Resea ch A icle
E ec o Mela onin as an An ioxidan D ug o Re e se Hepa ic
S ea osis: Expe imen al Model
Blanca Ma ´
ınez So iano ,
1
An onio G¨
uemes,
1
Guille mo Pola,
2
Azucena Gonzalo,
1
Pila Palacios Gas´
os,
1
Ana C. Na a o,
1
Robe o Ma ´
ınez-Beamon e,
3
Jes´
us Osada ,
4
and Jos´
eJ. Ga c´
ıa
5
1
Depa men o Su ge y, Uni e si y Hospi al Lozano Blesa, Za agoza, Spain
2
Depa men o Su ge y, Gene al De ense Hospi al, Za agoza, Spain
3
CIBER (Biomedical Ne In es iga ion Cen e), Obesi y and Nu i ion Physiopa hology, Mad id, Spain
4
Depa men o Biochemis y and Molecula and Cellula Biology, Uni e si y o Za agoza, Za agoza, Spain
5
Depa men o Physiology, Uni e si y o Za agoza, Za agoza, Spain
Co espondence should be add essed o Blanca Ma ´
ınez So iano; [email p o ec ed]
Recei ed 27 Sep embe 2019; Re ised 25 Decembe 2019; Accep ed 16 Janua y 2020; Published 5 June 2020
Academic Edi o : Jos´
eL. Mau iz
Copy igh ©2020 Blanca Ma ´
ınez So iano e al. This is an open access a icle dis ibu ed unde he C ea i e Commons
A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is
p ope ly ci ed.
In oduc ion. The hepa ic s ea osis o he nonalcoholic o igin o NAFLD is inc easing a p esen , pa icula ly in Wes e n coun ies,
pa allel o he inc ease in obesi y, cons i u ing one o he mos p e alen hepa ic p ocesses in he Wes e n socie y. Mela onin has
been success ully es ed in expe imen al models in mice as a d ug capable o e e sing s ea osis. The effec o mela onin on a
me abolism can be summa ized as a dec ease in lipid pe oxida ion and a dec ease in oxida i e s ess, biochemical phenomena
in ima ely ela ed o a deposi ion in he hepa ocy e. The e a e ha dly any s udies in la ge animals. Objec i e. In his s udy, we
in es iga e he effec s o mela onin adminis e ed o ally a a dose o 10 mg/kg/day o e e se es ablished hepa ic s ea osis induced
by a special die in a po cine animal model. Ma e ials and Me hods. We analyze he pa ame e s o oxida i e s ess: malon-
dialdehyde (MDA), 4-hyd oxyalkenals (4-HDA), and ca bonyls, deg ee o a infil a ion (analyzed by di ec ision by a pa-
hologis and by means o a compu e p og am o image ea men ), and se ological pa ame e s o lipid me abolism and hepa ic
damage. These pa ame e s we e analyzed in animals o which hepa ic s ea osis was induced by means o die a y modifica ions.
Resul s. We ha e no been able o demons a e globally a beneficial effec o mela onin in he imp o emen o e e sal o li e
s ea osis once es ablished, induced by die in a po cine animal model. Howe e , we ha e ound se e al signs o imp o emen a he
his ological le el, a he le el o lipid me abolism, and a he le el o oxida i e s ess pa ame e s. We ha e e ified in ou s udy ha ,
in he his ological analysis o he li e sample by means o he p og am image ea men ( ee o subjec i i y) o he animals ha
con inue wi h he die , hose ha consume mela onin do no inc ease s ea osis as much as hose ha do no consume i sig-
nifican ly (p�0.002). Rega ding he pa ame e s o oxida i e s ess, MDA modifies in a significan manne wi hin he g oup o
animals ha con inue wi h he die and ake mela onin (p�0.004). As o lipid me abolism, animals ha main ain he s ea o ic
die and ake mela onin lowe o al and LDL choles e ol le els and inc ease HDL le els, al hough hese esul s do no acqui e
s a is ical significance. Conclusions. In his s udy, i has no been possible o demons a e a beneficial effec o mela onin in he
imp o emen o e e sal o li e s ea osis once es ablished and induced by die in he po cine model. I is ue ha signs o
imp o emen ha e been ound a he his ological le el, a he le el o lipid me abolism, and a he le el o oxida i e s ess
phenomena, when compa ing animals wi h es ablished s ea osis ha a e ea ed wi h mela onin wi h hose who do no ake i . This
wo k is he fi s s udy conduc ed in a la ge animal model in which he effec o mela onin is s udied as a ea men in he e e sal o
es ablished hepa ic s ea osis.
Hindawi
Canadian Jou nal o Gas oen e ology and Hepa ology
Volume 2020, A icle ID 7315253, 12 pages
h ps://doi.o g/10.1155/2020/7315253
1. In oduc ion
Nonalcoholic a y li e disease (NAFLD) is cha ac e ized by
he accumula ion o a y acids, iglyce ides, and choles e ol
in he cy oplasm o he hepa ocy e. I occu s in subjec s who
do no d ink alcohol o d ink mode a ely (<20 g/day) and is
conside ed as he exp ession in he li e o a complex
synd ome called “me abolic synd ome.” NAFLD includes
wo clinical en i ies: nonalcoholic a y li e (NAFL), which
e e s o he p esence o hepa ic s ea osis wi hou e idence o
hepa ocellula damage o fib osis, and nonalcoholic s ea-
ohepa i is (NASH), which is he p esence o hepa ic s ea-
osis ha is associa ed wi h inflamma ion and li e damage
wi h o wi hou fib osis. NASH may p og ess o ci hosis,
li e ailu e, and hepa oca cinoma [1].
The p e alence o NAFLD is no well known and is
p obably unde es ima ed. This is because mos pa ien s
emain asymp oma ic o ha e disc e e biological al e a ions,
he absence o p ecise se ological ma ke s, and he need o
li e biopsy o defini i e diagnosis. Howe e , we know ha
s ea osis is one o he mos p e alen li e diseases in he
Wes e n wo ld, linked o he inc ease in obesi y and me -
abolic synd ome, and a ies widely depending on he
popula ion s udied. Two Japanese s udies [2, 3] published an
incidence in he gene al popula ion o 31 and 86 cases,
espec i ely, o suspec ed NAFLD pe 1000 pe sons/yea . In
se e ely obese pa ien s unde going ba ia ic su ge y, he
p e alence o NAFLD may exceed 90% and up o 5% may
ha e ci hosis.
The ea men o hepa ic s ea osis once es ablished is
achie ed p ima ily h ough die a y es ic ions and li es yle
changes [4–7]. D ugs used in he ea men o me abolic
synd ome (an idiabe ics, s a ins, e c.) ha e also been used
[8]. Recen ly, he efficacy o a ious an ioxidan agen s, such
as i amin E and mela onin [9–11], has been demons a ed
in he ea men o hepa ic s ea osis. The e is cu en ly no
specific ha mless and effec i e ea men o hepa ic
s ea osis.
Mela onin is a na u al ho mone syn hesized by he pi-
neal gland in animals. Mela onin syn hesis is no ound
exclusi ely in he pineal gland, as i s sec e ion has been
desc ibed in nume ous pe iphe al o gans such as he e ina,
bone ma ow, skin, and gas oin es inal ac and in some
cells such as lymphocy es and pla ele s [12, 13].
One o he main p ope ies o mela onin is i s powe ul
an ioxidan effec [12], which is a ibu ed o i s abili y o
neu alize ee adicals and i s indi ec de oxi ying ac ion by
s imula ing an ioxidan enzymes [14]. I has been p o en
ha mela onin supplemen s could p o ec agains some
diseases such as a he oscle osis, cance , and Alzheime ’s
disease. A he le el o hepa ic me abolism, mela onin has
been es ed as a d ug o p e en he p ocess o ischemia-
epe usion [15] and also as a ea men o li e damage by
oxins such as alcohol, ca bon e achlo ide, afla oxin, o
chemo he apy agen s [16–19].
Mela onin has been shown o be beneficial in he
ea men and p e en ion o NAFLD in mu ine expe i-
men al models. Expe imen al s udies ha e been ca ied ou
in oden s, he majo i y wi h he in en ion o p e en ing
die -induced hepa ic s ea osis, obse ing a dec ease in blood
lipids, an imp o emen in hepa ic enzymes, and a dec ease
in oxida i e s ess pa ame e s, as well as an imp o emen in
his ology [20–25]. The e a e ha dly any s udies in la ge o
small animals ha p o e his effec . In humans, s udies ha e
been ca ied ou ha demons a e he lipid lowe ing and
cy op o ec i e capaci y o mela onin in hepa ic s ea osis
once es ablished, al hough i is ue ha he e a e ha dly any
s udies ha demons a e his ological imp o emen [26–29].
Ou wo king hypo hesis is based on he ac ha mel-
a onin is an effec i e ea men o e e se es ablished and
induced hepa ic s ea osis by means o a special die in a
po cine animal model, as well as in he mu ine model. The
aim o ou s udy is o assess he effec o mela onin ad-
minis e ed o ally on lipid me abolism, hepa ic his ology, and
oxida i e s ess pa ame e s.
2. Ma e ial and Me hods
2.1. Model and Sample: An Expe imen al Animal. We use he
g ea English whi e pig as an expe imen al animal. This
model was chosen as such because i is consis en wi h he
hepa ic physiology o he human. The animals we e ob ained
om a b eeding a m o animal expe imen a ion, which
we e ee o diseases o pa asi es and wi h an app oxima e
weigh o 45 kg and an app oxima e age o 6 mon hs. All
animals ha did no mee a minimum weigh (30 kg) o ha
showed signs o diseases (dia hoea, adynamia, de ma osis,
and abno mal beha iou ) we e excluded om he expe i-
men . The animals we e kep in cages, sha ed by 3 o 4
animals, and p o ided wi h eede s and wa e . The s udy was
app o ed by he Ad iso y E hics Commission o Animal
Expe imen a ion o he Uni e si y o Za agoza.
2.2.Expe imen alDesignandS udyG oups. The induc ion o
s ea osis was ca ied ou by means o a special die , wi hou
using any pha macological agen . I s main cha ac e is ics
we e as ollows: high con en in sa u a ed a (25%), defi-
ciency in me hionine and choline, and supplemen ed wi h
2% choles e ol plus 0.5% sodium chola e. The animals be-
longing o s udy g oups 1 and 3 ecei ed mela onin doses o
10 mg/kg/day o ally o 4 weeks. The d ug was acqui ed in
powde o m, which was encapsula ed in a suc ose excipien .
I was adminis e ed in a single dose by means o capsules
ha we e inges ed a he same ime as he die desc ibed
abo e, mixing he d ug wi h pa o he die in such a way as
o ensu e comple e in ake.
A con ol g oup consis ed o 6 animals; heal hy animals
wi hou s ea osis unde wen a no mal nons ea osis die and
unde wen a single su gical in e en ion o he pu pose o
ob aining samples, whose alues se ed as a e e ence. 31
animals we e pa o he s udy. Fo 3 mon hs, hey we e on
an o iginal die o ou g oup specially designed o p oduce
s ea osis, a he end o which, hey unde wen an open
biopsy ( o ob ain sufficien samples). A e he su gical
in e en ion, he animals we e assigned o 4 s udy g oups:
g oup 1 (N�12): animals ha main ained a s ea o ic die o
1 mo e mon h and ecei ed concomi an ea men du ing
2Canadian Jou nal o Gas oen e ology and Hepa ology
his mon h wi h mela onin (10 mg/kg/day); g oup 2 (N�9):
animals ha main ained a s ea o ic die o one mo e mon h
and ecei ed no d ug; g oup 3 (N�5): animals ha did no
con inue wi h he s ea o ic die bu ecei ed mela onin
ea men o one mon h a he same dose; g oup 4 (N�5):
animals ha did no con inue wi h he s ea o ic die and did
no ecei e any d ug. A e his ime, he animals unde wen
a second su gical in e en ion o ob ain he same samples o
li e issue, blood, and se um. A e his second ope a ion,
he animals we e slaugh e ed.
In each su gical in e en ion, a minilapa o omy was
pe o med o obse e he mac oscopic cha ac e is ics o he
li e and o ake a sample o hepa ic issue om segmen IV.
This sample was di ided in o wo pa s: one o hem was kep
in o maldehyde o ana omopa hological s udy. Ano he
sample was ozen and p ese ed in liquid ni ogen o ob ain
oxida i e s ess pa ame e s: MDA and ca bonyls. Du ing he
in e en ion, a sample o 20 ml o enous blood ex ac ed om
he po al ein o he ena ca a was also ob ained. The blood
was cen i uged a 3000 pm o 3 min o ob ain se um.
Biochemical pa ame e s we e ob ained om he blood sample.
2.3. Induc ion o Hepa ic S ea osis. In he expe imen al
po cine model, hepa ic s ea osis can be induced by means o
a specially de eloped and p o en die ha esembles he
induc ion o s ea osis in humans. This is wi hou he need o
use d ugs o oxic p oduc s. The induc ion o s ea osis was
ca ied ou h ough a special die whose main cha ac e is ics
we e as ollows:
(i) High sa u a ed a con en (25%)
(ii) Deficien in me hionine (<1/4 o he needs) and
choline (1/6 o he needs)
(iii) Supplemen ed wi h 2% choles e ol and 0.5% so-
dium chola e.The ollowing able shows i s
composi ion
2.4. His ological Analysis o Li e Biopsy. To de e mine he
deg ee o hepa ic s ea osis, we used wo me hods. Me hod 1 is
a con en ional semiquan i a i e analysis in which he pa-
hologis es ima es he pe cen age o s ea osis unde di ec
ision (HEX ×1000) o each sample exp essed in %. Me hod 2
is a me hod o quan ifica ion ia compu e -assis ed analysis
consis ing o an es ima e o he a ea o compu e -assis ed
s ea osis o samples ob ained and p e iously selec ed.
The p og am measu es he deg ee o s ea osis by ana-
lyzing onali ies om pho og aphs aken o each sample o
hepa ic issue, wi hou a eas occupied by essels and o he
s uc u es. We use a specific so wa e assis ed by Ma lab®,
c ea ed by he esea ch g oup i sel o pe o m a digi al
analysis o he biopsy samples. I has h ee undamen al
unc ions:
(i) Cal-es ea.m: i is he main unc ion ha pe o ms
he calcula ion o s ea osis
(ii) Fil e -m: i is he unc ion o he g ay fil e , which
allows he p og am o be adap ed o he wide ange
o shades ha can be p esen ed o us in he sample
(iii) Region-m: i is he unc ion ha elimina es egions
ha do no co espond o a ( o example, eins
wi h whi e ones)
2.5. Measu emen o Oxida i e S ess Pa ame e s: MDA and
Ca bonyls. The concen a ion o MDA + 4-hyd oxyalkenals
(4-HDA) was de e mined by a colou ime ic me hod based
on he eac ion o a ch omogenic eagen , N-me hyl-2-
phenylindole, wi h MDA o wi h 4-HDA, a a empe a u e
o 45°C. The condensa ion o one MDA o 4-HDA molecule
wi h wo molecules o N-me hyl-2-phenylindole p oduces a
s able ch omopho e which, in he p esence o me h-
anesul onic acid, has a maximum abso bance a 586 nm [30].
In o de o e alua e he oxida i e damage o he p o eins
o he homogenized ones, he de e mina ion o he ca bonyl
emains o he p o eins was used. This me hod is based on
he eac ion o he ca bonyl emains o p o eins wi h
dini ophenylhyd azine (DNPH), o ming a de i a i e ha
is quan ified by measu ing i s abso bance in he ange
360–390 nm. Du ing he p ocedu e, ichlo oace ic acid
(TCA) is used o he p ecipi a ion o p o eins, washes o
he emo al o excess DNPH ha has no eac ed wi h
ca bonyl esidues, and guanidine o edissol e he p o eins
in o de o acili a e spec opho ome e eadings.
F om he abso bance ob ained, he concen a ion o
ca bonyl moie ies was calcula ed using he Bee –Lambe
law and he mola abso p ion coefficien o DNPH
(Ɛ�22,000 M-1 ×cm
−1
). Finally, a e he de e mina ion o
he o al p o eins, he esul s we e exp essed in nmol o
ca bonyl moie ies/mg o o al p o eins.
The labo a o y equipmen included a spec opho ome e
wi h disposable plas ic cu e es o measu e he isible ange
and qua z cu e es o measu e in he ul a iole (UV) ange.
2.6. Measu emen o Biochemical Pa ame e s. The mea-
su emen o se ological pa ame e s o hepa ic unc ion
(aspa a e amino ans e ase (AST), alanine amino ans e -
ase (ALT), gamma-glu amyl ans e ase (GGT), alkaline
phospha ase (FA), and bili ubin (B)) and plasma lipid
concen a ion ( o al choles e ol, HDL choles e ol, and LDL
choles e ol) was pe o med by au oma ic p ocesso s o
au oma ic eading o concen a ions o li e lipids and
enzymes.
2.7. Va iables o Be S udied. The ollowing a iables we e
s udied: he weigh o he animal, s ea osis eached, blood
lipids ( iglyce ides (TG), o al choles e ol, HDL choles e ol,
and LDL choles e ol), hepa ic enzymes (aspa a e amino-
ans e ase (AST), alanine amino ans e ase (ALT), gamma-
glu amyl ans e ase (GGT), alkaline-FA phospha ase, and
bili ubin), pa ame e s o oxida i e s ess (MDA and ca -
bonyls), and his ology (deg ee o a infil a ion).
2.8. S a is ic Analysis. To analyze he ela ionship be ween
he a iables o a s udy, a bi a ia e analysis is ca ied ou . To
s udy he linea ela ionship be ween wo quan i a i e
a iables, he Pea son o Spea man co ela ion coefficien is
Canadian Jou nal o Gas oen e ology and Hepa ology 3
used. The co ela ion coefficien can ake alues be ween −1
and 1, indica ing he ze o co ela ion be ween he a iables
unde s udy. To e alua e he diffe ences be ween he fi s
biopsy o p e and he second biopsy o pos , in ela ion o he
quan i a i e a iables o he s udy, means compa ison
me hods a e used o ela ed samples. Wilcoxon is used
when he a iable does no ollow he no mal dis ibu ion,
and S uden ’s - es is used when he e is no mali y.
To quan i y he diffe ence be ween bo h ime pe iods, we
calcula e he pe cen age o change. Posi i e esul s will in-
dica e inc eased alues in he pos biopsy and nega i e esul s
will indica e dec eased alues.
3. Resul s
3.1. S ea osis Reached. The o al o 31 (100%) animals in he
s udy g oup p esen ed significan hepa ic s ea osis in he fi s
biopsy, mode a e (s ea osis 30–59%) and se e e (s ea osis
>60%) a e being on a special die o 3 mon hs, bo h in he
measu emen made by he pa hologis and in he mea-
su emen made by he compu e p og am.
Figu e 1 shows he a e age deg ee o s ea osis o he
animals o each o he animals acco ding o pa hologis
analysis and digi al image analysis. The e a e no s a is ically
significan diffe ences in he a e age s ea osis eached pe
g oup.
I is obse ed ha he deg ee o s ea osis acco ding o he
digi al analysis p o ides in all cases a e age alues lowe
han hose p o ided acco ding o an analysis by a pa hol-
ogis . On a e age, he deg ee o s ea osis acco ding o a
pa hologis sco es 40.68 (DE �14.66) mo e han ha
acco ding o he digi al analysis a he p emomen .
Figu e 2 shows he op ical mic oscopy image o he
animal wi h he highes pe cen age o s ea osis being seen by
a pa hologis (95% o s ea osis) and he op ical mic oscopy
image o he animal wi h he lowes deg ee o s ea osis being
seen unde di ec ision by a pa hologis (40% o s ea osis).
3.2. Weigh . In all g oups, he e was an inc ease in a e age
weigh be ween he wo ime pe iods e alua ed, as shown in
Figu e 3, his inc ease being s a is ically significan in g oups
1, 3, and 4 (p�0.03, p�0.043, and p�0.043, espec i ely).
In all g oups, he pe cen age change (% diffe ence in
weigh a he end o he s udy wi h espec o he ini ial
weigh ) was s udied, calcula ed as ollows:
% weigh change �pos weigh −p eweigh
p eweigh .(1)
3.3. Biochemical Va iables: T iglyce ides, To al Choles e ol,
HDL, LDL, AST, ALT, GGT, and FA. Figu es 4–7 show he
mean p e alue and pos alue o each o he pa ame e s
e alua ed o each o he s udy g oups.
3.3.1. G oup 1. In g oup 1 ( he g oup ha con inued wi h
he s ea o ic die and was ea ed wi h mela onin), no pa-
ame e shows s a is ically significan diffe ences (p>0.05)
be o e and a e ea men .
3.3.2. G oup 2. In g oup 2 ( he g oup ha con inued wi h
he s ea o ic die and was no ea ed wi h mela onin), he
a e age AST dec eased significan ly (p�0.008), going om
75.11 UI/L (DE �47.29) o 32.67 UI/L (DE �13.93); he e is
also a significan dec ease (p�0.017) o he a e age B going
om 0.73 UI/L (DE �0.83) o 0.22 (DE �0.26).
3.3.3. G oup 3. In g oup 3 ( he g oup ha discon inued he
die and was ea ed wi h mela onin), he a e age alues o
o al choles e ol, HDL, LDL, GGT, and FA dec eased a he
end o he s udy pe iod in a s a is ically significan way,
compa ed wi h he alues a he beginning (p�0.043 in all
cases). Howe e , we obse e a significan inc ease in ALT
and AST (p�0.043 and p�0.042 espec i ely).
3.3.4. G oup 4. In g oup 4 ( he g oup ha discon inued
he s ea o ic die and was no ea ed wi h mela onin), he
a e age alues o TG, o al choles e ol, HDL, LDL, AST,
GGT, and FA also dec eased a he end o he s udy
pe iod, p esen ing significan diffe ences be ween he wo
s udy ime pe iods (p e and pos ) (p�0.043 in all cases).
Howe e he e is a significan inc ease in ALT
(p�0.042).
3.4. Analysis o Hepa ic Fa Infil a ion. Figu e 8 shows
g aphically he deg ee o a e age s ea osis by g oups in he
wo s udy ime pe iods analyzed by he di ec ision o he
pa hologis .
A dec ease in s ea osis was obse ed in g oups 3 and 4,
and an inc ease in s ea osis in g oups 1 and 2, wi h a sig-
nifican dec ease in g oup 4 (p�0.042), he animals ha
discon inued hei die and we e no ea ed wi h mela onin.
Figu e 9 shows g aphically he deg ee o a e age s ea osis
by g oups in he wo s udy ime pe iods digi ally analyzed by
he image p ocessing so wa e.
The e is a dec ease in s ea osis in g oups 3 and 4, and
he e is an inc ease in s ea osis in g oups 1 and 2. This
inc ease in g oup 2 is s a is ically significan (p�0.08), as is
he dec ease in g oup 4 (p�0.043).
The deg ee o s ea osis measu ed by he p og am
p o ides lowe a e age alues in ela ion o he deg ee o
s ea osis measu ed by he pa hologis . On a e age, he
deg ee o s ea osis measu ed by he di ec ision o he
pa hologis [31] sco es 40.68% mo e in he p emomen and
36.87% mo e in he pos momen . In Figu e 10, i is possible
o obse e, in a g aphical way, he a e age alues o he
deg ee o s ea osis o bo h measu emen s by g oups o
bo h pe iods o s udy (p e and pos ) and how he same
endency is obse ed o he wo me hods o measu emen
employed.
Figu e 11 shows he ela ionship be ween he pe cen age
o change ob ained wi h he p og am and he pe cen age o
change ob ained wi h he pa hologis .
The pe cen age o change o s ea osis be ween he p e-
ime and pos ime o he ollow-up o he subjec s shows a
s ong linea ela ionship be ween bo h me hods, ob aining
4Canadian Jou nal o Gas oen e ology and Hepa ology
45.98 47.69
56.7 57.83
48.03 48.01
66 65.7
0
10
20
30
40
50
60
70
80
1234
A e age weigh (kg)
S udy g oups
P e
Pos
∗
p = 0.03
∗
p = 0.043
∗
p = 0.043
Figu e 3: A e age p e-pos weigh (kg) by s udy g oups. G oup 1: animals ha main ained a s ea o ic die and ecei ed concomi an
ea men . G oup 2: animals ha main ained a s ea o ic die and ecei ed no d ug. G oup 3: animals ha did no con inue wi h he s ea o ic
die bu ecei ed mela onin ea men . G oup 4: animals ha did no con inue wi h he s ea o ic die and did no ecei e any d ug.
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
Pe cen age o s ea osis achie ed (%)
S udy animals
P epa hologis
P ep og am
Figu e 1: A e age s ea osis eached by each o he animals, acco ding o he pa hologis and p og am.
(a) (b)
Figu e 2: Op ical mic oscopy image, HE 2x dyeing wi h 95% s ea osis (a) and op ical mic oscopy image, HE 2x dyeing wi h 40%
s ea osis (b).
Canadian Jou nal o Gas oen e ology and Hepa ology 5

a co ela ion coefficien o 0.767 wi h a le el o significance
being lowe han 0.001.
3.5.MDA. Figu e 12 shows g aphically he deg ee o a e age
MDA by g oups in he wo s udy pe iods.
In all s udy g oups, he e was a dec ease in he a e age
alue o MDA, being s a is ically significan in g oup 1
(p�0.04), he subjec s ha con inued he s ea o ic die and
we e gi en mela onin.
3.6. Ca bonyls. Figu e 13 shows g aphically he a e age
ca bonyl deg ee by g oups in he wo s udy pe iods.
In g oups 2, 3, and 4, he e is an a e age inc ease o
ca bonyls, being only significan in g oup 4 (p�0.028). The
small a e age dec ease in g oup 1 (subjec s ha con inue on
a die and a e ea ed wi h mela onin) does no ha e s a-
is ical significance, as in he case o MDA.
4. Discussion
NAFLD is one o he mos p e alen hepa ic p ocesses in he
Wes e n socie y and one o he majo causes o li e ailu e
in he Wes e n wo ld. Usually associa ed wi h, bu no
exclusi ely o, obesi y and he me abolic synd ome, i could
lead o a eal epidemic in he u u e. Nume ous au ho s ha e
shown ha he educ ion o excess weigh and he conse-
quen esis ance o insulin can es o e li e physiology and
his ology [8,9]. Bu a he p esen ime, he e is no effec i e
pha macological ea men o NAFLD ha is no linked o a
li es yle change, including weigh loss. Many d ugs, an i-
oxidan s, hepa op o ec o s, i amin complexes, ee adical
sca enge s, e c. ha e been es ed wi h he idea o ei he
a oiding he accumula ion o lipids in he hepa ocy e o
ex ac ing lipids om cells, showing beneficial effec s.
Al hough mo e s udies a e needed o define hei use-
ulness in NAFLD [32–37], he disco e y o a d ug wi h hese
p ope ies could change he spec um and impac o he
disease.
Mela onin, he uni e sal ho mone ha is p oduced in
diffe en concen a ions, in almos all ou ana omy, has been
s udied as one o he possible solu ions o he al e a ion o
lipid me abolism and a deposi ion in he hepa ocy e [12].
Expe imen al s udies ha e shown an ac ion o mela onin on
a me abolism, which can be summa ized as a dec ease in
lipid pe oxida ion and a dec ease in oxida i e s ess [13],
biochemical phenomena in ima ely ela ed o he accumu-
la ion o lipids in he hepa ocy e. O he effec s ha ha e been
a ibu ed o mela onin a e as ollows: s imula ion o an i-
oxidan enzymes in hepa ocy es, egula ion o an ioxidan
enzyme ansc ip ion genes, sca enging o oxygen ee
27.3
252.58
66
186.58
28
225.83
73.08
152.75
–50
0
50
100
150
200
250
300
350
TG COL HDL LDL
MG/DL
P e
Pos
P e
Pos
0.34
58.33
17.75
56.58
200.25
0.26
41.67
17.42
53.42
162.08
–50
0
50
100
150
200
250
B AST ALT GGT FA
UI/L
Figu e 4: A e age alues o he p e-pos pa ame e s o g oup 1.
TG � iglyce ides, COL � o al choles e ol, HDL �HDL choles-
e ol, LDL �LDL choles e ol, B �bili ubin, AST �aspa a e ami-
no ans e ase, ALT �alanine amino ans e ase, GGT �gamma-
glu amyl ans e ase, FA �alkaline phospha ase.
30.22
186.87
65.89
120.78
28.67
158.78
49.78
136
–50
0
50
100
150
200
250
TG COL HDL LDL
MG/DL
0.73
75.11
16
52.44
212.78
0.22
32.67 11.44
49.89
232.56
–100
–50
0
50
100
150
200
250
300
B AST ALT GGT FA
UI/L
∗
p = 0.017
∗
p = 0.08
P e
Pos
P e
Pos
Figu e 5: A e age alues o he p e-pos pa ame e s o g oup 2.
TG � iglyce ides, COL � o al choles e ol, HDL �HDL choles-
e ol, LDL �LDL choles e ol, B �bili ubin, AST �aspa a e ami-
no ans e ase, ALT �alanine amino ans e ase, GGT �gamma-
glu amyl ans e ase, FA �alkaline phospha ase.
6Canadian Jou nal o Gas oen e ology and Hepa ology
adicals, s imula ion o glu a hione syn hesis, inc ease in he
ac i i y o o he an ioxidan molecules, dec ease in he
gene a ion o ee adicals in mi ochond ia, dec ease in he
exp ession o p o eins ha ha e an effec on he accumu-
la ion o lipids in he hepa ocy e cy oplasm, p o ec ion o cell
memb anes agains lipid pe oxida ion, and educ ion o
p oinflamma o y ac o s esponsible o he p og ession o
NAFLD o NASH.
Se e al expe imen al models ha e been desc ibed o
esemble human NAFLD. The ideal model would be one ha
eflec s bo h he his ology and he physiopa hology o he
disease in i s diffe en s ages. O cou se, his model should be
ep oducible, eliable, simple, p edic able, and economical.
Mos o hese models ha e been desc ibed in oden s. The e
a e ew models o hepa ic s ea osis in la ge animals, and in
mos o hem, li e damage has been induced by he ad-
minis a ion o oxins such as alcohol [38,39].
In 2009, Lee e al. desc ibed, o he fi s ime, a NASH
model in la ge animals p oduced by die a y manipula ion
using Ossabaw minia u e pigs [40]. This model is no e y
use ul due o he es ic ion o he use o hese animals. In
ou wo k, we used an expe imen al model, he pig Whi e-
Land ace, in which we ep oduced s ea osis. This model is
o iginal and sufficien ly p o en in p e ious s udies o ou
g oup and ha has he pa icula i y ha only by means o
die e ic manipula ion, wi hou using d ugs o oxics, con-
olled and e y high deg ees o s ea osis a e achie ed. We
ha e no ound any model published hus a ha induces,
wi hou he use o subs ances ha m ul o he li e o an
agg essi e die (excess o polyunsa u a ed, uc ose a s, o
he defici o elemen s necessa y o be a-oxida ion such as
choline and me hionine), a deg ee o mac o esicula s ea-
osis simila o ou s, in such a sho ime and wi h minimal
epe cussions on he physiology o he animal. Ano he
s eng h o ou expe imen al model is ha i allows us o
check li e damage and quan i y s ea osis by pe o ming
li e biopsies be o e and a e he de elopmen o s ea osis;
in many mu ine models, his is no possible, and in humans,
no au ho pe o ms hem.
All s ea ogenic die s desc ibed induce an inc ease in he
weigh o he animal. In ou s udy, all animals inc eased in
weigh a e he in oduc ion o he s ea ogenic die . The da a
is consis en wi h he da a ound in he li e a u e (Ossabaw
pigs). The effec s o mela onin we e null on he weigh o he
animal.
In o de o explo e he effec on he lipid me abolism o
mela onin, we s udied lipid pe oxida ion (LPO) on he
polyunsa u a ed a y acids o he hepa ocy e (du ing he
p ocess o accumula ion o lipids in he hepa ocy e, he
ac ion o ee adicals on he lipids akes place mainly on he
polyunsa u a ed a y acids o cellula samples, causing hei
30.2
341.4
47.4
294
23
77.6
24.8 52.8
–100
0
100
200
300
400
500
TG COL HDL LDL
MG/DL
0.24
33
14
55.2
193.6
0.16
55.8
34 43.8
103
–50
0
50
100
150
200
250
B AST ALT GGT FA
UI/L
∗
p = 0.043
∗
p = 0.043
∗
p = 0.043
∗
p = 0.043
∗
p = 0.042
∗
p = 0.043
∗
p = 0.043
P e
Pos
P e
Pos
Figu e 6: A e age alues o he p e-pos pa ame e s o g oup 3.
TG � iglyce ides, COL � o al choles e ol, HDL �HDL choles-
e ol, LDL �LDL choles e ol, B �bili ubin, AST �aspa a e ami-
no ans e ase, ALT �alanine amino ans e ase, GGT �gamma-
glu amyl ans e ase, FA �alkaline phospha ase.
59.2
389.6
39
350.6
26
82.4
25.8 56.6
–100
0
100
200
300
400
500
600
TG COL HDL LDL
MG/DL
∗
p = 0.043
0.2
65
14
49.4
232
0.18
39.8 34.4 36.8
128.6
–100
–50
0
50
100
150
200
250
300
B AST ALT GGT FA
UI/L
∗
p = 0.043
∗
p = 0.043
∗
p = 0.043
∗
p = 0.043
∗
p = 0.042
∗
p = 0.043
∗
p = 0.043
P e
Pos
P e
Pos
Figu e 7: A e age alues o he p e-pos pa ame e s o g oup 4.
TG � iglyce ides, COL � o al choles e ol, HDL �HDL choles-
e ol, LDL �LDL choles e ol, B �bili ubin, AST �aspa a e ami-
no ans e ase, ALT �alanine amino ans e ase, GGT �gamma-
glu amyl ans e ase, FA �alkaline phospha ase.
Canadian Jou nal o Gas oen e ology and Hepa ology 7
pe oxida ion). The final p oduc s o his p ocess o LPO a e
aldehydes, hyd oca bon gases, and a ious chemical esi-
dues, wi h MDA and 4-HDA being he majo i y. The e o e,
he concen a ion o MDA+4-HDA is an indica o o he
deg ee o pe oxida ion o he lipids o he biological
memb anes [41].
81.25
75.83
1.57
82.5
83.75
87.22
1.25
0.97
0
0.5
1
1.5
2
2.5
1234
A e age s ea osis (%)
S udy g oups
∗
p = 0.042
P e
Pos
Figu e 8: A e age p e-pos s ea osis by s udy g oups. G oup 1: animals ha main ained a s ea o ic die and ecei ed concomi an ea men .
G oup 2: animals ha main ained a s ea o ic die and ecei ed no d ug. G oup 3: animals ha did no con inue wi h he s ea o ic die bu
ecei ed mela onin ea men . G oup 4: animals ha did no con inue wi h he s ea o ic die and did no ecei e any d ug.
41.1
1.88
33.6
39.8
44.25
41.44
13.2
10.4
0
0.5
1
1.5
2
2.5
1234
A e age s ea osis p og am (%)
S udy g oups
∗
p = 0.08
∗
p = 0.043
P e
Pos
Figu e 9: Pe cen age o a e age p e-pos s ea osis (p og am) by s udy g oups. G oup 1: animals ha main ained a s ea o ic die and ecei ed
concomi an ea men . G oup 2: animals ha main ained a s ea o ic die and ecei ed no d ug. G oup 3: animals ha did no con inue wi h he
s ea o ic die bu ecei ed mela onin ea men . G oup 4: animals ha did no con inue wi h he s ea o ic die and did no ecei e any d ug.
041
030 034
040
081 076 074
083
0
20
40
60
80
100
1234
A e age s ea osis
S udy g oups
P ep og am
Pos p og am
P e
Pos
Figu e 10: Pe cen age o a e age p e-pos s ea osis (p og am) by s udy g oups. G oup 1: animals ha main ained a s ea o ic die and
ecei ed concomi an ea men . G oup 2: animals ha main ained a s ea o ic die and ecei ed no d ug. G oup 3: animals ha did no
con inue wi h he s ea o ic die bu ecei ed mela onin ea men . G oup 4: animals ha did no con inue wi h he s ea o ic die and did no
ecei e any d ug.
8Canadian Jou nal o Gas oen e ology and Hepa ology
In ou s udy, we ound ha bo h MDA and ca bonyls
dec eased in he g oups ha we e adminis e ed mela onin
wi h espec o hose who did no ecei e i ; he e o e, his
indica es an effec o he ho mone on lipid me abolism a he
in acellula le el.
Plasma le els o choles e ol and iglyce ides inc eased in
animals subjec ed o s ea ogenic die bu we e no modified
by he effec o mela onin wi h s a is ical significance.
Howe e , we can obse e ha animals ha con inue wi h he
die and ake mela onin (g oup 1) exhibi lowe ed LDL and
% change s ea osis pa hologis
% change s ea osis p og am
–100 100–50 500
100
–100
50
–50
0
Figu e 11: Sca e diag am. % change s ea osis p og am−% change s ea osis pa hologis .
2.13
1.88
1.57
1.18
1.27
1.64
1.25
0.97
0
0.5
1
1.5
2
2.5
1234
MDA (NMOL MDA/MG p o eins)
S udy g oups
∗
p = 0.04
P e
Pos
Figu e 12: A e age p e-pos MDA by s udy g oups. G oup 1: animals ha main ained a s ea o ic die and ecei ed concomi an ea men .
G oup 2: animals ha main ained a s ea o ic die and ecei ed no d ug. G oup 3: animals ha did no con inue wi h he s ea o ic die bu
ecei ed mela onin ea men . G oup 4: animals ha did no con inue wi h he s ea o ic die and did no ecei e any d ug.
8.64
8.26
4.89
4.99
7.22
9.2
5.3
6.69
0
0.5
1
1.5
2
2.5
1234
Ca bonyls (NMOL ca bonyls/mg p o eins)
S udy g oups
∗
p = 0.028
P e
Pos
Figu e 13: Medium p e-pos ca bonyls by s udy g oups. G oup 1: animals ha main ained a s ea o ic die and ecei ed concomi an
ea men . G oup 2: animals ha main ained a s ea o ic die and ecei ed no d ug. G oup 3: animals ha did no con inue wi h he s ea o ic
die bu ecei ed mela onin ea men . G oup 4: animals ha did no con inue wi h he s ea o ic die and did no ecei e any d ug.
Canadian Jou nal o Gas oen e ology and Hepa ology 9