Treadmill training combined with insulin suppress diabetic nerve
pain and cytokines in rat sciatic nerve
1. Yu-Wen Chen, Ph.D.
Title: Associate Professor
Affiliation: Department of Physical Therapy, Graduate Institute of
Rehabilitation Science, China Medical University, Taichung, Taiwan
Email: cywhwok@mail.cmu.edu.tw
Role: This author helped design the study, conduct the study, analyze the data,
and write the manuscript
Conflicts: Yu-Wen Chen reported no conflicts of interest
Attestation: Yu-Wen Chen has seen the original study data, reviewed the
analysis of the data, approved the final manuscript, and is the author responsible
for archiving the study files
2. Chong-Chi Chiu, M.D.
Title: Assistant Professor
Affiliation: Department of General Surgery, Chi-Mei Medical Center, Tainan
and Liouying, Taiwan; Department of Cosmetic Science, Chia Nan University of
Pharmacy and Science, Tainan, Taiwan
1. Department of General Surgery, Chi Mei Medical Center, Tainan and Liouying, Taiwan
2. Department of Electrical Engineering, Southern Taiwan University of Science and
Technology, Tainan, Taiwan
Email: chiuchongchi@yahoo.com.tw
Role: This author helped conduct the study, analyze the data, and write the
manuscript
Conflicts: Chong-Chi Chiu reported no conflicts of interest
Attestation: Chong-Chi Chiu has seen the original study data, reviewed the
analysis of the data, approved the final manuscript, and is the author responsible
for archiving the study files
3. Pei-Ling Hsieh, M.S.
Title: Student with Ph.D. program
Affiliation: Department of Physical Therapy, National Cheng Kung University,
Tainan, Taiwan
Email: akinosha@hotmail.com
Role: This author helped conduct the study, analyze the data, and write the
manuscript
1
Conflicts: Pei-Ling Hsieh reported no conflicts of interest
Attestation: Pei-Ling Hsieh has seen the original study data, reviewed the
analysis of the data, approved the final manuscript, and is the author responsible
for archiving the study files
4. Ching-Hsia Hung, Ph.D.
Title: Associate Professor
Affiliation: Department of Physical Therapy, National Cheng Kung University,
Tainan, Taiwan
Email: chhung@mail.ncku.edu.tw
Role: This author helped design the study, conduct the study, analyze the data,
and write the manuscript
Conflicts: Ching-Hsia Hung reported no conflicts of interest
Attestation: Ching-Hsia Hung has seen the original study data, reviewed the
analysis of the data, approved the final manuscript, and is the author responsible
for archiving the study files
5. Jhi-Joung Wang, M.D., Ph.D.
Title: Professor
Affiliation: Department of Medical Research, Chi-Mei Medical Center, Tainan,
Taiwan
Email: 400002@mail.chimei.org.tw
Role: This author helped conduct the study, analyze the data, and write the
manuscript
Conflicts: Jhi-Joung Wang reported no conflicts of interest
Attestation: Jhi-Joung Wang has seen the original study data, reviewed the
analysis of the data, approved the final manuscript, and is the author responsible
for archiving the study files
Institution: This work was done in National Cheng Kung University.
Short Title: Insulin and exercise reduce diabetic neuropathic pain
Funding: We gratefully acknowledge the financial support provided by the grant
from the Ministry of Science and Technology (MOST 103-2314-B-039 -004),
Taiwan.
Corresponding Author:
Ching-Hsia Hung, Ph.D.
Department of Physical Therapy, National Cheng Kung University, Tainan,
Taiwan
2
No.1 Ta-Hsueh Road, Tainan 70101, Taiwan
Phone: 886-6-2353535 ext 5939
FAX: 886-6-2370411
Email: chhung@mail.ncku.edu.tw
Information for LWW regarding depositing manuscript into PubMed Central: This
paper does not need to be deposited in PubMed Central.
Submitted as a Research Report
This manuscript was screened for plagiarism using We used turnitin to check our
manuscript before submitting..
Link to Title Page: http://www.aaauthor.org/pages/9524-2014-Oct-09
3
IMPLICATIONS STATEMENT
Combined insulin therapy and treadmill training alters the progression of diabetic
neuropathic pain. Streptozotocin-induced diabetes enhances TNF-α, IL-6, and MDA
expression, whereas combined insulin therapy and treadmill training shows a reversal
of TNF-α, IL-6, and MDA levels. The results will greatly enrich our understanding
for management of diabetic peripheral neuropathic pain in controlling the expression
of proinflammatory cytokines and malondialdehyde in the sciatic nerve.
4
ABSTRACT
BACKGROUND: Insulin therapy plays a critical role in managing type 1 diabetes.
Exercise produces the alterations in pain sensation. The experiment explored the
effects of insulin therapy combined with treadmill training on diabetic neuropathic
pain and on the expression of malondialdehyde (MDA) and cytokines.
METHODS: Four weeks of insulin (100 IU/kg) therapy and treadmill training (30-60
min/d of training at 20-25 m/min) were administered, daily, beginning on day 3 after
STZ (streptozotocin, 65 mg/kg, iv) injection and continued until day 27 in rats.
Sensitivity to heat and mechanical stimuli, and the expression of interleukin-10
(IL-10), IL-6, tumor necrosis factor-α (TNF-α) and MDA in the sciatic nerve were
estimated.
RESULTS: We showed that 2-4 weeks of treadmill training, insulin treatment or the
combination exhibited an increase in both paw withdrawal thresholds and latencies, as
compared to sedentary diabetic rats (all P < 0.0022). Treatment with insulin, but not
treadmill training, had great effects on glycemic control (P < 0.0001) and restored
body weight (P < 0.0001) in the diabetic rats. The diabetic rats demonstrated the
upregulation (all P < 0.009) of IL-6, MDA, and TNF-α in the sciatic nerve on days 14
and 28 after STZ treatment, whereas diabetic rats receiving insulin, treadmill training,
or the combination decreased (all P < 0.01) this upregulation. Insulin, treadmill
5
training, or the combination increased IL-10 expression (all P < 0.0051) in diabetic
rats.
CONCLUSION: Treadmill training combined with insulin therapy showed the
best improvements in tactile allodynia and thermal hyperalgesia among these three
treatment groups. The benefits of insulin intervention and treadmill training could be
related to chronic inflammation (proinflammatory cytokines) and oxidative stress
(MDA).
Keywords:
Insulin therapy; Treadmill training; Diabetic peripheral neuropathic pain;
Proinflammatory cytokines; Malondialdehyde; Streptozotocin
6
INTRODUCTION
Insulin therapy has several useful effects on numerous appearances of the
common sequelae of diabetic neuropathy, such as the suppression of the development
of tactile allodynia,1 an increase in nerve conduction velocity,2 a reversal of peripheral
and central neurophysiological alterations.3 As in the long period, insulin therapy does
not inhibit the progress of various complications,4-6 whereas the valuable treatment of
diabetes in type 1 diabetes mellitus patients needs a daily balancing of appropriate
diet, insulin injection, and exercise.7,8 Along with insulin administration, treadmill
training is enthroned for its beneficial actions on blood sugar control and its capacity
to postpone the development of various diabetic complications, including diabetic
neuropathy.9
Diabetic peripheral neuropathy is mediated with an inflammation that is involved
with changes in expressions of proinflammatory cytokines.9-12 Interleukin-6 (IL-6)
concentrations were significantly higher compared with controls in children with type
1 diabetes.12 Additionally, our previous study revealed that the levels of tumor
necrosis factor-α (TNF-α) and IL-6 in the peripheral nerves and the spinal cord were
significantly increased in a rat model of streptozotocin (STZ)-induced type 1 diabetes
when compared with normal rats.9 In the DRG of STZ-diabetic rats, Saleh et al.
declared that the cytokines (IL-1β and IL-1α) release were decreased 2-fold, while
7
IL-10 and IL-2 were unchanged.10
Apart from behavioral and functional changes there exist various biochemical
alterations like depletion of enhanced peroxynitrite generation, lipid peroxidation,
antioxidant enzymes, etc., that go with diabetic neuropathy.13,14 In addition,
resveratrol, a natural non-flavonoid antioxidant, has been known to attenuate the
expression of proinflammatory mediators in the diabetic rats.15 Recently, increasing
evidence suggested that the levels of oxidative markers (reactive oxygen species,
malondialdehyde (MDA), hydroperoxides) increases in the sciatic nerve of diabetic
rats.16,17
Evidence supports this involvement of inflammatory response, oxidative stress,
and energy depletion in the progress of diabetic neuropathy.18,19 No studies have
investigated a therapeutic intervention regiment combining insulin and treadmill
training to manage diabetic neuropathy. This purpose of the study was to estimate
body weight, blood glucose levels, tactile allodynia, heat hyperalgesia as well as
cytokine and MDA levels to assess the impact of the therapy on diabetic nerve pain in
STZ-induced rat model for studying diabetes.
8
METHODS
Animals
Male Wistar rats (290 to 340 g) were bought from the Laboratory Animal Center
of National Cheng Kung University and bred in the animal housing facility at
National Cheng Kung University, with controlled room temperature (24C), a 12-hour
(6:00 AM to 6:00 PM) light/dark cycle and humidity (~50% relative humidity). Our
investigative procedures were agreed via the Institutional Animal Care and Use
Committee of National Cheng Kung University, Tainan, Taiwan.
Groups and design
The experimenter was blind for animal assignment to five experimental groups.
The first group of SS rats was sedentary rats receiving streptozotocin (STZ) treatment.
The second group of NS rats was normal sedentary rats. The third group of SE rats
received treadmill training after STZ administration. The fourth group of SI rats
received insulin therapy after STZ treatment, and the fifth group of SEI animals was
received exactly like the STZ rats remedying insulin treatment and treadmill training.
The animals were considered for the behavioral examinations and the parameters of
blood glucose and body weight (n = 12 in each group), while several animals were
killed for cytokines (IL-6, IL-10 and TNF-α) (n = 5 in each group) and MDA (n = 5 in
each group) analyses on days 14 and 28 after STZ treatment.
9
On day 3 after STZ injection, the animals received three therapeutic methods
(insulin, treadmill training or insulin with treadmill training).
Insulin (100 IU/kg)
was injected to the STZ rats once per day beginning immediately on day 3 after STZ
treatment and then daily for the next 25 days. Treadmill training was performed to the
STZ animals for 30 minutes once per day starting immediately on day 3 after STZ
treatment and then daily for the next 4 weeks. The behavioral tests, body weight and
blood sugar concentration were evaluated in 3 days before inducement (STZ), the day
of inducement as well as 3, 7, 14, 21 and 28 days after inducement.
Type 1 diabetes inducement
Three days after the inducement (streptozotocin, 65 mg/kg; Sigma-Aldrich Co.,
St. Louis, MO), a blood sugar device (Accu-Check Active; Roche Boehringer
Mannheim Diagnostics, Mannheim, Germany) was used to examine whether blood
tests (procured via tail prick) exhibited the sugar concentration  300 mg/dL, which
was considered an achievement of streptozotocin-induced diabetes.20,21
Treadmill training program
The training program was modified in accordance with the previous method.22-24
Initially, the animals accommodated to this training protocol for 15 minutes at 10
m/min, for 3 days. Animals in the training plan run on the treadmill (treadmill
exerciser T510, Diagnostic & Research Instruments, Singa, Taiwan), daily for 4
10
weeks. The intensity and duration of the exercise were set to raise continuously so
that these rats were running for 30 minutes at 1.2 km/hr and 60 minutes at 1.5 km/hr
at first two and last two weeks of training, respectively. When necessary, the rat was
gently prodded in the hindquarters, to encourage the rat and to guarantee training
compliance.
Heat and mechanical sensitivity
After the 5-7 days of habituation to the experimental investigators and
environments, the rats were assessed for mechanical withdrawal threshold and
thermal withdrawal latency. For consonance, an expert experimenter, who was
blinded after assignment to interventions, was in control of handling the behavioral
examinations and rats. All behavioral examinations were tested between 9:00 A.M.
and 11:00 A.M.
Thermal withdrawal latency was evaluated through the Hargreaves’ Method.25
Briefly, the lateral-plantar area of rat hindpaw was undergone to the radiant thermal
source by the plantar test apparatus (Hargreaves' method) (Ugo Basile,
Comerio, Italy). The therrmal withdrawal latency (second) was taken down when the
rat exhibited its hindpaw withdrawal. The automatic cut-off latency was set at 20
seconds to prevent tissue damage.
Mechanical sensitivity was examined through
von Frey filaments (Anesthesiometer, Somedic AB, Sweden) when the rat was put
11
individually in the clear acrylic rectangular chamber with a wire mesh floor. Each
filament in ascending order of force was applied for 5 seconds vertically to the
lateral-plantar area of the rat hind paw. When the rat withdrew its hind leg to von Frey
stimulation, this mechanical withdrawal threshold (gram) was recorded.9,24
Cytokines analysis
The rats were anesthetized with urethane (1.67 g/kg, i.p.) and killed on days 14
and 28 after STZ treatment. The sciatic nerve (2 cm) was obtained. The concentration
of cytokines (IL-10, TNF-α and IL-6) in the supernatant of the homogenized sample
was detected via the DuoSet® ELISA Development Kit (R&D Systems, Minneapolis,
MN) through the manufacturer’s recommended procedures.26,27
Malondialdehyde (MDA) analysis
The oxidative stress parameter was evaluated in tissue (sciatic nerve)
homogenates. Lipid peroxidation was assessed in term of MDA by determining the
accumulation of thiobarbituric acid reactive substances (TBARS).28 Total protein was
determined through bovine serum albumin as a standard 29 in the sciatic nerve
homogenate.
Statistical analysis
The investigative values are presented as the mean ± S.E.M.
The differences
between data related to blood glucose (Fig. 1A), body weight (Fig. 1B), paw
12
withdrawal threshold (Fig. 3A) and paw withdrawal latency (Fig. 3B) were analyzed
by two-way analysis of variance (ANOVA) of repeated measures. If significant (P
< .05), the analyses were followed by post hoc t tests with Bonferroni correction to
evaluate group differences at specific time points. Exercise, insulin, and the
combination treatment was the between-subjects factor and time was the repeated
measure. The differences in TNF-α (Fig. 4A), IL-6 (Fig. 4B), IL-10 (Fig. 4C) and
MDA (Fig. 4D) between the sedentary streptozotocin (STZ)-group and others were
determined using one-way ANOVA followed by pairwise Tukey HSD test.
SPSS, a
statistical software for Windows (version 17.0; SPSS, Inc., Chicago, IL, USA), was
performed for all statistical analyses.
13
RESULTS
Treadmill training and/or insulin treatment suppresses diabetes-induced
hyperglycemia and body weight loss
ANOVA of repeated measures (including NS, SS, SE, SI and SEI groups) in Figs.
1A and 1B for blood glucose and body weight exhibited significant main effects for
the groups (F4,44 = 52.65, P< 0.0001; F4,44 = 48.92, P< 0.0001), time (F6,270 = 40.85,
P< 0.0001; F6,270 = 38.67, P< 0.0001) and significant interaction (F24,270 = 8.66, P<
0.0001; F24,270 = 7.48, P< 0.0001), respectively.
Post-hoc comparisons showed no
significant differences between SI and SEI for blood glucose (all P > 0.45, Bonferroni
post-hoc).
On days 3, 7, 14, 21 and 28 after receiving STZ, the rats exhibited a significant
elevation of blood sugar (> 300 mg/dL) in comparison with NS rats (P< 0.0001),
which exhibited the blood sugar concentrations between 90 and 100 mg/dL (Fig. 1A).
In most days after receiving STZ, Figure 1A demonstrated the marked differences
between the SE and SS rats in blood sugar (450-500 mg/dL versus 400-450 mg/dL).
The body weights developed normally in the NS rats during the four-week period
(Fig. 1B), whereas the SS rats displayed a regular decline in body weight gain (P<
0.0001). A parallel, but less extravagant decline was observed with SS rats
undergoing treadmill training (Fig. 1B). Furthermore, the body weight loss in the SI
14
or SEI groups was not as serious as that in the STZ-induced diabetic group (P<
0.0001; Fig. 1B). Additionally, the STZ rats receiving insulin treatment had a normal
blood glucose level in most of time (Fig. 2).
Treadmill training and/or insulin therapy postpones the progress of
diabetes-evoked allodynia
ANOVA of repeated measures (including NS, SS, SE, SI and SEI groups) in Fig.
3A for the mechanical withdrawal thresholds showed prominent main effects for the
groups (F4,44 = 23.78, P< 0.0001; F4,44 = 19.77, P< 0.0001), time (F6,270 = 33.21, P<
0.0001; F6,270 = 32.11, P< 0.0001) and significant interaction (F24,270 = 10.81, P<
0.0001; F24,270 = 14.69, P< 0.0001), respectively.
Post-hoc comparisons displayed
significant differences among five groups for the mechanical withdrawal threshold
(all P < 0.01, Bonferroni post-hoc).
Compared with the NS rats, the rats on day 3 after STZ administration displayed
the increased sensitivity to tactile von Frey stimulation (8.7 ± 0.3 g, n = 12) lasting 4
weeks (P< 0.0001; Fig. 3A). STZ-treated rats at second week underwent insulin alone
and treadmill training alone exhibited paw withdrawal thresholds of 7.8 ± 0.2 g (n =
12) and 8.1 ± 0.5 g (n = 12), respectively, lower than the values of NS rats (all P<
0.0022; Fig. 3A), but higher than the values of SS rats (P< 0.0001; Fig. 3A).
Moreover, combination therapy showed a short reversal of the values of paw
15
withdrawal thresholds in the diabetic rats on day 14 after inducement (Fig. 3A). The
therapeutic effect by insulin alone or combination of insulin therapy and treadmill
training remained on day 28 after inducement (STZ), but there was no marked
difference in paw withdrawal thresholds between the SE and SS rats (Fig. 3A).
Treadmill training and/or insulin injection inhibits the development of
diabetes-associated hyperalgesia
ANOVA of repeated measures (including NS, SS, SE, SI and SEI groups) in Fig.
3B for the thermal withdrawal latencies demonstrated predominant main effects for
the groups (F4,44 = 34.91, P< 0.0001; F4,44 = 36.89, P< 0.0001), time (F6,270 = 8.66, P<
0.0001; F6,270 = 9.71, P< 0.0001) and significant interaction (F24,270 = 27.29, P<
0.0001; F24,270 = 20.41, P< 0.0001), respectively.
Post-hoc comparisons exhibited
significant differences between groups (NS, SE, SI, or SEI) and SS group for the
thermal withdrawal latency (all P < 0.0081, Bonferroni post-hoc). There was no
significant difference among the NS, SI and SEI groups (all P > 0.56, Bonferroni
post-hoc).
The SS rats exhibited a marked reduction of the heat withdrawal latency on day
14 after receiving STZ (P < 0.0001; Fig. 3B) in comparison with the NS rats. The SE,
SI, or SEI rats had minimal alterations in the heat withdrawal latency on day 14 after
receiving STZ compared to NS rats (Fig. 3B). In contrast, the syndrome of heat
16
hyperalgesia (Fig. 3B) corresponded to harsh hyperglycemia in SS rats (Fig. 1A).
Furthermore, a 4-weeks therapeutic program did reverse the decrease in thermal
withdrawal latencies in the SI and SEI rats, but not the SE rats as compared to those in
the SS rats on day 28 after STZ injection (P = 0.0025; Fig. 3B).
Treadmill training and/or insulin treatment reduces MDA and cytokines levels in
the sciatic nerve
The Figure 4 A-D revealed the expression of IL-10, IL-6, TNF-α and MDA in
the sciatic nerve of SS, NS, SE, SI and SEI rats on days 14 and 28 after STZ treatment.
On days 14 and 28 after STZ injection, the level of TNF-α, IL-6 and MDA in the
sciatic nerve was raised in the SS group (all P < 0.009) when compared to the NS and
SEI group, respectively, as presented in the Figs. 4A, 4B, and 4D. Insulin therapy,
treadmill training, or the combination restrained the increased expression of IL-6,
TNF-α and MDA (all P < 0.01, Figs. 4A, 4B, and 4D). It can be seen that the IL-10
levels in the sciatic nerve markedly suppressed (all P < 0.006) in the SS group on
days 14 and 28 after inducement (STZ) in comparison with the NS group, whereas
these three therapeutic methods increased the IL-10 levels (all P < 0.0051, Fig. 4C).
17
DISCUSSION
In the current study we showed that insulin therapy, treadmill training, or the
combination apparently prevented abnormal weight loss, suppressed diabetes-induced
hyperglycemia, or attenuated the progress of heat hyperalgesia and mechanical
allodynia in the STZ-induced diabetic animals. This is in agreement with our previous
experiment that exercise delayed the process of diabetic peripheral neuropathic pain.9
Furthermore, the three treatment groups significantly suppressed excess IL-6, TNF-α,
and MDA expression in the sciatic nerve of the diabetic treated rats, in a time of
day-dependent manner, compared to the sedentary controls. In the sciatic nerves,
diabetes also induced a reduction in IL-10 expression that did inhibited by insulin
therapy, treadmill training, or the combination.
Overall, our results presumed that
insulin treatment alone, treadmill training alone, or combined treadmill training with
insulin therapy suppressed the progression of diabetic peripheral neuropathic pain.
This possibly pertains to a reversal of the upregulation of proinflammatory cytokines
and MDA in the sciatic nerves.
We demonstrated that the acute phase (within days) of continual hyperglycemia
was resulting in hypersensitivity to both thermal and mechanical stimuli. This can be
explained by that peripheral neuropathy grows in diabetes if hyperglycemia is poorly
controlled 7 and early phases of hyperglycemia may precipitate diabetic peripheral
18
neuropathy. Additionally, our data are in resemblance to that high blood sugar may
result in prolonged changes of pain threshold in the diabetic rats.30
To prevent the neuropathic and microcirculatory effects of hyperglycemia, the
concentration of blood sugar should be kept at a suitable level, which could be
achieved by adjustment of the entire therapeutic program and regular assessment.7,8,31
In the current study we revealed that treadmill exercise attenuated diabetic peripheral
neuropathic pain, including tactile allodynia and heat hyperalgesia, but hyperglycemia
remains (>300mg/dL). Our work of treadmill training confirms previous studies
demonstrating hyperglycemia sustained and mechanical allodynia declined with
low-dose insulin alone.32 Interestingly, we demonstrated that diabetic rats receiving
insulin therapy or the combination of insulin therapy and treadmill training displayed
normal sensitivity to heat stimuli with a normal level of blood sugar. Furthermore,
induction of diabetes by STZ in rats presented a regular body weight loss, whereas
treadmill training, insulin therapy, or the combination reduced the body weight loss.
In agreement with the results via Selagzi et al., exercise training reinstated body
weight as well as markedly decreased motor latency and increased the compound
muscle action potential (CMAP) amplitude in the diabetic rats.33
It has been known that STZ-induced β-cell death and hyperglycemia is able to
release pro-inflammatory cytokines and cause activation of microglia in the spinal
19
dorsal horn.34 The previous study demonstrated that STZ-diabetic rats showed an
up-regulation of TNF-α and IL-6 expression in the peripheral nerves and spinal cord.9
Our current study declared that the level of TNF-α and IL-6 of the sciatic nerve
increased after 2-weeks STZ-treatment, whereas the IL-10 level decreased. Our report
is in accord with a study via Kumar and Sharma,15 who reported an elevation of
TNF-α and IL-6 was induced in the sciatic nerves of STZ-induced diabetic rats;
however, moderate treadmill running did not regulate the plasma IL-10 levels,
probably relating to the short duration and/or low intensity of the exercise program
used in diabetic rats.35
The action or production of IL-10 has been presumed to be deficient in both
experimental animals and human patients of type 1 diabetes.36,37 For instance,
decreased IL-10 production is related to a higher risk of type 1 diabetes in human
subjects (e.g., identical twins).36 Otherwise, recombinant IL-10 administration in
nonobese diabetic mice inhibits the progress of diabetes,38 whereas IL-10–deficient
nonobese diabetic mice exhibits accelerated diabetes.39 Anti–IL-10 receptor therapy
promotes the onset of diabetes while endogenous IL-10 suppresses spontaneous
diabetes progress in BDC2.5 TCR-transgenic/nonobese diabetic mice.40 This is in
resemblance to our present study that diabetic rats showed a reduction of IL-10 levels
on days 14 and 28 after STZ injection. Nevertheless, cytokines IL-1α and IL-1β were
20
reduced 2-fold, but IL-2 and IL-10 were unchanged in DRG and/or nerve of 2 and
5 month STZ-diabetic rats.10 These results can be explained by that evidence
demonstrated a reduction of IL-10 protein early following peripheral nerve injury,41
and others showed a significant increase in IL-10 mRNA late (35 days) during
peripheral nerve injury,42 meaning a critical role of the cytokine in regeneration,
rather than in the early phase of nerve degeneration. On the basis of these researches,
IL-10 appears to stand a candidate for immunotherapy, and therefore prevents the
onset, recurrence and progress of autoimmune diabetes.
Physical training is known to be associated with the overall improvement of
systemic inflammation in both type 1 diabetes and healthy humans.43,44 It has been
presumed that moderate-intensity aerobic activity has significant anti-inflammatory
actions in STZ-induced diabetic rats,35 and serum IL-6 in response to aerobic exercise
is appears to be alleviated in patients with type 1 diabetes.45 Here, we revealed that
three therapeutic methods are useful to increase the IL-10 level and to suppress the
IL-6 and TNF-α content as well as to attenuate diabetic peripheral neuropathic pain.
We do not estimate the direct relationship between pain and cytokines, whereas
TNF-α plays a critical role in the development of diabetic neuropathy, and their
blockade via infliximab, the multiple TNF-α antagonists, reverses established diabetic
neuropathy.46
21
It has been shown that anti-inflammatory cytokines (e.g., IL-1ra, IL-10 and IL-6)
are upregulated, whereas pro-inflammatory cytokine production is downregulated
during endurance exercise.47-49
Antigen administration through oral or nasal routes
was effective in causing TGF-β-and IL-10-secreting CD4+ T cell and avoiding
becoming diabetes in nonobese diabetic mice.50 Moreover, the ratio of IL-6-to-IL-10
production following pathogen-associated molecular patterns (PAMP) stimulation was
significantly greater in dogs with type 1 diabetes mellitus undergoing insulin therapy
than healthy dogs.51 Therefore, these treatments constitute important components of
the anti-inflammatory and immunosuppressive effects of IL-10, while IL-10
suppressed TNF-α and IL-1 levels as well as the generation of free radical oxygen
products and increased IFN-γ contents by inhibiting IL-2 production of the
antigen-presenting cells.52,53
There is a growing body of evidence that systemic IL-10 treatment,54 viral IL-10
(vIL-10) gene therapy,55 intramuscular injection of IL-10 plasmid DNA,56 the
recombinant adenovirus vector containing mIL-10 genes (Ad-mIL-10) 57 and the
combination of IL-10 plus IGF-1 58 can prevent and/or therapy diabetes.
IL-10 may
have potential for either treatment of other autoimmune disorders 55 or tolerance
induction in diabetic neuropathic pain.
Insulin, exercise or the combination therapy
altered the expression of cytokines, suggesting that treatment to increase IL-10 and
22
decrease IL-6 and TNF-α contents with newly diagnosed type 1 diabetes.
The factors like oxidative stress, lipid peroxidation, and advanced glycation end
products formation (AGEs) which result from diabetes mellitus can stimulate
inflammatory processes.59 The previous study showed that pharmacological inhibition
of ROS through nitrosobenzene or 5,5-dimethylpyrroline-N-oxide, and
phenyl-N-t-butyl nitrone (PBN) suppressed mechanical hypersensitivity in
neuropathic pain model.60-62 Here we showed that there is a significant increase in
nerve lipid peroxidation (MDA) that may contribute to early neuroinflammation in the
sciatic/peripheral nerve. This is similar to the finding that MDA levels were
significantly raised to 3–4 times in STZ-induced diabetic rats when compared with
control rats.15
Our findings showed that decreased MDA and proinflammatory cytokine levels
are in parallel with a suppression of diabetes-induced neuropathic pain by three
therapeutic methods (insulin, treadmill training or insulin with treadmill training). The
present data are in agreement with the previous experiment that found that
administration of resveratrol exhibited a marked reduction of nerve MDA contents in
the diabetic rats which may contribute to the decrease in IL-6 and NF-alpha in the
sciatic nerves of acute STZ-induced diabetic rats as well.15 Additionally, Prasad and
Muralidhara reported that the property of geraniol (GE) was to regulate
23
neurotransmission, mitochondrial function, and the oxidative stress markers under
diabetic neuropathy conditions.16 Furthermore, a previous study showed that zinc
application attenuates the functional decline of peripheral nerves in diabetic rats and
the protective effect may be involved in its inhibitory effect on oxidative stress
through decreasing MDA expression and through modulating metallothionein
contents.17 The supplementation,15-17 exercise, insulin and the combination are worth
testing in future.
CONCLUSIONS
The present study shows that both treadmill training and insulin therapy are more
effective in improving diabetic neuropathic pain than insulin therapy and treadmill
training either solely. Each of insulin therapy & treadmill training separately, as well
as the combination (insulin therapy with treadmill training), reduces these animals
pain as measured by increases in the mechanical- and thermal-stimulation threshold
values, and attenuates the levels of TNF-α, IL-6, and MDA. Scientific and clinical
support for the applications of insulin administration or treadmill training in the
therapy of diabetic peripheral neuropathic pain can be contributing to annotating the
mechanism of action and the establishment of patient indications and appropriate
recommendations. Too often are considered an adjunct to insulin therapy, routine
treadmill training can be taken in mind as an overall therapeutic part of diabetic
24
intervention.
25
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FIGURE LEGENDS
Fig. 1. The time courses of blood glucose (A) and body weight (B) in the NS, SS, SE,
SI and SEI rats (NS: normal sedentary rats; SS: sedentary streptozotocin (STZ)-rats;
SE: STZ-rats receiving treadmill training; SI: STZ-rats receiving insulin treatment;
SEI: STZ-rats receiving insulin treatment and treadmill training). The values are
shown as mean ± S.E.M. and n =12 rats in each group. The plus symbol indicates P <
0.05 when compared with the SS group; the asterisk indicates P < 0.05 when
compared with the NS group (2-way ANOVA of repeated measures followed by post
hoc Bonferroni test).
Fig. 2. The time course of the blood glucose level in the STZ-rats receiving insulin
treatment. The values are presented as mean ± S.E.M. for 12 rats.
Fig. 3. The behavioral time course of the mechanical withdrawal threshold (A) and
the heat withdrawal latency (B) in the NS, SS, SE, SI and SEI groups, where NS =
normal sedentary rats; SS = sedentary streptozotocin (STZ)-rats; SE = STZ-rats
receiving treadmill training; SI = STZ-rats receiving insulin treatment; SEI = STZ-rats
receiving insulin treatment and treadmill training. The values are shown as mean ±
S.E.M. and n =12 rats in each group. The plus symbol indicates P < 0.05 when
37
compared with the SS group; the asterisk indicates P < 0.05 when compared with the
NS group (2-way ANOVA of repeated measures followed by post hoc Bonferroni
test).
Fig. 4. The levels of tumor necrosis factor (TNF)-α (A), interleukin (IL)-6 (B), IL-10
(C) and malondialdehyde (MDA) (D) on days 14 and 28 after STZ administration in
the sciatic nerve of the NS, SS, SE, SI and SEI rats (NS: normal sedentary rats; SS:
sedentary streptozotocin (STZ)-rats; SE: STZ-rats receiving treadmill training; SI:
STZ-rats receiving insulin treatment; SEI: STZ-rats receiving insulin treatment and
treadmill training). The values are shown as mean ± S.E.M. of the five separate
experiments. The plus symbol indicates P < 0.05 when compared with the SS group
(1-way ANOVA followed by pairwise Tukey HSD test).
38