studies

652A AASLD ABSTRACTS HEPATOLOGY, October, 2015
atohepatitis (NASH). We have also shown that nitro-oxidative
stress might play a key role in the pathogenesis of this dysfunction
and that activity of the NADPH oxidase is increased in the
liver of mice with NASH. The aim of this study was to evaluate
the role played by the NADPHox in the pathogenesis of the
OXPHOS dysfunction in high fat diet (HFD)-fed mice. Material
and Methods. Twenty four C57BL/6J mice were distributed in
four groups: (1) six wild-type (WT) mice fed a standard chow
diet (SCD) (WT/SCD); (2) six WT mice fed a HFD (WT/HFD);
(3) six NADPHox deficient mice on a SCD (NOX —/— /SCD); (4)
six NADPHox deficient mice on a HFD (NOX —/— /HFD). After
six months on these diets, we studied in the liver: histology,
MRC activity (spectrophotometry), fully assembled MRC complexes
(BN-PAGE), subunits of the MRC complexes (BN/SDS-
PAGE), gene expression of these subunits, as well as of TNFα,
IFNγ, MCP-1, caspase-3, collagen α1(I), and TGFβ1 (RT-PCR),
oxidative (thiobarbituric acid-reactive substances, glutathione)
and nitrosative (immunohistochemistry, nitration of MRC proteins)
stress, and oxidative DNA damage [8-hydroxy-2ʹ-deoxyguanosine
(8-OHdG)]. Results: (1) In HFD mice, we found:
severe steatosis, mild inflammatory infiltrates, ballooning
degeneration, pericellular fibrosis, 3-tyrosine nitrated proteins,
and marked increased gene expression of TNFα, IFNγ,
MCP-1, caspase-3, collagen α1(I), and TGFβ1. Activity of all
complexes of the MRC was decreased to about 50-60% of
activity in WT/SCD. Fully assembled complexes were reduced
to 50% to 60% of that found in control mice. The amount of
all studied subunits was markedly decreased, particularly, the
mitochondrial DNA-encoded subunits. Gene expression of
mitochondrial, but not of genomic, DNA-encoded subunits was
decreased to about 60% of control gene expression. 8-OHdG
was increased in mitochondrial, but not in genomic DNA. (2)
The liver of NOX —/— /HFD mice showed steatosis but not infiltrates,
ballooning degeneration pericellular fibrosis, 3-tyrosine
nitrated proteins, or increased gene expression of inflammatory,
apoptosis or fibrosis related proteins. Likewise, OXPHOS
activity, subunits, their gene expression, and assembly of these
subunits in OXPHOS complexes, and 8OHdG were normal in
the liver of these NOX —/— mice on a HFD. Conclusions. This
study shows that NADPHox plays a key role in the pathogenesis
of the OXPHOS dysfunction found in mice on a HFD, as
this dysfunction was not found in NADPHox deficient mice fed
a HFD.
Disclosures:
The following authors have nothing to disclose: José A. Solís-Herruzo, Pablo
Solis-Munoz, Daniel Fernández-Moreira, Teresa Muñoz-Yagüe, Inmaculada
García-Ruiz
897
Dual targeting of nuclear receptors ameliorates NAFLD
pathogenesis in mice
Pedro M. Rodrigues 2 , Marta B. Afonso 2 , André L. Simão 2 , Marta
Caridade 2 , Catarina C. Carvalho 3 , Alexandre Trindade 3 , António
Duarte 3 , Pedro M. Borralho 1 , Mariana V. Machado 4 , Helena Cortez-Pinto
4,5 , Cecília M. Rodrigues 1 , Rui E. Castro 1 ; 1 iMed.ULisboa
& Dep. of Biochemistry and Human Biology, Faculty of Pharmacy,
University of Lisbon, Lisboa, Portugal; 2 iMed.ULisboa, Faculty of
Pharmacy, University of Lisbon, Lisbon, Portugal; 3 Reproduction
and Development, Interdisciplinary Centre of Research in Animal
Health (CIISA), Faculty of Veterinary Medicine, University of Lisbon,
Lisbon, Portugal; 4 Gastrenterology, Hospital Santa Maria,
Lisbon, Portugal; 5 Faculty of Medicine, University of Lisbon, Lisbon,
Portugal
Non-alcoholic fatty liver disease (NAFLD) burden is rapidly
increasing and disease pathogenesis remains incomplete.
We and others have recently characterized miRNAs as novel
NAFLD pathogenic factors. In addition, nuclear receptors (NRs),
namely peroxisome proliferator-activated receptors (PPARs) and
the farnesoid X receptor (FXR) are currently under scrutiny as
promising therapeutic targets for non-alcoholic steatohepatitis
(NASH). In fact, the FXR agonist obeticholic acid (OCA) significantly
ameliorated NASH in a phase 2 clinical trial. We aimed
to: 1) elucidate the role of miR-21 during NAFLD pathogenesis
in mice, particularly regarding modulation of the miR-21 target
PPARα; and 2) evaluate the therapeutic potential of a dual
NR-targeting approach, specifically activation of PPARα and
FXR through miR-21 ablation and OCA administration, respectively.
C57BL/6 wild type (WT; n=24) and miR-21 knockout
(KO; n=24) mice were fed either a standard diet (SD; n=12)
or a fast food diet (FF; n=12) for 25 weeks. Six animals from
each group received OCA (10 mg/kg/day; Intercept Pharmaceuticals,
Inc.) as a dietary admixture. Human liver biopsies
were obtained from morbidly obese NAFLD patients at different
disease stages (n=28). Human and mice liver samples
were processed for histological analysis and for expression of
miR-21, pro-inflammatory cytokines, NRs and proteins of the
insulin signaling pathway, by qRT-PCR and immunoblotting.
Our results showed that WT FF-fed mice developed hepatomegaly,
with livers presenting macrovesicular steatosis and
inflammatory infiltrates, as well as deregulated insulin signaling.
mRNA levels of TNF-α, IL-6, TLR4, IL-1β and NLRP3 were
increased, further evidencing development of steatohepatitis.
miR-21 levels were increased in WT FF-fed mice concomitantly
with decreased expression of PPARα, a correlation also
found in NAFLD patients, increasing from steatosis to less and
more-severe NASH. OCA-fed animals displayed decreased
CYP7A1 and SREBP-1c liver expression. Further, WT FF+O-
CA-fed mice exhibited decreased steatosis and miR-21 expression,
compared with WT FF-fed mice. In addition, KO FF-fed
mice exhibited significantly reduced liver inflammation, as well
as steatosis severity, in parallel with increased PPARα, comparing
with WT FF-fed mice. Significantly, improvement of the
above histological, biochemical, metabolic and inflammatory
parameters was augmented in KO FF+OCA-fed mice. In conclusion,
our results indicate that miR-21 downregulation, leading
to increased PPARα, together with FXR activation by OCA,
strongly ameliorate NASH in mice, highlighting the therapeutic
potential of novel dual-targeting therapies for human NAFLD.
Disclosures:
Helena Cortez-Pinto - Advisory Committees or Review Panels: Norgine, Lundbeck;
Speaking and Teaching: Janssen, Gilead Janssen
The following authors have nothing to disclose: Pedro M. Rodrigues, Marta B.
Afonso, André L. Simão, Marta Caridade, Catarina C. Carvalho, Alexandre
Trindade, António Duarte, Pedro M. Borralho, Mariana V. Machado, Cecília M.
Rodrigues, Rui E. Castro
898
Alteration of Neutrophil-derived Lipocalin 2 in Bone
Marrow Cells Modulates Non-alcoholic Steatohepatitis
in Mice
Dewei Ye 1,2 , Yu Wang 2,1 , Karen S. L. Lam 1,2 , Aimin Xu 1,2 ; 1 State
Key Laboratory of Pharmaceutical Biotechnology, The University of
Hong Kong, Hong Kong, Hong Kong; 2 Department of Medicine,
The University of Hong Kong, Hong Kong, Hong Kong
Background and Aims: Hepatic infiltration of neutrophils has
been characterized as one of key histological features of nonalcoholic
steatohepatitis (NASH) in rodents and humans. Lipocalin-2
(LCN2) is a secretory glycoprotein originally identified
from neutrophil granules. This study aims to investigate the
potential role of LCN2-chimerism in bone marrow-derived cells
in the pathogenesis of NASH in mice. Methods: LCN2 knockout