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Long-term Voluntary Exercise Decreased the
Incidence of Apoplexy and Elongated the
Lifespan through Activation of eNOS and
Inhibition of Inflammatory Signaling Pathway
in Stroke-Prone SHR
Hideaki Higashino, Atsuko Niwa, Masaki Tabuchi,
Kana Ooshima, and Hiroshi Sakaue
Department of Pharmacology, Kinki University School of Medicine,
Osaka-Sayama, 589-8511, Japan.
Intervention Therapy for Hypertension
1. Guidance for Improvement of Life Style
a. Food (low salt, low calorie, much fiber)
b. Physical exercise
c. Save the body weight
d. To avoid much stress
e. Enjoy the daily life
2. Drug therapy
a. Early treatment
b. Select the appropriate drugs for prevention of AS
Objective:
Clinical evidences show that exercise
exerts atheroprotective or beneficial
effects on cardiovascular events.
Precisely causative mechanisms,
however, are still unknown.
Therefore, the hypothesis that endurance
voluntary exercise decreases the
inflammatory signaling through eNOS
induction and ROS inhibition was assessed
in SHRSP.
Methods
Animals:Male SHRSP aged 6-week-old at pre-hypertensive stage
Groups: 1. Voluntary wheel-running (WR): 2 to 3 km running/day
2. sedentary control (SED): in the cage without running
Duration:8 weeks
Analyses:
Thoracic Aortae: NOS expression, eNOS activity, oxidative stress
Akt, eNOS, phosphorylated ones by western blotting
NADPH oxidase mRNA by RT-PCR.
Activities of eNOS by using [3H]l-arginine
Blood: Superoxide (O2-) production by flow cytometer using DHE
Plasma: sICAM-1, MCP-1, 8-iso-PGF2αby ELISA
Observation of the occurrence of apoplexy: keeping them until the death
Changes of blood pressure in SEDENTARY
and EXERCISED SHRSP
Systolic blood pressure
(mmHg)
280.0
SEDENTARY
260.0
240.0
EXERCISE
220.0
* P<0.05 * P<0.05
200.0
** P<0.01
180.0
mean + SEM (n=6-12)
Significant difference compared with SED
160.0
～
140.0～
6
7
8
9
10
11
12
Age (weeks)
13
14
15
16
100
Stroke morbidity (%)
Incidents of Stroke determined by stroke
scores in SED and WR SHRSP
SEDENTARY (n=15)
50
EXERCISE (n=13)
EX vs. SED; P=0.016
Start of exercise
0
0
50
100
Age (Days)
150
200
Periods of Life-Span in Sedentary and Exercised SHRSP
1
Exercised Rats
Surviving
(n=10)
Sedentary rats
(n=9)
0.5
p<0.05 ; WR vs.SED
0
100
200
Age (days)
SEDENTARY
EXERCISE
Thickness of SM layers and Collagen area
in Thoratic aortae after exercise in SHRSP
Collagen area of
Thickness of media
vessel wall
(mm)
(%)
0.4
30
25
0.3
20
p<0.001
*
0.2
15
p<0.001
10
*
0.1
5
n=10
0
SED
12
WR
0
9
12
SED
WR
(Mean ± SEM)
Expression of angiotensin (AT)1 receptors and
AT2 receptors in the aortas of SED and WR
SHRSP
SEDENTARY AT1R
EXERCISE AT1R
SEDENTARY AT2R
EXERCISE AT2R
2
ACE
AT1R/AT2R
1.4
1.2
1.5
1
p<0.005
*
0.5
ACE (arb. units)
AT1R / AT2R (arb. units)
Levels of AT1 & AT2 receptors, and ACE
in the aortae between SED and WR
1
0.8
0.6
0.4
0.2
n=6
0
6
n=8
6
SED
WR
0
SED
WR
(Mean ± SEM)
NAD(P)H oxidase Subunit (Nox1) RNA in Aortas of EX SHRSP
Nox1
mRNA
1.2
mRNA/ 18S rRNA
1.2
1
0.8
0.6
0.4
0.2
4
4
Relative intensity (arb. units)
1.4
protein
1
P<0.01
*
0.8
0.6
0.4
0.2
5
8
8
SED
WR
0
0
SED
WR
Levels of eNOS, p-eNOS and p-Akt/Akt in the Aortae
eNOS protein
1.4
1.2
Ser1177-p-eNOS
p-Akt/Akt
1.8
p<0.001
Relative intensity (arb. units)
p<0.05
1.2
1.6
1
1.4
1
0.8
1.2
0.8
1
0.6
0.8
0.6
0.4
0.6
0.4
0.4
0.2
0.2
0.2
n=11
11
12
12
SED
WR
12
SED
WR
0
0
0
12
SED
WR
(Mean ± SEM)
ROS and NO Productions in the Aortae between SED and WR
2500
ROS production
NO production
x104
450
*
*
400
2000
P<0.05
P<0.01
350
300
1500
250
*
200
SED
150
100
500
5
5
0
50
5
5
5
EXERCISE
1000
P<0.01
5
5
5
0
SED
WR
Basal ACh 10-5M Insulin10-6M
Comparison of NOS activities and cGMP production
in the Aortae
NOS activity
cGMP
600
p<0.05
14
cGMP (fmol/mg protein)
[3H]l-citrulline (nmol/mg/min)
16
12
10
500
400
E
8
6
4
300
200
100
2
n=5
5
0
7
11
SED
WR
0
SED
WR
(Mean ± SEM)
Nitrotyrosine contents in Aortae and MFI by DHE in the Blood
Nitrotyrosine in Aortae
1.4
MFI in the Blood
140
p<0.001
120
1.2
Dihydroethidium (MFI)
Nitrotyrosine (arb. units)
p<0.05
1
0.8
0.6
0.4
80
60
40
20
0.2
n=7
0
100
SED
8
WR
4
5
SED
WR
0
(Mean ± SEM)
Changes of Phosphorylated Akt, and ERK1/2 levels
in the Aortae between SED and WR
p-Akt/Akt
p<0.001
*
1.6
p-Akt/Akt
1.4
1.2
1
0.8
0.6
p-ERK/ERK (arb. units)
1.6
1.8
1.4
1.2
1
p<0.05
0.8
*
0.6
0.4
0.4
0.2
p-ERK/ERK
n=12
12
0.2
8
7
SED
WR
0
0
SED
WR
(Mean ± SEM)
Changes of serum TGF-β levels after exercise
6
TGF-β(ng/ml)
5
4
3
2
1
0
n=6
4
SED
WR
(Mean ± SEM)
Comparison of inflammatory biomarkers in SED and EX
Plasma concentration
(mg/ml) high sensitive CRP
600
(pg/ml) soluble ICAM-1
25
p<0.01
500
*
20
400
15
p<0.05
300
*
200
100
7
6
10
5
22
22
SED
WR
0
0
SED
WR
Concentrations of PAI-1 and MCP-1 in the Plasma
MCP-1
(ng/ml)
(ng/ml) Complete PAI-1
10
7
Plasma concentration
6
8
5
6
4
p<0.05
3
*
p<0.05
*
4
2
1
6
4
2
8
7
SED
WR
0
0
SED
WR
Physical Exercise
Shear stress
Ang II, TNF-α
MCP-1
ICAM-1
AT1 Rs/AT2 Rs
PI3K
Mφ
PLC, PLD
Akt
NAD(P)H
oxidase
eNOS
NO
ROS
ONOO-
MAP kinases
cGMP
Hypertension
Apoplexy
Fibrosis
Prevention of Cardio-vasculitis
Vascular dilatation
Conclusions:
Data showed that exercise could protect
oxidative stress-induced cell injury or
inflammation by an interaction with
signaling molecules such as ASK1 /JNK/
p38MAPK through NO production and
inhibition of superoxide production.
Then, voluntary exercise significantly
attenuated the changes of vascular
remodeling, delayed stroke events and
elongated the lifespan in exercised rats.