الفهرس 
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Abstract
The current work was designed to investigate the potential
antifibrotic effect of honokiol in Con A immunological model of liver
fibrosis as well as its underlying molecular mechanisms by studying
its effects on some key events such as oxidative stress, inflammation
and fibrogenesis signaling pathway. To fulfill these goals, this study
was divided into two parts:
I. Screening the hepatoprotective dose of honokiol against
Con A induced acute hepatotoxicity: animals were divided
randomly into five groups and treated for 5 days as follow:
honokiol once daily at doses of (5, 10 & 20 mg/kg) orally
and/or Con A (20 mg/kg; IV) on day 5.
The following parameters were investigated:
1-Assessment of Liver enzymes: ALT&AST.
2-Histopathological examination of liver tissue using H&E stain.
The results of the present study can be summarized as follows:
1- A single Con A injection induced acute hepatotoxicity
represented by significant ALT and AST increase associated
with histopathological alterations such as hepatocytes
degeneration and inflammatory cells infiltration.
2- Screening different doses of honokiol (5, 10 & 20 mg/kg)
revealed that, all honokiol doses significanlty reduced serum
ALT and AST as compared to Con A group.
Summary and Conclusions
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3- Histopathological examination using H&E stain revealed that
10 mg/kg dose almost preserved the normal architecture of the
liver.
Collectively, the results of the acute study proved that, honokiol
at dose of 10 mg/kg is more protective than 5 & 20 mg/kg. Therefore
the chronic study was carried out using the 10 mg/kg dose.
II. Studying the potential antifibrotic mechanisms of honokiol
against chronic Con A model of liver fibrosis: animals were
divided randomly into four groups and treated for 4 weeks as
follow: honokiol five times weekly orally at dose (10 mg/kg)
and/or Con A (20 mg/kg; IV) once weekly.
The following parameters were investigated:
1-Hepatotoxicity indices: Liver index (liver weight / body weight)
x100, Alanine aminotransferase (ALT), Aspartate aminotransferase
(AST), and albumin.
2- Oxidative stress markers: Liver content of reduced glutathione
(GSH), Superoxide dismutase activity (SOD) and lipid peroxides
(MDA).
3- Inflammatory markers: Liver contents of Tumor necrosis factor
alpha (TNF-α), Interferon gamma (INF-γ), nuclear factor kappa b NF-
κB (p65) subunit expression and inducible nitric oxide synthase
enzyme expression (iNOS).
4-Liver fibrosis markers: Liver content of hydroxyproline and alpha
smooth muscle actin expression (α-SMA).
Summary and Conclusions
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5- TGF-β/SMAD pathway markers: Liver content of TGF-β and
p.samd2/3 expression.
6- Histopathological examination: Hematoxylin and Eosin for
routine histological examination, Masson’s trichrome for detection of
collagen fibers.
The results of the present study can be summarized as follows:
1- In Con A group, relative liver weight, serum ALT and AST
were significantly elevated while serum albumin was
significantly reduced when compared to control group.
However, co-treatment with honokiol restored all the levels of
these parameters.
2- chronic Con A injection induced oxidative stress as it
significantly reduced GSH level by 34 % and SOD activity by
36 % while significantly increased lipid peroxides level by 44%
as compared to control values. Treatment of animals with
honokiol concomitantly with Con A afforded significant
protection against Con A intoxication as it normalized the
values of GSH, SOD and lipid peroxides levels as compared to
control group.
3- Con A also showed pro-inflammatory response evidenced by
marked elevation in TNF-α and INF-γ levels by 159% and 89 %
respectively as compared to control group while concomitant
Summary and Conclusions
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treatment with honokiol prevented this elevation. The
inflammatory effect of Con A was further confirmed by the
immunohistochemical detection of NF-κB and iNOS as shown
by intense brown staining.
4- Liver fibrosis was evaluated histologically by visualizing the
blue color of collagen fibers using Masson’s trichrome stain
followed by morphometric analysis. In Con A group, collagen
fibers were heavily deposited around portal tract and central
vein forming pseudolobules. Morphometric analysis revealed
an area percent of 14.6%. Interestingly, honokiol co-treatment
prevented the abnormal collagen fibers deposition and induced
a significant decrease in the area percent as compared to Con A
group.
5- Biochemical measurement of hydroxyproline supported these
results in which Con A induced a significant increase in the
liver hydroxyproline content reaching 1.5 fold as compared to
control group. On the other hand, co-treatment with honokiol
induced a significant decrease the hydroxyproline content as
compared to Con A group. Additionally, immunohistochemical
detection of α-SMA revealed minimal staining in the blood
vessels of the control group while marked expression was
observed periportally and perisinusoidally in the Con A group
as shown by the intense brown staining. Honokiol co-treatment
markedly attenuated this elevated expression.
Summary and Conclusions
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6- Additionally, Con A activated TGF-β/SMAD signalling as
evidenced by high TGF-β levels as well as increased p.samd2/3
expression. In Con A group, TGF-β was significantly increased
by 40.5% as compared to control group. Moreover, p.samd2/3
was highly expressed as shown by intense nuclear brown
staining that quantitated by 55.5% increase in optical density as
compared to control group. On the other hand, honokiol cotreatment
attenuated the activation of the pathway.
7- Histopathological examination using H & E stain showed heavy
cells infiltration around most portal areas, sever congestion and
fibroblastic cells proliferation in the periductal tissue
surrounding the bile ducts in the portal area in the Con A group.
Interestingly, honokiol preserved the normal architecture of the
hepatic parenchyma when given concurrently with Con A.
In conclusion, the present study clearly demonstrates that honokiol
could be promising antifibrotic agent against Con A induced liver
fibrosis. Furthermore, this study sheds light on the mechanisms
involved in its antifibrotic effect. It guards against oxidative stress
induced by Con A by restoring cellular defense mechanism, through
restoring the liver GSH content, SOD activity and normalizing lipid
peroxidation. In addition, the antifibrotic effect was mediated through
attenuating the TGF-β/SMAD signalling as well as decreasing the
expression of NF-κB p65 and subsequent production of proSummary
and Conclusions
- 155 -
inflammatory enzyme iNOS and pro-inflammatory cytokines (TNF-α
and INF-γ) (Fig.30). Further investigations are warranted to establish
the clinical applicability of honokiol in patients with chronic liver
diseases, especially those associated with active fibrogenesis