 5 mg/kg, and $Po 0.05 for the comparison to diabetic rats treated with rutin at 50 mg/kg.

dose dependent manner (Po 0.05, Fig. 1E,F), indicating that rutin
is able to attenuate heat hyperalgesia and cold allodynia in dia-
betic rats. The beneficial effect of rutin on heat hyperalgesia and
cold allodynia lasted for 8 h in rutin group at 5 mg/kg and for 10 h
in rutin groups at 25 mg/kg and 50 mg/kg. In addition, the in-
creased number of paw withdrawal responses to mechanical sti-
muli, as well as the decreased paw withdrawal latency to heat and
cold stimuli in diabetic rats were partially inhibited by insulin and
BG-12 at 4 h after drugs administration (Po 0.05, Fig. 1G–I).
Whereas, the number of paw withdrawal responses to mechanical
stimuli was significantly higher, and the withdrawal latency to
heat and cold stimuli was significantly lower in insulin or BG-12
treatments than those receiving rutin at 50 mg/kg (P o0.05,
Fig. 1G–I), indicating that rutin at 50 mg/kg has a stronger ability
in ameliorating diabetic hyperalgesia and allodynia than insulin
and BG-12.
nerve conduction velocities in diabetic rats was significantly
lower than that in normal rats, confirming nerve conduction
impairment in diabetic rats (P o 0.05, Fig. 2A,B). Two weeks after
rutin application, the motor and sensory nerve conduction ve-
locities in diabetic rats was significantly increased by rutin
(P o 0.05, Fig. 2A,B). In addition, the motor and sensory nerve
conduction velocities in diabetic rats which received rutin at
25 mg/kg and 50 mg/kg were significantly higher than rutin at
5 mg/kg, indicating that rutin is able to improve the motor and
sensory nerve functions in diabetic rats in a dose dependent
manner. In addition, insulin and BG-12 significantly increased the
sciatic motor and sensory nerve conduction velocities in diabetic
rats (P o 0.05, Fig. 2A,B). Nevertheless, the sciatic motor and
sensory nerve conduction velocities in insulin and BG-12 groups
were significantly lower than those receiving rutin at 50 mg/kg
(P o 0.05, Fig. 2A,B).

3.3. Rutin increases nerve conduction velocities in diabetic rats 3.4. The effect of rutin on Na þ , K þ -ATPase and caspase-3 expression

Rutin is capable of increasing motor and sensory nerve con- Diabetic neuropathy was accompanied by decreased Na þ ,
þ
duction velocities in diabetic rats. The sciatic motor and sensory K -ATPase in sciatic nerves and caspase-3 expression in DRG,
88 R. Tian et al. / European Journal of Pharmacology 771 (2016) 84–92
Fig. 2. The beneficial effect of rutin on motor and sensory conduction velocities in
diabetic rats (n¼ 12 in each group). Administration of rutin significantly increased
the amplitude of motor (A) and sensory (B) conducting velocities in diabetic rats.
Insulin and BG-12 significantly increased the sciatic motor (A) and sensory
(B) nerve conduction velocities in diabetic rats. The sciatic motor and sensory nerve
conduction velocities in insulin and BG-12 groups were significantly lower than
those receiving rutin at 50 mg/kg. *P o0.05 for the comparison to the normal rats, #
Po 0.05 for the comparison to the vehicle-treated diabetic rats, &Po 0.05 for the
comparison to diabetic rats treated with rutin at 5 mg/kg, and $Po 0.05 for the
comparison to diabetic rats treated with rutin at 50 mg/kg.
which has been recognized as indicators for nerve damage in-
duced by diabetes mellitus (Kamboj et al., 2010; Liu et al., 2010). In
the present study, rutin is capable of increasing Na þ , K þ -ATPase
activities in sciatic nerves and decreasing caspase-3 expression in
DRG neurons in diabetic rats (P o0.05, Fig. 3A,B). In addition, the
Na þ , K þ -ATPase activities were significantly higher and caspase-3
expression were significantly lower in diabetic rats receiving rutin
at 25 mg/kg and 50 mg/kg than those receiving rutin at 5 mg/kg
(P o0.05, Fig. 3A,B), indicating that rutin is able to attenuating
nerve damage in diabetic rats in a dose dependent manner. Fur-
ther studies showed that insulin and BG-12 were also capable of
increasing Na þ , K þ -ATPase activities and decreasing caspase-3
expression in diabetic rats (Po 0.05, Fig. 3A,B). Rutin at 50 mg/kg
showed a stronger ability in increasing Na þ , K þ -ATPase activities
and decreasing caspase-3 expression in diabetic rats than insulin
and BG-12 (P o0.05, Fig. 3A,B).
Fig. 3. The effect of rutin on Na þ , K þ -ATPase in sciatic nerves and caspase-3 ex-
pression in DRG neurons in diabetic rats. Rutin significantly increased Na þ ,
K þ -ATPase (A) in sciatic nerves and caspase-3 expression (B) in DRG neurons in
diabetic rats. Insulin and BG-12 significantly increased Na þ , K þ -ATPase (A) and
caspase-3 expression (B) in diabetic rats. Rutin at 50 mg/kg showed a stronger
ability in increasing Na þ , K þ -ATPase (A) and caspase-3 expression (B) in diabetic
rats than insulin and BG-12. *Po 0.05 for the comparison to the normal rats, #
Po 0.05 for the comparison to the vehicle-treated diabetic rats, &Po 0.05 for the
comparison to diabetic rats treated with rutin at 5 mg/kg, and $Po 0.05 for the
comparison to diabetic rats treated with rutin at 50 mg/kg.
3.5. Rutin attenuates ROS and oxidative stress in diabetic nerves
Rutin is capable of attenuating oxidative stress in diabetic rats,
which has been shown to play a vital role in the pathogenesis of
diabetic neuropathy (Sayyed et al., 2006; Negi et al., 2011). The
elevated ROS and MDA levels in diabetic rats was significantly
attenuated by rutin in a dose dependent manner (P o0.05, Fig. 4A,
B), suggesting that rutin is capable of inhibiting ROS generation
and lipid peroxidation in diabetic rats. We next investigated the
effect of rutin on antioxidant enzymes in diabetic rats. It was
found that the activities of SOD, GPx, GST and catalase in sciatic
nerves were significantly inhibited in diabetic rats (Po 0.05,
Fig. 4C–F), suggesting an impaired antioxidant defense system.
Two weeks after rutin application, the activities of SOD, GPx, GST
and catalase were partially reversed by rutin (Po0.05, Fig. 4C–F).
 R. Tian et al. / European Journal of Pharmacology 771 (2016) 84–92 89

Fig. 4. The effect of rutin on oxidative stress in diabetic rats. Rutin significantly decreased ROS (A) and MDA (B) levels, and increased the activities of SOD (C), GPx (D), GST
(E) and catalase (F) in sciatic nerves in diabetic rats. Insulin and BG-12 significantly decreased the ROS (A) and MDA (B) levels, and increased the activities of SOD (C), GPx (D),
GST (E) and catalase (F). Rutin at 50 mg/kg showed a stronger ability in lowering ROS (A) and MDA (B) levels, as well as increasing the activities of antioxidant enzymes in
diabetic rats than insulin and BG-12. *P o 0.05 for the comparison to the normal rats, #P o 0.05 for the comparison to the vehicle-treated diabetic rats, &Po 0.05 for the
comparison to diabetic rats treated with rutin at 5 mg/kg, and $P o0.05 for the comparison to diabetic rats treated with rutin at 50 mg/kg.

The activities of antioxidants in diabetic rats receiving rutin at
25 mg/kg and 50 mg/kg were significantly higher than that at
5 mg/kg (P o0.05, Fig. 4C–F), indicating that rutin is capable of
restoring the impaired antioxidant defense system in a dose de-
pendent manner. In addition, insulin and BG-12 significantly de-
creased the ROS and MDA levels, and increased the activities of
SOD, GPx, GST and catalase in diabetic rats (Po 0.05, Fig. 4A–F).
Rutin at 50 mg/kg showed a stronger ability in lowering ROS and
MDA levels, as well as increasing the activities of antioxidant en-
zymes in diabetic rats than insulin and BG-12 (P o0.05, Fig. 4A–F).
3.6. Rutin activates H2S and Nfr2/HO-1 pathway in diabetic rats
Rutin dose dependently increased the level of H2S in DRG
neurons in diabetic rats (P o0.05, Fig. 5A), which has been shown
to scavenge ROS directly and activate endogenous antioxidant
enzymes. Since the effect of H2S on oxidative stress has been as-
sociated with Nrf2 dependent pathway, we further examined the
effect of rutin on the expression of Nrf2 and HO-1 in DRG neurons
in diabetic rats. It was found that the expression of Nrf2 and HO-1
was significantly inhibited in diabetic rats (P o0.05, Fig. 5B,C). Two
weeks after rutin application, the expression of Nrf2 and HO-1 was
significantly increased by rutin (Po0.05, Fig. 5B,C). In addition,
both insulin and BG-12 were able to up-regulate the expression of
Nrf2 and HO-1 in DRG neurons (P o0.05, Fig. 5B,C). Particularly,
the expression of Nrf2 and HO-1 was in the similar range between
rutin (50 mg/kg) and BG-12 (15 mg/kg). Rutin at 50 mg/kg had a
stronger ability in ameliorating diabetic neuropathy than BG-12 at
15 mg/kg, indicating that Nrf2/HO-1 pathway was only partially
responsible for the beneficial effect of rutin on diabetic
neuropathy.

Fig. 5. The effect of rutin on H2S, Nrf2 and HO-1 in DRG neurons in diabetic rats. Rutin significantly decreased the levels of H2S (A), Nrf2 (B) and HO-1(C) in DRG neurons in
diabetic rats. Both insulin and BG-12 were able to up-regulate H2S (A), Nrf2 (B) and HO-1(C) in diabetic rats. The expression of Nrf2 and HO-1 was in the similar range
between rutin (50 mg/kg) and BG-12 (15 mg/kg). *Po 0.05 for the comparison to the normal rats, # Po 0.05 for the comparison to the vehicle-treated diabetic rats, &Po 0.05
for the comparison to diabetic rats treated with rutin at 5 mg/kg, and $P o0.05 for the comparison to diabetic rats treated with rutin at 50 mg/kg.
90 R. Tian et al. / European Journal of Pharmacology 771 (2016) 84–92

Fig. 6. The effect of rutin on neuroinflammation in diabetic rats. Rutin is capable of decreasing the levels of NF-кB, IкBα, p-IкBα (A–D) and inflammatory cytokines (IL-6 and
TNF-α) in DRG neurons (E,F) in diabetic rats. Insulin and BG-12 were also found to significantly decrease the levels of NF-кB, IкBα, and p-IкBα (A–D) and inflammatory
cytokines in DRG neurons (E,F). Rutin at 50 mg/kg showed a stronger ability in decreasing the levels of neuroinflammation in diabetic rats. *P o0.05 for the comparison to
the normal rats, # P o0.05 for the comparison to the vehicle-treated diabetic rats, &Po 0.05 for the comparison to diabetic rats treated with rutin at 5 mg/kg, and $Po 0.05 for
the comparison to diabetic rats treated with rutin at 50 mg/kg.

3.7. The effect of rutin on neuroinflammation
Rutin is capable of attenuating neuroinflammation in diabetic
rats, which is known to be up-regulated after peripheral nerve
injury and are associated with neuropathic pain behaviors (Zhang
et al., 2013). The levels of NF-кB, IкBα and p-IкBα in DRG neurons
were significantly higher in diabetic rats than normal rats, in-
dicating the presence of neuroinflammation in diabetic nerves
(P o0.05, Fig. 6A–D). Rutin is capable of decreasing the levels of
NF-кB, IкBα, and p-IкBα in DRG neurons in diabetic rats (P o0.05,
Fig. 6A–D). In addition, the levels of NF-кB, IкBα, and p-IкBα in
DRG neurons were significantly lower in diabetic rats receiving
rutin at 50 mg/kg than those receiving rutin at 5 mg/kg and
25 mg/kg (P o0.05, Fig. 6A–D). All those findings indicate that ruin
is able to attenuate neuroinflammation in diabetic rats in a dose
dependent manner. Further studies showed that insulin and BG-12
were also capable of decreasing the levels of NF-кB, IкBα, and
p-IкBα in DRG neurons in diabetic rats (Po 0.05, Fig. 6A–D). Rutin
at 50 mg/kg showed a stronger ability in decreasing the levels of
NF-кB, IкBα, and p-IкBα in diabetic rats than insulin and BG-12
(P o0.05, Fig. 6A–D).
The levels of IL-6 and TNF-α in DRG neurons were also quan-
tified as indicators for diabetic neuroinflammation. The elevated
levels of IL-6 and TNF-α in diabetic rats were dose dependently
attenuated by rutin (Po0.05, Fig. 6 E,F). Insulin and BG-12 were
also found to significantly decrease the levels of IL-6 and TNF-α in
DRG neurons (P o0.05, Fig. 6E,F). Rutin at 50 mg/kg showed a
stronger ability in decreasing the levels of IL-6 and TNF-α in dia-
betic rats than insulin and BG-12 (Po 0.05, Fig. 6 E,F).
4. Discussion
The antinociceptive effect of rutin has been found in several
animal models, including pain induced by glutamate, formalin and
oxaliplatin (Azevedo et al., 2013; Lapa Fda et al., 2009; Hernandez-
Leon et al., 2015). In the present study, rutin has been found to be
capable of attenuating mechanical hyperalgesia, heat hyperalgesia
and cold allodynia in diabetic rats, indicating the antinociceptive
effect of rutin in painful diabetic neuropathy. In addition, rutin
significantly increased Na þ , K þ -ATPase in diabetic nerves and
decreased capspase-3 expression in DRG neurons, suggesting that
 R. Tian et al. / European Journal of Pharmacology 771 (2016) 84–92
less nerve damage and neuronal apoptosis was achieved by rutin
in diabetic rats. Further studies showed that rutin significantly
increased the amplitude of motor and sensory nerve conduction
velocities in diabetic rats, confirming the beneficial effect of rutin
on the impaired nerve functions in diabetes mellitus.
Chronic hyperglycemia has been recognized as the leading
factor initiating deficits in motor and sensory nerve conduction
velocities and other manifestations of peripheral diabetic neuro-
pathy (Singh et al., 2014). In the present study, rutin was started
when rats had successfully developed diabetic neuropathy (3
weeks after STZ). It was found that rutin is capable of decreasing
plasma glucose, as well as ameliorating symptoms related to dia-
betic neuropathy. To investigate the role of lowered plasma glu-
cose in the beneficial effect of rutin on diabetic neuropathy, insulin
was used in the present study, which has been shown to efficiently
lower plasma glucose in diabetic rats (Remor et al., 2011). It was
found that insulin achieved lower plasma glucose in diabetic rats
than rutin at 50 mg/kg. However, the stronger ability of insulin in
lowering plasma glucose was not accompanied by better perfor-
mance in ameliorating diabetic neuropathy. The beneficial effect of
insulin on diabetic neuropathy was inferior to that by rutin at
50 mg/kg. Therefore, part of the beneficial effect of rutin on dia-
betic neuropathy might be secondary to lowered plasma glucose
in diabetic rats. In addition, besides lowered plasma glucose, other
mechanisms might also be involved in the beneficial effect of rutin
on diabetic neuropathy.
Oxidative stress is the key pathological process in inducing
nerve damage in diabetes, which contribute to the abnormal pain
sensation in diabetic neuropathy (Sandireddy et al., 2014). It is
believed that neural tissue is vulnerable to oxidative attack due to
its relatively low antioxidant capacity, high consumption of oxy-
gen, and high polyunsaturated fatty acids content (Vincent et al.,
2005; Zhang et al., 2013). In addition, oxidative damage to mi-
tochondrial DNA would result in impaired energy regulation,
which is critically important in high-energy-requiring neurons
(Lin and Beal, 2006). The oxidative stress-induced damage in DRG
neurons and nerve tissues has been found to be responsible for
pain sensation in diabetes mellitus, since decreasing the oxidative
injury to neurons and nerve tissues is able to attenuate diabetic
neuropathic pain behaviors (Zhang et al., 2013). In the present
study, rutin significantly inhibited oxidative stress in diabetic rats,
which was evidenced by the decreased levels of ROS and MDA in
nerve tissues in diabetic rats. The decreased oxidative stress in
sciatic nerves might contribute to the antinociceptive effect of
rutin in diabetic neuropathy.
We further examined the H2S levels in both rutin and vehicle-
treated diabetic rats, since H2S has been found to scavenge ROS
directly and activate endogenous antioxidant defenses (Zhou et al.,
2014; 2015; Poole et al., 2015). It was found that rutin significantly
increased H2S level in diabetic rats, suggesting the possible in-
volvement of H2S in the activation of antioxidant defense system
by rutin in diabetic nerves. It has been shown that the effect of H2S
on oxidative stress was mainly through Nrf2-dependent pathway
(Calvert et al., 2009). We next examined the expression of Nrf2
and its downstream targets in the present study. It was found that
the Nrf2 and HO-1 in DRG neurons were significantly up-regulated
by rutin in diabetic rats. To further investigate the role of Nrf2
pathway in the beneficial effect of rutin on diabetic neuropathy, a
potent Nrf2 activator (BG-12) was used, which has little effect on
plasma glucose in diabetic rats. It was found that the ability in
activating Nrf2 pathway was in the similar range between BG-12
(15 mg/kg) and rutin (50 mg/kg). Whereas, the beneficial effect of
BG-12 on diabetic neuropathy was inferior to that of rutin at
50 mg/kg. All those evidences suggest that Nrf2 signaling pathway
was partially, not fully responsible for the beneficial effect of rutin
on diabetic neuropathy.
91
Hyperglycemia is believed to be underpinning for neuroin-
flammation in diabetic neuropathy (Sandireddy et al., 2014). The
hyperglycemia associated classical pathways like polyol pathway,
PKC, AGEs could directly or indirectly initiate and progress the
inflammatory process (Sandireddy et al., 2014; Singh et al., 2014).
In addition, oxidative stress and these classical pathways are able
to activate NF-κB, which is a transcription factor that up-regulates
the gene expression of proinflammatory cytokines and also is re-
sponsible for the induction of nerve damage in diabetes mellitus
(Sandireddy et al., 2014). Persistent hyperglycemia-induced in-
flammation also affects the structural features of neuron (Shi et al.,
2013). The neuroinflammation plays a vital role in structural and
functional damage of the peripheral nerve, which leads to the
painful diabetic neuropathy. In the present study, activation of NF-
κB, as well as increased inflammatory cytokines (IL-6 and TNF-α)
were found in DRG neurons in diabetic rats, indicating the pre-
sence of neuroinflammation in diabetic neuropathy. Further stu-
dies showed that rutin was capable of inhibiting NF-κB activation
and decreasing the levels of IL-6 and TNF-α in DRG neurons, in-
dicating the rutin is capable of attenuating neuroinflammation,
which might contribute to the beneficial effect of rutin on diabetic
neuropathy.
The oxidative stress and neuroinflammation do not work in-
dividually in the development of painful diabetic neuropathy. The
oxidative stress and inflammatory pathways interact at multiple
levels, which complicate the hyperglycemia-mediated neuronal
damage (Sandireddy et al., 2014). ROS are known to activate in-
hibitory kappa-B kinase which causes phosphorylation of IκB.
Release of free NF-κB heterodimer from IκB allows it to cross
nuclear membrane and bind with kappa region of genome, thus
enhancing the production of inflammatory cytokines such as TNF-
α, IL-6 etc. (Poli et al., 2004). In addition, activation of NF-κB also
suppresses the expression of antioxidant genes by down-regulat-
ing Nrf-2 pathway and thus indirectly weakening the innate an-
tioxidant defense, which increases the production of ROS (Ganesh
Yerra et al., 2013). In the present study, activation of NF-κB and
inhibition of Nrf-2 pathway were observed simultaneously after
establishment of diabetic neuropathy. Application of rutin is cap-
able of inhibiting NF-κB, as well as activating the Nrf-2 pathway in
DRG neurons in diabetic neuropathy. Those findings confirmed the
cross-talk between oxidative stress and neuroinflammation, and
helped to explain the mechanisms underlying the beneficial effect
of rutin on painful diabetic neuropathy.
Rutin appears to have direct effects on neuronal function that is
unrelated to diabetes, which is evidenced by the rise and decay of
its antinociceptive effect in the hours following the last rutin in-
jection. Rutin has been shown to ameliorate diabetic neuropathy
in a dose dependent manner. Rutin at 25 mg/kg and 50 mg/kg had
stronger antinociceptive effect than rutin at 5 mg/kg. Despite this
finding, it should be clear that the most commonly used dose of
rutin is 200–600 mg once or twice per day, which translates into
3–18 mg/kg in the assumption of 70 kg patient's body weight.
Therefore, rutin at 5 mg/kg has more clinical relevance to potential
human applications. In addition, rutin exhibited antinociceptive
effect after 2–10 h of the last treatment and the effect disappear at
12 h, which might be attributable to its sustained bioavailability
in vivo. It has been shown that peak concentrations were reached
at 7.07 2.9 h after ingestion of rutin and the t1/2 for rutin is
11.873.1 h after oral intake (Graefe et al., 2001). In the present
study, rutin was injected intraperitoneally. The maximum effect
was found at about 4 h after injection, which is much earlier than
the peak concentration achieved after oral administration.
5. Conclusion
Rutin produces significant protection in diabetic neuropathy as
92 R. Tian et al. / European Journal of Pharmacology 771 (2016) 84–92
evident from improvement in nociception and nerve conduction
velocity. The beneficial effects of rutin might be attributable to its
ability in lowering plasma glucose, inhibition of neuroinflamma-
tion, decreasing oxidative stress and augmenting antioxidant de-
fense system through increasing the levels of H2S and Nrf2. Rutin
holds the potential for the management of painful diabetic neu-
ropathy, which requires more evidences from more studies,
especially from clinical observations.
Conflict of interest
We declare no conflict of interest.
Acknowledgments
This work was supported by grants from the National Natural
Science Foundation of China (81172396).
References
Azevedo, M.I., Pereira, A.F., Nogueira, R.B., Rolim, F.E., Brito, G.A., Wong, D.V., Lima-
Júnior, R.C., de Albuquerque Ribeiro, R., Vale, M.L., 2013. The antioxidant effects
of the flavonoids rutin and quercetin inhibit oxaliplatin-induced chronic
painful peripheral neuropathy. Mol. Pain 9, 53.
Brownlee, M., 2001. Biochemistry and molecular cell biology of diabetic compli-
cations. Nature 414, 813–820.
Calvert, J.W., Jha, S., Gundewar, S., Elrod, J.W., Ramachandran, A., Pattillo, C.B., Kevil,
C.G., Lefer, D.J., 2009. Hydrogen sulfide mediates cardioprotection through Nrf2
signaling. Circ. Res. 105, 365–374.
Didangelos, T., Doupis, J., Veves, A., 2014. Painful diabetic neuropathy: clinical as-
pects. Handb. Clin. Neurol. 126, 53–61.
Ganesh Yerra, V., Negi, G., Sharma, S.S., Kumar, A., 2013. Potential therapeutic ef-
fects of the simultaneous targeting of the Nrf2 and NF-κB pathways in diabetic
neuropathy. Redox Biol. 1, 394–397.
Graefe, E.U., Wittig, J., Mueller, S., Riethling, A.K., Uehleke, B., Drewelow, B., Pforte,
H., Jacobasch, G., Derendorf, H., Veit, M., 2001. Pharmacokinetics and bioa-
vailability of quercetin glycosides in humans. J. Clin. Pharmacol. 41, 492–499.
Hargreaves, K., Dubner, R., Brown, F., Flores, C., Joris, J., 1988. A new and sensitive
method for measuring thermal nociception in cutaneous hyperalgesia. Pain 32,
77–88.
Hernandez-Leon, A., Fernández-Guasti, A., González-Trujano, M.E., 2015. Rutin
antinociception involves opioidergic mechanism and descending modulation of
ventrolateral periaqueductal grey matter in rats. Eur. J. Pain . http://dx.doi.org/
10.1002/ejp.720.
Hosseinzadeh, H., Nassiri-Asl, M., 2014. Review of the protective effects of rutin on
the metabolic function as an important dietary flavonoid. Endocrinol. Investig.
37, 783–788.
Kamalakkannan, N., Prince, P.S., 2006a. Antihyperglycaemic and antioxidant effect
of rutin, a polyphenolic flavonoid, in streptozotocin-induced diabetic wistar
rats. Basic Clin. Pharmacol. Toxicol. 98, 97–103.
Kamalakkannan, N., Stanely Mainzen Prince, P., 2006b. Rutin improves the anti-
oxidant status in streptozotocin-induced diabetic rat tissues. Mol. Cell. Bio-
chem. 293, 211–219.
Kamboj, S.S., Vasishta, R.K., Sandhir, R., 2010. N-acetylcysteine inhibits hypergly-
cemia-induced oxidative stress and apoptosis markers in diabetic neuropathy. J.
Neurochem. 112, 77–91.
Koda, T., Kuroda, Y., Imai, H., 2009. Rutin supplementation in the diet has protective
effects against toxicant-induced hippocampal injury by suppression of
microglial activation and pro-inflammatory cytokines: protective effect of rutin
against toxicant-induced hippocampal injury. Cell. Mol. Neurobiol. 29,
523–531.
Lapa Fda, R., Gadotti, V.M., Missau, F.C., Pizzolatti, M.G., Marques, M.C., Dafré, A.L.,
Farina, M., Rodrigues, A.L., Santos, A.R., 2009. Antinociceptive properties of the
hydroalcoholic extract and the flavonoid rutin obtained from Polygala panicu-
lata L. in mice. Basic Clin. Pharmacol. Toxicol. 104, 306–315.
Lin, M.T., Beal, M.F., 2006. Mitochondrial dysfunction and oxidative stress in neu-
rodegenerative diseases. Nature 443, 787–795.
Liu, Y., Wang, L., Li, X., Lv, C., Feng, D., Luo, Z., 2010. Tanshinone IIA improves im-
paired nerve functions in experimental diabetic rats. Biochem. Biophys. Res.
Commun. 399, 49–54.
Mascaraque, C., Aranda, C., Ocón, B., Monte, M.J., Suárez, M.D., Zarzuelo, A., Marín, J.
J., Martínez-Augustin, O., de Medina, F.S., 2014. Rutin has intestinal antiin-
flammatory effects in the CD4 þ CD62L þ T cell transfer model of colitis.
Pharmacol. Res. 90, 48–57.
Messinger, R.B., Naik, A.K., Jagodic, M.M., Nelson, M.T., Lee, W.Y., Choe, W.J., Orestes,
P., Latham, J.R., Todorovic, S.M., Jevtovic-Todorovic, V., 2009. In vivo silencing of
the Ca(V)3.2 T-type calcium channels in sensory neurons alleviates hyper-
algesia in rats with streptozocin-induced diabetic neuropathy. Pain 145,
184–195.
Negi, G., Kumar, A., Joshi, R.P., Sharma, S.S., 2011. Oxidative stress and Nrf2 in the
pathophysiology of diabetic neuropathy: old perspective with a new angle.
Biochem. Biophys. Res. Commun. 408, 1–5.
Padiya, R., Chowdhury, D., Borkar, R., Srinivas, R., Pal Bhadra, M., Banerjee, S.K.,
2014. Garlic attenuates cardiac oxidative stress via activation of PI3K/AKT/Nrf2-
Keap1 pathway in fructose-fed diabetic rat. PLoS One 9, e94228.
Poli, G., Leonarduzzi, G., Biasi, F., 2004. Chiarpotto E. Oxidative stress and cell sig-
nalling. Curr. Med. Chem. 11, 1163–1182.
Poole, K.M., Nelson, C.E., Joshi, R.V., Martin, J.R., Gupta, M.K., Haws, S.C., Kavanaugh,
T.E., Skala, M.C., Duvall, C.L., 2015. ROS-responsive microspheres for on demand
antioxidant therapy in a model of diabetic peripheral arterial disease. Bioma-
terials 41, 166–175.
Remor, A.P., de Matos, F.J., Ghisoni, K., da Silva, T.L., Eidt, G., Búrigo, M., de Bem, A.F.,
Silveira, P.C., de León, A., Sanchez, M.C., Hohl, A., Glaser, V., Gonçalves, C.A.,
Quincozes-Santos, A., Borba Rosa, R., Latini, A., 2011. Differential effects of in-
sulin on peripheral diabetes-related changes in mitochondrial bioenergetics:
involvement of advanced glycosylated end products. Biochim. Biophys. Acta
1812, 1460–1471.
Sandireddy, R., Yerra, V.G., Areti, A., Komirishetty, P., Kumar, A., 2014. Neuroin-
flammation and oxidative stress in diabetic neuropathy: futuristic strategies
based on these targets. Int. J. Endocrinol. 2014, 674987.
Sayyed, S.G., Kumar, A., Sharma, S.S., 2006. Effects of U83836E on nerve functions,
hyperalgesia and oxidative stress in experimental diabetic neuropathy. Life Sci.
79, 777–783.
Shi, X., Chen, Y., Nadeem, L., Xu, G., 2013. Beneficial effect of TNF-α inhibition on
diabetic peripheral neuropathy. J. Neuroinflamm. 10, 69.
Singh, R., Kishore, L., Kaur, N., 2014. Diabetic peripheral neuropathy: current per-
spective and future directions. Pharmacol. Res. 80, 21–35.
Vincent, A.M., McLean, L.L., Backus, C., Feldman, E.L., 2005. Short-term hypergly-
cemia produces oxidative damage and apoptosis in neurons. FASEB J. 19,
638–640.
Vogelaar, C.F., Vrinten, D.H., Hoekman, M.F., Brakkee, J.H., Burbach, J.P., Hamers, F.P.,
2004. Sciatic nerve regeneration in mice and rats: recovery of sensory in-
nervation is followed by a slowly retreating neuropathic pain-like syndrome.
Brain Res. 1027, 67–72.
Wild, S., Roglic, G., Green, A., Sicree, R., King, H., 2004. Global prevalence of dia-
betes: estimates for the year 2000 and projections for 2030. Diabetes Care 27,
1047–1053.
Zhang, Y.P., Eber, A., Yuan, Y., Yang, Z., Rodriguez, Y., Levitt, R.C., Takacs, P., Candiotti,
K.A., 2013. Prophylactic and antinociceptive effects of coenzyme Q10 on dia-
betic neuropathic pain in a mouse model of type 1 diabetes. Anesthesiology
118, 945–954.
Zhou, X., An, G., Lu, X., 2015. Hydrogen sulfide attenuates the development of
diabetic cardiomyopathy. Clin. Sci. (Lond.) 128, 325–335.
Zhou, X., Feng, Y., Zhan, Z., Chen, J., 2014. Hydrogen sulfide alleviates diabetic ne-
phropathy in a streptozotocin-induced diabetic rat model. J. Biol. Chem. 289,
28827–28834.