Melatonin attenuates inflammation and promotes regeneration in rabbits with fulminant hepatitis of viral origin.
ABSTRACT The objective of the present study was to investigate the effect of melatonin on the liver inflammatory and regenerative response in an animal model of fulminant hepatic failure (FHF) of viral origin. Rabbits were experimentally infected with 2 × 10(4) hemagglutination units of a rabbit hemorrhagic disease virus (RHDV) isolate and received melatonin at two concentrations of 10 or 20 mg/kg at 0, 12 and 24 hr postinfection. RHDV infection induced an inflammatory response, with increased expression of toll-like receptor 4, high-mobility group box (HMGB)1, interleukin (IL)-1β, IL-6, tumor necrosis factor-α, and C-reactive protein, and decreased expression of decay accelerating factor (DAF/CD55). These effects were significantly reduced by melatonin. Matrix metalloproteinase-9 expression was also lowered in melatonin-treated rabbits. RHDV infection inhibited the hepatic regenerative/proliferative response, with a reduced expression of hepatocyte growth factor (HGF), epidermal growth factor, platelet-derived growth factor (PDGF)-B and vascular endothelial growth factor and their receptors; these responses were prevented by melatonin administration. Melatonin treatment also resulted in reduced expression of phosphorylated Janus kinase and enhanced expression of extracellular mitogen-activated protein kinase (ERK) and signal transducer and activator of transcription (STAT) 3. Our findings show that anti-inflammatory effects and stimulation of regenerative mechanisms contribute to the beneficial effects of melatonin in rabbits with experimental infection by RHDV and support a potential hepatoprotective role of melatonin in FHF.
- [show abstract] [hide abstract]
ABSTRACT: Fulminant hepatic failure (FHF) is a severe, life-threatening disorder. Previous studies have suggested that intravenous prostaglandin treatment may improve survival in FHF. The present study was performed to further investigate the possible benefit of intravenous prostaglandin E1 (PGE1) for patients with FHF. A total of 18 patients, all excluded as candidates for hepatic transplantation, were studied. Thirteen of 18 participated in a randomized, double-blind, placebo-controlled trial. PGE1 was administered by continuous infusion at a dose of 10 to 40 microg/h as tolerated. After 48 hours of blinded treatment, 3 of 7 patients randomized to placebo were converted to open-label PGE1 for lack of biochemical and/or clinical improvement. Mean values for alanine transaminase, aspartate transaminase, total bilirubin, prothrombin time, factor V percent, factor VII percent, hepatic encephalopathy score, days from onset of symptoms to initiation of treatment, and cause of FHF were similar between treatment groups. Ten of 18 patients (55%) enrolled in this trial survived. However, survival was not different between PGE1-(60%) and placebo (50%) treated patients. The greatest predictor of survival was the number of days from onset of symptoms to hospitalization, which was significantly (P = .002) shorter for survivors (3.3 v 12.4 days), regardless of PGE1 treatment. Six of 8 patients (75%) who began PGE1 therapy and 4 of 5 placebo-treated patients (80%) hospitalized within 10 days of onset of symptoms survived. By contrast, all 5 patients who were hospitalized and subsequently began PGE1 treatment 10 days or longer after the onset of symptoms died. We conclude that early recognition and hospitalization is the most important factor in reduction of mortality from FHF. It is unclear whether PGE1 treatment is beneficial when administered during this period. However, it is apparent that PGE1 was not effective for treatment of FHF if treatment started more than 10 days after onset of this clinical syndrome.Liver transplantation and surgery: official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society 10/1998; 4(5):424-31.
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ABSTRACT: The influence of acetylcysteine, administered at presentation to hospital, on the subsequent clinical course of 100 patients who developed paracetamol-induced fulminant hepatic failure was analysed retrospectively. Mortality was 37% in patients who received acetylcysteine 10-36 h after the overdose, compared with 58% in patients not given the antidote. In patients given acetylcysteine, progression to grade III/IV coma was significantly less common than in those who did not receive the antidote (51% vs 75%), although the median peak prothrombin time was similar for both groups. Whether the beneficial effect is related to replenishment of glutathione stores or a consequence of another hepatic protective mechanism of acetylcysteine requires further study.The Lancet 01/1990; 335(8705):1572-3. · 39.06 Impact Factor
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ABSTRACT: In this study we sought to characterize a novel model of fulminant liver failure (FLF) by means of experimental infection of rabbits with the rabbit hemorrhagic disease virus (RHDV). Thirty-seven 9-week-old rabbits were injected intramuscularly with 2 x 10(4) hemagglutination units of an RHDV isolate. Eighty-five percent of rabbits died 36 to 54 hours after infection. From 36 hours after infection we noted marked increases in transaminases, lactate dehydrogenase, and total bilirubin. The rabbits exhibited hypoglycemia and coagulation abnormalities, with a significant decrease in factor V, factor VII, and prothrombin. Plasma aromatic amino acids and taurine showed progressive increases, and the Fischer index was significantly reduced. Expression of hepatocyte growth factor messenger RNA was inhibited from 36 hours after infection. Prostration and side recumbency were present at later stages, and neurologic symptoms rapidly progressed to coma. Onset of brain death was associated with a significant increase in intracranial pressure and blood ammonia. RHDV infection reproduces clinical, biochemical, and histologic features of the FLF syndrome and satisfies criteria for a suitable animal model. Rabbit hemorrhagic viral disease could provide a useful tool for the study of FLF and the evaluation of new liver-support technologies in human subjects.Journal of Laboratory and Clinical Medicine 05/2003; 141(4):272-8. · 2.62 Impact Factor
Melatonin attenuates inflammation and promotes regeneration in
rabbits with fulminant hepatitis of viral origin
Fulminant hepatic failure (FHF) is a life-threatening
clinical syndrome that results from severe impairment of
liver function induced by drugs, toxins or viral hepatitis.
Because FHF is associated with a high risk of lethal
results and orthotopic liver transplantation is not always
available in a timely fashion, there is an urgent need of
medical treatments for FHF patients . The mechanisms
responsible for liver injury in acute severe viral hepatitis
leading to FHF are complex and include the virus
cytopathic effect and a vigorous inflammatory response,
resulting in extensive hepatocellular cell damage that
surpasses the liver capacity to regenerate . Many
intracellular signaling pathways have been implicated in
regulating liver damage and regeneration . However,
although some therapeutic approaches to FHF have tried
to block the inflammatory response and to enhance
proliferative pathways [4, 5], pathogenic therapies able to
attenuate liver cell damage and stimulate regeneration are
Melatonin (N-acetyl-5-methoxytryptamine) plays a fun-
damental role in the neuro-immune-endocrine system, but
is also a potent antioxidant with therapeutic potential in
transcription factors, and
numerous cell and animal models of liver injury, such as
hepatic ischemia-reperfusion injury [6, 7], fatty liver disease
[8, 9], hepatocarcinoma [10, 11], or FHF [12, 13]. The rabbit
hemorrhagic disease virus (RHDV) infection is a unique
model that closely reproduces the histopathological, bio-
chemical, and clinical manifestations of human fulminant
viral hepatitis . In this model, we have shown the
presence of molecular mechanisms contributing to liver
damage and lack of regeneration [15, 16], which further
supports its usefulness to investigate novel therapeutical
modalities in FHF . Previous research from our group
has suggested a potential hepatoprotective role of melato-
nin in rabbits infected by the RHDV, partially mediated
through the abrogation of oxidative stress and the preven-
tion of the decreased activity of antioxidant enzymes via
Nrf2-dependent pathways , and through the inhibition
of apoptotic mechanisms . Although it has been recently
shown that melatonin reduces inflammation and modulates
regeneration in muscle  or new bone around implants
, it is unknown whether these effects may contribute
to amelioration of liver damage in FHF. In the present
investigation, we utilized the RHDV model of FHF to
evaluate the potential anti-inflammatory and regenera-
tive effects of melatonin. Our findings indicate that mela-
tonin inhibits different proinflammatory mechanisms and
Abstract: The objective of the present study was to investigate the effect of
melatonin on the liver inflammatory and regenerative response in an animal
model of fulminant hepatic failure (FHF) of viral origin. Rabbits were
experimentally infected with 2 · 104hemagglutination units of a rabbit
hemorrhagic disease virus (RHDV) isolate and received melatonin at two
concentrations of 10 or 20 mg/kg at 0, 12 and 24 hr postinfection. RHDV
infection induced an inflammatory response, with increased expression of
toll-like receptor 4, high-mobility group box (HMGB)1, interleukin (IL)-1b,
IL-6, tumor necrosis factor-a, and C-reactive protein, and decreased
expression of decay accelerating factor (DAF/CD55). These effects were
significantly reduced by melatonin. Matrix metalloproteinase-9 expression
was also lowered in melatonin-treated rabbits. RHDV infection inhibited the
hepatic regenerative/proliferative response, with a reduced expression of
hepatocyte growth factor (HGF), epidermal growth factor, platelet-derived
growth factor (PDGF)-B and vascular endothelial growth factor and their
receptors; these responses were prevented by melatonin administration.
Melatonin treatment also resulted in reduced expression of phosphorylated
Janus kinase and enhanced expression of extracellular mitogen-activated
protein kinase (ERK) and signal transducer and activator of transcription
(STAT) 3. Our findings show that anti-inflammatory effects and stimulation
of regenerative mechanisms contribute to the beneficial effects of melatonin
in rabbits with experimental infection by RHDV and support a potential
hepatoprotective role of melatonin in FHF.
Almudena Laliena1, Beatriz San
Miguel1,2, Irene Crespo1,2,
Marcelino Alvarez3, Javier
Gonza ´lez-Gallego1,2and Marı ´a
J. Tun ˜o ´n1,2
1Institute of Biomedicine (IBIOMED),
University of Leo ´n, Leo ´n;2Centro de
Investigacio ´n Biome ´dica en Red de
Enfermedades Hepa ´ticas y Digestivas
(CIBEREHD);3Department of Animal Health,
University of Leo ´n, Leo ´n, Spain
Key words: fulminant hepatic failure,
inflammation, melatonin, regeneration
Address reprint requests to Marı ´a Jesu ´s
Tun ˜o ´n Gonza ´lez, Institute of Biomedicine,
University of Leo ´n, 24071-Leo ´n, Spain.
Received January 16, 2012;
Accepted March 12, 2012.
J. Pineal Res. 2012; 53:270–278
? 2012 John Wiley & Sons A/S
Journal of Pineal Research
Molecular,Biological,Physiological and Clinical Aspects of Melatonin
enhances regenerative pathways, supporting its potential
therapeutical use in patients with FHF.
Materials and methods
Virus and experimental model
Nine-week-old New Zealand white rabbits were kept in the
animal facility of the University of Leon with 12-hr light
cycle at 21–22?C and 50% relative humidity. They were
given water and standard rabbit dry food ad libitum. The
study protocols were reviewed and approved by the
University of Leon Animal Care Committee. Eighteen
rabbits were injected intramuscularly with 2 · 104hemag-
glutination units of an RHDV isolate [14, 16] at 21 h pm.
At 0, 12 and 24 hr post infection (hpi), twelve animals
received an i.p. injection of melatonin (Sigma, St Louis,
MO, USA) at 10 and 20 mg/kg body weight (Group
RHDV + Mel10 and Group RHDV + Mel20, respec-
tively; n = 6 each). Melatonin was dissolved into absolute
ethanol and further dilutions were made in saline. The final
concentration of ethanol was 5%. Six infected rabbits
received the same volume of vehicle (Group RHDV) and
finally two group of noninfected rabbits (n = 6 each) were
used as healthy controls (Group Control received vehicle
and Group Control + Mel received 20 mg/kg body weight
i.p. of melatonin at 0, 12, and 24 hpi). We have previously
reported that during experimental RHDV infection, bio-
chemical data and expression of genes involved in injury
and regeneration change remarkably at 36–48 hpi [15, 16].
It was thus decided to study the effects of melatonin by
sacrificing two groups of control rabbits (n = 12) and
batches of infected animals (n = 18) at 36 hpi.
Blood samples were collected from the marginal ear vein of
animals in heparin tubes at 36 hpi, for determination of
aspartate aminotransferase (AST), alanine aminotransfer-
ase (ALT), lactate dehydrogenase (LDH), alkaline phos-
phatase (ALP), and c-glutamyltransferase (GGT). Analyses
were carried out in the Hospital of Leon Clinical Chemistry
Laboratory using standard techniques.
Total RNA was obtained using a Trizol reagent (Life
Technologies, Carlsbad, CA, USA) and quantified by the
fluorescent method Ribogreen RNA Quantitation Kit
(Molecular Probes, Leiden, The Netherlands). Residual
genomic DNA was removed by incubating RNA with
RQ1 RNase-free DNase (Promega, Madison, WI, USA).
RNA integrity was confirmed by formaldehyde gel
electrophoresis. cDNA was amplified using TaqMan
Universal PCR MasterMix [primers and probes for inter-
leukin (IL)-1b (GenBank accession no.: NM_001082201
NM_001082064 and Oc04097052_m1), tumor necrosis
factor (TNF)-a (GenBank accession no.: EF_472913 and
Oc03396507_m1), toll-like receptor (TLR) 4 (GenBank
matrix metalloproteinase (MMP)-9 (GenBank accession
no.: NM_001082203 and Oc03397520_m1), C-reactive
protein (CRP) (GenBank accession no.: NM_001082265
and Oc04097105_s1), vascular endothelial growth factor
Oc03395999_m1) and glyceraldehyde-3-phosphate dehy-
001082253 and Oc03823402_g1) (Applied Biosystems,
Foster City, CA, USA)] or QuantiTect SYBR Green PCR
master mix [primers for hepatocyte growth factor (HGF),
c-Met, epidermal growth factor (EGF), platelet-derived
growth factor (PDGF)-B, PDGFR, and high-mobility
group box (HMGB)-1 (Table 1)]. Relative changes in gene
expression levels were determined using the 2?DDCtmethod
. The cycle number at which the transcripts were
detectable (Ct) was normalized to the cycle number of
GAPDH detection, referred to as DCt.
Western blot analysis
Western blot analyses were performed on cytosolic extracts
prepared by liver tissue homogenization in 0.25 mm sucrose,
1 mm EDTA, 10 mm Tris, and a phosphatase and protease
inhibitor cocktail (Roche Diagnostic GmbH, Mannheim,
Germany). The homogenate was centrifuged at 4?C for
30 minat 13,000 g. Thesupernatantfraction wasrecollected
and stored at )80?C in aliquots until use. Protein concen-
tration of the cytosolic liver fractions was measured by the
Bradford assay. Equal amounts of protein (15–30 lg) were
separated by 9–12% sodium dodecyl sulfate (SDS)-poly-
acrylamide gel electrophoresis for 1.5 hr at 100 V and then
blotted on polyvinylidene fluoride membranes (Amersham
Pharmacia, Little Chalfont, UK). The membranes were then
blocked with 5% nonfat dry milk in phosphate-buffered
saline containing 0.05% Tween 20 (PBST) for 1 hr at room
temperature and probed overnight at 4?C with polyclonal
anti-TLR4, decreased expression of decay-accelerating fac-
tor (DAF), (HGF), c-Met (1:200; Santa Cruz Biotechnol-
ogy, Santa Cruz, CA, USA), Janus kinase (JNK), phospho-
JNK, signal transducer and activator of transcription
(STAT)3, phospho-STAT3, extracellular mitogen-activated
protein kinase (ERK), phospho-ERK (1:1000; Cell Signal-
ing Technology, Danvers, MA, USA) antibodies with PBST
containing 3% nonfat dry milk. Equal loading of protein
was demonstrated by probing the membranes with a rabbit
anti-b-actin polyclonal antibody (1:2000; Sigma). After
washing with TBST, the membranes were incubated for
1 hr at room temperature with secondary HRP-conjugated
using ECL detection kit (Amersham Pharmacia, Uppsala,
Sweden) [23, 24]. The density of the specific bands was
quantified with an imaging densitometer (Scion Image,
Maryland, MA, USA).
Results are expressed as mean values ± S.E.M. Data were
compared by analysis of variance (ANOVA); when the
analysis indicated the presence of a significant difference,
the means were compared with the Newman–Keuls
test. Significance was accepted at P < 0.05. Values were
Fulminant hepatic failure, regeneration and melatonin
analyzed using the statistical package Statistica 7.0 (Statsoft
Inc, Tulsa, OK, USA).
Serum transaminases, LDH, ALP, and GGT activities
experienced a striking elevation in RHDV-infected rab-
bits, but levels were significantly lower (versus RHDV
RHDV + Mel10, )53%; RHDV + Mel20, )81%; AST:
RHDV + Mel10,
LDH: RHDV + Mel10, )67%; RHDV + Mel20, )87%;
ALP: RHDV + Mel10, )25%; RHDV + Mel20, )56%;
GGT, RHDV + Mel10, )13%; RHDV + Mel20, )68%)
Activation of TLR4 may ignite cascades of proinflam-
matory mediators, thus aggravating hepatocellular damage
in severe forms of acute liver disease. We found that
melatonin administration significantly dampened the ele-
vation of TLR4 mRNA expression (RHDV + Mel10,
)20%; RHDV + Mel20, )57%) and TLR4 protein con-
centration (RHDV + Mel10, )32%; RHDV + Mel20,
)36%) that occurred in livers from RHDV-infected rabbits
(Fig. 1A,B). Liver mRNA expression of HMGB1, whose
binding to TLR4 mediates its effects in hepatic injury,
enhanced markedly in untreated infected rabbits while it
was maintained at significantly lower values in animals that
received melatonin therapy (RHDV + Mel10, )81%;
RHDV + Mel20, )83%) (Fig. 1A). An opposite effect
was observed for DAF, a molecule involved in modulating
the interaction between TLR signaling and complement.
DAF protein concentration was strongly reduced in RHDV
infection and melatonin administration prevented this effect
(RHDV + Mel10, +196%; RHDV + Mel20, +110%)
(Fig. 1B). High levels of proinflammatory mediators may
RHDV + Mel20,
induce the production of reactive oxygen species, leading to
persistent JNK phosphorylation, which in turn mediates
inflammatory processes by inducing the expression of
inflammatory cytokines. Interestingly, JNK phosphoryla-
tion was prominent in livers from RHDV-infected rabbits,
but was significantly attenuated in animals treated with
melatonin (RHDV + Mel10, )27%; RHDV + Mel20,
)48%) (Fig. 1B).
Proinflammatory cytokines have been shown to play a
key role in acute liver injury of various etiologies. We found
that mRNA levels of IL-6, IL-1b, and TNF-a were strongly
elevated in RHDV-infected livers compared to healthy
controls and that melatonin treatment resulted in a
significant reduction in the mRNA expression of all these
molecules (IL-6: RHDV + Mel10, )52%; RHDV +
Mel20, )71%; IL-1b: RHDV + Mel10, )21%; RHDV +
RHDV + Mel20, )67%) (Fig. 1A). CRP mRNA expres-
sion also increased significantly following experimental
infection, and this effect was partially prevented by mela-
tonin administration (RHDV + Mel10, )63%; RHDV +
Mel20, )62%). Activation of MMPs, particularly MMP-9,
is a critical event in the development of experimental FHF.
We observed a marked hepatic overexpression of MMP-9
in RHDV-infected animals; however, in the liver of RHDV-
infected rabbits that received melatonin, MMP-9 was
RHDV + Mel20, )83%) (Fig. 1A).
Growth factors have a huge potential to elicit liver
regeneration and to improve the management and outcome
of both acute and chronic hepatic disease. In the liver of
RHDV-infected rabbits, we found a marked decrease in the
mRNA expression of HGF and of its receptor c-Met. The
infected rabbits treated with melatonin exhibited higher
hepatic expression of HGF and of c-Met compared with
RHDV + Mel10,
(RHDV + Mel10,
Table 1. RT-PCR primers used in this study
Gene Sense primer (5¢-3¢) Antisense primer (5¢-3¢)
HGF, hepatocyte growth factor; EGF, epidermal growth factor; PDGF, platelet-derived growth factor.
Table 2. Effect of RHDV infection and melatonin treatment on blood biochemistry
ControlControl + MelRHDVRHDV + Mel10RHDV + Mel20
42.6 ± 9.6
33.9 ± 5.3
29.3 ± 1.3
25.9 ± 2.3
5.45 ± 0.85
41.3 ± 3.8
30.5 ± 5.9
28.0 ± 1.0
25.1 ± 1.0
2.60 ± 0.20
1964 ± 489a
6855 ± 2429a
3454 ± 137a
466 ± 26a
29.2 ± 4.2a
931 ± 277a
2771 ± 931a,b
1136 ± 140a
350 ± 13a
25.3 ± 4.9a
378 ± 153a,b,c
939 ± 326a,b,c
436 ± 41a,b,c
202 ± 16a,b
9.27 ± 1.30b,c
ALT, alanine aminotransferase; AST, aspartate aminotransferase; LDH, lactate dehydrogenase; ALP, alkaline phosphatase; GGT, c-
glutamyltransferase; RHDV, rabbit hemorrhagic disease virus. Values are expressed as means ± S.E.M. (n = 6).aP < 0.05, compared
with Control.bP < 0.05, compared with RHDV.cP < 0.05 compared with RHDV + Mel10.
Laliena et al.
Fig. 1. Effects of rabbit hemorrhagic disease virus (RHDV) infection and melatonin treatment on liver inflammation. (A) mRNA expression
of toll-like receptor (TLR)4, HMGB1, IL-6, tumor necrosis factor (TNF)-a, IL-1b, C-reactive protein (CRP), and matrix metalloproteinase
(MMP)-9. (B) Protein concentration of TLR4, decay accelerating factor (DAF), phospho-Janus kinase (JNK), and JNK. Equal loading of
proteins is illustrated by b-actin band. Values are expressed as means ± S.E.M. (n = 6).aP < 0.05, compared with Control.bP < 0.05,
compared with RHDV;cP < 0.05, compared with RHDV + Mel10.
Fulminant hepatic failure, regeneration and melatonin
untreated infected rabbits (HGF: RHDV + Mel10,
51%; RHDV + Mel20, +81%; c-Met: RHDV + Mel10,
+48%; RHDV + Mel20, +67%) (Fig. 2A). Positive
effect of melatonin was further confirmed by the increase
in both HGF and c-Met protein concentration (HGF:
RHDV + Mel10, +51%; RHDV + Mel20, +81%; c-
Met: RHDV + Mel10, +48%; RHDV + Mel20, +67%)
(Fig. 2B). mRNA expression of PDGF-B and PDGFRb in
liver tissue dropped intensely in RHDV-infected rabbits,
while those treated with melatonin showed significantly
(RHDV + Mel10,+59%;
RHDV + Mel10,+9%;
respectively) (Fig. 2A). Furthermore, melatonin therapy
prevented the significant decline in hepatic EGF, EGFR,
VEGF and VEGFR mRNA levels taking place in RHDV
infection (EGF: RHDV + Mel10, +27%; RHDV +
RHDV + Mel20, +75%;
+47%; RHDV + Mel20, 144%; VEGFR: RHDV +
Mel10, +50%; RHDV + Mel20, +217%, respectively)
To further examine the mechanisms underlying melato-
nin protection, we examined by Western blot the phos-
phorylation of ERK1/2 and STAT3, two key signaling
molecules involved in the regulation of hepatocyte cell
proliferation. Densitometric analysis revealed that relative
levels of phospho-ERK1/2 and phospho-STAT3 normal-
ized to that of b-actin were significantly enhanced in rabbits
treated with melatonin (phospho-ERK1/2: RHDV +
Mel10, +35%; RHDV + Mel20,
STAT3: RHDV + Mel10, +188%; RHDV + Mel20,
+204%, respectively) (Fig. 2B).
RHDV + Mel20,
RHDV + Mel20,
RHDV + Mel10,
RHDV + Mel10,
The transmembrane protein TLR4, which exists mainly in
macrophages such as Kupffer cells of the liver, plays an
important role in infectious and inflammatory disease states
. HMGB1 is a nuclear protein with cytokine-type
functions that is passively released during cell injury and
necrosis or actively secreted during immune cell activation
, and previous studies indicate that binding of HMGB1
to TLR4 mediates its effects in hepatic ischemia/reperfusion
and concanavalin A-mediated hepatic injury [27, 28]. Gene
deletion of TLR4 has been shown to palliate liver injury in
experimental models of acute liver damage , and it is
known that treatment with E5564, a TLR4 antagonist,
improves the overall survival rate of rats with d-galactos-
amine and lipopolysaccharide-induced FHF . Data in
the present study confirm that the HMGB1-TLR4 pathway
may contribute to inflammatory damage in RHDV-infected
rabbits, an effect that could be partly mediated by the
activation and nuclear translocation of nuclear factor jB
(NF-jB) previously shown to exist in this animal model of
FHF . Moreover, our results support an anti-inflam-
matory effect of melatonin, as both the liver expression of
HMGB1 and TLR4 were significantly reduced in infected
animals that received melatonin.
Regulation of complement activation is mediated by a
family of complement receptor and regulatory proteins,
including DAF , a molecule that has been recently
shown to reduce the inflammatory injury mediated by both
complement and TLR signaling . Viruses, bacteria and
parasites have developed many efficient strategies to avoid
clearance and destruction by complement. Thus, several
enveloped viruses, such as human immunodeficiency virus,
passively sequester DAF into the envelopes of newly
emerging viruses, thereby inhibiting the host?s ability to
regulate complement . This could also occur in RHDV-
infected rabbits, which show significantly decreased DAF
expression, an effect prevented by the administration of
melatonin. Activation of complement and TLR systems
results in the production of several biologically active
molecules that contribute to inflammation, including cyto-
kines and CRP, which play a key role in acute liver injury of
various etiologies . As expected, we found that mRNA
levels of IL-1b, IL-6, TNF-a and CRP were strongly
elevated in RHDV-infected rabbits. Treatment with mela-
tonin resulted in a significant reduction in the expression of
all these molecules, confirming the anti-inflammatory
action of the hormone.
In addition, we observed that melatonin treatment
elicited a significant reduction in the phosphorylation of
JNK, a MAP kinase that was upregulated in untreated
RHDV-infected animals. JNK mediates inflammatory
processes by inducing the expression of inflammatory
cytokines , and the induction of JNK represents the
strategic effector of cell death in the TNF-a-mediated
signaling pathway . Therefore, JNK is thought to play a
key role in acute hepatic injury. This chain of events
appears to be inhibited by melatonin administration. Our
results are supported by previous research, indicating that
suppression of the JNK pathway contributes to the
beneficial effects of melatonin in hepatic ischemia/reperfu-
sion injury . Activation of MMPs, particularly MMP-9,
is a critical event in the development of experimental FHF,
and it has been recently shown that IL-1-mediated MMP-9
induction plays an essential pathogenic role in models of
FHF by promoting degradation of extracellular matrix,
which leads to apoptosis of hepatocytes and triggers the
influx and activation of polymorphs that enhance tissue
damage . Similar to the previously described downre-
gulation of MMP-9 by cardiotrophin-1 in this animal
model of FHF , we found that melatonin elicited in
RHDV-infected rabbits a reduction in the expression of
MMP-9. In other experimental situations as well, such as
indomethacin- and ethanol-induced gastric ulcer or tran-
sient focal cerebral ischemia , melatonin was shown to
Fulminant hepatic failure arises as result of an extensive
hepatocellular cell injury that damages the liver capacity to
regenerate; however, pathogenic therapies able to stimulate
regeneration are lacking. Our data indicate that melatonin
triggers the expression of growth factors and growth factor
receptors, which have a huge potential to elicit liver
regeneration and to improve the management and outcome
of both acute and chronic liver disease . The HGF/
c-Met signaling pathway is a major survival pathway in
liver that operates during liver development, homeostasis
and regeneration . Liver-specific c-Met and HGF
conditional knock-out mice show an impairment of
Laliena et al.
Fig. 2. Effects of rabbit hemorrhagic disease virus (RHDV) infection and melatonin treatment on liver regeneration. (A) mRNA expression
of hepatocyte growth factor (HGF), c-Met, EGF, PDGF-B, PDGFR, EGF, EGFR, vascular endothelial growth factor (VEGF, and
VEGFR). (B) Protein concentration of HGF, c-Met, phospho-ERK1/2, ERK1/2, phospho-STAT3, and STAT3. Equal loading of proteins
is illustrated by b-actin band. Values are expressed as means ± S.E.M. (n = 6).aP < 0.05, compared with control;bP < 0.05, compared
with RHDV;cP < 0.05, compared with RHDV + Mel10.
Fulminant hepatic failure, regeneration and melatonin
the regenerative response . PDGF-B is endowed
with cytoprotective activity and promotes hepatocyte
proliferation . In patients with FHF, it has been shown
that PDGF-B plasma levels correlate positively with
survival , suggesting that upregulation of this growth
factor may foster recovery. Demonstration of a critical role
for EGFR in hepatocyte proliferation during the initial
phases of liver regeneration has recently been provided by
generating mice with a liver-specific EGFR deficiency .
Finally, VEGF expression increases markedly during liver
regeneration induced either by partial hepatectomy or drug
intoxication , and it has been suggested that VEGF
signaling facilitates liver recovery from acetaminophen
toxicity by promoting sinusoidal restoration . Melato-
nin therapy triggered the expression of the above growth
factors. We have recently found that cardiotrophin-1
induces a parallel increase in survival and expression of
PDGF-B, EGF, PDGFR, and c-Met in RHDV-infected
rabbits . Thus, it is likely that upregulation of growth
factors and their receptors by melatonin contribute to
stimulate regeneration following severe acute liver injury.
Wound repair activity in the liver is associated with an
orchestrated cellular response leading to tissue regenera-
tion, which involves coordinated activation of signaling
pathways, such as MAPKs and nuclear transcription
factors pathways . The MAPK ERK1/2 is employed
to support the replication of RNA viruses , and in our
experiments, p-ERK levels were significantly increased in
RHDV-infected rabbits. However, ERK1/2 also mediates a
number of cellular processes that include cell differentia-
tion, growth, and survival . ERK activation is a key
signaling pathway involved in the regulation of both
hepatocyte and endothelial cell proliferation, and activation
of the ERK pathway has been shown after partial hepa-
tectomy [50, 51]. Melatonin has been previously reported to
sustain the activation of this survival-promoting pathway in
UVB-stressed cells  and promotes a similar effect
following RHDV infection.
The role of the transcription factor STAT3 in liver
regeneration has also been extensively investigated. Rapid
activation of STAT3 has been well documented during liver
regeneration after partial hepatectomy or liver injury [53,
54], and it is known that HGF, EGF, and hepatitis viral
proteins can also activate STAT3 in hepatocytes .
Phosphorylated STAT3 then activates the transcription of
many target genes that play important roles in promoting
hepatocyte survival and liver regeneration, protect against
hepatocellular damage and reduce liver necrosis-associated
inflammation . We have previously reported that lack of
STAT3 activation, probably mediated by suppressor of
cytokine signaling (SOCS)3 overexpression, contributes to
the inhibition of the regenerative response in RHDV-
infected rabbits . Increased expression of STAT3 could
therefore be partly responsible for the beneficial effects of
melatonin in this animal model of FHF.
Overall, our findings indicate that melatonin prevents
inflammation and enhances regeneration in RHDV-induced
acute viral hepatitis. Thus, results from the present research
and from previous studies [18, 19] provide evidence that
melatonin might have some clinical application in the
management of patients with FHF and suggest that
inflammatory and regenerative mechanisms could be targets
for potential therapeutic use of melatonin, contributing to
temporary support while awaiting a liver transplant.
CIBEREHD is funded by Instituto de Salud Carlos III,
Spain. B. San Miguel is supported by a grant from Digna
Biotech, Spain. This work was partially supported by a
grant from the Ministerio de Ciencia e Innovacio ´ n
M. J. Tun ˜ o ´ n and J. Gonza ´ lez-Gallego were involved in the
concept/design, data analysis, interpretation, and drafting
of the manuscript; B. San Miguel, A. Laliena, I. Crespo and
M. Alvarez collected data and involved in data analysis and
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