Hideaki Higashino , Atsuko Niwa, Masaki Tabuchi, Kana Ooshima, and Hiroshi Sakaue Department of Pharmacology, Kinki University School o f Medicine, Osaka-Sayama, 589-8511, Japan. Long-term Voluntary Exercise Decreased the Incidence of Apoplexy and Elongate d the Lifespan through Activation of e NOS and Inhibition of Inflammatory Sig naling Pathway in Stroke-Prone SHR
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Hideaki Higashino , Atsuko Niwa, Masaki Tabuchi, Kana Ooshima, and Hiroshi Sakaue
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 - PowerPoint PPT Presentation
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Hideaki Higashino, Atsuko Niwa, Masaki Tabuchi, Kana Ooshima, and Hiroshi Sakaue
Department of Pharmacology, Kinki University School of Medicine, Osaka-Sayama, 589-8511, Japan.
Long-term Voluntary Exercise Decreased the Incidence of Apoplexy and Elongated the Lifespan through Activation of eNOS and Inhibition of Inflammatory Sig
naling Pathway in Stroke-Prone SHR
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 SHR
SP.
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 runningDuration: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 DHEPlasma: sICAM-1, MCP-1, 8-iso-PGF2αby ELISA
Observation of the occurrence of apoplexy: keeping them until the death
140.0
160.0
180.0
200.0
220.0
240.0
260.0
280.0
Age (weeks)
Syst
olic
blo
od p
ress
ure
6 7 8 9 10 11 12 13 14 15 16
(mmHg)
** P<0.01
* P<0.05* P<0.05
SEDENTARY
EXERCISE
Significant difference compared with SEDmean + SEM (n=6-12)
~~
Changes of blood pressure in SEDENTARY and EXERCISED SHRSP
0 50 100 150 200
0
50
100
Age (Days)
(%) SEDENTARY (n=15)
EXERCISE (n=13)
EX vs. SED; P=0.016
Stro
ke m
orbi
dity
Start of exercise
Incidents of Stroke determined by stroke scores in SED and WR SHRSP
0
0.5
1
100 200Age (days)
Periods of Life-Span in Sedentary and Exercised SHRSPSu
rviv
ing
p<0.05 ; WR vs.SED
Sedentary rats(n=9)
Exercised Rats(n=10)
SEDENTARY EXERCISE
0
0.1
0.2
0.3
0.4
SED WR
0
5
10
15
20
25
30
SED WR
(mm) (%)Thickness of media Collagen area of
vessel wall
*
*p<0.001
p<0.001
n=10 12 9 12
Thickness of SM layers and Collagen area in Thoratic aortae after exercise in SHRSP
(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
0
0.5
1
1.5
2
SED WR
AT 1
R /
AT 2
R
n=6 6
*p<0.005
(arb
. uni
ts)
(Mean ± SEM)
Levels of AT1 & AT2 receptors, and ACE in the aortae between SED and WR
0
0.2
0.4
0.6
0.8
1
1.2
1.4
WR
AC
E (a
rb. u
nits
)
SED
AT1R/AT2R ACE
n=8 6
Nox1
Rel
ativ
e in
tens
ity (a
rb. u
nits
)
0
0.2
0.4
0.6
0.8
1
1.2
1.4m
RN
A/ 1
8S rR
NA
SED WR
4 4
mRNA protein
0
0.2
0.4
0.6
0.8
1
1.2
SED WR
5 8
P<0.01
*
NAD(P)H oxidase Subunit (Nox1) RNA in Aortas of EX SHRSP
8
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Rel
ativ
e in
tens
ity (a
rb. u
nits
)
p-Akt/Akt
SED WR
p<0.001
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0
0.2
0.4
0.6
0.8
1
1.2Ser1177-p-eNOSeNOS protein
SED SEDWR WR
p<0.05
Levels of eNOS, p-eNOS and p-Akt/Akt in the Aortae
(Mean ± SEM)
12 12 n=11 12 11 12
ROS production
0
500
1000
1500
2000
2500
SED WR
5 5
P<0.01
*
0
50
100
150
200
250
300
350
400
450
Basal ACh 10-5M Insulin10-6M
NO production
55 5 5 55
P<0.01
*P<0.05
*
x104
SED
EXERC
ISE
ROS and NO Productions in the Aortae between SED and WR
0
2
4
6
8
10
12
14
16
[3 H]l-
citr
ullin
e (n
mol
/mg/
min
)
0
100
200
300
400
500
600
cGM
P (fm
ol/m
g pr
otei
n)
NOS activity
SED WR
cGMP
Comparison of NOS activities and cGMP production in the Aortae
SED WR
p<0.05
7 11n=5 5
(Mean ± SEM)
E
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Nitr
otyr
osin
e (a
rb. u
nits
)
0
20
40
60
80
100
120
140
Dih
ydro
ethi
dium
(MFI
)
Nitrotyrosine in Aortae
SED WR
MFI in the BloodMFI in the Blood
SED WR
Nitrotyrosine contents in Aortae and MFI by DHE in the Blood
p<0.05 p<0.001
(Mean ± SEM)
n=7 8 4 5
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
SED WR0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8p-Akt/Akt
p<0.001
n=12 12
SED WR
8 7
*
*p<0.05
p-ERK/ERK
Changes of Phosphorylated Akt, and ERK1/2 levels in the Aortae between SED and WR
p-A
kt/A
kt
p-ER
K/E
RK
(Mean ± SEM)
(arb
. uni
ts)
0
1
2
3
4
5
6
SED WR
TGF-
β(ng
/ml)
n=6 4
(Mean ± SEM)
Changes of serum TGF-β levels after exercise
0
100
200
300
400
500
600
SED WR
high sensitive CRPg/ml)
p<0.05
7 6
*
soluble ICAM-1
5
10
15
20
25
0SED WR
(pg/ml)
22 22
p<0.01
*
Plas
ma
conc
entr
atio
nComparison of inflammatory biomarkers in SED and EX
0
1
2
3
4
5
6
7
WR
Complete PAI-1
6 4
(ng/ml)(ng/ml)
0
2
4
6
8
10
SED WR
MCP-1
8 7
p<0.05
*
SED
p<0.05
*
Plas
ma
conc
entr
atio
nConcentrations of PAI-1 and MCP-1 in the Plasma
4
Physical Exercise
PI3K
Akt
eNOS
NO
cGMPPrevention of Cardio-vasculitisPrevention of Cardio-vasculitis
Ang II, TNF-α
PLC, PLD
NAD(P)Hoxidase
ROS
MAP kinasesONOO-
MCP-1ICAM-1
MφAT1 Rs/AT2 Rs
Vascular dilatation
Hypertension Apoplexy
Shear stress
Fibrosis
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.