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)

Sys

toli

c b

loo

d 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

Str

oke

mo

rbid

ity

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 SHRSPS

urv

ivin

g

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

1R

/ A

T2R

n=6 6

*p<0.005

(arb

. un

its)

(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 (

arb

. un

its)

SED

AT1R/AT2R ACE

n=8 6

Nox1

Rel

ativ

e in

ten

sity

(ar

b.

un

its)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

mR

NA

/ 18

S r

RN

A

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

ten

sity

(ar

b.

un

its)

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

555 5 55

P<0.01

*P<0.05

*

x104

SE

D

EX

ER

CIS

E

ROS and NO Productions in the Aortae between SED and WR

0

2

4

6

8

10

12

14

16

[3 H]l

-cit

rull

ine

(nm

ol/

mg

/min

)

0

100

200

300

400

500

600

cGM

P (

fmo

l/m

g p

rote

in)

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

Nit

roty

rosi

ne

(a

rb. u

nit

s)

0

20

40

60

80

100

120

140

Dih

ydro

eth

idiu

m (

MF

I)

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

1.8

p-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/

Ak

t

p-E

RK

/ER

K

(Mean ± SEM)

(arb

. un

its)

0

1

2

3

4

5

6

SED WR

TG

F-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

0

SED WR

(pg/ml)

22 22

p<0.01

*

Pla

sma

con

cen

trat

ion

Comparison 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

*

Pla

sma

con

cen

trat

ion

Concentrations 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 kinases

ONOO-

MCP-1ICAM-1

Mφ

AT1 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.