Running t i t l e : E f f e c t o f p r o b i o t i c s and ARB on l i v e r f i b r o s i s

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Combining p r o b i o t i c s and an a n g i o t e n s i n ‑ I I t y p e 1 r e c e p t o r b l o c k e r has b e n e f i c i a l e f f e c t s

on h e p a t i c f i b r o g e n e s i s i n a r a t model o f n o n a l c o h o l i c s t e a t o h e p a t i t i s

Running t i t l e : E f f e c t o f p r o b i o t i c s and ARB on l i v e r f i b r o s i s

Yasuhiko Sawada, Hideto Kawaratani, Takuya Kubo, Yukihisa F u j i n a g a , Masanori

Furukawa, S o i c h i r o Saikawa, Shin y a S a t o , K e n i c h i r o S e k i , Hiroaki T a k a y a , Ya s u s h i

Okura , Kosuke K a j i , Naotaka Shimozato , T s u y o s h i M a s h i t a n i , Mitsuteru K i t a d e , Kei

Mori y a , Tadashi Namisaki, ̲ Takemi Akahane , Akira M i t o r o , J u n i c h i Yamao and H i t o s h i

Y o s h i j i

Third Department o f In t e r n a l M e d i c i n e , Nara Medical U n i v e r s i t y , K a s h i h a r a , Nara , 634

^田

8522 , Japan

Address c o r r e s p o n d e n c e : Hid e t o Kawarata n i , M.D. P h . D . Third D epartme nt o f In t e r n a l

Medicine , Nara Medical U n i v e r s i t y , Kashihara , Nara, 6 3 4 ‑ 8 5 2 2 , Japan .

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Tel +81^目744223051,ext. 3415;

Fax: +81‑744247122

E‑mail: kawara@naramed‑u.ac.jp

Total word count: 2571 words

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ABSτ'RACT

Aim: I n t e s t i n a l e n d o t o x i n i s important f o r t h e p r o g r e s s i o n o f n o n a l c o h o l i c s t e a t o h e p a t i t i s

(NASH). C i r c u l a t i n g e n d o t o x i n l e v e l s a r e e l e v a t e d i n most animal models o f d i e t ‑ i n d u c e d

n o n a l c o h o l i c f a t t y l i v e r d i s e a s e (NAFLD) and NASH. F u r t h e r , plasma e n d o t o x i n l e v e l s

a r e sigm 五 c a n t l yh i g h e r i n NAFLD p a t i e n t s , which i s a s s o c i a t e d with sma l l i n t e s t i n a l

b a c t e r i a l overgrowth and i n c r e a s e d i n t e s t i n a l p e r m e a b i l i t y . B y improving t h e g u t

m i c r o b i o t a environment and r e s t o r i n g g u t ‑ b a r r i e r f u n c t i o n s , p r o b i o t i c s a r e e f f e c t i v e f o r

NASH treatment i n animal mod e l s . I t i s a l s o w i d e l y known t h a t hep a t i c f i b r o s i s and

s u p p r e s s i o n o f a c t i v a t e d h e p a t i c s t e l l a t e c e l l s CAc‑HSCs) can be a t t e n u a t e d using an

a n g i o t e n s i n ‑ I I (AT ‑ I I ) t y p e 1 r e c e p t o r b l o c k e r (ARB) . We thus e v a l u a t e d t h e e f f e c t o f

combination p r o b i o t i c s and ARB treatment on l i v e r f i b r o s i s using a r a t model o f NASH.

Me t h o d s : F i s h e r 344 ra t s w e r e f e d a c h o l i n e ‑ d e f i c i e n t / L ‑ amino a c i d ‑ d e f i n e d (CDAA) d i e t

f o r 8 weeks t o g e n e r a t e t h e NASH m o d e l . Animals were d i v i d e d i n t o ARB , p r o b i o t i c s ,

and ARB p l u s p r o b i o t i c s g r o u p s . T h e r a p e u t i c e f f i c a c y was a s s e s s e d by e v a l u a t i n g l i v e r

f i b r o s i s , t h e l i p opo l y s a c c h a r i d e ( L P S ) T o l l ‑ l i k e r e c e p t o r (TLR) 4 r e

伊

1 l a t o r yc a s c a d e , an d

i n t e s t i n a l b a r r i e r f u n c t i o n .

Results : Both p r o b i o t i c s a nd ARB inh i b i t ed l i v e r f i b r o s i s , with concom i t ant H SC

a c t i v a t i o n and suppr e s s i o n o f l i v e r ‑ s p e c i f i c t r a n s f o r m i n g growth f a c t o r ( T G F ) ‑ 8 and

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TLR4 e x p r e s s i o n . P r o b i o t i c s reduced i n t e s t i n a l p e r m e a b i l i t y by r e s c u i n g z o n u l a

o c c l u d e n s ‑ 1 ( Z 0 ‑ 1 ) d i s r u p t i o n induced by t h e CDAA d i e t . ARB was found t o d i r e c t l y

s u p p r e s s Ac‑HSCs.

C o n c l u s i o n s : P r o b i o t i c s and ARB a r e e f f e c t i v e i n s u p p r e s s i n g l i v e r f i b r o s i s v i a d i f f e r e n t

mechanisms. C u r r e n t l y both drugs a r e i n c l i n i c a l use ; t h e r e f o r e , th e combination o f

p r o b i o t i c s and ARB i s a promising new therapy f o r NASH .

Keywords N o n a l c o h o l i c s t e a t o h e p a t i t i s , h e p a t i c f i b r o s i s , p r o b i o t i c s , a n g i o t e n s i n ‑ 2 t y p e 1

r e c e p t o r b l o c k e r 仇 RB)

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INTRODUCTION

N o n a l c o h o l i c s t e a t o h e p a t i t i s (NASH) i s a p r o g r e s s i v e l i v e r d i s e a s e c h a r a c t e r i z e d by

h e p a t i c s t e a t o s i s t h a t l e a d s t o i n f l a m m a t i o n , f i b r o s i s , and c i r r h o s i s . However, c u r r e n t l y ,

t h e r e i s no w e l l ‑ e s t a b l i s h e d treatment f o r t h i s d i s e a s e . S e v e r a l mechanisms o f NASH

p r o g r e s s i o n have been r e p o r t e d ( l , 2 ) . Regarding t h e mechanism o f NASH p a t h o g e n e s i s ,

t h e t w o ‑ h i t t h e o r y has become w i d e l y a c c e p t e d . T h i s d i s e a s e i s thought t o b e g i n wi t h

e x c e s s i v e f a t accumulation i n t h e l i v e r ( f i r s t h i t ) , f o l l o w e d by a g g r a v a t i n g f a c t o r s such a s

o x i d a t i v e s t r e s s , inflammatory c y t o k i n e s , and e n d o t o x i n s ( s e c o n d h i t ) . ( 3 ) However, th e

p a t h o l o g y o f NASH i s more c o m p l i c a t e d and r e c e n t l y a t h e o r y p o s t u l a t i n g m u l t i p l e

p a r a l l e l h i t s has been p r e s e n t e d . ( 2 ) The p r o g r e s s i o n o f n o n a l c o h o l i c f a t t y l i v e r d i s e a s e

(NAFLD) i s known t o be dependent on both g e n e t i c and environmental f a c t o r s . ( 1 , 2 ) One

such f a c t o r i s b a c t e r i a l t r a n s l o c a t i o n through i n t e s t i n a l b a c t e r i a l overgrowth and

enhanced i n t e s t i n a l p e r m e a b i l i t y . L i p o p o l y s a c c h a r i d e ( L P S ) , s e c r e t e d from t h e i n t e s t i n a l

m i c r o b i o t a , i s d e l i v e r e d t o t h e l i v e r v i a t h e p o r t a l v e i n . ( 1 , 4 ) In p a t i e n t s with o b e s i t y ,

d i a b e t e s , m e t a b o l i c d i s o r d e r s , NAFLD, and NASH , e n d o t o x i n s from i n t e s t i n a l b a c t e r i a

c a u s e h e p a t i c i n f l a m m a t i o n . ( 1 , 2 ) M o r e o v e r , i n a mouse model o f NAFLD, b a c t e r i a l

o v e r growth w a s found t o m e d i a t e c o m p o s i t i o n a l ch a nges a nd i n c r e a s e i n t e s t i n a l

p e r m e a b i l i t y by down

^田

r e g u l a t i n gt h e e x p r e s s i o n o f t i g h t j u n c t i o n p r o t e i n s . ( 5 ) Serum

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e n d o t o x i n l e v e l s were a l s o determined t o i n c r e a s e i n an animal model o f NASH,(6) and

were s i m i l a r l y found t o be s i g n i f i c a n t l y h i g h e r i n h i s t o l o g i c a l l y ‑ s e v e r e NAFLD p a t i e n t s ,

which was accompanied by i n c r e a s e d i n t e s t i n a l b a c t e r i a l overgrowth and

p e r m e a b i l i t y . ( 7 ) Furthermore, s y s t e m i c inflammation , o x i d a t i v e s t r e s s , and i n s u l i n

r e s i s t a n c e

訂

eknown t o c a u s e c y t o t o x i c i t y and l i v e r d a m a g e . ( 1 , 2 , 8 ) M o r e o v e r , o b e s i t y ,

NAFLD, and NASH a r e a s s o c i a t e d with changes i n t h e i n t e s t i n a l b a c t e r i a l f l o r a , a nd

p r o b i o t i c s can i n t e r f e r e with t h e p r o g r e s s i o n o f NASH.

Butyrate i s a s h o r t c h a i n f a t t y a c i d t h a t i s produced by m i c r o b i o t a i n t h e c o l o n and

d i s t a l s m a l l i n t e s t i n e from i n d i g e s t i b l e s t a r c h , d i e t a r y f i b e r , and low d i g e s t i b l e

p o l y s a c c h a r i d e ; i t i s g e n e r a t e d through ferme

則的

i o nby microorganisms i n t h e c o l o n and

d i s t a l s m a l l i n t e s t i n e . ( 9 ) T h i s compound i s p a r t i c u l a r l y important f o r c o l o n h e a l t h , i t i s

t h e primary energy s o u r c e f o r c o l o n i c c e l l s , and i t ha s a n t i ‑ c a r c i n o g e n i c and a n t i ‑

inflammatory p r o p e r t i e s . C l i n i c a l t r i a l s have shown t h a t t h e a d m i n i s t r a t i o n o f b u t y r a t e

might be a promising treatment o p t i o n f o r u l c e r a t i v e c o l i t i s . ( 1 0 , 1 1

）

α o s t r i d i u m bu 砂 Ticumi s a g r a m ‑ p o s i t i v e , a n a e r o b i c bacterium t h a t p r o d u c e s b u t y r i c a c i d ; i t i s found

i n s o i l and t h e i n t e s t i n e s o f healthy animals and humans.(12) MIYAIRI 5 8 8 , a but y r i c

a c i d ‑ p r o d u c i n g , gram ‑ p o s i t i v e a n a e r o b e , and a

C.

but y ricum s t r a i n , i s u s e d a s a p r

油

i o t i c

f o r t h e t r ea tment and p r e v e n t i o n o f a n t i b i o t i cr e l a t e d d i a r r h e a i n h u m a n s . ( 1 3 , 1 4 )

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The r e n i n ‑ a n g i o t e n s i n ‑ a l d o s t e r o n e system p l a y s an important r o l e i n c h r o n i c l i v e r

d i s e a s e . ( 1 5 ‑ 1 7 ) We p r e v i o u s l y r e p o r t e d t h a t b l o c k i n g a n g i o t e n s i n ‑ I I (AT

^戸

I I )s i g n a l i n g

through t h e A T ‑ I I t y p e l r e c e p t o r (ATlR) s u p p r e s s e s l i v e r f i b r o s i s i n r a t s . ( 1 8 , 1 9 )

Furthermore, t h e i n h i b i t o r y e f f e c t o f ARB on h e p a t i c f i b r o s i s was c o n s i s t e n t with t h e

s u p p r e s s i o n o f a c t i v a t e d h e p a t i c s t e l l a t e c e l l s ( A c ‑ H S C s ) . ( 2 0 ) In a d d i t i o n , t h e

a d m i n i s t r a t i o n o f ARB was found t o improve l i v e r f i b r o s i s v i a A T ‑ I I ‑ m e d i a t ed L P S ‑ T o l l

l i k e r e c e p t o r (TLR) 4 s i g n a l i n g and s u p p r e s s TLR4 s i g n a l i n g i n A c ‑HS C s . ( 2 1 )

We thus h y p o t h e s i z e d t h a t MIYAIRI 588 might improve t h e i n t e s t i n a l f l o r a

environment and i n h i b i t t h e p r o g r e s s i o n o f NASH by p r e v e n t i n g d e s t r u c t i o n o f t h e

i n t e s t i n a l b a r r i e r . M o r e o v e r , combining MIYAIRI 588 with ARB was p r e d i c t e d t o improve

NASH v i a d i f f e r e n t mechanisms. As s u c h , we examined t h e e f f e c t o f MIYAIRI 588 and

ARB on NASH p r o g r e s s i o n using animals f e d a c h o l i n e ‑ d e f i c i e n t / L ‑ a m i n o a c i d ‑ d e f i n e d

(CDAA) d i e t .

METHODS

Animals and r e g e n t s

Male 6 ‑ w e e k ‑ o l d F i s h e r 344 ( F 3 4 4 ) r a t s were purchas e d from Japan SLC (Ham a matsu ,

S h i z u o k a , J a p a n ) . Rats were housed i n c o n t r o l l e d c o n d i t i o n s with a temperature o f 23

士

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3 ° c , a r e l a t i v e humidity o f 50

土

20%,a s t a i n l e s s s t e e l mesh, 10‑15 a i r changes p e r hour ,

and 12 hours o f l i g h t i r r a d i a t i o n per d a y . During the e x p e r i m e n t , animals were a l l o w e d

f r e e a c c e s s t o t a p water . MIYAIRI 588 was p r o v i d e d b y Miyarisan Pharmaceutical C o .

L t d . ( T o k y o , J a p a n ) . L o s a r t a n , was purchased from Merck ( T o k y o , J a p a n ) . C o n v e n t i o n a l

c h e m i c a l r e a g e n t s were purchased from N a c a l a i Te s que ( K y o t o , Japan ) . CDAA and

c h o l i n e s u f f i c i e n t IL‑amino a c i d ‑ d e f i n e d (CSAA) d i e t s were purchased from CLEA J apa n

I n c . ( T o k y o , J a p a n ) .

Experimental d e s i g n

A l l e x p e r i m e n t s were conducted o v e r 8 w e e k s . Rats were randomly d i v i d e d i n t o f i ve

g r o u p s . Groups r e c e i v i n g t h e CDAA d i e t , t o e s t a b l i s h t h e animal model o f d i e t

^田

induced

h e p a t i c s t e a t o s i s and f i b r o s i s , comprised t h e CDAA g r o u p . The group r e c e i v i n g t h e CDAA

d i e t c o n t a i n i g MIYAIRI 588 ( 8 . 5

×

10

9

CFU/g) was d e s i g n a t e d t h e p r o b i o t i c s g r o u p . ( 2 2 ）

Ten p e r c e n t o f t h e t o t a l CDAA d i e t was r e p l a c e d with an e x c i p i e n t c o n t a i n i n g MIY

孔

IRI

5 8 8 . Anim a l s r e c e i v i n g t h e CDAA d i e t c o n t a i n i n g l o s a r t a n ( 3 0 mg/kg / d a y ) was n amed

the ARB g r o u p . ( 2 3 , 2 4 ) F i n a l l ぁ r a t sr e c e i v i n g t h e CDAA d i e t c o n t a i n i n g M

町

: A I R I 58 8

and l o s a r t a n comprise d t he combination group o f p r o b i o t i c s and ARB. An a d d i t i o n a l

group was a d m i n i s t e r e d a CSAA d i e t . At t h e end o f t h e ex p e r i m e n t a l p e r i o d , t h e r a t s

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were a n e s t h e t i z e d with d i e t h y l e t h e r and d i f f e r e n t parameters wer e examined. A l l

animal p r o c e d u r e s were performed a c c o r d i n g t o D e c l a r a t i o n o f H e l s i n k i and i n

c o m p l i a n c e with standard recommendations f o r t h e p r o p e r c a r e and use o f l a b o r a t o r y

a n i m a l s . The p r o t o c o l was approved by t h e Committee o f Nara Medical U n i v e r s i t y .

H i s t o l o g i c a l and immunohistochemical a n a l y s e s

For a l l e x p e r i m e n t a l g r o u p s , 5‑μm t h i c k s e c t i o n s o f f o r m a l i n ‑ f i x e d and p a r a f f i n ‑

e mbedded l i v e r specimens wer e r o u t i n e l y s u b j e c t e d t o hematoxylin and e o s i n s t a i n i n g ,

o i l red 0 s t a i n i n g t o e v a l u a t e l i v e r s t e a t o s i s , S i r i u s Red ( S ‑ R ) s t a i n i n g t o e v a l u a t e l i v e r

f i b r o s i s , and immunohistochemical s t a i n i n g probing f o r alpha smooth mu s c l e a c t i n （ α

ー

SMA; DAKO, Kyoto , Japan) a s p r e v i o u s l y d e s c r i b e d ( 2 5 , 2 6 ) and f o r g l u t a t h i o n e S ‑

t r a n s f e r a s e p l a c e n t a l form (GST

、

P ) a s h e p a t i c p r e n e o p l a s t i c l e s i o n s . The s t a i n e d s e c t i o n s

were a n a l y z e d using Image ‑ J s o f t w a r e ( N a t i o n a l I n s t i t u t e s o f H e a l t h ) .

Q u a n t i t a t i v e RT‑PCR a n a l y s i s

mRNA was e x t r a c t e d from p u l v e r i z e d f r o z e n l i v e r and i n t e s t i n a l t i s s u e s using th e

RN ea sy Mini K i t (QIAGEN , To k y o , Japan) . T o t a l RN A ( 1 μ g ) from ea ch sample was

r e v e r s e t r a n s c r i b e d i n t o complementary DNA (cDNA) u s i n g a high c a p a c i t y RNA‑to‑

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cDNA k i t ( A p p l i e d Biosystems I n c . , F o s t e r C i t y , C a l i f . , USA). The e x p r e s s i o n l e v e l s o f

mRNA e n c o d i n g l i v e r t i s s u e ‑ d e r i v e d

_{TGF－ ~1,}

LPS

七

i n d i n gp r o t e i n ( L B P ) , and l i v e r

d e r i v e d TLR4 were a n a l y z e d using SYER Green and t h e S t e p One Sequence D e t e c t i o n

System ( A p p l i e d Biosystems I n c . , F o s t e r C i t y , C a l i f . , USA) by polymerase c h a i n r e a c t i o n

( P C R ) . The PCR p r o c e d u r e was a s f o l l o w s : t h e sample was heated a t 95 ° C . f o r 20 sand

s u b j e c t e d t o 40 c y c l e s o f denatur a t i o n a t 95 °C f o r 3 s and a n n e a l i n g a t 60 °C f o r 30 s .

For t h i s e x p e r i m e n t ,

~－Actin

was used a s an endogenous c o n t r o l . Primer s e quences used

were as f o l l o w s :

TGF－ ~1,

forward 5

^田

CGGCAG CTG TAC ATT GAC TT‑3 ' and r e v e r s e 5 ' ‑

AGC GCA CGA TCA TGT TGG AC‑3

；＇

α l ( I

『）

p r o c o l l a g e n , forward 5'‑AGC TCC TGG GCC

TAT CTG ATG A ‑ 3 ' and r e v e r s e 5'‑AAT GGT GCT CTG AAA CCC TGA T G ‑ 3 ' ; TLR4 ,

forward 5

九

CCG CTC TGG CAT CAT CTT CA‑3and r e v e r s e 5'‑CCC ACT CGA GGT AGG

TGT TTC T G ‑ 3 ' ; LBP , forward 5

ん

AACATC CGG CTG AAC ACC AAG‑3 ' and r e v e r s e 5

＇司

CAA GGA CAG ATT CCC AGG ACT G A ‑ 3 ' ;

~－actin,

forward 5 ' ‑ GGA GAT TAC TGC CCT

GGCTCCTA

^同

3 'and r e v e r s e 5'‑GAC TCA TCG TAC TCC TGC TTG CTG

・

3 ' .

Enzyme ‑ l i n k e d immune s o r b e n t assay (ELISA) ana l y s i s

Mmp‑9 c o n c e n t r a t i o n s were measured i n t h e supernatant o f t h e s n a p ‑ f r o z e n l i v e r t i s sue

using ELISA k i t s (R&D Systems , M i n n e a p o l i s , MN, USA) , a c c o r d i n g t o t h e

manufacturers i n s t r u c t i o n s .

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Immunofluorescence a n a l y s i s

Frozen s e c t i o n s o f i n t e s t i n a l t i s s u e (7‑μm t h i c k ) were prepared and f i x e d with 4 %

paraformaldehyde a t 4 °C f o r 10 min . A f t e r b l o c k i n g with 10% normal g o a t s e rum i n PBS ,

仕

ozens e c t i o n s were i n c u b a t e d with r a b b i t p o l y c l o n a l a n t i ‑ m o u s e ZO

・

1a n t i b o d y ( 1 : 100 ,

I n v i t r o g e n L i f e T e c h n o l o g i e s , C a r l s b a d , C a l i f . , USA) o ve r n i g h t a t 4 °C and then donkey

a n t i r a b b i t s e c o n d a r y a n t i b o d y a s s o c i a t e d with Dy L i g h t 488 f o r 1 h a t r oom t e mperature

with a n t i b o d y f l u o r e s c e n t dye ( J a c k son ImmunoResearch L a b o r a t o r i e s , West G r o v e ,

Pennsylvania USA) . S e c t i o n s were mounted u s i n g V e c t a s h i e l d l o a d e d medium with 4 ' , 6

・

d i a m i d i n o ‑ 2 ‑ p h e n y l i n d o l e Fluoromount‑G (DAPI) f o r f l u o r e s c e n t n u c l e i c a c i d s t a i n i n g

( V e c t o r L a b o r a t o r i e s , Burlingame, CA , USA). The s t a i n e d specimens were examined

using a c o n f o c a l scanning l a s e r m i c r o s c o p e equipped with a d i g i t a l camera ( L e i c a TCSNT,

L e i c a m i c r o s y s t e m s , W e t z l a r , German y ) . Green f l u o r e s c e n c e c o r r e s p o n d i n g t o Z0‑1

l o c a l i z e d t o t h e i n t e s t i n a l e p i t h e l i a l c e l l t i g h t j u n c t i o n s ( e x c i t a t i o n l i g h t w a v e l e n g t h , 490

nm; e m i s s i o n w a v e l e n g t h , 510 nm). For e ach s a m p l e , th e mean f l u o r e s c e nce i n t e n s i t y

(MFI) va l u e s from a r e a s o f e qu a l s i z e were m e a s ur e d f o r each im age u s i ng Im age J

S o f t w a r e . The MFI o f n eg a t i v e l y ‑ s t a i n e d s e c t i o n s was s u b t r a c t e d from t h e MFI o f

p o s i t i v e l y ‑ s t a i n ed s e c t i o n s .

(12)

S t a t i s t i c a l a n a l y s e s

Students t ‑ t e s t o r one‑way ANOVA f o l l o w e d by B o n f e r r o n is m u l t i p l e ‑ c o m p a r i s o n t e s t

were p e r f o r m e d . S t a t i s t i c a l a n a l y s e s were performed u s i n g GraphPad Prism v e r s i o n 6 . 0 4

(GraphPad S o f t w a r e , I n c . , La J o l l a , CA , USA). A l l t e s t s were two

^目

t a i l e dand p v a l u e s

<

0 . 0 5 were c o n s i d e r e d s t a t i s t i c a l l y s i g n i f i c a n t .

RESULTS

I n h i b i t o r y e f f e c t o f p r o b i o t i c s and ARB on h e p a t i c f i b r o s i s , c a r c i n o g e n e s i s and s t e

説。

s i s

F i r s t , we examined t h e e f f e c t o f c l i n i c a l l y comparable d o s e s o f p r o b i o t i c s and ARB on

l i v e r f i b r o s i s . L i v e r f i b r o s i s was suppressed i n both t h e p r o b i o t i c and ARB group ,

compared t o t h a t i n t h e CDAA group ( F i g . l A , B ) . A more p o t e n t i n h i b i t o r y e f f e c t on

h e p a t i c 五 b r o g e n e s i swas o b s e r v e d i n t h e combination group o f p r o b i o t i c s p l u s ARB ,

compared t o t h a t with e i t h e r drug a l o n e . We then performed immunohistochemistr y f o r

α

⁻

SMA , which c o r r e l a t e s with a c t i v a t e d h e p a t i c s t e l l a t e c e l l ( H S C s ) . In both t h e

p r o b i o t i c s a nd ARB g r o u p s , a s i g n i f i c a n t d e c r e a s e i n th e number o f a‑SMA

immunopositive Ac‑HSCs was o b s e r v e d ( F i g . 2 A ) . Compu t e r ‑ a s s i s t e d s e m i ‑ q u a n t i t a t i v e

a n a l y s i s o f a‑SMA immunohi s t och e mist ry sho we d a r e du c e d a r ea o f α

−

SMA s t a i n i n g i n

p a r a l l e l with t h e i n h i b i t i o n o f l i v e r f i b r o s i s ( F i g . 2 B ) . A s i g n i f i c a n t i n h i b i t i o n i n TGF‑8

(13)

e x p r e s s i o n was a l s o o b s e r v e d i n both t h e p r o b i o t i c and ARB g r o u p s , compared t o t h a t i n

t h e CDAA group ( F i g . 3 ) . S i m i l a r l y , t h e c o m b i n a t i o n o f p r o b i o t i c s and ARB r e s u l t e d i n a

s t r o n g e r i n h i b i t o r y e f f e c t than e i t h e r drug a l o n e . We performed immunohistochemistry

f o r G S T ‑ P , a s a s i g n i f i c a n t f a c t o r i n c a r c i n o g e n e s i s ( F i g . 4 ) . Treatment with e i t h e r

p r o b i o t i c s o r ARB r e s u l t e d i n a r n . a rked i n h i b i t o ry e f f e c t o n h e p a t i c GS

下

Pe x p r e s s i o n

compared t o t h e CDAA g r o u p . Combination treatment with both a g e n t s was e q u i v a l e n t

t o s i n g l e a d m i n i s t r a t i o n . No GST‑P c e l l s wer e o b s e r v e d i n l i v e r s e c t i o n s from t h e CS.AA

group . On t h e o t h e r h a n d , L i v e r s t e a t o s i s was s u p p r e s s e d i n t h e p r o b i o t i c g r o u p ,

compared t o t h a t i n t h e CDAA g r o u p , but n o t i n ARB group ( F i g . 5 ) .

I n h i b i t o r y e

首

e c to f p r o b i o t i c s and ARB on TLR4 s i g n a l i n g

N e x t , we e v a l u a t e d th e e f f e c t o f p r o b i o t i c s and ARB on h e p a t i c TLR4. H e p a t i c TLR4

e x p r e s s i o n i n c r e a s e d i n th e CDAA group compared t o t h a t i n th e CSAA group ( F i g . 6 ) . In

t h e p r o b i o t i c and ARB g r o u p s , h e p a t i c TLR4 e x p r e s s i o n d e c r e a s e d a s compared t o t h a t

i n th e CDAA g r o u p . Th e combination o f p r o b i o t i c s and ARB r e s u l t e d i n a s i g n i f i c a n t

a t t e nuation o f CDAA‑induced h e p a t i c TLR4 e x p r e s s i o n compared t o t h a t with p r o b i o t i c s

o r ARB a l o n e ( p

く

0 . 0 1 ) . Not a b l y , t h e i n h i b i t o r y e f f e c t o f p r o b i o t i c s a nd ARB on l i v e r

TLR4 e x p r e s s i o n was p r o p o r t i o n a l t o the i n h i b i t i o n o f l i v e r f i b r o s i s .

(14)

I n h i b i t o r y e f f e c t o f p r o b i o t i c s

但

idARBonLBP

The d i r e c t d e t e c t i o n o f LPS i s d i f f i c u l t , and thus we e v a l u a t e d hepa t i c LBP, a s t h i s

marker d i r e c t l y c o r r e l a t e s with LPS.( 2 7 ) We found t h a t l i v e r LBP m R NA was i n c r e a s ed

a f t e r 8 weeks o f CDAA a d m i n i s t r a t i o n ( p

<

0 . 0 5 ) . However , i n t h e p r o b i o t i c s group , LBP

mRNA d e c r e a s e d compared t o t h a t i n th e CDAA g r o up ( p

く

0 . 0 5 ; F i g . 7 ) . In c o n t r a s t , no

s i g n i f i c a n t e f f e c t on t h i s l i v e r LBP mRNA was o b s e r v e d i n t h e ARB g r o u p . Regarding t h e

c o mbination group o f p r o b i o t i c s and ARB , LBP mRN A was d e c r e a s e d compared t o th a t

i n t h e CDAA g r o u p ; h o w e v e r , t h e r e was no a d d i t i o n a l b e n e f i t compared t o p r o b i o t i c s

a l o n e . The s e d a t a s u g g e s t t h a t t h e i n h i b i t o r y e f f e c t on h e p a t i c f i b r o s i s w i t h r e s p e c t t o

LPS i s n o t a s s o c i a t e d with ARB, but r a t h e r p r o b i o t i c s .

Cor

て

l ・ e l a t i o n m

盟

p ‑ 9 c o n c e n t ra t i o n s and l i v e r f i b r o s i s

Mmp‑9 l ev e l s were h i g h e r i n CDAA g

・戸

ou p c ompa r e d t o t h e CS AA g roup ( p

く

0 . 0 1 ;Fig . 8 ) .

CDAA i n d u c ed i n c r e a s e i n mrn p ‑ 9 e x p r e s s i on v . r a s n o t r e du c ed i n p r o b i o t i c s g γou p,AR B

group , and both p r o b i o t i c s and .ARB g r o u p .

S e m i ‑ q u a n t i t a t i v e d e t e r m i n a t i o n o f i n t e s t i n a l t i g h t j u n c t i o n p r o t e i n e x p r e s s i o n

(15)

I n t e s t i n a l e p i t h e l i a l p e r m e a b i l i t y i s r e

思

1 l a t e dby t h e i n t e r c e l l u l a r t i g h t j u n c t i o n p r o t e i n

( T JP) complex c o n s i s t i n g o f many components i n c l u d i n g z o n u l a o c c u l u d e n s ‑ 1 ( Z 0 ‑ 1 ) . We

thus e v a l u a t e d t h e e f f e c t o f p r o b i o t i c s and ARB on Z0‑1 e x p r e s s i o n t o i d e n t i 命 t h e

p o t e n t i a l mechanism a s s o c i a t e d with i n t e s t i n a l p e r m e a b i l i t y . S e m i ‑ q u a n t i t a t i v e

immunofluorescence microscopy r e v e a l e d a marked r e d u c t i on i n Z0‑1 e x p r e s s i o n i n t h e

CDAA group compared t o t h a t i n t he CSAA group ( F i g . 9 A ) . M

百五

IRI 588 s i g n i f i c a n t l y

improved Z0‑1 e x p r e s s i o n compared t o t h a t i n t h e CDAA g r o u p . In c o n t r a s t , t h i s was

not s i g n i f i c a n t l y changed with ARB a d m i n i s t r a t i o n com pared t o t h a t i n t he CDAA g r o u p .

Upon combining p r o b i o t i c s with ARB, Z0‑1 e x p r e s s i o n s i g n i f i c a n t l y improved compared

t o t h a t i n t h e CDAA g r o u p ; howev e r , t here was no d i f f e r e nce compared t o e x p r e s s i o n i n

the p r o b i o t i c s o n l y group ( F i g . 9 B ) . These r e s u l t s i n d i c a t e t h a t the i n h i b i t o r y e f f e c t o f

p r o b i o t i c s , but n o t ARB, on l i v e r f i b r o s i s i n CDAA‑induced NASH p o t e n t i a l l y o c c u r s

through t h e r e s t o r a t i o n o f TJP e x p r e s s i o n , which c o u l d c o n t r i b u t e t o t h e regu l a t i o n o f

e n d o t o x i n i n f l u x .

DISCUSSION

We demon s t r a t e d t h a t t r eatment w i t h c l i n i c a l l y equiva l e n t d o s e s o f l o s a r t a n

(30

m g / k g / d a y ) and MIYAIRI 588 s u c c e s s f u l l y a m e l i o r a t e d h e p a t i c f i b r o s i s and suppressed

(16)

Ac‑HSCs i n a r a t model o f NASH. I n t h i s m o d e l , combining MIY AIRI 588 with losm

「

tan

treatment r e s u l t e d i n a s y n e r g i s t i c i n h i b i t o r y e f f e c t and almost c o m p l e t e l y a t t e n u a t e d

h e p a t i c f i b r o g e n e s i s . V a r i o u s f a c t o r s ha ve been r e p o r t e d t o cause NASH. Two o f th e s e

i n c l u d e inflammation and e n d o t o x i n s . L i v e r inflammation c a u s e s the a c t i v a t i o n o f HS C s ,

which i s a c e n t r a l e v e n t during h e p a t i c f i b r o s i s . Thi s i n v o l v e s a complex network o f

a u t o c r i n e / p a r a c r i n e f i b r o g e n i c s i g n a l s t h a t promote the t r a n s d i f f e r e n t i a t i o n o f q u i e s c e n t

HSCs t o Ac‑HSCs ( m y o f i b r o b l a s t i c p h e n o t y p e s ) , which a r e c h a r a c t e r i z e d by abundant

ex p r e s s i o n ofαSMA.

The i n t e r a c t i o n between A T ‑ I I and v a s c u l a r e n d o t h e l i a l growth f a c t o r (V EGF) p l a y s a n

important r o l e i n l i v e r f i b r o g e n e s i s a nd c a r c i n o g e n e s i s . ARB was p r e v i o u s l y shown t o

s i g n i f i c a n t l y s u p p r e s s t h e development o f l i v e r f i b r o s i s a l o n g with VEGF e x p r e s s i o n and

n e o v a s c u l a r i z a t i on i n t h e l i v e r . ( 2 8 ) We p r e v i o u s l y r e p o r t e d th a t ARB d i r e c t l y i n h i b i t s

Ac‑HSC a c t i va t i o n , and t hat AT‑ I I i s important f o r the up

^四

r e g u l a t i o no f TLR4 ex pr e s s i o n

through t h e s timul a t i on o f ATlR i n Ac‑HSCs. ( 2 1 ) Cro s s t a l k betw e e n A T ‑ I I and TLR4

s i g n a l i n g p l a y s a s ubst a n t i a l r o l e i n the deve lopm e n t o f l i ve r f i b r o s i s by regu l a t i n g T G F ‑

~1

p r o d u c t i o n inAc‑HSCs. Our r e s u l t s showe d t h a t A RB a d m i n i s t r a t i o n i n a r a t mod e l

o f N A SH r e d u c e s α

−

SMA ‑ p o s i t i v i t y , TLR 4 , and

TGF－~，

w h i c h l e ads t o a n imp rovement

i n hεp a t i c f i b r o g enes i s and c a r c i n o g ene s i s .

(17)

As p r o b i o t i c s c o n t r o l i n t e s t i n a l b a c t e r i a and e n d o t o x i n p r o d u c t i o n , i t was e x p e c t e d t o be

e f f e c t i v e f o r s u p p r e s s i n g NASH. There a r e numerous s t u d i e s on t h e e f f e c t i v e n e s s o f

p r o b i o t i c s f o r NASH,(29) h o w e v e r , s u f f i c i e n t e f f e c t i v e n e s s has not been e s t a b l i s h e d

using p r o b i o t i c s . Long term a d m i n i s t r a t i o n o f b u t y r i c a c i d

同

producingp r o b i o t i c

C

butyricum s t r a i n MIYAIRI 588 was shown t o d e c r e a s e h e p a t i c f i b r o u s depo s i t i o n and t h e

development o f GST‑P p o s i t i v e f o c i i n CDAA‑induced NAFLD.(22) The TJ P h a s b e e n

r e p o r t e d t o l o c a l i z e t o t h e a p i c a l plasma membrane o f e p i t h e l i a l c e l l s t o maintain

e p i t h e l i a l b a r r i e r i n t e g r i t y . ( 3 0 ) Endotoxins d e r i v e d from i n t e s t i n a l b a c t e r i a can c a u s e

l i v e r inflammation v i a TLR4 a c t i v a t i o n . TLR4 i s a p a t t e r n r e c o g n i t i o n r e c e p t o r t h a t

r e c o g n i z e s e n d o t o x i n and s i g n a l s through myeloid d i f f e r e n t i a t i o n p r i m a r y ‑ r e s p o n s e

p r o t e i n 88 (MyD 8 8 ) and T o l l / i n t e r l e u k i n l r e c e p t o r ‑ d o m a i n ‑ c o n t a i n i n g adaptor p r o t e i n

i n d u c i n g

interferon-~

(TR I F ) t o a c t i va t e t r a n s c r i p t i o n f a c t o r s i n v o l ve d i n i n n a t e

i m m u n i t y . ( 3 1 ) TLR4 i s e x p r e s s e d i n v a r i o u s h e p a t i c c e l l s i n c l u d i n g l i v e r v a s c u l a r

e n d o t h e l i a l c e l l s , Kupffer c e l l s , and HSCs. ( 3 2 , 3 3 ) In human and animal s t u d i e s , NASH

was shown t o be a s s o c i a t e d with p o r t a l LPS l e v e l s v i a mechanisms i n v o l v i n g b a c t e r i a l

t r a n s l o c a t i o n . ( 2 2 , 3 4 ) I n a d d i t i o n , e n d o t o x i n produced by g u t m i c r o b i o t a might c a u s e

inflammation i n p a t i e n t s with o b e s i t y , d i a b e t e s m e l l i t u s , met abo l i c d i s o r d e r , NAFLD ,

and NASH.(1, 3 5 ) M o r e o v e r , plasma LPS l e v e l s a r e a s s o c i a t e d with s m a l l i n t e s t i n a l

(18)

b a c t e r i a l o v e r g r o w t h , changes t o t h e c o m p o s i t i o n o f t h e m i c r o b i o t a , and i n c r e a s e d

i n t e s t i n a l p e r m e a b i l i t y . ( 7 ) As s u c h , t h e d i s r u p t i o n o f i n t e s t i n a l b a c t e r i a c o n t r i b u t e s t o

the p a t h o g e n e s i s o f NAFLD. A m e t h i o n i n e ‑ c h o l i n e d e f i c i e n t d i e t and f r u c t o s e uptake was

shown t o c a u s e NAFLD i n m i c e . I n t h e s e m o d e l s , i n t e s t i n a l e n d o t o x i n l e v e l s i n c r e a s e d

i n t h e p o r t a l v e i n ( 3 6 ‑ 3 8 ) An imbalance between p r o l i f e r a t i o n and a p o p t o s i s , i n a d d i t i o n

t o i n t e s t i n a l mucosal a t r o p h y and edema, which i s a s s o c i a t e d with p o r t a l h y p e r t e n s i o n

o r t h e a b s e n c e o f b i l e a c i d s , r e s u l t s i n i n c r e a s e d produdion o f inflammatory c y t o k i n e s

and enhanced o x i d a t i v e s t r e s s i n t h e l i v e r . ( 2 7 , 3 9 , 4 0 ) A c c o r d i n g l y , we performed

immunohistochemistry f o r t h e i n t e s t i n a l t i g h t j u n c t i o n p r o t e i n Z 0 ‑ 1 . The e x p r e s s i o n o f

i n t e s t i n a l ZO

・

1 was reduced i n t h e CDAA model compared t o t h a t i n t h e CSAA g r o u p ;

h o w e v e r , i t was i n c r e a s e d with t h e a d m i n i s t r a t i o n o f MIY

孔

IRI5 8 8 . In c o n t r a s t , ARB d i d

n o t i n f l u e n c e i n t e s t i n a l Z0‑1 e x p r e s s i o n . Taken t o g e t h e r , t h i s s u g g e s t s t h a t MIY

孔

IRI

5 8 8 , but n o t ARB, has t h e p o t e n t i a l t o improve i n t e s t i n a l t i g h t j u n c t i o n i n t e g r i t y . The

i n h i b i t o r y e f f e c t o f MIYAIRI 588 on hepa t i c f i b r o g e n e s i s and c a r c i n o g e n e s i s was

me d i a t e d by pr eve n t i n g i n t e s t i n a l p e r m e a b i l i t y through th e r e s t o r a t i o n o f TJP

e x p r e s s i o n and i n h i b i t i o n o f t h e s y s t e m i c inflammatory r e s p o n s e caused by LPS

t r a n s l o c a t i o n .

The mechanism through which MIYAIRI588 improves i n t e s t i n a l b a r r i e r f u n c t i o n has

(19)

been s u g g e s t e d ; i t was p o s t u l a t e d t h a t b u t y r a t e enhances t h i s p r o p e r t y by a c t i v a t i n g

adenosine monophosphatea c t i v a t e d p r o t e i n k i n a s e (AMPK) s i g n a l i n g . ( 4 1 ) D i e t a r y

supplementation o f b u t y r a t e i n d u c e s t h e a c t i v a t i o n o f AMPK, t h e r e b y p r e v e n t i n g and

i n h i b i t i n g h i g h ‑ f a t ‑ d i e t ‑ i n d u c e d o b e s i t y and i n s u l i n r e s i s t a n c e i n m i c e . ( 4 2 ) AMPK

r e g u l a t e s e n e r

白r

h o m e o s t a s i s through i t s e f f e c t s on g l u c o s e and l i p i d m e t a b o l i s m , ( 4 3 )

c o n t r o l s f a t t y a c i d o x i d a t i o n by r e g u l a t i ng m i t o c h o n d r i a l b i o g e n e s i s , and s u p p r e s s e s

l i p o g e n i c gene e x p r e s s i o n by r e d u c i n g t h e a c t i v i t y o f t h e t r a n s c r i p t i o n f a c t o r s t e r o l ‑

r e g u l a t o r y e l e m e n t ‑ b i n d i n g p r o t e i n l c (SREBP‑lc) . ( 4 4 ) Hepatic AMPK a l s o d e c r e a s e s

h e p a t i c l i p o g e n e s i s , and i t s a c t i v i t y can i n h i b i t r e a c t i v e o x i d a t i v e s t r e s s and

i n f l a m m a t i o n . ( 4 5 ) I n v i t r o experiments have shown t h a t NaB treatment can i n c r e a s e

AMPK a c t i v i t y and a c c e l e r a t e t h e assembly o f TJPs i n t h e C a c o ‑ 2 c o l o n i c e p i t h e l i a l c e l l

l i n e . ( 4 1 )

In t h i s r e p o r t , t h e combined use o f p r o b i o t i c s and ARB r e s u l t e d i n a s t r onger a n t i

f i b r o t i c e f f e c t a s compared t o t h a t with e i t h e r drug a l o n e . S e v e r a l i n v e s t i g a t o r s i n c l u d i n g

our

gi^噌

oup h a v e shown t h a t ACE ‑ I a n d ARB p o s s e s s s t r o n g ant i ψ a n g i ogen i c a c t i v i t y , a nd

t h a t t h e s e a g e n t s c o u l d i n h i b i t t h e growth o f s e v e r a l t y p e s o f t umors i n c l u d i n g HCC a t

c l i n i c a l l y c o mpara b l e l ow d o s e s

。（

4 6 ,4 7 )

M1'f~AIRI

588 wa s shown t o d e c r e a s e t he

development o f GST‑P p o s i t i v e f o c i i n CDAA‑induced NAF LD . ( 2 2 ) P r o b i o t i c s and ARB

(20)

have t h e i n h i b i t o r y e f f e c t on h e p a t i c c a r c i n o g e n e s i s , but i n c o m b i n a t i o n t h e r e was no

b e t t e r e f f e c t than s i n g l e adm i n i s t r a t i o n . I n our exp e r i m e n t s , t h e p e r i o d g i v e n CDAA was

8 w e e k s , which was s h o r t , s o GST‑P p o s i t i v e f o c i was s m a l l .

If

we g i v e CDAA f o r a l o n g

p e r i o d o f time we may have seen t h e combined e f f e c t o f p r o b i o t i c s and ARB.

M

町

AIRI588 improved t h e i n t e g r i t y o f i n t e s t i n a l t i g h t j u n c t i o n s , whereas ARB s uppressed t h e a c t i v a t i o n o f HSCs. I n o t h e r wo r d s , t hese a g e n t s improved h e p a t i c

f i b r o s i s through two d i f f e r e n t mechanisms, one t a r g e t i n g t h e i n t e s t i n a l t r a c t and t h e

o t h e r a f f e c t i n g HSCs.

We i n v e s t i g a t e d t h e involvement o f f i b r o l y s i s i n c l a r i f y i n g t h e mechanism o f

improvement o f f i b r o s i s . I n t h i s experiment , t he r e wa s no change i n mmp‑9 e x p r e s s i o n

by p r o b i o t i c s and ARB a d m i n i s t r a t i o n .

It

seems t h a t f i b l o l y s i s i s n o t i n v o l v e d i n t h e

mechan i s m o f im p rovement o f f i b r o s i s i n t h i s experimen t .

In c o n c l u s i o n , using our e x p e r i m e n t a l m o d e l s , s i m u l t a n e o u s a d m i n i s t r a t i o n ofMIYAIRI

588 and l o s a r t a n e x e r t e d a more p o t e n t and s y n e r g i s t i c i n h i b i t o r y e f f e c t on h e p a t i c

f i b r o g e n e s i s than e i t h e r a g e n t a l o n e by a l l e v i a t i n g e n d o t o x i n ‑ i n d u c ed g u t b a r r i e r

d y s f u n c t i o n and s u p p r e s s i n g A c ‑HSC p r o l i f e r a t i o n , r e s p e c t i v e l y ( F i g . 1 0 ) . S i n c e both

drugs a r e c l i n i c a l l y s a f e , t h e combination o f p r o b i o t i c s and ARB c o u l d be us e f u l f o r

s l o w i n g NASH p r o g r e s s i o n i n f u t u r e c l i n i c a l a p p l i c a t i o n s .

(21)

(22)

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i g n a l i n g i n me

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4 4 . L i Y , Xu S , Mihaylova MM, Zheng B , Hou X , Jian

g

B

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e t a l . AMPK p h o s p h o r y l a t e s

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and i n h i b i t s SREBP a c t i v i t y t o a t t e n u a t e h e p a t i c s t e a t o s i s and a t h e r o s c l e r o

sis i

n d i e t ‑

induced i n s u l i n ‑ r e s i s t a n

t

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4 5 . L i XN, Song J

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Zhang L , LeMair

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vat

i

o

n o f the

AMPK

‑

FOX03 pathway reduces f a t t y a c i d ‑ i n d u c e d i n c r e a s e i n

i

ntra

ce

l l u l a r r e a c t i v e

oxyge

n

s p e c i e s

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. Y o s h i j i H,

Kuriyama

S , Noguchi

R,

Y o s h i i J , Ikenaka

Y,

Yanase

K, et al.

Combination o f vitamin K

2

and t h e a n g i o t e n s i n ‑ c o n v e r t i n g enzyme i n h i

b

i t

or, p

e r i n d o p r i l ,

a t t e n u a t e s t h

e

l i

ve

r

e

nz

y

m e ‑ a l t

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p r e n e o p l a s t i c l

esio

ns i n r a t s

v

i

a angiogenesis

s u p p r e s s i o n . J H e p a t o l .

20

0 5 ; 4 2 ( 5 ) : 6 8 7

‑93

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4 7 . Yo

shiji

H, Kuriyama S , Y o s h i i J , Ikenaka Y , Noguchi R

,

Hi

cklin

DJ,

et al. Vascu

l

ar

e n d o t h e l i a l growth f a c t

or

and r e c e p t o r i n t e r a c t i o n i s a p r e r e q u i s i t e f o r murine h e p a t i c

f i b r o g e n e s i s . G u t . 2 0 0 3 ; 5 2 ( 9 ) : 1 3 4 7

・

54

.

(30)

F i g u r e l e g e n d s

F i g . I

(A)

Microphotographs o f l i v e r s e c t i o n s with S i r i u s r e d . No f i b r o s i s was o b s e r v e d i n

c h o l i n e ‑ s u f f i c i e n t amino a c i d (CSAA)‑fed c o n t r o l r a t s . L i v e r f i b r o s i s was o b s e r v e d i n t h e

c h o l i n e ‑ d e f i c i e n t L‑amino a c i d (CDAA) treatment group . Monotherapy with P r o b i o t i c s

demonstrated a l i t t l e i n h i b i t o r y e f f e c t ( p = 0 . 0 8 ) . Monotherapy with a n g i o t e n s i n I I t y p e I

r e c e p t o r b l o c k e r 仏 RB)demonstrated a s i g n i f i c a n t i n h i b i t o r y e f f e c t . Combined p r o b i o t i c s

and ARB ( P r o b i o t i c s + A R B ) e x e r t e d a more i n h i b i t o r y e f f e c t i v e than e i t h e r monotherapy.

( B ) S e m i q u a n t i t a t i v e a n a l y s i s con 五 rmedh i s t o l o g i c a l f i n d i n g s . Values r e p r e s e n t mean

土

SD.

六

P<0 . 0 5

，六六

P<0 . 0 1 .

F i g . 2

(A)

Immunohistochemical a n a l y s i s o f h e p a t i c

・

a‑smoothmuscle a c t i n ( a ‑SMA )

e x p r e s s i o n . ( B ) S e m i q u a n t i t a t i v e a n a l y s i s o f t h e a‑SMA immunohistochemistry were

performed by using image a n a l y s i s s o f t w a r e . Treatment with e i t h e r P r o b i o t i c s o r ARB

r e s u l t e d i n a s i g n i f i c a n t i n h i b i t o r y e 町 e c t on h e p a t i c α

−

SMA e x p r e s s i o n compared t o t h e

CDAA‑fed group . Combined p r o b i o t i c s and ARB ( P r o b i o t i c s + A R B ) e x e r t e d a s t r o n g e r

i n h i b i t o r y e f f e c t . No a‑SMAcells were o b s e r v e d i n l i v e r s e c t i o n s from t h e CSAA‑fed group .

Values r e p r e s e n t m ea n

土

SD. *P < 0 . 0 5

，付

P

く