Animal models used to study the
pathogenesis of non-alcoholic fatty liver disease
(NAFLD) are, in general, either genetically altered, or
fed with a diet that is extremely high in fat or
carbohydrates. Recent findings support the role of
oxidative stress, lipid peroxidation and inflammation as
probable causative factors. We hypothesize that not only
the amount of dietary fat, but the quality of fat is also
important in inducing NAFLD. Based on previous
observations that female rats fed a diet comprising
unsaturated fatty acids are susceptible to liver injury, we
proposed that female rats fed with a diet containing fish
oil and dextrose would develop pathological and
biochemical features of NAFLD. We fed a highly
unsaturated fat diet (30% fish oil) to female SpragueDawley rats (180-200g), consumed ad libitum for 8
weeks (NAFLD; n=6-8 ). Control animals (CF; n=6-8)
were fed with an isocaloric regular rat chow. At killing,
blood and liver samples were collected for serum alanine
aminotransferase (ALT), histology and molecular
analysis. Each histological sample was evaluated for
fatty liver (graded from 0 to 4+ according to the amount
of fatty change), necrosis (number of necrotic foci
(no./mm2
) and inflammation (cells per mm2
). The
amount of collagen formation was estimated based on
the amount of Sirius Red staining. Reverse transcriptase
polymerase chain reaction (RT-PCR) was carried out for
tumor necrosis factor alpha (TNF-α), cyclooxygenase-2
(COX-2), inducible nitric oxide synthase (iNOS),
adiponectin, glutathione peroxidase (GPx), superoxide
dismutase (Cu/Zn SOD) and catalase (CAT). Western
Blot analysis was done for cyclooxygenases-2 (COX-2),
inducible nitric oxide synthase (iNOS) and nitrotyrosine.
Electrophoretic mobility shift assay was performed for
nuclear factor-kappa B (NF-κB) activity. NAFLD rats
had a significantly higher serum ALT level, amount of
collagen formation, fatty liver, necrosis and
inflammation when compared with the chow-fed control
rats. mRNA and protein levels of NF-κB regulated
genes, which included TNF-alpha, COX-2 and iNOS
were also significantly (p<0.01; p<0.01; p<0.05
respectively) upregulated in the NAFLD group when
compared with the chow-fed control rats. mRNA levels
of antioxidants CAT and GPX were reduced by 35% and
50% respectively in the NAFLD group. However, Cu/Zn
SOD mRNA was similar in both groups. The mRNA
level of adiponectin was also reduced in NAFLD group.
NF-κB activity was markedly increased in the NAFLD
rats (p<0.01). The level of oxidative stress, represented
by the formation of nitrotyrosine, was significantly
elevated in the NAFLD rats (p<0.01). We conclude that
NAFLD rats demonstrated several features of NAFLD,
which included fatty liver, inflammation, necrosis,
increased oxidative stress, an imbalance between pro and
antioxidant enzymes mRNAs, reduced adiponectin
levels and upregulation of pro-inflammatory mediators.
We propose that female rats fed with a diet containing
highly unsaturated fatty acids are an extremely useful
model for the study of NAFLD