Download Effect of Interleukin-6 (IL-6) on the Vascular Smooth Muscle

Chinese Journal of Physiology 54(5): 318-323, 2011
DOI: 10.4077/CJP.2011.AMM069
Effect of Interleukin-6 (IL-6) on the Vascular
Smooth Muscle Contraction in Abdominal
Aorta of Rats with Streptozotocin-Induced
Diabetes
Wen-Bo Tang 1, Yu-Qin Zhou 1, Ting Zhao 1, Jing-Li Shan 1, Peng Sun 1, Ting-Ting Yang 1,
Xin-Wen Chang 2, Sen Li 2, Paulus S. Wang 3, and Dong-Ping Xie 2
1
Institute of Physiology, School of Medicine, Shandong University, Jinan 250012,
Shandong, P.R. China
2
Department of Physiology, School of Medicine, Tongji University,
Shanghai 200092, P.R. China
and
3
Department of Physiology, School of Medicine, National Yang-Ming University,
Taipei 11221, Taiwan, R.O.C.
Abstract
Patients with type 1 diabetes are at a risk of hypertension. However, the mechanisms behind the
findings are not completely known. The aim of the present study was to investigate involvement of
interleukin-6 (IL-6) on the contraction of abdominal aorta in rats with type 1 diabetes. IL-6 levels in the
plasma of rats with streptozotocin (STZ)-induced diabetes were determined by ELISA. The abdominal
aorta was dissected free of fat and connective tissues and then cut into spiral rings. The endotheliumdenuded strip was vertically suspended in tissue chambers containing 5 ml Krebs solution at 37°C and
bubbled continuously with 95% O 2-5% CO 2. The effects of phenylephrine (Phe) on the contractile
responses of abdominal aorta were recorded. The effects of IL-6 and anti-rat IL-6 antibody on the Pheinduced response were also examined. Plasma levels of IL-6 increased time-dependently in rats with
STZ-induced diabetes. Phe caused concentration-dependent contraction in aortic rings. Phe-induced
contractions were higher in vascular strips of STZ-induced diabetic rats than that of control rats.
Pretreatment of vascular strips with IL-6 for 1 h did not cause contraction but enhanced the contraction
in response to Phe. Treatment of the vascular strips with an anti-IL-6 antibody for 1 h decreased the
Phe-induced contractions. These results suggest that IL-6 causes vascular smooth muscle contraction
in abdominal aorta of rats with type 1 diabetes.
Key Words: abdominal aorta, smooth muscle contraction, interleukin-6, type 1 diabetes
Introduction
peripheral resistance thereby contributing to diabetic
retinopathy, nephropathy, neuropathy and a higher
rate of hypertension. Patients with type 1 diabetes are
also at a substantially increased risk of hypertension.
However, the cellular and molecular mechanisms of
these effects are not completely known.
Type 1 diabetes is associated with increased
Vascular complications are the major causes of
increased mortality and morbidity in diabetic patients
(2). Abnormalities in blood vessel constriction or
dilatation (vasomotion), detected in early diabetes,
can result in blood flow dysregulation and increased
Corresponding author: Dr. Dong-ping Xie, Department of Physiology, School of Medicine, Tongji University, Shanghai 200092, P.R. China.
Tel: +86-021-6598-5448, Fax: +86-021-6598-5448, E-mail: [email protected].
Received: July 20, 2010; Revised (Final Version): November 6, 2010; Accepted: November 15, 2010.
2011 by The Chinese Physiological Society and Airiti Press Inc. ISSN : 0304-4920. http://www.cps.org.tw
318
IL-6 and Abdominal Aorta Motility
cytokine-mediated inflammation. Monocyte interleukin (IL)-6 levels are significantly elevated in type
1 diabetic subjects (4), and plasma concentrations of
IL-6 are high in type 1 diabetic patients (1, 10, 16).
IL-6 is increased during hyperglycemia in young and
children patients with type 1 diabetes (7, 15). Lower
heart rate variability is associated with higher plasma
concentrations of IL-6 in type 1 diabetes (6). Hypertensive patients have high circulating levels of IL-6.
IL-6 overexpression induces pulmonary hypertension
(17). A role for IL-6 in hypertension is suggested by
its contractile effects on smooth muscle, including
vascular smooth muscle.
IL-6 enhances α(1)-adrenergic receptor-mediated
contraction in the corpus cavernosum of the rat (12).
IL-6 directly impairs endothelium-dependent relaxation
and enhances vascular contraction in systemic vessels
of pregnant rats (13). IL-6 might play a role in the
vascular contraction of type 1 diabetes. In this study,
we have investigated the involvement of IL-6 on the
contraction of abdominal aorta in rats with type 1
diabetes.
Materials and Methods
Animals
Experiments were performed on male Wistar
rats. Animals were 4-6 weeks old and weighed
180 ± 10 grams at the beginning of the experiment.
Rats were housed in a temperature (22°C)-controlled
environment. The use and treatment of animals
followed the guidelines of the International Animal
Care and Use Committee of Shandong University.
All animals were cared for in compliance with the
Principles of Laboratory Animal Care and the Guide
for the Care and Use of Laboratory Animals published by the National Natural Science Foundation of
PRC.
Diabetes was induced with an intraperitoneal
injection of streptozotocin (STZ, 60 mg/kg) in sodium
citrate buffer (0.1 M, pH 4.2). Control animals were injected with the sodium citrate buffer alone. Body
weight and blood glucose levels were measured at the
beginning of the experiment. Glucose levels were measured with glucose test strips (Bayer, Elkhart, IN, USA).
Blood glucose levels were measured again, after 1
week, to confirm the development of hyperglycemia.
Four weeks after the induction of diabetes, body
weight and blood glucose levels were measured, the
animals were sacrificed, and tissue samples were collected for in vitro studies. A second group of animals
was killed after 16 weeks to measure IL-6 levels in
blood. A third group of untreated animals was killed
at 10-12 weeks of age. Aortic samples from these
animals were used to study the effect of IL-6 on con-
319
tractile responses.
Measurement of Plasma IL-6
IL-6 levels in blood were measured with an
ELISA system (Cytoscreen, Biosource International,
Camarillo, CA, USA). The assay is a solid-phase
sandwich-type system that utilizes a specific anti-rat
IL-6 antibody coated onto the wells of microtiter
plates. Serum samples (50 µl) and standards were
pipetted in triplicate into appropriate microtiter wells,
and the assay was performed according to manufacturer’s instructions. The sensitivity of this IL-6 ELISA
system is 0.7 pM, and the upper limit of detection is
150 pM.
Tissue Culture
Strips of the abdominal aorta were placed in
Krebs solution containing 119 mM NaCl, 4.75 mM
KCl, 1.2 mM KH 2 PO 4 , 25 mM NaHCO 3 , 1.5 mM
MgSO 4 , 2.5 mM CaCl 2 and 11 mM glucose. They
were dissected free of fat and connective tissues and
then cut into 2 × 8 mm spiral strips. The endothelium
was removed by rubbing the vessel interior with a wet
cotton swab. Removal of the endothelium was verified
by the absence of ACh relaxation in vascular strips
precontracted with phenylephrine (Phe).
Vascular strips were studied in an apparatus in
which one end of the loop was attached to a glass hook
and the other end was connected to an external
isometric force transducer (JH-2B, Beijing, PRC).
They were stretched to an initial tension of 1 g and
allowed to equilibrate for 30 min in a tissue bath filled
with 5 ml Krebs solution continuously bubbled with
95% O 2-5% CO 2 at 37°C. The changes in isometric
force were recorded using an MFlab system with
SMUP-PC amplifier (Fudan University, Shanghai,
PRC).
Dose-Response Effects of Phe on the Contraction of
Abdominal Aorta in STZ-Diabetic Rats
Aortic rings from 10- to 12-week-old STZdiabetic and age-matched control rats were mounted
on the apparatus, left to equilibrate for 30 min, and
then contracted using increasing concentrations of
Phe (0.001-3 µM). Concentration-contraction curves
were then constructed.
Effects of IL-6 on Phe-Induced Abdominal Aorta
Contraction in Normal Rats
In another set of experiments, intact 10- to 12week-old male Wistar rats were killed and aortic
rings were prepared. The strips were either nontreated,
320
Tang, Zhou, Zhao, Shan, Sun, Yang, Chang, Li, Wang and Xie
A
Plasma Glucose Concentration (mM)
25
20
Control
Diabetes
*
or treated with IL-6 (0.1 µg/ml) for 1 h. Then, increasing concentrations of Phe (0.001-3 µM) were
applied, and the effects on Phe-induced contraction
were measured.
Effects of Anti-Rat IL-6 Antibody on Phe-Induced
Abdominal Aorta Contraction in STZ-Diabetic Rats
15
10
5
0
0 Week
4 Weeks
The effect of anti-IL-6 anitbody was tested on
aortic rings from 10- to 12-week-old STZ-diabetic
and age-matched controls. Aortic rings were either
nontreated or treated with an anti-rat IL-6 antibody
(10 ng/ml) for 1 h, following which the dose-response
curves were recorded following Phe administration.
Chemicals
B
500
Body Weight (g)
400
Control
Diabetes
300
*
STZ and Phe were obtained from Sigma Co.
Ltd. (St. Louis, MO, USA). Recombinant rat IL-6 and
anti-rat IL-6 antibody were purchased from Peprotech
Inc. (Rocky Hill, NJ, USA). STZ was dissolved in
sodium citrate buffer (0.1 M, pH 4.2). Phe, IL-6 and
anti-rat IL-6 antibody were prepared in distilled water.
200
Data Analysis
100
0
0 Week
4 Weeks
C
Plasma IL-6 Concentration (pg/ml)
100
80
Control
Diabetes
#
*
Data are presented as means ± SEM, with n indicating the number of rats. A paired t-test was used
to test for a significant difference between two treatments in one group. ANOVA repeat measures were
used to evaluate differences among more than two
groups. A probability level of P < 0.05 was considered
to be significant for statistical analysis.
Results
60
Blood Glucose and Body Weight Levels in STZ-Diabetic
and Control Rats
*
40
20
0
4 Weeks
16 Weeks
Fig. 1. Plasma concentration of blood glucose (A), the body
weight (B) and the plasma concentration of IL-6 (C) in
STZ-treated and the sodium citrate buffer-treated rats.
Control, gender/age-matched sodium citrate buffertreated rats; Diabetes, gender/age-matched STZ-treated
rats. Zero week, the time before the injection of STZ; 4
weeks, the time after hyperglycemia lasted for 4 weeks;
16 weeks, the time after hyperglycemia lasted for 16
weeks. *P < 0.05 vs. control at the same time point;
#
P < 0.05 vs. STZ induced diabetes rat hyperglycemia
lasted for 4 weeks. Data are means ± SEM of 6-12
samples.
Four weeks after induction of hyperglycemia,
plasma glucose concentrations in STZ-treated rats
were significantly greater than in the control animals,
while body weight of the diabetic animals was significantly less than the controls (Fig. 1, A and B).
Plasma Concentrations of IL-6 in STZ-Diabetic and
Control Rats
In the 4-week STZ diabetic animals, the plasma
concentration of IL-6 was significantly higher than that
of the controls (the value in STZ rats was 44.58 ± 3.18
pg/ml; the value in control rats was 11.99 ± 1.9 pg/ml.
P < 0.05). In the 16-week STZ animals, the plasma
concentration of IL-6 in type 1 diabetic rats increased
to 81.97 ± 5.00 pg/ml which was significantly higher
than the 4-week group (Fig. 1C, P < 0.05).
IL-6 and Abdominal Aorta Motility
A
10-9
10-8
[Phe] (M)
10-7
321
A
10-9
10-6
[Phe] (M)
10-7
10-8
g
g
0.5
Control
0.5
Control
2.5
2.5
g
Diabetes
0.5
1725
g
IL-6
0.5
3450
1725
Time (s)
Time (s)
B
3450
B
2.4
2.0
Control
Diabetes
2.2
*
2.0
*
*
*
*
1.8
*
1.6
1.4
1.2
1.0
.8
-9
-8.5
-8
-7.5
-7
-6.5
-6
-5.5
Log [Phe] (M)
Fig. 2. The contractile response to increasing doses of Phe
(0.001-3 µM) of one aortic strip (A) and dose-response
curve (B). Phe, phenylephrine. Control, the gender/agematched sodium citrate buffer treated rats; Diabetes, the
gender/age-matched STZ induced diabetes rats. Drug
was applied at points marked by the downward-pointing
arrows. Each point represents the means ± SEM. *P <
0.05 compared with the control (n = 6-12).
Effects of Phe on the Contraction of Abdominal Aorta in
STZ-Diabetic Rats
The contractile responses of one aortic strip to
increasing doses of Phe are shown in Fig. 2A. Data
from 6-12 rats are summarized in Fig. 2B. Low concentrations of Phe (0.001 µM) failed to elicit effects
on contraction of abdominal aorta strips. Phe (0.013 µM) dose-dependently increased the contractions
of abdominal aorta strips. Phe (0.01-3 µM)-induced
contraction appeared to be higher in the vascular
strips isolated from STZ-diabetic rats (P < 0.05 compared with gender/age-matched control rat).
Effect of IL-6 on Phe-Induced Abdominal Aorta
Contraction in Normal Rats
IL-6 (0.1 µg/ml) had no effect on the tension of
abdominal aorta strips (P > 0.05, n = 6). However,
pretreating rings with IL-6 for 1 h increased the response to Phe (Fig. 3, P < 0.05).
Tension of Abdominal Aorta (g)
Tension of Abdominal Aorta (g)
10-6
2.5
2.5
Control
IL-6
1.8
*
1.6
*
*
*
1.4
1.2
1.0
.8
-9
-8.5
-8
-7.5 -7 -6.5
Log [Phe] (M)
-6
-5.5
Fig. 3. Effects of IL-6 (0.1 µg/ml) on Phe (0.001-3 µM)-induced
contraction of abdominal aorta in normal rats. (A)
Representative of the recordings of aortic motility. (B)
Dose-dependent response curves induced by Phe and
effect of IL-6 on Phe-induced contraction of abdominal
aorta. Phe, phenylephrine. Control, the gender/agematched normal rats. Drug was applied at points marked
by the downward-pointing arrows. Each point represents the means ± SEM. *P < 0.05 compared with the
contraction in the presence of IL-6 at the same time
points (n = 6).
Effect of Anti-Rat IL-6 Antibody on Phe-Induced
Contraction in STZ-Diabetic Rats
To test the effect of anti-rat IL-6 antibody on
contractile responses of aortic strips, strips were treated
with an anti-rat IL-6 antibody (10 ng/ml) for 1 h
following which Phe (0.001-3 µM)-induced contractions of the vascular strips in type 1 diabetes rats
were studied (Fig. 4A). Antibody treatment alone had
no effect on the tension of the abdominal aorta strips
(P > 0.05, n = 6). However, the IL-6 antibody treatment reduced the Phe contractile response in the STZ
rats (Fig. 4B). At a dose of 0.01 µM, Phe-induced
contraction of the STZ vascular strips was 1.16 ± 0.09
g, which was lower than the Phe response in controls
(1.62 ± 0.14 g, P < 0.05). At 0.03 µM, the Phe-induced
response of the anti-rat IL-6 group was almost the
same with that of controls (1.17 ± 0.09 g, P > 0.05).
But when the concentrations were higher than 0.03 µM
322
Tang, Zhou, Zhao, Shan, Sun, Yang, Chang, Li, Wang and Xie
A
[Phe] (M)
2.5
10-9
10-8
10-7
10-6
g
g
Control
0.5
2.5
Diabetes
0.5
2.5
g
Diabetes + Anti-IL-6
0.5
1725
3450
Time (s)
B
2.8
Tension of Abdominal Aorta (g)
2.6
2.4
Control
Diabetes
Diabetes + Anti-IL-6
2.2
2.0
1.8
1.6
1.4
*
*
1.2
1.0
.8
-9.5 -9.0 -8.5 -8.0 -7.5 -7.0 -6.5 -6.0 -5.5 -5.0
Log [Phe] (M)
Fig. 4. Effects of anti-rat IL-6 antibody (anti-IL-6, 10 ng/ml) on
Phe (0.001-3 µM)-induced contraction of abdominal
aorta in STZ-diabetic rats. (A) Representative of the
recordings of aortic motility. (B) Dose-dependent response curves induced by Phe and effect of anti-IL-6
on Phe-induced contraction of abdominal aorta. Phe,
phenylephrine. Control, the gender/age-matched sodium citrate buffer treated rats. Diabetes, the gender/
age-matched STZ treated rats; Diabetes + anti-IL-6,
treatment of anti-IL-6 antibody in the abdominal aorta
strips isolated from diabetic rats. Drug was applied at
arrow-marked points. Each point represents the means ±
SEM. *P < 0.05 compared with the contraction of
abdominal aorta strips isolated from diabetic rats in the
presence of anti-IL-6 antibody at the same time points
(n = 6).
(0.1-3 µM), the anti-IL-6 antiboy was without effect:
Phe-induced responses of abdominal aorta strips in
STZ rats were not significantly different from those of
the STZ rats in the absence of anti-rat IL-6 antibody at
the same time points (P > 0.05).
Discussion
The main findings of the present study are: [1]
Concentrations of plasma IL-6 in type 1 diabetic rats
are increased, [2] IL-6 enhances the contraction of
abdominal aorta strips in rats, and [3] IL-6 is involved
in the enhanced vascular contraction of rats with type
1 diabetes.
In diabetes, early inflammation causes the
release of chemokines such as monocyte chemoattractant protein 1 (MCP-1) in vascular endothelium
and in adipose tissues. These factors increase expression of interstitial and vascular cellular adhesion
molecules and attract monocytes and immunocytes.
They undergo chemokine-induced proliferation and
proinflammatory gene activation producing cytokines
like tumor necrosis factor α (TNFα), interleukin-1
(IL-1), IL-6, IL-18, interferon (IFN) and others. Many
of these molecules enter the circulation where their
levels correlate with the degree of inflammatory activity. Among these cytokines, IL-6 appears to be a
very important cytokine associated with type 1 diabetic
(5). Consistent with previous studies on diabetic
patients (1, 10, 16), we found that blood level of IL6 elevates in the rats with type 1 diabetes.
The risk of peripheral vascular disease is increased in diabetic patients in whom it occurs earlier
and is often more severe and diffuse (2). Data in
patients with type 1 diabetes mellitus show a significant increase in arterial pressure (11). Endothelial
dysfunction, vascular smooth muscle cell dysfunction,
inflammation and hypercoagubility are the key factors
in diabetic arteriopathy (8). We compared the Pheinduced vascular contraction between type 1 diabetic
and control rats. The results showed that the vascular
contractions were higher in type 1 diabetic rats than in
the control rats suggesting that diabetes-associated
vascular smooth muscle shows hyper-contractility.
Derosa et al. (3) evaluated systolic blood pressure (SBP), diastolic blood pressure (DBP) and IL-6
in the diabetic hypertensive (DH) patients. They
found that SBP, DBP and IL-6 were significantly
higher in the DH group. After 6 months of candesartan
therapy, IL-6 decreased. Furthermore, there was a
significant decrease of SBP and DBP values in the
DH group. The results suggested the involvement of
IL-6 in the diabetic hypertensive patients. Iversen
et al. (9) investigated the effect of IL-6 on vascular
tone in isolated human arterial and venous segments
from various organs. They found that IL-6 induced
contraction of arterial but not of venous segments.
Studies in pregnant rats showed that IL-6 increased
both systolic blood pressure and isometric contraction
of isolated aortic strips (14). IL-6 was also reported
to enhance vasomotor reactivity of the corpus cavernosum in rats (12). In our studies, we first observed
the effect of IL-6 on Phe-induced abdominal aorta
contraction in normal rats, and that IL-6 increased the
Phe-induced contraction. We further observed the
effect of anti-rat IL-6 antibody treatment on Pheinduced contraction in type 1 diabetic rats, and that
IL-6 and Abdominal Aorta Motility
anti-rat IL-6 antibody partly decreased Phe-induced
contraction. These results suggest that IL-6 is involved
in the vascular hyper-contractility of rats with type 1
diabetes. IL-6 might increase the rate of diabetic hypertension via vascular hyper-contractility.
In conclusion, IL-6 excites the vascular smooth
muscle contraction in abdominal aorta of the rats with
type 1 diabetes.
7.
8.
9.
Acknowledgments
10.
We thank Peter Grigg for his critical reading of
the manuscript. This work was supported by National
Natural Science Foundation of PRC (30872475),
National Undergraduates Innovating Experimentation
Project and the 985 Foundation of Tongji University
in PRC.
11.
12.
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