3 research outputs found
Overexpression of iNOS and down-regulation of BMPs-2, 4 and 7 in retinoic acid induced cleft palate formation
The present work studied the induction of
cleft palate formation in embryos developed from
pregnant BALB/c mice treated orally with retinoic acid
(RA). Previous studies on mature somatic cell types
showed that RA exerted inhibitory effects on inducible
nitric oxide synthase (iNOS) production. For the first
time, our study has shown that RA actually stimulates
significant expression of iNOS at specific zones of the
affected embryonic palatal tissues at three consecutive
stages, from gestation day 13 (GD13) to day 16 (GD16).
Enzymatically, iNOS facilitates intracellular nitric oxide
(NO) synthesis from L-arginine. When NO reacts with
reactive superoxides it may result in irreparable cell
injury. NO was also reported to induce apoptosis in some
mammalian cell systems. Based on our findings, we
propose that such an increase in NO production might be
associated with apoptosis in the embryonic palatal
tissues in the RA-treated mice. The detrimental effects of
NO resulted in a reduction in proliferating palatal cells
and therefore disturbed the normal plasticity of the
palatal shelves. With iNOS overexpression, our findings
also showed that there was significant concomitant
down-regulation in the expressions of Bone
Morphogenetic Proteins (BMPs) –2, 4, and 7 with
regional variations particularly in the palatal
mesenchymal cells for those embryos developing cleft
palate. Since specific spatial and temporal expressions of BMPs –2, 4, and 7 are critical during normal palatal
morphogenesis, any deficiency in the epithelialmesenchymal interaction may result in retarding growth
at the embryonic palatal shelves. Taken together, our
study has demonstrated cleft palate formation in the
BALB/c embryos involved overexpression of iNOS and
down-regulation of BMPs- 2, 4 and 7
Voluntary oral feeding of rats not requiring a very high fat diet is a clinically relevant animal model of non-alcoholic fatty liver disease (NAFLD)
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
Inhibitors of inducible nitric oxide (NO) synthase are more effective than an NO donor in reducing carbon-tetrachloride induced acute liver injury
The exact functional role of nitric oxide
(NO) in liver injury is currently a source of controversy.
NO is enzymatically synthesized by nitric oxide
synthase (NOS). In this study, we assessed the role of
inducible NOS (iNOS) in carbon tetrachloride (CCl4)-
induced acute liver injury using inhibitors of iNOS, and
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hemisulfate [SMT] and l-N6-(1-iminoethyl)-lysine [LNIL])
and an NO donor (Sodium Nitroprusside [SNP]).
Blood and liver tissues were collected for analysis.
Immunohistochemistry (IHC), serum alanine
aminotransferase (ALT), serum total 8-isoprostane
analysis, RT-PCR, Western Blotting (WB) and EMSA
were done. Our results showed increased levels of ALT,
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these effects but the effect was more pronounced with
SMT and L-NIL. RT-PCR, WB and IHC in CCl4–treated
mice demonstrated upregulation of TNF-a, iNOS, and
COX-2. The administration of iNOS inhibitors with
CCl4 diminished the expression of these
proinflammatory mediators. NF-kB was also
upregulated in CCl4-treated mice and was reversed in
mice pretreated with iNOS inhibitors. SNP pretreated
mice also showed a lower expression of COX-2 when
compared with CCl4 treated mice but TNF-a, iNOS and NF-kB activity were unaffected. We propose that a high
level of nitric oxide is associated with CCl4-induced
acute liver injury and the liver injury can be ameliorated
by decreasing the NO level with iNOS inhibitors and an NO donor with the former more effective in reducing
CCl4-induced liver injury