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Eicosanoids in the progression of liver steatosis to cancer
(USC Thesis Other)
Eicosanoids in the progression of liver steatosis to cancer
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壹
E i c os a noi ds i n t he P r og r e s s i on of L i ve r S t e a t os i s t o C a nc e r
by
Y uny i J i a
A T he s i s P r e s e nt e d t o t he
F A U L T Y O F T H E U S C M A N N S C H O O L O F P H A R M A C Y A N D P H A R M A C E U T I C A L
S C I E N C E S
U N I V E R S I T Y O F S O U T H E R N C A L I F O R N I A
I n P a r t i a l F ul f i l l m e nt of t he
R e qui r e m e nt s f or t he D e gr e e
M A S T E R O F P H A R M A C E U T I C A L S C I E N C E S
A ugus t 2023
C opy R i g ht 2023 Y uny i J i a
i i
A c know l e dge m e nt s
F i r s t of a l l , I w a nt t o de e pl y t ha nk m y m e nt or , D r . B a ngy a n S t i l e s , w ho ha s he l pe d m e de ve l op
m y r e s e a r c h di r e c t i on a nd g ui de m e w he n I ha ve que s t i ons i n m y m a s t e r c a r e e r . I a m g r a t e f ul t o
be one s t ude nt a nd r e s e a r c he r i n t he S t i l e s L a b. I a l s o w a nt t o t ha nk D r . S t a n L o ui e a nd B r a ndon
E br i ght f or pr ovi di ng t he L C M S e qui pm e nt a nd ki ndl y t e a c hi ng m e t a r ge t e d l i pi dom i c s s ki l l s . I
w a nt t o t ha nk M a r i o A l ba f or t e a c hi ng m e m a s s s pe c t r om e t r y t e c hni que a nd bi oi nf or m a t i c s t o
a na l y z e da t a . I n a ddi t i on, I w a nt t o t ha nk L i na H e , T a oj i a n T u, Q i T a ng f or pa t i e nt l y t e a c hi ng m e
R T - qP C R t e c hni que a nd ot he r ba s i c s ki l l s .
I w a nt t o t ha nk K oi c hi U g a f r om A r t c e nt e r C ol l e g e of D e s i gn f or t e a c hi ng m e P hot os hop s ki l l s
t o g e ne r a t e t he m e t hod di a gr a m s a nd t he e i c os a noi ds pa t hw a y s .
L a s t but not l e a s t , I w oul d l i ke t o t ha nk m y pa r e nt s a nd a l l m y f r i e nds f or t he i r w a r m s uppor t a nd
e nc our a ge m e nt t o a l l ow m e t o c om pl e t e m y r e s e a r c h a nd s t udy f or t w o y e a r s .
i i i
T a bl e of C ont e nt s
A c know l e dge m e nt s ......................................................................................................................... i i
L i s t of F i gur e s ................................................................................................................................ i v
A bs t r a c t ........................................................................................................................................... v
C ha pt e r I : B a c kgr ound a nd I n t r oduc t i on ......................................................................................... 1
1. L i ve r D i s e a s e ............................................................................................................................... 1
1.1 N on- a l c ohol i c f a t t y l i ve r di s e a s e ( N A F L D ) .............................................................................. 1
1.2 H e pa t oc e l l ul a r c a r c i nom a ( H C C ) .............................................................................................. 2
1.3 I n f l a m m a t i on i n L i ve r D i s e a s e .................................................................................................. 3
2. E i c os a noi ds .................................................................................................................................. 5
2.1 E i c os a noi ds bi os y n t he s i s ........................................................................................................... 6
2.2 E i c os a noi ds F unc t i on i n I n f l a m m a t i on ................................................................................... 10
2.3 R e gul a t i on of E i c os a noi ds ....................................................................................................... 11
3. H y p ot he s i s a nd O bj e c t i ve s ........................................................................................................ 12
C ha pt e r I I : M e t hods a nd M a t e r i a l s ................................................................................................ 13
2.1 L i pi d E x t r a c t i on ....................................................................................................................... 14
2.2 M a s s S pe c t r om e t r y B a s e d T a r ge t e d L i pi dom i c s ..................................................................... 15
2.3 T i s s ue hom og e ni z a t i on a nd R N A E xt r a c t i on .......................................................................... 16
2.4 P r i m e r T e s t i ng ......................................................................................................................... 17
2.5 R e a l - T i m e R T - P C R ................................................................................................................. 18
2.6 S t a t i s t i c a l A na l y s i s .................................................................................................................. 19
C ha pt e r I I I : R e s ul t s ....................................................................................................................... 20
3.1 L i ve r T i s s ue f r om P t e n D e l e t e d M i c e ..................................................................................... 20
3.2 I d e nt i f i c a t i on of S pe c i f i c L i pi d S pe c i e s .................................................................................. 22
3.3 D e c r e a s i ng L e ve l s of E i c os a noi ds i n t he L i ve r a s M i c e A ge .................................................. 23
3.4 P t e n D e l e t i on A l t e r e d t he E i c os a noi d L e ve l s .......................................................................... 30
3.4.1 B i oa c t i ve l i pi d m e t a bol i t e c ha nge due t o P T E N l os s ........................................................... 30
3.4.2 T he e f f e c t s of P T E N on t he e x pr e s s i on of C O X a nd L O X e nz y m e s ................................... 34
3.5 E i c os a noi ds i n t he L i ve r D i s e a s e P r og r e s s i on ......................................................................... 37
C ha pt e r I V D i s c us s i on a nd F ut ur e P e r s pe c t i ve s ........................................................................... 44
R e f e r e nc e s ..................................................................................................................................... 49
i v
L i s t of F i gur e s
F i gur e 1: T he pr ogr e s s of N A F L D t o H C C ( F i gur e i s g e ne r a t e d i n B i oR e nde r ) ............................ 2
F i gur e 2: H e pa t i c i nf l a m m a t i on- f i br os i s - c a nc e r a x i s ...................................................................... 4
F i gur e 3: B i os y n t he s i s pa t hw a y s of E i c os a noi ds ( L O X , C O X a nd C y p 450 e nz y m e s )
1 5
................ 7
F i gur e 4: T a r ge t e d l i pi dom i c s a nd R e a l t i m e qP C R on W T a nd P T E N de l e t e d m i c e m ode l . ...... 13
F i gur e 5: L i ve r s a m pl e s of W T a nd P t e n de l e t e d m i c e m ode l s . ................................................... 21
F i gur e 6: P e a k S e l e c t i on a nd D a t a N or m a l i z a t i on ........................................................................ 22
F i gur e 7: H e a t m a p of t he e i c os a noi d l e ve l s i n w i l d t y p e a nd P t e n de l e t e d m ous e m ode l s .......... 24
F i gur e 8: H e a t m a ps of t he L O X a nd C O X m e t a bol i t e s i n w i l d t y p e m i c e i n 3 t i m e pe r i ods . ..... 25
F i gur e 9: T a r ge t e d l i pi d l e ve l s of s om e e x c e pt i ons a nd r e pr e s e nt a t i ons i n t he W T m ode l s ......... 26
F i gur e 10: m R N A e x pr e s s i on l e ve l s of L O X - 5, L O X - 15 a nd C O X e nz y m e s i n t he W T m ous e
m ode l ........................................................................................................................... 28
F i gur e 11: P U F A s l e ve l of i m por t a nt pr e c ur s or s of a l l m e t a bol i t e s ............................................. 29
F i gur e 12: P r o a nd a nt i - i nf l a m m a t or y e i c os a noi ds l e ve l f r om t a r ge t e d l i pi dom i c s . ..................... 31
F i gur e 13: I n t e r m e di a t e e i c os a noi ds l e ve l s f r om t a r ge t e d l i pi dom i c s . .......................................... 32
F i gur e 14: P r os t a noi d l e ve l s c om pa r i s ons be t w e e n W T a nd P t e n de l e t e d m ode l s . ...................... 33
F i gur e 15: m R N A e x pr e s s i on l e ve l of L i pox y ge n a s e 5 a nd 15 of w i l d t y p e a nd P T E N de l e t e d
m i c e i n t he pr oc e s s of l i ve r s t e a t os i s t o c a nc e r . .......................................................... 35
F i gur e 16: m R N A e x pr e s s i on l e ve l of C y c l oox y ge n a s e 1 a nd 2 of w i l d t y p e a nd P T E N de l e t e d
m i c e i n t he pr oc e s s of l i ve r s t e a t os i s t o c a nc e r . .......................................................... 36
F i gur e 17: A A a nd D P A l e ve l s f r om t a r ge t e d l i pi dom i c s ............................................................. 37
F i gur e 18: H e a t m a ps of t he L O X - 5, L O X - 15 a nd C O X m e t a bol i t e s i n w i l d t y p e a nd P T E N
de l e t e d m ode l s i n 3 t i m e pe r i ods . ............................................................................... 39
F i gur e 19: E i c os a noi ds l e ve l s of s om e e xc e pt i ons i n t he P t e n de l e t e d m ode l s ............................. 40
F i gur e 20: m R N A e xpr e s s i on l e ve l s of L O X - 5, L O X - 15 a nd C O X e nz y m e s i n t he P t e n de l e t e d
m ous e m ode l . .............................................................................................................. 42
F i gur e 21: E i c os a noi d l e ve l s of pr e c ur s or s i n t he P t e n de l e t e d m ode l i n 3 t i m e pe r i ods . ............. 43
F i gur e 22: S um m a r y of L O X a nd C O X E i c os a noi ds a nd t he i r bi ol og i c a l f unc t i ons . ................... 47
v
A bs t r a c t
H e pa t oc e l l ul a r c a r c i nom a ( H C C ) i s t he m os t c om m on pr i m a r y l i ve r c a nc e r . T he c ur r e nt
t r e a t m e nt s i nc l ude s ur gi c a l r e s e c t i on, c he m ot he r a py a nd i m m unot he r a py . H ow e ve r , t he l ong
t e r m s ur vi va l r a t e i s r e l a t i ve l y l ow . E i c os a noi ds a r e s i g na l i ng m ol e c ul e s pr oduc e d f r om
pol y u ns a t ur a t e d f a t t y a c i ds , us ua l l y A r a c hi doni c a c i d. E i c os a noi ds ha ve bot h pr o a nd a nt i -
i nf l a m m a t or y a c t i ons i n t he i nf l a m m a t i on. T he g l oba l l e ve l s of e i c os a noi ds m a y be a l t e r e d
dur i ng t he pr ogr e s s i on of l i ve r s t e a t os i s t o c a nc e r . I n t hi s s t udy , w e a na l y z e d t he c ha nge of
e i c os a noi d l e ve l s i n l i ve r di s e a s e us i ng a m ous e m ode l l a c ki ng P T E N i n t he l i ve r . W e s e l e c t e d
m i c e a t t hr e e a ge s : 3, 9 a nd 15 m ont hs of a ge . T he s e t hr e e t i m e pe r i ods r e pr e s e nt t hr e e s t a g e s of
t he H C C t um or i g e ne s i s be g i nni ng w i t h l i ve r s t e a t os i s , l i ve r s t e a t os i s a c c om pa ni e d by
i nf l a m m a t i on a nd ul t i m a t e l y t um or t r a ns f or m a t i on. T a r ge t e d l i pi dom i c s a r e pe r f or m e d t o
de t e r m i ne t he e i c os a noi d l e ve l s i n t he s e m i c e us i ng a g e - m a t c he d w i l d t y p e m i c e a s c ont r ol s . W e
r e por t he r e a g l oba l de c r e a s e of e i c os a noi ds i n r e l a t i on t o a gi ng of t he m i c e i n w i l d- t y p e ( W T )
l i ve r s . P t e n de l e t i on r e duc e s t he r a t e of e i c os a noi d de c l i ne w he n c om pa r e d t o a ge - m a t c he d W T
l i ve r s . U ne x pe c t e dl y , m os t e i c os a noi ds de c r e a s e d i n t he P t e n de l e t e d m ode l c om pa r e d t o t he a ge -
m a t c he d W T c ont r ol s . A f e w e x c e pt i ons a r e di s c ove r e d, i n pa r t i c ul a r i nf l a m m a t or y pr os t a noi ds
f ound i n t he t um or m i c r oe nvi r onm e nt . M ol e c ul a r a na l y s i s f ound a l t e r a t i on of l i poxy ge na s e
( L O X ) a nd c y c l oox y ge na s e ( C O X ) g e ne e x pr e s s i on t ha t s uppor t t he l i pi dom i c c ha nge s . I n
a ddi t i on, a l t e r a t i ons i n e i c os a noi d pr e c ur s or s m a y a l s o be a m a j or f a c t or c ont r i but i ng t o t he
obs e r ve d c ha nge s .
1
C ha pt e r I : B a c kgr ound a nd I n t r oduc t i on
1. L i ve r D i s e a s e
1.1 N on- a l c ohol i c f a t t y l i ve r di s e a s e ( N A F L D )
T he l i ve r i s t he l a r ge s t or ga n i n t he body w he r e i t pe r f or m s e s s e nt i a l r ol e s i n m e t a bol i s m , bi l e
pr oduc t i on a nd g l y c o ge n s t or a ge
1
. I n t he pa s t 20 y e a r s , t he pr e va l e nc e of l i ve r s t e a t os i s ha s
g r e a t l y i nc r e a s e d t hr oughout t he w or l d
2
, t og e t he r w i t h obe s i t y , dy s l i pi de m i a , hy p e r t e ns i on a nd
t y p e 2 di a be t e s
3 , 4
. L i ve r s t e a t os i s i s t he a c c um ul a t i on of f a t e x c e e di ng 5% of t he w e t w e i ght of
t he he a l t hy l i ve r
2
. I t i s a c hr oni c di s or de r , w hi c h i s a s s oc i a t e d w i t h e i t he r a l c ohol i c or
nona l c ohol i c f a c t or s
2
. N ona l c ohol i c f a t t y l i ve r di s e a s e ( N A F L D ) i s t he m os t c om m on c hr oni c
l i ve r di s e a s e a nd a ppr ox i m a t e l y 25% of a dul t s i n t he U S A ha ve be e n a f f e c t e d
2
.
D ur i ng N A F L D , e x c e s s f a t a c c um ul a t i on i n t he l i ve r r e s ul t s i n c hr oni c i nf l a m m a t i on a nd c e l l
de a t h t ha t l e a ds t o t he de ve l opm e nt of a m or e s e ve r e c ondi t i on na m e d non- a l c ohol i c
s t e a t ohe pa t i t i s ( N A S H )
3
. A ppr ox i m a t e l y 20% of N A F L D pa t i e nt s a r e e s t i m a t e d t o de ve l op
N A S H
4
. T he pe r s i s t e nt i nf l a m m a t i on a nd t i s s ue r e pa i r r e s ul t s i n t he f or m a t i on of l i ve r f i br os i s ,
w he r e a n a bnor m a l l y l a r ge a m ount of s c a r t i s s ue i s f or m e d i n t he l i ve r dur i ng t he f i br os i s s t a ge
3
.
T he s c a r t i s s ue di m i ni s he s t he bl ood f l ow t hr oug hout t he l i ve r a nd s l ow s t he a bi l i t y of t he l i ve r t o
m e t a bol i z e dr ug m ol e c ul e s a nd br e a k dow n na t ur a l t oxi ns
3
. I f t he f i br os i s i s not m a na ge d, m or e
l i ve r t i s s ue s w i l l g r a dua l l y be r e pl a c e d by c onne c t i ve t i s s ue , l e a di ng t o c i r r hos i s , a n e nd- s t a ge
l i ve r di s e a s e ( F i g. 1) .
2
F i gur e 1: T he pr ogr e s s of N A F L D t o H C C ( F i gur e i s g e ne r a t e d i n B i oR e nde r )
I n t he e a r l y N A F L D , e x c e s s f a t s t a r t s a c c um ul a t i ng i n t he he pa t oc y t e s , l e a di ng t o i nc r e a s e d
c y t oki ne l e ve l s . I n f l a m m a t i on oc c ur s i n he pa t oc y t e s i n N A S H , l e a di ng t o g r ow t h of c onne c t i ve
t i s s ue i n t he l i ve r . I n l i ve r c a nc e r , m or e i nf l a m m a t i on oc c ur s i n he pa t oc y t e s a nd t um or s a r e
f or m e d.
1.2 H e pa t oc e l l ul a r c a r c i nom a ( H C C )
H e pa t oc e l l ul a r c a r c i nom a ( H C C ) i s t he f i f t h m os t c om m on c a us e of c a nc e r i n t he w or l d. I t i s t he
m os t c om m on pr i m a r y l i ve r c a nc e r a nd oc c ur s i n a ppr ox i m a t e l y 80- 90% of c i r r hos i s pa t i e nt s .
T he a nnua l i nc i de nc e of H C C i n c i r r hos i s pa t i e nt s i s 2- 4%
4 , 5
. T he c ur r e nt t r e a t m e nt of H C C i s
be s t pe r f or m e d i n a m ul t i di s c i pl i na r y s e t t i ng , i nc l udi ng s ur gi c a l r e s e c t i on, l i ve r t r a ns pl a nt a t i on,
c he m ot he r a py a nd i m m unot he r a py
8
. H ow e ve r , t he c l i ni c a l out c om e s a r e f a r f r om s a t i s f a c t or y
be c a us e of dr ug r e s i s t a nc e a nd di s e a s e r e l a ps e
8
. S ur g i c a l r e s e c t i on a nd l i ve r t r a ns pl a nt a t i on g i ve
H C C pa t i e nt s t he m oda l i t y w i t h c ur a t i ve out c om e , but t he y ha ve l i m i t e d a ppl i c a bi l i t y be c a us e of
t he t um or s i z e a nd l i ve r f unc t i on
8
. O nl y a ppr ox i m a t e l y 5% of H C C pa t i e nt s a r e s ui t a bl e f or l i ve r
t r a ns pl a nt a t i on
9
. T he ove r a l l pr og nos i s f or s ur vi va l i s r e l a t i ve l y l ow , w hi c h i s t he s e c ond t o
pa nc r e a t i c c a nc e r w hi c h ha s t he poor e s t s ur vi va l out c om e
1 0
. T he ove r a l l s ur vi va l r a t e w i t hi n 5
y e a r s i s a bout 18.4% . W he n s t r a t i f i e d by di s e a s e s t a g i ng, t he r e l a t i ve 5- y e a r s ur vi va l i s 32.6% i n
pa t i e nt s w i t h l oc a l i z e d di s e a s e s , 10.8% w i t h r e gi ona l di s e a s e s a nd onl y 2.4% i n pa t i e nt s w i t h
3
m e t a s t a t i c di s e a s e s
1 0
. I n r e c e nt y e a r s , i m m unot he r a py ha s pr ovi de d l ong t e r m c a nc e r c ont r ol t o
H C C pa t i e nt s w i t hout l a r ge s i de e f f e c t s , a nd i t g r e a t l y i nc r e a s e d t he s ur vi va l r a t e
3 0
. H ow e ve r , i t
i s ur g e nt a nd i m por t a nt t o unde r s t a nd t he unde r l y i ng m ol e c ul a r c a us e of H C C a nd di s c ove r ne w
m ol e c ul a r t a r ge t s f or t he t r e a t m e nt of H C C .
1.3 I n f l a m m a t i on i n L i ve r D i s e a s e
D ur i ng t he pr ogr e s s i on of N A F L D t o l i ve r f i br os i s , t he a c c um ul a t i on of i nf l a m m a t i on a nd t i s s ue
da m a ge i nc r e a s e s t he r a t e of r e ge ne r a t i on, c e l l pr ol i f e r a t i on a nd m i t ot i c a c t i vi t i e s . H ow e ve r , t hi s
t i s s ue e nvi r onm e nt c a n a l s o s e r ve t o pr om ot e t he de ve l opm e nt of he pa t oc e l l ul a r c a r c i nom a
( H C C ) . A s s uc h, i nf l a m m a t i on pl a y s a n i m por t a nt r ol e i n t he pr ogr e s s i on of l i ve r s t e a t os i s t o
c a nc e r
4 , 6
.
D ur i ng a c ut e i nf l a m m a t or y s t a t e , K upf f e r c e l l s , t he r e s i de nt m a c r opha ge s a r e a c t i va t e d a s a r e s ul t
of i nj ur y - i nduc e d a popt os i s a nd ne c r os i s of he pa t oc y t e s
6
. I n t he m ous e m ode l , N F - κ B w a s s how n
t o be a c t i va t e d i n t he K upf f e r c e l l s by t he ba c t e r i a l a nd vi r a l a nt i g e ns a nd i t i nduc e s t he
t r a ns c r i pt i on of pr oi nf l a m m a t or y c y t oki ne s , T N F - α a nd I L - 6
6
. T he s e pr oi nf l a m m a t or y c y t oki ne s
bi nd t o t he i r g e ne r i c m e m br a ne r e c e pt or s on he pa t oc y t e s a nd he pa t i c s t e l l a t e c e l l s ( H S C s ) a nd
a c t i va t e N F - κ B f or f ur t he r s i g na l i ng pa t hw a y s
6
. I n H S C s , t he a c t i va t i on of t he N F - κ B i nduc e s
c e l l di f f e r e nt i a t i on, w hi c h di r e c t l y a nd i ndi r e c t l y l e a ds t o t he s e c r e t i on of pr of i br oge ni c a nd pr o-
pr ol i f e r a t i ve f a c t or s
6
. T he s e pr of i br oge ni c f a c t or s , i n t ur n, pr om ot e t i s s ue r e m ode l i ng a nd
de pos i t i on of a c ol l a ge n- r i c h e x t r a c e l l ul a r m a t r i x ( E C M ) , w hi c h pr ovi de s a n i de a l
m i c r oe nvi r onm e nt f or H C C
6
( F i g. 2) . I n he pa t oc y t e s , s uc h a c t i va t i on of N F - κ B a l s o pr e ve nt s
a popt os i s a nd i nduc e s c e l l ul a r pr ol i f e r a t i on
6
. T he l os s of a popt os i s a nd unc ont r ol l e d c e l l di vi s i on
4
a l l ow c a nc e r c e l l s t o s ur vi ve f or a l ong e r t i m e
3 4
. T he a c c um ul a t i on of m ut a t i ons f i na l l y l e a ds t o
t he f or m a t i on of t um or s a nd H C C
6 , 3 4
.
F i gur e 2: H e pa t i c i nf l a m m a t i on- f i br os i s - c a nc e r a xi s
I l l us t r a t i on of t he f unc t i on of N F - κ B i n t he he pa t i c i nf l a m m a t i on f i br os i s c a nc e r a x i s . A popt os i s
a nd ne c r os i s of he pa t oc y t e s a c t i va t e kupf f e r c e l l s a nd t he N F - κ B . T he t r a ns c r i pt i on of
pr oi nf l a m m a t or y c y t oki ne s T N F - α a nd I L - 6 i s i nduc e d by t he N F - κ B . T N F - α a nd I L - 6 bi nd t o
m e m br a ne r e c e pt or s on bot h he pa t oc y t e s a nd H S C s . T hi s a c t i va t e s t he N F - κ B i n he pa t oc y t e s a nd
H S C s . I n he pa t oc y t e s , N F - κ B i nduc e s c e l l ul a r pr ol i f e r a t i on a nd pr e ve nt s a popt os i s . I n H S C s ,
N F - κ B i nduc e s t he s e c r e t i on of pr of i br oge ni c a nd pr o- pr ol i f e r a t i ve f a c t or s , w hi c h l e a ds t o
f i br os i s a nd H C C i n t he l ong t e r m .
5
2. E i c os a noi ds
F a t t y a c i ds a r e l ong a l ky l c ha i ns w i t h a t e r m i na l c a r box y l i c a c i d m oi e t y
1 1
. T he i r a l ky l m oi e t y c a n
be e i t he r s a t ur a t e d or uns a t ur a t e d ba s e d on t he pr e s e nc e / a bs e nc e of t he doubl e bonds . S a t ur a t e d
f a t t y a c i ds ( S F A s ) a r e a l ky l c ha i ns w i t h no doubl e bonds
1 1
. S a t ur a t e d f a t t y a c i ds pl a y i m por t a nt
r ol e s i n hor m one pr oduc t i on, e ne r gy s t or a ge a nd c e l l ul a r m e m br a ne s t r uc t ur e
3 1
. U ns a t ur a t e d f a t t y
a c i ds c ont a i n one or m or e doubl e bonds a l ong t he a l ky l c ha i n
1 1
. U ns a t ur a t e d f a t t y a c i ds pl a y
s i g ni f i c a nt r ol e s i n i nf l a m m a t i on a nd s i g na l t r a ns duc t i on
3 2
.
E i c os a noi ds a s a c l a s s of bi oa c t i ve s i g na l i ng l i pi ds a r e de r i ve d f r om pol y u ns a t ur a t e d f a t t y a c i ds
( P U F A s )
1 3
. T he s e bi oa c t i ve l i pi ds a r e t r a ns por t e d out of t he c e l l s t ha t pr oduc e t he m a nd bi nd t o
t he G pr ot e i n c oupl e d r e c e pt or s ( G P C R s ) on t he t a r ge t c e l l s t o e x e r t t he i r bi ol og i c a l f unc t i ons
1 3
.
A l t houg h e i c os a noi ds a r e l i pi d c om pounds , t he y a r e not a bl e t o m ove a c r os s t he c e l l m e m br a ne
f r e e l y due t o t he c ha r ge s r e s ul t i ng f r om t he oxi da t i on r e a c t i on
1 3
. T r a ns por t e r s s uc h a s
pr os t a gl a ndi n t r a ns por t e r ( P G T ) a nd m ul t i dr ug - r e s i s t a nc e pr ot e i n 1 ( M R P 1) a r e r e qui r e d t o
t r a ns por t e i c os a noi ds out of t he c e l l s
1 3
.
C ol l e c t i ve l y , e i c os a noi ds ha ve be e n s how n t o pos s e s s bot h pr o a nd a nt i - i nf l a m m a t or y a c t i ons .
T he y m odul a t e i m m une r e s pons e s , a nd t hus pl a y i m por t a nt r ol e s i n r e gul a t i ng i nf l a m m a t i on
1 4
.
V i a t he i r a c t i ons a s i nf l a m m a t or y m e di a t or s , e i c os a noi ds a r e s t r ong l y l i nke d t o di f f e r e nt
phy s i ol ogi c a l a nd pa t hol og i c a l pr oc e s s e s , i nc l udi ng t i s s ue r e pa i r , i nf l a m m a t i on a nd di s e a s e
i nc l udi ng c a nc e r
1 4
.
6
2.1 E i c os a noi ds bi os y n t he s i s
S t r uc t ur a l l y di s t i nc t e i c os a noi ds a r e m a de f r om t he 20 c a r bon P U F A s s uc h a s a r a c hi doni c a c i d
( A A , C 20: 4 ω - 6) a nd ot he r ω - 6 P U F A s , i nc l udi ng di hom o- γ - l i nol e ni c a c i d
1 3
. E i c os a noi ds c a n
a l s o be de r i ve d f r om ω - 3 P U F A s s uc h a s e i c os a pe nt a e noi c a c i d ( E P A , C 22: 5 ω - 3) ,
doc os a pe nt a e noi c a c i d ( D P A , C 22: 6 ω - 3) a nd doc os a he x a e noi c a c i d ( D H A , C 22: 6 ω - 3)
1 3 , 1 5
. T he
di f f e r e nc e be t w e e n ω - 6 a nd ω - 3 P U F A s i s t he pos i t i on of t he f i r s t doubl e bond i n t he c a r bon
c ha i n. I n ω - 6 P U F A s , t he f i r s t doubl e bond oc c ur s on t he s i xt h c a r bon a t om
3 3
. I n ω - 3 P U F A s , t he
f i r s t doubl e bond oc c ur s on t he t hi r d c a r bon a t om
3 3
. T he m ul t i pl i c i t y of t he pos i t i ons a nd
num be r s of doubl e bonds of P U F A s l e d t o a di ve r s e f a m i l y of c om pounds t ha t c om pos e
bi oa c t i ve l i pi ds
3 3
.
E i c os a noi ds a r e de r i ve d f r om t he oxi da t i on of A A a nd ot he r r e l a t e d P U F A s by t hr e e m a j or
e nz y m a t i c pa t hw a y s : l i poxy ge na s e ( L O X ) , c y c l oox y ge na s e ( C O X ) a nd c y t oc hr om e P 450 ( C Y P )
e nz y m e s
1 5
. L e ukot r i e ne s a nd l i poxi ns a r e f or m e d t hr oug h t he a c t i on of 5- L O X
1 5
. I n c ont r a s t ,
pr os t a gl a ndi ns a nd t hr om boxa ne s a r e f or m e d vi a t he C O X pa t hw a y
1 5
. E poxy e i c os a t r i e noi c a c i ds
( E E T s ) a nd hy d r ox y e i c os a t e t r a e noi c a c i ds ( H E T E s ) a r e f or m e d t hr ough C Y P - m e di a t e d
m e t a bol i s m
1 5
. E i c os a noi ds c a n a l s o be f or m e d by non- e nz y m a t i c f r e e r a di c a l pa t hw a y s
1 5
. F i gur e
3 s how s t he pr oduc t i on of e i c os a noi ds f r om A A a nd t he i r r e c e pt or s .
7
F i gur e 3: B i os y n t he s i s pa t hw a y s of E i c os a noi ds ( L O X , C O X a nd C y p 450 e nz y m e s )
1 5
T he s um m a r y of t he e i c os a noi d pa t hw a y s a nd t he i r bi ol og i c a l f unc t i ons . T he r e d c ol or r e pr e s e nt s
pr o- i nf l a m m a t or y e i c os a noi ds , a nd t he bl ue c ol or r e pr e s e nt s t he a nt i - i nf l a m m a t or y e i c os a noi ds .
N um be r s 1- 10 r e pr e s e nt t he G pr ot e i n c oupl e d r e c e pt or s of di f f e r e nt e i c os a noi ds a nd t he i r
s i g na l i ng pa t hw a y s .
8
2.1.1 L i pox y ge n a s e pa t hw a y
L i pox y ge n a s e s ( L O X ) a r e t he e nz y m e s t ha t c a t a l y z e t he oxy ge n a t i on r e a c t i ons of P U F A s
t hr ough t he oxi da t i on of t he doubl e bond f ound i n t he a l ky l c ha i n
7
. S i x L O X i s of or m s ha ve be e n
i de nt i f i e d i n m a m m a l s
2 7
. A s t he na m e i ndi c a t e d, 5- L O X oxi di z e s t he P U F A c ha i n a t c a r bon- 5 t o
pr oduc e 5- H E T E a nd t he n t o l e ukot r i e ne s
2 7
. 15- L O X i s a n e nz y m a t i c i s of or m c a t a l y z i ng t he
oxy ge n a t i on of P U F A s t o g e ne r a t e 15- H pE T E a nd t he n t o 15- H E T E
2 7
. 12- L O X i s a not he r
i s of or m t ha t i s e x pr e s s e d i n e pi t he l i a l c e l l s a nd m y e l oi d c e l l s i nc l udi ng pl a t e l e t s t ha t m e t a bol i z e s
A A t o 12- H pE T E t he n t o 12- H E T E
2 7
. 5- L O X , 15- L O X a nd 12- L O X pl a y i m por t a nt r ol e s i n
i nf l a m m a t i on. T he di f f e r e nc e be t w e e n 5- L O X , 15- L O X a nd 12- L O X i s r e l a t e d t o t he pos i t i on
a l ong t he a l ky l c ha i n t ha t t he y be oxi di z e d
7
. 15- L O X t y p e I I a nd 12- L O X t y p e I I ha ve s i m i l a r
f unc t i ons c om pa r e d t o 15- L O X a nd 12- L O X but w i t h l ow e r c a t a l y t i c a c t i vi t y
2 7
. E - L O X i s
di f f e r e nt f r om a l l ot he r l i poxy ge na s e s . I t i s a hy d r ope r ox i de i s om e r a s e , w hi c h m e t a bol i t e s
uns a t ur a t e d hy d r ope r ox y f a t t y a c i ds i nt o e pox y a l c ohol a nd e pox y ke t o m e t a bol i t e s
2 7
.
T he i m m e di a t e m e t a bol i t e s of t he L O X e nz y m e s , hy d r ope r ox y e i c os a t e t r a e noi c a c i ds ( H pE T E s )
a r e hi g hl y uns t a bl e c om pounds r a pi dl y c onve r t e d i nt o H E T E s a nd l e ukot r i e ne s
1 5
. F or a s pe c i f i c
e x a m pl e , 5- L O X i nt e r a c t s w i t h 5- L O X - a c t i va t i ng pr ot e i n ( F L A P ) t ha t phy s i c a l l y i nt e r a c t s w i t h
5- L O X t o r e gul a t e i t s a c t i vi t y t o f or m H pE T E s f r om A A
2 8
. 5- H pE T E i s t he n r a pi dl y c onve r t e d
i nt o 5- H E T E or l e ukot r i e ne A 4 ( L T A 4)
1 5
. L T A 4 a r e t he n s pont a ne ous l y c onve r t e d i nt o L T B 4,
l i poxi ns a nd ot he r a c t i ve c y s t e i ny l l e ukot r i e ne s , s uc h a s L T C 4, L T D 4 a nd L T E 4 by s pe c i f i c
s y n t ha s e s
1 5
. W hi l e l i poxi ns a nd l e ukot r i e ne s a r e t he c ha r a c t e r i z e d m e t a bol i t e s f r om t he L O X
pa t hw a y , ot he r i nt e r m e di a t e pr oduc t s m a y a l s o ha ve t he i r ow n f unc t i ons e ve n t houg h t he y a r e
not r e a di l y de t e c t e d due t o t he i r s hor t ha l f - l i f e i n vi vo.
9
2.1.2 C y c l oox y ge n a s e P a t hw a y
C y c l oox y ge n a s e s ( C O X ) a r e e nz y m e s t ha t c a t a l y z e t he f or m a t i on of a s ubg r oup of e i c os a noi ds
c ol l e c t i ve l y t e r m e d pr os t a noi ds
1 5 , 2 7
. T he s e e nz y m e s a r e he m e - c ont a i ni ng pr ot e i ns w i t h bot h
oxy ge n a s e s a nd pe r ox i da s e s a c t i vi t i e s
2 6
. T he pe r ox y l r a di c a l i s pr oduc e d t o i ni t i a t e t he C O X
r e a c t i on a nd i t i s t he n r e duc e d t o t he hy d r ope r ox i de t o f or m t he pr os t a g l a ndi n
2 6
. T he y a r e t he
i m por t a nt r a t e l i m i t i ng e nz y m e s t ha t c a t a l y z e t he f i r s t c om m i t t e d s t e ps i n t he s y n t he s i s of
pr os t a noi ds f r om P U F A s , l i ke A A s
1 5
. T he r e a r e t w o C O X i s of or m s , w hi c h a r e know n a s C O X - 1
a nd C O X - 2. C O X - 1 e x pr e s s i on i s c ons t i t ut i ve i n a l l t i s s ue s , e s pe c i a l l y G I t r a c t
2 7
. C O X - 1 i s
g e ne r a l l y r e s pons i bl e f or m a i nt a i ni ng t he i nt e r na l hom e os t a s i s of t he body
2 7
. H ow e ve r , t he
e x pr e s s i on of C O X - 2 i s us ua l l y unde t e c t a bl e i n t he he a l t hy c ondi t i on, but i nduc i bl e by
i nf l a m m a t or y s t i m ul i , hor m one s a nd g r ow t h f a c t or s
2 7
. A s pi r i n a nd ot he r non- s t e r oi da l a nt i -
i nf l a m m a t or y dr ugs ( N S A I D s ) c a n bi nd t o C O X e nz y m e s a nd i nhi bi t t he pr oduc t i on of
e i c os a noi ds ( pr os t a gl a ndi ns a nd l e ukot r i e ne s ) dur i ng t he i nf l a m m a t or y pr oc e s s
1 4
.
I n t he C O X pa t hw a y , A A i s m e t a bol i z e d t o pr os t a gl a ndi n G 2 ( P G G 2) a f t e r be i ng r e l e a s e d f r om
m e m br a ne g l y c e r ophos phol i pi ds by c y t os ol i c phos phol i pa s e A 2 ( c P L A 2)
2 6
. T he uns t a bl e P G G 2
w i l l be r a pi dl y r e duc e d i nt o P G H 2
2 6
. A s t he i m por t a nt pr e c ur s or of ot he r pr os t a noi ds , P G H 2 i s
t he n c onve r t e d i nt o ot he r m or e s t a bl e m e t a bol i t e s l i ke P G D 2, P G E 2 a nd P G F 2a by t he
c a t a l y z a t i on of di f f e r e nt s y n t ha s e s
1 5 , 2 7
. T he s e m e t a bol i t e s a r e t r a ns por t e d i nt o e x t r a c e l l ul a r s pa c e
a nd bi nd t o s pe c i f i c r e c e pt or s on t he s ur f a c e of t he t a r ge t e d c e l l t o pe r f or m t he i r phy s i ol ogi c a l
a nd pa t hophy s i ol og y f unc t i ons
1 5
. P G H 2 c a n a l s o be c onve r t e d i nt o P G I 2 a nd t hr om boxa ne A 2
( T X A 2) , a n uns t a bl e pr oduc t t ha t i s r a pi dl y c onve r t e d i nt o T X B 2, s o na m e d due t o i t s
i nvol ve m e nt i n pl a t e l e t a ggr e ga t i on a nd va s oc ons t r i c t i on
2 6
.
1 0
2.2 E i c os a noi ds F unc t i on i n I n f l a m m a t i on
E i c os a noi ds m e di a t e t he i m m une r e s pons e a nd pl a y e i t he r pr o or a nt i - i nf l a m m a t or y f unc t i ons
dur i ng t he i nf l a m m a t or y s t a ge . H e r e , P G E 2 a nd L T B 4 a r e e x a m pl e s of pr o- i nf l a m m a t or y
e i c os a noi ds t ha t pr ol ong t he i nf l a m m a t i on.
P G E 2 i s one of t he m os t s t udi e d e i c os a noi ds a nd i s r e l e a s e d dur i ng t he i nf l a m m a t or y pr oc e s s
1 9
.
D ur i ng ba c t e r i a l i nf e c t i ons , P G E 2 m e di a t e s t he i nf l a m m a t or y r e s pons e by s t i m ul a t i ng t he
pr oduc t i on of pr o- i nf l a m m a t or y c y t oki ne s s uc h a s I L - 1 β , I L - 23 a nd I L - 8
1 9
. I n m ous e m ode l s ,
L P S t r e a t m e nt i nduc e s t he e x pr e s s i on of C O X - 2, w hi c h l e a ds t o t he f or m a t i on a nd s e c r e t i on of
P G E 2
1 5 , 1 9
. P G E 2 f unc t i ons by bi ndi ng t o i t s r e c e pt or s E P r e c e pt or s 1- 4, w hi c h ha ve i s of or m
s pe c i f i c dow ns t r e a m e f f e c t s i n r e s pons e t o a c t i va t i on
1 9
. A f t e r bi ndi ng , t he G - l i nke d r e c e pt or s
c ont r ol l e ve l s of s e c onda r y m e s s e nge r s i n t he c e l l , s uc h a s c A M P , I P 3 a nd c a l c i um
1 9
. T he s e
s e c onda r y m e s s e nge r s m e di a t e t he c e l l ul a r r e s pons e t o P G E 2 vi a r e gul a t i on of t r a ns c r i pt i on
f a c t or s , s uc h a s N F - κ B a nd C - J U N
1 9
. T hus , t he a c t i va t i on of t he s e E P r e c e pt or s i g na l i ng l e a ds t o
di ve r s e e f f e c t s i nc l udi ng s hi f t s i n m a c r opha ge pol a r i z a t i on, dow nr e gul a t i on of ba c t e r i c i da l
c a pa bi l i t i e s i n ne ut r ophi l s
1 9
.
L T B 4 i s a not he r i m por t a nt e i c os a noi d t ha t ha s be e n f ound t o pl a y i m por t a nt r ol e s i n t he e a r l y
s t a ge of i nf l a m m a t i on dur i ng i nf e c t i on
2 0
. L T B 4 i s s y n t he s i z e d a nd s e c r e t e d i n m a c r opha ge s a nd
m onoc y t e s by t he c a t a l y t i c a c t i on of 5- L O X i n r e s pons e t o ba c t e r i a l i nf e c t i on
1 5 , 2 0
. L T B 4 bi nds t o
i t s r e c e pt or , B L T 1 r e c e pt or , t o r e c r ui t ne ut r ophi l s . A t t he i nf l a m m a t or y s i t e s , t he r e c r ui t e d
ne ut r ophi l s pha g oc y t i z e a nd de gr a de i nva di ng pa t hog e ns
2 0
. I n a ddi t i on, L T B 4 a l s o i nduc e s t he
m i g r a t i on of N F - κ B t o i ni t i a t e t he t r a ns c r i pt i on of pr o- i nf l a m m a t or y c y t oki ne s s uc h a s I L - 1 β a nd
T N F - α , w hi c h a r e i m por t a nt t o t he hos t i m m une r e s pons e t o t he i nva di ng pa t hog e ns
2 0
. T og e t he r ,
L T B 4 a nd P G E 2 pl a y i m por t a nt r ol e s i n t he hos t i m m une r e s pons e t o t he i nva di ng pa t hog e ns .
1 1
2.3 R e gul a t i on of E i c os a noi ds
W hi l e ne gl i g i bl e a m ount s of e i c os a noi ds a r e pr oduc e d i n m os t c e l l t y p e s , t he bi os y n t he s i s of
e i c os a noi ds i s dr a m a t i c a l l y i nc r e a s e d f ol l ow i ng c P L A 2 a c t i va t i on a nd ot he r dow ns t r e a m
e nz y m e s dur i ng t he i nf l a m m a t or y pr oc e s s
1 3 , 2 3
. A s t he m a j or pr e c ur s or s f or e i c os a noi ds , di e t a r y
l i pi ds c a n r e gul a t e t he pr oduc t i on of e i c os a noi ds
2 3 , 2 4
. I nc r e a s i ng t he a m ount of di e t a r y A A w i l l
l e a d t o a n e l e va t e d s t or a ge of A A i n t i s s ue phos phol i pi ds
2 4
. I n a ddi t i on, di e t a r y l i nol e i c a c i d i s
t he m a j or s our c e of t he A A pr oduc t i on. L i nol e i c a c i d c a n be de s a t ur a t e d a nd e l onga t e d t o
pr oduc e A A a nd i nc r e a s e t he A A s t or a ge i n t he t i s s ue . H ow e ve r , A A i s not f r e e l y a va i l a bl e i n t he
c e l l s a nd i s s t or e d i n a n e s t e r i f i e d f or m
2 1
. S t i m ul a t i on of t he T ol l - l i ke r e c e pt or 4 ( T L R 4) w i t h
L P S i nduc e s r a pi d phos phor y l a t i on of c P L A 2 vi a a c t i va t i on of M A P K i na s e ( M A P K ) . A c t i va t e d
c P L A 2 c l e a ve s t he m e m br a ne phos phol i pi ds t o r e l e a s e f r e e A A . O t he r e i c os a noi ds pr e c ur s or s
s uc h a s E P A , D P A a nd D H A a r e r e l e a s e d by i P L A 2
2 1 , 2 2
. I n a ddi t i on, t he e x pr e s s i on of C O X - 2
a nd m i c r os om a l pr os t a gl a ndi n E s y n t ha s e - 1 ( m P G E S - 1) a r e a l s o upr e gul a t e d dur i ng t he
i nf l a m m a t or y r e s pons e t o i nduc e e i c os a noi d bi os y n t he s i s . A ddi t i ona l l y , L O X e nz y m e s a r e a l s o
e x pr e s s e d i n i m m une , e pi t he l i a l a nd t um or c e l l s t o pr oduc e l e ukot r i e ne s a nd l i poxi ns , w he r e a s
t he C O X e nz y m e s a r e e x pr e s s e d i n t he f i br obl a s t s , e ndot he l i a l a nd i nf l a m m a t or y c e l l s
2 3
. T he
e x pr e s s i on of s pe c i f i c e i c os a noi d bi os y n t he t i c e nz y m e s i n s pe c i f i c c e l l t y p e s g r e a t l y l i m i t s t he
pr oduc t i on a nd s e c r e t i on of e i c os a noi ds , pr ovi di ng s pe c i f i c i t y f or t he r ol e of t he s e e i c os a noi ds i n
i nf l a m m a t or y r e s pons e
2 3
.
1 2
3. H y p ot he s i s a nd O bj e c t i ve s
I n f l a m m a t i on pl a y s i m por t a nt r ol e s i n t he pr ogr e s s i on of l i ve r s t e a t os i s t o c a nc e r . W i t h l e s s
i nf l a m m a t i on i n t he l i ve r , t he r i s k of de ve l opi ng f i br os i s , c i r r hos i s a nd H C C a r e dr a m a t i c a l l y
de c r e a s e d. A s e i c os a noi ds a s a w hol e pl a y ke y r ol e s i n t he i nf l a m m a t or y pr oc e s s , w e hy pot he s i z e
he r e t hat e i c os anoi d l e v e l s c hange i n t he pr ogr e s s i on of l i v e r s t e at os i s and pr om ot e
t um or i ge ne s i s . I n t hi s s t udy , w e a na l y z e d t he g l oba l c ha nge of e i c os a noi d l e ve l s i n a m ous e
m ode l of l i ve r di s e a s e i n a n e f f or t t o i de nt i f y s pe c i f i c di s e a s e s t a ge c ha nge s of t he e i c os a noi d
s pe c i e s . W e us e d t he g e ne t i c m ous e m ode l t ha t m i m i c ke d hum a n l i ve r di s e a s e pr og r e s s i on a t 3
di f f e r e nt a ge s : 3, 9 a nd 15 m ont hs . T he s e 3 t i m e pe r i ods r e pr e s e nt t hr e e s t a ge s of t he H C C
pr ogr e s s i on: l i ve r s t e a t os i s , l i ve r s t e a t os i s a c c om pa ni e d w i t h i nf l a m m a t i on a nd l i ve r c a nc e r .
U s i ng m a s s s pe c t r om e t r y ba s e d t a r ge t e d l i pi dom i c s , w e i de nt i f i e d bi oa c t i ve l i pi ds t ha t l i ke l y pl a y
c r i t i c a l r ol e s a nd t he i r l e ve l c ha nge s i n t he pr ogr e s s i on of l i ve r s t e a t os i s t o c a nc e r . O ur a na l y s e s
i ndi c a t e d t ha t e i c os a noi d l e ve l s de c r e a s e a s t he m i c e a ge . W e f ur t he r i de nt i f i e d di s t i nc t
di f f e r e nc e s of e i c os a noi ds t ha t a r e a l t e r e d i n t he P t e n de l e t e d m i c e dur i ng t he pr ogr e s s i on of l i ve r
s t e a t os i s t o c a nc e r . T hi s s t udy i de nt i f i e d t he pot e nt i a l m e t a bol i t e s a nd e nz y m a t i c pa t hw a y s t ha t
m i g ht be r e s pons i bl e f or t he pr og r e s s i on f r om one s t a ge t o t he ne x t dur i ng t he pr ogr e s s i on of
l i ve r di s e a s e . W e a l s o r e por t a nove l f i ndi ng of a ge - r e l a t e d de c l i ne of e i c os a noi ds i n t he l i ve r
t i s s ue .
1 3
C ha pt e r I I : M e t hods a nd M a t e r i a l s
F i gur e 4 s how s t he w or kf l ow of t he e x pe r i m e nt s i n t hi s s t udy . L i ve r s a m pl e s of W T a nd P T E N
m i c e a t 3 di f f e r e nt a ge s w e r e i s ol a t e d a nd t he l i pi ds w e r e e x t r a c t e d. T a r ge t e d l i pi dom i c s w e r e
pe r f or m e d t o a na l y z e t he g l oba l l e ve l c ha nge s of t he bi oa c t i ve l i pi ds . A f t e r s t udy i ng t he
bi ol og i c a l pa t hw a y of t he pr oduc t i on of e i c os a noi ds , s om e c a t a l y t i c e nz y m e s w e r e s e l e c t e d f or
f ur t he r a na l y s i s . R T - qP C R w a s pe r f or m e d t o unde r s t a nd t he e x pr e s s i on l e ve l c ha nge s of t he s e
c a t a l y t i c e nz y m e s ( F i g. 4) .
F i gur e 4: T a r ge t e d l i pi dom i c s a nd R e a l t i m e qP C R on W T a nd P T E N de l e t e d m i c e m ode l .
T he w or kf l ow of t a r ge t e d l i pi dom i c s a nd R T - qP C R pe r f or m e d i n t hi s s t udy . L i pi d w a s e x t r a c t e d
f r om t he l i ve r s a m pl e s of P T E N de l e t e d m i c e f or t he t a r ge t e d l i pi dom i c s . A f t e r t he e i c os a noi ds
l e ve l s i n t he l i pi d pr of i l e w e r e a na l y z e d, t he m R N A e x pr e s s i on of L O X a nd C O X w e r e va l i da t e d
by t he R T - qP C R .
1 4
2.1 L i pi d E x t r a c t i on
C r y o - pr e s e r ve d l i ve r t i s s ue s w e r e w e i ghe d, a nd t he w e i ght r e c or de d. T he t i s s ue s w e r e
hom og e ni z e d by a ddi ng one s poonf ul of s t a i nl e s s - s t e e l be a ds ( 0.9 - 2.0 m m ) t o t he E ppe ndor f
t ube s c ont a i ni ng t he t i s s ue . T o e x t r a c t l i pi d c om pone nt s , 450 μ L of c ol d M e O H w a s t he n a dde d
t o e a c h t ube . W e t he n a dde d 50 μ L of 50 ng / m L i nt e r na l s t a nda r d ( I S ) m i xt ur e t o e a c h s a m pl e .
T he I S m i xt ur e c ont a i ne d l i pi d s t a nda r ds d5- R vD 2, d8- 5S - H E T E , d4- P G E 2, d5- L X A 4, d4- L T B 4.
T he s a m pl e s w e r e t he n hom og e ni z e d us i ng 2.5- m i nut e pul s e i nt e r va l s a t 30 hz f or a t ot a l of 7.5
m i nut e s . T he hom og e ni z e d s a m pl e s w e r e c l a r i f i e d by c e nt r i f ugi ng a t 10,000 r pm f or 15 m i nut e s
t o g e t r i d of c e l l de br i s . T he c l a r i f i e d s upe r na t a nt s w e r e t r a ns f e r r e d i nt o ne w t ube s a nd di l ut e d
w i t h w a t e r t o a f i na l 10% M e O H s ol ut i on. T o f ur t he r e x t r a c t t he l i pi ds , s a m pl e s w e r e t he n l oa de d
ont o t he e xt r a c t i on c ol um n. T he e x t r a c t i on c ol um n w a s pr e - c ondi t i one d w i t h a g r a di e nt e l ut i on
c ont a i ni ng 1 m L of pur e M e O H a nd f ol l ow e d by 1 m L of w a t e r . A f t e r s a m pl e s w e r e a ppl i e d, t he
c ol um ns w e r e w a s he d w i t h 2 m L of w a t e r a nd c ol l e c t e d a s f l ow t hr oug h f r a c t i on. S a m pl e s w e r e
t he n e l ut e d w i t h 500 μ L of pur e M e O H . T he e l ue nt f r a c t i on w a s dr i e d unde r ni t r og e n g a s . T he
s a m pl e w a s r e c ons t i t ut e d t o 50 μ L w i t h 50% M e O H . 30 μ L of t he f i na l m i xt ur e w a s t r a ns f e r r e d
i nt o L C / M S vi a l s a nd r e a dy t o be us e d f or t he l i pi dom i c s .
1 5
2.2 M a s s S pe c t r om e t r y B a s e d T a r ge t e d L i pi dom i c s
A t r i pl e qua dr upol e m a s s s pe c t r om e t e r ( M S ) w a s us e d f or a t a r ge t e d l i pi dom i c s a na l y s i s f or
pol y u ns a t ur a t e d f a t t y a c i d. D ur i ng t he a na l y s i s , a l l m a s s e s e x c e pt t he t a r ge t e d pa r e nt i ons w e r e
r e j e c t e d i n t he f i r s t qua dr upol e . T he t a r ge t e d pa r e nt i ons w e r e t he n f r a gm e nt e d i n t he s e c ond
qua dr upol e by c ol l i s i on- i nduc e d di s s oc i a t i on. P r e - s e l e c t e d num be r s of pa r e nt t o t r a ns i t i on i ons
w e r e c ol l e c t e d i n t he t hi r d qua dr upol e a nd a na l y z e d t hr ough t he m a s s s pe c t r om e t r y . N i t r og e n g a s
w a s us e d t o br e a k t he pa r e nt a l i ons i nt o s m a l l e r f r a gm e nt s or da ught e r i ons . B y s e l e c t i ng
m ul t i pl e l i pi ds a nd f r a gm e nt i ng t he r e s pe c t i ve pr oduc t i vi t y l i pi ds , t he r e l a t i ve qua nt i t a t i ve
i nf or m a t i on of t he s e l e c t e d l i pi ds w a s a c qui r e d. T he r e s ul t w a s a uni que pa r e nt t o da ught e r i on
pa i r di s t i nc t t o a l i pi d of i nt e r e s t .
R a w da t a f r om t he t a r ge t e d l i pi dom i c s w a s a na l y z e d us i ng M ul t i Q ua nt 3.0. P e a ks of e a c h
r e a di ng w e r e s e l e c t e d ba s e d on r e t e nt i on t i m e t o c a l c ul a t e a r e a unde r t he c ur ve ( A U C ) f or
r e l a t i ve a bunda nc e of t he l i pi d i n t he e x t r a c t s . T he w e i ght of t he l i ve r s a m pl e w a s t he n us e d f or
nor m a l i z a t i on. A f t e r nor m a l i z a t i on, t he g l oba l l e ve l of e a c h 95 l i pi ds w e r e t he n m e a s ur e d.
1 6
2.3 T i s s ue hom og e ni z a t i on a nd R N A E x t r a c t i on
P r e vi ous l y c r y o - pr e s e r ve d l i ve r t i s s ue s w e r e us e d f or t hi s s t udy . F or R N A i s ol a t i on, 20m g of
l i ve r t i s s ue w a s c ut a nd put on t he dr y i c e . T he l i ve r t i s s ue s w e r e t r a ns f e r r e d i nt o a 5 m L r ound
bot t om t ube w i t h 1 m L T R I Z O L . L i ve r t i s s ue s w e r e c om pl e t e l y hom og e ni z e d w i t h t he r e a ge nt
by a s oni c a t or f or 10- 15s . T he hom og e ni z e d t i s s ue w a s i nc uba t e d a t r oom t e m pe r a t ur e f or 5
m i nut e s t o pe r m i t t he c om pl e t e di s s oc i a t i on of nuc l e opr ot e i n c om pl e xe s . T he s a m pl e w a s
c e nt r i f uge d a t 12000 R P M a t 4
o
C f or 5 m i nut e s t o r e m ove t i s s ue de br i s . S upe r na t a nt s w e r e
t r a ns f e r r e d t o ne w 15m L t ube s . 0.2 m L of c hl or of or m pe r 1 m L of T R I Z O L w a s a dde d t o e a c h
t ube . T he t ube s w e r e c a ppe d s e c ur e l y a nd vor t e x e d vi g or ous l y f or 15 s e c onds . T he t ube s w e r e
t he n i nc uba t e d a t r oom t e m pe r a t ur e f or 15 m i nut e s . T he s a m pl e w a s c e nt r i f uge d a t 12,000 R P M
a t 4
o
C f or 5 m i nut e s . F ol l ow i ng t he c e nt r i f uga t i on, t hr e e di s t i nc t l a y e r s : l ow e r r e d, phe nol -
c hl or of or m pha s e ; i nt e r pha s e ; a nd c ol or l e s s uppe r a que ous pha s e w e r e f ound i n t ube s . R N A i s
f ound i n t he t op l a y e r . D N A c oul d be f ound i n t he m i ddl e l a y e r a nd pr ot e i n c oul d be f ound i n t he
bot t om l a y e r .
R N A r e m a i ne d e x c l us i ve l y i n t he a que ous pha s e of t he t ube a f t e r t he c e nt r i f uga t i on. T hus , t he
uppe r a que ous pha s e w a s c a r e f ul l y t r a ns f e r r e d i nt o ne w t ube s w i t hout di s t ur bi ng t he i nt e r pha s e .
0.5 m L of c ol d i s opr opy l a l c ohol w a s t he n us e d t o pr e c i pi t a t e t he R N A f r om t hi s a que ous pha s e .
T he m i xt ur e w a s t he n c e nt r i f uge d a t 12000 x g a t 4
o
C f or 10 m i nut e s . R N A f or m s a g e l - l i ke
pe l l e t a t t he bot t om of t he t ube . S upe r na t a nt w a s di s c a r de d a nd t he R N A pe l l e t w a s w a s he d w i t h
a t l e a s t 1 m L of 75% of e t ha nol . T he m i xt ur e w a s vor t e x e d a nd c e nt r i f uge d a t 7500 x g a t 4
o
C
f or 5 m i nut e s . T hi s s t e p w a s r e pe a t e d onc e . T he 75% e t ha nol w a s r e m ove d by a i r dr y or
c e nt r i f uge f r om t he R N A pe l l e t f or 5- 10 m i nut e s . T he R N A pe l l e t w a s di s s ol ve d by D E P C -
1 7
t r e a t e d w a t e r a nd w e l l m i xe d. T he R N A c onc e nt r a t i on w a s m e a s ur e d us i ng t he N a nodr op
m a c hi ne .
2.4 P r i m e r T e s t i ng
A l l pr i m e r s e que nc e s w e r e de s i g ne d a nd ve r i f i e d us i ng N C B I a nd c us t om m a de by I n t e gr a t e d
D N A T e c hnol ogi e s . T w o s e t s of pr i m e r s w e r e or de r e d f or e a c h t a r ge t e d g e ne a nd t e s t e d. P r i m e r s
w i t h l ow e r C T va l ue s a nd g ood m e l t i ng c ur ve s w e r e s e l e c t e d f or f ur t he r qP C R e x pe r i m e nt s .
T a bl e 1 s how s a l l t he pr i m e r s us e d i n t he r e a l t i m e qP C R of t hi s s t udy .
T a bl e 1 O l i g onuc l e ot i de P r i m e r s U s e d f or R e a l - T i m e qP C R
G e ne F or w a r d R e ve r s e
A L O X - 5 5’ - A G T C A A G A A C C A C T G G C A G G 5’ - G A G T A C T G G G T T G C A G C C A T
A L O X - 15 5’ - C A C T T G G T G G C T G A G G T C T T 5’ - T G T A G G T C A G G A A G G C T C C A
C O X - 1 5’ - G A G A T C T G G A C C T G G C T T C G 5’ - G C G C A T G A G T A C T T C T C G G A
C O X - 2 5’ - A G C C C A T T G A A C C T G G A C T G 5’ - A C C C A A T C A G C G T T T C T C G T
18S 5’ - A A A C G G C T A C C A C A T C C A A G 5’ - C A A T T A C A G G G C C T C G A A A G
1 8
2.5 R e a l - T i m e R T - P C R
A t ot a l of 1000 ng of R N A s a m pl e w a s t r a ns f e r r e d i nt o a R T t ube . D E P C w a t e r w a s a dde d t o
br i ng t he vol um e up t o a t ot a l of 16.5 μ L . 1 μ L of 50 μ M r a ndom he x a m e r pr i m e r ( T he r m oF i s he r
S c i e nt i f i c ) w a s t he n a dde d t o t he R T t ube . T he s a m pl e w a s i nc uba t e d i n t he R T m a c hi ne a t 70 ℃
f or 10 m i nut e s t o br e a k up t he R N A t e m pl a t e s a nd a l l ow a nne a l i ng t o oc c ur . A f t e r i nc uba t i on, 5
μ L of M - M L V R T 5X buf f e r ( P R O M E G A ) , 1 μ L of dN T P s ( B i ol a nd S c i e nt i f i c L L C ) , 1 μ L of
R N a s e i nhi bi t or a nd 0.5 μ L of M - M L V R e ve r s e T r a ns c r i pt a s e ( P R O M E G A ) w e r e m i xe d a nd
a dde d t o t he R T t ube f or r e ve r s e t r a ns c r i pt i on t o oc c ur . T he c D N A s a m pl e s w e r e c ol l e c t e d a f t e r
90 m i nut e s a nd s t or e d i n t he - 20
o
C f r i dge .
T o pe r f or m t he P C R r e a c t i on f ol l ow i ng R T , 1 μ L of f or w a r d pr i m e r , 1 μ L of ba c kw a r d pr i m e r ,
10 μ L of S Y B R g r e e n r e a ge nt w i t h T a q D N A pol y m e r a s e a nd dN T P s ( A P E x B I O ) , 7 μ L of
D E P C t r e a t e d w a t e r a nd 1 μ L of c D N A w e r e m i xe d c om pl e t e l y i n a t ube a nd t he n t r a ns f e r r e d
i nt o t he 96 w e l l pl a t e . S t e pO ne P l us R e a l T i m e S y s t e m w a s us e d f or t he P C R r e a c t i on.
T o c a l c ul a t e t he r e l a t i ve c onc e nt r a t i on of t he t a r ge t g e ne , t he C T va l ue of t he t a r ge t e d g e ne i s
s ubt r a c t e d by t he C T va l ue of t he c ont r ol ( 18s ) . T he n 2
- Δ C T
i s c a l c ul a t e d t o g e t t he r e l a t i ve
c onc e nt r a t i on of t he t a r ge t g e ne . T he a ve r a ge c onc e nt r a t i on w a s c a l c ul a t e d i n t he c ont r ol g r oup.
T o obt a i n t he r e l a t i ve f ol d c ha nge of t a r ge t g e ne e x pr e s s i on i n di f f e r e nt e x pe r i m e nt a l g r oups , t he
r e l a t i ve c onc e nt r a t i on of t he t a r ge t g e ne e x pr e s s i on i n e a c h s a m pl e ( e x pe r i m e nt a l a nd c ont r ol
g r oups ) w a s di vi de d by t he a ve r a ge c onc e nt r a t i on i n t he c ont r ol . T he r e s ul t i ng va l ue w a s
r e por t e d a s f ol d c ha nge .
1 9
2.6 S t a t i s t i c a l A na l y s i s
A l l da t a w e r e pr e s e nt e d a s m e a n ± t he s t a nda r d e r r or of t he m e a n ( S E M ) . S t ude nt ’ s t - t e s t w a s
us e d t o a na l y z e m e a n di f f e r e nc e s be t w e e n t w o s a m pl e g r oups a nd one - w a y A N O V A t e s t w i t h
m ul t i pl e c om pa r i s ons w a s us e d f or a na l y z i ng m e a n di f f e r e nc e s a m ong t hr e e or m or e g r oups . P -
va l ue i s s how n i n t he f i gur e i f t he r e s ul t i s s t a t i s t i c a l l y s i g ni f i c a nt .
2 0
C ha pt e r I I I : R e s ul t s
3.1 L i ve r T i s s ue f r om P t e n D e l e t e d M i c e
T o e x pl or e t he c ha nge s of e i c os a noi ds dur i ng t he pr ogr e s s i on of N A F L D t o H C C , w e c hos e a
m ous e m ode l w he r e t he s t a ge s of l i ve r di s e a s e a r e c l e a r l y de f i ne d. O ur l a b pr e vi ous l y de ve l ope d
a m ous e m ode l w he r e P t e n ( P hos pha t a s e a nd t e ns i n hom ol og de l e t e d on c hr om os om e 10) i s
de l e t e d
4 8
. P T E N pl a y s a n i m por t a nt r ol e i n t he phos pha t i dy l - i nos i t ol - 3- ki na s e ( P I 3 K ) pa t hw a y
4 9
.
I t de phos phor y l a t e s t he phos pha t i dy l i nos i t ol - ( 3,4,5) - t r i phos pha t e pr oduc e d by P I 3K a nd i nhi bi t s
t he a c t i on of pr ot e i n ki na s e B t o r e duc e c e l l r e ge ne r a t i on a nd pr ol i f e r a t i on. L o s s of P T E N i n t he
l i ve r l e a ds t o t he a c t i va t i on of t he P I 3K - A K T pa t hw a y a nd r e s ul t s i n a c c um ul a t i on of f a t ,
a bnor m a l c e l l pr ol i f e r a t i on, i nc r e a s e of t he pe r s i s t e nt i nf l a m m a t i on l e a di ng t o f i br os i s , c i r r hos i s
a nd f i na l l y l i ve r c a nc e r
5 0
. W e us e d t he s e P t e n de l e t e d m i c e a t t hr e e di f f e r e nt a ge s : 3 m ont hs , 9
m ont hs a nd 15 m ont hs . T he s e t hr e e a ge s r e pr e s e nt l i ve r s t e a t os i s , f i br os i s a nd H C C . A ge -
m a t c he d m i c e w i t h i nt a c t P T E N a r e us e d a s c ont r ol s . T he us e of t he s e m i c e w i t h c l e a r l y de f i ne d
pa t hol og y s t a g e s i s de s i g ne d t o he l p us unde r s t a nd how e i c os a noi d l e ve l s c ha nge dur i ng t he
de ve l opm e nt of l i ve r c a nc e r .
2 1
3 M ont h 9 M ont h 15 M ont h
W T
P M
F i g ur e 5: L i ve r s a m pl e s of W T a nd P t e n de l e t e d m i c e m ode l s .
F i gur e 5 s how s pi c t ur e s of l i ve r s a m pl e s f r om W T a nd P t e n de l e t e d m i c e i n 3, 9 a nd 15 m ont hs .
T op t hr e e s a m pl e s a r e w i l d t y p e a nd bot t om t hr e e s a m pl e s a r e P t e n de l e t e d. I n 3- , 9- a nd 15-
m ont hs w i l d t y p e s a m pl e s s how ve r y s i m i l a r phe not y p e s . C om pa r e d t o t he 3- m ont h W T s a m pl e ,
t he 3- m ont h P t e n de l e t e d s a m pl e ha s m or e f a t . C om pa r e d t o t he 3- m ont h P t e n de l e t e d s a m pl e ,
t he 9- m ont h P t e n de l e t e d s a m pl e ha s m or e f i br ot i c t i s s ue . C om pa r e d t o t he 9- m ont h P t e n de l e t e d
s a m pl e , t um or s f or m e d i n t he 15- m ont h P t e n de l e t e d s a m pl e .
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3.2 I d e nt i f i c a t i on of S pe c i f i c L i pi d S pe c i e s
W e pe r f or m e d t a r ge t e d l i pi dom i c s f or P U F A s a nd t he i r m e t a bol i t e s , t o m e a s ur e t he g l oba l l e ve l
c ha nge s of e i c os a noi ds ( n: 6) a nd doc os a noi ds ( n: 3) i n t he l i ve r s of 3- , 9- a nd 15- m ont h w i l d t y p e
a nd P t e n de l e t e d m i c e ( F i g. 6) . F or t he t a r ge t e d l i pi dom i c s , a s e t of i nt e r na l s t a nda r ds ( I S ) a nd
e a c h of t he s pe c i f i c m e t a bol i t e s a r e i nc l ude d t o he l p us de t e r m i ne t he i de nt i t y s pe c i f i c
e i c os a noi ds . S pe c i f i c e i c os a noi ds w e r e i de nt i f i e d us i ng t he i r s i g na t ur e m ul t i pl e - r e a c t i on
m oni t or i ng ( M R M ) a s pr e vi ous l y de s c r i be d by E br i ght B e t a l
5 0
. T o obt a i n t he r e l a t i ve
a bunda nc e of t he s pe c i f i c e i c os a noi ds i n t he l i ve r l i pi d e x t r a c t i on, A U C i s c a l c ul a t e d f or e a c h
e i c os a noi d a nd doc os a noi d pe a ks us i ng m a nua l i nt e g r a t i on ( F i g 6) . A s a n e xa m pl e , F i gur e 6
s how s one of t he l i pi d s t a nda r ds , d4- P G E 2 ( t op r ow ) , a nd s e l e c t e d P G E 2 pe a ks ( bot t om r ow ) i n
t hr e e 3- m ont h P t e n de l e t e d s a m pl e s . O nl y di f f e r e nc e be t w e e n d4- P G E 2 a nd P G E 2 i s t he f our
de ut e r i um a t om s , t hus ha vi ng s i m i l a r c he m i c a l pr ope r t i e s , a nd w e us e d4- P G E 2 a s t he r e f e r e nc e
t o f i nd P G E 2 i n our s a m pl e s . T he s pe c i f i c M R M f or P G E 2 ha s a r e t e nt i on t i m e of 10.78 m i n.
T he r e w a s onl y one pe a k c l os e t o t hi s r e t e nt i on t i m e , s o i t w a s s e l e c t e d a s t he s i g na l pe a k of
P G E 2, a nd t he A U C w a s c a l c ul a t e d. T he r e w a s a s m a l l s hi f t of t he a c t ua l s i g na l f r om 10.78 m i n
t o 10.93 m i n. T he A U C w a s di vi de d by t he m a s s of t he l i ve r s a m pl e t o obt a i n t he nor m a l i z e d
da t a f or t he f ur t he r a na l y s i s .
F i gur e 6: P e a k S e l e c t i on a nd D a t a N or m a l i z a t i on
2 3
I n t e r na l s t a nda r ds ( I S ) a r e us e d t o m e a s ur e l i pi d s i g na l s a nd M R M a r e us e d t o i de nt i f y t he
s pe c i f i c e i c os a noi d m e t a bol i t e s . d4- P G E 2 ( t op) i s us e d a s t he I S t o f i nd t he r e a l s i g na l pe a k of
P G E 2 i n t he l i pi d pr of i l e .
3.3 D e c r e a s i ng L e ve l s of E i c os a noi ds i n t he L i ve r a s M i c e A ge
T o a na l y z e t he pa t t e r ns of e i c os a noi d a nd doc os a noi d c ha nge s a c r os s a l l s pe c i e s , a ge s a nd
g e not y p e g r oups , w e pe r f or m e d uns upe r vi s e d c l us t e r i ng of t he de t e c t e d e i c os a noi ds a nd
doc os a noi ds . I n t e r e s t i ng l y , uns upe r vi s e d c l us t e r i ng a na l y s i s of t he e i c os a noi d l e ve l s s how e d a
g e ne r a l de c r e a s i ng t r e nd i n bot h w i l d t y p e a nd t he P T E N de l e t e d g r oup a s t he m i c e a ge ( F i g. 7) .
I n pa r t i c ul a r , a l l de t e c t a bl e e i c os a noi ds de c r e a s e d i n t he W T l i ve r s a s a f unc t i on of a ge w i t h t he
e x c e pt i on of R vD 2 a nd 14- H D P A . W e f ur t he r a na l y z e d t he s e a ge - de pe nde nt c ha nge s of s pe c i f i c
e i c os a noi ds ba s e d on t he i r m e t a bol i c pa t hw a y s ( F i g.8) . T hi s a na l y s i s s how s s i m i l a r a ge -
de pe nde nt de c r e a s e s of 5- L O X , 12/ 15- L O X a nd C O X m e t a bol i t e s i n t he a gi ng m i c e w i t h a f e w
e x c e pt i ons obs e r ve d i n t he W T l i ve r s i nc l udi ng : R vD 2, 14- H D H A a nd P G E 2 ( F i g. 8) . A f e w
ot he r s a l s o s how e d va r i e d pa t t e r ns i nc l udi ng R vD 1 a nd R vE 1 of w hi c h t he l e ve l s a r e hi g he r i n
t he 9 m ont h t ha n t he 3 m ont h ol d l i ve r s but t he i r l e ve l s de c r e a s e d i n 15 m ont hs of a ge . T he s e
e i c os a noi ds ( e x c e pt P G E 2) a r e pr o- r e s ol vi ng m e t a bol i t e s de r i ve d f r om n- 3 P U F A s . T he e l e va t e d
l e ve l s of t he s e pr o- r e s ol vi ng/ pr e - r e pa i r m e t a bol i t e s m a y i ndi c a t e a n e f f or t f or t he l i ve r t o c ope
w i t h t he a g i ng- r e l a t e d m e t a bol i c or ot he r s t r e s s .
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F i gur e 7: H e a t m a p of t he e i c os a noi d l e ve l s i n w i l d t y p e a nd P t e n de l e t e d m ous e m ode l s
T he he a t m a p i s m a de ba s e d on t he t a r ge t e d l i pi dom i c s da t a . T he r e d c ol or r e pr e s e nt s
upr e gul a t i on of t he e i c os a noi ds a nd t he bl ue c ol or r e pr e s e nt s t he dow nr e gul a t i on of t he
e i c os a noi ds . E i c os a noi ds s how a g e ne r a l de c r e a s i ng pa t t e r n i n bot h W T a nd P M m ode l s a s t he
m i c e a ge .
2 5
T o f ur t he r c onf i r m t hi s a ge - de pe nde nt de c r e a s e of e i c os a noi ds a nd doc os a noi ds , w e c om pa r e d
t he r e l a t i ve l e ve l s of r e pr e s e nt a t i ve pr oduc t s ve r s us m e t a bol i t e s ( 15- H E T E a nd L X A 4) f r om e a c h
e nz y m a t i c pa t hw a y . C ons i s t e nt w i t h t he g e ne r a l a ge - de pe nde nt de c l i ne t r e nd obs e r ve d f or l i pi d
m e t a bol i t e s , our da t a c onf i r m e d t ha t t he l e ve l s of 15- H E T E a nd L X A 4 g r e a t l y de c r e a s e d i n t he
l i ve r a s t he W T m i c e a ge d ( F i g. 9A ) . W e a l s o a na l y z e d t he l e ve l s of R vE 1, 14- H D H A a nd P G E 2,
w hi c h w e r e s how n t o be e l e va t e d on t he uns upe r vi s e d he a t m a p c l us t e r i ng a na l y s i s ( F i g 7 a nd 8) .
E ve n t houg h R vD 2 l e ve l s a r e m uc h hi g he r i n t he 15- m ont h l i ve r s a s s how n f r om t he he a t m a p,
s i g na l s f or R vD 2 a r e ve r y w e a k. A s 14- H D H A s e r ve s a s t he pr e c ur s or f or R vD 2, w e e va l ua t e d
t he l e ve l s of 14- H D H A a nd not R vD 2. F or bot h 14- H D H A a nd P G E 2 l e ve l s , one out l i e r s e e m s
A. B.
C.
F i gur e 8: H e a t m a p s o f t h e L O X a n d C O X
m e t a b o l i t e s i n w i l d t y p e m i c e i n 3 t i m e
p e r i o d s .
P a n e l A i n c l u d e s a l l t h e L O X - 5
m e t a b o l i t e s . P a n e l B i n c l u d e s a l l t h e
L O X - 1 5 a n d L O X - 1 2 m e t a b o l i t e s . P a n e l
C i n c l u d e s a l l t h e C O X m e t a b o l i t e s . T h e
r e d c o l o r r e p r e s e n t s t h e u p r e g u l a t i o n o f
t h e e i c o s a n o i d m e t a b o l i t e s a n d t h e b l u e
c o l o r r e p r e s e n t s t h e d o w n r e g u l a t i o n o f
t h e e i c o s a n o i d m e t a b o l i t e s .
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t o c ont r i but e t o t he obs e r ve d de c l i ne f or t he i r l e ve l s i n t he 15- m ont h- ol d l i ve r s ( F i g. 9B ) . W he n
t he out l i e r i s di s c a r de d, a n a ge - de pe nde nt de c l i ne i s a l s o
B .
C .
A .
F i gur e 9: T a r ge t e d l i pi d l e ve l s of s om e
e x c e pt i ons a nd r e pr e s e nt a t i ons i n t he W T
m ode l s
S om e e x c e pt i ons ( B a nd C ) s how di f f e r e nt
pa t t e r ns of t he l i pi d l e ve l f r om t he
r e pr e s e nt a t i on ( A ) i n t he W T m ode l s .
R e pr e s e nt a t i on l i pi ds s how a de c r e a s i ng
pa t t e r n a s t he m i c e a ge . 14- H D H A a nd P G E 2
i nc r e a s e d f r om 3 t o 9 m ont h t he n de c r e a s e d
f r om 9 m ont h t o 15 m ont h. R vE 1 i nc r e a s e d
f r om 3 m ont h t o 9 m ont h t he n de c r e a s e d f r om
9 m ont h t o 15 m ont h.
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obs e r ve d f or t he s e m e t a bol i t e s s i m i l a r t o ot he r s . F or R vE 1, a l t houg h t he r e w a s a n i nc r e a s e f r om
3 m ont h t o 9 m ont h, a de c r e a s e f r om 9 m ont h t o 15 m ont h i s obs e r ve d ( F i g. 9C ) . O ve r a l l , t hi s
a na l y s i s i s c ons i s t e nt w i t h t he pa t t e r n f r om F i gur e s 7 a nd 8 s ug ge s t t ha t e i c os a noi ds l e ve l s
de c l i ne w i t h a ge i n t he m ous e l i ve r .
T o de t e r m i ne i f t hi s a ge - de pe nde nt de c l i ne of m e t a bol i t e s i s due t o t he r e duc e d e xpr e s s i on of t he
e nz y m e s r e s pons i bl e f or t he i r pr oduc t i on, w e a na l y z e d t he e x pr e s s i on of t he L O X a nd C O X
e nz y m e s a s r e pr e s e nt a t i ve s ( F i g. 10) . O ur da t a s how s t ha t t he C O X - 1 m R N A e x pr e s s i on w a s
i nc r e a s e d a s m i c e a ge d ( F i g. 10C ) . T he e x pr e s s i on of t he t hr e e e nz y m e s A L O X - 5, A L O X - 15 a nd
C O X - 2 s how e d s i m i l a r pa t t e r ns a s m i c e a ge d. I n c r e a s e d m R N A e x pr e s s i on w a s s e e n f r om 3
m ont h t o 9 m ont h, t he n de c r e a s e d f r om 9 m ont h t o 15 m ont h w i t h t he m os t s i g ni f i c a nt c ha nge
obs e r ve d w i t h A L O X - 15. T he l e ve l of A L O X - 15 i s 20- f ol d hi g he r i n t he 9- m ont h- ol d l i ve r s
c om pa r e d t o 3 m ont h a nd 15- m ont h- ol d m i c e . I n t he 15 m ont hs ol d m i c e , i t s e x pr e s s i on i s
c om pa r a bl e t o t ha t of t he 3 m ont hs ol d m i c e ( F i g. 10B ) . S i m i l a r l y , t he m R N A e x pr e s s i on of
C O X - 2 a l s o di s pl a y e d a s i m i l a r pa t t e r n a s t ha t of A L O X - 15 ( F i g. 10D ) . R vE 1 i s t he m e t a bol i t e
de r i ve d f r om E P A by t he a c t i on of C O X - 2. T he i nduc t i on of C O X - 2 i n t he 9 m ont hs ol d l i ve r s
m a y e x pl a i n w hy t he R vE 1 l e ve l i s i nc r e a s e d a t t hi s a ge .
A s m os t l i pi ds s how e d a n a ge de pe nde nt de c l i ne , but t he m R N A e x pr e s s i on of t he e nz y m e s
r e s pons i bl e f or t he i r pr oduc t i on a r e g e ne r a l l y i nduc e d or unc ha nge d w i t h a ge , w e a na l y z e d t he
s ubs t r a t e a va i l a bi l i t y f or t he pr oduc t i on of t he s e m e t a bol i t e s . F i gur e 11 s how s t ha t t he g l oba l
l e ve l s of a l l f our pr e c ur s or s of e i c os a noi d m e t a bol i t e s a r e r e duc e d i n t he l i ve r s a s t he m i c e a ge
s ug ge s t i ng a de c r e a s e i n c P L A 2 a nd i P L A 2 a c t i vi t i e s ( F i g. 11) . T he s e pr e c ur s or s a r e t he
2 8
i m por t a nt s ubs t r a t e s of L O X a nd C O X e nz y m e s . T he dr a m a t i c de c l i ne of t he s e pr e c ur s or s
s ug ge s t s t he r e a r e not e nough s ubs t r a t e s f or L O X a nd C O X t o be us e d f or t he pr oduc t i on of
dow ns t r e a m m e t a bol i t e s . T hus , w hi l e t he e x pr e s s i on of t he e i c os a noi ds pr oduc i ng e nz y m e s di d
not r e duc e w i t h a ge , t he de c l i ne of s ubs t r a t e s l i m i t e d t he a bi l i t y of t he l i ve r t o pr oduc e
e i c os a noi ds i n t he a gi ng m i c e .
A . B .
C . D .
F i gur e 10: m R N A e x pr e s s i on l e ve l s of L O X - 5, L O X - 15 a nd C O X e nz y m e s i n t he W T m ous e
m ode l
2 9
L O X - 5, L O X - 15 a nd C O X - 2 s how s i m i l a r e x pr e s s i on l e ve l s a s t he m i c e a ge i n t he W T m ode l s .
T he y a l l i nc r e a s e f r om 3- m ont h t o 9- m ont h a nd t he n de c r e a s e f r om 9- m ont h t o 15- m ont h. C O X -
1 s how s a c ont i nuous l y i nc r e a s i ng pa t t e r n f r om 3 m ont h t o 15 m ont h.
F i gur e 11: P U F A s l e ve l of i m por t a nt pr e c ur s or s of a l l m e t a bol i t e s
A l l P U F A s s how de c r e a s i ng pa t t e r ns i n t he W T m ode l a s m i c e a ge . T he l e ve l s of s ubs t r a t e s
de c r e a s e pr oba bl y c a us e t he de c r e a s e i n t he g l oba l l e ve l s of t he e i c os a noi ds .
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3.4 P t e n D e l e t i on A l t e r e d t he E i c os a noi d L e ve l s
3.4.1 B i oa c t i ve l i pi d m e t a bol i t e c ha nge due t o P T E N l os s
C om pa r e d t o t he W T g r oups , a ppr ox i m a t e l y ha l f of t he de t e c t a bl e l i pi ds a r e a l t e r e d i n t he P t e n
de l e t e d g r oups a t di f f e r e nt a ge s ( F i g. 7) . T he s e e i c os a noi ds be l ong t o t w o c l us t e r s : t he f i r s t
c l us t e r i nc l ude s pr os t a noi ds a nd t he s e c ond c l us t e r i nc l ude s t he l e ukot r i e ne . W hi l e t hi s a ge -
r e l a t e d t r e nd i s g e ne r a l l y obs e r ve d i n t he P t e n de l e t e d g r oups , a f e w e x c e pt i ons a r e not e d,
i nc l udi ng 21- H D P A , T X B 2, P G D 2, 12- H E T E , 11,12- E E T , P G E 2, D P A , 5,15- di H E T E a nd
L T B 4 ( F i g. 7) . T he m os t obvi ous di f f e r e nc e s obs e r ve d be t w e e n t he a ge - m a t c he d c ont r ol a nd
P t e n de l e t e d g r oups a r e t he l ow e r l e ve l s of a ppr ox i m a t e l y 1/ 3 of t he m e t a bol i t e s i n t he 3- m ont h-
ol d l i ve r s ( F i g. 7) . S e ve r a l m e t a bol i t e s a l s o s how e d dr a m a t i c e l e va t e d l e ve l s i n t he P t e n de l e t e d
g r oups a t 15 m ont hs of a ge . W hi l e m ode r a t e c ha nge s a r e a l s o obs e r ve d i n ot he r g r oups a nd
m e t a bol i t e s , t he i r c ha nge s a r e l e s s pr of ound. A t 3 m ont hs of a ge , t he l e ve l s of m os t e i c os a noi ds
e i t he r de c r e a s e d or unc ha nge d i n t he P t e n de l e t e d m i c e c om pa r e d t o t he W T m i c e . T w o m a j or
e x c e pt i ons a r e 20- C O O H - L T B 4 a nd R vE s ( F i g. 7) . T he s e t w o e i c os a noi ds s how e d i nc r e a s i ng
pa t t e r ns i n t he P t e n de l e t e d m ode l . I n t he 9- m ont h- ol d m i c e , no m a j or c ha nge s w e r e obs e r ve d
be t w e e n t he P t e n de l e t e d l i ve r s a nd c ont r ol s ba s e d on t he he a t m a p, e xc e pt R vD 2. T he l e ve l s of
s e ve r a l m e t a bol i t e s a ppe a r e d t o i nc r e a s e i n t he 15- m ont h l i ve r s l a c ki ng P t e n w hi l e a l l ot he r
e i c os a noi ds di d not c ha nge . T he s e i nc r e a s e d m e t a bol i t e s i nc l ude d T X B 2, P G E 2/ D 2, R vD 1,
L T B 4 a nd 12- H E T E a s w e l l a s s e ve r a l ot he r i nt e r m e di a t e m e t a bol i t e s ( F i g. 7) .
S i m i l a r t o t he a na l y s i s pe r f or m e d on t he a ge - de pe nde nt c ha nge s of e i c os a noi ds i n t he W T m i c e ,
w e c hos e s e ve r a l r e pr e s e nt a t i ve m e t a bol i t e s a nd c om pa r e d t he i r l e ve l s be t w e e n W T a nd P t e n
de l e t e d l i ve r s . C ons i s t e nt w i t h t he he a t m a p, P t e n de l e t i on s i g ni f i c a nt l y r e duc e d t he l e ve l s of
L X A 4 a nd 5- H E T E by 44% a nd 51% i n t he 3- m ont h- ol d l i ve r s ( F i g. 12 & 13) . A l t houg h a t r e nd
3 1
of de c r e a s e w a s obs e r ve d f or 15- H E T E , t he hi g h i ndi vi dua l va r i a t i ons pr e c l ude d a ny s i g ni f i c a nt
c ha nge s f r om be i ng obs e r ve d ( F i g. 13) .
F i gur e 12: P r o a nd a nt i - i nf l a m m a t or y e i c os a noi ds l e ve l f r om t a r ge t e d l i pi dom i c s .
L T B 4 a nd L X A 4 a r e i m por t a nt pr o- a nd a nt i - i nf l a m m a t or y e i c os a noi ds . L T B 4 di d not s how a
not i c e a bl e di f f e r e nc e be t w e e n t he W T a nd P M m ode l s i n a l l t hr e e t i m e pe r i ods . L X A 4 s how s a
g r e a t de c r e a s e i n t he P M m ode l c om pa r e d t o t he W T m ode l i n t he 3 m ont h, but not not i c e a bl e
di f f e r e nc e i n 9 m ont h a nd 15 m ont h. V a l ue s t ha t a r e g r e a t e r t ha n + 2.5 S D or l e s s t ha n - 2.5 S D
f r om t he m e a n a r e i nc l ude d a s out l i e r s a nd a r e di s c a r de d.
3 2
F i gur e 13: I n t e r m e di a t e e i c os a noi ds l e ve l s f r om t a r ge t e d l i pi dom i c s .
5- H E T E i s t he i nt e r m e di a t e be t w e e n L T B 4 a nd A A , 15- H E T E i s t he i nt e r m e di a t e be t w e e n
L X A 4 a nd A A . 15- H E T E a nd 12- H E T E de c r e a s e i n t he P M m ode l c om pa r e d t o t he W T m ode l
a s m i c e a ge . 5- H E T E i nc r e a s e s i n t he P M m ode l c om pa r e d t o t he W T m ode l i n 9 m ont h, but
de c r e a s e s i n 3 m ont h a nd 15 m ont h. V a l ue s t ha t a r e g r e a t e r t ha n + 1.5 S D or l e s s t ha n - 1.5 S D
f r om t he m e a n a r e i nc l ude d a s out l i e r s a nd a r e di s c a r de d.
T hi s i s l i ke l y due t o t he l ow a bunda nc e of t he s e m e t a bol i t e s i n t he l i ve r . A l t houg h 20- C O O H -
L T B 4 a nd R vE s w e r e i de nt i f i e d on t he he a t m a p a s t he i nc r e a s e d m e t a bol i t e s , t he i r l e ve l s w e r e
ve r y l ow f or s pe c i f i c c om pa r i s on. W e di d not obs e r ve di f f e r e nc e s i n L T B 4 i t s e l f a t t hi s a ge ( F i g.
12) . F or t he C O X f a m i l y of m e t a bol i t e s , w e a na l y z e d a l l f our m a j or m e t a bol i t e s ( F i g. 14) .
A l t houg h a m ode r a t e i nc r e a s e of a l l f our m e t a bol i t e s , P G D 2, P G E 2, P G F 2 α a nd T X B 2 w e r e
obs e r ve d i n t he 3- m ont h- ol d P t e n de l e t e d l i ve r s c om pa r e d w i t h t he c ont r ol g r oups , i ndi vi dua l
va r i a t i ons m a ke i t di f f i c ul t t o c onc l ude on t he s pe c i f i c r ol e of P T E N on t he pr oduc t i on of t he s e
m e t a bol i t e s .
3 3
F i gur e 14: P r os t a noi d l e ve l s c om pa r i s ons be t w e e n W T a nd P t e n de l e t e d m ode l s .
P G D 2, P G E 2 a nd P G F 2 α a r e t he m e t a bol i t e s of P G H a nd T X B 2 i s t he s t a bl e f or m of T X A 2.
T he y i nc r e a s e i n t he P M m ode l s c om pa r e d t o t he W T m ode l s i n bot h 3 m ont h a nd 15 m ont h, but
de c r e a s e i n t he 9 m ont h. V a l ue s t ha t a r e g r e a t e r t ha n + 1.5 S D or l e s s t ha n - 1.5 S D f r om t he m e a n
a r e i nc l ude d a s out l i e r s a nd a r e di s c a r de d. T he pa t t e r ns i n 3- m ont h m ode l s a r e di f f e r e nt f r om t he
he a t m a p ( F i g. 7) .
W hi l e t he he a t m a p ( F i g. 7) s how s hi g he r l e ve l s of 12- H E T E i n t he 15- m ont h P t e n de l e t e d l i ve r s ,
t he l e ve l s of 12- H E T E i n t he s e ol de r m i c e ( bot h c ont r ol a nd P t e n de l e t e d m i c e ) a r e t oo l ow t o
obs e r ve a ny a ppr e c i a bl e di f f e r e nc e s be t w e e n t w o g e not y p i ng g r oups . I n a ddi t i on, no s i g ni f i c a nt
di f f e r e nc e s w e r e obs e r ve d f or 15- H E T E , L T B 4 or L X A 4 be t w e e n t he t w o g e not y p e g r oups i n
t he t w o ol de r a ge g r oups l i ke l y r e s ul t i ng f r om t he i r r e duc e d l e ve l s obs e r ve d i n t he a g e d c ont r ol
l i ve r s ( F i g. 12 & 13) . S i m i l a r l y , w hi l e a t r e nd of hi g he r C O X m e t a bol i t e s w a s f ound f or t he 3-
3 4
m ont h- ol d P t e n de l e t e d l i ve r s vs . c ont r ol s . T hi s di f f e r e nc e w a s not obs e r ve d i n t he 9 a nd 15-
m ont h g r oups . O ne e x c e pt i on i n t he ol de r a ge g r oups i s t he l e ve l of 5- H E T E a t 9 m ont hs of a g e .
A s i g ni f i c a nt i nc r e a s e of 5- H E T E w a s obs e r ve d i n t he P t e n de l e t e d l i ve r s c om pa r e d w i t h t he
c ont r ol s a t t hi s a ge ( F i g. 13) .
T oge t he r , t he s e da t a s ug ge s t t ha t P T E N m a y m a i nt a i n t he l e ve l s of L O X m e t a bol i t e s . I n ol de r
m i c e w he n t he l e ve l s of e i c os a noi d m e t a bol i t e s dr oppe d i n t he c ont r ol m i c e , t he i r l e ve l s i n t he
P t e n de l e t e d l i ve r s a r e no l ong e r di f f e r e nt f r om t ha t of t he c ont r ol s . T hus , P T E N l os s w hi l e
de c r e a s e d t he L O X m e t a bol i t e s , a l s o f unc t i one d t o m a i nt a i n t he i r l e ve l s i n t he ol d m i c e .
H ow e ve r , i t i s unc l e a r i f t hi s i s a c e l l a ut onom ous e f f e c t or i nvol ve s ot he r c e l l t y p e s . F i na l l y ,
P T E N l os s a ppe a r s t o pos i t i ve l y i m pa c t t he C O X m e t a bol i t e s t hr oug hout t he a ge s e ve n t houg h
e l e va t e d m e t a bol i t e l e ve l s w e r e onl y obs e r ve d f or t he 3 m ont h a ge g r oup. T he l a c k of a ny
obs e r va bl e di f f e r e nc e s i n t he ol de r m i c e i s l i ke l y due t o t he a ge - r e l a t e d de c l i ne obs e r ve d w i t h t he
W T c ont r ol l i ve r s .
3.4.2 T he e f f e c t s of P T E N on t he e x pr e s s i on of C O X a nd L O X e nz y m e s
T o ha ve a be t t e r unde r s t a ndi ng on t he e f f e c t s of P t e n de l e t i on on e i c os a noi d m e t a bol i s m , w e
c om pa r e d t he e x pr e s s i on of t he e nz y m e s t ha t a r e r e s pons i bl e f or t he pr oduc t i on of t he s e
m e t a bol i t e s . F i gur e 16 s how s t he m R N A e x pr e s s i on of A L O X - 5 a nd A L O X - 15 i n bot h W T a nd
P t e n de l e t e d m ode l s i n t he t hr e e a ge g r oups . R e l a t i ve t o t he a g e - m a t c he d e x pr e s s i on of t he
e nz y m e s , P T E N l os s a ppe a r s t o r e d uc e t he e x pr e s s i on of a l l e nz y m e s i n 3- a nd 9- m ont h- ol d m i c e ,
w he r e i t l e a ds t o i nc r e a s e d e x pr e s s i on of t he s e e nz y m e s i n t he 15- m ont h- ol d m i c e . F or t he 3- a nd
9- m ont h- ol d m i c e , e x pr e s s i ons of A L O X - 5 a nd A L O X - 15 w e r e bot h dr a m a t i c a l l y de c r e a s e d i n
3 5
t he P t e n de l e t e d m i c e c om pa r e d t o t he W T m i c e . T hi s t r e nd w a s r e ve r s e d f or t he 15- m ont h- ol d
m i c e . I n t he s e ol d m i c e c a r r y i ng P t e n de l e t i on, e x pr e s s i ons of A L O X - 5 a nd A L O X - 15 w e r e 3
f ol ds a nd 2 f ol ds hi g he r w he n c om pa r e d t o t he w i l d- t y p e m ode l r e s pe c t i ve l y ( F i g. 15) . T he s e
e l e va t e d e x pr e s s i ons obs e r ve d i n t he ol de r m i c e l a c ki ng P T E N w e r e c ons i s t e nt w i t h t he he a t m a p
a na l y s i s s how i ng m a j or e l e va t i on i n e i c os a noi d m e t a bol i t e s w e r e obs e r ve d a t 15 m ont hs w he n
c om pa r i ng t he a g e - m a t c he d P t e n de l e t e d g r oups t o t he c ont r ol g r oups .
F i gur e 15 : m R N A e x pr e s s i on l e ve l of L i pox y ge n a s e 5 a nd 15 of w i l d t y p e a nd P T E N de l e t e d
m i c e i n t he pr oc e s s of l i ve r s t e a t os i s t o c a nc e r .
E x pr e s s i ons of A L O X - 5 a nd A L O X - 15 a r e l ow e r i n t he P M m ode l c om pa r e d t o W T m ode l i n
bot h 3 m ont h a nd 9 m ont h. H ow e ve r , t he r e i s a dr a m a t i c i nc r e a s e i n t he P M m ode l c om pa r e d t o
t he W T m ode l i n 15 m ont h.
F i gur e 16 s how s t he m R N A e x pr e s s i on of C O X - 1 a nd C O X - 2 i n t he t w o g r oups of m i c e . S i m i l a r
t o t he t w o L O X e nz y m e s , e x pr e s s i ons of bot h C O X - 1 a nd C O X - 2 a r e l ow e r i n t he P t e n de l e t e d
m ode l a s c om pa r e d t o t he W T m ode l a t 3 m ont hs of a ge . T hi s P T E N l os s a s s oc i a t e d w i t h l ow
e x pr e s s i on pe r s i s t e d f or C O X - 2 a t 9 m ont hs of a ge but be c a m e l e s s pr of ound f or C O X - 1. T hi s
3 6
r e duc e d e x pr e s s i on a s s oc i a t e d w i t h P T E N l os s w a s r e ve r t e d w he n t he m i c e r e a c he d 15 m ont hs of
a ge w he r e no di f f e r e nc e w a s obs e r ve d f or C O X - 1 e x pr e s s i on be t w e e n t he t w o g e not y p e g r oups
w hi l e t he e x pr e s s i on of C O X - 2 a ppe a r e d t o i nc r e a s e w i t h P T E N l os s ( F i g. 17) . I n F i gur e 7,
he a t m a p a na l y s i s i ndi c a t e d t ha t e i c os a noi d m e t a bol i t e s i nc l udi ng T X B 2, P G D 2, P G E 2, L T B 4
a nd L X A 4 w e r e hi g he r i n t he P t e n de l e t e d m ode l c om pa r e d t o t he W T m ode l i n 15 m ont h ol d
m i c e . E ve n t houg h w e a r e una bl e t o s ubs t a nt i a t e t hi s w he n a na l y z i ng t he s pe c i f i c m e t a bol i t e s
be c a us e of t he hi g h i ndi vi dua l va r i a t i ons , t he hi g he r e x pr e s s i on obs e r ve d f or C O X - 1 a nd 2 m a y
ha ve c ont r i but e d t o t he pa t t e r ns obs e r ve d on t he he a t m a p ( F i g. 7) .
F i gur e 16: m R N A e x pr e s s i on l e ve l of C y c l oox y ge n a s e 1 a nd 2 of w i l d t y p e a nd P T E N de l e t e d
m i c e i n t he pr oc e s s of l i ve r s t e a t os i s t o c a nc e r .
E x pr e s s i ons of C O X - 1 a nd C O X - 2 a r e l ow e r i n t he P M m ode l c om pa r e d t o t he W T m ode l i n
bot h 3 m ont h a nd 9 m ont h. H ow e ve r , t he r e i s a dr a m a t i c i nc r e a s e i n t he P M m ode l c om pa r e d t o
t he W T m ode l i n 15 m ont h.
I n a ddi t i on, F i gur e 7 a l s o s how e d hi g he r l e ve l s of A A , D H A , D P A , pr e c ur s or s of m e t a bol i t e s , i n
t he P t e n de l e t e d m ode l a t 15 m ont hs of a ge . I n pa r t i c ul a r , l e ve l s of A A a nd D P A w e r e 2 a nd 3-
3 7
f ol d hi g he r r e s pe c t i ve l y i n t he P t e n de l e t e d m ode l c om pa r e d t o t he c ont r ol s a t t hi s a ge ( F i g. 17) .
T he l e ve l of D H A w a s m ode r a t e l y hi g he r i n t he P t e n de l e t e d l i ve r s a t t hi s a ge . T he s e e l e va t e d
s ubs t r a t e l e ve l s m a y a ddi t i ona l l y c ont r i but e t o t he obs e r ve d hi g he r l e ve l of C O X m e t a bol i t e s i n
t he 15- m ont h- ol d P t e n de l e t e d m i c e vs . t he a ge - m a t c he d c ont r ol s .
F i gur e 17: A A a nd D P A l e ve l s f r om t a r ge t e d l i pi dom i c s
A A a nd D P A l e ve l s s how s i m i l a r pa t t e r ns i n t he a ge m a t c he d g r oups . T he y de c r e a s e d i n t he P M
m ode l c om pa r e d t o t he W T m ode l a t 3 m ont hs of a ge . T he r e i s no bi g di f f e r e nc e be t w e e n P M
a nd W T m ode l s a t 9 m ont hs of a ge . T he r e i s a hug e i nc r e a s e i n t he P M m ode l c om pa r e d t o t he
W T m ode l a t 15 m ont hs of a ge .
3.5 E i c os a noi ds i n t he L i ve r D i s e a s e P r og r e s s i on
T o i nve s t i g a t e how e i c os a noi d m e t a bol i s m c ha nge s a s l i ve r di s e a s e pr ogr e s s i on, w e c om pa r e d
t he l e ve l s of t he s e a na l y t e s i n t he P t e n de l e t e d m ur i ne l i ve r s a t 3 m ont hs of a ge ( r e pr e s e nt i ng
s t e a t os i s ) , 9 m ont hs of a g e ( r e pr e s e nt i ng i nf l a m m a t i on/ f i br os i s ) a nd 15 m ont hs of a ge
( r e pr e s e nt i ng c a nc e r / t um or s ) . A na l y s i s of a l l m e t a bol i t e s t og e t he r s how e d a g e ne r a l t r e nd of
de c l i ne f or L O X m e t a bol i t e s but e l e va t i on f or C O X m e t a bol i t e s i n t he t um or be a r i ng m i c e ( F i g.
3 8
18) . C ons i s t e nt w i t h t hi s t r e nd, e x pr e s s i on of bot h C O X - 1 a nd C O X - 2 w e r e i nc r e a s e d i n t he 15-
m ont h t um or m i c e w he n c om pa r e d t o 3- m ont h s t e a t os i s m i c e w he n s t a nda r di z e d t o t he a ge -
m a t c he d c ont r ol s . F or C O X - 1, t hi s s t a nda r di z e d e x pr e s s i on i s 1- a nd 2- f ol d hi g he r i n t he 9 m ont h
f i br os i s a nd 15 m ont h t um or be a r i ng m i c e w he n c om pa r e d t o t he 3- m ont h- ol d s t e a t os i s m i c e ,
r e s pe c t i ve l y . T hi s g r a dua l i nc r e a s e f r om s t e a t os i s t o f i br os i s / i nf l a m m a t i on t o t um or s ug ge s t s t ha t
C O X m e t a bol i s m m a y be i m por t a nt f or t he pr og r e s s i on of l i ve r di s e a s e . E xpr e s s i on of C O X - 2
a l s o i nc r e a s e d i n t he 15- m ont h- ol d t um or m i c e c om pa r e d t o t he s t e a t os i s m i c e . H ow e ve r , w e di d
not obs e r ve a n i nc r e a s e i n t he f i br os i s / i nf l a m m a t i on m i c e . T hi s l a c k of c ha nge w i t h C O X - 2 i n
t he f i br os i s / i nf l a m m a t i on s t a ge i s s ur pr i s i ng g i ve n t he w e l l c ha r a c t e r i z e d f unc t i ons of C O X - 2
a nd i t s m e t a bol i t e s i n i nf l a m m a t i on
2 3
. T og e t he r , t he da t a s ug ge s t s t ha t C O X - 1 m a y a l s o pl a y a
r ol e i n t r i g ge r i ng t he i nf l a m m a t or y r e s pons e dur i ng t he pr ogr e s s i on of l i ve r di s e a s e a nd C O X - 2
i nduc t i on m a y be a l a t e r e ve nt t ha t s us t a i ns t um or pr om ot i on a s r e por t e d pr e vi ous l y .
C ons i s t e nt w i t h t he i nc r e a s e d e xpr e s s i on of C O X - 1 a nd C O X - 2, l e ve l s of a l l 5 C O X m e t a bol i t e s
w e r e e l e va t e d i n t he 15- m ont h- ol d t um or be a r i ng l i ve r s c om pa r e d t o y o unge r m i c e w i t h s t e a t os i s
a nd i nf l a m m a t i on ( F i g. 18C ) . T hi s pa t t e r n w a s di f f e r e nt f r om t ha t obs e r ve d i n t he W T l i ve r s
w he r e onl y P G E 2 l e ve l s i nc r e a s e d w i t h a ge . W e pe r f or m e d a na l y s i s on s pe c i f i c C O X m e t a bol i t e s
a nd s t a nda r di z e d t he m t o t he a g e - m a t c he d c ont r ol s . T hi s a na l y s i s s how e d t ha t P G D 2 a nd P G F 2 α
w e r e bot h i nc r e a s e d i n t he t um or l i ve r s vs . t he s t e a t os i s a nd f i br os i s l i ve r s ( F i g. 19) e ve n t houg h
one s a m pl e w i t h s i g ni f i c a nt l y hi g he r l e ve l s t ha n ot he r s s e e m e d t o c ont r i but e t o t hi s obs e r va t i on.
S a m pl i ng i s s ue s m i g ht c ont r i but e t o t hi s va r i a t i on a s t um or l i ve r s w e r e hi g hl y he t e r oge ne ous
w i t h va r i e d de gr e e s of s t r om a a nd i nf l a m m a t or y c e l l s . S pe c i f i c t um or vs . non- t um or s a m pl i ng
m a y be ne e de d t o c onc l ude on t he s pe c i f i c c ha nge s of t he m e t a bol i t e s .
3 9
F i gur e 18: H e a t m a ps of t he L O X - 5, L O X - 15 a nd C O X m e t a bol i t e s i n w i l d t y p e a nd P T E N
de l e t e d m ode l s i n 3 t i m e pe r i ods .
M e t a bol i t e l e ve l s of L O X - 5, L O X - 15 a nd C O X e nz y m e s i n P M m ode l s w e r e g r oupe d a nd t hr e e
he a t m a ps w e r e m a de . T he r e d c ol or i ndi c a t e s t he upr e gul a t i on of t he m e t a bol i t e s , a nd t he bl ue
c ol or i ndi c a t e s t he dow nr e gul a t i on of t he m e t a bol i t e s . ( A ) . T he l e ve l s of L O X - 5 m e t a bol i t e s
s how a g e ne r a l de c r e a s i ng pa t t e r n a s t he m i c e a g e i n t he P M m ode l . T he r e a r e s om e e x c e pt i ons
i nc l udi ng L T B 4, R vD 1, R vD 2 a nd 5- H E T E . ( B ) . M os t L O X - 15 m e t a bol i t e s de c r e a s e a s t he m i c e
A. B.
C.
4 0
a ge i n t he P M m ode l w i t h e x c e pt i ons of 12- H E T E , 15- H E T E , H D H A s a nd M a r e s i ns . ( C ) . A l l
C O X m e t a bol i t e s i nc r e a s e a s t he m i c e a ge i n t he P M g r oup.
F i gur e 19: E i c os a noi ds l e ve l s of s om e e x c e pt i ons i n t he P t e n de l e t e d m ode l s
L T B 4 de c r e a s e s f r om t he s t e a t os i s s t a ge t o f i br os i s s t a ge t he n i nc r e a s e s i n t he t um or s t a ge .
L X A 4 i nc r e a s e s f r om t he s t e a t os i s s t a ge t o f i br os i s s t a ge t he n de c r e a s e s i n t he t um or s t a ge .
P G D 2 a nd P G F 2 α s how a dr a m a t i c i nc r e a s e i n t he l a t e r s t a ge of l i ve r di s e a s e . H ow e ve r , t hi s
i nc r e a s e c a n be c ont r i but e d by t he e x t r e m e va l ue .
4 1
T he e x pr e s s i on of A L O X - 5 a nd A L O X - 15 di d not f ol l ow t he i nc r e a s e d pr ogr e s s i on a s obs e r ve d
f or t he C O X e nz y m e s ( F i g. 20) . T he e x pr e s s i on of bot h A L O X - 5 a nd A L O X - 15 s how e d a hi g he r
t r e nd i n t he f i br os i s a nd c a nc e r s t a t e w he n c om pa r e d t o t he s t e a t os i s s t a t e , w hi c h m a y s ug ge s t a
c om pe ns a t or y r e s pons e f ol l ow i ng di s e a s e pr ogr e s s i on. T he e x pr e s s i on l e ve l of A L O X - 15
s uppor t s t he g l oba l l e ve l c ha nge of L X A 4 a s L X A 4 i nc r e a s e d f r om t he s t e a t os i s s t a t e t o t he
f i br os i s s t a t e a nd de c r e a s e d i n t he c a nc e r s t a t e . L e ve l s of L T B 4 di d not s how t he s a m e pa t t e r n a s
A L O X - 5 a nd A L O X - 15 ( F i g. 19) . L T B 4 pl a y s a pr o- i nf l a m m a t or y r ol e a nd i s e x pe c t e d t o be
a l t e r e d i n t he f i br os i s / i nf l a m m a t i on s t a ge of t he di s e a s e . T he pos s i bi l i t y f or t hi s l a c k of c ha nge i s
t ha t w e do not ha ve t he g l oba l l e ve l of L T A 4 ( i nt e r m e di a t e be t w e e n L T B 4 a nd A A ) i n our
t a r ge t e d l i pi dom i c da t a a nd t he e x pr e s s i on l e ve l of L T A 4 hy d r ol a s e , t he e nz y m e t ha t c a t a l y z e s
t he pr oduc t i on of L T B 4 f r om L T A 4. O ve r a l l , no m a j or a ppr e c i a bl e c ha nge s or t r e nd w e r e
obs e r ve d f or e i t he r t he L O X e nz y m e s or m e t a bol i t e s a s t he di s e a s e pr ogr e s s e s f r om s t e a t os i s t o
c a nc e r .
C om pa r e d t o t he w i l d t y p e , a not a bl e di f f e r e nc e i n t he P t e n de l e t e d l i ve r s i s t he a va i l a bi l i t y of t he
P U F A s ubs t r a t e s i n t he f i br os i s / t um or m i c e . L e ve l s of a l l f our de t e c t e d P U F A pr e c ur s or s ( e .g.,
A A , E P A , D P A a nd D H A ) a r e r e duc e d w i t h a ge i n t he W T l i ve r s ( F i g. 8) . T he l e ve l s of A A a nd
D P A a r e m uc h hi g he r i n t he t um or - be a r i ng m i c e w he n c om pa r e d t o t he s t e a t os i s a nd f i br os i s
m i c e ( F i g. 21) . C ol l e c t i ve l y , t he s e da t a i ndi c a t e t ha t P U F A s a r e e nr i c he d i n t he H C C l i ve r s t ha t
de ve l ope d i n t he P t e n de l e t e d m i c e a nd t he C O X m e t a bol i t e s de r i ve d e i c os a noi ds f r om t he s e
P U F A s m a y pl a y a r ol e i n t he pr ogr e s s i on of t he t um or s .
4 2
F i gur e 20: m R N A e x pr e s s i on l e ve l s of L O X - 5, L O X - 15 a nd C O X e nz y m e s i n t he P t e n de l e t e d
m ous e m ode l .
L O X - 5 a nd L O X - 15 s how s i m i l a r e x pr e s s i on l e ve l s i n t he P M m ode l . B ot h L O X - 5 a nd L O X - 15
i nc r e a s e f r om 3 m ont h t o 9 m ont h t he n de c r e a s e f r om 9 m ont h t o 15 m ont h. C O X - 1 s how s a
c ont i nuous i nc r e a s e i n t he P M m ode l a s t he m i c e a ge . C O X - 2 doe s not s how a not i c e a bl e c ha nge
be t w e e n 3 m ont h a nd 9 m ont h, but s how a dr a m a t i c i nc r e a s e i n 15 m ont h. A l m os t a l l e nz y m e s
s how pa t t e r ns i n t he P M m ode l a s t he m i c e a ge .
4 3
F i gur e 21: E i c os a noi d l e ve l s of pr e c ur s or s i n t he P t e n de l e t e d m ode l i n 3 t i m e pe r i ods .
A A a nd D P A i nc r e a s e i n t he pr ogr e s s i on of l i ve r di s e a s e . T he i nc r e a s i ng l e ve l s of s ubs t r a t e s c a n
be one e x pl a na t i on f or t he i nc r e a s e of P r os t a noi ds i n t he l a t e s t a g e of l i ve r di s e a s e .
4 4
C ha pt e r I V D i s c us s i on a nd F ut ur e P e r s pe c t i ve s
I n t hi s s t udy , w e de t e r m i ne d t he e i c os a noi ds m e t a bol i t e c ha nge s dur i ng t he pr og r e s s i on of l i ve r
di s e a s e . W e us e d a m ous e m ode l t ha t r e f l e c t s t he di s e a s e pr ogr e s s i on obs e r ve d i n pa t i e nt s . H e r e ,
w e r e por t a nove l di s c ove r y t ha t l e ve l s of e i c os a noi ds de c r e a s e w i t h a ge i n m ous e l i ve r s . M os t
e i c os a noi ds s how e d de c r e a s i ng pa t t e r ns a s t he m i c e a ge d i n bot h t he W T a nd P t e n de l e t e d
m ode l s . P t e n de l e t i on c a n a l t e r bot h e i c os a noi d l e ve l s a nd e nz y m e e x pr e s s i on l e ve l s ( L O X a nd
C O X ) i n a l l t hr e e t i m e pe r i ods , e s pe c i a l l y 3 m ont h a nd 15 m ont h. M os t e i c os a noi ds de c r e a s e d
dur i ng t he pr ogr e s s i on f r om s t e a t os i s t o c a nc e r . H ow e ve r t he r e w e r e s om e e x c e pt i ons i nc l udi ng
pr os t a noi ds a nd L X A 4. T he s e pr o a nd a nt i - i nf l a m m a t or y e i c os a noi ds w e r e i nduc e d i n t he c a nc e r
s t a t e pr oba bl y be c a us e t he y a r e t r y i ng t o r e s ol ve t he i nf l a m m a t i on.
T he di s c ove r y t ha t e i c os a noi d m e t a bol i t e s de c l i ne i n t he l i ve r a s t he m i c e a ge i s a hi g hl y
s i g ni f i c a nt a nd i nt e r e s t i ng f i ndi ng . A gi ng i s a n i m por t a nt f a c t or t ha t a f f e c t s t he g l oba l l e ve l of
e i c os a noi ds i n t he body . I n m a c r opha ge s , e i c os a noi ds l i ke P G E 2 i nc r e a s e d a s m i c e a ge d
4 0
. I n
l ung s , e i c os a noi ds i nc l udi ng P G I 2 a nd T X B 2 g r e a t l y i nc r e a s e d a s t he m i c e a ge d
4 1
. I n t he l i ve r ,
a n a ge - r e l a t e d e i c os a noi d l e ve l c ha nge ha s not be e n r e por t e d pr e vi ous l y . I n t hi s s t udy , w e r e por t
a g e ne r a l t r e nd of de c l i ne f or m os t e i c os a noi ds a s m i c e a ge d i nc l udi ng P G E 2 a nd T X B 2. T he
onl y e x c e pt i on i s a f e w r e s ol vi ng m e t a bol i t e s . D e c l i ne of e i c os a noi ds m a y be one i m por t a nt
f a c t or t ha t c ont r i but e s t o t he de ve l opm e nt of l i ve r di s e a s e .
I n t he P t e n de l e t e d m ode l , t he g e ne r a l t r e nd of de c l i ne i n e i c os a noi d l e ve l s i s a t t e nua t e d. T hi s
m a y be r e l a t e d t o t he i nf l a m m a t or y c ondi t i ons t ha t de ve l op i n t he P t e n de l e t e d m i c e , pa r t i c ul a r l y
a s t he y de ve l op f i br os i s a nd t um or s a t 9 a nd 15 m ont hs of a ge . I n t he s e l a t e r s t a ge s of l i ve r
4 5
di s e a s e , s i g ni f i c a nt a m ount s of i nf l a m m a t or y c e l l s w e r e f ound a c c om pa ny i ng t he pr ogr e s s i on of
t he di s e a s e
4 2
. T he e l e va t e d l e ve l s of e i c o s a no i d m e t a b ol i t e s ( r e l a t i ve t o t he a ge - m a t c he d c ont r ol s )
m a y be a n i ndi c a t i on of or r e s ul t f r om t he i nf i l t r a t i on of t he i nf l a m m a t or y c e l l s .
E i c os a noi ds a r e c ha r a c t e r i z e d a s i m por t a nt i nf l a m m a t or y m e di a t or s . D ur i ng i nf l a m m a t i on,
pa t hoge n- a s s oc i a t e d m ol e c ul a r pa t t e r ns ( P A M P s ) a nd da nge r - a s s oc i a t e d m ol e c ul a r pa t t e r ns
( D A M P s ) bi nd t o pa t t e r n r e c ogni t i on r e c e pt or s ( P R R s ) i nc l udi ng T ol l - l i ke r e c e pt or s ( T L R s ) a nd
N od- l i ke r e c e pt or s ( N L R s )
3 7
. I n t he l i ve r , P A M P s a nd D A M P s a c c um ul a t e d due t o pa t hol og i c a l
c ondi t i ons s uc h a s s t e a t os i s s t i m ul a t e t he a c t i va t i on of m a c r opha ge s
3 7
. M a c r opha ge s a r e
pol a r i z e d t o M 1 ( pr o- i nf l a m m a t or y ) or M 2 ( a nt i - i nf l a m m a t or y ) m a c r opha ge s m a r ke d by s pe c i f i c
c y t oki ne pr oduc t i ons
3 8
. M 1 m a c r opha ge s s e c r e t e pr o- i nf l a m m a t or y c y t oki ne s s uc h a s T N F - α a nd
I L - 6, w hi c h pr om ot e s l i ve r i nj ur y a t t he e a r l y s t a ge
3 8 , 3 9
. M 2 m a c r opha ge s pr oduc e a nt i -
i nf l a m m a t or y c y t oki ne s s uc h a s I L - 10 a nd T G F - β t o he l p r e s ol ve t he i nf l a m m a t i on dur i ng t he
l a t e r s t a ge
3 8 , 3 9
. W hi l e t he s pe c i f i c r ol e s of i nf l a m m a t i on a nd m a c r opha ge on l i ve r di s e a s e
pr ogr e s s i on de pe nds on t he c y t oki ne s pr oduc e d a nd t he pa r t i c ul a r pa t hol og i c a l / e x pe r i m e nt a l
c ondi t i ons , unr e s ol ve d i nf l a m m a t i on a nd w ound he a l i ng c a n l e a d t o l i ve r f i br os i s a nd f i na l l y
de ve l op i nt o H C C
3 8 , 3 9
. S pe c i f i c a l l y , a m a j or r e s pons i bi l i t y of m a c r opha ge s i s t o c l e a n t he
pa t hoge ns / da m a ge d c e l l de br i s . S pe c i f i c pr or e s ol vi ng e i c os a noi ds he l p m a c r opha ge s w i t h t he
r e s ol ut i on of i nf l a m m a t i on. F or e x a m pl e , L X A 4 a nd L X B 4 bi nd t o F P R 2 a nd ha ve a nt i -
i nf l a m m a t or y e f f e c t s . P G D 2, P G F 2 α bi nd t o D P a nd F P a nd ha ve a nt i - i nf l a m m a t or y e f f e c t s ( F i g.
22) . B e s i de s , L T B 4 a nd P G E 2 bi nd B L T s a nd E P t o ha ve pr o- i nf l a m m a t or y e f f e c t s ( F i g. 22) .
O ur da t a r e por t s P G D 2 a nd P G F 2 α a r e bot h i nc r e a s e d i n t he l a t e r s t a ge s of l i ve r di s e a s e . T he s e
4 6
c ha nge s m a y c ont r i but e t o t he pr og r e s s i on f r om s t e a t os i s t o c a nc e r a s P G D 2 a nd P G F 2 α bot h
pr ol ong t he i nf l a m m a t i on.
T he P T E N r e gul a t e d P I 3K / A K T s i g na l i ng m e di a t e d i ns ul i n s i g na l i ng . A m a j or c ons e que nc e of
i ns ul i n s i g na l s i n t he l i ve r i s t o i nduc e l i pog e ne s i s
4 3
. I n a ddi t i on, t he P T E N r e gul a t e d P I 3K / A K T
s i g na l i ng a l s o l e a ds t o i nc r e a s e d oxi da t i ve phos phor y l a t i on a nd m i t oc hondr i a l f unc t i on
4 4
. O ur l a b
ha s s how n t ha t t he i nc r e a s e d O X P H O S c ont r i but e s t o t he N A F L D / N A S H c ondi t i ons de ve l ope d
i n t he P t e n de l e t e d m i c e
4 7
. A s l i pi ds , pa r t i c ul a r l y P U F A s a r e r e a di l y oxi di z e d, a nd t he
e i c os a noi ds a r e oxi da t i on pr oduc t s of l i pi ds , w e e x pe c t e d a n i nc r e a s e i n t he P U F A s i n t he l a t e r
s t a ge of l i ve r di s e a s e . O ur da t a pr e s e nt e d he r e s how e d i nc r e a s e d l e ve l s of A A a nd D P A i n t he
c a nc e r s t a t e a s w e l l a s i nc r e a s e s on s pe c i f i c m e t a bol i t e s . H ow e ve r , i t i s unc l e a r w he t he r t he
P I 3K / A K T s i g na l i ng pa t hw a y m a y di r e c t l y r e gul a t e t he e x pr e s s i on of L O X a nd C O X e nz y m e s .
O ne m a j or obs e r va t i on w e di s c ove r e d he r e i s t he de c l i ne of P U F A w i t h a ge i n t he w i l d t y p e
l i ve r s . I n a ddi t i on, P T E N l os s a ppe a r s t o s l ow dow n t hi s de c l i ne . T he a va i l a bi l i t y of P U F A s i s
r e gul a t e d by di e t i nput a nd de novo s y n t he s i s . A s bot h W T a nd P t e n de l e t e d m i c e a r e f e d t he
s a m e c how di e t a nd no s i g ni f i c a nt di f f e r e nc e s ha ve be e n r e por t e d f or f ood c ons um pt i on
pr e vi ous l y
4 8
, t he P T E N l os s i nduc e d do novo l i pog e ne s i s m a y ha ve c ont r i but e d t o t he s l ow e d
de c l i ne of P U F A s obs e r ve d w i t h P t e n de l e t e d m i c e a s t he y a ge . I t ha s be e n r e por t e d pr e vi ous l y
t ha t P T E N l os s l e a ds t o r e duc t i ons of P U F A s
4 5
. T hus , e ve n t houg h P T E N l os s i nduc e s ove r a l l
c ont e nt i n t he l i ve r , a n i nc r e a s e i n t ot a l P U F A l e ve l s m a y or m a y not ha ve l e d t o t he s l ow i ng
dow n of t he a ge - de pe nde nt de c l i ne of e i c os a noi ds obs e r ve d i n t he P t e n de l e t e d l i ve r s . T o a ddr e s s
4 7
t hi s que s t i on s pe c i f i c a l l y , unt a r ge t e d l i pi dom i c s w oul d be ne e de d t o de c i phe r s pe c i f i c l i pi d
c ha nge s i n t he f ut ur e .
I n a ddi t i on, P U F A s e x i s t i n c om pl e x l i pi ds i n t i s s ue s . c P L A 2 pl a y s a n i m por t a nt r ol e i n r e l e a s i ng
P U F A s f r om t he phos phol i pi d m e m br a ne . c P L A 2 hy d r ol y z e s t he s n2 pos i t i on of t he m e m br a ne
phos phol i pi ds a nd r e l e a s e s A A f or t he pr oduc t i on of dow ns t r e a m m e t a bol i t e s . A K T / P I 3 K
pa t hw a y i nduc e s t he C a
2 +
l e ve l a nd m a y upr e gul a t e t he c P L A 2 e xpr e s s i on
2 2
. W i t h t he i nc r e a s e of
c P L A 2, m or e f r e e A A c a n be r e l e a s e d i nt o t he c e l l a nd m or e e i c os a noi ds c a n be pr oduc e d
4 6
.
F i gur e 22: S um m a r y of L O X a nd C O X E i c os a noi ds a nd t he i r bi ol og i c a l f unc t i ons .
E i c os a noi ds a r e pr oduc e d f r om P U F A s by t he a c t i on of A L O X a nd C O X e nz y m e s . T he i r
m e t a bol i t e s bi nd t o t he s pe c i f i c G pr ot e i n- c oupl e d r e c e pt or s a nd t he n pe r f or m t he i r pr o a nd a nt i -
i nf l a m m a t or y f unc t i ons .
4 8
A m a j or hur dl e f or t hi s s t udy i s t he va r i a t i ons f r om m ous e t o m ous e . T hi s i s obs e r ve d on bot h t he
m e t a bol i t e l e ve l s a nd t he m R N A e x pr e s s i on of L O X a nd C O X e nz y m e s . W he n t a r ge t e d
l i pi dom i c s r e s ul t s w e r e s e l e c t e d f or f ur t he r a na l y s i s , I f ound t ha t out l i e r s i n t he nor m a l i z a t i on
da t a g r e a t l y a f f e c t e d t he e i c os a noi d l e ve l s a nd ove r a l l pa t t e r ns i n t he he a t m a ps . O ne f a c t or
c ont r i but e d t o t hi s va r i a t i on i s t he l ow s i gna l t o noi s e r a t i o ( S N R ) obs e r ve d f or m a ny m e t a bol i t e s .
T he S N R i s one i m por t a nt f a c t or t ha t a f f e c t s de t e c t i on l e ve l s of e i c os a noi ds . I n our a na l y s i s ,
s om e e i c os a noi ds ha ve be t t e r S N R s t ha n ot he r s , but ot he r s ha ve unr e l i a bl e S N R s . T he
ba c kgr ounds of t he r a w s i g na l pe a ks of t he s e m e t a bol i t e s w e r e ve r y noi s y a nd t he i nt e ns i t y of t he
t a r ge t pe a k w a s l ow ( s m a l l e r t ha n 1000) . T hi s m a de t he pe a k s e l e c t i on pr oc e s s r e a l l y c ha l l e ngi ng
a nd t he r e s ul t s be t w e e n s a m pl e s w e r e not c ons i s t e nt .
I n a ddi t i on, w e onl y ha d 4 s a m pl e s f or e a c h c ont r ol g r oup a nd P T E N de l e t e d g r oup f or t he
t a r ge t e d l i pi dom i c s . T he s m a l l s a m pl e s i z e c om pounde d w i t h t he va r i a t i ons due t o l ow S N R s
m a y a l s o c ont r i but e d t o t he l a c k of s i g ni f i c a nc e w i t h t he da t a . T hus , f ut ur e a na l y s i s ne e ds t o be
pe r f or m e d us i ng t he s e pr e l i m i na r y r e s ul t s . F or t hos e w i t h g ood S N R s , pow e r a na l y s i s s houl d be
c a l c ul a t e d us i ng va r i a nc e c ol l e c t e d he r e . F ut ur e s t udi e s w i l l i nc r e a s e t he s a m pl e s i z e ba s e d on
t he s e pow e r a na l y s e s .
4 9
R e f e r e nc e s
1. G r a nt D M . D e t ox i f i c a t i on pa t hw a y s i n t he l i ve r . J our nal of I nhe r i t e d M e t abol i c D i s e as e .
1991: 421- 430. doi : 10.1007/ 978- 94- 011- 9749- 6_2
2. W u J , Y ou J , Y e r i a n L , S hi ba A , S c ha ue r P R , S e s s l e r D I . P r e va l e nc e of l i ve r s t e a t os i s a nd
f i br os i s a nd t he di a gnos t i c a c c ur a c y of ul t r a s ound i n B a r i a t r i c s ur ge r y pa t i e nt s . O be s i t y
Sur ge r y . 2011; 22( 2) : 240- 247. doi : 10.1007/ s 11695- 011- 0509- 8
3. T he . ht t ps : / / w w w .t he - na s h- e duc a t i on- pr ogr a m .c om / w ha t - i s - na s h/ . A c c e s s e d M a r c h 15,
2023.
4. L u c a C i c a l e s e M D . H e pa t oc e l l ul a r c a r c i nom a ( H C C ) . P r a c t i c e E s s e nt i a l s , A na t om y ,
P a t hophy s i ol ogy . ht t ps : / / e m e di c i ne .m e ds c a pe .c om / a r t i c l e / 197319- ove r vi e w . P ubl i s he d
D e c e m be r 22, 2022. A c c e s s e d M a r c h 12, 2023.
5. I o a nnou G N , S pl a n M F , W e i s s N S , M c D ona l d G B , B e r e t t a L , L e e S P . I n c i de nc e a nd
pr e di c t or s of he pa t oc e l l ul a r c a r c i nom a i n pa t i e nt s w i t h c i r r hos i s . C l i ni c al
G as t r oe nt e r ol ogy and H e pat ol ogy . 2007; 5( 8) . doi : 10.1016/ j .c g h.2007.02.039
6. E l s ha r ka w y A M , M a nn D A . N uc l e a r f a c t or - Κ B a nd t he he pa t i c i nf l a m m a t i on- f i br os i s -
c a nc e r a x i s . H e pat ol ogy . 2007; 46( 2) : 590- 597. doi : 10.1002/ he p.21802
7. M a s hi m a , R ., & O kuy a m a , T . ( 2015) . T he r ol e of l i poxy ge na s e s i n pa t hophy s i ol ogy ; ne w
i ns i g ht s a nd f ut ur e pe r s pe c t i ve s . R e dox B i ol ogy , 6, 297–310.
ht t ps : / / doi .or g / 10.1016/ j .r e dox .2015.08.006
8. de L o pe C R , T r e m os i ni S , F or ne r A , R e i g M , B r ui x J . M a na ge m e nt of H C C . J our nal of
he pat ol ogy . 2012; 56( 1) : S 75- S 87. doi : 10.1016/ S 0168- 8278( 12) 60009- 9
9. H . E , M a ns our H , G hobr i a l M . L i ve r t r a ns pl a nt a t i on f or he pa t oc e l l ul a r c a r c i nom a .
H e pat oc e l l ul ar C ar c i nom a - F ut ur e O ut l ook . 2013. doi : 10.5772/ 55374
10. J e m a l A , W a r d E M , J ohns on C J , e t a l . A nnua l r e por t t o t he na t i on on t he s t a t us of c a nc e r ,
1975–2014, f e a t ur i ng s ur vi va l . J N C I : J our nal of t he N at i onal C anc e r I ns t i t ut e .
2017; 109( 9) . doi : 10.1093/ j nc i / dj x030
11. R us t a n A C , D r e von C A . F a t t y a c i ds : S t r uc t ur e s a nd pr ope r t i e s . e L S . 2001.
doi : 10.1038/ npg .e l s .0000715
12. G R O O T P H E , S C H O L T E H R , H Ü L S M A N N W C . F a t t y a c i d a c t i va t i on: S pe c i f i c i t y ,
l oc a l i z a t i on, a nd f unc t i on. A dv anc e s i n L i pi d R e s e ar c h . 1976: 75- 126. doi : 10.1016/ b978-
0- 12- 024914- 5.50009- 7
13. D e nni s E A , N or r i s P C . E i c os a noi d s t or m i n i nf e c t i on a nd i nf l a m m a t i on. N at ur e R e v i e w s
I m m unol ogy . 2015; 15( 8) : 511- 523. doi : 10.1038/ nr i 3859
5 0
14. S he ppe A E , E de l m a nn M J . R ol e s of e i c os a noi ds i n r e gul a t i ng i nf l a m m a t i on a nd
ne ut r ophi l m i g r a t i on a s a n i nna t e hos t r e s pons e t o ba c t e r i a l i nf e c t i ons . I nf e c t i on and
I m m uni t y . 2021; 89( 8) . doi : 10.1128/ i a i .00095- 21
15. A l ba M M , E br i ght B , H ua B , e t a l . E i c os a noi ds a nd ot he r oxy l i pi ns i n l i ve r i nj ur y ,
i nf l a m m a t i on a nd l i ve r c a nc e r de ve l opm e nt . F r ont i e r s i n P hy s i ol ogy . 2023; 14.
doi : 10.3389/ f phy s .2023.1098467
16. E nge l ki ng L R . T e x t book of V e t e r i nar y P hy s i ol ogi c al C he m i s t r y . L ondon: A c a de m i c
P r e s s ; 2015.
17. S t r a us s J F . Y e n and J af f e ' s R e pr oduc t i v e E ndoc r i nol ogy ( E i ght h E di t i on) . E l s e vi e r
M a s s on; 2019.
18. B a l a z y M . E i c os a nom i c s : T a r ge t e d l i pi dom i c s of e i c os a noi ds i n B i ol og i c a l S y s t e m s .
P r os t agl andi ns & O t he r L i pi d M e di at or s . 2004; 73( 3- 4) : 173- 180.
doi : 10.1016/ j .pr os t a g l a ndi ns .2004.03.003
19. R i c c i ot t i E , F i t z G e r a l d G A . P r os t a g l a ndi ns a nd i nf l a m m a t i on. A r t e r i os c l e r os i s ,
T hr om bos i s , and V as c ul ar B i ol ogy . 2011; 31( 5) : 986- 1000.
doi : 10.1161/ a t vba ha .110.207449
20. L e B e l M , B r une t A , G os s e l i n J . L e ukot r i e ne B 4, a n e ndoge nous s t i m ul a t or of t he i nna t e
i m m une r e s pons e a ga i ns t pa t hoge ns . J our nal of I nnat e I m m uni t y . 2013; 6( 2) : 159- 168.
doi : 10.1159/ 000353694
21. F i t z pa t r i c k F A , S obe r m a n R . R e gul a t e d f or m a t i on of e i c os a noi ds . J our nal of C l i ni c al
I nv e s t i gat i on . 2001; 107( 11) : 1347- 1351. doi : 10.1172/ j c i 13241
22. K r a m e r R M , S ha r p J D . S t r uc t ur e , f unc t i on a nd r e gul a t i on of C a 2+ - s e ns i t i ve c y t os ol i c
phos phol i pa s e A 2 ( c P L A 2) . F E B S L e t t e r s . 1997; 410( 1) : 49- 53. doi : 10.1016/ s 0014-
5793( 97) 00322- 0
23. F ont - N i e ve s M , S a ns - F ons M G , G or i na R , e t a l . I nduc t i on of C O X - 2 e nz y m e a nd dow n-
r e gul a t i on of C O X - 1 e x pr e s s i on by l i popol y s a c c ha r i de ( L P S ) c ont r ol pr os t a gl a ndi n E 2
pr oduc t i on i n a s t r oc y t e s . J our nal of B i ol ogi c al C he m i s t r y . 2012; 287( 9) : 6454- 6468.
doi : 10.1074/ j bc .m 111.327874
24. K e l l e y D S , T a y l or P C , N e l s on G J , S c hm i dt P C , M a c ke y B E , K y l e D . E f f e c t s of di e t a r y
a r a c hi doni c a c i d on hum a n i m m une r e s pons e . L i pi ds . 1997; 32( 4) : 449- 456.
doi : 10.1007/ s 11745- 997- 0059- 3
25. P e i s e l e r M , T a c ke F . I n f l a m m a t or y m e c ha ni s m s unde r l y i ng nona l c ohol i c s t e a t ohe pa t i t i s
a nd t he t r a ns i t i on t o he pa t oc e l l ul a r c a r c i nom a . C anc e r s . 2021; 13( 4) : 730.
doi : 10.3390/ c a nc e r s 13040730
5 1
26. R ouz e r , C . A ., & M a r ne t t , L . J . ( 2009) . C y c l oox y ge n a s e s : S t r uc t ur a l a nd f unc t i ona l
i ns i g ht s . J our nal of L i pi d R e s e ar c h , 50. ht t ps : / / doi .or g / 10.1194/ j l r .r 800042- j l r 200
27. X u, Y .- J ., Z he ng, Z ., C a o, C ., L i , J ., & L i u, Y . ( 2018) . B i oa na l y t i c a l i ns i g ht s i nt o t he
a s s oc i a t i on be t w e e n e i c os a noi ds a nd pa t hoge ne s i s of he pa t oc e l l ul a r c a r c i nom a . C anc e r
and M e t as t as i s R e v i e w s , 37( 2- 3) , 269–277. ht t ps : / / doi .or g / 10.1007/ s 10555- 018- 9747- 8
28. R å dm a r k, O ., & S a m ue l s s on, B . ( 2009) . 5- l i poxy ge n a s e : M e c ha ni s m s of r e gul a t i on.
J our nal of L i pi d R e s e ar c h , 50. ht t ps : / / doi .or g / 10.1194/ j l r .r 800062- j l r 200
29. M a s hi m a R , O kuy a m a T . T he r ol e of l i poxy ge na s e s i n pa t hophy s i ol og y ; ne w i ns i g ht s a nd
f ut ur e pe r s pe c t i ve s . R e dox B i ol ogy . 2015; 6: 297- 310. doi : 10.1016/ j .r e dox.2015.08.006
30. M a ndl i k D S , M a ndl i k S K , C houdha r y H B . I m m unot he r a py f or he pa t oc e l l ul a r c a r c i nom a :
C ur r e nt s t a t us a nd f ut ur e pe r s pe c t i ve s . W o r l d J our nal of G as t r oe nt e r ol ogy .
2023; 29( 6) : 1054- 1075. doi : 10.3748/ w j g .v29.i 6.1054
31. L e gr a nd P , R i oux V . T he c om pl e x a nd i m por t a nt c e l l ul a r a nd m e t a bol i c f unc t i ons of
s a t ur a t e d f a t t y a c i ds . L i pi ds . 2010; 45( 10) : 941- 946. doi : 10.1007/ s 11745- 010- 3444- x
32. N a ka m ur a M T , C ho H P , X u J , T a ng Z , C l a r ke S D . M e t a bol i s m a nd f unc t i ons of hi g hl y
uns a t ur a t e d f a t t y a c i ds : A n upda t e . L i pi ds . 2001; 36( 9) : 961- 964. doi : 10.1007/ s 11745- 001-
0806- 5
33. B a l i ć A , V l a š i ć D , Ž už ul K , M a r i novi ć B , B ukvi ć M okos Z . O m e ga - 3 ve r s us om e ga - 6
pol y u ns a t ur a t e d f a t t y a c i ds i n t he pr e ve nt i on a nd t r e a t m e nt of i nf l a m m a t or y s ki n di s e a s e s .
I nt e r nat i onal J our nal of M ol e c ul ar Sc i e nc e s . 2020; 21( 3) : 741. doi : 10.3390/ i j m s 21030741
34. W ong R S Y . A popt os i s i n c a nc e r : F r om pa t hoge ne s i s t o t r e a t m e nt . J our nal of
E x pe r i m e nt al & C l i ni c al C anc e r R e s e ar c h . 2011; 30( 1) . doi : 10.1186/ 1756- 9966- 30- 87
35. L a ne C S , W a ng Y , B e t t s R , G r i f f i t hs W J , P a t t e r s on L H . C om pa r a t i ve c y t oc hr om e P 450
pr ot e om i c s i n t he l i ve r s of i m m unode f i c i e nt m i c e us i ng 18O s t a bl e i s ot ope l a be l i ng.
M ol e c ul ar & C e l l ul ar P r ot e om i c s . 2007; 6( 6) : 953- 962. doi : 10.1074/ m c p.m 600296-
m c p200
36. R e na ud H J , C ui J Y , K ha n M , K l a a s s e n C D . T i s s ue di s t r i but i on a nd g e nde r - di ve r ge nt
e x pr e s s i on of 78 c y t oc hr om e P 450 m R N A s i n m i c e . T ox i c ol ogi c al Sc i e nc e s .
2011; 124( 2) : 261- 277. doi : 10.1093/ t oxs c i / kf r 240
37. R oh J S , S ohn D H . D a m a ge - a s s oc i a t e d m ol e c ul a r pa t t e r ns i n i nf l a m m a t or y di s e a s e s .
I m m une N e t w or k . 2018; 18( 4) . doi : 10.4110/ i n.2018.18.e 27
38. S un Y - Y , L i X - F , M e ng X - M , H ua ng C , Z ha ng L , L i J . M a c r opha ge phe not y p e i n l i ve r
i nj ur y a nd R e pa i r . Sc andi nav i an J our nal of I m m unol ogy . 2017; 85( 3) : 166- 174.
doi : 10.1111/ s j i .12468
5 2
39. B udhu A , W a ng X W . T he r ol e of c y t oki ne s i n he pa t oc e l l ul a r c a r c i nom a . J our nal of
L e uk oc y t e B i ol ogy . 2006; 80( 6) : 1197- 1213. doi : 10.1189/ j l b.0506297
40. B e ha r ka , A . A ., W u, D ., H a n, S . N ., & M e y d a ni , S . N . ( 1997) . M a c r opha ge pr os t a gl a ndi n
pr oduc t i on c ont r i but e s t o t he a ge - a s s oc i a t e d de c r e a s e i n T c e l l f unc t i on w hi c h i s r e ve r s e d
by t he di e t a r y a nt i oxi da nt vi t a m i n E . M e c hani s m s of A ge i ng and D e v e l opm e nt , 93( 1 – 3) ,
59–77. ht t ps : / / doi .or g / 10.1016/ s 0047- 6374( 96) 01819- 2
41. M e y d a ni S N , S ha pi r o A C , M e y d a ni M , B l um be r g J B . L u ng e i c os a noi d s y n t he s i s i s
a f f e c t e d by a ge , di e t a r y f a t a nd vi t a m i n E . T he J our nal of N ut r i t i on . 1992; 122( 8) : 1627-
1633. doi : 10.1093/ j n/ 122.8.1627
42. D e be be A , M e di na V , C he n C - Y , e t a l . W nt / β - c a t e ni n a c t i va t i on a nd m a c r opha ge
i nduc t i on dur i ng l i ve r c a nc e r de ve l opm e nt f ol l ow i ng s t e a t os i s . O nc oge ne .
2017; 36( 43) : 6020- 6029. doi : 10.1038/ onc .2017.207
43. H e L , H ou X , K a ne l G , e t a l . T he c r i t i c a l r ol e of A K T 2 i n he pa t i c s t e a t os i s i nduc e d by
P T E N l os s . T he A m e r i c an J our nal of P at hol ogy . 2010; 176( 5) : 2302- 2308.
doi : 10.2353/ a j pa t h.2010.090931
44. L i Y , H e L , Z e ng N , e t a l . P hos pha t a s e a nd t e ns i n hom ol og de l e t e d on c hr om os om e 10
( P T E N ) s i g na l i ng r e gul a t e s m i t oc hondr i a l bi og e ne s i s a nd r e s pi r a t i on vi a e s t r oge n- r e l a t e d
r e c e pt or α ( e r r α ) . J our nal of B i ol ogi c al C he m i s t r y . 2013; 288( 35) : 25007- 25024.
doi : 10.1074/ j bc .m 113.450353
45. V l oc k E M , K a r a nj i t S , T a l m on G , F a r a z i P A . R e duc t i on of pol y u ns a t ur a t e d f a t t y a c i ds
w i t h t um or pr ogr e s s i on i n a l e a n non- a l c ohol i c s t e a t ohe pa t i t i s - a s s oc i a t e d he pa t oc e l l ul a r
c a r c i nom a m ous e m ode l . J our nal of C anc e r . 2020; 11( 19) : 5536- 5546.
doi : 10.7150/ j c a .48495
46. G hi g o A , L a f f a r gue M , L i M , H i r s c h E . P I 3K a nd c a l c i um s i g na l i ng i n c a r di ova s c ul a r
di s e a s e . C i r c ul at i on R e s e ar c h . 2017; 121( 3) : 282- 292. doi : 10.1161/ c i r c r e s a ha .117.310183
47. C he n C , L i Y , Z e ng N , e t a l . I n hi bi t i on of e s t r oge n- r e l a t e d r e c e pt or α bl oc ks l i ve r
s t e a t os i s a nd s t e a t ohe pa t i t i s a nd a t t e nua t e s t r i g l y c e r i de bi os y n t he s i s . T he A m e r i c an
J our nal of P at hol ogy . 2021; 191( 7) : 1240- 1254. doi : 10.1016/ j .a j pa t h.2021.04.007
48. G a l i c i a V A , K a ne l G , H e L , V e ndr y e s C , S t i l e s B L . A bs t r a c t 4260: A c t i va t i on of c a nc e r
s t e m c e l l s i n he pa t i c pt e n de f i c i e nt m i c e r e qui r e s l i ve r i nj ur y . C anc e r R e s e ar c h .
2010; 70( 8_S uppl e m e nt ) : 4260- 4260. doi : 10.1158/ 1538- 7445.a m 10- 4260
49. C a r ne r o A , B l a nc o- A pa r i c i o C , R e nne r O , L i nk W , L e a l J . T he P T E N / P I 3K / a kt
s i g na l l i ng pa t hw a y i n c a nc e r , t he r a pe ut i c i m pl i c a t i ons . C ur r e nt C anc e r D r ug T ar ge t s .
2008; 8( 3) : 187- 198. doi : 10.2174/ 156800908784293659
5 3
50. D ong- D ong L , X i - R a n Z , X i a ng- R ong C . E x pr e s s i on a nd s i g ni f i c a nc e of ne w t um or
s uppr e s s or g e ne P T E N i n pr i m a r y l i ve r c a nc e r . J our nal of C e l l ul ar and M ol e c ul ar
M e di c i ne . 2003; 7( 1) : 67- 71. doi : 10.1111/ j .1582- 4934.2003.t b00204.x
51. E br i ght B , A s s a nt e I , P obl e t e R A , e t a l . E i c os a noi d l i pi dom e a c t i va t i on i n pos t - m or t e m
br a i n t i s s ue s of i ndi vi dua l s w i t h A P O E 4 a nd A l z he i m e r ' s de m e nt i a . A l z he i m e r ’ s
R e s e ar c h & a m p; T he r apy . 2022; 14( 1) . doi : 10.1186/ s 13195- 022- 01084- 7
Abstract (if available)
Abstract
Hepatocellular carcinoma (HCC) is the most common primary liver cancer. The current treatments include surgical resection, chemotherapy and immunotherapy. However, the long term survival rate is relatively low. Eicosanoids are signaling molecules produced from polyunsaturated fatty acids, usually Arachidonic acid. Eicosanoids have both pro and anti-inflammatory actions in the inflammation. The global levels of eicosanoids may be altered during the progression of liver steatosis to cancer. In this study, we analyzed the change of eicosanoid levels in liver disease using a mouse model lacking PTEN in the liver. We selected mice at three ages: 3, 9 and 15 months of age. These three time periods represent three stages of the HCC tumorigenesis beginning with liver steatosis, liver steatosis accompanied by inflammation and ultimately tumor transformation. Targeted lipidomics are performed to determine the eicosanoid levels in these mice using age-matched wild type mice as controls. We report here a global decrease of eicosanoids in relation to aging of the mice in wild-type (WT) livers. Pten deletion reduces the rate of eicosanoid decline when compared to age-matched WT livers. Unexpectedly, most eicosanoids decreased in the Pten deleted model compared to the age-matched WT controls. A few exceptions are discovered, in particular inflammatory prostanoids found in the tumor microenvironment. Molecular analysis found alteration of lipoxygenase (LOX) and cyclooxygenase (COX) gene expression that support the lipidomic changes. In addition, alterations in eicosanoid precursors may also be a major factor contributing to the observed changes.
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Creator
Jia, Yunyi
(author)
Core Title
Eicosanoids in the progression of liver steatosis to cancer
School
School of Pharmacy
Degree
Master of Science
Degree Program
Pharmaceutical Sciences
Degree Conferral Date
2023-08
Publication Date
07/31/2023
Defense Date
07/31/2023
Publisher
University of Southern California. Libraries
(digital)
Tag
eicosanoid,hepatocellular carcinoma,Inflammation,liver disease,liver steatosis,OAI-PMH Harvest,polyunsaturated fatty acids
Language
English
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Electronically uploaded by the author
(provenance)
Advisor
Stiles, Bangyan (
committee chair
), Louie, Stan (
committee member
), Okamoto, Curtis (
committee member
)
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yunyij@hotmail.com,yunyijia@usc.edu
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https://doi.org/10.25549/usctheses-oUC113291582
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UC113291582
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Jia, Yunyi
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texts
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20230731-usctheses-batch-1076
(batch),
University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
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Tags
eicosanoid
hepatocellular carcinoma
liver disease
liver steatosis
polyunsaturated fatty acids